Effect of rotavirus genetic diversity on vaccine impact.

Group A rotaviruses (RVAs) are the leading cause of gastroenteritis, causing 0.2 million deaths and several million hospitalisations globally each year. Four rotavirus vaccines (RotarixTM , RotaTeqTM , Rotavac® and ROTASIIL® ) have been pre-qualified by the World Health Organization (WHO), but the two newly pre-qualified vaccines (Rotavac® and ROTASIIL® ) are currently only in use in Palestine and India, respectively. In 2009, WHO strongly proposed that rotavirus vaccines be included in the routine vaccination schedule of all countries around the world. By the end of 2019, a total of 108 countries had administered rotavirus vaccines, and 10 countries have currently been approved by Gavi for the introduction of rotavirus vaccine in the near future. With 39% of global coverage, rotavirus vaccines have had a substantial effect on diarrhoeal morbidity and mortality in different geographical areas, although efficacy appears to be higher in high income settings. Due to the segmented RNA genome, the pattern of RVA genotypes in the human population is evolving through interspecies transmission and/or reassortment events for which the vaccine might be less effective in the future. However, despite the relative increase in some particular genotypes after rotavirus vaccine use, the overall efficacy of rotavirus mass vaccination worldwide has not been affected. Some of the challenges to improve the effect of current rotavirus vaccines can be solved in the future by new rotavirus vaccines and by vaccines currently in progress.

Rotavirus gastroenteritis in Pakistan, 2018: updated disease burden.

OBJECTIVE: Rotavirus A (RVA) is a significant cause of severe diarrheal illness and one of the common causes of death in children under the age of five. This study was aimed at detecting the prevalence of RVA in Pakistan after rotavirus vaccines were introduced. Fecal samples were obtained from 813 children from different hospitals in Rawalpindi and Islamabad, Pakistan, from January 2018 to December 2018. To obtain additional information from the parents / guardians of the children, a standard questionnaire was used. RESULTS: Using an enzyme-linked immunosorbent assay kit (ELISA), rotavirus antigen was detected and ELISA positive samples were subjected to reverse transcription PCR (RT-PCR). The findings showed 22% prevalence of RVA in children with acute gastroenteritis (AGE) via ELISA and 21% prevalence via RT-PCR in children with AGE. There was no statistically significant difference between gender, age and RVA infections. The winter, spring and fall/autumn seasons were statistically significant for RVA prevalence. CONCLUSION: The present study will provide post vaccine prevalence data for the health policy makers. The implementation of rotavirus vaccines, along with adequate nutrition for babies, clean water supply and maternal hygienic activities during infant feeding, is recommended. Furthermore, continuous surveillance is mandatory in the whole country to calculate the disease burden caused by RVA.

Magnitude of Rotavirus A and Campylobacter jejuni infections in children with diarrhea in Twin cities of Rawalpindi and Islamabad, Pakistan.

BACKGROUND: Acute diarrhea is a leading cause of morbidity and mortality in children particularly in developing countries of Asia and Africa. The present study was conducted to detect the two most important pathogens, rotavirus and Campylobacter Jejuni in children suffering with diarrhea in Rawalpindi and Islamabad, Pakistan in 2014. The clinical and epidemiological aspects of the disease were also investigated. METHODS: A total of 500 stool samples were collected from children presented with clinical signs and symptoms of acute diarrhea. The samples were initially screened for the presence of rotavirus A (RVA) via ELISA (Enzyme-linked immunosorbent assay) and RT-PCR (Reverse Transcriptase PCR) and then were analysed for C. jejuni by using species specific PCR assay. RESULTS: The detection rate of RVA was 26.4% (132/500) while, Campylobacter was detected in 52% (260/500) of samples with C. jejuni accounted for 48.2% (241/500) of all study cases. Co-infection of C. jejuni with RVA was identified in 21.8% of all cases. Children with RVA and C. jejuni co-infection showed a higher probability (p = 0.01) to be dehydrated. A significant association (p = 0.02) was found between C. jejuni positive status and fever in children. The median age of children with both RVA and C. jejuni infection was 6-11 months. The RVA detection rate was high in winter months of the year while, C. jejuni infections were documented high in summer over 1 year study period. CONCLUSIONS: The overall results have demonstrated the high prevalence of C. jejuni in Rawalpindi, Islamabad, Pakistan in 2014. The results of present study will not only help to calculate disease burden caused by C. jejuni and rotavirus but also will provide critical information to health authorities in planning public health care strategies against these pathogens.

