Dengue Viral Infection : World Health Organisation (WHO) Guidelines

Dengue Virus Source
Dengue.jpg
A TEM micrograph showing Dengue virus virions (the cluster of dark dots near the center). Source

Brief Information for Public in General

Dengue is a mosquito-borne viral disease that has rapidly spread in all regions of WHO in recent years. Dengue virus is transmitted by female mosquitoes mainly of the species Aedes aegypti and, to a lesser extent, A. albopictus. The disease is widespread throughout the tropics, with local variations in risk influenced by rainfall, temperature and unplanned rapid urbanization.

Severe dengue (also known as Dengue Haemorrhagic Fever) was first recognized in the 1950s during dengue epidemics in the Philippines and Thailand. Today, severe dengue affects most Asian and Latin American countries and has become a leading cause of hospitalization and death among children in these regions.

There are 4 distinct, but closely related, serotypes of the virus that cause dengue (DEN-1, DEN-2, DEN-3 and DEN-4). Recovery from infection by one provides lifelong immunity against that particular serotype. However, cross-immunity to the other serotypes after recovery is only partial and temporary. Subsequent infections by other serotypes increase the risk of developing severe dengue.

Aedes aegypti; adult female mosquito taking a blood meal on human skin.

Dengue
Aedes aegypti

Transmission

The Aedes aegypti mosquito is the primary vector of dengue. The virus is transmitted to humans through the bites of infected female mosquitoes. After virus incubation for 4–10 days, an infected mosquito is capable of transmitting the virus for the rest of it’s life.

Infected humans are the main carriers and multipliers of the virus, serving as a source of the virus for uninfected mosquitoes. Patients who are already infected with the dengue virus can transmit the infection (for 4–5 days; maximum 12) via Aedes mosquitoes after their first symptoms appear.

The Aedes aegypti mosquito lives in urban habitats and breeds mostly in man-made containers. Unlike other mosquitoes Ae. aegypti is a day-time feeder; its peak biting periods are early in the morning and in the evening before dusk. Female Ae. aegypti bites multiple people during each feeding period.

Aedes albopictus, a secondary dengue vector in Asia, has spread to North America and Europe largely due to the international trade in used tyres (a breeding habitat) and other goods (e.g. lucky bamboo). Ae. albopictus is highly adaptive and, therefore, can survive in cooler temperate regions of Europe. It’s spread is due to its tolerance to temperatures below freezing, hibernation, and ability to shelter in micro-habitats.

Characteristics

Dengue fever is a severe, flu-like illness that affects infants, young children and adults, but seldom causes death.

Dengue should be suspected when a high fever (40°C/104°F) is accompanied by 2 of the following symptoms: severe headache, pain behind the eyes, muscle and joint pains, nausea, vomiting, swollen glands or rash. Symptoms usually last for 2–7 days, after an incubation period of 4–10 days after the bite from an infected mosquito.

Severe dengue is a potentially deadly complication due to plasma leaking, fluid accumulation, respiratory distress, severe bleeding, or organ impairment.

Warning signs occur 3–7 days after the first symptoms in conjunction with a decrease in temperature (below 38°C/100°F) and include: severe abdominal pain, persistent vomiting, rapid breathing, bleeding gums, fatigue, restlessness and blood in vomit. The next 24–48 hours of the critical stage can be lethal; proper medical care is needed to avoid complications and risk of death.

Treatment

There is no specific treatment for dengue fever.

For severe dengue, medical care by physicians and nurses experienced with the effects and progression of the disease can save lives – decreasing mortality rates from more than 20% to less than 1%. Maintenance of the patient’s body fluid volume is critical to severe dengue care.

Immunization

There is no vaccine to protect against dengue. However, major progress has been made in developing a vaccine against dengue/severe dengue. Three tetravalent live-attenuated vaccines are under development in phase II and phase III clinical trials, and 3 other vaccine candidates (based on subunit, DNA and purified inactivated virus platforms) are at earlier stages of clinical development. WHO provides technical advice and guidance to countries and private partners to support vaccine research and evaluation.

Prevention and control

Havana: A local health worker uses a torch to check for signs of water and mosquito eggs inside tyres in a tyre depot.

 At present, the only method to control or prevent the transmission of dengue virus is to combat vector mosquitoes through:
  • preventing mosquitoes from accessing egg-laying habitats by environmental management and modification;
  • disposing of solid waste properly and removing artificial man-made habitats;
  • covering, emptying and cleaning of domestic water storage containers on a weekly basis;
  • applying appropriate insecticides to water storage outdoor containers;
  • using of personal household protection such as window screens, long-sleeved clothes, insecticide treated materials, coils and vaporizers;
  • improving community participation and mobilization for sustained vector control;
  • applying insecticides as space spraying during outbreaks as one of the emergency vector-control measures;
  • active monitoring and surveillance of vectors should be carried out to determine effectiveness of control interventions.
Reference
  1. Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL et.al. The global distribution and burden of dengue. Nature;496:504-507.
  2. Brady OJ, Gething PW, Bhatt S, Messina JP, Brownstein JS, Hoen AG et al. Refining the global spatial limits of dengue virus transmission by evidence-based consensus. PLoS Negl Trop Dis. 2012;6:e1760. doi:10.1371/journal.pntd.0001760.

Dengue Viruses

Viruses are tiny agents that can infect a variety of living organisms, including bacteria, plants, and animals. Like other viruses, the dengue virus is a microscopic structure that can only replicate inside a host organism.

Discovery of the Dengue Viruses

 The dengue viruses are members of the genus Flavivirus in the family Flaviviridae. Along with the dengue virus, this genus also includes a number of other viruses transmitted by mosquitoes and ticks that are responsible for human diseases. Flavivirus includes the yellow fever, West Nile, Japanese encephalitis, and tick-borne encephalitis viruses.

In 1943, Ren Kimura and Susumu Hotta first isolated the dengue virus. These two scientists were studying blood samples of patients taken during the 1943 dengue epidemic in Nagasaki, Japan. A year later, Albert B. Sabin and Walter Schlesinger independently isolated the dengue virus. Both pairs of scientists had isolated the virus now referred to as dengue virus 1 (DEN-1).

Is DEN-1 the only type of dengue virus?

The Dengue Serotypes

 Dengue infections are caused by four closely related viruses named DEN-1, DEN-2, DEN-3, and DEN-4. These four viruses are called serotypes because each has different interactions with the antibodies in human blood serum. The four dengue viruses are similar — they share approximately 65% of their genomes — but even within a single serotype, there is some genetic variation. Despite these variations, infection with each of the dengue serotypes results in the same disease and range of clinical symptoms.

Are these four viruses all found in the same regions of the world?

In the 1970s, both DEN-1 and DEN-2 were found in Central America and Africa, and all four serotypes were present in Southeast Asia. By 2004, however, the geographical distribution of the four serotypes had spread widely. Now all four dengue serotypes circulate together in tropical and subtropical regions around the world. The four dengue serotypes share the same geographic and ecological niche.

Where did the dengue viruses first come from?

Scientists hypothesize that the dengue viruses evolved in non-human primates and jumped from these primates to humans in Africa or South-east Asia between 500 and 1,000 years ago.

After recovering from an infection with one dengue serotype, a person has immunity against that particular serotype.

Does infection with one serotype protect against future dengue infections with the other serotypes?

Individuals are  protected from infections with the remaining three serotypes for two to three months after the first dengue infection.

Unfortunately, it is not long-term protection.

After that short period, a person can be infected with any of the remaining three dengue serotypes. Researchers have noticed that subsequent infections can put individuals at a greater risk for severe dengue illnesses than those who have not been previously infected.

Dengue Virus Genome and Structure

 The dengue virus genome is a single strand of RNA. It is referred to as positive-sense RNA because it can be directly translated into proteins. The viral genome encodes ten genes. The genome is translated as a single, long polypeptide and then cut into ten proteins.
A diagram shows the dengue virus RNA genome with its structural and non-structural regions labeled. The RNA is depicted as a horizontal cylinder separated into several colored sections of varying sizes. A thin black coiled line representing untranslated RNA extends from the cylinder's lefthand terminus and is labeled the 5 prime UTR. From left to right, the genes encoded by the dengue virus genome are: the capsid, labeled C and colored light brown; the membrane, labeled M and colored orange; the envelope, labeled E and colored blue; and several non-structural genes, including NS1 (green), NS2A (red), NS2B (dark brown), NS3 (yellow), NS4A (dark orange), NS4B (teal), and NS5 (purple). NS5 is the longest gene; NS2A and NS2B have the shortest lengths. A region of untranslated RNA at the cylinder's righthand terminus is labeled the 3 prime UTR.
Dengue virus genome The dengue virus genome encodes three structural (capsid [C], membrane [M], and envelope [E]) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. © 2010 Nature Publishing Group Guzman, M. G. et al. Dengue: A continuing global threat. Nature Reviews Microbiology 8, S7–S16 (2010). doi:10.1038/nrmicro2460 All rights reserved.

What are the roles of these ten proteins?

Three are structural proteins: the capsid (C), envelope (E), and membrane (M) proteins. Seven are nonstructural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. These nonstructural proteins play roles in viral replication and assembly.

The structure of the dengue virus is roughly spherical, with a diameter of approximately 50 nm (1 nm is one millionth of 1 mm) (Figure). The core of the virus is the nucleocapsid, a structure that is made of the viral genome along with C proteins. The nucleocapsid is surrounded by a membrane called the viral envelope, a lipid bilayer that is taken from the host. Embedded in the viral envelope are 180 copies of the E and M proteins that span through the lipid bilayer. These proteins form a protective outer layer that controls the entry of the virus into human cells.

A schematic of the dengue virus shows its primary structural features. The virus is depicted as an orange hexagon encapsulated inside a light brown circle. The hexagon is the nucleocapsid and the brown circle is the viral envelope. Thin red material coiled up inside the nucleocapsid represents the viral genome. Seven red lines radiate outward from the viral envelope in a symmetrical orientation. Each line has a green sphere at the end. These protrusions are E and M proteins.
Dengue virus structure The dengue virus has a roughly spherical shape. Inside the virus is the nucleocapsid, which is made of the viral genome and C proteins. The nucleocapsid is surrounded by a membrane called the viral envelope, a lipid bilayer that is taken from the host. Embedded in the viral envelope are E and M proteins that span through the lipid bilayer. These proteins form a protective outer layer that controls the entry of the virus into human cells. © 2011 Nature Education All rights reserved.

Dengue Virus Replication and Infectious Cycle

 How does the virus behave once it enters the human body?
The dengue viral replication process begins when the virus attaches to a human skin cell (Figure). After this attachment, the skin cell’s membrane folds around the virus and forms a pouch that seals around the virus particle. This pouch is called an endosome. A cell normally uses endosomes to take in large molecules and particles from outside the cell for nourishment. By hijacking this normal cell process, the dengue virus is able to enter a host cell.

Dengue virus replication The dengue virus attaches to the surface of a host cell and enters the cell by a process called endocytosis. Once deep inside the cell, the virus fuses with the endosomal membrane and is released into the cytoplasm. The virus particle comes apart, releasing the viral genome. The viral RNA (vRNA) is translated into a single polypeptide that is cut into ten proteins, and the viral genome is replicated. Virus assembly occurs on the surface of the endoplasmic reticulum (ER) when the structural proteins and newly synthesized RNA bud out from the ER. The immature viral particles are transported through the trans-Golgi network (TGN), where they mature and convert to their infectious form. The mature viruses are then released from the cell and can go on to infect other cells. © 2005 Nature Publishing Group Mukhopadhyay, S., Kuhn, R. J., & Rossmann M. G. A structural perspective of the flavivirus life cycle. Nature Reviews Microbiology 3, 13–22 (2005). doi:10.1038/nrmicro1067 All rights reserved.

Once the virus has entered a host cell, the virus penetrates deeper into the cell while still inside the endosome.

How does the virus exit the endosome, and why?

Researchers have learned that two conditions are needed for the dengue virus to exit the endosome:

  1. The endosome must be deep inside the cell where the environment is acidic.
  2. The endosomal membrane must gain a negative charge.

These two conditions allow the virus envelope to fuse with the endosomal membrane, and that process releases the dengue nucleo-capsid into the cytoplasm of the cell.

Once it is released into the cell cytoplasm, how does the virus replicate itself?

In the cytoplasm, the nucleocapsid opens to uncoat the viral genome. This process releases the viral RNA into the cytoplasm. The viral RNA then hijacks the host cell’s machinery to replicate itself. The virus uses ribosomes on the host’s rough endoplasmic reticulum (ER) to translate the viral RNA and produce the viral polypeptide. This polypeptide is then cut to form the ten dengue proteins.

The newly synthesized viral RNA is enclosed in the C proteins, forming a nucleo-capsid. The nucleo-capsid enters the rough ER and is enveloped in the ER membrane and surrounded by the M and E proteins. This step adds the viral envelope and protective outer layer. The immature viruses travel through the Golgi apparatus complex, where the viruses mature and convert into their infectious form. The mature dengue viruses are then released from the cell and can go on to infect other cells.

Summary

 The dengue virus is a tiny structure that can only replicate inside a host organism. The four closely related dengue viruses — DEN-1, DEN-2, DEN-3, and DEN-4 — are found in the same regions of the world. The dengue virus is a roughly spherical structure composed of the viral genome and capsid proteins surrounded by an envelope and a shell of proteins. After infecting a host cell, the dengue virus hijacks the host cell’s machinery to replicate the viral RNA genome and viral proteins. After maturing, the newly synthesized dengue viruses are released and go on to infect other host cells.
Source:  www.nature.com

Dengue case classification

Dengue has a wide spectrum of clinical presentations, often with unpredictable clinical evolution and outcome. While most patients recover following a self-limiting non-severe clinical course, a small proportion progress to severe disease, mostly characterized by plasma leakage with or without haemorrhage.

Intravenous rehydration is the therapy of choice; this intervention can reduce the case fatality rate to less than 1% of severe cases. The group progressing from non-severe to severe disease is difficult to define, but this is an important concern since appropriate treatment may prevent these patients from developing more severe clinical conditions.

Triage, appropriate treatment, and the decision as to where this treatment should be given (in a health care facility or at home) are influenced by the case classification for dengue. This is even more the case during the frequent dengue outbreaks worldwide, where health services need to be adapted to cope with the sudden surge in demand.

Symptomatic dengue virus infections were grouped into three categories:

undifferentiated fever,

dengue fever (DF) 

and

dengue haemorrhagic fever (DHF).

DHF was further classified into four severity grades, with grades III and IV being defined as dengue shock syndrome (DSS).

There have been many reports of difficulties in the use of this classification , which were summarized in a systematic literature review. Difficulties in applying the criteria for DHF in the clinical situation, together with the increase in clinically severe dengue cases which did not fulfil the strict criteria of DHF, led to the request for the classification to be reconsidered. Currently the classification into DF/DHF/DSS continues to be widely used.

A WHO/TDR-supported prospective clinical multi-centre study across dengue-endemic regions was set up to collect evidence about criteria for classifying dengue into levels of severity. The study findings confirmed that, by using a set of clinical and/or laboratory parameters, one sees a clear-cut difference between patients with severe dengue and those with non-severe dengue.

However, for practical reasons it was desirable to split the large group of patients with non-severe dengue into two subgroups — patients with warning signs and those without them. Criteria for diagnosing dengue (with or without warning signs) and severe dengue are presented in Figure. It must be kept in mind that even dengue patients without warning signs may develop severe dengue.

Expert consensus groups in Latin America (Havana, Cuba, 2007), South-East Asia (Kuala  Lumpur, Malaysia, 2007), and at WHO headquarters in Geneva, Switzerland in 2008 agreed that:

“dengue is one disease entity with different clinical presentations and often with unpredictable clinical evolution and outcome”;

the classification into levels of severity has a high potential for being of practical use in the clinicians’ decision as to where and how intensively the patient should be observed and treated (i.e. triage, which is particularly useful in outbreaks), in more consistent reporting in the national and international surveillance system, and as an end-point measure in dengue vaccine and drug trials.

Suggested Dengue Case Classification And Levels of Severity

This model for classifying dengue has been suggested by an expert group (Geneva, Switzerland, 2008) and is currently being tested in 18 countries by comparing it’s performance in practical settings to the existing WHO case classification.

Dengue slide

TRANSMISSION

The host

Dengue hmg fdengue hmg fev

After an incubation period of 4–10 days, infection by any of the four virus serotypes can produce a wide spectrum of illness, although most infections are asymptomatic or sub-clinical. Primary infection is thought to induce lifelong protective immunity to the infecting serotype [Halstead SB. Etiologies of the experimental dengues of Siler and Simmons. American Journal of Tropical Medicine and Hygiene, 1974, 23:974–982.]

Individuals suffering an infection are protected from clinical illness with a different serotype within 2–3 months of the primary infection but with no long-term cross-protective immunity.

Individual risk factors determine the severity of disease and include secondary infection, age, ethnicity and possibly chronic diseases (bronchial asthma, sickle cell anaemia and diabetes mellitus). Young children in particular may be less able than adults to compensate for capillary leakage and are consequently at greater risk of dengue shock.

Sero-epidemiological studies in Cuba and Thailand consistently support the role of secondary heterotypic infection as a risk factor for severe dengue, although there are a few reports of severe cases associated with primary infection.

[Halstead SB, Nimmannitya S, Cohen SN. Observations related to pathogenesis of dengue hemorrhagic fever. IV. Relation of disease severity to antibody response and virus recovered. Yale Journal of Biology and Medicine, 1970, 42:311–328.]

[Sangkawibha N et al. Risk factors in dengue shock syndrome: a prospective epidemiologic study in Rayong, Thailand. I. The 1980 outbreak. American Journal of Epidemiology, 1984;120:653–669.]

[Guzman MG et al. Epidemiologic studies on dengue in Santiago de Cuba, 1997. American Journal of Epidemiology, 2000, 152(9):793–799.]

[Halstead SB. Pathophysiology and pathogenesis of dengue haemorrhagic fever. In: Thongchareon P, ed. Monograph on dengue/dengue haemorrhagic fever. New Delhi, World Health Organization, Regional Office for South-East Asia, 1993 (pp 80–103).]

The time interval between infections and the particular viral sequence of infections may also be of importance.

For instance, a higher case fatality rate was observed in Cuba when DEN-2 infection followed a DEN-1 infection after an interval of 20 years compared to an interval of four years.

Severe dengue is also regularly observed during primary infection of infants born to dengue-immune mothers.

Antibody-dependent enhancement (ADE) of infection has been hypothesized as a mechanism to explain severe dengue in the course of a secondary infection and in infants with primary infections.

[Halstead SB. Antibody, macrophages, dengue virus infection, shock, and hemorrhage: a pathogenetic cascade. Reviews of Infectious Diseases, 1989, 11(Suppl4):S830–S839][Halstead SB, Heinz FX. Dengue virus: molecular basis of cell entry and pathogenesis, 25-27 June 2003, Vienna, Austria. Vaccine, 2005, 23(7):849–856]

In this model, non-neutralizing, cross-reactive antibodies raised during a primary infection, or acquired passively at birth, bind to epitopes on the surface of a heterologous infecting virus and facilitate virus entry into Fc-receptor-bearing cells. The increased number of infected cells is predicted to result in a higher viral burden and induction of a robust host immune response that includes inflammatory cytokines and mediators, some of which may contribute to capillary leakage. During a secondary infection, cross-reactive memory T cells are also rapidly activated, proliferate, express cytokines and die by apoptosis in a manner that generally correlates with overall disease severity. Host genetic determinants might influence the clinical outcome of infection [Kouri GP, Guzman MG. Dengue haemorrhagic fever/dengue shock syndrome: lessons from the Cuban epidemic, 1981. Bulletin of the World Health Organization, 1989, 67(4):375–380.][Sierra B, Kouri G, Guzman MG. Race: a risk factor for dengue hemorrhagic fever. Archives of Virology, 2007, 152(3):533–542.], though most studies have been unable to adequately address this issue. Studies in the American region show the rates of severe dengue to be lower in individuals of African ancestry than those in other ethnic groups.

The dengue virus enters via the skin while an infected mosquito is taking a blood-meal.

During the acute phase of illness the virus is present in the blood and its clearance from this compartment generally coincides with defervescence. Humoral and cellular immune responses are considered to contribute to virus clearance via the generation of neutralizing antibodies and the activation of CD4+ and CD8+ T lymphocytes. In addition, innate host defence may limit infection by the virus. After infection, serotype-specific and cross-reactive antibodies and CD4+ and CD8+ T cells remain measurable for years.

Plasma leakage, haemoconcentration and abnormalities in homeostasis characterize severe dengue. The mechanisms leading to severe illness are not well defined but the immune response, the genetic background of the individual and the virus characteristics may all contribute to severe dengue.

Recent data suggest that endothelial cell activation could mediate plasma leakage.

[Avirutnan P et al. Dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis. Journal of Immunology, 1998, 161:6338–6346]

[Cardier JE et al. Proinflammatory factors present in sera from patients with acute dengue infection induce activation and apoptosis of human microvascular endothelial cells: possible role of TNF-alpha in endothelial cell damage in dengue. Cytokine,
2005, 30(6):359–365.]

Plasma leakage is thought to be associated with functional rather than destructive effects on endothelial cells. Activation of infected monocytes and T cells, the complement system and the production of mediators, monokines, cytokines and soluble receptors may also be involved in endothelial cell dysfunction.

