Massab Umair

Dengue Virus Serotypes Circulating in Khyber Pakhtunkhwa Province, Pakistan, 2013-2015

From 2013 to 2015, the National Institute of Health, Pakistan, received 1,270 blood samples of suspected dengue cases reported from inpatient and outpatient departments of various hospitals in Khyber Pakhtunkhwa (KPK) province. In this study, we determined the circulating dengue virus (DENV) serotypes using real-time reverse transcriptase (RT)-PCR to understand the serotype-based epidemiology of DENV. All four serotypes (DENV-1 [6%], DENV-2 [33%], DENV-3 [47%], and DENV-4 [0.1%]) were found circulating during the study period. Our findings suggest the need for an active surveillance system coupled with the laboratory diagnosis, especially in the chronic endemic areas of the country. Public awareness programs are needed for effective control and prevention of outbreaks in the future.

Preliminary Seroepidemiological survey of dengue infections in Pakistan, 2009-2014

BackgroundDengue virus is the causative agent of dengue fever, a vector borne infection which causes self-limiting to life threatening disease in humans. A sero-epidemiological study was conducted to understand the current epidemiology of dengue virus in Pakistan which is now known as a dengue endemic country after its first reported outbreak in 1994.MethodsTo investigate the prevalence of dengue virus in Pakistan during 2009-2014, a total of 9,493 blood samples were screened for the detection of anti-dengue IgM antibodies using ELISA. Clinical and demographic features available with hospital records were reviewed to ascertain mortalities related to dengue hemorrhagic shock syndrome.ResultsOut of 9,493 samples tested, 37% (3,504) were found positive for anti-dengue IgM antibodies. Of the seropositive cases, 73.6% (2,578/3,504) were male and 26.4% (926/3,504) were female. The highest number (382/929; 41.1%) of sero-positive cases was observed among the individuals of age group 31-40 years. The highest number of symptomatic cases was reported in October (46%; 4,400/9,493), and the highest number of sero-positive cases among symptomatic cases was observed in November (45.7%; 806/1,764). Mean annual patient incidence (MAPI) during 2009-2014 in Pakistan remained 0.30 with the highest annual patient incidence (11.03) found in Islamabad. According to the available medical case record, 472 dengue related deaths were reported during 2009-2014.ConclusionThe data from earlier reports in Pakistan described the dengue virus incidence from limited areas of the country. Our findings are important considering the testing of clinical samples at a larger scale covering patients of vast geographical regions and warrants timely implementation of dengue vector surveillance and control programs.Trial registration numberIt is an epidemiological research study, so trial registration is not required.

Molecular analysis of group A rotaviruses detected in hospitalized children from Rawalpindi, Pakistan during 2014

As a part of strategy to control diarrheal diseases, World Health Organization (WHO) recommends to include rotavirus vaccines in national immunization programs. Sentinel surveillance networks have been established to monitor rotavirus disease burden and genotype distribution in both pre and post vaccine era in many countries. Unfortunately, due to lack of proper surveillance programs, data on rotavirus disease burden and genotype distribution from Pakistan is scarce. We investigated 502 stool samples from children (<5years) hospitalized due to gastroenteritis in Rawalpindi, Pakistan during 2014 for the presence of group A rotavirus (RVA) and its genotypic diversity. Among 147 ELISA positive samples, 131 were successfully genotyped for RVA. Common G types detected were G1 (23.6%), followed by G3 (22.9%), G12 (19.8%), G2 (19.08%) and G9 (9.9%). The most common P-type was P[8] (41.2%), followed by P[6] (29%) and P[4] (28.24%). G3P[8] (17.55%) was the most prevalent genotype combination followed by G12P[6] (16.7%), G2P[4] (15.2%) and G1P[8] (14.5%). Mixed infection of rotavirus G-P types was also observed in 6% of samples. Phylogenetic analysis of VP7 and VP4 genes of Pakistani strains showed that G1, G2, G9 and P[4], P[6], P[8] were closely related to strains circulating worldwide as well as previously reported strains from Pakistan. Pakistani G12P[8] strains NIH-BBH-3981 and NIH-BBH-4003 belonged to lineage 3 cluster 3a along with strains from USA and Italy whereas G12P[6] strains NIH-BBH-3978, NIH-BBH-4052 and NIH-BBH-4444 were closely related to strains from Italy, Thailand, United Kingdom and with previously reported G12 strains from Pakistan within lineage 3 cluster 3b. This pre-vaccination data supports the need for RVA vaccine inclusion at our national level and will be helpful in assessing the effect of vaccination on RVA genotype diversity due to vaccine selection pressure once post-vaccination data becomes available.

