|Year : 2014 | Volume
| Issue : 3 | Page : 277-280
Serological diagnosis of dengue in laboratory practice in Kolkata
N Bhattacharya1, H Mukherjee1, R Naskar1, S Talukdar2, G Das1, N Pramanik3, AK Hati4
1 Medical Entomology Unit, Calcutta School of Tropical Medicine, Kolkata, West Bengal, India
2 Department of Biochemistry and Medical Biotechnology, , Calcutta School of Tropical Medicine, Kolkata, West Bengal, India
3 Department of Tropical Medicine, Medical Entomology, , Calcutta School of Tropical Medicine, Kolkata, West Bengal, India
4 Calcutta School of Tropical Medicine, Calcutta School of Tropical Medicine, Kolkata, West Bengal, India
|Date of Submission||24-Jan-2013|
|Date of Acceptance||02-Dec-2013|
|Date of Web Publication||10-Jul-2014|
A K Hati
Calcutta School of Tropical Medicine, Calcutta School of Tropical Medicine, Kolkata, West Bengal
Source of Support: Science and Technology Department, Govt. of
West Bengal, Bikash Bhawan, Kolkata,, Conflict of Interest: None
Purpose: To find out the most suitable serological investigative procedures to diagnose dengue cases effectively in the laboratory practice identifying primary and secondary cases as well as period of suffering. Materials and Methods: Dengue suspected cases sent to the laboratory in 2012 in central Kolkata by the local physicians were categorised into seven panels according to the investigations asked for such as (1) only dengue-specific NS1 antigen (2) only IgM antibodies, (3) NS1 + IgM + IgG antibodies, (4) only IgM and IgG, (5) NS1 + IgM, (6) NS1 + IgG and (7) only IgG. Results: Out of 1892 suspected cases, dengue was diagnosed in 725 (38.3%). Through panels I, II, III, IV, V, VI and VII, it was possible to diagnose dengue in (I) 35.98% (435/1209), (II) 37.5% (24/60), (III) 49% (173/354), (IV) 30.8% (68/221), (V) 60.5% (23/38), (VI) 40% (2/5) and (VII) 0 of cases respectively. Detail information such as confirmed diagnosis, duration of the disease (whether early or prolonged) and classification of primary and secondary dengue in such early or prolonged stages would only be possible in panel III, which information would be helpful for effective monitoring and treatment of dengue patients. In all other panels, merely fragmentary information would be obtained. Conclusions: Serodiagnostic tests dengue-specific NS1 antigen and IgM and IgG antibodies when conducted simultaneously would be able to diagnose confirmed dengue cases categorising primary and secondary dengue along with the duration of the disease, whether early or prolonged.
Keywords: Dengue, dengue diagnosis, laboratory practice, serological diagnosis
|How to cite this article:|
Bhattacharya N, Mukherjee H, Naskar R, Talukdar S, Das G, Pramanik N, Hati A K. Serological diagnosis of dengue in laboratory practice in Kolkata. Indian J Med Microbiol 2014;32:277-80
|How to cite this URL:|
Bhattacharya N, Mukherjee H, Naskar R, Talukdar S, Das G, Pramanik N, Hati A K. Serological diagnosis of dengue in laboratory practice in Kolkata. Indian J Med Microbiol [serial online] 2014 [cited 2019 Aug 22];32:277-80. Available from: http://www.ijmm.org/text.asp?2014/32/3/277/136563
| ~ Introduction|| |
Dengue is endemic in Kolkata. ,,, During the transmission season that extends generally from July to November,  patients suspected to be suffering from dengue are referred to the clinical laboratories for confirmatory diagnosis of dengue cases. Tests usually asked for are dengue-specific NS1 antigen and IgM and IgG antibodies, either alone or in some combination including or excluding one or the other parameter.
