- Email: [email protected]
Rational investigations in a suspected case of dengue
p e d i a t r i c i n f e c t i o u s d i s e a s e 5 ( 2 0 1 3 ) 9 2 e9 7
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/pid
Notes from the Lab
Rational investigations in a suspected case of dengue Nupur Ganguly* Associate Professor, Department of Pediatric Medicine, Institute of Child Health, 11, Dr Biresh Guha Street, Kolkata 700017, India
article info Article history: Received 5 June 2013 Accepted 3 July 2013 Available online 19 July 2013
1.
Introduction
Dengue virus infection produces a broad spectrum of symptoms, many of which are non-specific and can be easily confused with other non-dengue illnesses such as malaria, typhoid, rickettsial diseases, Japanese encephalitis, chikungunya, leptospirosis, measles, influenza and influenzalike illnesses. Thus, a diagnosis based only on clinical symptoms is unreliable, and laboratory diagnosis is important early in illness to prevent complications as some patients progress from Dengue Fever (DF) to Severe Dengue (severe plasma leakage/severe hemorrhage/severe organ impairment) over a short period of time. Dengue fever should be considered in any patient presenting with fever, generalised skin flushing, leukopenia and thrombocytopaenia. Early diagnosis and intervention may be lifesaving and will help to differentiate from other infectious disease. Unlike other diseases, lab investigations are different in different phases of illness in dengue fever and the investigations are to be done in accordance with the timeline of the disease. Hence understanding of the timeline of the disease (Fig. 1) is important for the appropriate investigation and optimum result. However requisition for laboratory
investigation should further depend upon the epidemiology, the type of laboratory facilities available, technical expertise, costs, and the time of sample collection.
2. Laboratory investigations are divided in two groups 2.1. Laboratory investigations for assessment and management of dengue viral infection 2.1.1.
Haematology, complete blood count (CBC)
The most important laboratory investigation is a full blood count at the first visit in case of suspected dengue fever which will detect the patient’s own baseline value. A haematocrit test in the early febrile phase establishes the patient’s own baseline haematocrit. A decreasing white blood cell count is suggestive of dengue. 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.1 In the absence of the patient’s baseline, agespecific population haematocrit levels could be used as a surrogate during the critical phase. A haematocrit level increase greater than 20% is a sign of haemoconcentration and
* Tel.: þ91 3322893242. E-mail address: [email protected]. 2212-8328/$ e see front matter Copyright ª 2013, Indian Academy of Pediatrics, Infectious Disease Chapter. All rights reserved. http://dx.doi.org/10.1016/j.pid.2013.07.002
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thousands in patients with severe A ratio of AST: ALT >2 supports diagnosis of severe dengue. Hypoproteinemia, Hypoalbuminaemia (i.e., serum albumin <35 g/L [3.5 g/dL] suggests plasma leakage) and haemoconcentration.
2.1.4.
In the critical phase DIC panel is recommended
Coagulation studies may help to guide therapy in patients with severe hemorrhagic manifestations. Prolonged prothrombin time, activated partial thromboplastin time, low fibrinogen and elevated fibrin degradation product levels are signs of disseminated intravascular coagulation. Signs of early coagulopathy may be subtle. Positive guaiac test for occult blood in the stool is a sign of early coagulopathy Guaiac testing should be performed on all patients in whom dengue virus infection is suspected.1
Fig. 1 e Time line of dengue viral infection.
precedes shock. The haematocrit level should be monitored at least every 24 h to facilitate early recognition of dengue hemorrhagic fever and every 3e4 h in severe cases of dengue.
2.1.1.1. Thrombocytopaenia. The characteristic findings in dengue fever are thrombocytopaenia and with the symptoms and epidemiological clue the decreasing platelet count makes dengue very likely. It has been demonstrated in up to 50% of patients. Falling platelet counts less than 100,000 cells/mL are seen in severe dengue and occur before defervescence and the onset of shock. The platelet count should be monitored at least every 24 h to facilitate early recognition of plasma leakage. It is important to correlate and compare the low platelet count done on cell counter with the manual count on the peripheral smear. This is due to the fact that platelets might clump in EDTA anticoagulated blood, and the automatic analyser may give a falsely low platelet count. During manual count the person viewing the slide will see clumps of platelets and can estimate if there are low, normal, or high numbers of platelet. 2.1.1.2. Leukopenia. Leukopenia (WBC 5000 cell/cmm) and thrombocytopaenia may occur as early as second day of fever2 leukopenia with positive tourniquet test in dengue-endemic area has a positive predictive value of 70e80%.3 Lymphocytosis, with atypical lymphocytes, commonly develops before defervescence or shock A systematic review found that patients with dengue had significantly lower total WBC, neutrophil, and platelet counts than patients with other febrile illnesses in dengue-endemic populations.