Rotavirus: Genetics, pathogenesis and vaccine advances.

Since its discovery 40 years ago, rotavirus (RV) is considered to be a major cause of infant and childhood morbidity and mortality particularly in developing countries. Nearly every child in the world under 5 years of age is at the risk of RV infection. It is estimated that 90% of RV-associated mortalities occur in developing countries of Africa and Asia. Two live oral vaccines, RotaTeq (RV5, Merck) and Rotarix (RV1, GlaxoSmithKline) have been successfully deployed to scale down the disease burden in Europe and America, but they are less effective in Africa and Asia. In April 2009, the World Health Organization recommended the inclusion of RV vaccination in national immunization programs of all countries with great emphasis in developing countries. To date, 86 countries have included RV vaccines into their national immunization programs including 41 Global Alliance for Vaccines and Immunization eligible countries. The predominant RV genotypes circulating all over the world are G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8], while G12[P6] and G12[P8] are emerging genotypes. On account of the segmented genome, RV shows an enormous genetic diversity that leads to the evolution of new genotypes that can influence the efficacy of current vaccines. The current need is for a global RV surveillance program to monitor the prevalence and antigenic variability of new genotypes to formulate future vaccine development planning. In this review, we will summarize the previous and recent insights into RV structure, classification, and epidemiology and current status of RV vaccination around the globe and will also cover the status of RV research and vaccine policy in Pakistan.

Rotavirus genotype dynamics in Pakistan: G9 and G12 emerging as dominant strains in vaccinated children (2019).

Rotavirus A (RVA) is a leading cause of severe gastroenteritis in children worldwide, and vaccination has become a pivotal strategy to reduce the associated morbidity and mortality. This study presents a molecular characterization of RVA genotypes circulating among vaccinated children in Pakistan during the year 2019. A total of 510 stool samples were collected from children of up to five years of age presenting with acute gastroenteritis symptoms in Rawalpindi, Islamabad regions of Pakistan. The RVA antigen was detected using ELISA on these samples. RVA G/P genotyping was performed on ELISA positive samples using Multiplex semi-nested reverse transcriptase PCR. RVA was found in 130 fecal samples, with an overall prevalence of 25.4 %. G9P[8] (20 %) is the most prevalent genotype, followed by G12P[6] (17 %), G3P[8] (14 %), G1P[8] (12 %), G2P[4] (10 %), G12P[8] (7 %), G9P[6] (7 %), G3P[6] (6 %), G3P[4] (4 %) and G1P[6] (3 %) respectively. There is a statistically significant difference (p < 0.05) found in the group age (in months) of RVA gastroenteritis cases as detected by RT-PCR. The highest number of positive cases was found in the age range from 0 to 6 months, followed by 7-12 months, 13-24 months, and 25-60 months, respectively. Dehydration is statistically significantly associated (p˂ 0.05) in RVA gastroenteritis cases compared to those who tested negative. This study emphasizes the significance of maintaining a continuous surveillance system and conducting genomic analysis of RVA genotypes in children upto the age of 5 years. This is essential for tracking the circulation of RVA genotypes. The results from this research enhance our comprehension of how RVA genotypes are changing over time in Pakistan, underscoring the ongoing necessity for improving vaccine coverage and effectiveness. This, in turn, can help reduce the impact of RVA-related illnesses in children.

Antigenic epitope analysis of Pakistani G3 and G9 rotavirus strains compared to vaccine strains revealed multiple amino acid differences.

