Serodiagnosis of Dengue by Particle Agglutination Assay

Dengue Viruses (DV) are transmitted to humans by infected mosquitoes, mainly Aedes aegypti and Aedes albopictus1. There are four serotypes, dengue virus types 1, 2, 3, and 4.2 The secondary infection by a different dengue serotype in patients harboring pre-existing, heterologous dengue antibodies is a risk factor.3,4 However there is evidence that primary infection of dengue can cause severe manifestation leading to DHF/ DSS indicating important of viral virulence suggested that molecular differences in dengue is also risk factor for manifesting.5 DF is an emerging disease affecting Nepal since 2004.6 There was an concern of the disease in Nepal after the Indian and Pakistani epidemic of DF/DHF, which claimed more than 100 deaths and more increasingly several thousand cases in the year 2006.7 There was an outbreak which was observed in 9 districts of Tarai region in Nepal in 2006.8 At present, diagnosis and management of dengue, JE and other infectious diseases in Nepal is based on patient’s clinical symptoms due to lack of limited diagnostic facility.9 The laboratory diagnosis of dengue therefore, usually relies on serology. The simple and rapid ELISA methods have been adapted to detect antibodies against DENV.10 The antigen capturing ELISA J Nepal Health Res Counc 2009 Apr;7(14):29-32


INTRODUCTION
Dengue Viruses (DV) are transmitted to humans by infected mosquitoes, mainly Aedes aegypti and Aedes albopictus 1 .There are four serotypes, dengue virus types 1, 2, 3, and 4. 2 The secondary infection by a different dengue serotype in patients harboring pre-existing, heterologous dengue antibodies is a risk factor. 3,4owever there is evidence that primary infection of dengue can cause severe manifestation leading to DHF/ DSS indicating important of viral virulence suggested that molecular differences in dengue is also risk factor for manifesting. 5 is an emerging disease affecting Nepal since 2004. 6here was an concern of the disease in Nepal after the Indian and Pakistani epidemic of DF/DHF, which claimed more than 100 deaths and more increasingly several thousand cases in the year 2006. 7There was an outbreak which was observed in 9 districts of Tarai region in Nepal in 2006. 8At present, diagnosis and management of dengue, JE and other infectious diseases in Nepal is based on patient's clinical symptoms due to lack of limited diagnostic facility. 9The laboratory diagnosis of dengue therefore, usually relies on serology.The simple and rapid ELISA methods have been adapted to detect antibodies against DENV. 10 The antigen capturing ELISA for virus detection is the most useful procedure currently available and it is widely recommended for Virological Surveillance. 11Now a days Particle Agglutination (PA) system, which does not require specifi c laboratory facilities, has been developed for detecting DEV IgM. 12 In the present study we validated the PA assay in more serum samples.We applied the PA system to serum samples collected from febrile patients clinically suspected to suffer from dengue, in Tarai region of Nepal.We compared the results with those obtained by IgM-capture ELISA.

METHODS
One hundred and eighty-three blood samples were collected from the suspected patients having clinical symptoms of dengue suspected on the basis of fever, severe headache and rashes in Dengue-epidemic areas.The specimen was collected from hospitals of Chitwan, Hetauda, Birgung and Biratnagar in Tarai region of Nepal in 2007.Before collecting blood specimen, demographic information was recorded and informed consent was obtained from each patient or guardian.The samples were kept in the ice compartment of the refrigerator, brought to Everest International Clinic and Research Center in Kathmandu and they were stored at -70 0 C in deep freeze until use.
The ELISA Kit is used to this report (PanBio Dengue Duo, Australia) to diagnose of dengue infection.The required number of the well are determined for the assay.Ten μl antigens were mixed with 2.5 ml antigen diluent.Then required volume of dilute antigen was removed from the mixture.The equal volume of Monoclonal antibody tracer were added to diluent antigen and mixed it left in a room that temperature between 20-25 0 C. Immediately one hundred μl of diluted serum (1:100) were added into wells contained anti-human IgM plate.The plates were incubated at 37 0 C for 30 min.The plates were washed six times with diluted wash buffer.One hundred μl of Ag-Mab tracer were mixed to plate.Again incubated at 37 0 C for 30 min.The plates were washed six times with diluted wash buffer.One hundred μl Tetramethylbenzidine was Pipete and added in each well.This time incubation was done at 20-25 0 C for only 10 min.Finally one hundred μl of stop solution was added and the color developed was closely observed.With in 30 min the absorbance of each well were taken at a wavelength of 450 nm as a reference to wave length by using Microplate ELISA Reader Model 700 (Cam Tech USA).If Panbio Unit exceeds 11, then Sample was considered as positive one and lesser as 9 were considered as negative one.
The samples were diluted with serum dilution buffer at 1:100.The human IgM (Red coloured plate) capture micro plates that were cut by a cutter for a number of strips needed and removed the seal from the strip.In addition two wells were required for positive and negative controls.The strips were washed thrice with wash buffer and taped on the paper towel to remove excess liquid from them.
The fi fty μl of diluted samples were added to the wells.Samples were added immediately after washing strips to prevent surface of the wells from drying.Otherwise the wells may lose the ability to capture antibodies if dried completely and the wells were incubated for 30 min at room temperature.The strip was washed thrice with wash buffer and taped on paper towels to remove any liquid as previously.The Dengue antigen coated bead slurry (red colored) was gently mixed.The one hundred μl of the beads slurry was added into wells and allowed to settle for one hour at room temperature.When the Ha-Ny beads formed a button pattern at the bottom of well, the reaction was defi ned as negative.In contrast to this adhesion of Ha-Ny beads on the wall of the well was taken as positivity.Statistical analysis was done using a software SSPS 11.5 version.

