Prevalence of dengue virus serotypes in the 2017 outbreak in Peshawar, KP, Pakistan

Abstract Background Dengue is a viral disease, transmitted by infected Aedes aegypti and Aedes albopictus female mosquitoes. Worldwide, 96 million infections were estimated in 2010. The dengue virus comprises four distinct serotypes (DENV‐1, DENV‐2, DENV‐3, and DENV‐4) which belong to the genus Flavivirus. Determining the serotypes during dengue outbreaks is crucial for its effective management in terms of diagnostics improvement and polyvalent vaccine development. The aim of the present study is to determine the prevalence rate of dengue virus serotypes in the samples collected from patients during the 2017 outbreak in Khyber Pakhtunkhwa, Pakistan. Methods A total of 800 ELISA‐positive samples were collected, of which 513 (290 males, 223 females) samples were confirmed positive by PCR. Results Out of 513, 25 were found serotype 1 (5%), 196 were serotype 2 (38%), 192 were serotype 3 (37%), 56 were serotype 4 (11%), and 44 (8%) were found to have mix serotypes. Conclusion We can conclude that serotypes 2 and 3 of dengue virus were the predominated serotypes of dengue virus in the 2017 outbreak in Peshawar, capital city of Khyber Pakhtunkhwa, Pakistan.


| INTRODUC TI ON
Dengue is mosquito-borne viral infection, which affects about 390 million people every year. 1 In the last decade, 120 countries are facing great challenge to prevent dengue virus (DENV) transmission and 2.5 billion world populations are at risk of dengue infection according to WHO report. 3 During the dengue outbreaks in Pakistan, more than 55 000 individuals were hospitalized and many of them are children. Since 2006, dengue epidemics have occurred every year and have become a major health problem in Pakistan. 2 Dengue is endemic in most tropical and subtropical countries, 4 and most of the dengue spread has caused by large number of human migration. Aedes aegypti mosquito is native to Africa but worldwide migration caused the spread of dengue, and Ae. Aegypti became one of major vector of dengue spread. 4 The epidemiology and spread of DENV became a serious burden in South East Asia after Second World War. 5 The first recorded epidemic of dengue hemorrhagic fever (DHF) was recorded in Philippines (Manila) in 1953-1954, followed by second emergence in 1956, 5 in Bangkok in 1958, and in Thailand in 1960s. 5,6 Until 1970s, only nine countries reported the presence of DHF which increased in 1995. 7 The first case of dengue in Pakistan was reported from Hub, in Baluchistan in 1960, at that time Pakistan population was 45.9 million. From 1960 to 1980, there were only 12 dengue cases reported in Pakistan. 8 In Pakistan, the first outbreak of DENV was detected in 1994, in which two distinct serotypes DENV-2 and DENV-3 had been identified in Karachi. 9 Dengue virus is a positive single-stranded RNA virus composed of about 11 kilobase (kb) that codes for 3411 amino acids polypeptide molecule. The polypeptide gives rise to structural proteins (membrane, envelope, and capsid) which are responsible for the basic structure of virus and are not take part in the viral genome replication and to non-structural proteins (NS5, NS4B, NS4A, NS3, NS2B, NS2A, NS1) which are expressed inside the infected cells. 10 Globally, the variants of dengue virus have been classified into four serotypes named DENV-1, DENV-2, DENV-3, and DENV-4, belonging to family named "Flaviviridae" of "Flavivirus" genus. The four serotypes of dengue virus have been originated from sylvatic strains, found in South East Asia forests. 11 Each serotype of DENV consists of different genetic composition and undergoes genetic mutation that makes difficult to produce vaccines against all four serotypes. If patients gain immunity against one serotype, then immunity will be maintained against that serotype, but immunity against another serotype would be temporary. 12 Dengue virus infection can cause a wide spectrum of clinical manifestations, which differ from dengue fever (DF) and severe dengue disease (SDD), which includes dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). 2 Dengue fever (DF) is characterized with high fever onset. In young children, neurological disturbances caused by dehydration may occur during febrile phase. 13 DF can cause extreme weakness along with myalgia, leucopenia, petechiae rash, arthralgia, and eye pain. 14 DF is also called breakbone fever due to severe pain in muscles and joints. There is no difference between the symptoms of DF and DHF during early stages. Although DHF leads to manifestations of viral hemorrhagic, vascular permeability that causes plasma leakage and thrombocytopenia. 15 On the other hand, dengue shock syndrome (DSS) is different from DHF because it causes plasma leakage, a process in which the protein-rich fluid component of the blood leaks from blood vessels into interstitial spaces and leads to shock. 16 Plasma leakage is the major cause of mortality and morbidity in patients with DSS.
The conditions of about 5%-10% of patients with DF can become severe; hospitalization is required for the patients suffering from DHF. About 30%-40% of children suffering from DHF can contract DSS. Treatment is symptomatic and supportive. Fatality rate due to DSS/DHF can reach 5%. The patients recovered from DF acquired immunity for the life time, against the serotype of dengue that had caused infection in the patient. 17 Determining the dengue serotypes during a particular outbreak of the disease is very important not only for its effective management but above all for improving diagnostics and for the develop-

| Sample collection
The study was approved by the Ethics Committee of the IBGE, UAP.
A total of 800 patients from various healthcare centers, showing DF symptoms during 2017 outbreak, were enrolled in the study. About 4 mL blood was collected from DF-positive samples in EDTA tubes after the approval of informed consent from the selected patients.
Collected blood samples were screened for the presence of dengue virus active infection according to standard biosafety protocol. The demographic and clinical history was recorded on a proforma.

