A Newcastle disease live virus vaccine is safe and efficacious at various storage conditions.

Pure, potent and efficacious vaccines could help in the control of Newcastle disease (ND). The present study was designed to evaluate the thermo-stability of a live-attenuated ND virus vaccine containing the Mukteswar strain and to genetically characterize the seed virus. Moreover, the presence of extraneous agents (Fowl adenovirus, Mycoplasma, Salmonella Pullorum, and Salmonella Gallinarum) was assessed using polymerase chain reactions (PCR) optimized for detection in a panel. The vaccine was evaluated for its potency and efficacy after storage at 4°C, 25°C and 37°C for 36, 48, 96 and 144 hours. A total of 100 commercial broiler chickens were randomly divided into six groups and immunized with the vaccine stored at specified temperatures for the given times. Blood samples were collected on days 0, 7, 14, 21 and 28 post-vaccination, sera were separated and antibody titers were assessed using hemagglutination inhibition (HI) assay. The data were analyzed by two-way analysis of variance (ANOVA) and multivariate analysis of variance (MANOVA). Reverse-transcription  PCR targeting the F gene of Newcastle disease virus (NDV) and subsequent sequence analysis confirmed the presence of NDV in the vaccine seed (deposited to GenBank Acc. Nos. MK310260 and MK310261). Phylogenetic analysis revealed a close resemblance of the vaccine virus with other Avian Avulaviruses (NDV class II Genotype III viruses and more specifically with NDV Mukteswar vaccine strains), yet it was distinct from NDV class II Pakistani field isolates, which grouped into genotype XIII.2.1. The PCR testing confirmed that the vaccine was free from extraneous agents. The present study's findings propose an alternative rapid PCR-based method to evaluate the purity of NDV live vaccines. Together these data suggest that the tested vaccine is pure, potent and efficacious, yet continuous maintenance of the cold chain for vaccine storage is recommended to maintain its potency and efficacy.

A retrospective analysis of tuberculosis in livestock farmers in Lahore district, Pakistan.

INTRODUCTION: Mycobacterium tuberculosis is the main cause of tuberculosis in humans, accounting for numerous illnesses and thousands of fatalities globally. Data regarding the association of various risk factors and TB in livestock farmers in Pakistan is scarce. METHODOLOGY: A retrospective matched case-control study of TB cases was performed in Lahore, Pakistan to investigate the potential risk factors that lead to the development of TB in Pakistani livestock farmers. A total of 170 participants were included in the study. The case was matched with control based on neighborhood and the case-control ratio was kept 1:1. Data were statistically analyzed using R version 4.2.1. Conditional logistic regression was conducted to identify biologically and statistically plausible risk factors associated with the TB outcome among livestock farmers. RESULTS: In univariable analysis, 10 risk factors were identified (p < 0.05). Gender, age, being married, family type, living in a big family, BCG vaccination status, history of smoking, working at a cattle farm, co-housing with cattle at night, , consumption of raw milk. The multivariable model identified four risk factors i.e., consumption of raw milk (Odds Ratio [OR]: 7.7; 95% Confidence Interval [CI]: 1.95-30.68), living in big family (OR: 6.2; 95% CI: 1.25-30.82) and working at cattle farm (OR: 4.2; 95% CI: 1.08-16.56), while gender was found to be a protective factor with OR < 1 (OR: 0.06; 95% CI: 0.01-0.26). CONCLUSIONS: This study demonstrated that sociodemographic risk factors and exposure to infected cattle can influence the development of TB in farmers.

Risk factors associated with influenza A (H1N1)pdm09: a nested case control study of TB patients with ILI in Lahore District, Pakistan.

