Comparison of Serology, Virus Isolation and RT-PCR in the Diagnosis of Dengue Viral Infections in Sri Lanka V.G.N.S. Velathanthiria , Sirimali Fernandoa,d, Rohan Fernandob, G. Neelika Malavigea,c, Mallika Peelawaththagea, S.D. Jayaratnee and John Aaskovf a b
Department of Microbiology, Faculty of Medical Sciences, University of Sri Jayewardenepura, Sri Lanka
Department of Veterinary & Biomedical Sciences, University of Nebraska-Lincoln, Lincoln NE 68504,USA c
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, UK d
National Science Foundation, 47/5, Vidaya Mawatha, Colombo 7, Sri Lanka
e
Department of Medicine, Faculty of Medical Sciences, University of Sri Jayewardenepura, Sri Lanka f
School of Life Sciences, Faculty of Science, Queensland University of Technology, Australia
Abstract Currently, the methods for the diagnosis of dengue infections include: antibody detection, virus isolation and antigen detection methods such as RT-PCR. To compare the usefulness of each of these methods in the diagnosis of dengue infections at various time points of illness, 226 patients with suspected dengue virus infections were enrolled and serum-tested by all the three methods during 1999 to 2001. Serological methods were most useful in the diagnosis of dengue infections at all time points of clinical disease and the percentage of detection of dengue virus-specific antibodies increased with the duration of illness. However, virus isolation and molecular detection methods were also sensitive in the detection of the dengue virus and its serotypes, especially during days 3 to 5 of illness. The dengue virus was detected in 26 (18.2%) of our patients and DENV-2 was the predominant virus serotype that was isolated. Virus isolation was not possible in patients with dengue-specific antibodies. In a majority of the patients (64%), dengue-specific IgM was only seen in their serum indicative of a primary dengue infection. Although serological methods appear to be most useful in the diagnosis of dengue infections, virus detection methods are vital in gathering epidemiological data regarding the evolution of dengue epidemics in Sri Lanka. Keywords: Serology, virus isolation, RT-PCR, dengue viral infections.
Introduction Dengue is the most prevalent mosquito-borne viral infection worldwide. Annually, a 100 million of dengue fever (DF) and half a million chm@nsf.ac.lk Dengue Bulletin – Volume 30, 2006
of dengue haemorrhagic fever (DHF) cases occur.[1] Sri Lanka has been affected by dengue for over two decades now and dengue epidemics are becoming more regular with the number of cases rising with each epidemic.
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Dengue infections may be asymptomatic or give rise to undifferentiated fever, dengue fever, dengue haemorrhagic fever or dengue shock syndrome (DSS). Although a diagnosis is usually made based on clinical features, laboratory diagnosis of dengue infections is necessary as it may simulate many other viral and bacterial infections. [2] Furthermore, complications due to dengue infections such as encephalopathy and myocarditis, etc., are thought to be on the rise and laboratory diagnosis in these instances is vital.[3] Currently, the methods for the diagnosis of dengue infections include: virus isolation, serology and molecular techniques such as RTPCR. However, the diagnosis of dengue infections using each of these methods depends on the time of illness, their availability and cost.[4] The usefulness of each of these methods in the diagnosis of dengue infections at various time points of illness is poorly defined. Therefore, we set to compare the sensitivity of each of these methods in the diagnosis of dengue infections at various time points of clinical disease.
Serology Dengue viral-specific antibodies were measured using the PANBIOTM Dengue duo IgM and IgG rapid strip test.[5] This test is said to have a high sensitivity (76% for primary dengue infections and 88% for secondary dengue infections) and specificity (88-99%) and thus it can be used for the differentiation of primary and secondary dengue viral infections. The detection of dengue virus-specific IgG by this assay indicates an IgG antibody titre equivalent to an antibody titre of >2560 by the haemaglutination inhibition (HI) test. The presence of an antibody titre of >2560 by the HI assay is thought to be suggestive of a secondary dengue viral infection.[1] If dengue virus-specific IgM antibodies were only detectable in the test sample, the patient was considered to be having a primary dengue infection, whereas the presence of both IgM and IgG or IgG alone was considered to indicate a secondary dengue infection.