2.1.2. Biochemistry 2.1.2.1. Serum electrolytes. Hyponatremia is the most common electrolyte abnormality in patients with dengue hemorrhagic fever or dengue shock syndrome. Metabolic acidosis and elevated blood urea nitrogen (BUN) is observed in those with shock and must be corrected rapidly.
2.1.3.
Liver function test and kidney function test if required
Liver function test should be done in all symptomatic cases Transaminase levels may be elevated into the several
2.1.5.
Imaging
Imaging studies are only required if severe dengue is suspected Chest X-ray may be done to look for pleural effusions and bronchopneumonia. Right-sided pleural effusion is typical during plasma leakage. Bilateral pleural effusions are common in patients with dengue shock syndrome. Ultrasonography is a cost-effective, non-invasive procedure in the evaluation of potential dengue fever with capillary leak or gallbladder wall oedema. It will detect pleural effusion, pericardial effusion, presence of ascites and other pathological changes in abdominal organs including the liver, gallbladder (i.e., oedema may precede plasma leakage), and kidneys.1,2
3. Lab investigations for confirmation of diagnosis There are 3 types of diagnostic test available for confirmation of dengue virus infection: Virus isolation Viral nucleic acid detection (molecular diagnosis) Serology Viral antigen detection Detection of antibody response
3.1.
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). However, this is only available in some locations and results are usually not available in a clinically required time frame, hence it is generally not recommended.
3.2.
Viral nucleic acid detection (molecular diagnosis)
For molecular diagnosis Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) is the method of choice and can be ordered in the first 5 days of onset of fever. It detects DENV serotypes 1, 2, 3 or 4 from serum or plasma in patients with signs and symptoms of dengue infection. Tissue, whole blood, serum, or plasma specimen can be used. The CDC DENV-1-4
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Real-Time RT-PCR assay is the first nucleic acid diagnostic device for detection and typing of DENV nucleic acid in suspected, and symptomatic patients which is approved by the FDA for use in the United States A positive result confirms diagnosis. It is the most sensitive and specific test in early infection, which will help in early diagnosis, detect serotype and facilitate early management. However it is expensive, requires laboratory facilities and expertise, not rapid (takes 24e48 h), cannot differentiate between primary and secondary infection, and there is potential for false-positive result due to contamination.2 Molecular diagnosis based on reverse transcription (RT)-PCR, has gradually replaced the virus isolation method as the new standard for the detection of dengue virus in acute-phase serum samples.
3.3.
Serology: serological diagnosis
3.3.1.
Detection of viral antigen
NS1 is 55 kD a structurally conserved glycoprotein secreted by DENV infected cells in vitro and in vivo. And detection of this viral protein, NS1, represents a new approach to dengue diagnosis. Assay of this non-structural protein (NS1) using ELISA or rapid kits in serum sample is useful in early diagnosis.1 High concentration of this NS1 antigens is detected in the acute-phase sera of both patients with primary and secondary dengue virus infections up to 9 days after the onset of illness (Fig. 2).4,5 This could be a potential means for the early diagnosis of dengue virus infection. However as it does not provide a differentiation between primary and secondary dengue infection. Differentiation can only be achieved with the use of (IgM Antibody Captured ELISA) IgM MAC ELISA and IgG antibody detection tools. Two types of tests are commercially available for the detection of dengue NS1 antigen in human serum and plasma. 1) Dengue NS1 Ag micro-ELISA (Microwell ELISA TEST for detection of dengue antigen)/(Platelia) assay 2) Rapid visual test for the detection of dengue NS1 Ag & differential detection of IgM & IgG antibodies in human serum and plasma (rapid chromatographic test)/Lateral Flow Rapid Test (LFRT)
3.4.
Sensitivity and specificity of NSI antigen
The sensitivity and specificity of the NS1 micro-ELISA or Platelia assay and an NS1 lateral Flow Rapid Test (LFRT) or the
Fig. 2 e Positivity of NS1 antigen in dengue fever.
rapid chromatographic test were compared against a gold standard reference RT-PCR diagnostic algorithm in various studies in children and adults. NS1 positivity was associated with the underlying viraemia, as NS1-positive samples were found to be having significantly higher viraemia than NS1negative samples matched for duration of illness. It has been found that sensitivity declines with increasing time since the onset of symptoms and this may be due to the decreasing viral load. The increased amount of DENV-reactive IgG and to a lesser extent IgM in the test samples were also found to be associated with a significantly lower rate of NS1 positivity in both the assays. This may be a limiting factor for its use in case of secondary dengue viral infection where NS1 antigen may be negative due to increased IgG levels. Hence both ELISA and chromatographic assays were more sensitive for primary than secondary dengue, and for specimens collected within 3 days of onset of illness than later presentation. Overall, the Platelia or the NS1 ELISA was modestly more sensitive (82%) than the NS1-chromatographic test (72%) in confirmed dengue cases against the gold standard reference RT-PCR. Both the Elisa and NS1 chromatographic test were 100% specific, being negative in all febrile patients without evidence of recent dengue, as well as in patients with enteric fever, malaria, Japanese encephalitis and leptospirosis.6e8 The advantage of the NS1-chomatographic test is, it is less time consuming (15e30 min) and can diagnose quickly as compared to NS1 ELISA where the time required is 2 h.6 Thus NS1 assays deserve inclusion in the diagnostic evaluation of dengue patients, but with due consideration for the limitations in patients who present late in their illness or have a secondary immune response in presence of increased level of IgG antibody, where NS1 antigen may be negative and one should not exclude dengue as a diagnosis and interpret the result with caution.