BACKGROUND: Co-morbidity with respiratory viruses including influenza A, cause varying degree of morbidity especially in TB patients compared to general population. This study estimates the risk factors associated with influenza A (H1N1)pdm09 in TB patients with ILI. METHODS: A cohort of tuberculosis (TB) patients who were admitted to and enrolled in a TB Directly Observed Therapy Program (DOTs) in tertiary care hospitals of Lahore (Mayo Hospital and Infectious Disease Hospital) were followed for 12 weeks. At the start of study period, to record influenza-like illness (ILI), a symptom card was provided to all the participants. Every participant was contacted once a week, in person. When the symptoms were reported by the participant, a throat swab was taken for the detection of influenza A (H1N1)pdm09. A nested case control study was conducted and TB patients with ILI diagnosed with influenza A (H1N1)pdm09 by conventional RT-PCR were selected as cases, while those who tested negative by conventional RT-PCR were enrolled as controls. All cases and controls in the study were interviewed face-to-face in the local language. Epidemiological data about potential risk factors were collected on a predesigned questionnaire. Logistic analysis was conducted to identify associated risk factors in TB patients with ILI. RESULTS: From the main cohort of TB patients (n = 152) who were followed during the study period, 59 (39%) developed ILI symptoms; of them, 39 tested positive for influenza A (H1N1)pdm09, while 20 were detected negative for influenza A (H1N1)pdm09. In univariable analysis, four factors were identified as risk factors (p < 0.05). The final multivariable model identified one risk factor (sharing of towels, P = 0.008)) and one protective factor (wearing a face mask, p = < 0.001)) for influenza A (H1N1)pdm09 infection. CONCLUSION: The current study identified the risk factors of influenza A (H1N1)pdm09 infection among TB patients with ILI.

Burden and factors associated with occupational tuberculosis infection among high-risk workers in Lahore District, Pakistan.

INTRODUCTION: The main objective of the study was to estimate the burden of occupational tuberculosis infection in high-risk occupational workers and to identify risk factors associated with the prevalence of Mycobacterium tuberculosis complex (MTBC). METHODOLOGY: An analytical cross-sectional study was conducted among high-risk occupational workers including veterinarians, abattoir workers, animal handlers, livestock farmers, and microbiology laboratory workers. Sputum samples were collected from 100 participants and polymerase chain reaction (PCR) tests were done to diagnose tuberculosis (TB) infection. Data on potential risk factors was collected in a pre-designed questionnaire. The MTBC prevalence ratio was estimated. Logistic regression analysis was conducted to identify risk factors and the crude odds ratio (OR) was calculated. RESULTS: Among the 100 enrolled high risk occupational workers, the prevalence of MTBC was 46% (95% CI: 35.98-56.25). Living in a joint family (OR 3.85, 95% CI: 1.58-9.37), and use of unpasteurized milk (OR 3.42, 95% CI: 1.4-8.39), were significantly associated with MTBC infection. CONCLUSIONS: Tuberculosis is a significant health burden in high-risk occupational groups, especially animal handlers and laboratory workers, in Lahore, Pakistan. The study also emphasized the need for formal work-related training, and enhanced zoonotic TB awareness among occupational workers.

Milk microbiota of Holstein Friesian cattle reared in Lahore: Association with mastitis.

The dairy industry is reshaping itself and becoming commercialized in Pakistan due to the increased demand for milk to overcome the shortage. Exotic breeds such as Holstein Friesian, a high milk producing breed have started being reared more on farms in Pakistan. Along with other issues, mastitis does affects the milk production of this breed. The objective of this study was to evaluate the milk composition in terms of bacterial communities in Holstein Friesian reared in Punjab, Pakistan and alteration in microbial composition with healthy and mastitic udder. Milk samples (n = 36) from farms rearing Holstein Friesian were collected. Among these samples, 05 samples from each three groups, HHF(healthy), CHF (clinical mastitis) and SHF (subclinical mastitis), based on their udder health condition, were processed using the 16 S r=RNA gene based technique. Diversity assessment as carried out by alpha diversity indices showed that milk samples from the udder infected with clinical mastitis were the least diverse and those from the healthy udder were more diverse. Beta diversity across samples showed a scattered pattern suggesting overlap amongst bacterial communities across different groups samples as depicted by PCA plots of beta diversity indices. The taxonomic profile revealed that Proteobacteria Firmicutes, Bacteroidota and Actinobacteriota were the major phyla detected across all groups. Proteobacteria dominated the HHF and SHF group while abundance of Firmicutes was higher in CHF group. Differences at other levels including order, genus and species were also recorded. The overall picture concludes that diverse microbiota is associated with different udder health conditions.