Virus isolation 100 µl of each serum sample was added to a monolayer of C6/36 Aedes albopictus cell lines maintained in RPMI 1640 (Gibco™) in 25 ml tissue culture flasks and incubated for 14 days at 30 °C in 2.5% CO2. Dengue virus serotypes were identified by indirect immunofluorescence assay[6] by staining each cell line with each of the flavivirus group reactive monoclonal antibodies (MAb) 4G2, dengue complex reactive MAb 2H2, dengue 1-specific MAb 15F3, dengue 2-specific MAb 3H5, dengue 3-specific MAb 5D4 and dengue 4-specific MAb 14H10 (kindly supplied by Dr John Aaskov). One spot of cells were not stained with any of the MAbs and was used as the control. The cells were stained with conjugated rabbit anti-mouse immunoglobulins (FITC-Dako™) and observed under a fluorescent microscope for the detection of the dengue virus.
Materials and methods The study was carried out in a general medical unit of a tertiary care hospital in Colombo from September 1999 to August 2002. A total of 226 patients with suspected dengue infections (fever without any other identifiable cause for it) were recruited during the periods of dengue outbreaks in Colombo district. 5 ml of venous blood was obtained from each patient, following informed written consent. Demographic data was obtained using an interviewer-administered questionnaire and relevant clinical and laboratory data were recorded. Ethical approval for the study was obtained from the Ethical Review Committee of the University of Sri Jayewardenepura.
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RT-PCR RNA was extracted from serum using a commercial kit according to the manufacturer’s instructions (QIAamp Viral RNA Mini Kit, QIAGEN™- Germany). One µl (1 unit) of random primers (RocheTM) and 14 µl of DEPCtreated water were added to 10 µl of RNA and incubated at 72 °C for 10 minutes and then on ice for 2-3 minutes. RNase inhibitor 0.8 µl, DEPC water 8.2 µl was added to each tube containing the RNA and random primer and the tubes heated at 55 °C for 10 minutes, followed at 45°C for 1 hour in an automated thermal cycler (GeneAmp™ version II). The PCR was performed with the Universal primers and with specific primers. PCR was carried out in a thermal cycler (Gene Amp™ PCR System, 9200Ver 2.10, PE Applied Bio System). DEPC-treated water and supernatant from culture of uninfected C6/36 cells were used as negative controls in the RT-
PCR. PCR products were separated on 1% W/ V agarose TBE gels (Sigma™ Type II-A)[7] and stained with 1% ethidium bromide (Sigma™). Molecular weight markers (0.019-1.11kbp) (Roche™ VIII) were included with each analysis. The products were visualized under a U.V. transilluminator (Fotodyne™).
Results A total of 226 patients (98 females) with clinical features suggestive of dengue infections were included in the study. The mean age of our cohort was 18.8 years (range 7 months to 70 years). Of these, 95 (42.03%) blood samples were obtained from patients with fever for 4 days or less, 71 (31.5%) on day 5 of illness, and 60 (26.5%) samples between day 6 and 8 of illness. Dengue infections were confirmed in 143 (63%) of the patients either by serology, virus isolation or PCR (Table 1).
Table 1: Comparison of serology, RT-PCR and virus isolation in the diagnosis of dengue viral infection at different time points of illness Duration of fever/days 1 2 3 4 5 6 7 8 Total Total number tested 1 9 33 52 71 38 21 1 226 Serology No. positive (%) 1 (100) 6 (66.7) 9 (27.3) 24 (46) 42 (59) 23 (60.5) 12 (57) – 117 (51.8) Virus isolation No. positive (%) – – 9 (27.2) 7 (13.5) 8 (11.3) – 2 (9.5) – 26 (11.5) RT-PCR No. positive (%) – – 9 (27.2) 7 (13.5) 8 (11.3) 1 (2.6) 2 (9.5) – 27 (11.9)
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Table 2: Dengue virus-specific antibodies in 117 serology-positive patients Total Total number detected Number (%) 40 (42.1) 42 (59) 35 (58.3) 117 (51.8) IgM only Number (%) 25 (62.5) 24 (33.8) 26 (43.3) 75 (33.2) IgG only Number (%) 8 (8.4) 13 (18.3) 6 (0.1) 27 (11.9) Both IgM and IgG Positive Number (%) 7 (7.3) 5 (7.0) 2 (3.3) 14 (6.2)
Fever < 5 days Fever for 5 days Fever > 5 days Total
95 71 60 226
Serological diagnosis of dengue infections Dengue viral infections were confirmed by serology in 117 (81.8%) of our study patients. 40 (34%) of the patients had detectable antibody responses on day 4 of illness and even before this. Interestingly, 7 (5.9%) patients had anti-dengue virus antibodies even on day 1 and 2 of their illness. However, dengue virus could not be detected in these individuals by either virus isolation or by RT-PCR. Although dengue virus-specific antibodies could be detected in patients before day 5 of illness, the detection rates were higher after day 5 of illness (Table 2). 75 (64%) of the patients who were positive by serology only had dengue virus-specific IgM antibodies in their serum, thus indicating a primary dengue virus infection (Table 2). Surprisingly, 42 (55.2%) of those who had only IgM antibodies were adults and 34 (44.7%) were children.
