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ORIGINAL ARTICLE
Year : 2014  |  Volume : 41  |  Issue : 2  |  Page : 85-88

Challenges in the early diagnosis of dengue: A practical approach


Department of Microbiology, Tirunelveli Medical College, Highgrounds, Tirunelveli, Tamil Nadu, India

Date of Web Publication20-May-2014

Correspondence Address:
Poongodi S. Lakshmi
Department of Microbiology, Tirunelveli Medical College, Highgrounds, Tirunelveli, Tamilnadu - 627 011
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0974-5009.132833

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  Abstract 

Introduction: Mosquito borne diseases such as dengue, chikungunya, and malaria are clinically indistinguishable and diagnosis is solely upon laboratory findings. In an endemic area, early diagnosis of dengue requires detection of all the three markers (NS1, immunoglobulin (Ig) M and G simultaneously to distinguish primary and secondary infection. Aim: To assess the value of all dengue markers by rapid immunochromatography test (ICT) in early diagnosis. Materials and Methods: A total of 120 blood samples collected from patients with fever were tested for dengue markers by rapid ICT. Results: Of the 120 samples tested, 41 (34%) were positive for one or more of the three markers. Of the 41 samples, NS1 only was positive in 10 (24%), IgM only in two (5%), IgG only in 10 (24%), NS1 and IgM in two (5%), NS1 and IgG in one (2%), IgM and IgG in eight (20%), and triple positive (NS1, IgM, and IgG) in eight (20%). Of the 41 cases, NS1 alone was detected in two patients (5%) who came on 1-2 days of illness. Conclusion: Rapid ICT detecting NS1, IgM, and IgG is much valuable in emergencies as a screening test for early diagnosis and management. Rapid tests should be confirmed by enzyme-linked immunosorbent assay (ELISA). Still, "gold standard" tests in the diagnosis of dengue are virus isolation or molecular methods.

Keywords: Dengue, rapid, early diagnosis, NS1, primary, secondary


How to cite this article:
Lakshmi PS, Nainar P. Challenges in the early diagnosis of dengue: A practical approach. J Sci Soc 2014;41:85-8

How to cite this URL:
Lakshmi PS, Nainar P. Challenges in the early diagnosis of dengue: A practical approach. J Sci Soc [serial online] 2014 [cited 2020 Aug 12];41:85-8. Available from: http://www.jscisociety.com/text.asp?2014/41/2/85/132833


  Introduction Top


In the last 2 years, Tamil Nadu has witnessed an upsurge in various mosquito borne diseases such as dengue, chikungunya, and malaria. All these diseases were once considered sporadic, now turned into epidemic. They are clinically indistinguishable and diagnosis is solely upon laboratory findings. These viral diseases like dengue and chikungunya are self-limiting and treatment is only symptomatic. Since vaccines are not available prevention is mainly by vector control in endemic areas. Further, mosquitoes are also resistant to insecticides.

Of these three, dengue creates more serious threat to human. It infects 50-100 million people worldwide annually. [1] Population-based studies suggest that asymptomatic infections are the main outcome of dengue virus exposure. Dengue fever/dengue hemorrhagic fever (DF/DHF) is caused by infection with four dengue virus serotypes DEN-1, DEN-2, DEN-3, and DEN-4; which are closely related to each other antigenically. This results in extensive cross-reactivity in serological tests, but infection with one serotype does not provide cross-protective immunity against the others; thus, persons living in an endemic area can be infected with each of the four dengue serotypes during their lifetime. [2]

Further, subsequent infections with another serotype may increase the risk of developing DHF/dengue shock syndrome (DSS) owing to the cocirculation of various DEN serotypes. [3] Till date, diagnosis is mainly by dengue immunoglobulin (Ig)M capture enzyme-linked immunosorbent assay (ELISA) even in tertiary care hospitals and Infectious Diseases Surveillance Programme (IDSP) reference laboratories. IgM appears only after 3-5 days of illness in primary infection and persist for 2-3 months, whereas in secondary infection it is not always positive. [4]

There are practical constraints to run ELISA tests with few samples on and off even during epidemic. Thisresults in delayed diagnosis, misdiagnosis, and underreporting. Keeping all this in mind, this study was done in a private multispecialty hospital using a commercial rapid immunochromatography test (ICT) detecting dengue NS1, IgM, and IgG simultaneously for early diagnosis.


  Materials and methods Top


A total of 120 blood samples were collected from patients with fever attending a private multispecialty hospital from October 2011 to February 2012. Informed consent and Institutional Ethical Committee approval were obtained. Sera were separated and tested for dengue by ICT as per manufacturer's instructions. (Bioline, Dengue Duo manufactured by SD Standard Diagnostics, Inc., Korea) which detects NS1, IgM, and IgG. Along with this, blood-total count (TC) and differential count (DC), erythrocyte sedimentation rate (ESR), hemoglobin (Hb), platelet count, renal and liver parameters, ultra sonogram, X-ray chest, and tests to rule out other causes of fever were done wherever necessary. Statistical analysis was done by using chi-square test and Student's paired t-test.