Thrombocytopenia may be associated with alterations in megakaryocytopoieses by the infection of human haematopoietic cells and impaired progenitor cell growth, resulting in platelet dysfunction (platelet activation and aggregation), increased destruction or consumption (peripheral sequestration and consumption).

Haemorrhage may be a consequence of the thrombocytopenia and associated platelet dysfunction or disseminated intravascular coagulation. In summary, a transient and reversible imbalance of inflammatory mediators, cytokines and chemokines occurs during severe dengue, probably driven by a high early viral burden, and leading to dysfunction of vascular endothelial cells, derangement of the haemocoagulation system then to plasma leakage, shock and bleeding.

Transmission of the dengue virus

Humans are the main amplifying host of the virus. Dengue virus circulating in the blood of viraemic humans is ingested by female mosquitoes during feeding. The virus then infects the mosquito mid-gut and subsequently spreads systemically over a period of 8–12 days. After this extrinsic incubation period, the virus can be transmitted to other humans during subsequent probing or feeding. The extrinsic incubation period is influenced in part by environmental conditions, especially ambient temperature. Thereafter the mosquito remains infective for the rest of its life. Ae. aegypti is one of the most efficient vectors for arboviruses because it is highly anthropophilic, frequently bites several times before completing oogenesis, and thrives in close proximity to humans.

Vertical transmission (transovarial transmission) of dengue virus has been demonstrated in the laboratory but rarely in the field. The significance of vertical transmission for maintenance of the virus is not well understood. Sylvatic dengue strains in some parts of Africa and Asia may also lead to human infection, causing mild illness. Several factors can influence the dynamics of virus transmission — including environmental and climate factors, host pathogen interactions and population immunological factors. Climate directly influences the biology of the vectors and thereby their abundance and distribution; it is consequently an important determinant of vector-borne disease epidemics.

CLINICAL MANAGEMENT AND DELIVERY OF CLINICAL SERVICES

OVERVIEW

Dengue infection is a systemic and dynamic disease. It has a wide clinical spectrum that includes both severe and non-severe clinical manifestations [Rigau-Perez JG et al. Dengue and dengue haemorrhagic fever. Lancet, 1998, 352:971–977.] After the incubation period, the illness begins abruptly and is followed by the three phases — febrile, critical and recovery.

dengue hmg fe

For a disease that is complex in its manifestations, management is relatively simple, inexpensive and very effective in saving lives so long as correct and timely interventions are instituted. The key is early recognition and understanding of the clinical problems during the different phases of the disease, leading to a rational approach to case management and a good clinical outcome. An overview of good and bad clinical practices is given in Textbox.

Good clinical practice and bad clinical practice

Activities (triage and management decisions) at the primary and secondary care levels (where patients are first seen and evaluated) are critical in determining the clinical outcome of dengue. A well-managed front-line response not only reduces the number of unnecessary hospital admissions but also saves the lives of dengue patients. Early notification of dengue cases seen in primary and secondary care is crucial for identifying outbreaks and initiating an early response. Differential diagnosis needs to be considered.

The Course of Dengue Illness [Yip WCL. Dengue haemorrhagic fever: current approaches to management. Medical Progress, October 1980.]

Febrile phase

Patients typically develop high-grade fever suddenly. This acute febrile phase usually lasts 2–7 days and is often accompanied by facial flushing, skin erythema, generalized body-ache, myalgia, arthralgia and headache [Rigau-Perez JG et al. Dengue and dengue haemorrhagic fever. Lancet, 1998, 352:971–977.]

Some patients may have sore throat, injected pharynx and conjunctival injection. Anorexia, nausea and vomiting are common. It can be difficult to distinguish dengue clinically from non-dengue febrile diseases in the early febrile phase. A positive tourniquet test in this phase increases the probability of dengue.

[Kalayanarooj S et al. Early clinical and laboratory indicators of acute dengue illness. Journal of Infectious Diseases, 1997, 176:313–321.]

[Phuong CXT et al. Evaluation of the World Health Organization standard tourniquet
test in the diagnosis of dengue infection in Vietnam. Tropical Medicine and International Health, 2002, 7:125–132.]

dengue pic           dengue picss IJD-55-79-g002               IJD-55-79-g003

In addition, these clinical features are indistinguishable between severe and non-severe dengue cases. Therefore monitoring for warning signs and other clinical parameters  is crucial to recognizing progression to the critical phase. Mild haemorrhagic manifestations like petechiae and mucosal membrane bleeding (e.g. nose and gums) may be seen [Kalayanarooj S et al. Early clinical and laboratory indicators of acute dengue illness. Journal of Infectious Diseases, 1997, 176:313–321.][Balmaseda A et al. Assessment of the World Health Organization scheme for classification of dengue severity in Nicaragua. American Journal of Tropical Medicine and Hygiene, 2005, 73:1059–1062.]

Massive vaginal bleeding (in women of childbearing age) and gastrointestinal bleeding may occur during this phase but is not common [Balmaseda A et al. Assessment of the World Health Organization scheme for classification of dengue severity in Nicaragua. American Journal of Tropical Medicine and Hygiene, 2005, 73:1059–1062.]

The liver is often enlarged and tender after a few days of fever [Kalayanarooj S et al. Early clinical and laboratory indicators of acute dengue illness. Journal of Infectious Diseases, 1997, 176:313–321.]

The earliest abnormality in the full blood count is a progressive decrease in total white cell count, which should alert the physician to a high probability of dengue.

Dengue slide 2

Critical phase

1 DHF complication and unusual manifestations

Around the time of defervescence, when the temperature drops to 37.5–38 degree C or less and remains below this level, usually on days 3–7 of illness, an increase in capillary permeability in parallel with increasing haematocrit levels may occur

[Srikiatkhachorn A et al. Natural history of plasma leakage in dengue hemorrhagic fever: a serial ultrasonic study. Pediatric Infectious Disease Journal, 2007, 26(4):283– 290.]

[Nimmannitya S et al. Dengue and chikungunya virus infection in man in Thailand, 1962–64. Observations on hospitalized patients with haemorrhagic fever. American Journal of Tropical Medicine and Hygiene, 1969, 18(6):954–971.]

This marks the beginning of the critical phase. The period of clinically significant plasma leakage usually lasts 24–48 hours.

[Kalayanarooj S et al. Early clinical and laboratory indicators of acute dengue illness. Journal of Infectious Diseases, 1997, 176:313–321.]

Progressive leukopenia followed by a rapid decrease in platelet count usually precedes plasma leakage. At this point patients without an increase in capillary permeability will improve, while those with increased capillary permeability may become worse as a result of lost plasma volume. The degree of plasma leakage varies. Pleural effusion and ascites may be clinically detectable depending on the degree of plasma leakage and the volume of fluid therapy. Hence chest x ray and abdominal ultrasound can be useful tools for diagnosis. The degree of increase above the baseline haematocrit often reflects the severity of plasma leakage.

Shock occurs when a critical volume of plasma is lost through leakage. It is often
preceded by warning signs. The body temperature may be subnormal when shock occurs. With prolonged shock, the consequent organ hypoperfusion results in progressive organ impairment, metabolic acidosis and disseminated intravascular coagulation. This in turn leads to severe haemorrhage causing the haematocrit to decrease in severe shock. Instead of the leukopenia usually seen during this phase of dengue, the total white cell count may increase in patients with severe bleeding. In addition, severe organ impairment such as severe hepatitis, encephalitis or myocarditis and/or severe bleeding may also develop without obvious plasma leakage or shock.

[Martinez-Torres E, Polanco-Anaya AC, Pleites-Sandoval EB. Why and how children with dengue die? Revista cubana de medicina tropical, 2008, 60(1):40–47.]

Those who improve after defervescence are said to have non-severe dengue. Some patients progress to the critical phase of plasma leakage without defervescence and, in these patients, changes in the full blood count should be used to guide the onset of the critical phase and plasma leakage.

Those who deteriorate will manifest with warning signs. This is called dengue with warning signs. Cases of dengue with warning signs will probably recover with early intravenous rehydration. Some cases will deteriorate to severe dengue.

Recovery phase

If the patient survives the 24–48 hour critical phase, a gradual reabsorption of extravascular compartment fluid takes place in the following 48–72 hours. General well-being improves, appetite returns, gastrointestinal symptoms abate, haemodynamic status stabilizes and diuresis ensues. Some patients may have a rash of “isles of white in the sea of red”

[Nimmannitya S. Clinical spectrum and management of dengue haemorrhagic fever. Southeast Asian Journal of Tropical Medicine and Public Health, 1987, 18(3):392–397.]

Some may experience generalized pruritus. Bradycardia and electrocardiographic changes are common during this stage. The haematocrit stabilizes or may be lower due to the dilutional effect of reabsorbed fluid. White blood cell count usually starts to rise soon after defervescence but the recovery of platelet count is typically later than that of white blood cell count.

Respiratory distress from massive pleural effusion and ascites will occur at any time if excessive intravenous fluids have been administered. During the critical and/or recovery phases, excessive fluid therapy is associated with pulmonary oedema or congestive heart failure.

Febrile, Critical and Recovery Phases in Dengue

  2 DHF compli
Dengue fev case def

dengue fever case defi     Grading severity 
GuidUntitled

Dengue slide 4

Severe dengue

Severe dengue is defined by one or more of the following:

(i) plasma leakage that may lead to shock (dengue shock) and/or fluid accumulation, with or without respiratory distress, and/or

(ii) severe bleeding, and/or

(iii) severe organ impairment.

Spectrum of dengue hmg fever

As dengue vascular permeability progresses, hypovolaemia worsens and results in shock. It usually takes place around defervescence, usually on day 4 or 5 (range days 3–7) of illness, preceded by the warning signs. During the initial stage of shock, the compensatory mechanism which maintains a normal systolic blood pressure also produces tachycardia and peripheral vasoconstriction with reduced skin perfusionresulting in cold extremities and delayed capillary refill time. Uniquely, the diastolic pressure rises towards the systolic pressure and the pulse pressure narrows as the peripheral vascular resistance increases. Patients in dengue shock often remain conscious and lucid. The inexperienced physician may measure a normal systolic pressure and misjudge the critical state of the patient.

dss

Finally, there is decompensation and both pressures disappear abruptly. Prolonged hypotensive shock and hypoxia may lead to multi-organ failure and an extremely difficult clinical course.

The patient is considered to have shock if the pulse pressure (i.e. the difference between the systolic and diastolic pressures) is ≤ 20 mm Hg in children or he/she has signs of poor capillary perfusion (cold extremities, delayed capillary refill, or rapid pulse rate).

In adults, the pulse pressure of ≤ 20 mm Hg may indicate a more severe shock.

Hypotension is usually associated with prolonged shock which is often complicated by major bleeding. Patients with severe dengue may have coagulation abnormalities, but these are usually not sufficient to cause major bleeding. When major bleeding does occur, it is almost always associated with profound shock since this, in combination with thrombocytopaenia, hypoxia and acidosis, can lead to multiple organ failure and advanced disseminated intravascular coagulation. Massive bleeding may occur without prolonged shock in instances when acetylsalicylic acid (aspirin), ibuprofen or corticosteroids have been taken. Unusual manifestations, including acute liver failure and encephalopathy, may be present, even in the absence of severe plasma leakage or shock.

criteria dd

Cardiomyopathy and encephalitis are also reported in a few dengue cases. However, most deaths from dengue occur in patients with profound shock, particularly if the situation is complicated by fluid overload. Severe dengue should be considered if the patient is from an area of dengue risk presenting with fever of 2–7 days plus any of the following features:

  •  There is evidence of plasma leakage, such as:
    – high or progressively rising haematocrit;
    – pleural effusions or ascites;
    – circulatory compromise or shock (tachycardia, cold and clammy extremities, capillary refill time greater than three seconds, weak or undetectable pulse, narrow pulse pressure or, in late shock, unrecordable blood pressure).
  • There is significant bleeding.
  • There is an altered level of consciousness (lethargy or restlessness, coma, convulsions).
  • There is severe gastrointestinal involvement (persistent vomiting, increasing or intense abdominal pain, jaundice).
  • There is severe organ impairment (acute liver failure, acute renal failure, encephalopathy or encephalitis, or other unusual manifestations, cardiomyopathy) or other unusual manifestations.

DHF lab

Delivery of clinical services and case management

Introduction

Reducing dengue mortality requires an organized process that guarantees early recognition of the disease, and its management and referral when necessary. The key component of the process is the delivery of good clinical services at all levels of health care, from primary to tertiary levels. Most dengue patients recover without requiring hospital admission while some may progress to severe disease. Simple but effective triage principles and management decisions applied at the primary and secondary care levels, where patients are first seen and evaluated, can help in identifying those at risk of developing severe disease and needing hospital care. This should be complemented by prompt and appropriate management of severe dengue in referral centres.

Activities at the first level of care should focus on:

  • recognizing that the febrile patient could have dengue;
  • notifying early to the public health authorities that the patient is a suspected case of dengue;
  • managing patients in the early febrile phase of dengue;
  • recognizing the early stage of plasma leakage or critical phase and initiating fluid therapy;
  • recognizing patients with warning signs who need to be referred for admission and/or intravenous fluid therapy to a secondary health care facility;
  • recognizing and managing severe plasma leakage and shock, severe bleeding and severe organ impairment promptly and adequately.

Primary and secondary health care centres

At primary and secondary levels, health care facilities are responsible for emergency/ambulatory triage assessment and treatment.

Triage is the process of rapidly screening patients soon after their arrival in the hospital or health facility in order to identify those with severe dengue (who require immediate emergency treatment to avert death), those with warning signs (who should be given priority while waiting in the queue so that they can be assessed and treated without delay), and non-urgent cases (who have neither severe dengue nor warning signs).

During the early febrile phase, it is often not possible to predict clinically whether a patient with dengue will progress to severe disease. Various forms of severe manifestations may unfold only as the disease progresses through the critical phase, but the warning signs are good indicators of a higher risk of developing severe dengue. Therefore, the patient should have daily outpatient health care assessments for disease progression with careful checking for manifestations of severe dengue and warning signs.

A Stepwise Approach to The Management of Dengue

Referral centres

Referral centres receiving severely ill dengue patients must be able to give prompt attention to referred cases. Beds should be made available to those patients who meet the admission criteria, even if elective cases have to be deferred. If possible, there should be a designated area to cohort dengue patients, and a high-dependency unit for closer monitoring of those with shock. These units should be staffed by doctors and nurses who are trained to recognize high-risk patients and to institute appropriate treatment and monitoring.

A number of criteria may be used to decide when to transfer a patient to a high dependency unit. These include:

  • early presentation with shock (on days 2 or 3 of illness);
  • severe plasma leakage and/or shock;
  • undetectable pulse and blood pressure;
  • severe bleeding;
  • fluid overload;
  • organ impairment (such as hepatic damage, cardiomyopathy, encephalopathy,
    encephalitis and other unusual complications).

Resources needed

In the detection and management of dengue, a range of resources is needed to deliver good clinical services at all levels.

Resources include

[Martinez E. A Organizacao de Assistencia Medica durante uma epidemia de FHD-SCD. In: Dengue. Rio de Janeiro, Editorial Fiocruz, 2005 (pp 222–229)]:

  • Human resources: The most important resource is trained doctors and nurses. Adequate health personnel should be allocated to the first level of care to help in triage and emergency management. If possible, dengue units staffed by experienced personnel could be set up at referral centres to receive referred cases, particularly during dengue outbreaks, when the number of personnel main need to be increased.
  • Special area: A well equipped and well staffed area should be designated for giving immediate and transitory medical care to patients who require intravenous fluid therapy until they can be transferred to a ward or referral health facility.
  • Laboratory resources: The most important laboratory investigation is that of serial haematocrit levels and full blood counts. These investigations should be easily accessible from the health centre. Results should be available within two hours in severe cases of dengue. If no proper laboratory services are available, the minimum standard is the point-of-care testing of haematocrit by capillary (finger prick) blood sample with the use of a micro-centrifuge.
  •  Consumables: Intravenous fluids such as crystalloids, colloids and intravenous giving sets should be available.
  • Drugs: There should be adequate stocks of antipyretics and oral rehydration salts. In severe cases, additional drugs are necessary (vitamin K1, Ca gluconate, NaHCO3, glucose, furosemide, KCl solution, vasopressor, and inotropes).
  • Communication: Facilities should be provided for easy communication, especially between secondary and tertiary levels of care and laboratories, including consultation by telephone.
  • Blood bank: Blood and blood products will be required by only a small percentage of patients but should be made readily available to those who need them.

Education and training

To ensure the presence of adequate staffing at all levels, the education and training of doctors, nurses, auxiliary health care workers and laboratory staff are priorities.

Educational programmes that are customized for different levels of health care and that reflect local capacity should be supported and implemented widely. The educational programmes should develop capacities for effective triage and should improve recognition, clinical management and laboratory diagnosis of dengue.

National committees should monitor and evaluate clinical management and outcomes.

Review committees at different levels (e.g. national, state, district, hospital) should review all dengue deaths, and, if possible, all cases of severe dengue, evaluate the health care delivery system, and provide feedback to doctors on how to improve care. In dengue-endemic countries, the knowledge of dengue, the vectors and transmission of disease should be incorporated into the school curriculum. The population should also be educated about dengue in order to empower patients and their families in their own care – so that they are prepared to seek medical care at the right time, avoid self-medication, identify skin bleedings, consider the day of defervescence (and during 48 hours) as the time when complications usually occur, and look for warning signs such as intense and continuous abdominal pain and frequent vomiting.

The mass media can give an important contribution if they are correctly briefed.

Workshops and other meetings with journalists, editors, artists and executives can contribute to drawing up the best strategy for health education and communication without alarming the public.

During dengue epidemics, nursing and medical students together with community activists can visit homes with the double purpose of providing health education and actively tracing dengue cases. This has been shown to be feasible, inexpensive and effective [Lemus ER, Estevez G, Velazquez JC. Campana por la Esparanza. La Lucha contra el Dengue (El Salvador, 2000). La Habana, Editors Politica, 2002.] and must be coordinated with the primary health care units. It is useful to have printed information about dengue illness and the warning signs for distribution to members of the community. Medical care providers must include health education activities such as disease prevention in their daily work.

Recommendations For Treatment

A stepwise approach to the management of dengue

Step I—Overall assessment

History

The history should include:

  • date of onset of fever/illness;
  • quantity of oral intake;
  • assessment for warning signs;
  • diarrhoea;
  • change in mental state/seizure/dizziness;
  • urine output (frequency, volume and time of last voiding);
  • other important relevant histories, such as family or neighbourhood dengue, travel to dengue endemic areas, co-existing conditions (e.g. infancy, pregnancy, obesity, diabetes mellitus, hypertension), jungle trekking and swimming in waterfall (consider leptospirosis, typhus, malaria), recent unprotected sex or drug abuse (consider acute HIV sero-conversion illness).

Physical examination

The physical examination should include:

  • assessment of mental state;
  • assessment of hydration status;
  • assessment of haemodynamic status;
  • checking for tachypnoea/acidotic breathing/pleural effusion;
  • checking for abdominal tenderness/hepatomegaly/ascites;
  • examination for rash and bleeding manifestations;
  • tourniquet test (repeat if previously negative or if there is no bleeding
    manifestation).

Investigation

A full blood count should be done at the first visit. A haematocrit test in the early febrile phase establishes the patient’s own baseline haematocrit. A decreasing white blood cell count makes dengue very likely. A rapid decrease in platelet count in parallel with a rising haematocrit compared to the baseline is suggestive of progress to the plasma leakage/critical phase of the disease. In the absence of the patient’s baseline, age specific population haematocrit levels could be used as a surrogate during the critical phase.

Laboratory tests should be performed to confirm the diagnosis. However, it is not necessary for the acute management of patients, except in cases with unusual manifestations.

Additional tests should be considered as indicated (and if available). These should include tests of liver function, glucose, serum electrolytes, urea and creatinine, bicarbonate or lactate, cardiac enzymes, ECG and urine specific gravity.

Step II—Diagnosis, assessment of disease phase and severity

On the basis of evaluations of the history, physical examination and/or full blood count and haematocrit, clinicians should be able to determine whether the disease is dengue, which phase it is in (febrile, critical or recovery), whether there are warning signs, the hydration and haemodynamic status of the patient, and whether the patient requires admission.

Step III—Management

Disease notification

In dengue-endemic countries, cases of suspected, probable and confirmed dengue should be notified as soon as possible so that appropriate public health measures can be initiated.

Laboratory confirmation is not necessary before notification, but should be obtained. In non-endemic countries, usually only confirmed cases will be notified.

Suggested criteria for early notification of suspected cases are that the patient lives in or has travelled to a dengue-endemic area, has fever for three days or more, has low or decreasing white cell counts, and/or has thrombocytopaenia ± positive tourniquet test.

In dengue-endemic countries, the later the notification, the more difficult it is to prevent dengue transmission.

Management decisions

Depending on the clinical manifestations and other circumstances, patients may [Martinez E. Preventing deaths from dengue: a space and challenge for primary health care. Pan American Journal of Public Health, 2006, 20:60–74.] be sent home (Group A), be referred for in-hospital management (Group B), or require emergency treatment and urgent referral (Group C).