Rotaviruses belong to genotype VP4-P[8] are a significant cause of severe loose diarrhea in infants and young children. In the present study, we characterised the complete genome of three of the Pakistani P[8]b RVA strains by Illumina HiSeq sequencing technology to determine the complete genotype constellation providing insight into the evolutionary dynamics of their genes using maximum likelihood analysis. The maximum genomic sequences of our study strains were similar to more recent human Wa-Like G1P[8]a, G3P[8]a, G4P[6], G4P[8], G9P[4], G9P[8]a, G11P[25],G12P[8]a and G12P[6] strains circulating around the world. Therefore, strains PAK274, PAK439 and PAK624 carry natively distinctive VP4 gene with universally common human Wa-Like genetic backbone. Comparing our study P[8]b strains with vaccines strains RotarixTM and RotaTeqTM, multiple amino acid differences were examined between vaccine virus antigenic epitopes and Pakistani isolates. Over time, these differences may result in the selection for strains that will escape the vaccine-induced RVA-neutralizing-antibody effect.

Molecular characterization of plasma virome of hepatocellular carcinoma (HCC) patients.

Hepatocellular carcinoma (HCC) stands as the most common cancer type, arising from various causes, and responsible for a substantial number of cancer-related fatalities. Recent advancements in viral metagenomics have empowered scientists to delve into the intricate diversity of the virosphere, viral evolution, interactions between viruses and their hosts, and the identification of viral causes behind disease outbreaks, the development of specific symptoms, and their potential role in altering the host's physiology. The present study had the objective of "Molecular Characterization of HBV, HCV, anelloviruses, CMV, SENV-D, SENV-H, HEV, and HPV viruses among individuals suffering from HCC." A total of 381 HCC patients contributed 10 cc of blood each for this study. The research encompassed the assessment of tumor markers, followed by molecular characterization of HBV, HCV, Anelloviruses (TTV, TTMV, and TTMDV), SENV-H and SENV-D viruses, HEV, CMV, and HPV, as well as histopathological examinations. The outcomes of this study revealed that majority of the HCC patients 72.4% (276/381) were male as compared to females. HCV infection, at 76.4% (291 out of 381), exhibited a significant association (p < 0.05) with HCC. Most patients displayed singular lesions in the liver, with Child Pugh Score Type B being the predominant finding in 45.2% of cases. Plasma virome analysis indicated the prevalence of TTMDV (75%), followed by TTMV (70%) and TTV (42.1%) among anelloviruses in HCC patients. Similarly, SENV-H (52%) was followed by SENV-D (20%), with co-infections at 15%. The presence of CMV and HEV among the HCC patients was recorded 5% each however 3.5% of the patients showed the presence of HPV. In conclusion, this study underscores that HCC patients serve as reservoirs for various pathogenic and non-pathogenic viruses, potentially contributing to the development, progression, and severity of the disease.

Rotavirus in developing countries: molecular diversity, epidemiological insights, and strategies for effective vaccination.

Rotavirus (RV) causes the loss of numerous children's lives worldwide each year, and this burden is particularly heavy in low- and lower-middle-income countries where access to healthcare is limited. RV epidemiology exhibits a diverse range of genotypes, which can vary in prevalence and impact across different regions. The human genotypes that are most commonly recognized are G1P[8], G2P[4], G3P[8], G4P[8], G8P[8], G9P[8], and G12P[8]. The diversity of rotavirus genotypes presents a challenge in understanding its global distribution and developing effective vaccines. Oral, live-attenuated rotavirus vaccines have undergone evaluation in various contexts, encompassing both low-income and high-income populations, demonstrating their safety and effectiveness. Rotavirus vaccines have been introduced and implemented in over 120 countries, offering an opportunity to assess their effectiveness in diverse settings. However, these vaccines were less effective in areas with more rotavirus-related deaths and lower economic status compared to wealthier regions with fewer rotavirus-related deaths. Despite their lower efficacy, rotavirus vaccines significantly decrease the occurrence of diarrheal diseases and related mortality. They also prove to be cost-effective in regions with a high burden of such diseases. Regularly evaluating the impact, influence, and cost-effectiveness of rotavirus vaccines, especially the newly approved ones for worldwide use, is essential for deciding if these vaccines should be introduced in countries. This is especially important in places with limited resources to determine if a switch to a different vaccine is necessary. Future research in rotavirus epidemiology should focus on a comprehensive understanding of genotype diversity and its implications for vaccine effectiveness. It is crucial to monitor shifts in genotype prevalence and their association with disease severity, especially in high-risk populations. Policymakers should invest in robust surveillance systems to monitor rotavirus genotypes. This data can guide vaccine development and public health interventions. International collaboration and data sharing are vital to understand genotype diversity on a global scale and facilitate the development of more effective vaccines.