RESULTS
The 183 serum samples collected from the febrile patients with the clinical symptoms of dengue, in DFepidemic area of the Tarai region were tested for Dspecifi c IgM by two serological methods namely IgMcapture PA and ELISA.Fifty-fi ve (30%) of the 183 sample were IgM positive by the PA (Tab 1).Fifty (90%) out of the fi fty-fi ve PA IgM-positive samples were also IgM positive by IgM capture ELISA (Table 2).This PA assay has the Sensitivity of 98% and Specifi city of 96%, a positive prediction value of 90% and negative prediction value of 99% in comparison with IgM-capture ELISA.Five samples were positive by PA assay, but negative by IgM captured ELISA.One hundred twenty seven cases were Dengue IgM negative by both IgM-captured ELISA and PA.These results suggest that there is high level of compatibility between the PA assay and IgM-captured ELISA.Sensitivity was 98% and Specifi city was 96%.

DISCUSSION
Dengue is a major public health problem that is responsible for millions of cases of illness and thousands of deaths in tropical countries every year. 13The increasing importance of Dengue and DHF in Asia, South America, and the Caribbean countries underlines the importance of early detection in controlling the spread of the disease. 13Dengue occurred mainly in the Tarai region of Nepal during past few years.In Nepal, sporadic cases were noticed in foreigners in Nineties and the fi rst case of DF was reported in the year 2004. 6In previous report by Scherchanda et al., the prevalence of dengue antibodies in the Southwestern region of Nepal was 10.4%. 14Then, a confi rmed outbreak i.e eleven cases, which was observed in nine districts of Tarai region in Nepal in 2006. 8This report suggests that dengue virus have been circulating in Nepal for several years.A study by Epidemiology and Disease control Division, Department of Health Service of Nepal on A.aegypti between September and December 2006, indicated that A. aegypti has been introduced in the country and that its densities were high enough for successful transmission. 15 present, Dengue is diagnosed based on patient's clinical symptoms: high fever, headache, muscle and joint pain.Dengue infections are being misdiagnosed for other related infections and its importance is underestimated.Despite the available clinical guidelines, dengue can be misdiagnosed due to limited information on it among medical community.Given the lack of specifi city of the symptoms of dengue, clinicians can confuse dengue with other similar infections, such as infl uenza, enterococus, chikungunya, viral haemorrhagic fevers, leptospirosis, malaria, or typhoid. 16Moreover, dengue infection as an undifferentiated febrile illness may represent a large proportion of all the symptomatic cases of dengue. 17lthough there is currently no effective antiviral treatment; rapid diagnosis of Dengue to detect the antibody provides the opportunity to treat Dengue Infection.
The laboratory diagnosis of Dengue usually relies on serology.This is simple and rapid, ELISA methods which have been adapted to detect antibodies against DENV. 10 ELISA based methods using specifi c MABs can also lead to defi nite diagnosis.The antigen capture ELISA for virus detection is the most useful procedure currently available and it is widely recommended for virological surveillance. 11However, IgM-capture ELISA requires relatively more sophisticated equipments, and highly trained personnel and is more useful in the referral diagnostic centers in developing countries.
The development of PA assay, which does not require such specifi c equipment and is relatively economical too and therefore, be benefi cial for the rural areas with limited facilities and where trained personnel are not available.We did the PA assay of serum samples collected from Tarai region of Nepal.The sensitivity and specifi city of the PA assay is high and will be useful in rural areas of Nepal.The discrepancies between the PA and IgM-combo ELISA need to evaluate further considering the patients histories in-depth.It may not be specifi c compare to IgM ELISA leading to false positive result for other fl avivirus or other infectious disease.
Serially collected serum samples could provide more valuable information.Although cross-reactivity of antifl aviviral IgG has been well documented.IgM is known to be specifi c antibodies to dengue and is the antibody assay by ELISA in the study. 18The data suggest that the PA assay for DV is quick, easy to perform and specifi c.This assay system is useful especially in the rural areas of Nepal to support the clinical diagnosis, management, and epidemiological studies of Dengue.

CONCLUSION
The fi ndings of this study showed that dengue is fi rmly established in low endemic Tarai region of Nepal.The sensitivity and specifi city of PA assay is acceptably high and determined to be useful in rural areas of Nepal.

Table 1 .
The Prevalence of IgM Cases by PA tests in symptomatic Patients.

Table 2 .
Comparison of the Results between PA Assay and IgM-capture ELISA