Treatment according to groups A–C

Group A – patients who may be sent home

These are patients who are able to tolerate adequate volumes of oral fluids and pass urine at least once every six hours, and do not have any of the warning signs, particularly when fever subsides.

Ambulatory patients should be reviewed daily for disease progression (decreasing white blood cell count, defervescence and warning signs) until they are out of the critical period. Those with stable haematocrit can be sent home after being advised to return to the hospital immediately if they develop any of the warning signs and to adhere to the following action plan:

  • Encourage oral intake of oral rehydration solution (ORS), fruit juice and other fluids containing electrolytes and sugar to replace losses from fever and vomiting. Adequate oral fluid intake may be able to reduce the number of hospitalizations [Harris E et al. Fluid intake and decreased risk for hospitalization for dengue fever, Nicaragua. Emerging Infectious Diseases, 2003, 9:1003–1006.]. [Caution: fluids containing sugar/glucose may exacerbate hyperglycaemia of physiological stress from dengue and diabetes mellitus.]
  • Give paracetamol for high fever if the patient is uncomfortable. The interval of paracetamol dosing should not be less than six hours. Tepid sponge if the patient still has high fever. Do not give acetylsalicylic acid (aspirin), ibuprofen or other non-steroidal anti inflammatory agents (NSAIDs) as these drugs may aggravate gastritis or bleeding. Acetylsalicylic acid (aspirin) may be associated with Reye’s Syndrome.
  • Instruct the care-givers that the patient should be brought to hospital immediately if any of the following occur: no clinical improvement, deterioration around the time of defervescence, severe abdominal pain, persistent vomiting, cold and clammy extremities, lethargy or irritability/restlessness, bleeding (e.g. black stools or coffee-ground vomiting), not passing urine for more than 4–6 hours.

Patients who are sent home should be monitored daily by health care providers for temperature pattern, volume of fluid intake and losses, urine output (volume and frequency), warning signs, signs of plasma leakage and bleeding, haematocrit, and white blood cell and platelet counts (see group B).

Group B – patients who should be referred for in-hospital management

Patients may need to be admitted to a secondary health care centre for close observation, particularly as they approach the critical phase. These include patients with warning signs, those with co-existing conditions that may make dengue or its management more complicated (such as pregnancy, infancy, old age, obesity, diabetes mellitus, renal failure, chronic haemolytic diseases), and those with certain social circumstances (such as living alone, or living far from a health facility without reliable means of transport).

If the patient has dengue with warning signs, the action plan should be as follows:

Obtain a reference haematocrit before fluid therapy. Give only isotonic solutions such as 0.9% saline, Ringer’s lactate, or Hartmann’s solution. Start with 5–7 ml/kg/hour for 1–2 hours, then reduce to 3–5 ml/kg/hr for 2–4 hours, and then reduce to 2–3 ml/kg/hr or less according to the clinical response.

Reassess the clinical status and repeat the haematocrit. If the haematocrit remains the same or rises only minimally, continue with the same rate (2–3 ml/kg/hr) for another 2–4 hours. If the vital signs are worsening and haematocrit is rising rapidly, increase the rate to 5–10 ml/kg/hour for 1–2 hours. Reassess the clinical status, repeat the haematocrit and review fluid infusion rates accordingly.

Give the minimum intravenous fluid volume required to maintain good perfusion and urine output of about 0.5 ml/kg/hr. Intravenous fluids are usually needed for only 24–48 hours. Reduce intravenous fluids gradually when the rate of plasma leakage decreases towards the end of the critical phase. This is indicated by urine output and/or oral fluid intake that is/are adequate, or haematocrit decreasing below the baseline value in a stable patient.

Patients with warning signs should be monitored by health care providers until the period of risk is over. A detailed fluid balance should be maintained. Parameters that should be monitored include vital signs and peripheral perfusion (1–4 hourly until the patient is out of the critical phase), urine output (4–6 hourly), haematocrit (before and after fluid replacement, then 6–12 hourly), blood glucose, and other organ functions (such as renal profile, liver profile, coagulation profile, as indicated.

If the patient has dengue without warning signs, the action plan should be as follows:

Encourage oral fluids. If not tolerated, start intravenous fluid therapy of 0.9% saline or Ringer’s lactate with or without dextrose at maintenance rate. For obese and overweight patients, use the ideal body weight for calculation of fluid infusion. Patients may be able to take oral fluids after a few hours of intravenous fluid therapy. Thus, it is necessary to revise the fluid infusion frequently. Give the minimum volume required to maintain good perfusion and urine output. Intravenous fluids are usually needed only for 24–48 hours.

Patients should be monitored by health care providers for temperature pattern, volume of fluid intake and losses, urine output (volume and frequency), warning signs, haematocrit, and white blood cell and platelet counts. Other laboratory tests (such as liver and renal functions tests) can be done, depending on the clinical picture and the facilities of the hospital or health centre.

Group C – patients who require emergency treatment and urgent referral when
they have severe dengue

Patients require emergency treatment and urgent referral when they are in the critical phase of disease, i.e. when they have:

  • severe plasma leakage leading to dengue shock and/or fluid accumulation with respiratory distress;
  • severe haemorrhages;
  • severe organ impairment (hepatic damage, renal impairment, cardiomyopathy, encephalopathy or encephalitis).

All patients with severe dengue should be admitted to a hospital with access to intensive care facilities and blood transfusion. Judicious intravenous fluid resuscitation is the essential and usually sole intervention required. The crystalloid solution should be isotonic and the volume just sufficient to maintain an effective circulation during the period of plasma leakage. Plasma losses should be replaced immediately and rapidly with isotonic crystalloid solution or, in the case of hypotensive shock, colloid solutions. If possible, obtain haematocrit levels before and after fluid resuscitation.

There should be continued replacement of further plasma losses to maintain effective circulation for 24–48 hours. For overweight or obese patients, the ideal body weight should be used for calculating fluid infusion rates. A group and cross-match should be done for all shock patients. Blood transfusion should be given only in cases with suspected/severe bleeding.

Fluid resuscitation must be clearly separated from simple fluid administration. This is a strategy in which larger volumes of fluids (e.g. 10–20 ml boluses) are administered for a limited period of time under close monitoring to evaluate the patient’s response and to avoid the development of pulmonary oedema. The degree of intravascular volume deficit in dengue shock varies. Input is typically much greater than output, and the input/output ratio is of no utility for judging fluid resuscitation needs during this period.

The goals of fluid resuscitation include improving central and peripheral circulation (decreasing tachycardia, improving blood pressure, pulse volume, warm and pink extremities, and capillary refill time <2 seconds) and improving end-organ perfusion

– i.e. stable conscious level (more alert or less restless), urine output ≥ 0.5 ml/kg/hour, decreasing metabolic acidosis.

Treatment of shock

The action plan for treating patients with compensated shock is as follows.

  • Start intravenous fluid resuscitation with isotonic crystalloid solutions at 5–10 ml/kg/hour over one hour. Then reassess the patient’s condition (vital signs, capillary refill time, haematocrit, urine output). The next steps depend on the situation.
  • If the patient’s condition improves, intravenous fluids should be gradually reduced to 5–7 ml/kg/hr for 1–2 hours, then to 3–5 ml/kg/hr for 2–4 hours, then to 2–3 ml/kg/hr, and then further depending on haemodynamic status, which can be maintained for up to 24–48 hours.
  • If vital signs are still unstable (i.e. shock persists), check the haematocrit after the first bolus. If the haematocrit increases or is still high (>50%), repeat a second bolus of crystalloid solution at 10–20 ml/kg/hr for one hour. After this second bolus, if there is improvement, reduce the rate to 7–10 ml/kg/hr for 1–2 hours, and then continue to reduce as above. If haematocrit decreases compared to the initial reference haematocrit (<40% in children and adult females, <45% in adult males), this indicates bleeding and the need to cross-match and transfuse blood as soon as possible (see treatment for haemorrhagic complications).
  • Further boluses of crystalloid or colloidal solutions may need to be given during the next 24–48 hours.
Algorithm For Fluid Management in Compensated Shock.

Patients with hypotensive shock should be managed more vigorously. The action plan for treating patients with hypotensive shock is as follows:

  •  Initiate intravenous fluid resuscitation with crystalloid or colloid solution (if available) at 20 ml/kg as a bolus given over 15 minutes to bring the patient out of shock as quickly as possible.
  • If the patient’s condition improves, give a crystalloid/colloid infusion of 10 ml/kg/hr for one hour. Then continue with crystalloid infusion and gradually reduce to 5–7 ml/kg/hr for 1–2 hours, then to 3–5 ml/kg/hr for 2–4 hours, and then to 2–3 ml/kg/hr or less, which can be maintained for up to 24–48 hours.
  • If vital signs are still unstable (i.e. shock persists), review the haematocrit obtained before the first bolus. If the haematocrit was low (<40% in children and adult females, <45% in adult males), this indicates bleeding and the need to crossmatch and transfuse blood as soon as possible (see treatment for haemorrhagic complications).
  • If the haematocrit was high compared to the baseline value (if not available, use population baseline), change intravenous fluids to colloid solutions at 10–20ml/kg as a second bolus over 30 minutes to one hour. After the second bolus, reassess the patient. If the condition improves, reduce the rate to 7–10ml/kg/hr for 1–2 hours, then change back to crystalloid solution and reduce the rate of infusion as mentioned above. If the condition is still unstable, repeat the haematocrit after the second bolus.
  • If the haematocrit decreases compared to the previous value (<40% in children and adult females, <45% in adult males), this indicates bleeding and the need to cross-match and transfuse blood as soon as possible (see treatment for haemorrhagic complications). If the haematocrit increases compared to the previous value or remains very high (>50%), continue colloid solutions at 10–20 ml/kg as a third bolus over one hour. After this dose, reduce the rate to 7–10 ml/kg/hr for 1–2 hours, then change back to crystalloid solution and reduce the rate of infusion as mentioned above when the patient’s condition improves.
  • Further boluses of fluids may need to be given during the next 24 hours. The rate and volume of each bolus infusion should be titrated to the clinical response. Patients with severe dengue should be admitted to the high-dependency or intensive care area.

Patients with dengue shock should be frequently monitored until the danger period is over. A detailed fluid balance of all input and output should be maintained.

Parameters that should be monitored include vital signs and peripheral perfusion (every 15–30 minutes until the patient is out of shock, then 1–2 hourly). In general, the higher the fluid infusion rate, the more frequently the patient should be monitored and reviewed in order to avoid fluid overload while ensuring adequate volume replacement.

If resources are available, a patient with severe dengue should have an arterial line placed as soon as practical. The reason for this is that in shock states, estimation of blood pressure using a cuff is commonly inaccurate. The use of an indwelling arterial catheter allows for continuous and reproducible blood pressure measurements and frequent blood sampling on which decisions regarding therapy can be based. Monitoring of ECG and pulse oximetry should be available in the intensive care unit.

Urine output should be checked regularly (hourly till the patient is out of shock, then 1–2 hourly). A continuous bladder catheter enables close monitoring of urine output. An acceptable urine output would be about 0.5 ml/kg/hour. Haematocrit should be monitored (before and after fluid boluses until stable, then 4–6 hourly). In addition, there should be monitoring of arterial or venous blood gases, lactate, total carbon dioxide/bicarbonate (every 30 minutes to one hour until stable, then as indicated), blood glucose (before fluid resuscitation and repeat as indicated), and other organ functions (such as renal profile, liver profile, coagulation profile, before resuscitation and as indicated).

Changes in the haematocrit are a useful guide to treatment. However, changes must be interpreted in parallel with the haemodynamic status, the clinical response to fluid therapy and the acid-base balance. For instance, a rising or persistently high haematocrit together with unstable vital signs (particularly narrowing of the pulse pressure) indicates active plasma leakage and the need for a further bolus of fluid replacement. However, a rising or persistently high haematocrit together with stable haemodynamic status and adequate urine output does not require extra intravenous fluid. In the latter case, continue to monitor closely and it is likely that the haematocrit will start to fall within the next 24 hours as the plasma leakage stops.
A decrease in haematocrit together with unstable vital signs (particularly narrowing of the pulse pressure, tachycardia, metabolic acidosis, poor urine output) indicates major haemorrhage and the need for urgent blood transfusion. Yet a decrease in haematocrit together with stable haemodynamic status and adequate urine output indicates haemodilution and/or reabsorption of extravasated fluids, so in this case intravenous fluids must be discontinued immediately to avoid pulmonary oedema.
 
Treatment of haemorrhagic complications
Mucosal bleeding may occur in any patient with dengue but, if the patient remains stable with fluid resuscitation/replacement, it should be considered as minor. The bleeding usually improves rapidly during the recovery phase. In patients with profound thrombocytopaenia, ensure strict bed rest and protect from trauma to reduce the risk of bleeding. Do not give intramuscular injections to avoid haematoma. It should be noted that prophylactic platelet transfusions for severe thrombocytopaenia in otherwise haemodynamically stable patients have not been shown to be effective and are not necessary. [Lum L et al. Preventive transfusion in dengue shock syndrome – is it necessary? Journal of Pediatrics, 2003, 143:682–684.]
If major bleeding occurs it is usually from the gastrointestinal tract, and/or vagina in adult females. Internal bleeding may not become apparent for many hours until the first black stool is passed.
Patients at risk of major bleeding are those who:
  • have prolonged/refractory shock;
  • have hypotensive shock and renal or liver failure and/or severe and persistent metabolic acidosis;
  • are given non-steroidal anti-inflammatory agents;
  • have pre-existing peptic ulcer disease;
  • are on anticoagulant therapy;
  • have any form of trauma, including intramuscular injection.
 Patients with haemolytic conditions are at risk of acute haemolysis with haemoglobinuria
and will require blood transfusion.
Severe bleeding can be recognized by:
  • persistent and/or severe overt bleeding in the presence of unstable haemodynamic status, regardless of the haematocrit level;
  • a decrease in haematocrit after fluid resuscitation together with unstable haemodynamic status;
  • refractory shock that fails to respond to consecutive fluid resuscitation of 40-60 ml/kg;
  • hypotensive shock with low/normal haematocrit before fluid resuscitation;
  • persistent or worsening metabolic acidosis ± a well-maintained systolic blood pressure, especially in those with severe abdominal tenderness and distension.
Blood transfusion is life-saving and should be given as soon as severe bleeding is suspected or recognized. However, blood transfusion must be given with care because of the risk of fluid overload. Do not wait for the haematocrit to drop too low before deciding on blood transfusion. Note that haematocrit of <30% as a trigger for blood transfusion, as recommended in the Surviving Sepsis Campaign Guideline [Dellinger RP, Levy MM, Carlet JM. Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008. Critical Care Medicine, 2008, 36:296–327.], is not applicable to severe dengue. The reason for this is that, in dengue, bleeding usually occurs after a period of prolonged shock that is preceded by plasma leakage. During the plasma leakage the haematocrit increases to relatively high values before the onset of severe bleeding. When bleeding occurs, haematocrit will then drop from this high level. As a result, haematocrit levels may not be as low as in the absence of plasma leakage.
The action plan for the treatment of haemorrhagic complications is as follows:
  •  Give 5–10ml/kg of fresh-packed red cells or 10–20 ml/kg of fresh whole blood at an appropriate rate and observe the clinical response. It is important that fresh whole blood or fresh red cells are given. Oxygen delivery at tissue level is optimal with high levels of 2,3 di-phosphoglycerate (2,3 DPG). Stored blood loses 2,3 DPG, low levels of which impede the oxygen-releasing capacity of haemoglobin, resulting in functional tissue hypoxia. A good clinical response includes improving haemodynamic status and acid-base balance.
  • Consider repeating the blood transfusion if there is further blood loss or no appropriate rise in haematocrit after blood transfusion. There is little evidence to support the practice of transfusing platelet concentrates and/or fresh-frozen plasma for severe bleeding. It is being practised when massive bleeding can not be managed with just fresh whole blood/fresh-packed cells, but it may exacerbate the fluid overload.
  • Great care should be taken when inserting a naso-gastric tube which may cause severe haemorrhage and may block the airway. A lubricated oro-gastric tube may minimize the trauma during insertion. Insertion of central venous catheters should be done with ultra-sound guidance or by a very experienced person.
Treatment of complications and other areas of treatment
Fluid overload
Fluid overload with large pleural effusions and ascites is a common cause of acute respiratory distress and failure in severe dengue. Other causes of respiratory distress include acute pulmonary oedema, severe metabolic acidosis from severe shock, and Acute Respiratory Distress Syndrome (ARDS) (refer to standard textbook of clinical care for further guidance on management).
Causes of fluid overload are:
  • excessive and/or too rapid intravenous fluids.
  •  incorrect use of hypotonic rather than isotonic crystalloid solutions;
  • inappropriate use of large volumes of intravenous fluids in patients with unrecognized severe bleeding;
  • inappropriate transfusion of fresh-frozen plasma, platelet concentrates and   cryoprecipitates;
  • continuation of intravenous fluids after plasma leakage has resolved (24–48 hours from           defervescence);
  • co-morbid conditions such as congenital or ischaemic heart disease, chronic lung and renal     diseases.
Early clinical features of fluid overload are:
  • respiratory distress, difficulty in breathing.
  • rapid breathing.
  •  chest wall in-drawing;
  • wheezing (rather than crepitations);
  • large pleural effusions.
  • tense ascites.
  •  increased jugular venous pressure (JVP).
Late clinical features are:
  • pulmonary oedema (cough with pink or frothy sputum ± crepitations, cyanosis);
  • irreversible shock (heart failure, often in combination with ongoing hypovolaemia).
 Additional investigations are:
  • the chest x-ray which shows cardiomegaly, pleural effusion, upward
    displacement of the diaphragm by the ascites and varying degrees of
    “bat’s wings” appearance ± Kerley B lines suggestive of fluid overload and
    pulmonary oedema;
  • ECG to exclude ischaemic changes and arrhythmia;
  • arterial blood gases;
  • echocardiogram for assessment of left ventricular function, dimensions and regional wall dyskinesia that may suggest underlying ischaemic heart disease;
  • cardiac enzymes
The action plan for the treatment of fluid overload is as follows:
  • Oxygen therapy should be given immediately.
  • Stopping intravenous fluid therapy during the recovery phase will allow fluid in the pleural and peritoneal cavities to return to the intravascular compartment. This results in diuresis and resolution of pleural effusion and ascites. Recognizing when to decrease or stop intravenous fluids is key to preventing fluid overload. When the following signs are present, intravenous fluids should be discontinued or reduced to the minimum rate necessary to maintain euglycaemia:
  • signs of cessation of plasma leakage;
  • stable blood pressure, pulse and peripheral perfusion;
  • haematocrit decreases in the presence of a good pulse volume;
  • afebrile for more than 24–48 days (without the use of antipyretics);
  • resolving bowel/abdominal symptoms;
  • improving urine output.
  • The management of fluid overload varies according to the phase of the disease and the patient’s haemodynamic status. If the patient has stable haemodynamic status and is out of the critical phase (more than 24–48 hours of defervescence), stop intravenous fluids but continue close monitoring. If necessary, give oral or intravenous furosemide 0.1–0.5 mg/kg/dose once or twice daily, or a continuous infusion of furosemide 0.1 mg/kg/hour.
  • Monitor serum potassium and correct the ensuing hypokalaemia.
  • If the patient has stable haemodynamic status but is still within the critical phase, reduce the intravenous fluid accordingly. Avoid diuretics during the plasma leakage phase because they may lead to intravascular volume depletion.
  • Patients who remain in shock with low or normal haematocrit levels but show signs of fluid overload may have occult haemorrhage. Further infusion of large volumes of intravenous fluids will lead only to a poor outcome. Careful fresh whole blood transfusion should be initiated as soon as possible. If the patient remains in shock and the haematocrit is elevated, repeated small boluses of a colloid solution may help.
Other complications of dengue
Both hyperglycaemia and hypoglycaemia may occur, even in the absence of diabetes mellitus and/or hypoglycaemic agents. Electrolyte and acid-base imbalances are also common observations in severe dengue and are probably related to gastrointestinal losses through vomiting and diarrhoea or to the use of hypotonic solutions for resuscitation and correction of dehydration. Hyponatraemia, hypokalaemia, hyperkalaemia, serum calcium imbalances and metabolic acidosis (sodium bicarbonate for metabolic acidosis is not recommended for pH ≥ 7.15) can occur. One should also be alert for co-infections and nosocomial infections.
Supportive care and adjuvant therapy
Supportive care and adjuvant therapy may be necessary in severe dengue. This may
include:
  • renal replacement therapy, with a preference to continuous veno-venous haemodialysis (CVVH), since peritoneal dialysis has a risk of bleeding;
  • vasopressor and inotropic therapies as temporary measures to prevent lifethreatening hypotension in dengue shock and during induction for intubation, while correction of intravascular volume is being vigorously carried out;
  • further treatment of organ impairment, such as severe hepatic involvement or encephalopathy or encephalitis;
  • further treatment of cardiac abnormalities, such as conduction abnormalities, may occur (the latter usually not requiring interventions).
  • In this context there is little or no evidence in favour of the use of steroids and intravenous immunoglobulins, or of recombinant Activated Factor VII.
Refer to standard textbooks of clinical care for more detailed information regarding the treatment of complications and other areas of treatment.