An intense dengue infection swept Kolkata in 2012. Many suspected persons were referred by the local physicians to our laboratory for serological diagnosis of dengue. An opportunity was obtained to investigate, analyse and categorise the cases who were actually suffering from dengue as well as to note which serological investigation procedure was superior to others to diagnose a dengue patient in the laboratory practice along with the status of the patient related to duration of the disease and detection of primary or secondary infection. So the purpose of this paper was to find out the most suitable serological investigative procedure to diagnose dengue cases effectively in the laboratory practice identifying primary and secondary cases along with the period of suffering.
| ~ Materials and Methods|| |
The investigations were carried out in our laboratory in central Kolkata. The study period extended from 1 st January to 31 st December 2012. The blood was collected from the individual patient, suspected to be suffering from dengue, the serum was separated, the required test was done on the same date of collection, and the report was delivered to the person concerned. Serological tests requested by the physicians to diagnose dengue were dengue-specific NS1 antigen and IgM and IgG antibodies, either alone or in various combination as stated below:
- Panel I only NS1 antigen
- Panel II only IgM antibody
- Panel III NS1 antigen + IgM and IgG antibodies
- Panel IV only IgM and IgG antibodies
- Panel V NS1 antigen and IgM antibodies
- Panel VI NS1 antigen and IgG antibodies
- Panel VII only IgG antibodies.
To test dengue-specific NS1 antigen, ELISA method was employed using BIORAD kits  MAC ELISA was performed using Vircell kits  to detect IgM antibodies. For detection of IgG antibodies BIORAD kits were used.  The instructions of the manufacturers were meticulously followed while performing the tests. When asked for testing IgM alone (Panel II) or both IgM and IgG antibodies (Panel IV) the blood was taken 5 days after the onset of fever. To test other panels no such discrimination was done.
| ~ Results|| |
A total of 1892 persons, suspected to be suffering from dengue were investigated, of whom 1209 (63.9%), 64 (3.4%), 354 (18.7%), 221 (11.7%), 38 (2%), 5 (0.3%) and 1 (0.05%) were in Panel I, II, III, IV, V, VI and VII, respectively [Table 1].
|Table 1: Classification of suspected Dengue cases according to different serological tests in Kolkata in 2012 |
Click here to view
Out of 1892 suspected persons dengue was diagnosed in 725, comprising 38.3% of total cases [Table 1].
In panel I, sera of 1209 persons were tested for dengue-specific NS1 antigen alone, of which 435 (35.98%) were reactive. In panel II sera of 64 persons were tested for dengue-specific IgM antibodies alone, of which 24 (37.5%) were reactive. In panel III 354 persons were asked to test both dengue-specific NS1 antigen and IgM and IgG antibodies simultaneously, out of those persons 173 (48.87%) were diagnosed as dengue cases. In panel IV, sera of 221 persons were tested for both IgM and IgG antibodies of which 68 (30.8%) were reactive. In panel V, 38 persons were asked to examine NS1 antigen and IgM antibodies, of whom 23 (60.5%) were diagnosed as dengue cases. In panel VI, 5 persons were tested for NS1 antigen and IgG antibodies, of whom 2 (40%) were diagnosed as dengue cases.
In panel III, 354 persons investigated were further classified into eight categories as follows:
In this panel the cases were subdivided into six groups as follows:
(a) Not suffered from dengue 60 (16.9%), (b) suffered from dengue previously i.e. old dengue 121 (34.2%), (c) early primary dengue 46 (13.0%), (d + g) late primary dengue 15 + 31 = 46 (13.0%), (f) early secondary dengue 42 (11.9%) and (g + h) late secondary dengue 10 + 29 = 39 (11.0%).
The cases of panel IV were subdivided into following 4 groups: (a) not suffering from dengue 67 (30.3%), (b) old dengue 86 (38.9%), late primary dengue 31 (14.0%) and late secondary dengue 37 (16.7%).
The cases of panel V were subdivided into following 3 groups: (a) not dengue 15 (33.5%), (b) early dengue 13 (34.2%), late dengue 10 (26.3%) (either IgM alone reactive or both NS1 + IgM reactive).
The cases of panel VI were subdivided into four groups as follows: (a) not dengue 2, old dengue 1 (b) early dengue 1 and early secondary dengue 1.