3.5.
Viral antibody detection
Two patterns of serological response are observed in patients with dengue virus infection, primary and secondary antibody responses, depending on the immunological status of the infected individuals antibody response to infection differs according to the immune status of the host.1 In primary infection when dengue infection occurs in persons who have not previously been infected with a dengue or flavivirus or immunized with a flavivirus vaccine (e.g. for yellow fever, Japanese encephalitis, tick-borne encephalitis), the patients develop a primary antibody response. Anti-dengue virus IgM antibody is produced transiently during primary and secondary infections. In patients with primary dengue virus infections, IgM antibodies develop rapidly and are detectable on days 3e5 of illness in half of the patients, peaks at about 2 weeks and then decline to undetectable level by 2e3 months9,10 anti-dengue virus IgG appears thereafter. In patients with secondary dengue virus infections, i.e. a dengue infection in a host who 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
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Fig. 3 e Various antigen and antibody response in primary and secondary dengue infection.
in the acute-phase, before or simultaneously with the IgM responses and persists for periods lasting from 10 months to life. Though the IgM production are similar to those observed in patients with primary infections, IgM levels are significantly lower in secondary infection and may be undetectable in some cases (Fig. 3).9,10 To distinguish primary and secondary dengue infections, IgM/IgG antibody ratios are now more commonly used than the haemagglutination-inhibition test (HI).
3.6.
Dengue IgM/IgG ratio
This method is very useful in the detection of immune status of patients with dengue and can be used to distinguish primary from secondary 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 optical density 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 1.4.11 However, ratios may vary between laboratories, thus indicating the need for better standardization of test performance.
3.7.
Methods for detection of antibody
Dengue virus is a positive-stranded encapsulated RNA virus. The genomic RNA is approximately 11 kb in length and is composed of three structural protein genes that encode the nucleocapsid or core protein (C), a membrane-associated protein (M), an envelope protein (E), and seven non-structural (NS) protein genes. Several methods have been described for the serological detection of dengue virus-specific antibodies against these antigen proteins.
Many commercial kits with good sensitivities and specificities are now available.
3.9. ELISA
Many rapid test kits that use the principle of immunochromatography are commercially available. Most of these kits can simultaneously detect IgM and IgG antibodies to dengue virus in human whole blood, serum, or plasma within 5e30 min. Some of these kits claim that it is possible to differentiate primary and secondary dengue virus infections, but they are not always reliable. Several evaluations that offer conclusions in favour or against these commercial kits are available.13e18 Preliminary evaluations of five rapid test kits (unpublished data) showed that these kits generally have higher sensitivities for IgG detection but lower sensitivities for IgM detection and varied specificities as compared to the E/M-specific capture IgM and IgG ELISA. Although the rapid test has the advantages of easy performance and the rapid provision of results, it should be used as a screening test. According to WHO guidelines, these kits should not be used in the clinical settings to guide management of severe dengue cases because many serum samples taken in the first five days after the onset of illness will not have detectable IgM antibodies. The tests would thus give a false negative result. Reliance on such tests in clinical management might lead to an increase in case fatality rate. Furthermore, these kits should not be used for surveillance for dengue disease in public health settings or in seroepidemiological studies due to the high sensitivity of this assay for the detection of IgG and the long persistence of cross-reactive flavivirus IgG antibodies in the general population in many areas where dengue is endemic.
3.10. 3.8.
Immunochromatographic assay for IGM and IgG
Anti-dengue IgA and IgE
The E/M-specific capture IgM and IgG ELISA
It has become the most robust and powerful assay for the serodiagnosis of dengue virus infection due to its high sensitivity, specificity, simplicity, and feasibility for automation.12
Measurement of dengue IgA by anti-dengue virus capture ELISA (AAC-ELISA) becomes positive 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. However
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no differences in IgA titres are found 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. This approach is not used very often and requires additional evaluation. Some investigators have studied dengue virus-specific IgA and IgE antibody responses. Talarmin et al reported on the use of an IgA- and IgM-specific capture ELISA for the diagnosis of dengue virus infection. The results showed that IgM appears more rapidly and lasts longer (over 2e3 months) than IgA (about 40 days). They concluded that the capture IgA ELISA is a simple method that can be performed together with the capture IgM ELISA and that can help in interpreting the serology of DF.19 More recently, Balmaseda et al20 reported on the detection of specific IgM and IgA antibodies in serum and saliva. They concluded that dengue virus-specific IgA in serum is a potential diagnostic target. According to Koraka et al21 capture IgE ELISA for the detection of total and dengue virus-specific IgE antibody responses might be helpful for early diagnosis of DHF/DSS It has been seen that dengue virus-specific IgE titres are significantly higher in patients with DHF and/or DSS than in patients with DF and non-dengue virus infections. They concluded that analysis of dengue virus-specific IgE ELISA might contribute to the understanding of the pathogenesis of dengue virus.