LABORATORY DIAGNOSIS AND DIAGNOSTIC TESTS

 

OVERVIEW
Efficient and accurate diagnosis of dengue is of primary importance for clinical care (i.e. early detection of severe cases, case confirmation and differential diagnosis with other infectious diseases), surveillance activities, outbreak control, pathogenesis, academic research, vaccine development, and clinical trials.
Laboratory diagnosis methods for confirming dengue virus infection may involve detection of the virus, viral nucleic acid, antigens or antibodies, or a combination of these techniques. After the onset of illness, the virus can be detected in serum, plasma, circulating blood cells and other tissues for 4–5 days. During the early stages of the disease, virus isolation, nucleic acid or antigen detection can be used to diagnose the infection. At the end of the acute phase of infection, serology is the method of choice for diagnosis.
Antibody response to infection differs according to the immune status of the host [Vorndam V, Kuno G. Laboratory diagnosis of dengue virus infections. In: Gubler DJ, Kuno G, eds. Dengue and dengue hemorrhagic fever. New York, CAB International, 1997:313–333.]
When dengue infection occurs in persons who have not previously been infected with a flavivirus or immunized with a flavivirus vaccine (e.g. for yellow fever, Japanese encephalitis, tick-borne encephalitis), the patients develop a primary antibody response characterized by a slow increase of specific antibodies. IgM antibodies are the first immunoglobulin isotype to appear. These antibodies are detectable in 50% of patients by days 3-5 after onset of illness, increasing to 80% by day 5 and 99% by day 10. IgM levels peak about two weeks after the onset of symptoms and then decline generally to undetectable levels over 2–3 months. Anti-dengue serum IgG is generally detectable at low titres at the end of the first week of illness, increasing slowly thereafter, with serum IgG still detectable after several months, and probably even for life. [Innis B et al. An enzyme-linked immunosorbent assay to characterize dengue infections where dengue and Japanese encephalitis co-circulate. American Journal of Tropical Medicine and Hygiene, 1989, 40:418–427.]
[PAHO. Dengue and dengue hemorrhagic fever in the Americas: guidelines for
prevention and control. Washington, DC, Pan American Health Organization, 1994
(Scientific Publication No. 548).]
[WHO. Dengue haemorrhagic fever: diagnosis, treatment, prevention and control,
2nd ed. Geneva, World Health Organization, 1997.]
During a secondary dengue infection (a dengue infection in a host that has previously been infected by a dengue virus, or sometimes after non-dengue flavivirus vaccination or infection), antibody titres rise rapidly and react broadly against many flaviviruses. The dominant immunoglobulin isotype is IgG which is detectable at high levels, even in the acute phase, and persists for periods lasting from 10 months to life. Early convalescent stage IgM levels are significantly lower in secondary infections than in primary ones and may be undetectable in some cases, depending on the test used.[Chanama S et al. Analysis of specific IgM responses in secondary dengue virus infections: levels and positive rates in comparison with primary infections. Journal of Clinical Virology, 2004, 31:185–189.]
To distinguish primary and secondary dengue infections, IgM/IgG antibody ratios are now more commonly used than the haemagglutination-inhibition test (HI) [Kuno G, Gomez I, Gubler DJ. An ELISA procedure for the diagnosis of dengue infections. Journal of Virological Methods, 1991, 33:101–113.]
[Shu PY et al. Comparison of a capture immunoglobulin M (IgM) and IgG ELISA and non-structural protein NS1 serotype-specific IgG ELISA for differentiation of primary and secondary dengue virus infections. Clinical and Diagnostic Laboratory Immunology, 2003, 10:622–630.]
[Falconar AK, de Plata E, Romero-Vivas CM. Altered enzyme-linked immunosorbent assay immunoglobulin M (IgM)/IgG optical density ratios can correctly classify all primary or secondary dengue virus infections 1 day after the onset of symptoms, when all of the viruses can be isolated. Clinical and Vaccine Immunology, 2006, 13:1044–1051.]
A range of laboratory diagnostic methods has been developed to support patient management and disease control. The choice of diagnostic method depends on the purpose for which the testing is done (e.g. clinical diagnosis, epidemiological survey, vaccine development), the type of laboratory facilities and technical expertise available,costs, and the time of sample collection.
In general, tests with high sensitivity and specificity require more complex technologies and technical expertise, while rapid tests may compromise sensitivity and specificity for the ease of performance and speed. Virus isolation and nucleic acid detection are more labour intensive and costly but are also more specific than antibody detection using serologic methods. Figure shows a general inverse relationship between the ease of use or accessibility of a diagnostic method and the confidence in the results of the test.
CONSIDERATIONS IN THE CHOICE OF DIAGNOSTIC METHODS
Clinical Management
Dengue virus infection produces a broad spectrum of symptoms, many of which are non-specific. Thus, a diagnosis based only on clinical symptoms is unreliable. Early laboratory confirmation of clinical diagnosis may be valuable because some patients progress over a short period from mild to severe disease and sometimes to death. Early intervention may be life-saving.
Before day 5 of illness, during the febrile period, dengue infections may be diagnosed by virus isolation in cell culture, by detection of viral RNA by nucleic acid amplification tests (NAAT), or by detection of viral antigens by ELISA or rapid tests. Virus isolation in cell culture is usually performed only in laboratories with the necessary infrastructure and technical expertise. For virus culture, it is important to keep blood samples cooled or frozen to preserve the viability of the virus during transport from the patient to the laboratory. The isolation and identification of dengue viruses in cell cultures usually takes several days. Nucleic acid detection assays with excellent performance characteristics may identify dengue viral RNA within 24–48 hours. However, these tests require expensive equipment and reagents and, in order to avoid contamination, tests must observe quality control procedures and must be performed by experienced technicians.

NS1 antigen detection kits now becoming commercially available can be used in laboratories with limited equipment and yield results within a few hours. Rapid dengue antigen detection tests can be used in field settings and provide results in less than an hour. Currently, these assays are not type-specific, are expensive and are under evaluation for diagnostic accuracy and cost-effectiveness in multiple settings.

Summary of operating characteristics and comparative costs of dengue diagnostic methods. Pelegrino JL. Summary of dengue diagnostic methods. World Health Organization, Special Programme for Research and Training in Tropical Diseases, 2006 (unpublished report).

After day 5, dengue viruses and antigens disappear from the blood coincident with the appearance of specific antibodies. NS1 antigen may be detected in some patients for a few days after defervescence. Dengue serologic tests are more available in dengueendemic countries than are virological tests. Specimen transport is not a problem as immunoglobulins are stable at tropical room temperatures.

For serology, the time of specimen collection is more flexible than that for virus isolation or RNA detection because an antibody response can be measured by comparing a sample collected during the acute stage of illness with samples collected weeks or months later. Low levels of a detectable dengue IgM response – or the absence of it – in some secondary infections reduces the diagnostic accuracy of IgM ELISA tests. Results of rapid tests may be available within less than one hour. Reliance on rapid tests to diagnose dengue infections should be approached with caution, however, since the performance of all commercial tests has not yet been evaluated by reference laboratories.[Hunsperger EA et al. Evaluation of commercially available anti–dengue virus immunoglobulin M tests. Emerging Infectious Diseases (serial online), 2009, March (date cited). Accessible at http://www.cdc.gov/EID/content/15/3/436.htm]

A four-fold or greater increase in antibody levels measured by IgG ELISA or by haemagglutination inhibition (HI) test in paired sera indicates an acute or recent flavivirus infection. However, waiting for the convalescent serum collected at the time of patient discharge is not very useful for diagnosis and clinical management and provides only
a retrospective result.

Differential diagnosis

Dengue fever can easily be confused with non-dengue illnesses, particularly in nonepidemic
situations. Depending on the geographical origin of the patient, other etiologies – including non-dengue flavivirus infections – should be ruled out. These include yellow fever, Japanese encephalitis, St Louis encephalitis, Zika, and West Nile, alphaviruses (such as Sinbis and chikungunya), and other causes of fever such as malaria, leptospirosis, typhoid, Rickettsial diseases (Rickettsia prowazeki, R. mooseri, R. conori, R. rickettsi, Orientia tsutsugamushi, Coxiella burneti, etc.), measles, enteroviruses, influenza and influenza-like illnesses, haemorrhagic fevers (Arenaviridae: Junin, etc.; Filoviridae: Marburg, Ebola; Bunyaviridae: hantaviruses, Crimean-Congo haemorrhagic fever, etc.).

Both the identification of virus/viral RNA/viral antigen and the detection of an antibody
response are preferable for dengue diagnosis to either approach alone.

Interpretation of dengue diagnostic tests [adapted from Dengue and Control (DENCO) study]
Unfortunately, an ideal diagnostic test that permits early and rapid diagnosis, is affordable
for different health systems, is easy to perform, and has a robust performance, is not yet
available.
Outbreak investigations
During outbreaks some patients may be seen presenting with fever with or without rash
during the acute illness stage; some others may present with signs of plasma leakage or
shock, and others with signs of haemorrhages, while still others may be observed during
the convalescent phase.
One of the priorities in a suspected outbreak is to identify the causative agent so that
appropriate public health measures can be taken and physicians can be encouraged to
initiate appropriate acute illness management. In such cases, the rapidity and specificity
of diagnostic tests is more important than test sensitivity. Samples collected from febrile patients could be tested by nucleic acid methods in a well-equipped laboratory or a broader spectrum of laboratories using an ELISA-based dengue antigen detection kit. If specimens are collected after day 5 of illness, commercial IgM ELISA or sensitive dengue IgM rapid tests may suggest a dengue outbreak, but results are preferably confirmed with reliable serological tests performed in a reference laboratory with broad arbovirus diagnostic capability. Serological assays may be used to determine the extent of outbreaks.

Surveillance

Dengue surveillance systems aim to detect the circulation of specific viruses in the human
or mosquito populations. The diagnostic tools used should be sensitive, specific and
affordable for the country. Laboratories responsible for surveillance are usually national
and/or reference laboratories capable of performing diagnostic tests as described
above for dengue and for a broad range of other etiologies.

Vaccine trials

Vaccine trials are performed in order to measure vaccine safety and efficacy in vaccinated
persons. The plaque reduction and neutralization test (PRNT) and the microneutralization
assays are commonly used to measure protection correlates.
Following primary infections in non-flavivirus immunes, neutralizing antibodies as measured by PRNT may be relatively or completely specific to the infecting virus type.[Morens DM et al. Simplified plaque reduction neutralization assay for dengue viruses by semimicro methods in BHK-21 cells: comparison of the BHK suspension test with standard plaque reduction neutralization. Journal of Clinical Microbiology, 1985,22(2):250–254.]
[Alvarez M et al. Improved dengue virus plaque formation on BHK21 and LLCMK2
cells: evaluation of some factors. Dengue Bulletin, 2005, 29:1–9.]
This assay is the most reliable means of measuring the titre of neutralizing antibodies in the serum of an infected individual as a measure of the level of protection against an infecting virus. The assay is based on the principle that neutralizing antibodies inactivate the virus so that it is no longer able to infect and replicate in target cells.
After a second dengue virus infection, high-titre neutralizing antibodies are produced against at least two, and often all four, dengue viruses as well as against non-dengue flaviviruses. This cross reactivity results from memory B-cells which produce antibodies directed at virion epitopes shared by dengue viruses. During the early convalescent stage following sequential dengue infections, the highest neutralizing antibody titre is often directed against the first infecting virus and not the most recent one. This phenomenon is referred to as “original antigenic sin”.[Halstead SB, Rojanasuphot S, Sangkawibha N. Original antigenic sin in dengue. American Journal of Tropical Medicine and Hygiene, 1983, 32:154–156.]
The disadvantages of PRNT are that it is labour-intensive. A number of laboratories recently developed high through-put neutralization tests that can be used in large-scale surveillance studies and vaccine trials. Variable results have been observed in PRNTs performed in different laboratories. Variations can be minimized if tests are performed on standard cell lines using the same virus strains and the same temperature and time for incubation of virus with antibody. Input virus should be carefully calculated to avoid plaque overlap. Cell lines of mammalian origin, such as VERO cells, are recommended for the production of seed viruses for use in PRNT.
The microneutralization assay is based on the same principle as PRNT. Variable methods exist. In one, instead of counting the number of plaques per well, viral antigen is stained using a labelled antibody and the quantity of antigen measured colorimetrically. The test may measure nucleic acid using PCR. The microneutralization assay was designed to use smaller amounts of reagents and for testing larger numbers of samples. In viral antigen detection tests the spread of virus throughout the cells is not limited because, in PRNTs using
semisolid overlays, the time after infection must be standardized to avoid measuring growth after many cycles of replication. Since not all viruses grow at the same rate, the incubation periods are virus-specific. As with standard PRNTs, antibodies measured by micro methods from individuals with secondary infections may react broadly with all four dengue viruses.
Advantages and limitations of dengue diagnostic methods. Summary of dengue diagnostic methods. World Health Organization, Special Programme for Research and Training in Tropical Diseases, 2006 (unpublished report).

In drug trials, patients should have confirmed etiological diagnosis.

Current dengue diagnostic methods

Virus isolation

Specimens for virus isolation should be collected early in the course of the infection, during the period of viraemia (usually before day 5). Virus may be recovered from serum, plasma and peripheral blood mononuclear cells and attempts may be made from tissues collected at autopsy (e.g. liver, lung, lymph nodes, thymus, bone marrow).

Because dengue virus is heat-labile, specimens awaiting transport to the laboratory should be kept in a refrigerator or packed in wet ice. For storage up to 24 hours, specimens should be kept at between +4 °C and +8 °C. For longer storage, specimens should be frozen at -70 °C in a deep-freezer or stored in a liquid nitrogen container. Storage even for short periods at –20 °C is not recommended.

Cell culture is the most widely used method for dengue virus isolation. The mosquito cell line C6/36 (cloned from Ae. albopictus) or AP61 (cell line from Ae. pseudoscutellaris) are the host cells of choice for routine isolation of dengue virus. Since not all wild type dengue viruses induce a cytopathic effect in mosquito cell lines, cell cultures must be screened for specific evidence of infection by an antigen detection immunofluorescence assay using serotype-specific monoclonal antibodies and flavivirus group-reactive or dengue complex-reactive monoclonal antibodies. Several mammalian cell cultures, such as Vero, LLCMK2, and BHK21, may also be used but are less efficient.

Virus isolation followed by an immunofluorescence assay for confirmation generally requires 1–2 weeks and is possible only if the specimen is properly transported and stored to preserve the viability of the virus in it.

When no other methods are available, clinical specimens may also be inoculated by intracranial route in suckling mice or intrathoracic inoculation of mosquitoes. Newborn animals can develop encephalitis symptoms but with some dengue strains mice may exhibit no signs of illness. Virus antigen is detected in mouse brain or mosquito head squashes by staining with anti-dengue antibodies.

Nucleic acid detection

RNA is heat-labile and therefore specimens for nucleic acid detection must be handled
and stored according to the procedures described for virus isolation.

RT-PCR

Since the 1990s, several reverse transcriptase-polymerase chain reaction (RT-PCR) assays have been developed. They offer better sensitivity compared to virus isolation with a much more rapid turnaround time. In situ RT-PCR offers the ability to detect dengue RNA in paraffin-embedded tissues.

All nucleic acid detection assays involve three basic steps: nucleic acid extraction and purification, amplification of the nucleic acid, and detection and characterization of the amplified product. Extraction and purification of viral RNA from the specimen can be done by traditional liquid phase separation methods (e.g. phenol, chloroform) but has been gradually replaced by silica-based commercial kits (beads or columns) that are more reproducible and faster, especially since they can be automated using robotics systems. Many laboratories utilize a nested RT-PCR assay, using universal dengue primers targeting the C/prM region of the genome for an initial reverse transcription and amplification step, followed by a nested PCR amplification that is serotype-specific.[Lanciotti RS et al. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. Journal of Clinical Microbiology, 1992, 30:545–551.]

A combination of the four serotype-specific oligonucleotide primers in a single reaction tube (one-step multiplex RT-PCR) is an interesting alternative to the nested RT-PCR.[Harris E et al. Typing of dengue viruses in clinical specimens and mosquitoes by single tube multiplex reverse transcriptase PCR. Journal of Clinical Microbiology, 1998,36:2634–2639.] The products of these reactions are separated by electrophoresis on an agarose gel, and the amplification products are visualized as bands of different molecular weights in the agarose gel using ethidium bromide dye, and compared with standard molecular weight markers. In this assay design, dengue serotypes are identified by the size of their bands.

Compared to virus isolation, the sensitivity of the RT-PCR methods varies from 80% to 100% and depends on the region of the genome targeted by the primers, the approach used to amplify or detect the PCR products (e.g. one-step RT-PCR versus two-step RTPCR), and the method employed for subtyping (e.g. nested PCR, blot hybridization with specific DNA probes, restriction site-specific PCR, sequence analysis, etc.). To avoid false positive results due to non-specific amplification, it is important to target regions of the genome that are specific to dengue and not conserved among flavi- or other related viruses. False-positive results may also occur as a result of contamination by amplicons from previous amplifications. This can be prevented by physical separation of different steps of the procedure and by adhering to stringent protocols for decontamination.

Real-time RT-PCR

The real-time RT-PCR assay is a one step assay system used to quantitate viral RNA and using primer pairs and probes that are specific to each dengue serotype. The use of a fluorescent probe enables the detection of the reaction products in real time, in a specialized PCR machine, without the need for electrophoresis. Many real-time RT-PCR assays have been developed employing TaqMan or SYBR Green technologies. The TaqMan real-time PCR is highly specific due to the sequence-specific hybridization of the probe. Nevertheless, primers and probes reported in publications may not be able to detect all dengue virus strains: the sensitivity of the primers and probes depends on their homology with the targeted gene sequence of the particular virus analyzed. The SYBR green real-time RT-PCR has the advantage of simplicity in primer design and uses universal RT-PCR protocols but is theoretically less specific.

Real-time RT-PCR assays are either “singleplex” (i.e. detecting only one serotype at a time) or “multiplex” (i.e. able to identify all four serotypes from a single sample). The multiplex assays have the advantage that a single reaction can determine all four serotypes without the potential for introduction of contamination during manipulation of the sample. However the multiplex real-time RT-PCR assays, although faster, are currently less sensitive than nested RT-PCR assays. An advantage of this method is the ability to determine viral titre in a clinical sample, which may be used to study the pathogenesis of dengue disease.[Vaughn DW et al. Dengue viremia titer, antibody response pattern and virus serotype correlate with disease severity. Journal of Infectious Diseases, 2000, 181:2–9.]

Isothermal amplification methods

The NASBA (nucleic acid sequence based amplification) assay is an isothermal RNA specific amplification assay that does not require thermal cycling instrumentation. The initial stage is a reverse transcription in which the single-stranded RNA target is copied into a double-stranded DNA molecule that serves as a template for RNA transcription. Detection of the amplified RNA is accomplished either by electrochemiluminescence or in real-time with fluorescent-labelled molecular beacon probes. NASBA has been adapted to dengue virus detection with sensitivity near that of virus isolation in cell cultures and may be a useful method for studying dengue infections in field studies.[Shu PY, Huang JH. Current advances in dengue diagnosis. Clinical and Diagnostic Laboratory Immunology, 2004, 11(4):642–650.]

Loop mediated amplification methods have also been described but their performance compared to other nucleic acid amplification methods are not known.[Parida MM et al. Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay. Journal of Clinical Microbiology, 2005, 43:2895–2903 (doi: 10.1128/JCM.43.6.2895-2903.2005).]

Detection of antigens

Until recently, detection of dengue antigens in acute-phase serum was rare in patients with secondary infections because such patients had pre-existing virus-IgG antibody immunocomplexes. New developments in ELISA and dot blot assays directed to the envelop/membrane (E/M) antigen and the non-structural protein 1 (NS1) demonstrated that high concentrations of these antigens in the form of immune complexes could be detected in patients with both primary and secondary dengue infections up to nine days after the onset of illness.

The NS1 glycoprotein is produced by all flaviviruses and is secreted from mammalian cells. NS1 produces a very strong humoral response. Many studies have been directed at using the detection of NS1 to make an early diagnosis of dengue virus infection.

Commercial kits for the detection of NS1 antigen are now available, though they do not differentiate between dengue serotypes. Their performance and utility are currently being evaluated by laboratories worldwide, including the WHO/TDR/PDVI laboratory network. Fluorescent antibody, immunoperoxidase and avidin-biotin enzyme assays allow detection of dengue virus antigen in acetone-fixed leucocytes and in snap-frozen or formalin-fixed tissues collected at autopsy.

Serological tests

MAC-ELISA

For the IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) total IgM in patients’ sera is captured by anti-μ chain specific antibodies (specific to human IgM) coated onto a microplate. Dengue-specific antigens, from one to four serotypes (DEN-1, -2, -3, and -4), are bound to the captured anti-dengue IgM antibodies and are detected by monoclonal or polyclonal dengue antibodies directly or indirectly conjugated with an enzyme that will transform a non-coloured substrate into coloured products. The optical density is measured by spectrophotometer.