| Viral RNA isolation
The RNA was extracted from 150 µL serum sample using Favorgen viral RNA extraction kit (Favorgen Biotech Corp), following the manufacturer's instructions. Extracted RNA was either stored at −80°C or processed immediately. Method introduced by Lanciotti and colleagues in 1992 was used for single-tube multiplex PCR, rapid detection, and typing of dengue virus in clinical samples. 18  was as follows: initial denaturing for 42 minutes at 42°C, annealing at 60°C for 1 minutes, final temperature 92°C for 2 minutes, and holding temperature 22°C for 2 minutes. Two-step PCR (1st and 2nd round) was carried out to amplify the synthesized cDNA and dengue virus serotyping. cDNA was amplified using forward and reverse primers: D1 represents the forward (sense) primers while D2 represents the reverse (antisense) primers. In the 2nd round of PCR, the reverse primers were replaced with four serotype-specific primers. D1 and type specific 1 (TS1) were used for the identification of serotype 1, which amplify a region of 482 bp; D1 along with type specific 2 (TS2) was used to identify serotype 2, which amplify a region of 119 bp; and D1 and type specific 3 (TS3) amplified a region of 290 bp. Serotype 4 was amplified by D1 and type specific 4 (TS4), and a region of 392 bp was amplified. The primers used are shown in Table 1. PCR-amplified products were detected by using agarose gel. 2% agarose gel was prepared in TBE buffer (1×), which was pre-stained with 3 µL ethidium bromide that was used as a florescent dye. After electrophoresis, photographs were taken under ultraviolet light transilluminator. A 100 bp of DNA ladder was used as a DNA marker. Using serotype-specific primers, specific serotypes were determined, as illustrated in Figure 3. Out of 513 patients, serotype 1 was the least prevalent serotype with only 4% cases, serotypes 2 and 3 were predominant serotypes accounting for 38% and 37%, respectively, whereas 10% positive DENV patients have serotype 4. Mixed circulating serotype was detected in approximately 9% patients.

| D ISCUSS I ON
Dengue is a fast emerging pandemic-prone viral disease in many parts of the world. Dengue is caused by virus of Flaviviridae family, and there are 4 distinct, but closely related, serotypes that cause dengue (DENV-1, DENV-2, DENV-3, and DENV-4). 19 The aim of our study is to investigate which DENV serotype was prevalent in the The KP population is exposed to all kinds of deadly diseases because the etiological factors for most diseases are found mainly in this province. The country has been battered by the war, the last one has been fought against terrorism, and therefore, there have been outbreaks of many deadly diseases. However, many diseases have been controlled and their spread has been overcome, but adequate planning is needed to combat a deadly disease. Efficient strategy should not only neutralize the viral spread but should also include eliminating or reducing risk factors. Although the province's health system is constantly improving, there are many problems to be solved. Dengue flourishes in urban poor areas, in the suburbs and in the countryside, but it also affects the more affluent neighborhoods in countries located in the tropics and subtropics.
In Pakistan, people are exposed to comorbidities, infected blood products, and many risk factors. The risk factors are influenced by local spatial variations of rainfall, temperature, relative humidity, degree of urbanization, and quality of vector control services in urban areas.
The recommendation to combat the disease is to inform the local community about risk factors and potentially fatal complications of dengue virus. Awareness seminars and workshops should be held to educate the population. In particular, healthcare workers should be employed for the cause so that they can carry out a massive campaign and inform people to take preventive measures to combat dengue.
In conclusion, the incidence of dengue has grown dramatically in the last decade in Pakistan. In our study, serotypes 2 and 3 were the predominated serotypes of dengue virus in the 2017 outbreak in Peshawar. To prevent and combat the spread of dengue is necessary an efficient monitoring and evaluation strategy. This includes the disease surveillance through the identification and molecular characterization of infected human cases, and the vector surveillance by tracking mosquito populations in areas of potential. In addition, monitoring of behavioral impact is essential for observing whether the strategies aimed to reduce dengue transmission are adopted and supported by the community.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interests regarding the publication of this paper.

E TH I C A L A PPROVA L
The Ethics Committee of the IBGE, UAP, granted approval for the study that was conducted according to the principles of ICH-GCP