Outbreak of dengue virus type-3 in Malakand, Pakistan 2015; A laboratory perspective

An outbreak of dengue fever was reported in Malakand district, Khyber Pakhtunkhwa (KP) province of Pakistan during 2015. Detection of viral RNA by real-time PCR confirmed dengue virus serotype-3 (DENV-3) to be the causative agent causing the outbreak. Phylogenetic analysis based on partial E-NS1 gene sequences showed that the DENV-3 viruses belonged to genotype III with maximum homology with the dengue-3 strains previously reported from Pakistan and India. Our current report provides updated information on molecular epidemiology and phylogenetic analysis of dengue virus serotypes responsible for 2015 outbreak in KP.

Distribution of Cytomegalovirus Genotypes among Neonates Born to Infected Mothers in Islamabad, Pakistan

Background Congenital cytomegalovirus (cCMV) infection contributes to considerable long-term sequelae in neonates and children all over the world. The association between viral genotypes and severity of clinical cytomegalovirus (CMV) infection is yet to be defined. The objective of this study was to find the impact of active CMV infection during pregnancy and the clinical significance of genotypes in neonates with congenital cytomegalovirus infections in Pakistan. Methods A total of 409 blood samples from pregnant women seeking health care services at the two antenatal hospitals of Islamabad during January to December 2012 were tested by ELISA and nested-PCR. Pregnant women with active infection (detected as IgM positive, PCR positive or positive on both assays) were followed until delivery, to detect the outcome of overt cCMV infection in neonates. Genetic characterization of CMV strains was performed by sequence analysis of envelope glycoproteins: gB, gN and gH to detect the contributing CMV genotypes. Results The seroprevalence of anti-CMV IgG and IgM was 97.5% (399 out of 409) and 12.7% (52 out of 409), respectively, while 20% (82/409) pregnant women were found positive for CMV DNA by PCR. Logistic regression analysis showed a significant association of active infection with parity [OR = 2.56, 95% CI = 1.82–2.62, p = 0.04], febrile illness [OR = 1.84, 95% CI = 1.76–3.65, p = 0.01] and jaundice [OR = 22.5, 95% CI = 4.53–85.02, p = 0.002]. We were able to isolate virus in 41 out of 70 neonates; 36.6% (15 out of 41) of them were symptomatic at birth while 63.4% (26 out of 41) were asymptomatic. The most prominent clinical feature observed in symptomatic neonates was hepatosplenomegaly (26.6%; 4 out of 15). All three genotypes gB, gN and gH were found with the highest frequency of gB1 genotype, found in 75% infants with hepatic damage. Phylogenetic analysis of Pakistani strains showed 96%-100% homology to their prototype strains. Conclusions Active CMV infection during pregnancy is a major cause of congenital CMV infection with comparable distribution of all three genotypes: gB, gN and gH in symptomatic and asymptomatic neonates. Our findings emphasize to conduct a comprehensive large scale survey and introduction of country wide routine screening at maternity clinics for early diagnosis of CMV to reduce its associated devastating outcomes.

Rotavirus surveillance in Pakistan during 2015-2016 reveals high prevalence of G12P[6]

The G12 rotavirus genotype has emerged globally since their first detection in 1987 from the Philippines; however it remains a rare cause of gastroenteritis in Pakistan. Rotavirus surveillance conducted during 2015‐2016, assessed 3446 children <5 years hospitalized for gastroenteritis and found 802 (23.2%) positive on ELISA. Genotyping of a subset of positive samples (n = 319) revealed G12P[6] (11.28%) as the third most common G/P combination following G3P[8] (28.5%) and G1P[8] (12.5%); G2P[4] (10.65%) and G3P[6] (8.15%) were other frequently detected strains. Phylogenetic analysis of G12 strains from Pakistan revealed high genetic similarity to G12 strains from Italy, Thailand, Korea, and Great Britain as well as local strains within G12 lineage III. In conclusion, G12P[6] was a major contributor of RVA gastroenteritis in Pakistani children. Robust surveillance after the introduction of rotavirus vaccines will help determine the evolution of G12 and other circulating genotypes in the country.