3.11.
Haemagglutination-inhibition test
Optimally the HI test requires paired sera obtained during 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 flaviviruses (e.g. between dengue virus and Japanese encephalitis virus or West Nile virus) nor between immunoglobulin isotypes. The response in 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 response Haemagglutination-inhibition (HI) test is useful for diagnosing secondary dengue infection (i.e., titre 1:1280). Disadvantages: lower specificity compared to other tests, requires 2 serum samples, delays confirmation of diagnosis.2
4.
Conclusion
Laboratory diagnosis of dengue virus infection is very important for early diagnosis and management and differentiate it from various other infections. The haematological investigation will help in the early detection of progression to severe dengue. The advances in molecular and serological diagnostic methods have greatly improved the sensitivity and specificity of the diagnosis However no single diagnostic assay in isolation is adequately sensitive and specific enough to diagnose all acute cases of dengue While direct methods such as, viral isolation, viral nucleic acid or viral antigen detection are more
specific, they are more costly and labour-intensive. Indirect methods (i.e., serology) are less specific, but are more accessible, faster, and less expensive.2,22 Viral antigen detection is positive in the first 5 days of illness, and serological tests becomes positive after the fifth day. Thus, appropriate application of these tests when combined together will be of significant help for the clinical management, etiologic investigation, and control of dengue virus infections.
Conflicts of interest The author has none to declare.
references
1. World Health Organization. Comprehensive Guidelines for Prevention and Control of Dengue and Dengue Haemorrhagic Fever e Revised and Expanded Edition. Regional Office for South-East Asia. http://www.searo.who.int/. Accessed 19.04.13. 2. World Health Organization. Special Programme for Research and Training in Tropical Diseases (TDR). Dengue: Guidelines for Diagnosis, Treatment, Prevention and Control; 2009. http://www. cdc.gov/Dengue/clinicalLab/index.html. Accessed 19.04.13. 3. Teixeira MG, Barreto M. Diagnosis and management of dengue: clinical review. BMJ. 2009;339:1189e1193. 4. Kalayanarooj S, Nimmanitya S, Suntayakorn S, et al. Can doctors make an accurate diagnosis of dengue infection at an early stage? Dengue Bull. 1999;23:1e9. 5. Lima Mda R, Nogueira RM, Schatzmayr HG, et al. Comparison of three commercially available dengue NS1 antigen capture assays for acute diagnosis of dengue in Brazil. PLoS Negl Trop Dis. 2010;4:e738. 6. Hang Vu Ty, Nguyet Nguyen Minh, Trung Dinh The, Tricou Vianney, Yoksan Sutee, Nguyen AL. Diagnostic accuracy of NS1 ELISA and lateral Flow rapid tests for dengue sensitivity, specificity and relationship to viraemia and antibody responses. PLoS Negl Trop Dis. 2009 January;3(1):e360. 7. Dussart P, Petit L, Labeau B, et al. Evaluation of two new commercial tests for the Diagnosis of acute dengue virus infection using NS1 antigen detection in human serum. PLoS Negl Trop Dis. 2008;2:e280. 8. Bessoff K, Delorey M, Sun W, et al. Comparison of two commercially available dengue virus (DENV) NS1 capture enzyme-linked immunosorbent assays using a single clinical sample for diagnosis of acute DENV infection. Clin Vaccine Immunol. 2008 Oct;15(10):1513e1518. http://dx.doi.org/10.1128/ CVI.00140-08. Epub 2008 Aug 6. 9. Innis BL, Nisalak A, Nimmannitya S, et al. An enzyme-linked immunosorbent assay to characterize dengue infection where dengue and Japanese encephalitis co circulate. Am J Trop Med Hyg. 1989 Apr;40(4):418e427. 10. World Health Organization. Dengue Haemorrhagic Fever: Diagnosis, Treatment, Prevention and Control. 2nd ed. Geneva, Switzerland: World Health Organization; 1997. 11. Shu PY, K Chen L, Chang SF, et al. Comparison of capture immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assay (ELISA) and nonstructural protein NS1 serotype-specific IgG ELISA for differentiation of primary and secondary dengue virus infections. Clin Diagn Lab Immunol. 2003;10:622e630. [PubMed]. 12. Kuno G, Gomez I, Gubler DJ. An ELISA procedure for the diagnosis of dengue infections. J Virol Methods. 1991;33:101e113. [PubMed].