Serum, blood on fi lter paper and saliva, but not urine, can be used for detection of IgM if samples are taken within the appropriate time frame (fi ve days or more after the onset of fever). Serum specimens may be tested at a single dilution or at multiple dilutions. Most of the antigens used for this assay are derived from the dengue virus envelope protein (usually virus-infected cell culture supernatants or suckling mouse brain preparations). MAC-ELISA has good sensitivity and specifi city but only when used fi ve or more days after the onset of fever. Different commercial kits (ELISA or rapid tests) are available but have variable sensitivity and specifi city. A WHO/TDR/PDVI laboratory network recently evaluated selected commercial ELISAs and fi rst-generation rapid diagnostic tests, finding that ELISAs generally performed better than rapid tests.

Cross-reactivity with other circulating fl aviviruses such as Japanese encephalitis, St Louis encephalitis and yellow fever, does not seem to be a problem but some false positives were obtained in sera from patients with malaria, leptospirosis and past dengue infection.[Hunsperger EA et al. Evaluation of commercially available anti–dengue virus immunoglobulin M tests. Emerging Infectious Diseases (serial online), 2009, March (date cited). Accessible at http://www.cdc.gov/EID/content/15/3/436.htm]

These limitations have to be taken into account when using the tests in regions where these pathogens co-circulate. It is recommended that tests be evaluated against a panel of sera from relevant diseases in a particular region before being released to the market. It is not possible to use IgM assays to identify dengue serotypes as these antibodies are broadly cross-reactive even following primary infections. Recently, some authors have described MAC-ELISA that could allow serotype determination but further evaluations are required.[Vazquez S et al. Serological markers during dengue 3 primary and secondary infections. Journal of Clinical Virology, 2005, 33(2):132–137.]

IgG ELISA

The IgG ELISA is used for the detection of recent or past dengue infections (if paired sera are collected within the correct time frame). This assay uses the same antigens as the MAC ELISA. The use of E/M-specific capture IgG ELISA (GAC) allows detection of IgG antibodies over a period of 10 months after the infection. IgG antibodies are lifelong as measured by E/M antigen-coated indirect IgG ELISA, but a fourfold or greater increase in IgG antibodies in acute and convalescent paired sera can be used to document recent infections. Test results correlate well with the haemagglutination-inhibition test.

An ELISA inhibition method (EIM) to detect IgG dengue antibodies is also used for the serological diagnosis and surveillance of dengue cases. This system is based in the competition for the antigen sites by IgG dengue antibodies in the sample and the conjugated human IgG anti-dengue.[Fernandez RJ, Vazquez S. Serological diagnosis of dengue by an ELISA inhibition method (EIM). Memórias do Instituto Oswaldo Cruz, 1990, 85(3):347-351.]

This method can be used to detect IgG antibodies in serum or plasma and filter-paper stored blood samples and permits identification of a case as a primary or secondary dengue infection.

[Fernandez RJ, Vazquez S. Serological diagnosis of dengue by an ELISA inhibition method (EIM). Memórias do Instituto Oswaldo Cruz, 1990, 85(3):347–351.]

[Vazquez S, Fernandez R, Llorente C. Usefulness of blood specimens on paper strips for serologic studies with inhibition ELISA. Revista do Instituto de Medicina Tropical de São Paulo, 1991, 33(4):309–311.]

[Vazquez S et al. Kinetics of antibodies in sera, saliva, and urine samples from adult patients with primary or secondary dengue 3 virus infections. International Journal of Infectious Diseases, 2007, 11:256–262.]

In general, IgG ELISA lacks specificity within the flavivirus serocomplex groups. Following viral infections, newly produced antibodies are less avid than antibodies produced months or years after infection. Antibody avidity is used in a few laboratories to discriminate primary and secondary dengue infections. Such tests are not in wide use and are not available commercially.

IgM/IgG ratio

A dengue virus E/M protein-specific IgM/IgG ratio can be used to distinguish primary from secondary dengue virus infections. IgM capture and IgG capture ELISAs are the most common assays for this purpose. In some laboratories, dengue infection is defined as primary if the IgM/IgG OD ratio is greater than 1.2 (using patient’s sera at 1/100 dilution) or 1.4 (using patient’s sera at 1/20 dilutions). The infection is secondary if the ratio is less than 1.2 or 1.4. This algorithm has also been adopted by some commercial vendors. However, ratios may vary between laboratories, thus indicating the need for better standardization of test performance.[Falconar AK, de Plata E, Romero-Vivas CM. Altered enzyme-linked immunosorbent assay immunoglobulin M (IgM)/IgG optical density ratios can correctly classify all primary or secondary dengue virus infections 1 day after the onset of symptoms, when all of the viruses can be isolated. Clinical and Vaccine Immunology, 2006, 13:1044 -1051.]

IgA

Positive detection for serum anti-dengue IgA as measured by anti-dengue virus IgA capture ELISA (AAC-ELISA) often occurs one day after that for IgM. The IgA titre peaks around day 8 after onset of fever and decreases rapidly until it is undetectable by day 40. No differences in IgA titres were found by authors between patients with primary or secondary infections. Even though IgA values are generally lower than IgM, both in serum and saliva, the two methods could be performed together to help in interpreting dengue serology.

[Vazquez S et al. Kinetics of antibodies in sera, saliva, and urine samples from adult patients with primary or secondary dengue 3 virus infections. International Journal of Infectious Diseases, 2007, 11:256–262.]

[Nawa M. Immunoglobulin A antibody responses in dengue patients: a useful
marker for serodiagnosis of dengue virus infection. Clinical and Vaccine Immunology, 2005, 12:1235–1237.]

This approach is not used very often and requires additional evaluation.

Haemagglutination-inhibition test

The haemagglutination-inhibition (HI) test is based on the ability of dengue antigens to agglutinate red blood cells (RBC) of ganders or trypsinized human O RBC. Anti-dengue antibodies in sera can inhibit this agglutination and the potency of this inhibition is measured in an HI test. Serum samples are treated with acetone or kaolin to remove non-specific inhibitors of haemagglutination, and then adsorbed with gander or trypsinized type O human RBC to remove non-specific agglutinins. Each batch the use of multiple different pH buffers for each serotype. Optimally the HI test requires paired sera obtained upon hospital admission (acute) and discharge (convalescent) or paired sera with an interval of more than seven days. The assay does not discriminate between infections by closely related fl aviviruses (e.g. between dengue virus and Japanese encephalitis virus or West Nile virus) nor between immunoglobulin isotypes.

The response to a primary infection is characterized by the low level of antibodies in the acute-phase serum drawn before day 5 and a slow elevation of HI antibody titres thereafter. During secondary dengue infections HI antibody titres rise rapidly, usually exceeding 1:1280. Values below this are generally observed in convalescent sera from patients with primary responses.

Haematological tests

Platelets and haematocrit values are commonly measured during the acute stages of dengue infection. These should be performed carefully using standardized protocols, reagents and equipment.

A drop of the platelet count below 100 000 per μL may be observed in dengue fever but it is a constant feature of dengue haemorrhagic fever. Thrombocytopaenia is usually observed in the period between day 3 and day 8 following the onset of illness.

Haemoconcentration, as estimated by an increase in haematocrit of 20% or more compared with convalescent values, is suggestive of hypovolaemia due to vascular permeability and plasma leakage.

Future test developments

Microsphere-based immunoassays (MIAs) are becoming increasingly popular as a serological option for the laboratory diagnosis of many diseases. This technology is based on the covalent bonding of antigen or antibody to microspheres or beads. Detection methods include lasers to elicit fluorescence of varying wavelengths. This technology is attractive as it is faster than the MAC-ELISA and has potential for multiplexing serological tests designed to identify antibody responses to several viruses. MIAs can also be used to detect viruses.

Rapid advances in biosensor technology using mass spectrometry have led to the development of powerful systems that can provide rapid discrimination of biological components in complex mixtures. The mass spectra that are produced can be considered a specific fingerprint or molecular profile of the bacteria or virus analysed. The software system built into the instrument identifies and quantifies the pathogen in a given sample by comparing the resulting mass spectra with those in a database of infectious agents,
and thus allows the rapid identification of many thousands of types of bacteria and viruses. Additionally, these tools can recognize a previously unidentified organism in the sample and describe how it is related to those encountered previously. This could be useful in determining not only dengue serotypes but also dengue genotypes during an outbreak. Identification kits for infectious agents are available in 96-well format and can be designed to meet specific requirements. Samples are processed for DNA extraction, PCR amplification, mass spectrometry and computer analysis.

Microarray technology makes it possible to screen a sample for many different nucleic acid fragments corresponding to different viruses in parallel. The genetic material must be amplified before hybridization to the microarray, and amplification strategy can target conserved sequences as well as random-based ones. Short oligonucleotides attached on the microarray slide give a relatively exact sequence identification, while longer DNA fragments give a higher tolerance for mismatches and thus an improved ability to detect diverged strains. A laser-based scanner is commonly used as a reader to detect amplified fragments labelled with fluorescent dyes. Microarray could be a useful technology to test, at the same time, dengue virus and other arboviruses circulating in the region and all the pathogens responsible for dengue-like symptoms.

Other approaches have been tested but are still in the early stages of development and evaluation. For instance, the luminescence-based techniques are becoming increasingly popular owing to their high sensitivity, low background, wide dynamic range and relatively inexpensive instrumentation.

QUALITY ASSURANCE

Many laboratories use in-house assays. The main weakness of these assays is the lack of standardization of protocols, so results cannot be compared or analysed in aggregate. It is important for national or reference centres to organize quality assurance programmes to ensure the profi ciency of laboratory staff in performing the assays and to produce reference materials for quality control of test kits and assays.

For nucleic acid amplifi cation assays, precautions need to be established to prevent contamination of patient materials. Controls and profi ciency-testing are necessary to ensure a high degree of confidence.[Lemmer K et al. External quality control assessments in PCR diagnostics of dengue virus infections. Journal of Clinical Virology, 2004, 30:291–296.]

BIOSAFETY ISSUES

The collection and processing of blood and other specimens place health care workers at risk of exposure to potentially infectious material. To minimize the risk of infection, safe laboratory techniques (i.e. use of personal protective equipment, appropriate containers for collecting and transporting samples, etc.) must be practised as described in WHO’s Laboratory biosafety manual.[WHO. Laboratory biosafety manual, 3rd ed. Geneva, World Health Organization, 2004 (ISBN 92 4 154650 6, WHO/CDS/CSR/LYO/2004.11, http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf).]

ORGANIZATION OF LABORATORY SERVICES

In a disease-endemic country, it is important to organize laboratory services in the context of patients’ needs and disease control strategies. Appropriate resources should be allocated and training provided.

Proposed model for organisation of laboratory services.
Dengue laboratory diagnosis: examples of good and bad practice
 

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Dr. House (a Television Character) An Enigma And His Demeanours


Gregory House, M.D. — typically referred to simply as House — is the title character of the American medical drama House. House was the most-watched television program in the world in 2008.

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Portrayed by English actor Hugh Laurie, he leads a team of diagnosticians as the Head of Diagnostic Medicine at the fictional Princeton-Plainsboro Teaching Hospital in New Jersey based on Yale-New Haven Hospital.
House’s character, created by David Shore, has been described as a

misanthrope (someone who dislikes people in general),

cynic, (someone who is critical of the motives of others)

narcissist, (someone in love with themselves)

and

curmudgeon (A crusty irascible cantankerous old person full of stubborn ideas)(An ill-tempered (and frequently old) person full of stubborn ideas or opinions.),


which was named one of the top television words of 2005 in honour of the character.


“There’s a cranky curmudgeon working at the hospital who gives all the patients and other doctors flak.”


He is the only character to appear in all 177 episodes and except for Wilson’s brief appearance, is the only regular character to appear in the season six premiere.


  • The humor itself is glorious, more than enough to draw in fans despite a repetitious plot. House’s deadpan humor and caustic verbal strikes entertain the viewers while also shedding some light on the history of a broken man. Still, the most entertaining part of the show was simply watching House at his work, which, in retrospect, was the whole point of the show. The entire show, based on Sherlock Holmes (did you catch the name pun), was meant to show off the brilliance of a detective at his work–his medical work.
  • House describes the story of a doctor in Japan, who was a social outcast by birthright. The doctor was brilliant and was able to garner the respect of other doctors simply because he was needed. In this story, I see an example of how beautiful it is to watch a professional at his job. Some will say that baseball can be a boring sport to watch. However, watching a brilliant pitcher who can control the wind up manipulate the ball is fascinating. A brilliant mathematician weaves ideas and algorithms to create new discoveries. Many people can think brilliant and artistic thoughts, but professional writers are the ones who weave them onto paper and into stories. People often talk about the beauty of art, but there is beauty in any profession.
  • In one single word, I see beauty. Whether it’s restraint of the body, manipulation of words, or simply control of a tool, the control of the craft is what sparks this beauty. Yes, “House M.D.” is fiction. However, it is fascinating to watch his redirection of ideas and his attention to details, details that everybody sees, but that only he notices. I’m not pleading for a sudden binge-watch of the “House” series. I’m making a small request: next time that you have a chance, watch a professional and notice the fluidity with which they can attack their work. Source: What “House M.D.” has to Say About The Beauty Of Your Craft

In the series, the character’s unorthodox diagnostic approaches, radical therapeutic motives, and stalwart rationality have resulted in much conflict between him and his colleagues. House often clashes with his fellow physicians, including his own diagnostic team, because many of his hypotheses about patients’ illnesses are based on subtle or controversial insights. His flouting of hospital rules and procedures frequently leads him into conflict with his boss, hospital administrator and Dean of Medicine Dr. Lisa Cuddy (Lisa Edelstein). House’s only true friend is Dr. James Wilson (Robert Sean Leonard), head of the Department of Oncology.

House is also often portrayed as lacking sympathy for his patients, a practice that allots him time to solve pathological enigmas.

The character is partly inspired by Sherlock Holmes. A portion of the show’s plot centers on House’s habitual use of Vicodin (Hydrocodone/paracetamol, hydrocodone/acetaminophen, or hydrocodone/APAP (or under brand names such as Lortab, Norco and Vicodin) is a combination opioid narcotic analgesic drug consisting of hydrocodone and paracetamol (acetaminophen) used to relieve moderate to severe pain.) to manage pain stemming from a leg infarction involving his quadriceps muscle some years earlier, an injury that forces him to walk with a cane. This addiction is also one of the many parallels to Holmes, who was a habitual user of cocaine. Source: Gregory House

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References to Sherlock Holmes

References to the fact that Gregory House was based on the famous fictional detective Sherlock Holmes created by Sir Arthur Conan Doyle appear throughout the series.

Shore explained that he was always a Holmes fan and found the character’s indifference to his clients unique.
  • The resemblance is evident in House’s reliance on deductive reasoning and psychology, even where it might not seem obviously applicable, and his reluctance to accept cases he finds uninteresting. His investigatory method is to eliminate diagnoses logically as they are proved impossible; Holmes used a similar method.
  • Both characters play instruments (House plays the piano, the guitar, and the harmonica; Holmes, the violin) and take drugs (House is dependent on Vicodin; Holmes uses cocaine recreationally).
  • House’s relationship with Dr. James Wilson echoes that between Holmes and his confidant, Dr. John Watson. Robert Sean Leonard, who portrays Wilson, said that House and his character—whose name is very similar to Watson’s—were originally intended to work together much as Holmes and Watson do; in his view, House’s diagnostic team has assumed that aspect of the Watson role. Wilson even has a dead-beat brother who may be dead, like Watson’s dead alcoholic brother. (season 1, episode 10) Shore said that House’s name itself is meant as “a subtle homage” to Holmes. House’s address is 221B Baker Street, a direct reference to Holmes’s street address. Wilson’s address is also 221B.

Gregory House, M.D., often construed as a misanthropic medical genius, heads a team of diagnosticians at the Princeton-Plainsboro Teaching Hospital in New Jersey. Most episodes revolve around the diagnosis of a primary patient and start with a precredits scene set outside the hospital, showing events ending with the onset of the patient’s symptoms. The typical episode follows the team in their attempts to diagnose and treat the patient’s illness, which often fail until the patient’s condition is critical. They usually treat only patients whom other doctors have not accurately diagnosed, and House routinely rejects cases that he does not find interesting. The story lines tend to focus on his unconventional medical theories and practices, and on the other characters’ reactions to them, rather than on the details of the treatments.
The team employs the differential diagnosis method, listing possible etiologies on a whiteboard, then eliminating most of them, usually because one of the team (most often House) provides logical reasons for ruling them out. Typically, the patient is misdiagnosed at least once and accordingly receives some treatments that are at best useless; this usually causes further complications, but—as the nature of the complications often provides valuable new evidence—eventually these help them diagnose the patient correctly. House often tends to arrive at the correct diagnosis seemingly out of the blue, often inspired by a passing remark made by another character. Diagnoses range from relatively common to very rare diseases.
The team faces many diagnostic difficulties from patients’ concealment of symptoms, circumstances, or personal histories, so House frequently proclaims during the team’s deliberations, “The patient is lying”, or mutters “Everybody lies”; such an assumption guides House’s decisions and diagnoses, and makes the countermeasure of housebreaking a routine procedure. Because many of his hypotheses are based on epiphanies or controversial insights, he often has trouble obtaining permission for medical procedures he considers necessary from his superior, who in all but the final season is hospital administrator Dr. Lisa Cuddy. This is especially the case when the proposed procedures involve a high degree of risk or are ethically questionable. Frequent disagreements occur between House and his team, especially Dr. Allison Cameron, whose standards of medical ethics are more conservative than those of the other characters.
Like all of the hospital’s doctors, House is required to treat patients in the facility’s walk-in clinic. His grudging fulfillment of this duty, or his creative methods of avoiding it, constitute a recurring subplot, which often serves as the series’ comic relief. During clinic duty, House confounds patients with unwelcome observations into their personal lives, eccentric prescriptions, and unorthodox treatments. However, after seeming to be inattentive to their complaints, he regularly impresses them with rapid and accurate diagnoses. Analogies with some of the simple cases in the clinic occasionally inspire insights that help solve the team’s case.

“It’s not a show about addiction, but you can’t throw something like this into the mix and not expect it to be noticed and commented on. There have been references to the amount of his consumption increasing over time. It’s becoming less and less useful a tool for dealing with his pain, and it’s something we’re going to continue to deal with, continue to explore.”
—Shore on House’s Vicodin addiction

House first attended Johns Hopkins University as an undergraduate. Before fully committing to medicine as his discipline, he considered getting a Ph.D. in Physics, researching dark matter. He was accepted to the Johns Hopkins School of Medicine and excelled during his time there. He was a front runner for a prestigious and competitive internship at the Mayo Clinic; however, another student, Philip Weber, caught him cheating, resulting in his expulsion from Johns Hopkins and rejection from the internship. While appealing his expulsion, he studied at the University of Michigan Medical School, and worked at a bookstore, where he met his future employer and love interest Lisa Cuddy (Lisa Edelstein).

House eventually became a Board certified diagnostician with a double specialty in infectious disease and nephrology.

House’s character frequently shows off his cunning and biting wit and enjoys picking people apart and mocking their weaknesses. House accurately deduces people’s motives and histories from aspects of their personality, appearance, and behaviour. His friend and colleague Wilson says some doctors have the “Messiah complex” — they need to “save the world” — but House has the “Rubik’s complex” — he needs to “solve the puzzle”. House typically waits as long as possible before meeting his patients. When he does, he shows an unorthodox bedside manner and uses unconventional treatments. However, he impresses them with rapid and accurate diagnoses after seemingly not paying attention. This skill is demonstrated in a scene where House diagnoses an entire waiting room full of patients in little over one minute on his way out of the hospital clinic. Critics have described the character as “moody”, “bitter”, “antagonistic”, “misanthropic”, “cynical”, “grumpy”, “maverick”,[40] “anarchist”, “sociopath”, and a “curmudgeon”. The Global Language Monitor chose the word “curmudgeon” as the best way to describe the character.

I infact like him, but characters like him wouldn’t survive in Indian conditions.

Sometimes we forget goodness of a person in the din of negativity. There are many good character traits of Dr. House,

First, he  always involves his team members to arrive at a diagnosis, sometimes even forcefully.

Secondly, he always try to take a point of view, independent of higher authorities, and accepts full responsibility for all his demeanours and decisions.

Thirdly, he never tries to take credit for what duly belongs to others.

Fourthly, though his methods and strategies are highly questionable, he always tries to help his friends, keeping an eye over them protecting them from harm, but sometimes, putting them in trouble, most of the times, without their permission, and without their knowledge.

Fifthly, he never punishes or reprimands his team members for arriving at a wrong diagnosis, but does so, when they dare not think differently.

Sixthly, and the most important reason being that most of the criticisms that Dr. House face, is because of the way he handles his patients, no emotional attachment of any sorts, no sympathy, no clinical examination. But then, he always gets rarest of rare cases/diseases and patients with an illness at a terminal stage, where a patient has already been rejected for a possible cure by many doctors, he neither has the time nor resources to even ponder over such luxuries. Furthermore, he has his protégés to do that job for him. He dislikes attending to his patients individually, mainly because he thinks doing so would develop an emotional bond between the two, which as a result would hinder his critical thinking process in arriving at a correct diagnosis.

Seventhly, Dr. House has  a keen sense of observation, and his memory, which would not let him forget even the very fine details.

Hugh Laurie (Dr. House) portrays a character that is adept at finding solutions to complex or obscure diseases.The only potential problem is his tendency to break the rules. Dr. House might not care much to preserve patients’ sanity, but this allows him to calculate the course of action that guarantees the highest possible chance of survival.

