Dengue and Chikungunya virus co-infection in major metropolitan cities of provinces of Punjab and Khyber Pakhtunkhwa: A multi-center study

Dengue has become endemic in Pakistan with annual recurrence. A sudden increase in the dengue cases was reported from Rawalpindi in 2016, while an outbreak occurred for the first time in Peshawar in 2017. Therefore, a multi-center study was carried out to determine the circulating dengue virus (DENV) serotypes and Chikungunya virus (CHIKV) co-infection in Lahore, Rawalpindi, and Peshawar cities in 2016-18. A hospital-based cross-sectional study was carried out in Lahore and Rawalpindi in 2016-18, while a community-based study was carried out in Peshawar in 2017. The study participants were tested for dengue NS1 antigen using an immunochromatographic device while anti-dengue IgM/IgG antibodies were detected by indirect ELISA. All NS1 positive samples were used for DENV serotyping using multiplex real-time PCR assay. Additionally, dengue samples were tested for CHIKV co-infection using IgM/IgG ELISA. A total of 6291 samples were collected among which 8.11% were NS1 positive while 2.5% were PCR positive. DENV-2 was the most common serotype (75.5%) detected, followed by DENV-1 in 16.1%, DENV-3 in 3.9% and DENV-4 in 0.7% while DENV-1 and DENV-4 concurrent infections were detected in 3.9% samples. DENV-1 was the predominant serotype (62.5%) detected from Lahore and Rawalpindi, while DENV-2 was the only serotype detected from Peshawar. Comorbidities resulted in a significant increase (p-value < 0.001) in the duration of hospital stay of the patients. Type 2 diabetes mellitus substantially (p-value = 0.004) contributed to the severity of the disease. Among a total of 590 dengue positive samples, 11.8% were also positive for CHIKV co-infection. Co-circulation of multiple DENV serotypes and CHIKV infection in Pakistan is a worrisome situation demanding the urgent attention of the public health experts to strengthen vector surveillance. Author summary Mosquitoes are responsible for the transmission of many different types of infectious pathogens and parasites to humans. Some of these pathogens include viruses like dengue and chikungunya viruses which cause diseases in humans. Dengue virus has four different types (1, 2, 3, and 4) present in nature. In this study, we detected all four dengue virus types in hospitalized patients in Lahore and Rawalpindi. While only dengue virus type-2 was detected from Peshawar city. The secondary infections with different dengue virus types than the previous one might result in a more severe form of the disease. Therefore, the population of Peshawar is at risk of developing a severe form of dengue fever in the future in case of infection from another dengue virus type. The data of hospitalized patients showed that other diseases in addition to dengue fever result in an increased hospital stay. Comorbidities like diabetes also increase the risk of developing a more severe form of dengue fever. Combined infections of both dengue and chikungunya viruses were detected from Lahore, Rawalpindi, and Peshawar. The transmission of dengue and chikungunya viruses and the co-circulation of multiple dengue virus types not only impose public health burden but also result in economic losses.