Liver Disease and Sickle Cell Disease: Auto-Immune Hepatitis more than a Coincidence; A Systematic Review of the Literature.

In patients with SCD, chronic liver damage is a common manifestation. More than 50% of SCD patients have elevated liver enzymes. Common underlying aetiologies include sickle cell hepatic crisis, viral hepatitis, sickle cell intrahepatic cholestasis and hepatic sequestration in the acute setting, and cholelithiasis and iron overload in the chronic setting. Autoimmune hepatitis (AIH) is a rare disease that appears to occur more commonly in the sickle cell disease (SCD) population than in the general population. There are many schools of thought as to why this is the case, including the phosphatidylserine hypothesis, the heme inflammatory hypothesis, the complement generation hypothesis, and the transfusion alloimmunization hypothesis. Due to the natural history of the two illnesses, SCD is almost always diagnosed first in cases of dual pathology. Symptoms such as jaundice, fatigue, and abdominal pain are common in SCD, as are abnormal liver function tests (LFTs). These abnormalities, attributed to the other more frequent liver involvements in SCD, can lead to delays in AIH diagnosis in this population. Corticosteroids, sometimes with other immunosuppressive agents, such as azathioprine, are the cornerstone of acute AIH treatment. However, corticosteroid use in the SCD population has been shown to carry an increased risk of vaso-occlusive crises, providing a treatment dilemma. The following is a review of AIH in the SCD population, where we explore the pathophysiology behind the association between the two disorders, discuss an approach to investigating abnormal LFTs in SCD, and examine treatment options in this population with co-existing diseases.

Evolutionary Dynamics of Foot and Mouth Disease Virus Serotype A and Its Endemic Sub-Lineage A/ASIA/Iran-05/SIS-13 in Pakistan.

Foot and mouth disease (FMD) causes severe economic losses to the livestock industry of endemic countries, including Pakistan. Pakistan is part of the endemic pool 3 for foot and mouth disease viruses (FMDV), characterized by co-circulating O, A, and Asia 1 serotypes, as designated by the world reference laboratory for FMD (WRL-FMD). FMDV serotype A lineage ASIA/Iran-05 is widespread in buffalos and cattle populations and was first reported in Pakistan in 2006. This lineage has a high turnover, with as many as 10 sub-lineages reported from Pakistan over the years. In this study, we reconstructed the evolutionary, demographic, and spatial history of serotype A and one of its sub-lineages, A/ASIA/Iran-05/SIS-13, prevalent in Pakistan. We sequenced nearly complete genomes of three isolates belonging to sub-lineage A/ASIA/Iran-05/SIS-13. We estimated recombination patterns and natural selection acting on the serotype A genomes. Source and transmission routes in Pakistan were inferred, and the clustering pattern of isolates of the SIS-13 sub-lineage were mapped on a tree. We hereby report nearly complete genome sequences of isolates belonging to sub-lineage A/ASIA/Iran-05/SIS-13, along with purported recombinant genomes, and highlight that complete coding sequences can better elucidate the endemic history and evolutionary pressures acting on long-term co-circulating FMDV strains.

Seroprevalence of Anti-tTg-IgA among Symptomized Celiac Disease Patients and Their Correlation with Rotavirus Infection.