If he followed all the rules, most of his patients would have died.

House doesn’t think invading privacy is an issue, so long as the patients are able to live. This applies to countless other rules that he breaks in order to get the job done.

This series gives enough evidence, proving that even doctors are humans and have their own set of problems, which have to be dealt with simultaneously along with daily chores.

Quality of Being Tangible: A Story of Different Attitudes – Different Perception

What is the meaning of the word “Tangible”?

As per the Oxford English dictionary, it means “Clear and definite; real.”

or  “A thing that is perceptible by touch.”

Source: http://www.oxforddictionaries.com/definition/english/tangible#

Today (12/09/2015) I came across a story on facebook in my newsfeed from the account of Dr Prashant Gautam ( https://www.facebook.com/drprashantgautam?fref=nf).

I liked it, as it was based on the strong scientific foundation and not merely a fable or a part of gossip mongering.

So, I gave myself a thought,“If what is mentioned in the story is true, why can’t I prove this scientifically, it won’t be and shouldn’t be that difficult if indeed it was correct”.

So here I go.

Kindly read it with patience as I have made a few deliberations.

The story is in the language ‘Hindi’ and is as follows (kindly use the site  translate.google.com for a rough translation into any other language):

एक राजा का दरबार लगा हुआ था क्योंकि सर्दी का दिन था, इसलिये राजा का दरवार खुले मे बैठा था पूरी आम सभा सुबह की धूप मे बैठी थी । महाराज ने सिंहासन के सामने एक टेबल जैसी कोई कीमती चीज रखी थी पंडित लोग दीवान आदि सभी दरबार मे बैठे थे,  राजा के परिवार के सदस्य भी बैठे थे । उसी समय एक व्यक्ति आया और प्रवेश मांगा । राजा ने बुलाया और कहा,” क्या बात हैं ?” प्रवेश मिल गया तो उसने कहा, 

  • [English translation- Once a king was holding a darbaar (court). As it was during winters, it was being held in open and every person was sitting under open skies under the morning sun. A precious table was kept in front of the throne of the king, and all his courtiers, his family along with his prime minister, were also sitting in that darbar. Suddenly, came along a traveller, who asked for king’s permission to present himself in front of the king. King asked him, “What is the matter?” When permission was granted, he said,]

“मेरे पास दो वस्तुए है मै हर राज्य के राजा के पास जाता हूँ और अपनी बात रखता हूँ।  कोई परख नही पाता सब हार जाते हैंं और मै विजेता बनकर घूम रहा हूँ। अब आपके नगर मे आया हूँ।”

तब उसने दोनो वस्तुये टेबल पर रख दी बिल्कुल समान आकार, समान रुप रंग, समान प्रकाश सब कुछ नख सिख समान। राजा ने कहा,” ये दोनो वस्तुए एक हैं।”

  • ” I have two items, whenever I go to a new place or a kingdom, I keep these items in front of the king. No one has ever been able to examine these two items and tell their worth, as yet. Now I have come to your kingdom with the same problem and an air of invincibility.”  Then, the traveller kept those two items on a precious table which was kept in front of the king. Both were of the same size, look, shape, colour etc. Superficially, both looked as exact copies of each other. The king also said, “These two items are one and the same.”

तब उस व्यक्ति ने कहा,“हाँ दिखाई तो एक सी देती हैं लेकिन हैं भिन्न। इनमे से एक हैं बहुत कीमती हीरा हैं और एक हैं काँच का टुकडा। लेकिन रूप रंग सब एक हैं। कोइ आज तक परख नही पाया की कौन सा हीरा हैं और कौन सा काँच। कोइ परख कर बताये की ये हीरा हैं या ये काँच। अगर परख खरी निकली तो मे हार जाउँगा और यह कीमती हीरा मैं आपके राज्य की तिजोरी मे जमा करवा दूंगा । यदि कोइ न पहचान पाया तो इस हीरे की जो कीमत हैं उतनी धनराशि आपको मुझे देनी होगी। इसी प्रकार मे कइ राज्यो से जीतता आया हूँ।”

  • Then that traveller too replied, “Yes, superficially they look the same, but they are different. One of these items is a diamond and the other one is made of glass, but their colour, size, shape etc. is same. No one, till date has ever been able to examine them and tell the difference, if someone succeeds, I would accept defeat and deposit the diamond in your treasure. If no one can tell the difference, I would like to have the actual price of diamond be given to me as a prize. This is the same deal I have presented, before the others, and winning all these years in the kingdoms I have travelled to.”

राजा ने कहा,“मैं तो नही परख सकूंगा”, दीवान बोले,“हम भी हिम्मत नही कर सकते क्योंकि दोनो बिल्कुल समान हैं ।” कोइ हिम्मत नही जुटा पाया। हारने पर पैसे देने पड़ेंगे, इसका कोई सवाल नही था क्योकि राजा के पास बहुत धन हैं । राजा की प्रतिष्ठा गिर जायेगी, इसका सबको भय था, अगर कोइ व्यक्ति पहचान नही पाया। आखिरकार पीछे थोड़ी हलचल हुई। एक अंधा आदमी हाथ मे लाठी लेकर उठा। उसने कहा,“मुझे महाराज के पास ले चलो मैने सब बाते सुनी हैं । और यह भी सुना कि कोइ परख नही पा रहा हैं । एक अवसर मुझे भी दो।”

  • The king replied,” I will not be able to do the same”, Minister also said, “I cannot also dare to tell the difference, as both of them look the same.” No one present was able to gather enough courage to come forward to take that challenge. As the king had lots of money, a monetary loss was not amongst his first and foremost priority but if none could tell the difference, losing pride and self-respect of the kingdom were what everyone feared. Then suddenly one could hear some commotion at the back. A blind (having little or no visibility; one who lacks vision) person with a cane in his hand got up from his seat and entered the premises. He said, “Take me to the king, I have heard everything. Give me a chance.” 

एक आदमी के सहारे वह राजा के पास पहुँचा अौर उसने राजा से प्रार्थना की,“मैं तो जनम से अंधा हूँ फिर भी मुझे एक अवसर दिया जाये। मैं भी एक बार अपनी बुद्धि को परखू और हो सकता हैं  कि सफल भी हो जाऊ, और यदि सफल न भी हुआ तो वैसे भी आप तो हारे ही हैं।” राजा को लगा कि इसे अवसर देने मे क्या हरज़ है। राजा ने कहा,“ठीक हैं ।” तब उस अंधे आदमी को दोनो चीज़े छुआ दी गयी और पूछा गया,“इसमे कौन सा हीरा हैं  और कौन सा काँच, यही परखना हैं ।”

  • With assistance from another person, he reached near the king, where the table with two items was kept and there he requisitioned the king,“I am blind since birth, still I would like to request you to give me an opportunity. I would like to use my intellect to examine these items, it is a possibility that I may eventually tell the difference, even if I am unsuccessful it’s of no consequence to you as you’ve already accepted defeat.” The king said,“You have my permission.” Then that blind man was asked to touch those items and enquired from,“Kindly examine them and inform us which is diamond and which is not.”

कथा कहती हैं कि उस आदमी ने एक मिनट मे कह दिया कि,“यह हीरा हैं और यह काँच”, जो आदमी इतने राज्यों को जीतकर आया था । आदमी नतमस्तक हो गया और बोला,“सही हैं । आपने पहचान लिया, धन्य हो आप अपने वचन के मुताबिक यह हीरा मैं आपके राज्य की तिजोरी मे दे रहा हूँ।” सब बहुत खुश हो गये और जो आदमी आया था वह भी बहुत प्रसन्न हुआ कि कम से कम कोई तो मिला परखने वाला। वह राजा और अन्य सभी लोगो ने उस अंधे व्यक्ति से एक ही जिज्ञासा जताई कि,“तुमने यह कैसे पहचाना कि यह हीरा हैं और वह काँच?” उस अंधे व्यक्ति ने कहा,

  • As the story proceeds, it was believed that within a minute, that blind man was able to tell the difference and identified exactly which one of them was a diamond and which one was a fake or glass, he triumphantly said,“this is a diamond and the other one is made of glass.” The traveller who had visited so many kingdoms and previously won this bet accepted defeat and informed the blind man,“You are right, you could indeed tell the difference, may GOD bless you, and as per my promise, I would like to deposit this diamond in the treasure of king.” Everyone present there was gratified including the traveller, who could take solace in the fact that at least there was someone on this planet who could tell the difference. But the king and the traveller were curious to know how could a blind man achieve what none else could even think of. So they asked the blind man,“How could you tell the difference between a diamond and a glass.” The blind man replied,

“सीधी सी बात हैं। मालिक! धूप मे हम सब बैठे हैं। मैने दोनो को छुआ। जो ठंडा रहा वह हीरा जो गरम हो गया वह काँच ।”

जीवन मे भी देखना जो बात बात मे गरम हो जाये उलझ जाये वह काँच।

जो विपरीत परिस्थिति मे भी ठंडा रहे वह हीरा है।

  • “It’s very simple sire, all of us are sitting under direct sunlight. Hence, when I touched these two items, the one which was the same temperature as the environment, was diamond and the one which warmed up due to the heat of sunlight was glass,”

Diamond Ornaments

Glass Ornaments

Here’s another informative story I would like to share with you.

  • The first day I walked into my 10th-grade chemistry class, the lights were off. The teacher was waiting at the front of the room, holding a small candle in front of him. The candle’s tiny flame was the only source of light in the room.

    The class murmured a bit, confused and excited. Once we’d all quieted down, the teacher said, “Tell me some of the observations you can make about this candle.”

    We paused for a moment. That seemed a little too easy for chemistry class.

    “But keep in mind,” the teacher added, “that observations are the things you observe, empirically. Inferences are the things you conclude… and inferences can be wrong.”

    We stared at him, wary now.

    At last, someone piped up, “Um…The flame is yellow?”

    “The flame gives off heat.”

    “The wick is burning.”

    “The wax is cylindrical!”

    The teacher raised his eyebrows at that one. “Are you sure?”

    We stared harder at the candle, not sure how to respond to that one. It definitely looked cylindrical.

    The teacher shrugged. Then he put the candle into his mouth and started chewing.

    The class stared at him.

    At this point, I was wondering if I ought to call the emergency room or something, but the teacher just kept chewing as though nothing was wrong. Eventually, he swallowed, shrugged again, and said, “Actually tastes okay.”

    As it turns out, the so-called “candle” wasn’t made out of wax at all. It was actually just a slice of banana, with an almond slice for the wick.

    The student’s “wax” statement had actually been an inference.

    After explaining this to us, the teacher turned on the lights and said, “If there’s one thing I want you to learn in this class, it’s the first rule of scientific thinking: Never assume that your mind can’t lie to you.”

    And that’s the story of how my 10th-grade chemistry teacher swallowed a candle to teach us the first principle of scientific thinking. Here’s to you, Mr. Miller. Hannah Yang

https://www.quora.com/What-was-the-most-interesting-thing-your-teacher-said/answer/Hannah-Yang-4?srid=dP5R&share=0d62bd1c

Here teacher also draws a wrong inference when she says,“If there’s one thing I want you to learn in this class, it’s the first rule of scientific thinking: Never assume that your mind can’t lie to you.”

The mind rarely lies. It’s not the fault of mind but that of intellect, and how one uses it. The inference is always drawn after careful observation, reasoning and analyses of the given problem, wherein one should use all the five senses, and sometimes even our sixth sense. Sometimes the limiting factor (or senses) can act as a facilitator (as in the story Quality of Being Tangible: A Story of Different Attitudes – Different Perception) and sometimes (as in above example wherein vision and touch is a limiting factor) act as an impediment. Kindly read this story wherein absence of vision acts as a facilitator in arriving at a solution. Quality of Being Tangible: A Story of Different Attitudes – Different Perception

Scientific Explanation

glass_is_a_liquid

Although glass appears to be solid, it isn’t. Technically, glass is considered a liquid. In a liquid, the molecules are connected in no special way. Solids, like sand, have an ordered molecular structure. http://www.acmecompany.com/Pages/glass.html

250px-diamond_cubic-f_lattice_animation

Unit cell of the diamond cubic crystal structure

The diamond cubic crystal structure is a repeating pattern of 8 atoms that certain materials may adopt as they solidify. While the first known example was diamond, other elements in group 14 also adopt this structure, including α-tin, the semiconductors silicon and germanium, and silicon/germanium alloys in any proportion. https://en.wikipedia.org/wiki/Diamond_cubic

The diamond structure, which is made up of a carbon atom joined to 4 other carbon atoms, this is an example of a giant covalent structure. http://lucychemistry.blogspot.in/2013/05/triple-144-draw-diagrams-representing.html



tetincube4aNumerous mineral structures are based on the fact that tetrahedra can be inscribed in a cube. If atoms have a face-centered arrangement, we can join a corner atom to the three nearest face-centered atoms to create a tetrahedron. Four similarly-oriented tetrahedra can be created in the cube. Image source

tetincube4b There are two ways to orient tetrahedra in a face-centered cubic array.Image source

diamond

Diamond is one mineral that employs this structure. There are carbon atoms in a face-centered array (dark gray) plus an extra one (light gray) at the center of each tetrahedron. At left, the relationship of the carbon atoms to the tetrahedra is shown. On the right, the carbon-carbon bonds are shown. Image source

diamond2

Above is the diamond structure in a different orientation showing the tetrahedral structure a bit more clearly. As above, carbon atoms at the corners of the tetrahedra are dark gray, those in the middle are light gray. Image source

diamond3

Above is the diamond structure showing the carbon-carbon bonding. Bonds closer to the viewer are shown thicker. Image source



Very well said, the above-mentioned fact, has even been proven scientifically,  the tight, evenly-packed crystalline structure of diamonds makes them disperse heat quickly; thus, real diamonds will not heat up easily.

Same principle is used behind “diamond testers”. Commercial pen-size probes, called simply “diamond testers,” can test the thermal conductivity of the gem.

(In physics, thermal conductivity (often denoted k, λ, or κ) is the property of a material to conduct heat. Heat transfer occurs at a lower rate across materials of low thermal conductivity than across materials of high thermal conductivity. Correspondingly, materials of high thermal conductivity are widely used in heat sink applications and materials of low thermal conductivity are used as thermal insulation.)

https://en.wikipedia.org/wiki/Thermal_conductivity

Of course, these tests don’t tell us anything about the VALUE of the available diamond. According to the Gemological Institute of America, jewellers grade diamonds on the “4Cs” cut, clarity, colour and carat weight.

It also proves another philosophical point.

Having all our senses intact, sometimes can be a handicap in real life, where in, we get so used to using all of them, simultaneously, that we forget their individual importance.

Diamond Tester

Furthermore, heat test can be used to differentiate between a fake and a real diamond, but still, it can not decide the true value of a diamond, or a person, or an individual.

I would also discuss one more fact here, the specific heat (or the heat required for raising the temperature of a given mass of substance). Heat capacity or thermal capacity is a measurable physical quantity equal to the ratio of the heat added to (or removed from) an object to the resulting temperature change. The SI unit of heat capacity is joule per kelvin or J/K and the dimensional form is L2MT−2Θ−1. Specific heat is the amount of heat needed to raise the temperature of a certain mass 1 degree Celsius. https://en.wikipedia.org/wiki/Heat_capacity

Diamond (carbon) = 516 J/kg degree C and

for Glass = 670 to 753 J/kg degree C.

[Source: http://www.engineeringtoolbox.com/specific-heat-capacity-d_391.html#]

This means that in a controlled environment, and if evenly heated, the energy required for raising the temperature of diamond (1 kg) by 1-degree centigrade is less compared to that of glass (1 kg). Diamond (carbon) has a lower specific heat, and thus requires the least heat in order to have an increase in temperature. It’s (diamond) temperature will thus increase the most for a given the amount of heat. Temperature reflects the average randomised kinetic energy of constituent particles of matter (e.g. atoms or molecules) relative to the centre of mass of the system, while heat is the transfer of energy across a system boundary into the body other than by work or matter transfer. Translation, rotation and vibration of atoms represent the degrees of freedom of motion which classically contribute to the heat capacity of gases, while only vibrations are needed to describe the heat capacities of most solids, as shown by the Dulong–Petit law [Dulong–Petit law, statement that the gram-atomic heat capacity (specific heat times atomic weight) of an element is a constant; that is, it is the same for all solid elements, about six calories per gram atom]. Other, more exotic contributions can come from magnetic and electronic degrees of freedom in solids, but these rarely make substantial contributions. [https://en.wikipedia.org/wiki/Heat_capacity]

Source: http://chemwiki.ucdavis.edu/Physical_Chemistry/Thermodynamics/Chemical_Thermochemistry/18.4_Entropy_Changes_and_the_Third_Law_of_Thermodynamics

File:Thermally Agitated Molecule.gif

Shown here is the thermal motion of a segment of protein alpha helix. Molecules have various internal vibrational and rotational degrees of freedom. This is because molecules are complex objects; they are a population of atoms that can move about within a molecule in different ways. This makes molecules distinct from the noble gases such as helium and argon, which are monatomic (consisting of individual atoms). Heat energy is stored in molecules’ internal motions which give them an internal temperature. Even though these motions are called “internal,” the external portions of molecules still move—rather like the jiggling of a water balloon.

Glass absorbs more heat for a given temperature rise, compared to the diamond of the same mass, when the energy/heat is provided evenly.

Extrapolating the above findings one can also opine that a people/group of individuals who has been likened to have a diamond like quality would withstand more stresses or difficulties in life when stressed or when energy is provided to it on a singular basis rather than uniformly, as they have a property to uniformly distribute that singular energy amongst it’s constituents (high thermal conductivity) more evenly. Though they will be able to tolerate less stress/energy on group basis (as a whole) when compared to glass. (diamond=low specific heat)

On the contrary,  people or a group of individuals who have been likened to have a glass like quality would be able to tolerate more stress/energy on a group basis compared to diamond when done on a uniform basis i.e. every individual is stressed with equal amount of heat/energy/stress. (glass=high specific heat) but less or lower when the same is done on individual basis.(glass=low thermal conductivity)

http://www.livescience.com/29172-how-to-test-a-diamond.html and http://www.wikihow.com/Tell-if-a-Diamond-is-Real

Ultrasound Mediated Bio-effects And Sonoporation.

What are ultrasounds?

Ultrasound is defined by the American National Standards Institute as “sound at frequencies greater than 20 kHz.”

Ultrasounds are sound waves with frequencies higher than the upper audible limit of human hearing. Ultrasound is not different from ‘normal’ (audible) sound in its physical properties, only in that humans cannot hear it. This limit varies from person to person and is approximately 20 kilohertz (20,000 hertz) in healthy, young adults. Ultrasound devices operate with frequencies from 20 kHz up to several gigahertz. [https://en.wikipedia.org/wiki/Ultrasound]

Therapeutic applications of ultrasound, including microbubble-enhanced sonoporation, are stimulating widespread research activity aimed at both the characterization and control of ultrasonically mediated bioeffects.

Modern medical ultrasonics may be viewed as having evolved through three generations of applications.

The first generation emphasizes diagnostic imaging, employing ultrasound fields not intended to have tissue effects.

The second generation has deliberately exploited more aggressive ultrasound regimes for direct interventional approaches, including lithotripsy (of ductal calculi), phacoemulsification (of cataracts), and high intensity focused ultrasound (HIFU) for tumour ablation, thrombolysis, and haemostatis.

A third and emerging area involves an indirect therapeutic application of ultrasound to sensitize tissue actively, or otherwise enhance the efficacy for parallel administration of biotherapeutics. Here, particular progress has been achieved with ultrasound assisted transdermal delivery. However, this has been facilitated, in part, because the target tissue (i.e., stratum corneum) is non-viable. Perhaps the most challenging avenue for therapeutic ultrasound is to facilitate molecular delivery whilst retaining tissue viability, the criteria necessary for drug- and gene-based therapies. Excitingly, initial in vitro demonstrations of enhanced transfection, and also increased sensitivity to chemotherapeutic agents, have now also been realized with compelling in-vivo validations.

Evidently, this latter category of ultrasound-mediated therapy holds promise for a diversity of potential uses. However, reducing the multiplicity of these abstract possibilities to the more refined base of concrete realizations that are best suited to ultrasonic enhancement requires strategic action. Targeting research with the greatest impact requires an understanding of the strengths and weaknesses of ultrasonic bioeffects, which remain poorly understood at a mechanistic level. This situation hinders insight and indeed foresight into the future of this field.

Source: Paul Campbell and Mark R. Prausnitz: Ultrasound Med Biol. 2007 Apr; 33(4): 657. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1934612/

Lack of efficient drug and gene delivery is one of the major problems of cancer chemo- and bio-therapy. Different non-viral approaches have been proposed for drug and gene delivery, such as electroporation, chemical methods, liposomal delivery, gene gun mediated transfer. These techniques show a potential for drug and gene delivery, however, site-specific and efficient delivery still remains a difficult problem.

Recently, novel ultrasound-mediated techniques have been proposed for drug and gene delivery. In this approach, ultrasound radiation is used to induce cavitation, which can be applied to different organs. Therefore, this technique may have an advantage over other methods for delivery of drugs and genes to specific sites including tumours.