Investigation of milk microbiota of healthy and mastitic Sahiwal cattle.

BACKGROUND: Sahiwal cattle is an indigenous cattle breed of Pakistan and mastitis is one of the major problems faced by Sahiwal cattle which hinders its production potential. The study was designed to investigate the milk microbiota of healthy and mastitic Sahiwal cattle as part of a multistep project to develop probiotics for the mitigation and control of mastitis. Milk samples of Sahiwal cattle (healthy clinical mastitis and subclinical mastitis) reared under similar husbandry and management practices were processed for 16S rRNA gene base metagenomics analysis. RESULTS: Results revealed that Proteobacteria were dominant in the healthy group and subclinical mastitis group (56.48% and 48.77%, respectively) as compared to the clinical mastitis group (2.68%). In contrast, Firmicutes were abundant in the clinical mastitis group (64%) as compared to the healthy and subclinical mastitis groups (15.87% and 38.98%, respectively). Dominant species assigned in the healthy group were Ignavibacterium album, Novosphingobium capsulatum, Akkermansia muciniphila and Lactobacillus fermentum.The clinical mastitis group was dominated by Streptococcus dysgalactiae and Corynebacterium bovis, while subclinical mastitis group included Lactobacillus fermentum and uncultured acidobacteriales and Akkermansia muciniphila as dominant species. Alpha diversity indices showed higher microbial diversity in the healthy group compared to the clinical and sub-clinical mastitis groups. CONCLUSION: It is concluded that the milk microbiota of healthy sahiwal cattle has higher diversity and dominant taxa in the different groups may be used as signature microbes for mastitis susceptibility. Akkermansia muciniphila is one of candidate specie that was identified and may be used for development of probiotics.

Exploring the Milk Microbiota of Healthy and Mastitic Nili Ravi Buffalo Using 16S rRNA Gene Base Metagenomic Analysis.

The Nili Ravi, a buffalo breed from Pakistan, significantly contributes to the dairy industry. Mastitis is one of the major challenges affecting milk production in this breed. The objective of the current study was to identify the bacterial communities and diversity in healthy and mastitic milk of this breed. Milk samples (n = 14) were collected from Nili Ravi buffaloes with different udder health statuses, i.e., healthy (5), subclinical mastitis (4), and clinical mastitis (5). The DNAs were extracted, subjected to partial amplification of 16S rDNA (V3 and V4 regions), and sequenced using the Illumina platform. The results revealed variations in the bacterial communities in the milk of animals with different udder health statuses. Proteobacteria was the predominant phylum in the healthy group, while clinical and subclinical mastitis milk had a higher abundance of Firmicutes. Dominant bacterial genera in the healthy group were Streptococcus (11.60%), Herbaspirillum (7.65%), and Staphylococcus (4.70%), whereas the clinical mastitis group was dominated by Streptococcus (33.96%), Staphylococcus (7.87%), and Corynebacterium (2.68%), and the subclinical mastitis group was dominated by Bacillus (15.70%), Corynebacterium (6.70%), and Staphylococcus (6.58%). Assignment of operational taxonomic units at the species level resulted in most species being assigned to uncultured or unknown bacteria or remaining unassigned. Alpha diversity indices indicated lower microbial diversity in the clinical mastitis group, while beta diversity indices showed a scattered pattern of sample clustering in PCA plots among different groups. It is concluded that bacterial diversity in the milk of Nili Ravi buffaloes suffering from clinical mastitis is lower compared to healthy and subclinical mastitis cases. It is concluded that the variations in the microbiota of healthy and mastitic milk may be further investigated and exploited as signature microbes associated with the udder health status of Nili Ravi buffalo.

Genomic Diversity and Evolution of SARS-CoV-2 Lineages in Pakistan.