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13. Groen J, Koraka P, Velzing J, Copra C, Osterhaus AD. Evaluation of six immunoassays for detection of dengue virus-specific immunoglobulin M and G antibodies. Clin Diagn Lab Immunol. 2000 Nov;7(6):867e871. 14. Branch SL, Levett PN. Evaluation of four methods for detection of immunoglobulin M antibodies to dengue virus. Clin Diagn Lab Immunol. 1999;6:555e557. [PubMed]. 15. Charrel RN, de Lamballerie X. Low specificity of an immunochromatographic serological assay for diagnosis of dengue fever in travelers returning with malaria. Clin Diagn Lab Immunol. 2002;9:1400. [PubMed]. 16. Lam SK, Devine PL. Evaluation of capture ELISA and rapid immunochromatographic test for the determination of IgM and IgG antibodies produced during dengue infection. Clin Diagn Virol. 1998;10:75e81. 17. Vaughn DW. Serological investigation of a febrile outbreak in Delhi, India, using a rapid immunochromatographic test. J Clin Microbiol. 1998;36:2795e2796.
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18. Vaughn DW, Nisalak A, Kalayanarooj S, et al. Evaluation of a rapid immunochromatographic test for diagnosis of dengue virus infection. J Clin Microbiol. 1998;36:234e238. 19. Talarmin A, Labeau B, Lelarge J, Sarthou JL. Immunoglobulin A-specific capture enzyme-linked immunosorbent assay for diagnosis of dengue fever. J Clin Microbiol. 1998;36:1189e1192. 20. Balmaseda AMG, Guzman S, Hammond G, Carolina, Flores Y. Diagnosis of dengue virus infection by detection of specific immunoglobulin M (IgM) and IgA antibodies in serum and saliva. Clin Diagn Lb Immunol. 2003;10:317e322. 21. Koraka P, Murgue B, Deparis X, et al. Elevated levels of total and dengue virus-specific immunoglobulin E in patients with varying disease severity. J Med Virol. 2003;70:91e98. 22. Nimmannitya Suchitra. Management of DF/DHF-WHO Regional Publication No. 22 e Monograph on Dengue/DHF. New Delhi: WHO/SEARO;55e61.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/pid
Notes from the Lab
Rational investigations in a suspected case of dengue Nupur Ganguly* Associate Professor, Department of Pediatric Medicine, Institute of Child Health, 11, Dr Biresh Guha Street, Kolkata 700017, India
article info Article history: Received 5 June 2013 Accepted 3 July 2013 Available online 19 July 2013
1.
Introduction
Dengue virus infection produces a broad spectrum of symptoms, many of which are non-specific and can be easily confused with other non-dengue illnesses such as malaria, typhoid, rickettsial diseases, Japanese encephalitis, chikungunya, leptospirosis, measles, influenza and influenzalike illnesses. Thus, a diagnosis based only on clinical symptoms is unreliable, and laboratory diagnosis is important early in illness to prevent complications as some patients progress from Dengue Fever (DF) to Severe Dengue (severe plasma leakage/severe hemorrhage/severe organ impairment) over a short period of time. Dengue fever should be considered in any patient presenting with fever, generalised skin flushing, leukopenia and thrombocytopaenia. Early diagnosis and intervention may be lifesaving and will help to differentiate from other infectious disease. Unlike other diseases, lab investigations are different in different phases of illness in dengue fever and the investigations are to be done in accordance with the timeline of the disease. Hence understanding of the timeline of the disease (Fig. 1) is important for the appropriate investigation and optimum result. However requisition for laboratory
investigation should further depend upon the epidemiology, the type of laboratory facilities available, technical expertise, costs, and the time of sample collection.
2. Laboratory investigations are divided in two groups 2.1. Laboratory investigations for assessment and management of dengue viral infection 2.1.1.
Haematology, complete blood count (CBC)
The most important laboratory investigation is a full blood count at the first visit in case of suspected dengue fever which will detect the patient’s own baseline value. A haematocrit test in the early febrile phase establishes the patient’s own baseline haematocrit. A decreasing white blood cell count is suggestive of dengue. 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.1 In the absence of the patient’s baseline, agespecific population haematocrit levels could be used as a surrogate during the critical phase. A haematocrit level increase greater than 20% is a sign of haemoconcentration and
* Tel.: þ91 3322893242. E-mail address: [email protected]. 2212-8328/$ e see front matter Copyright ª 2013, Indian Academy of Pediatrics, Infectious Disease Chapter. All rights reserved. http://dx.doi.org/10.1016/j.pid.2013.07.002
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thousands in patients with severe A ratio of AST: ALT >2 supports diagnosis of severe dengue. Hypoproteinemia, Hypoalbuminaemia (i.e., serum albumin <35 g/L [3.5 g/dL] suggests plasma leakage) and haemoconcentration.
2.1.4.