Ultrasound-induced cavitation has been shown to deliver macromolecules and genes in cells in vitro and in vivo and to improve cancer chemotherapy in nude mice. Since ultrasound-induced drug and gene delivery has a great potential for in vivo application in cancer chemoand bio-therapy, optimization of ultrasound-induced delivery of macromolecular drugs and DNA in cancer cells in vitro may provide a protocol which could routinely be used in anti-cancer drug design and in in vivo applications.

Source: Irina V, Larina B, Mark Evers, and Rinat O. Esenaliev: Optimal Drug and Gene Delivery in Cancer Cells by Ultrasound-Induced Cavitation. Anticancer Res. 25: 149-156 (2005). http://ar.iiarjournals.org/content/25/1A/149.long

Now scientists are developing innovative ways to adapt noninvasive ultrasound technology to deliver drugs and genes to specific organs and tissues in the body. Some traditional methods of drug delivery are often not suitable for large molecules such as proteins and DNA, underscoring the need for improved drug delivery strategies. Ultrasound holds considerable clinical promise in this realm.

Ultrasound is a very effective modality for drug delivery and gene therapy because energy that is non-invasively transmitted through the skin can be focused deeply into the human body in a specific location and employed to release drugs at that site. Ultrasound cavitation, enhanced by injected microbubbles, perturbs cell membrane structures to cause sonoporation and increases the permeability to bioactive materials. Cavitation events also increase the rate of drug transport in general by augmenting the slow diffusion process with convective transport processes. Drugs and genes can be incorporated into microbubbles, which in turn can target a specific disease site using ligands such as the antibody. Drugs can be released ultrasonically from microbubbles that are sufficiently robust to circulate in the blood and retain their cargo of drugs until they enter an insonated volume of tissue. Local drug delivery ensures sufficient drug concentration at the diseased region while limiting toxicity for healthy tissues.

Ultrasound-mediated gene delivery has been applied to heart, blood vessel, lung, kidney, muscle, brain, and tumour with enhanced gene transfection efficiency, which depends on the ultrasonic parameters such as acoustic pressure, pulse length, duty cycle, repetition rate, and exposure duration, as well as microbubble properties such as size, gas species, shell material, interfacial tension, and surface rigidity. Microbubble-augmented sonothrombolysis can be enhanced further by using targeting microbubbles.

Source: Liang, H.,D., Tang, J., Halliwell, M. Sonoporation, drug delivery, and gene therapy. Proc Inst Mech Eng H. 2010;224(2):343-61.


Transmission electron micrograph showing a prostate cancer cell immediately after exposure to ultrasound. The image has been colour enhanced to show the spot where the cell membrane has been removed. Image courtesy of Robyn Schlicher, Robert Apkarian, and Mark Baran.


Scientists have known for sometime now that exposure of cells to ultrasound at higher intensity levels and different frequencies than those used for diagnostic purposes can drive molecules into the living cells, thereby increasing the effects of drugs and the expression of genes. The mechanism by  which ultrasound increases permeability of the protective outer membrane of cells, however, was uncertain until Dr. Mark Prausnitz, Professor of Chemical and Biomedical Engineering at Georgia Institute of Technology, and his team directed their research toward this phenomenon. With the aid of various microscopy techniques, the researchers showed that ultrasound can cause bubbles to oscillate and violently collapse – a process known as cavitation – in a cell suspension, producing a shock wave which, in turn, causes fluid to move and open up holes in the cell membranes. The holes allow macromolecules like proteins to enter the cell before the holes close in a matter of minutes by an internal cellular patching process.

Prausnitz and his team have seen that the extent to which the cellular membrane is  disrupted can vary depending on the ultrasound parameters used. Through study of the physical, acoustic and biological conditions in which molecules are driven into cells, they found that the impact of ultrasound can also kill cells.

“The cell has it’s plasma membrane to regulate what is inside the cell, so when you disrupt the membrane all sorts of molecules can go inside the cell and that regulation is disrupted. The cell can be highly stressed by that, and therefore actively tries to repair the membrane. If the membrane breach is not that big, the membrane is able to reseal itself. But if it’s too big, we see evidence of various biochemical pathways of programmed cell death that kick into gear due to that stress.” says Prausnitz.

A major challenge that researchers face is determining the optimal ultrasound qualities that will open holes in cell membranes without killing the cells, so that drug delivery can be maximised while cell viability is maintained. The Prausnitz team is focussing it’s research on determining the mechanisms by which cellular bio-effects of ultrasound occur.

“If we can learn more about how cells respond to plasma membrane disruptions, we will have a valuable tool in designing a controllable therapeutic ultrasound system,” says Hutcheson, a graduate student in Prausnitz lab.

 There is another method by which ultrasound can be used for drug or material delivery at the desired areas. Ultrasound is a method that was widely used for the synthesis of various nanomaterials, for example coating carbon nanotubes and nobel metals. In the biomedical field, ultrasound has been used in various application: destruction and fragmentation of contrast agents, gas release, destruction of polymers, albumin or lipid shells of microbubbles and in drug delivery (Unger, 1997). Recent studies showed that ultrasound can be used for destruction of Polyelectrolyte multilayer or PEM capsules (Shchukin, Gorin, &  Möhwald,2006, Skirtach, De Geest, Mamedov, Antipov, Kotov, & Sukhorukov, 2007). Here, nanoparticles were used to increase the density of the shells of microcapsules, the ultrasound serves as a trigger to release encapsulated materials. It is worthwhile to notice that upon propagation an ultrasound wave experiences viscous and thermal absorption, and scattering in the medium. However, it is the cavitation microbubble which occur as a result of the collapse of the generated microbubble and the shear forces which cause the destruction of the polyelectrolyte capsules. Powers in the range of 100-500 W at frequencies of 20 KHz were applied for destruction of the capsules. It was found that nanoparticles adsorbed on microcapsules affect the action of ultrasound on their shells.

[Page 563, Biotechnology: Pharmaceutical Aspects. Nanotechnology in Drug delivery ©2009]

Polyelectrolyte multilayer (PEM) capsules engineered with active elements for targeting, labeling, sensing and delivery hold great promise for the controlled delivery of drugs and the development of new sensing platforms. PEM capsules composed of biodegradable polyelectrolytes are fabricated for intracellular delivery of encapsulated cargo (for example peptides, enzymes, DNA, and drugs) through gradual biodegradation of the shell components. PEM capsules with shells responsive to environmental or physical stimuli are exploited to control drug release. In the presence of appropriate triggers (e.g., pH variation or light irradiation) the pores of the multilayer shell are unlocked, leading to the controlled release of encapsulated cargos. By loading sensing elements in the capsules interior, PEM capsules sensitive to biological analytes, such as ions and metabolites, are assembled and used to detect analyte concentration changes in the surrounding environment.

[del Mercato, L., L., Ferraro M., M., Baldassarre F., Mancarella, S., Greco,V., Rinaldi, R., Leporatti S. Biological applications of LbL multilayer capsules: from drug delivery to sensing: Adv Colloid Interface Sci. 2014 May;207:139-54. doi: 10.1016/j.cis.2014.02.014. Epub 2014 Feb 21.]

Clinical Possibilities

Prausnitz perceives ultrasound-mediated drug delivery as having a potentially broad range of applications within therapeutic medicine, as this process is independent of both cell and drug type. He also envisions other applications in which the goal might not necessarily be to get molecules into cells, but rather to penetrate deeper into a multicellular tissue to sensitize it. “Ultrasound might be able to open up the permeability of the tissue as a whole and in that way drive drugs more generally into that tissue,” Prausnitz explains. The team is studying the effect of ultrasound on the uptake and viability of cells in explanted carotid arteries from pigs as a three-dimensional tissue model.

Widespread research activity is also under way to understand the bio-effects of ultrasound in the context of gene therapy. “It appears that ultrasound is doing other things to the cell [ in addition to enhancing DNA delivery ] that might further enhance that transfection efficiency of a cell,” says Prausnitz.

Challenges to Overcome

Brightfield (above) and confocal/fluorescence (below) microscopy of prostate cancer cells before (left), immediately after (center), and long after (right) exposure to ultrasound. Holes in the cell membranes are highlighted with an arrow. Image Courtesy of Robyn Schlicher.

 Previous applications of ultrasound as a diagnostic tool and as a direct interventional approach for the pulverization of kidney stones and tumour ablation, among other uses, have contributed to the current standing of medical ultrasonics as a well-developed, non invasive technology. The therapeutic application of ultrasound to enhance efficacy of drugs and gene based therapy, however, is still in early stages of development.

“If the ultrasonic parameters can be well controlled, drugs could be targeted to a specific region in the body. For example, a tumour site could be targeted so that drugs are preferentially delivered to the tumour cells. This could minimize the side-effects commonly associated with traditional chemotherapeutic treatments.” explains Hutcheson. Thus, non invasively focussed ultrasound has the potential to improve the delivery of drugs and genes to targeted tissues, and increasing efficacy.

 Prausnitz believes that a number of challenges need to be overcome before ultrasound-mediated therapy can be used in humans. Researchers need to determine the optimum cavitational activity, as well as other physical and chemical parameters, within the body for each given application. To control the impact of ultrasound on cells, researchers first need to better understand the pathways that mediate cell death so that maximum cell viability can be preserved. Further studies are needed to determine whether other cellular and physiological pathways are affected by ultrasound. There is still a long way to go to fully validate initial demonstrations that the ultrasound bioeffects that are effective and desirable in vitro – enhanced transfection and increased sensitivity to chemotherapeutics and other agents – are reproducible in vivo.

Evidence from other research fields suggests that cell membranes are continually ripped open and repaired inside the body without long-term effects. Mechanical impacts such as the beating of the heart, the motility of the gut, and the movement of muscles rip open cells in our bodies, and a similar mechanism of membrane repair as that shown by the Prausnitz team was demonstrated earlier with these mechanical stresses. While these data would suggest that cells may similarly withstand the effects of ultrasound, thorough studies are needed to corroborate this hypothesis and address other safety concerns.
Despite these obstacles, Prausnitz and the ultrasound research community remain optimistic that the day will come when ultrasound-mediated therapy will be widely applied in humans.

New Research

In the past two decades, research has underlined the potential of ultrasound and microbubbles to enhance drug delivery. However, there is less consensus on the biophysical and biological mechanisms leading to this enhanced delivery. Sonoporation, i.e. the formation of temporary pores in the cell membrane, as well as enhanced endocytosis is reported. Because of the variety of ultrasound settings used and corresponding microbubble behavior, a clear overview is missing. Therefore, in this review, the mechanisms contributing to sonoporation are categorized according to three ultrasound settings:

i) low intensity ultrasound leading to stable cavitation of microbubbles,

ii) high intensity ultrasound leading to inertial cavitation with microbubble collapse, and

iii) ultrasound application in the absence of microbubbles.

Using low intensity ultrasound, the endocytotic uptake of several drugs could be stimulated, while short but intense ultrasound pulses can be applied to induce pore formation and the direct cytoplasmic uptake of drugs. Ultrasound intensities may be adapted to create pore sizes correlating with drug size. Small molecules are able to diffuse passively through small pores created by low intensity ultrasound treatment. However, delivery of larger drugs such as nanoparticles and gene complexes, will require higher ultrasound intensities in order to allow direct cytoplasmic entry.

Source: Lentacker I, De Cock I, Deckers R, De Smedt SC, Moonen CT. Understanding ultrasound induced sonoporation: definitions and underlying mechanisms. Adv Drug Deliv Rev. 2014 Jun;72:49-64. doi: 10.1016/j.addr.2013.11.008. Epub 2013 Nov 21.

Image for unlabelled figure

Source: http://www.nibib.nih.gov/news-events/newsroom/ultrasound-mediated-therapy-are-we-there-yet

Fluorescent Light-Emitting Diode (LED) Microscopy For Diagnosis of Tuberculosis (WHO)

This is an acid fast stain of Mycobacterium tuberculosis (MTB). Note the red rods–hence the terminology for MTB in histologic sections or smears: acid fast bacilli.

Image Source: http://library.med.utah.edu/WebPath/INFEHTML/INFEC033.html

A sputum smear containing Mycobacterium tuberculosis bacteria, as seen under the LED fluorescence microscope

LED fluorescence microscope

Source: http://www.finddiagnostics.org/programs/tb/find_activities/led_microscopy.html

Source: http://www.olympusmicro.com/primer/techniques/fluorescence/anatomy/fluoromicroanatomy.html

Mercury vapour lamp fluorescence microscopy

Direct sputum smear microscopy is the most widely used means for diagnosing pulmonary TB and is available in most primary health-care laboratories at health-centre level. Most laboratories use conventional light microscopy to examine Ziehl-Neelsen-stained direct smears; this has been shown to be highly specific in areas with a high prevalence of TB but with varying sensitivity (20–80%).

Fluorescence microscopy is more sensitive (10%) than conventional Ziehl-Neelsen microscopy, and examination of fluorochrome-stained smears takes less time.

Uptake of fluorescence microscopy has, however, been limited by its high cost, due to expensive mercury vapour light sources, the need for regular maintenance and the requirement for a dark room. 

LED microscopy was developed mainly to give resource-limited countries access to the benefits of fluorescence microscopy.

  1. First, existing fluorescence microscopes were converted to LED light sources. Considerable research and development subsequently resulted in inexpensive, robust LED microscopes or LED attachments for routine use in resource-limited settings.
  2. In comparison with conventional mercury vapour fluorescence microscopes, LED microscopes are less expensive, require less power and can run on batteries; furthermore, the bulbs have a long half-life and do not pose the risk of releasing potentially toxic products if broken, and LED microscopes are reported to perform equally well in a light room. These qualities make LED microscopy feasible for use in resource-limited settings, bringing the benefits of fluorescence microscopy (improved sensitivity and efficiency) where they are needed most.
  3. The LED microscope lamp is also inexpensive when compared to the mercury vapor or halogen lamp used in regular fluorescent microscopy and has a life span of more than 10,000-50,000 hours.

Accuracy of LED in comparison with reference standards

LED microscopy showed 84% sensitivity (95% confidence interval [CI], 76–89%) and 98% specificity (95% CI, 85–97%) against culture as the reference standard. When a microscopic reference standard was used, the overall sensitivity was 93% (95% CI, 85–97%), and the overall specificity was 99% (95% CI, 98–99%). A significant increase in sensitivity was reported when direct smears were used rather than concentrated smears (89% and 73%, respectively).

Accuracy of LED in comparison with Ziehl-Neelsen microscopy

LED microscopy was statistically significantly more sensitive by 6% (95% CI, 0.1–13%), with no appreciable loss in specificity, when compared with direct Ziehl-Neelsen microscopy.

Accuracy of LED in comparison with conventional fluorescence microscopy

LED microscopy was 5% (95% CI, 0–11%) more sensitive and 1% (95% CI, -0.7% – 3%) more specific than conventional fluorescence microscopy.

In qualitative assessments of user characteristics and outcomes in relation to implementation, such as time to reading, cost-effectiveness, training and smear fading, the main findings were:

  1. In comparison with Ziehl-Neelsen, LED showed similar gains in time for reading as conventional fluorescence microscopy, with about half the time for smear examination.
  2. Cost assessments predict better cost-effectiveness with LED than with Ziehl-Neelsen microscopy, with improved efficiency.
  3. Qualitative assessments of LED microscopy confirmed many anticipated advantages, including use of the devices without a dark room, durability and portability (in the case of attachment devices); user acceptability in all field studies was reported as excellent.
  4. LED may be useful for diagnosing other diseases, e.g. malaria and trypanosomiasis, reducing the costs involved in providing integrated laboratory services.

    A thin blood smear showing trypanosomes stained with Acridine Orange, as seen under the LED fluorescence microscope

  5. Possible barriers to widescale use of LED include training of laboratory staff unfamiliar with fluorescence microscopy and the fading of inherently unstable fluorochrome stains. Evidence from standardized training suggests that full proficiency in LED microscopy can be achieved within 1 month.
  6. Adequate evidence is available to recommend the use of auramine stains for LED microscopy. Other commercial and in-house fluorochrome stains are not recommended.
  7. Evidence on the effect of fading of fluorochrome stains on the reproducibility of smear results over time suggests that current external quality assurance programmes should be adapted.
  8. The introduction of LED might affect the cost of other diagnostic modalities, e.g. light microscopy for examining urine, stools and blood, which should be retained at peripheral health laboratory level.
  9.  No studies were found on the direct effect of LED microscopy on outcomes important to patients, such as cure and treatment completion.
  10. Further research is required on the outcomes of LED microscopy that are important to patients and on combinations of LED microscopy with novel approaches for early case detection or sputum processing.

Source: WHO Policy Statement http://apps.who.int/iris/bitstream/10665/44602/1/9789241501613_eng.pdf

Green Fluorescent Protein (GFP’s)- Inspirational And Motivational Story of Sheer Resilience.

Lessons from the jellyfish’s green light

che_illpress_2008_aequorea

 

Off the west coast of North America, floats the jellyfish Aequorea victoria. In its light-emitting organs resides the green fluorescent protein, GFP, which glows intensely under ultraviolet light. GFP now revolutionizes the life sciences, and the scientists responsible for its development have been awarded Nobel Prize in Chemistry for the year 2008. The green light enables scientists to track, amongst other things, how cancer tumours form new blood vessels, how Alzheimer’s disease kills brain neurons and how HIV infected cells produce new viruses.

An unexpected catch for O. Shimomura

che_illpress_2008_shimomura_fishing

 

Throughout the summers of the 1960s, Osamu Shimomura (rearmost in the picture above), his family and various assistants, caught tens of thousands of jellyfish in the Pacific Ocean. From the edge, which emits green light when the jellyfish is agitated, Shimomura isolated a protein. Surprisingly, that protein did not shine in green. It was blue. Shimomura assumed that additional proteins were involved, and indeed found one more. It was not luminescent but did glow bright green under the light of an ultraviolet lamp.

che_illpress_2008_gfp_protein

 

A green guiding star for biosciences

Today, scientists use GFP to understand the function of cells and proteins in living creatures. Proteins are the chemical tools of life – they control most of what happens within a living cell. Every human being functions thanks to the well-oiled machinery of thousands of proteins, like haemo­globin, antibodies and insulin. If something malfunctions, illness and disease often follows. Therefore it is fundamental for the biosciences to map out the role of various proteins. Using DNA-technology, scientists connect GFP to interesting, but otherwise invisible, proteins. GFP functions like a little lantern, which is activated by ultraviolet light. The green glow helps scientists  track these proteins in the body.

che_illpress_2008_dna_rna

 

Tsien creates a palette with all the colours of the rainbow

During the 1990s Roger Tsien explored and changed GFP. His playful research resulted in proteins that glowed cyan, blue and yellow. However, he did not manage to produce any red colours. Red light penetrates the skin and other biological tissue more easily, and so is especially useful for research.

che_illpress_2008_rainbow

 

In 1999, Russian scientists isolated a red fluorescent protein, DsRED, from a coral. This protein was larger and more cumbersome than GFP. Tsien, however, managed to decrease the size of DsRED. From DsRED, Tsien also developed proteins with mouth-watering names like mPlum, mCherry, mStrawberry, mOrange and mCitrine.

A brilliant experiment by Chalfie

che_illpress_2008_gfpworm

 

When Martin Chalfie first heard about GFP in 1988, he was delighted. He realised that GFP could possibly be used to colour cells and proteins. If that was the case, it would revolutionise the biosciences. The picture above shows Chalfie’s successful experiment. The touch receptor neurons of the millimetre-sized roundworm Caenorhabditis elegans fluoresces green. We can see the round bodies and the long slender projection of the nerve cells.

GFP-gene

How cells become green
Chalfie positioned the GFP-gene behind a promoter (a gene switch), which is active in the touch receptor neurons of the round worm. He injected the gene construct into the gonads of a mature worm. The worm is a hermaphrodite and fertilizes itself. The GFP gene is passed onto the eggs that the worm lays. The eggs divide, forming new individuals. The GFP-gene is then present in all cells of the new generation of roundworms, but only the touch receptor neurons will produce GFP. When they fill up with GFP, they start to glow green under ultraviolet light.

The brainbow

The brainbow
Scientists have used three fluorescent proteins in cyan, yellow and red – colours similar to those used by a computer printer – to colour the brain of a mouse. Different neurons randomly produce different amounts of the proteins. We can distinguish single neurons interlaced within the dense network.

Tumour surrounded by nourishing blood vesselsTumour surrounded by nourishing blood vessels Scientists have coloured a breast cancer tumour with DsRED and the surrounding blood vessels with GFP. In this experiment, scientists discovered two proteins which help breast cancer cells spread. If scientists can neutralize these proteins, they might also be able to stop the cells from breaking away from the tumour area.

Osamu Shimomura                      Roger Y. Tsien              Martin Chalfie

This is an inspirational and motovational story of Osamu Shimomura.

Osamu Shimomura

Photo: Ulla Montan

Born: 27 August 1928, Kyoto, Japan.

Osamu Shimomura went on to share the Nobel Prize for Chemistry in the field of Biochemistry, year 2008, for the discovery and development of the green fluorescent protein, GFP.

Affiliation at the time of the award: Marine Biological Laboratory (MBL), Woods Hole, MA, USA, Boston University Medical School, Massachusetts, MA, USA

He shared his prize with Martin Chalfie (Columbia University, New York, NY, USA) and Roger Y. Tsien (University of California, San Diego, CA, USA, Howard Hughes Medical Institute). (Source: http://www.nobelprize.org)

Following are the excerpts from Nobel Lecture, December 8, 2008 by Osamu Shimomura.