Celiac disease (CD) is a chronic inflammatory disorder in the intestinal tract as a response to the use of gluten in genetically predisposed individuals. It is a worldwide problem, with a high prevalence rate in North America. This is a descriptive cross-sectional study involving 1090 samples collected from different hospitals of Rawalpindi and Islamabad, Pakistan, from January 2019 to December 2019. In this study, 1090 blood samples screened for seroprevalence of anti-tTG antibodies in CD symptomatic patients via ELISA (enzyme-linked immunosorbent assay). 1090 fecal samples from the same CD patients were collected and tested for the presence of rotavirus (RV) via ELISA and RT-PCR. Of the 1090 patients tested for seroprevalence of anti-tTG antibodies, 112/1090 (10.3%) were found to be positive. Of the 112 anti-tTG-positive patients, 78/112 (70%) were positive for RV via ELISA and 74/112 (66.1%) were RV positive via RT-PCR. A statistically significant association was reported between rotavirus infection and celiac disease (p˂0.05). Anti-tTG antibodies were higher in age group 6 (12-18 years) patients (18.4%) and at minimum in age group 3 (1-3 years) patients (4.8%). However, there was a statistically insignificant relationship between group age and CD prevalence (p > 0.05). The highest CD prevalence was noted during winter season (19.6%) and the lowest (3.0%) during fall/autumn. Our study findings demonstrate that Pakistan has a high prevalence of CD compared to other studies. Further studies in the fields of environmental risk factors and treatment with more advanced serological and histopathological studies are needed in the future.

Whole genome analysis of Aichivirus A, isolated from a child, suffering from gastroenteritis, in Pakistan.

Viruses are the primary cause of acute gastroenteritis in children all over the world. Understanding the emergence and genetic variation of these viruses may help to prevent infections. Aichivirus (AiV) is a member of the Kobuvirus genus, which currently contains six officially recognized species: Aichivirus A-F. The species AiV A contains six types including Aichivirus 1 (AiV 1) and eventually, three genotypes have been identified in the human AiV 1 (named A to C). The present study describes the identification and sequencing of the polyprotein gene of a human AiV 1 strain PAK419 via NGS in Pakistani children with acute gastroenteritis. Our study strain PAK419 was classified as AiV 1 genotype A, most commonly found in Japan and Europe, and closely related to non-Japanese and European strains on the phylogenetic tree. PAK419 showed 95-98 % nucleotide sequence identity with strains isolated from Ethiopia (ETH/2016/P4), Australia (FSS693) and China (Chshc7). On phylogenetic observation PAK419 formed a distinct cluster in the AiV 1 genotype A with the above mentioned and other human AiV strains detected around the world (Germany, Brazil, Japan, Thailand, Korea and Vietnam). The data clearly showed that Pakistani AiV strains and human strains identified from all over the world are distinct from Aichivirus strains found in bovine, swine, canine, feline, caprine, ferret, bat, and environmental samples. The distinguishing characteristics of the AiV genome showed a lower probability of inter-genotypic recombination events, which may support the lack of AiV serotypes. PAK419 also had a high content of C nucleotide (37.4 %), as found in previous studies, which could also restrict the possible genetic variation of AiV. This study demonstrate the power of NGS in uncovering unknown gastroenteric etiological agents circulating in the population.

Molecular characterization of the complete genome sequence of human Parechovirus 1 in Pakistan.