samples which had dengue virus-specific antibodies. Out of the 109 seronegative samples, the dengue virus was detected in 26 (23.8%) cases by virus isolation and in 27 (24.7%) cases by RT-PCR. Virus isolation was more successful in samples in obtained in the early phase of clinical disease (< day 4 of illness) (Table 3). Of the 26 viruses isolated, 15 were DENV-2 and 11 DENV-3. The same dengue viral serotype was identified with RT-PCR. In addition, DENV-2 virus was detected in one sero-negative sample by RT-PCR but not by virus isolation.
Discussion We compared three different laboratory methods used for the diagnosis of dengue infections and found that the serological Table 3: Virus isolation according to day of clinical illness Total number of seronegative samples Fever ≤ 4 days Fever > 4 days Total 56 87 109 Total number of viruses isolated Number (%) 16 (28.6) 11 (12.6) 26 (23.8)
Virological detection of dengue by isolation in cell culture and by RT-PCR We could identify the virus in 26 (18.2%) of the patients, either by RT-PCR or by virus isolation. The virus was not isolated from any of the 194
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methods were most useful in the diagnosis of dengue infections at all time points of clinical disease. However, we were quite surprised in detecting dengue virus-specific antibodies in patients’ serum even on day 1 and day 2 of illness. Our definition of day 1 was the day on which symptoms first occurred and this may be different to that of the onset of viraemia. It is possible that in these few patients, a subclinical viraemia may have occurred prior to the onset of symptoms. However, it would be interesting to investigate if those who mounted a detectable antibody response on day 1 or 2 (with clearance of viraemia) had milder disease. Although dengue infections were confirmed by the presence of dengue virus-specific antibodies in some patients in early disease, the percentage of detection of dengue virus-specific antibodies increased with the duration of illness. As we did not repeat serology, virus isolation or RT-PCR, in those who were ‘negative for dengue virus infections’ (i.e. no detectable dengue-specific antibodies, negative RT-PCR and virus isolation), there is a possibility that at least some of them might have had a dengue infection. Therefore, it is possible that we may have underestimated the number of patients who actually did have a dengue infection. The dengue virus was detected in 26 (18.2%) of our patients during day 3 to 5 of illness. As expected, the virus could not be detected by RT-PCR or by virus isolation in any of the patients with dengue virus-specific antibodies.[4] Although the usefulness of the RT-PCR and virus isolation in the diagnosis of dengue infections appears to be lower than antibody detection methods, these methods are vital in isolating the virus in other clinical samples such as cerebrospinal fluid (CSF) and also in epidemiological studies. Of the 26 viruses isolated during our study period, DENV-2 was the predominant virus
serotype. As we collected serum samples from patients only during the epidemic periods, it is likely that DENV-2 was the predominant serotype and thus responsible for the epidemics during the years 1999 to 2001. Epidemiological studies done in Sri Lanka prior to 1989 and from 1989 to 1998 also suggest that DENV-2 was the predominant serotype.[8] However, some researchers have implicated that DENV-3 serotype is associated with more severe form of the disease and thus hospitalization.[9,10] Although, DENV-3 virus was the second commonest serotype isolated by us, we could not establish if it resulted in more severe disease than patients infected with DENV-2. In a majority of our patients (64%), denguespecific IgM was only seen in their serum indicative of a primary dengue infection. In secondary dengue infection, the IgG antibodies for the previous infecting serotype rise substantially while the IgM antibody titres are usually low. [11,12] In primary dengue viral infections, IgM antibodies develop rapidly and are detectable on days 3 to 5 of illness in a majority of patients.[13] Therefore, it is unlikely that these patients had secondary dengue infections considering the fact that IgM was detectable while IgG was not. Interestingly, 55.2% of those who had only dengue-specific IgM antibodies were adults, which suggested that a significant proportion of our adult patients had a primary dengue infection. Although one would expect that a majority of adults in Sri Lanka would have been exposed to the dengue virus, a recent study has shown that anti-dengue virus antibodies were only detected in 45.5% of children at 17 years of age.[14] Furthermore, another study done in adult patients in 2004 shows similar results, with a significant proportion of adults with primary dengue infections.[10] In conclusion, serological methods appear to be most useful in confirming a diagnosis of
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dengue infections. However, virus isolation and molecular detection methods also appeared to be sensitive in the detection of the virus and its serotype, especially during days 3 to 5 of
illness. In addition, these virus detection methods are vital in gathering epidemiological data regarding the evolution of dengue epidemics in Sri Lanka.