Source: http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/shimomura_lecture.pdf

I discovered the green fluorescent protein GFP from jellyfish Aequorea aequorea in 1961 as a byproduct of the Ca-sensitive photoprotein aequorin [Shimomura, O., Johnson, F. H., and Saiga, Y. (1962), “Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea,” J. Cell. Comp. Physiol. 59: 223–239.], and identified it’s chromophore (an atom or group whose presence is responsible for the colour of a compound) in 1979 [Shimomura, O. (1979), “Structure of the chromophore of Aequorea green fluorescent protein,” FEBS Lett. 104, 220–222.]


GFP was a beautiful protein but it remained useless for the next 30 years after the discovery. Now GFP and it’s homologues are indispensable in biomedical research, due to the fact that these proteins self contain a fluorescent chromophore in their peptide chains and they can be expressed in living bodies. The identification of the fluorescent chromophore, however, depended on the GFP that had been accumulated for many years in our study of aequorin. Without the study of aequorin, the chromophore of GFP would have remained unknown and the flourishing of fluorescent proteins would not have occurred.


In 1967, Ridgeway and Ashley microinjected aequorin into single muscle fibres of barnacles, and observed transient calcium ion-dependent signals during muscle contraction. They were the first to directly measure the free Calcium ions (Ca2+) in a muscle cell. They achieved this by injecting a large cell from barnacle muscle with aequorin, a Calcium ions (Ca2+) activated photoprotein. Three important points were made in these studies: 

  1. the Calcium ions (Ca2+) in the resting muscle cell is ∼0.1μM and rises transiently to no more than 10 μM when the cell is stimulated to contract.
  2. the rise in Calcium ions (Ca2+) follows the electrical stimulation but preceeds the onset of contraction.
  3. the Calcium ions (Ca2+) begins to fall before maximal tension is actually achieved.

Source: Ridgway EB, Ashley CC. Calcium transients in single muscle fibers. Biochem Biophys Res Commun. 1967 Oct 26;29(2):229-34.

An example of just how important the potential of GFP’s is found in the recent work of Jeff Litchman and Joshua Sanes, researchers at the Harvard Brain Center. They have created transgenic mice with fluoroscent multicoloured neurons. But it is not their colourful splendour that makes these genetically modified mice so amazing, it is their potential to revolutionise neurobiology that has scientists across the globe so excited. Using individual colour derived from GFP’s, researchers will now be able to map the neural circuit of brain. By creating a wire diagram, researchers hope to identify “defective” wiring found in the neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease.

Brainbow is the process by which individual neurons in the brain can be distinguished from neighbouring neurons using fluorescent proteins. By randomly expressing different ratios of red, green, and blue derivatives of green fluorescent protein in individual neurons, it is possible to flag each neuron with a distinctive color. This process has been a major contribution to the field of connectomics, or the study of neural connections in the brain. The study of neural pathways is also known as hodology by earlier neuroanatomists.
The technique was originally developed in the spring of 2007 by a team led by Jeff W. Lichtman and Joshua R. Sanes, both professors of Molecular & Cellular Biology at Harvard University. Their demonstration of the technique in mice first appeared in the November 1, 2007 issue of the journal Nature.The original technique has recently been adapted for use with other model organisms including Drosophila melanogaster and Caenorhabditis elegans.

While older techniques were only able to stain cells with a constricted range of colors, often utilizing bi- and tri-color transgenic mice to unveil limited information in regards to neuronal structures, Brainbow is much more flexible in that it has the capacity to fluorescently label individual neurons with up to approximately 100 different hues so that scientists can identify and even differentiate between dendritic and axonal processes.

Source: https://en.wikipedia.org/wiki/Brainbow

File:Brainbow (Lichtman 2008).jpg

a) A motor nerve innervating ear muscle. b) An axon tract in the brainstem. c) The hippocampal dentate gyrus. In the Brainbow mice from which these images were taken, up to ~160 colours were observed as a result of the co-integration of several tandem copies of the transgene into the mouse genome and the independent recombination of each by Cre recombinase. The images were obtained by the superposition of separate red, green and blue channels. From Lichtman and Sanes, 2008.  Jeff W. Lichtman and Joshua R. Sanes.



My story begins in 1945, the year the city of Nagasaki was destroyed by an atomic bomb and the World War II ended. At that time I was a 16 year old high school student, and I was working at a factory about 15 Km north-east of Nagasaki. I watched the B-29 that carried the atomic bomb heading towards Nagasaki, then soon I was exposed to a blinding bright flash and a strong pressure wave that were caused by gigantic explosion. I was lucky to survive the war. In the mess after the war, however, I could not find any school to attend. I idled for 2 years, and then I learned that the pharmacy school of Nagasaki Medical College, which had been completely destroyed by the atomic bomb, was going to open a temporary campus near my home. I applied to the pharmacy school and was accepted. Although I didn’t have any interest in pharmacy, it was the only way that I could have some education.

After graduating from the primary school, I worked as a teaching assistant at the same school, which was re-organised as a part of Nagasaki University. My boss Professor Shungo Yasunga was a gentle and very kind person. In 1955, when I had worked for four years on the job, he arranged for me a paid leave of absence for one year, and he sent me to Nagoya University, to study at the laboratory of Professor Yoshimasa Hirata.

Cypridina Luciferin

The research subject that Professor Hirata gave me was the bioluminescence of the crustacean ostracod Cypridina hilgendorfii. Cypridina emits blue light when it’s luciferin is oxidised in the presence of an enzyme luciferase and molecular oxygenFigure imgb0001Mg2+acts as an inhibitor.

The ostracod Cypridina hilgendorfi freshly caught, and placed on a dark surface.

Source: http://www.nobelprize.org

Source: https://www.tumblr.com/search/sea%20firefly


Vargula hilgendorfii, sometimes called the sea-firefly, one of three bioluminescent species known in Japan as umi-hotaru, is a species of ostracod crustacean. In 1962, the name of the species was changed from Cypridina hilgendorfii to Vargula hilgendorfii. The species was first described by Gustav Wilhelm Müller in 1890. He named the species after the zoologist Franz Martin Hilgendorf (1839–1904). Dried sea-firefly were sometimes used as a light source by the Japanese army during World War II to read maps in the dim lightV. hilgendorfii is a small animal, only 3 millimetres long. It is nocturnal and lives in the sand at the bottom of shallow water. At night, it feeds actively.

Source: https://en.wikipedia.org/wiki/Vargula_hilgendorfii


How Bioluminescence Works

Source: http://animals.howstuffworks.com/animal-facts/bioluminescence3.htm


The luciferin had been studied for many years at Newton Harvey’s laboratory at Princeton University [Harvey, E. N., (1952). Bioluminescene, Academic Press, New York.], but it had never been completely purified, due to it’s extreme instability. Prof. Hirata wanted to determine the structure of the luciferin of Cypridina, and he asked me to purify the luciferin and to crystallize it, because crystallisation was the only way to confirm the purity  of substances at the time.

Using 500 gm of dried Cypridina (about 2.5 kg before drying), I began the extraction and purification of luciferin in an atmosphere of purified hydrogen using a large specially made Soxhlet apparatus.


A Soxhlet extractor is lab equipment designed for processing certain kinds of solids. These devices allow for continuous treatment of a sample with a solvent over a period of hours or days to extract compounds of interest. Typically, a Soxhlet extraction is only required where the desired compound has a limited solubility in a solvent, and the  impurity is insoluble in that solvent.

   Crystals of Cypridina luciferin

Mg2+ acts as an inhibitor. Bioluminescence vs. Fluorescence. Bioluminescence (left) is emitted from the reaction of luciferase enzyme and its substrate, such as firefly luciferase and luciferin, respectively. Cofactor requirements (e.g., ATP, O2) vary depending on the luciferase used. Fluorescence (right) is the product of a fluorophore (e.g., FITC, DyLight dyes) absorbing the energy from a light source and emitting the light energy at a different wavelength.


After 5 days of day-and-night work, 500 gm of dried Cypridina yielded about 2 mg of luciferin after purification. I tried to crystallise the purified luciferin, but all my efforts ended up with amorphous precipitates, and any leftover luciferin became useless by oxidation by the next morning. So I had to repeat the extraction and purification, again and again. I worked very hard, and tried every method of crystallisation that I could think of, without success. Ten months later, however, I finally found that the luciferin could be crystallised in a highly unusual solvent. [Shimomura, O., Goto, T., and Hirata, Y. (1957), “Crystalline Cypridina luciferin,” Bull. Chem. Soc. Japan 30: 929–933.] The solvent I found was high concentration of hydrochloric acid. Using the crystallised luciferin, we were able to determine the chemical structures of the luciferin and it’s oxidation products [Kishi, Y., Goto, T., Hirata, Y., Shimomura, O., and Johnson, F. H. (1966), “Cypridina bioluminescence I: structure of Cypridina luciferin,” Tetrahedron Lett., 3427–3436.] Those data became essential later in the study of aequorin.

THE JELLYFISH AEQUOREA AND THE PHOTOPROTEIN AEQUORIN

Early in the summer of 1961, we travelled from Princeton, NJ, to Friday Harbour, WA, driving 5,000 km. Friday Harbour was a quiet, peaceful small village at the time. The jellyfish were abundant in water. At the University of Washington laboratory there, we carefully scooped up the jellyfish one by one using a shallow dip net. The light organs of Aequorea aequorea are located along the edge of the umbrella, which we called a ring. The ring could be cut off with a pair of scissors, eliminating most of the unnecessary body part.

The jellyfish Aequorea aequorea in nature.

At the time, it was a common belief that the light of all bioluminescent organisms was produced by the reaction of luciferin and luciferase. Therefore, we tried to extract luciferin and luciferase from the rings of the jellyfish. We tried every method we could think of, but all our efforts failed. After only a few days  of work, we ran out of ideas.

I was convinced that the cause of our failure was the luciferin-luciferase hypothesis that dominated our mind. I suggested to Dr. Johnson that we forget the idea of extracting luciferin and luciferase and, instead, try to extract a luminescent substance whatever it might be. However, I was unable to convince him. Because of the disagreement  on experimental method, I started to work alone at one side of a table, while, on the other side, Dr. Johnson and his assistant continued their efforts to extract a luciferin. It was an awkward, uncomfortable situation.

Since the emission of light means the consumption (loss) of active bioluminescent substance, the extraction of bioluminescent substances from light organs must be performed under a condition that reversibly inhibits the luminescence reaction. Therefore, I tried to reversibly inhibit luminescence with various kinds of inhibitors of enzymes and proteins. I tried very hard, but nothing worked. I spent the next several days soul searching, trying to find out something missing in my experiments and in my thought. I thought day and night. I often took a rowboat out to the middle of the bay to avoid interference by people. One afternoon, an idea suddenly struck me on the boat. It was a very simple idea: “Luminescence reaction probably involves a protein. If so, luminescence might be reversibly inhibited at a certain pH.”

I immediately went back to the lab and tested the luminescence of light organs at various pHs. I clearly saw luminescence at pH 7, 6 and 5, but not at pH 4. I ground the light organs in a pH 4 buffer, and then filtered the mixture. The cell free filtrate was nearly dark. But it regained luminescence when it was neutralised by sodium bicarbonate. The experiment showed that I could extract the luminescence substance, at least in principle.

But a big surprise came the next moment. When I threw the extract into a sink, the inside of the sink lit up with a bright blue flash. The overflow of an aquarium was flowing into the sink, so I figured out that sea water had caused the luminescence. Because the composition of sea water is known, I easily found out that calcium ions (Ca2+activated the luminescence. The discovery of Calcium ions (Ca2+)  as the activator suggested that the luminescence material could be extracted utilizing the Ca-chelator EDTA, and we devised an extraction method of the luminescent substance.

The process was ⇒ Rings of jelly fish ( tissue of light organs) ⇒ shaken in saturated  (NH4)2SO4 ⇒ squeezed through gauze ⇒ filtered ⇒ Granular light organs observed ⇒ shaken in EDTA solution ⇒ Filtration ⇒ Crude aequorin solution ⇒             PURIFICATION ⇒ Aequorin and GFP.AequorinCa2+ sensitive luminescent protein – Aequorea aequorea- Inhibited by Mg2+ and also triggered by Eu2+, Sr2+ and Ba...Source

During the rest of the summer of 1961, we extracted the luminescent substance from about 10,000 jellyfish. After returning to Princeton, we purified the luminescent substance and obtained a few milligrams of purified protein. The protein emitted blue light in the presence of a trace of Calcium ions (Ca2+). We named the protein aequorin Shimomura, O., Johnson, F. H., and Saiga, Y. (1962), “Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea,” J. Cell. Comp. Physiol. 59: 223–239].

Aequorin was the first example of photoproteins discovered [Shimomura, O. (1985), “Bioluminescence in the sea: photoprotein systems,” Symp. Soc. Exp. Biol. 39: 351–372]. During the purification of aequorin, we found another protein that exhibited a bright green fluorescence. It was only in a trace amount, but we purified this protein too, and called it “green protein”. The protein was renamed “green fluorescent protein” by Morin and Hastings (1971).[Morin, J. G., and Hastings, J. W. (1971), “Energy transfer in a bioluminescent system,” J. Cell. Physiol., 77: 313–318.]

We wanted to understand the mechanism of the aequorin bioluminescence reaction; because it became clear in 1967 that aequorin was highly useful and important as a calcium probe in biological studies (Ridgeway and Ashley, 1967).[Ridgway, E. B., and Ashley, C. C. (1967), “Calcium transients in single muscle fibers,” Biochem. Biophys. Res. Commun. 29: 229–234.]

First, we tried to isolate the light-emitting chromophore of aequorin. However, there was no way to extract the native chromophore [Shimomura, O., and Johnson, F. H. (1969), “Properties of the bioluminescent protein aequorin,” Biochemistry 8: 3991–3997.][Shimomura, O., Johnson, F. H., and Morise, H. (1974), “Mechanism of the luminescent intramolecular reaction of aequorin,” Biochemistry 13: 3278–3286.], because any attempt to extract the chromophore always resulted in an intramolecular reaction of aequorin that triggered the emission of light, destroying the original chromophore. Indeed, the secret of light emission of Aequora was well protected.

We nevertheless found that a fluorescent compound was formed when aequorin was denatured with urea in the presence of 2-mercaptoethanol [Shimomura, O., and Johnson, F. H. (1969), “Properties of the bioluminescent protein aequorin,” Biochemistry 8: 3991–3997]. We named this fluorescent compound AF-350, based on it’s absorption maximum at 350nm. We decided to determine the structure of AF-350. However, to obtain the 1 mg of AF-350 needed for a single experiment toward the structural study of this compound, about 150 mg of purified aequorin was needed, and that meant we had to collect and extract atleast 50,000 jellyfish. Considering that we probably would need several milligrams of AF-350, the structure determination was a huge undertaking for us. 

The jellyfish ring cutting machine constructed by Frank H. Johnson in 1969. A specimen is placed on the black Plexiglas platform and rotated to spread the edge of the umbrella. While rotating, the specimen is pushed toward the rotating blade (10-inch meat cutting blade) to cut off a 2–3 mm wide strip containing the light organs. The strip drops into a container below.

In processing a large number of jellyfish to obtain a sufficient amount of AF-350, we found that cutting rings with a pair of scissors was too slow. To speed up the process, Dr. Johnson constructed a jellyfish cutting machine, which enabled one person to cut more than 600 rings per hour, or 10 times more than by hand. We started to collect jellyfish at 6 a.m, and a part of our group began to cut off rings at 8 a.m. We spent all afternoon extracting aequorin from the rings. Then, we collected more jellyfish in the evening, 7 p.m to 9 p.m, for the next day. Our laboratory looked like a jellyfish factory and was filled with the jellyfish smell.

After five years of hard work, we determined the chemical structure of AF-350 in 1972 [Shimomura, O., and Johnson, F. H. (1972), “Structure of the light-emitting moiety of aequorin,” Biochemistry 11: 1602–1608]

The result was surprising. The structure of AF-350 contained the skeleton of 2-aminopyrazine that was previously found in the oxidation products of Cypridina luciferin, although the side chains are different. This finding suggested a close relationship between the luminescence systems of Aequorea and Cypridina. Based on that information, we were able to determine the structure of the chromophore of aequorin to be coelenterazine.

Luminescence

The luminescence and regeneration of aequorin.

The photoprotein aequorin binds with two calcium ions [Shimomura, O. (1995), “Luminescence of aequorin is triggered by the binding of two calcium ions,” Biochem. Biophys. Res. Commun. 211: 359–363.][Shimomura, O., and Inouye, S. (1996), “Titration of recombinant aequorin with calcium chloride,” Biochem. Biophys. Res. Commun. 221: 77–81.], and decomposes into coelenteramide, CO2 and apoaequorin accompanied by the emission of light (emission maximum at 465 nm). Apoaequorin accompanied by the emission of light (emission maximum at 465 nm). Apoequorin can be regenerated into the original aequorin by incubation with coelenterazine in the presence of oxygen.

The chemical structures of AF-350 (coelenteramine), coelenteramide (o product of luminescence reaction of aequorin) and coelenterazine, compared with those of Cypridina oxyluciferin and luciferin.
b713374j-s1gfp

Lighting mechanisms of marine luciferases and fluorescent proteins. (A) Marine luciferases oxidize coelenterazine (CTZ) to emit bioluminescence; (B) Chromophore of GFP, 65SYG67, is matured by oxidation. The chemical structural backbone is similar to that of CTZ. Abbreviations: CTZ, coelenterazine; GFP, green fluorescent protein; RLuc, Renilla luciferase. This figure was obtained from a reference by Dr. Kim [Kim, S.B. Labor-effective manipulation of marine and beetle luciferases for bioassays. Protein Eng. Des. Sel 2012, 25, 261–269.]

Source: http://www.mdpi.com/1422-0067/13/12/16986/htm

GREEN FLUORESCENT PROTEIN

In a live specimen of Aequora, the light organs contain GFP in addition to aequorin, and the energy of the blue light produced by the aequorin molecule is transferred to the GFP molecule, and GFP emits green light [Morise, H., Shimomura, O., Johnson, F. H., and Winant, J. (1974), “Intermolecular energy transfer in the bioluminescent system of Aequorea,” Biochemistry 13: 2656–2662.]

GFP (100mg) ⇒ Denature at 90°C ⇒ Digest with papain ⇒ Extraction with butanol at pH 1 ⇒ TLC purification ⇒ Isolated chromophore (0.1 mg)

Although GFP is highly visible and easily crystallisable, the yield of GFP from the jellyfish is extremely low, much lower than that of aequorin. Therefore, to study GFP, we had to accumulate GFP little by little for many years while we studied the chemistry of aequorin luminescence. The amount of GFP we accumulated reached a sufficient amount to study this protein in 1979. Thus, we tried find out the nature of the GFP chromophore by a series of experiments, using 100 mg of the protein in one experiment.

We first cut the molecule of GFP into small pieces of peptide by enzymic digestion. We isolated and purified the peptide that contained the chromophore, and then analysed the structure of the chromophore. I was surprised when I measured the absorption spectrum of the peptide. The spectrum was nearly identical to that of a compound that I had synthesized in my study of Cypridia luciferin 20 years earlier. Based on the spectral resemblance and some other properties, I could quickly identify the chromophore structure of GFP [Shimomura, O. (1979), “Structure of the chromophore of Aequorea green fluorescent protein,” FEBS Lett. 104, 220–222].

Fluorescent proteins are usually a complex of a protein and a fluorescent compound. However, GFP was a very special fluorescent protein that contained a fluorescent chromophore within the protein molecule.

GFP picture

[Cody, C. W., Prasher, D. C., Westler, W. M., Prendergast, F. G., and Ward, W. W. (1993), “Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein,” Biochemistry 32: 1212–1218.]

The finding was extremely important because it showed that the chromophore is a part of the peptide chain, and thus it opened the possibility of cloning GFP. The chromophore structure was later confirmed by Cody et al. (1993)

When I found the chromophore of GFP in 1979, I  thought I had done all I could do with GFP, and decided to terminate my work on GFP in order to concentrate my efforts in the study of bioluminescence, my  lifework. Then a mysterious thing happened. The population of Aequora in the Friday Harbor area drastically decreased after 1990, thus making it practically impossible to prepare any new samples of natural aequorin or GFP. Fortunately, however, aequorin had been cloned by Inouye et al. [Inouye, S., Noguchi, M., Sakaki, Y., Takagi, Y., Miyata, T., Iwanaga, S., Miyata, T., and Tsuji, F. I. (1985), “Cloning and sequence analysis of cDNA for the luminescent protein aequorin,” Proc. Natl. Acad. Sci. USA 82: 3154–3158.][Inouye, S., Sakaki, Y., Goto, T., and Tsuji, F. I. (1986), “Expression of apoaequorin complementary DNA in Escherichia coli,” Biochemistry 25: 8425–8429] and Prasher et al.] [Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., and Cormier, M. J. (1992), “Primary structure of the Aequorea victoria green fluorescent protein,” Gene 111: 229– 233], thus making  the natural proteins unessential. In 1994, GFP was successfully expressed in living organisms by Chalfie et al. and it was further developed into it’s present prosperous state by Roger Tsien.