  Results Top


Of the 120 serum samples tested, 41 (34%) were positive for one or more of the three markers (NS1, IgM, and IgG). Of the 41 samples, NS1 only was positive in 10 (24%), IgM only in two (5%), IgG only in 10 (24%), NS1 and IgM in two (5%), NS1 and IgG in one (2%), IgM and IgG in eight (20%), and triple positive (NS1, IgM, and IgG) in eight (20%). NS1 alone or in combination with IgM, IgG, or both were detected in 21 cases (51%) [Table 1].
Table 1: Dengue markers in pediatric and adult population

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Of the 19 pediatric (<14 years) cases, 11 (58%) were positive, of the 101 adult (>14 years) cases, 30 (30%) were positive for one or more markers. There was significant difference in the incidence of dengue between the pediatric and adult population (P < 0.05). Of this 11 pediatric cases, eight (73%) were positive for IgG [Table 1].

Of the 41 cases, 14 (34%) had primary infection and 27 (66%) had secondary infection. The difference in the incidence of primary and secondary dengue infection is statistically significant (P < 0.001). Among these 41 cases, NS1 alone was detected in two patients (5%) who came within 1-2 days of illness and IgM in eight patients (20%) who came on 3-5 days of illness. Difference in the proportion of detection of primary infection of dengue was different between NS1 and IgM (P < 0.01) [Table 2].
Table 2: Primary and secondary dengue based on markers

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Thrombocytopenia (platelet count <1 lakh/μl) was noted in 32 cases (78%) and leukopenia (white blood cell (WBC) count <4,000/mm 3 ) in nine cases (22%) who were positive for either one or more of the dengue markers. There was significant difference in the platelet count between dengue positive and negative cases (P < 0.001). There was significant difference in the WBC count between dengue positive and negative cases (P < 0.01) [Table 3]. Seasonal trend was noted during post monsoon season, that is, October-February.
Table 3: Dengue markers and cell count (WBC and platelets)

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  Discussion Top


Diagnosis of acute stage of dengue by IgM capture ELISA alone is very difficult, which is the only available test even in tertiary care centers. Simultaneous detection of NS1, IgM, and IgG is essential to distinguish primary and secondary dengue depending upon the time of collection of specimen. All these three markers by ELISA is time consuming and costlier. Further it requires costly equipment, expertise, and microbiologist. This is not feasible in primary healthcare settings, hence rapid ICT detecting these three markers is an efficient alternative. In the present study, NS1 alone was positive in 10 (24%). This was observed as 23% by Datta et al., Shrivastava et al., as 16%, and Kulkarni et al., as 30%. [5],[6],[7] This implies that all these cases would be misdiagnosed if NS1 is not included in the test panel.

NS1 alone or in combination with IgM, IgG, or both were detected in 21 cases (51%). The nonstructural proteins are NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5. [8] NS1 is a highly conserved glycoprotein essential for viral replication. [9] Libraty et al., demonstrated that the high level of NS1 in plasma was correlated with viremia levels in patients with DHF than in patients within 72 h of illness onset. [10] High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of DHF. [10] This indicates quantitative NS1 can be used as a prognostic indicator also. NS1 positive patients are infectious for mosquito. Further, viral strain and host immune status have also been suggested as a risk factor for DHF/DSS.

Triple positive (NS1, IgM, and IgG) implies, they are either in the late stage of primary or secondary infection. But in the present study, this was observed in eight (20%) cases and they were detected within 7 days indicating secondary infection.

In this study, 11 pediatric (<14 years) cases (58%) were positive for one or more dengue markers. This indicates that dengue is more common in children as observed in previous studies. [3],[11]

During the acute phase of the disease, presence of dengue IgM denotes primary infection and concomitant detection of dengue IgM and IgG is suggestive of secondary infection. Absence of dengue IgG in samples collected between days 0 and 8 makes it possible to define the case as primary infection. Of the 41 cases, 14 (34%) had primary infection and 27 (66%) had secondary infection. Differentiation of primary and secondary dengue infection is essential to reduce morbidity and mortality due to DHF/DSS, particularly in children and elderly. Hence, this distinction is useful in prognostic and epidemiological purpose. [12],[13] Studies reported that NS1 is detectable for shorter period in secondary infection because IgG may serve to mask NS1 from detection or rapid clearance of NS1 in the form of immune complexes. [14] Of the 41 cases, NS1 alone was detected in two patients (5%) who came on 1-2 days of illness. Datta et al., also reported that NS1 is detectable from day 1 of fever both in primary and secondary infections. [5]

Thrombocytopenia (platelet count <1 lakh/ml) was noted in 32 cases (78%) among positive patients. This was supported by Kulkarni et al., who observed this as 69%. [7] Fever and leukopenia, plus one or more of the list of symptoms in the World Health Organization (WHO) 2009 classification could be used for a clinical diagnosis of probable dengue. Leukopenia (WBC count <4,000/mm 3 ) was observed in 9 cases (22%) of positive patients in this study.