References [1] World Health Organization. Prevention and control of dengue and dengue haemorrhagic fever: comprehensive guidelines. WHO Regional Publication, SEARO; 1999. [2] Malavige GN, Fernando S, Fernando DJ, Seneviratne SL. Dengue viral infections. Postgrad Med J 2004 Oct;80(948):588-601. [3] Pancharoen C, Kulwichit W, Tantawichien T, Thisyakorn U, Thisyakorn C. Dengue infection: a global concern. J Med Assoc Thai 2002 Jun;85 Suppl 1:S25-33. [4] Guzman MG, Kouri G. Advances in dengue diagnosis. Clin Diagn Lab Immunol 1996 Nov;3(6):621-7. [5] Cuzzubbo AJ, Vaughn DW, Nisalak A, Solomon T, Kalayanarooj S, Aaskov J, Dung NM, Devine PL. Comparison of PanBio dengue duo enzyme-linked immunosorbent assay (ELISA) and MRL dengue fever virus immunoglobulin M capture ELISA for diagnosis of dengue virus infections in Southeast Asia. Clin Diagn Lab Immunol 1999 Sep;6(5):705-12. [6] Henchal EA, McCown JM, Seguin MC, Gentry MK, Brandt WE. Rapid identification of dengue virus isolates by using monoclonal antibodies in an indirect immunofluorescence assay. Am J Trop Med Hyg 1983 Jan;32(1):164-9. [7] Sambrook J, Fritch EF , Maniatis T. In: Molecular cloning: a laboratory manual. 2nd ed. New York (NY): Cold Sping Harbor Laboratory Press; 1989. [8] Messer WB, Vitarana UT, Sivananthan K, Elvtigala J, Preethimala LD, Ramesh R, et al. Epidemiology of dengue in Sri Lanka before and after the emergence of epidemic dengue hemorrhagic fever. Am J Trop Med Hyg 2002 Jun;66(6):765-73. [9] Messer WB, Gubler DJ, Harris E, Sivananthan K, de Silva AM. Emergence and global spread of a dengue serotype 3, subtype III virus. Emerg Infect Dis 2003 Jul;9(7):800-9. [10] Malavige GN, Velathanthiri VG, Wijewickrama ES, Fernando S, Jayaratne SD, Aaskov J, Seneviratne SL. Patterns of disease among adults hospitalized with dengue infections. QJM 2006 May;99(5):299-305. [11] Branch SL, Levett PN. Evaluation of four methods for detection of immunoglobulin M antibodies to dengue virus. Clin Diagn Lab Immunol 1999 Jul;6(4):555-7. [12] Sa-Ngasang A, Anantapreecha S, ANuegoonpipat A, Chanama S, Wibulwattanakij S, Pattanakul K, Sawanpanyalert P , Kurane I. Specific IgM and IgG responses in primary and secondary dengue virus infections determined by enzyme-linked immunosorbent assay. Epidemiol Infect 2006 Aug;134(4):820-5. [13] Shu PY, Huang JH. Current advances in dengue diagnosis. Clin Diagn Lab Immunol 2004 Jul;11(4):642-50. [14] Malavige GN, Fernando S, Aaskov J, Sivayogan S, Dissanayaka T, Peelawattage MK, et al. Seroprevalence of anti-dengue virus antibodies in children in the Colombo district. In: Annual Scientific Congress of the College of Paediatricians, 2004; Sri Lanka; 2004.
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