In the critical phase DIC panel is recommended
Coagulation studies may help to guide therapy in patients with severe hemorrhagic manifestations. Prolonged prothrombin time, activated partial thromboplastin time, low fibrinogen and elevated fibrin degradation product levels are signs of disseminated intravascular coagulation. Signs of early coagulopathy may be subtle. Positive guaiac test for occult blood in the stool is a sign of early coagulopathy Guaiac testing should be performed on all patients in whom dengue virus infection is suspected.1
Fig. 1 e Time line of dengue viral infection.
precedes shock. The haematocrit level should be monitored at least every 24 h to facilitate early recognition of dengue hemorrhagic fever and every 3e4 h in severe cases of dengue.
2.1.1.1. Thrombocytopaenia. The characteristic findings in dengue fever are thrombocytopaenia and with the symptoms and epidemiological clue the decreasing platelet count makes dengue very likely. It has been demonstrated in up to 50% of patients. Falling platelet counts less than 100,000 cells/mL are seen in severe dengue and occur before defervescence and the onset of shock. The platelet count should be monitored at least every 24 h to facilitate early recognition of plasma leakage. It is important to correlate and compare the low platelet count done on cell counter with the manual count on the peripheral smear. This is due to the fact that platelets might clump in EDTA anticoagulated blood, and the automatic analyser may give a falsely low platelet count. During manual count the person viewing the slide will see clumps of platelets and can estimate if there are low, normal, or high numbers of platelet. 2.1.1.2. Leukopenia. Leukopenia (WBC 5000 cell/cmm) and thrombocytopaenia may occur as early as second day of fever2 leukopenia with positive tourniquet test in dengue-endemic area has a positive predictive value of 70e80%.3 Lymphocytosis, with atypical lymphocytes, commonly develops before defervescence or shock A systematic review found that patients with dengue had significantly lower total WBC, neutrophil, and platelet counts than patients with other febrile illnesses in dengue-endemic populations.
2.1.2. Biochemistry 2.1.2.1. Serum electrolytes. Hyponatremia is the most common electrolyte abnormality in patients with dengue hemorrhagic fever or dengue shock syndrome. Metabolic acidosis and elevated blood urea nitrogen (BUN) is observed in those with shock and must be corrected rapidly.
2.1.3.
Liver function test and kidney function test if required
Liver function test should be done in all symptomatic cases Transaminase levels may be elevated into the several
2.1.5.
Imaging
Imaging studies are only required if severe dengue is suspected Chest X-ray may be done to look for pleural effusions and bronchopneumonia. Right-sided pleural effusion is typical during plasma leakage. Bilateral pleural effusions are common in patients with dengue shock syndrome. Ultrasonography is a cost-effective, non-invasive procedure in the evaluation of potential dengue fever with capillary leak or gallbladder wall oedema. It will detect pleural effusion, pericardial effusion, presence of ascites and other pathological changes in abdominal organs including the liver, gallbladder (i.e., oedema may precede plasma leakage), and kidneys.1,2
3. Lab investigations for confirmation of diagnosis There are 3 types of diagnostic test available for confirmation of dengue virus infection: Virus isolation Viral nucleic acid detection (molecular diagnosis) Serology Viral antigen detection Detection of antibody response
3.1.
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). However, this is only available in some locations and results are usually not available in a clinically required time frame, hence it is generally not recommended.
3.2.
Viral nucleic acid detection (molecular diagnosis)
For molecular diagnosis Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) is the method of choice and can be ordered in the first 5 days of onset of fever. It detects DENV serotypes 1, 2, 3 or 4 from serum or plasma in patients with signs and symptoms of dengue infection. Tissue, whole blood, serum, or plasma specimen can be used. The CDC DENV-1-4
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Real-Time RT-PCR assay is the first nucleic acid diagnostic device for detection and typing of DENV nucleic acid in suspected, and symptomatic patients which is approved by the FDA for use in the United States A positive result confirms diagnosis. It is the most sensitive and specific test in early infection, which will help in early diagnosis, detect serotype and facilitate early management. However it is expensive, requires laboratory facilities and expertise, not rapid (takes 24e48 h), cannot differentiate between primary and secondary infection, and there is potential for false-positive result due to contamination.2 Molecular diagnosis based on reverse transcription (RT)-PCR, has gradually replaced the virus isolation method as the new standard for the detection of dengue virus in acute-phase serum samples.
3.3.
Serology: serological diagnosis
3.3.1.
Detection of viral antigen
NS1 is 55 kD a structurally conserved glycoprotein secreted by DENV infected cells in vitro and in vivo. And detection of this viral protein, NS1, represents a new approach to dengue diagnosis. Assay of this non-structural protein (NS1) using ELISA or rapid kits in serum sample is useful in early diagnosis.1 High concentration of this NS1 antigens is detected in the acute-phase sera of both patients with primary and secondary dengue virus infections up to 9 days after the onset of illness (Fig. 2).4,5 This could be a potential means for the early diagnosis of dengue virus infection. However as it does not provide a differentiation between primary and secondary dengue infection. Differentiation can only be achieved with the use of (IgM Antibody Captured ELISA) IgM MAC ELISA and IgG antibody detection tools. Two types of tests are commercially available for the detection of dengue NS1 antigen in human serum and plasma. 1) Dengue NS1 Ag micro-ELISA (Microwell ELISA TEST for detection of dengue antigen)/(Platelia) assay 2) Rapid visual test for the detection of dengue NS1 Ag & differential detection of IgM & IgG antibodies in human serum and plasma (rapid chromatographic test)/Lateral Flow Rapid Test (LFRT)
3.4.
