Detection of Cytomegalovirus in Pregnant Women of Lakki Marwat and Bannu, Khyber Pakhtunkhwa, Pakistan

The present study was carried out in districts Lakki Marwat and Bannu of Southern Khyber Pakhtunkhwa, Pakistan. Ethical approval was obtained from ethical review committee of the Kohat University of Science and Technology, Kohat.

A total of 188 blood samples (3ml) were randomly collected in sterilized vacationer from pregnant women of the study area and were transported to Molecular Parasitology and Virology laboratory, department of Zoology, Kohat University of Science and Technology Kohat. Sera was separated and tested for CMV-specific immunoglobulin G (IgG) through ELISA using Cenix diagnostic kit (Dresden Germany) following the manufacturer protocol.

DNA was extracted from blood samples in a biosafety hood type II cabinet using ultra script DNA extraction kit (Anagen, Inc. USA) as per manufacture protocol and stored at -20°C till for further analysis. Regular PCR was performed in a total reaction volume of 50µl by using 10X PCR buffer, 2.5mM magnesium chloride (MgCl2), 10µM of each deoxynucleotide triphosphates (dNTPs), 5 units of Taq DNA polymerase (Fermentas, USA) and 10pmol of each forward and reverse primer: (B1: 5´-CAAGARGTGAACATGTCCGA-3´ and B2: 5´-GTCACGCAGCTGGCCAG-3´). The cycling conditions for PCR were denaturation at 94°C for 2 minutes followed by 35 cycles of 94°C for 30 seconds, 58°C for 40sec, 72°C for 50 sec, followed by final extension at 72°C for 3 min (Shams et al., 2011; Revathy et al., 2018).

Second round of amplification (nested PCR) was carried out using 5µl of a previous PCR product obtained in regular PCR. Amplification was done with a nested inner sense B3 (5´-TGGAACTGGAACGTTTGGC-3´) and antisense B4(5´-GAAACGCGCGGCAATCGG-3´) primers. The reaction mixture and the optimal conditions for nested PCR were the same as standard for regular PCR. The amplified PCR product was run on 2% agarose gel and visualized under ultraviolet (UV) light (Shams et al., 2011; Revathy et al., 2018).

Data was analyzed using SPSS version 18 used for data entry and analysis. P value was calculated, where P< 0.05 was considered significant at 95% CI (Confidence Interval).

Among 188 pregnant women, 138(73.40%) and 108(57.44%) were found positive through ELISA and PCR respectively for CMV infection. A similar type of study conducted in India showed 8.5% positivity for CMV infection. In Afghanistan CMV was 99.79 % positive through IgG and 1.24% through IgM antibodies test (Husseini et al., 2019). Variation of the present study may be due to the different sample population, vaccination status, climate and geographic conditions from the previous studies.

We also analyzed CMV infection reference to socio-economic status of the study participants (Tables I and II). Results of the present study showed that women of low socio-economic status of both districts; Lakki Marwat (29.79%) and Bannu (17.02%) were at higher risk of getting infection as compared to those from the middle and high socio-economic level. Women of low income and congested family also had a high risk of transmission of CMV infection in the study area.

CMV infection in rural areas was higher in both district Bannu (24.47%) and district Lakki Marwat (30.32%). The high prevalence rate of CMV infection in rural may attribute due to unhygienic and unhealthy environmental condition at homes.

The sensitivity of PCR and ELISA was analyzed (Table III) and ELISA was found more sensitive for CMV infection as compared to PCR which is contrary with the finding of Revathy et al. (2018) where PCR was found more sensitive compared to ELISA.

It was concluded from the present study that significant number of the women were positive for CMV infection in the study areas. Education can play a major rule in CMV control and prevention. Individuals with lower socioeconomic status were at higher risk of CMV infection. Overall district Bannu has a low prevalence of CMV infection as compared to Lakki Marwat. In Bannu a low prevalence of CMV infection may be due to high literacy rate compared to district Lakki Marwat. Further studies will be required by taking more number of the women from the study area in order to get a clearer picture of the disease status in the study area and to know better about the sensitivity of diagnostic methods whether it is PCR or ELISA for CMV detection.

We are indeed very thankful to medical staff: doctors, nurses andwomen of study DHQ hospitals and Women and Children hospital of both districts for helping in blood samples and data collection.

Cl, Confidence interval; one sample t- test; p-value < 0.05.

Cl, Confidence interval; one sample t- test; p-value < 0.05.

The authors have declared no competing interests.