Seasonal trend in post monsoon (October-February) was observed in this study. This is in concordance with a study by Bharaj et al. [15] In the present study, the results of the rapid tests correlated with clinical and other laboratory parameters, but they were not evaluated against ELISA and molecular methods.

This study highlights prompt early diagnosis and appropriate treatment prevents the transmission. Effective anti-mosquito measures should be taken during post monsoon season. If effective vaccines are also developed, all these integrated approach will reduce the incidence of dengue.


  Conclusion Top


Specific IgM detection alone is useful in most of the acute infections. But simultaneous detection of NS1, IgM, and IgG is vital for the diagnosis of dengue, otherwise it would be missed. Rapid ICT detecting all the three markers is much valuable in emergencies as a screening test for early diagnosis and management. Rapid tests should be confirmed by ELISA. Dengue IgG can be used for seroepidemiological surveillance. False positive serology can occur in dengue antibody detection by ELISA due to cross reaction with other arboviruses. Hence "gold standard" tests in the diagnosis of dengue are virus isolation or molecular methods.


  Acknowledgement Top


All persons who have made substantial contributions to the preparation of the manuscript, but who are not authors are named in the Acknowledgement section and have agreed in writing to be named. No contributor has been omitted.

 
  References Top

1.WHO. Fact sheets Media centre. Dengue and severe dengue. No.117 January 2012. Available from: http://www.who.int/mediacentre/factsheets/fs117/en/ [Last accessed on 2012 Aug].  Back to cited text no. 1
    
2.Gubler DJ. Epidemic dengue/dengue haemorrhagic fever: A global public health problem in the 21st century. Dengue Bull 1997;21:1.  Back to cited text no. 2
    
3.Gubler DJ. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 1998;11:480-96.  Back to cited text no. 3
[PUBMED]    
4.WHO. Dengue hemorrhagic fever: Diagnosis, treatment, prevention and control. World Health Organization, Geneva, Switcherland. 2 nd ed. 1997. Available from: http://www.who.int/csr/resources/publications/dengue/Dengue publication/en/inbex.tml [Last accessed on 2012 Mar].  Back to cited text no. 4
    
5.Datta S, Wattal C. Dengue NS1 antigen detection: A useful tool in early diagnosis of dengue infection. Indian J Med Microbiol 2010;28:107-10.  Back to cited text no. 5
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6.Shrivastava A, Dash PK, Tripati NK, Sahni AK, Gopalan N, Laksmana Rao PV. Evaluation of a commercial dengue NS1 enzyme linked immunosorbent assay for early diagnosis of dengue infection. Indian J Med Microbiol 2011;29;51-5.  Back to cited text no. 6
    
7.Kulkarni RD, Patil SS, Ajantha GS, Upadhya AK, Kalabhavi AS, Shubhada RM, et al. Association of platelet count and serological markers of dengue infection-importance of NS1 antigen. Indian J Med Microbiol 2011;29:359-62.  Back to cited text no. 7
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8.Chambers TJ, Hahn CS, Galler R, Rice CM. Flavivirus genome organization, expression and replication. Annu Rev Microbiol 1990;44:649-88.  Back to cited text no. 8
    
9.Lindenbach BD, Rice CM. Trans-complementation of yellow fever virus NS1 reveals a role in early RNA replication. Virol J 1997;71:9608-17.  Back to cited text no. 9
    
10.Libraty DH, Young PR, Pickering D, Endy TP, Kalyanrooj S, Green S, et al. High circulating levels of the dengue virus non structural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever. J Infect Dis 2002;186:1165-8.  Back to cited text no. 10
    
11.Gunasekaran P, Kavery K, Mohana S, Arunagiri K, Babu BV, Priya PP, et al. Dengue disease status in Chennai (2006-2008): A retrospective analysis. Indian J Med Res 2011;133:322-5.  Back to cited text no. 11
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12.Shu PY, Huang JH. Current advances in dengue diagnosis. Clin Diagn Lab Immunol 2004;11:642-50.  Back to cited text no. 12
    
13.Shu PY, Chen IK, Chang SF, Yuch YY, Chow L, Chien LJ, 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 Diag Lab Immunol 2003;10:622-30.  Back to cited text no. 13
    
14.Hang VT, Nguyet NM, Trung DT, Tricon V, Yoksan S, Dung NM, et 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;3:e360. Available from: www.plosntds.org [Last accessed on 2012 Feb 11.  Back to cited text no. 14
    
15.Bharaj P, Chahar HS, Pandey A, Diddi K, Dar L, Guleria R, et al. Concurrent infections by all four dengue virus serotypes during an outbreak of dengue in 2006 in Delhi, India. Virol J 2008;5:1-5.  Back to cited text no. 15
    



 
 
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  [Table 1], [Table 2], [Table 3]



 

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