Sensitivity and specificity of NSI antigen
The sensitivity and specificity of the NS1 micro-ELISA or Platelia assay and an NS1 lateral Flow Rapid Test (LFRT) or the
Fig. 2 e Positivity of NS1 antigen in dengue fever.
rapid chromatographic test were compared against a gold standard reference RT-PCR diagnostic algorithm in various studies in children and adults. NS1 positivity was associated with the underlying viraemia, as NS1-positive samples were found to be having significantly higher viraemia than NS1negative samples matched for duration of illness. It has been found that sensitivity declines with increasing time since the onset of symptoms and this may be due to the decreasing viral load. The increased amount of DENV-reactive IgG and to a lesser extent IgM in the test samples were also found to be associated with a significantly lower rate of NS1 positivity in both the assays. This may be a limiting factor for its use in case of secondary dengue viral infection where NS1 antigen may be negative due to increased IgG levels. Hence both ELISA and chromatographic assays were more sensitive for primary than secondary dengue, and for specimens collected within 3 days of onset of illness than later presentation. Overall, the Platelia or the NS1 ELISA was modestly more sensitive (82%) than the NS1-chromatographic test (72%) in confirmed dengue cases against the gold standard reference RT-PCR. Both the Elisa and NS1 chromatographic test were 100% specific, being negative in all febrile patients without evidence of recent dengue, as well as in patients with enteric fever, malaria, Japanese encephalitis and leptospirosis.6e8 The advantage of the NS1-chomatographic test is, it is less time consuming (15e30 min) and can diagnose quickly as compared to NS1 ELISA where the time required is 2 h.6 Thus NS1 assays deserve inclusion in the diagnostic evaluation of dengue patients, but with due consideration for the limitations in patients who present late in their illness or have a secondary immune response in presence of increased level of IgG antibody, where NS1 antigen may be negative and one should not exclude dengue as a diagnosis and interpret the result with caution.
3.5.
Viral antibody detection
Two patterns of serological response are observed in patients with dengue virus infection, primary and secondary antibody responses, depending on the immunological status of the infected individuals antibody response to infection differs according to the immune status of the host.1 In primary infection when dengue infection occurs in persons who have not previously been infected with a dengue or flavivirus or immunized with a flavivirus vaccine (e.g. for yellow fever, Japanese encephalitis, tick-borne encephalitis), the patients develop a primary antibody response. Anti-dengue virus IgM antibody is produced transiently during primary and secondary infections. In patients with primary dengue virus infections, IgM antibodies develop rapidly and are detectable on days 3e5 of illness in half of the patients, peaks at about 2 weeks and then decline to undetectable level by 2e3 months9,10 anti-dengue virus IgG appears thereafter. In patients with secondary dengue virus infections, i.e. a dengue infection in a host who 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
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Fig. 3 e Various antigen and antibody response in primary and secondary dengue infection.
in the acute-phase, before or simultaneously with the IgM responses and persists for periods lasting from 10 months to life. Though the IgM production are similar to those observed in patients with primary infections, IgM levels are significantly lower in secondary infection and may be undetectable in some cases (Fig. 3).9,10 To distinguish primary and secondary dengue infections, IgM/IgG antibody ratios are now more commonly used than the haemagglutination-inhibition test (HI).
3.6.
Dengue IgM/IgG ratio
This method is very useful in the detection of immune status of patients with dengue and can be used to distinguish primary from secondary 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 optical density 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 1.4.11 However, ratios may vary between laboratories, thus indicating the need for better standardization of test performance.
3.7.
Methods for detection of antibody
Dengue virus is a positive-stranded encapsulated RNA virus. The genomic RNA is approximately 11 kb in length and is composed of three structural protein genes that encode the nucleocapsid or core protein (C), a membrane-associated protein (M), an envelope protein (E), and seven non-structural (NS) protein genes. Several methods have been described for the serological detection of dengue virus-specific antibodies against these antigen proteins.
Many commercial kits with good sensitivities and specificities are now available.