Identification of measles virus genotype B3 associated with outbreaks in Islamabad, Pakistan, 2013–2015

BACKGROUND Measles virus infection remains a significant cause of childhood mortality and morbidity despite continued global efforts and the availability of a safe and effective vaccine. Molecular analysis of indigenous measles viruses could provide critical information on outbreak linkages and transmission pathways that can aid the implementation of appropriate control programs in Pakistan. METHODS Blood samples and throat swabs were collected from subjects suspected with measles in Islamabad, Pakistan from 2013 to 2015. Serum samples were tested for the presence of measles immunoglobulin M (IgM) antibodies using enzyme-linked immunosorbent assay (ELISA) while throat swabs were used for the isolation (Vero/SLAM cell line) and subsequent characterization and phylogenetic analysis of measles strains. RESULTS Of 373 blood samples, 66% tested positive for measles IgM. Male subjects were more often infected (58%) than female (42%) with the highest frequency of positive cases (63%) in the 0-5-years age group. Among the positive cases, only 13% had received one or two doses of the measles vaccine, while 87% were unvaccinated. Of 80 throat swabs, 29 (36%) showed a measles virus-specific cytopathic effect (CPE) and were characterized as genotype B3 through partial sequencing of the nucleoprotein (N) gene. Phylogenetic analysis revealed the Pakistani B3 strains to be closely related to strains from neighboring countries (Iran and Afghanistan) as well as with B3 viruses from the USA, Germany, and the UK. CONCLUSIONS The study results showed that despite the availability of an effective vaccine, the burden of measles infections is very high in Pakistan due to poor routine immunization coverage even in major cities, including the capital city of Islamabad. It is imperative that national health authorities take urgent strategic steps to improve routine immunization and implement adequate molecular identification methods to tackle future measles outbreaks.

NS1 antigen: A new beam of light in the early diagnosis of dengue infection

OBJECTIVE To evaluate NS1 antigen detection ELISA for the early laboratory diagnosis of dengue virus infection. METHODS The present study was conducted to evaluate the overall positivity of NS1 antigen detection ELISA and its comparison with viral RNA detection via real time PCR and IgM antibodies detection by ELISA. RESULTS A total of 1270 serum samples were tested 86% (1097/1270) were detected positive by one or more than one diagnostic test. Out of 1 270, 64% (807/1270) were positive by NS1 ELISA and 52% (662/1270), 51% (646/1270) were positive by real-time RT-PCR and IgM ELISA respectively. CONCLUSIONS NS1 antigen detection ELISA is highly suitable diagnostic tools and it also has great value for use in outbreak and epidemic situation.

High prevalence of G3 rotavirus in hospitalized children in Rawalpindi, Pakistan during 2014

Rotavirus A species (RVA) is the leading cause of severe diarrhea among children in both developed and developing countries. Among different RVA G types, humans are most commonly infected with G1, G2, G3, G4 and G9. During 2003–2004, G3 rotavirus termed as “new variant G3” emerged in Japan that later disseminated to multiple countries across the world. Although G3 rotaviruses are now commonly detected globally, they have been rarely reported from Pakistan. We investigated the genetic diversity of G3 strains responsible RVA gastroenteritis in children hospitalized in Rawalpindi, Pakistan during 2014. G3P[8] (18.3%; n = 24) was detected as the most common genotype causing majority of infections in children less than 06 months. Phylogenetic analysis of Pakistani G3 strains showed high amino acid similarity to “new variant G3” and G3 strains reported from China, Russia, USA, Japan, Belgium and Hungary during 2007–2012. Pakistani G3 strains belonged to lineage 3 within sub-lineage 3d, containing an extra N-linked glycosylation site compared to the G3 strain of RotaTeqTM. To our knowledge, this is the first report on the molecular epidemiology of G3 rotavirus strains from Pakistan and calls for immediate response measures to introduce RV vaccine in the routine immunization program of the country on priority.

Epidemiological and molecular investigation of a measles outbreak in Punjab, Pakistan, 2013-2015: ZAIDI et al.

Despite the availability of an effective vaccine, the measles virus continues to cause significant morbidity and mortality in children worldwide. Molecular characterization of wild‐type measles strains is an invaluable component of epidemiological studies or surveillance systems that provides important information pertinent to outbreak linkages and transmission pathways. Serum samples and throat swabs were collected from suspected measles cases from the Punjab province of Pakistan (2013‐2015) and further tested for measles immunoglobulin M (IgM) through enzyme‐linked immunosorbent assay and reverse‐transcriptase polymerase chain reaction for molecular characterization. Among the total of 5415 blood samples, 59% tested positive for measles IgM. Males had a higher infection rate (55%) than females (45%), and the highest frequency of positive cases (63%) was found in the age group of 0 to 5 years. Partial sequencing of the nucleoprotein gene showed that 27 strains belonged to the B3 genotype, whereas 2 viruses were identified as D4. On phylogenetic analysis, Pakistani B3 strains were found to be closely related to previously reported indigenous strains and those from neighboring countries of Iran and Qatar. This is the first report on the detection of the measles B3 genotype from Punjab, Pakistan. The current study shows a high burden of measles infections in Punjab province owing to poor routine immunization coverage in major cities. It is imperative that national health authorities adopt strategic steps on an urgent basis for improvement of routine immunization coverage. Molecular epidemiology of the measles viruses circulating in different parts of the country can provide useful data to manage future outbreaks.