Comparative Analysis of Immunological and Genomic Outcomes of Dengue Virus Outbreak in Pakistan

A total of 720 suspected blood serum samples were collected along with information on demographic characteristics, dengue specific symptoms, area, hematological and biochemical features and duration of onset of disease from commencement of September till end of December 2013 at Division of Molecular Virology, CEMB, University of the Punjab Lahore for the molecular characterization and detection of different serotypes of dengue virus from Pakistan. An informed consent was taken from all hospitals and enrolled patients to conduct the study in accordance with the Declaration of Helsinki, 1964 and Directions for Virtuous Medical Research Practice in Pakistan.

All of the suspected patients having characteristic dengue symptoms were screened for the existence of dengue specific NS1, IgG and IgM antibodies according to the manufacturer code of practice. 100 µl of samples was used to detect dengue IgG and IgM using kits from Calbiotech USA according to manufacture’s protocol. Both positive and negative controls were run alongside the samples.

Viral nuclear RNA was isolated from 150 μl of blood serum sample and dengue virus was confirmed by RT-PCR as described earlier (Fatima et al., 2013). cDNA was synthesized using Moloney Murine Leukiemia Virus (MMLV) Reverse Transcriptase (RTse) (Invitrogen Biotechnology, USA). Dengue detection by using nested PCR procedure as developed by our laboratory (Fatima et al., 2011) was done and the oligonucleotide sequences used to amplify C-prM gene junction of dengue virus were 5’TCAATATGCTGAAACGCGWGAGAAACCG3’and 5’TTGCACCARCARTCWATGTCTTCWGGYTC3’. After performing 30 cycles of PCR amplification, product was run at 2% agarose gel stained with ethidium bromide and visualized under UV light. The size of amplified PCR product was 511bp.For further serotyping analysis of Dengue the primers and procedure was followed as mentioned earlier (Fatima et al., 2011). SPSS V.17 software was used for Statistical analysis and all data were presented as number of subjects and percentage. P-value was adjusted at 0.05.

Out of our 720 collected samples, 703 were positive for dengue virus through ELISA and/or PCR detection methods and were further used for our study. We evaluated the incidence of infectivity in both males and females, 70% of samples were obtained from males and 30% from females, suggesting a higher incidence in males than females. The higher number of samples from young patients suggests a greater susceptibility to infection in 16-35 years old age group (Table I), whereas, the two parameters (infectivity and age/gender) were not correlated. Age did not tend to have any specific correlation with IgM positivity (p=0.55), IgG positivity (p=0.23) and NS1 positivity (p=0.89). Similarly, gender could not be associated with IgM positivity (p=0.49), IgG positivity (p=0.89) and NS1 positivity (p=0.72).

Out of 703 positive samples, 658 (93.5%) were positive to IgM and only 261 (39.66%) RNA positives were identified by PCR. In these 261 PCR positive subjects, 2 (0.76%), 145 (55.5%) and 42 (16%) were infected with any one dengue serotype- 1, 2 and 3, respectively. On the other hand 72 (27.58%) IgM positive patients had concurrent infection with serotypes 2 and 3.

Five hundred and three (71.55%) samples were identified dengue positive through ELISA in which 202 (40.1%) were dengue virus RNA positive. From these PCR positive samples 112 (55.4%) and 34 (16.8%) had infections of either serotype 2 or 3, respectively. Only 56 (27.72) IgG positive subjects had concurrent infection with both serotypes 2 and 3.

From 703 studied serum samples 572 (81.7%) exhibited NS1antigen and 211 (36.8%) of these NS1 positive subjects had RNA positivity by PCR. Out of the 211 PCR positive subjects 2 (0.74%), 122 (57.8%) and 37 (17.5%) had serotype 1, 2 and 3 infections, respectively, but 50 (23.69%) NS1 positive subjects had concurrent viral infection with both serotypes 2 and 3 (Table I). The 2:1 male: female ratio in studied population remains the same as already reported (Fatima et al., 2013). As 39.66% of anti-dengue IgM positive samples were dengue viral RNA positive, so these results confirm a current infection because viral RNA can be isolated from day one of post-infection till 7-8 days (Marinho et al., 2017).

*RT-PCR positive samples in given group.

Some of the important findings and limitations of this study are described here. Firstly, anti-dengue IgM was performed, IgM may stay in blood of patient till 2-3 months after infection and the antigens used for anti-dengue IgM ELISA are derived from the envelope protein of the virus. One of the limitation of this test is the cross reactivity between other circulating flaviviruses. This limitation must be considered when working in regions where multiple RNA viruses co-circulate. Anti- dengue IgM detection may not be 100% useful for dengue serotype determination due to cross-reactivity of the antibody. The result with negative anti-dengue IgM and positive with other methods may be due to compromised or low sensitivity of the ELISA based test which was used to perform IgM detections. According to the Pan American Health Organization (PAHO) guidelines 80% of all dengue cases have detectable IgM antibody by day five of illness, and 93-99% of cases have detectable IgM by day six to ten of illness, which may then remain detectable for over 90 days (Marinho et al., 2017).

Secondly, we have performed anti-dengue IgG ELISA on our collected samples. This information of anti-dengue IgG can be used to detect primary and secondary infections by using simple algorithms (Marinho et al., 2017). For this early sampling must be done in acute and convalescent phases, but under the settings of our society it may become difficult to collect samples twice because most of the patients, if not admitted to the hospital, do not prefer to give second blood samples. As dengue fever can present a range of symptoms so patients take painkillers to relieve themselves of the pain till the symptoms are gone.

Thirdly, the use of anti-dengue NS1 is considered specific and reliable in identifying current infections as NS1 levels can be detected from day one of post infection with specificity among different flaviviruses. According to CDC laboratory protocol the PCR can give positive results up to 5 days post infection and NS1 can give positive results up to 18 days post infection. So the difference noted in our studied samples may be due to the difference in post infection days at the time of testing (Marinho et al., 2017).

The present study suggests that many of the infections were few months previous or not current ones. As dengue fever has a range of symptoms so during an epidemic, few of the symptoms if not due to dengue virus, may lead the doctors to checking for dengue virus detection. The treatment of dengue fever is prophylactic so all of the medical care depends upon laboratory diagnosis i.e. ELISA or PCR detection tests.

Another important finding of this study was that the serotype-2 remains the main causative serotype of this outbreak of dengue fever as reported in the previous outbreaks in Pakistan (Shahid et al., 2017).

After performing IgM, IgG antibodies, NS1 and PCR detection, this immunological and genomic study reveals that there are more seropositive patients than RNA positive ones during the study. So we concluded that the detection of RNA through PCR, and NS1 detection are considered more sensitive and specific methods for current infection of dengue virus. Hence they should be incorporated more in detecting the circulating virus in blood of patient.

We would like to acknowledge all the patients and clinicians who helped us in getting the samples for this study.

We declare that we have no conflict of interest.