Human parechoviruses (HPeVs) are highly common pathogens in children under 2 years of age. Of the 19 distinct HPeV genotypes identified worldwide, HPeV1 is still the most prevalent type associated with respiratory and gastrointestinal symptoms in infants and young children. Pakistan's previous studies have focused only on the detection and partial sequencing of HPeV genotypes. In the present study, we have obtained the complete genomes of 2 HPeV1 strains (PAK419 and PAK663) from children using NGS method on Illumina Hiseq Platform. These samples were collected from children suffering from acute gastroenteritis in Rawalpindi, Pakistan during 2016. The near complete genome sequences obtained for two HPeV1 strains (PAK419 and PAK663) consist of total 6877 nucleotides with a single, large open reading frame (ORF) encoding a polyprotein gene. Phylogenetic analysis showed that both HPeV1 strains exhibited maximum amino acid similarity (97 %) to HPeV1 strains from The Nederlands (2007-863, GQ183034) and clustered closely with this and with other HPeV1 strains isolated from other countries in the world (Ethiopia, Taiwan, Russia and Brazil). A motif of arginine-glycine-aspartic acid (RGD) in the VP1 (Outer capsid protein) C-terminus region that is suggested to help virus entry into the host cell also identified in PAK419 and PAK663. SimPlot analysis revealed that intergenotypic recombination events may have take place in the non-structural region between both HPeV1 strains (PAK419, PAK663), two major strains of HPeV1 (GQ183034 and MG873157) and four minor strains of HPeV4 (AM235750), HPeV7 (EU556224), HPeV15 (MN265386) and HPeV18 (KT879915). The full genome of HPeV1 strains characterized in the current study will provide complete information on these newly isolated strains for further preventive or treatment measures.

Corrigendum: Comparative Analysis of G1P[8] Rotaviruses Identified Prior to Vaccine Implementation in Pakistan With Rotarix™ and RotaTeq™ Vaccine Strains.

[This corrects the article DOI: 10.3389/fimmu.2020.562282.].

Comparative Analysis of G1P[8] Rotaviruses Identified Prior to Vaccine Implementation in Pakistan With Rotarix™ and RotaTeq™ Vaccine Strains.

Group A rotavirus (RVA) is the leading cause of severe childhood diarrhea globally, even with all effective interventions, particularly in developing countries. Among the diverse genotypes of RVA, G1P[8] is a common genotype that has continued to pervade around the world, including Pakistan. Two universally accepted rotavirus vaccines-Rotarix™ and RotaTeq™ contain the genotype G1P[8]. The current work was aimed at identifying differences between antigenic epitopes of Pakistan's G1P[8] strains and those of the two licensed vaccines. We sequenced 6 G1P[8] rotavirus strains previously reported in Rawalpindi, Islamabad, Pakistan in 2015 and 2016 for their outer capsid genes (VP7 and VP4). Phylogenetic analysis was then conducted in order to classify their specific lineages and to detect their association with strains isolated throughout world. Compared with the Rotarix™ and RotaTeq™ vaccine strains (G1-lineage II, P[8]-lineage III), our study G1-lineage I, P[8]-lineage IV strains showed 3 and 5 variations in the VP7 epitopes, respectively, and 13 and 11 variations in the VP4 epitopes, respectively. The G1 lineage II strains showed no single amino acid change compared to Rotarix™ (lineage II), but exhibited changes at 2 positions compared to RotaTeq™ (lineage III). So, this has been proposed that these G1 strains exist in our natural setting, or that they may have been introduced in Pakistan from other countries of the world. The distinct P[8]-lineage IV (OP354-like) strains showed twelve and thirteen amino acid variations, with Rotarix™ and RotaTeq™ (lineages II and III) strains, respectively. Such findings have shown that the VP4-P[8] component of the G1P[8] strains circulating in Pakistan differs considerably from that of the vaccine viruses compared to that of the VP7-G1. To monitor the long-term effects of vaccines on the emergence of G1P[8] strains with different lineages, routine and successful monitoring of these strains will be crucial.