The cases of panel VII were subdivided into two groups (a) old dengue 0 and (b) not old dengue 1 [Table 1].
| ~ Discussion|| |
Effective and accurate diagnosis of dengue is of primary importance for clinical care, early detection of severe cases, case confirmation and differential diagnosis.  In clinical practice to diagnose dengue serological tests, such as dengue-specific NS1 antigen and IgM and IgG antibodies are now often performed. According to WHO  NS1 antigen can be detected up to 9 days after the onset of illness. IgM antibodies are detectable in 50% of patients by days 3-5 after the onset of illness, increasing to 80% by day 5 and 99% by day 10. IgM levels peak about 2 weeks after the onset of symptoms and then decline generally to undetectable level after 2-3 months. Anti-dengue serum IgG is generally detectable at low titres at the end of the week of illness, increasing slowly thereafter with serum IgG still detectable after several months and probably even for the life. 
Time of collection of the blood after the onset of fever is 1-6 days and after five days for NS1 antigen and IgM antibodies, respectively.  In the present study, panel I could detect dengue in early stage, facilitating monitoring of the patient, but it did not indicate whether the patient was suffering from primary or secondary dengue. Previous dengue cases (old cases) and primary or secondary cases could also not be identified.
Panel II diagnosed dengue in its late stage at least 5 days after the onset of fever, here also no information related to primary and secondary dengue was obtained. Again, early appraisal or monitoring of a case would be out of question in such cases. Here also, previous dengue cases could not be detected.
Cases not suffering from dengue, previous old cases and late primary and late secondary cases could be detected in panel IV, but not early primary or secondary cases. So early monitoring would not be possible and the disease might take a serious turn by the time of diagnosis.
Panel V could diagnose the disease either in the early or in late stage. Here information about primary or secondary infection and previous dengue infection could not be obtained.
Panel VI diagnosed secondary dengue cases in early stage (early secondary dengue) only.
In all those above panels except panel IV, one or more vital important information for effective early and accurate diagnosis of severe cases - vis a vis care, treatment and monitoring of a patient suffering from a complex disease like dengue was lacking.
A comprehensive information, giving clue to the above-mentioned points was, however, obtained through the analysis of panel III when both dengue-specific antigen NS1 and antibodies IgM and IgG were tested. Confirmed (a) early primary (of the three tests only NS1 was reactive), (b) early secondary (of the three tests NS1 and IgM were reactive) (c) early secondary (of the three tests NS1 and IgM were reactive only) and (d) late secondary (NS1, IgM and IgG were all reactive or IgM and IgG only were reactive) cases could be diagnosed. Only IgG reactive cases were previous dengue cases. Cases nonreactive to NS1, IgM and IgG were not suffering from dengue.
In panel III, both NS1 antigen and IgM antibodies were detected in 31/354 (8.8%). In them in all probability the duration of fever would be more than 5 days but less than 9 days.
Although 285 (15.1%) had the history of fever for more than 5 days. Other 1607 (84.9%) patients were suffering for fever for less than 5 days. In the panel III, 354 (18.7%) persons were suffers for fever less than 5 days, of whom 46 (13%) and 39 (11%) were detected no late primary and late secondary cases, respectively. Patients coming in late primary infection can be labeled as secondary.
There was no much difference between the number of primary (92) and secondary (81) dengue cases. Out of a total of 173 dengue cases in panel III 42 (24.3%) cases of secondary dengue were diagnosed in early stage of the disease. Of all secondary dengue cases (81), 42 (51.9%) cases could be diagnosed at an early period, facilitating better care and monitoring.
Although only NS1 test was the most favourite one (63.9%), next favourite test (NS1 + IgM and IgG) (18.7%) would provide all the information required for a dengue patient. Detection of primary or secondary dengue cases in early or late stage would be possible with the help of panel III, fulfilling the ultimate goal of diagnosis. Information regarding primary and secondary infection in early stage of the disease seemed to be very important due to the possibility of secondary dengue cases to turn to DHS or DSS.  Considering this fact, in laboratory practice the serological procedure of panel III would be superior to any other procedure that would yield desired result.
| ~ Acknowledgement|| |
The work done in this study has been financially supported by Department of Science and Technology, Government of West Bengal. Also the help rendered by the Directors of Gautam Laboratories, Kolkata in this study is thankfully acknowledged.
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