3.9. ELISA
Many rapid test kits that use the principle of immunochromatography are commercially available. Most of these kits can simultaneously detect IgM and IgG antibodies to dengue virus in human whole blood, serum, or plasma within 5e30 min. Some of these kits claim that it is possible to differentiate primary and secondary dengue virus infections, but they are not always reliable. Several evaluations that offer conclusions in favour or against these commercial kits are available.13e18 Preliminary evaluations of five rapid test kits (unpublished data) showed that these kits generally have higher sensitivities for IgG detection but lower sensitivities for IgM detection and varied specificities as compared to the E/M-specific capture IgM and IgG ELISA. Although the rapid test has the advantages of easy performance and the rapid provision of results, it should be used as a screening test. According to WHO guidelines, these kits should not be used in the clinical settings to guide management of severe dengue cases because many serum samples taken in the first five days after the onset of illness will not have detectable IgM antibodies. The tests would thus give a false negative result. Reliance on such tests in clinical management might lead to an increase in case fatality rate. Furthermore, these kits should not be used for surveillance for dengue disease in public health settings or in seroepidemiological studies due to the high sensitivity of this assay for the detection of IgG and the long persistence of cross-reactive flavivirus IgG antibodies in the general population in many areas where dengue is endemic.
3.10. 3.8.
Immunochromatographic assay for IGM and IgG
Anti-dengue IgA and IgE
The E/M-specific capture IgM and IgG ELISA
It has become the most robust and powerful assay for the serodiagnosis of dengue virus infection due to its high sensitivity, specificity, simplicity, and feasibility for automation.12
Measurement of dengue IgA by anti-dengue virus capture ELISA (AAC-ELISA) becomes positive 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. However
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no differences in IgA titres are found 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. This approach is not used very often and requires additional evaluation. Some investigators have studied dengue virus-specific IgA and IgE antibody responses. Talarmin et al reported on the use of an IgA- and IgM-specific capture ELISA for the diagnosis of dengue virus infection. The results showed that IgM appears more rapidly and lasts longer (over 2e3 months) than IgA (about 40 days). They concluded that the capture IgA ELISA is a simple method that can be performed together with the capture IgM ELISA and that can help in interpreting the serology of DF.19 More recently, Balmaseda et al20 reported on the detection of specific IgM and IgA antibodies in serum and saliva. They concluded that dengue virus-specific IgA in serum is a potential diagnostic target. According to Koraka et al21 capture IgE ELISA for the detection of total and dengue virus-specific IgE antibody responses might be helpful for early diagnosis of DHF/DSS It has been seen that dengue virus-specific IgE titres are significantly higher in patients with DHF and/or DSS than in patients with DF and non-dengue virus infections. They concluded that analysis of dengue virus-specific IgE ELISA might contribute to the understanding of the pathogenesis of dengue virus.
3.11.
Haemagglutination-inhibition test
Optimally the HI test requires paired sera obtained during 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 flaviviruses (e.g. between dengue virus and Japanese encephalitis virus or West Nile virus) nor between immunoglobulin isotypes. The response in 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 response Haemagglutination-inhibition (HI) test is useful for diagnosing secondary dengue infection (i.e., titre 1:1280). Disadvantages: lower specificity compared to other tests, requires 2 serum samples, delays confirmation of diagnosis.2
4.
Conclusion
Laboratory diagnosis of dengue virus infection is very important for early diagnosis and management and differentiate it from various other infections. The haematological investigation will help in the early detection of progression to severe dengue. The advances in molecular and serological diagnostic methods have greatly improved the sensitivity and specificity of the diagnosis However no single diagnostic assay in isolation is adequately sensitive and specific enough to diagnose all acute cases of dengue While direct methods such as, viral isolation, viral nucleic acid or viral antigen detection are more
specific, they are more costly and labour-intensive. Indirect methods (i.e., serology) are less specific, but are more accessible, faster, and less expensive.2,22 Viral antigen detection is positive in the first 5 days of illness, and serological tests becomes positive after the fifth day. Thus, appropriate application of these tests when combined together will be of significant help for the clinical management, etiologic investigation, and control of dengue virus infections.
Conflicts of interest The author has none to declare.
references
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18. Vaughn DW, Nisalak A, Kalayanarooj S, et al. Evaluation of a rapid immunochromatographic test for diagnosis of dengue virus infection. J Clin Microbiol. 1998;36:234e238. 19. Talarmin A, Labeau B, Lelarge J, Sarthou JL. Immunoglobulin A-specific capture enzyme-linked immunosorbent assay for diagnosis of dengue fever. J Clin Microbiol. 1998;36:1189e1192. 20. Balmaseda AMG, Guzman S, Hammond G, Carolina, Flores Y. Diagnosis of dengue virus infection by detection of specific immunoglobulin M (IgM) and IgA antibodies in serum and saliva. Clin Diagn Lb Immunol. 2003;10:317e322. 21. Koraka P, Murgue B, Deparis X, et al. Elevated levels of total and dengue virus-specific immunoglobulin E in patients with varying disease severity. J Med Virol. 2003;70:91e98. 22. Nimmannitya Suchitra. Management of DF/DHF-WHO Regional Publication No. 22 e Monograph on Dengue/DHF. New Delhi: WHO/SEARO;55e61.