Methicillin-Resistant Staphylococcus aureus (MRSA) in Slaughter Houses and Meat Shops in Capital Territory of Pakistan During 2018-2019.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is a major concern in many parts of the world, including Pakistan. The aim of this study was to investigate the prevalence of MRSA in slaughterhouses and meat shops in Rawalpindi-Islamabad, Pakistan, 2018-2019. A total of 300 samples were collected: 40 from each of working area, tools (knives, hooks), butcher hands and beef, 30 from each of chicken and mutton, 20 from each of nasal and rectal swabs. S. aureus was phenotypically identified by performing gram staining and biochemical tests. 150 of the 300 samples were confirmed to be S. aureus by phenotypic identification. MRSA was identified among S. aureus positive isolates by performing disk diffusion test and by detecting S. aureus-specific genes such as 16s rRNA, nuc, mecA, spa, and coa. Out of 150 isolates 96 (63%) showed resistance to antibiotic cefoxitin, known as a potential marker for detecting MRSA. While all 150 isolates have shown complete resistance to the four antibiotics neomycin, methicillin, ciprofloxacin and tetracycline. The nuc and 16s rRNA genes were detected in all 150 S. aureus-positive isolates and 118 (79%) were confirmed to be MRSA through the detection of the mecA gene. MRSA prevalence was highest in chicken (23/30, 77%) followed by beef (25/40, 63%), mutton (15/30, 50%), knives (18/40, 45%), nasal swabs (7/20, 35%), working area (11/40, 28%), rectal swabs (5/20, 25%), hooks (7/40, 18%), and butcher hands (7/40, 18%). 50 MRSA-positive isolates were chosen to identify two virulence factors (spa and coa gene). Of the 50 MRSA isolates subject to coa and spa gene typing, 27 (54%) were positive for the coa gene and 18 (36%) were positive for the spa gene, respectively. To the best of our knowledge, this was the first study on the molecular identification of MRSA in meat samples from Pakistan. High prevalence of MRSA in meat samples demand for implementation of proper hygienic practices and procedures during the slaughtering, transport and marketing of meat and meat products in order to prevent the spread of these bacteria to the human population.

G3 and G9 Rotavirus genotypes in waste water circulation from two major metropolitan cities of Pakistan.

Rotavirus A (RVA) is a diarrheal pathogen affecting children under age five, particularly in developing and underdeveloped regions of the world due to malnutrition, poor healthcare and hygienic conditions. Water and food contamination are found to be major sources of diarrheal outbreaks. Pakistan is one of the countries with high RVA related diarrhea burden but with insufficient surveillance system. The aim of this study was to gauge the RVA contamination of major open sewerage collecting streams and household water supplies in two major metropolitan cities of Pakistan. Three concentration methods were compared using RNA purity and concentration as parameters, and detection efficiency of the selected method was estimated. Water samples were collected from 21 sites in Islamabad and Rawalpindi in two phases during the year 2014-2015. Meteorological conditions were recorded for each sampling day and site from Pakistan Meteorological Department (PMD). Nested PCR was used to detect the presence of RVA in samples targeting the VP7 gene. Logistic regression was applied to assess the association of weather conditions with RVA persistence in water bodies. Statistical analysis hinted at a temporal and seasonal pattern of RVA detection in water. Phylogenetic analysis of selected isolates showed a close association of environmental strains with clinical RVA isolates from hospitalized children with acute diarrhea during the same period. This is the first scientific report cataloging the circulating RVA strains in environmental samples from the region. The study highlights the hazards of releasing untreated sewerage containing potentially infectious viral particles into collecting streams, which could become a reservoir of multiple pathogens and a risk to exposed communities. Moreover, routine testing of these water bodies can present an effective surveillance system of circulating viral strains in the population.

Whole Genome Analysis of Selected Human Group A Rotavirus Strains Revealed Evolution of DS-1-Like Single- and Double-Gene Reassortant Rotavirus Strains in Pakistan During 2015-2016.

Acute gastroenteritis due to group A rotaviruses (RVAs) is the leading cause of infant and childhood morbidity and mortality particularly in developing countries including Pakistan. In this study we have characterized the whole genomes of five RVA strains (PAK56, PAK419, PAK585, PAK622, and PAK663) using the Illumina HiSeq platform. The strains PAK56 and PAK622 exhibited a typical Wa-like genotype constellation (G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 and G3-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, respectively), whereas PAK419, PAK585, and PAK663 exhibited distinct DS-1-like genotype constellations (G3P[4]-I2-R2-C2-M2-A2-N2-T1-E2-H2, G1P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2, and G3P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2, respectively). Despite their DS-1-like genotype constellation, strain PAK585 possessed the typical Wa-like G1P[8] genotypes, whereas both PAK419 and PAK663 possessed the G3 genotype. In addition, PAK419 also possessed the Wa-like NSP3 genotype T1, suggesting that multiple reassortments have occurred. On Phylogenetic analysis, all of the gene segments of the five strains examined in this study were genetically related to globally circulating human G1, G2, G3, G6, G8, G9, and G12 strains. Interestingly, the NSP2 gene of strain PAK419 showed closest relationship with Indian bovine strain (India/HR/B91), suggesting the occurrence of reassortment between human and bovine RVA strains. Furthermore, strains PAK419, PAK585, and PAK663 were closely related to one another in most of their gene segments, indicating that these strains might have been derived from a common ancestor. To our knowledge this is the first whole genome-based molecular characterization of human rotavirus strains in Pakistan. The results of our study will enhance our existing knowledge on the diversity and evolutionary dynamics of novel RVA strains including DS-1-like intergenogroup reassortant strains spreading in Asian countries including Pakistan, in the pre-vaccine era. Therefore, continuous surveillance is recommended to monitor the evolution, spread and genetic stability of novel reassortant rotavirus strains derived from such events.

Molecular characterization of human group A rotavirus genotypes circulating in Rawalpindi, Islamabad, Pakistan during 2015-2016.

Group A rotaviruses (RVA) are one of the major causes of acute gastroenteritis (AGE) in young children worldwide. Owing to lack of proper surveillance programs and health facilities, developing countries of Asia and Africa carry a disproportionately heavy share of the RVA disease burden. The aim of this hospital-based study was to investigate the circulation of RVA genotypes in Rawalpindi and Islamabad, Pakistan in 2015 and 2016, prior to the implementation of RVA vaccine. 639 faecal samples collected from children under 10 years of age hospitalized with AGE were tested for RVA antigen by ELISA. Among 171 ELISA positive samples, 143 were successfully screened for RT-PCR and sequencing. The prevalence of RVA was found to be 26.8% with the highest frequency (34.9%) found among children of age group 6-11 months. The most predominant circulating genotypes were G3P[8] (22.4%) followed by G12P[6] (20.3%), G2P[4] (12.6%), G1P[8] (11.9%), G9P[6] (11.9%), G3P[4] (9.1%), G1P[6] (4.2%), G9P[8] (4.2%), and G3P[6] (0.7%). A single mixed genotype G1G3P[8] was also detected. The findings of this study provide baseline data, that will help to assess if future vaccination campaigns using currently available RVA vaccine will reduce RVA disease burden and instigate evolutionary changes in the overall RVA biology. The high prevalence of RVA infections in Pakistan require to improve and strengthen the surveillance and monitoring system for RVA. This will provide useful information for health authorities in planning public health care strategies to mitigate the disease burden caused by RVA.

Analytical Method for the Identification and Assay of Kojic Acid, Methylparaben, and Propylparaben in Cosmetic Products Using UPLC: Application of ISO 12787:2011 Standard.

A straightforward, fast UPLC method is developed for the identification and quantification of kojic acid (KA), methylparaben (MP), and propylparaben (PP) in 15 cosmetic products (skin whitening creams and lotions). Chromatographic separations for KA, MP, and PP were obtained in 3.5 min on an Acquity BEH-C18 column (100 × 2.1 mm, 1.7 µm particle size) as the stationary phase at 260 nm (diode-array detector), with the mobile phase comprising a mixture of 0.01 M dibasic potassium phosphate and methanol-acetonitrile (50 + 50). Validation studies were performed according to in-house established criteria. There was a linear function of concentrations over the range of 0.4-1.6 µg/mL for KA, MP, and PP. The LOQ for all components was 0.2 µg/mL using the S/N method. Good separation of analytes was observed, with acceptable values of resolution and tailing. The analytical approach defined in the ISO 12787:2011 standard ("Cosmetics-Analytical methods-Validation criteria for analytical results using chromatographic techniques") was used for the assay of cosmetic samples.