The emergence of SARS-CoV-2 variants has posed a challenge to disease control efforts worldwide. This study explored the genomic diversity and phylogenetic relationship of SARS-CoV-2 variants reported in Pakistan. Our objective was to understand the transmission dynamics of different lineages within the country. We retrieved and analyzed spike protein sequences from Pakistan and compared them with reference sequences reported worldwide. Our analysis revealed the clustering of Pakistan-origin isolates in nine different clades representing different regions worldwide, suggesting the transmission of multiple lineages within the country. We found 96 PANGO lineages of SARS-CoV-2 in Pakistan, and 64 of these corresponded to 4 WHO-designated variants: Alpha, Beta, Delta, and Omicron. The most dominant variants in Pakistan were Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2, AY.108), and Omicron (BA.2.75, BA.5.2), and the N-terminal domain and receptor binding regions were the most hypervariable regions of the spike gene. Compared to the reference strain, characteristic substitutions were found in dominant variants. Our findings emphasize the importance of continuously monitoring and assessing nucleotide and residue substitutions over time to understand virus evolutionary trends better and devise effective disease control interventions.

In Vitro Characterization of Probiotic Potential of Limosilactobacillus fermentum against Salmonella Gallinarum Causing Fowl Typhoid.

Fowl typhoid, a septicaemic disease of poultry, is caused by Salmonella Gallinarum and leads to severe economic losses. The aim of the present study was to isolate, select and characterize indigenous probiotic lactobacilli with anti-Salmonella Gallinarum activity. A total 55 lactobacilli were isolated from the caeca and ileum parts of healthy chickens and identified to species level by 16S rDNA sequencing. All the isolates were initially screened for antimicrobial activity and selected isolates were further subjected to in vitro evaluation of probiotic properties. Lactobacilli isolates (n = 21) showed varying levels of activity (08-18 mm) against Salmonella Gallinarum. These selected isolates also showed tolerance to acidic conditions (pH 3 and 4). Out of these 21 isolates, 13 showed growth (>0.5 OD at 600 nm) 0.3% bile salts. Moreover, these isolates also had the ability of auto-aggregation (20.05 ± 0.62%-50.70 ± 1.40%), and co-aggregation with Salmonella Gallinarum (5.22 ± 0.21%-42.07 ± 0.70%). Results revealed that lactobacilli had a higher level of resistance to vancomycin (100%), streptomycin (100%), ciprofloxacin (95%), gentamicin (90%), doxycycline (90%), oxytetracycline (85%), and bacitracin (80%), and a lower level of resistance to penicillin (33%), erythromycin (28%), chloramphenicol (23%), fusidic acid (23%) and amoxicillin (4%). The Limosilactobacillus fermentum PC-10 and PC-76 were sensitive to most of the antibiotics. The overall results revealed that two Limosilactobacillus fermentum strains (PC-10 and PC-76) fulfill the in vitro selection criteria of probiotics, i.e, tolerance to low pH, resistance to bile salts, auto-aggregation, co-aggregation with Salmonella Gallinarum, and absence of acquired antibiotic resistance. The Limosilactobacillus fermentum PC-10 and PC-76 also inhibited the (>5 log10) growth of Salmonella Gallinarum in co-culture assay. It is concluded that Limosilactobacillus fermentum PC-10 and PC-76 may be further investigated and developed as anti-Salmonella Gallinarum probiotics for poultry.

Molecular Characterization and Selection of Indigenous SARS-CoV-2 Delta Variant for the Development of the First Inactivated SARS-CoV-2 Vaccine of Pakistan.

Vaccines are one of the efficient means available so far for preventing and controlling the infection rate of COVID-19. Several researchers have focused on the whole virus's (SARS-CoV-2) inactivated vaccines which are economically efficient to produce. In Pakistan, multiple variants of SARS-CoV-2 have been reported since the start of the pandemic in February 2020. Due to the continuous evolution of the virus and economic recessions, the present study was designed to develop an indigenous inactivated SARS-CoV-2 vaccine that might help not only to prevent the COVID-19 in Pakistan, it will also save the country's economic resources. The SARS-CoV-2 were isolated and characterized using the Vero-E6 cell culture system. The seed selection was carried out using cross-neutralization assay and phylogenetic analysis. The selected isolate of SARS-CoV-2 (hCoV-19/Pakistan/UHSPK3-UVAS268/2021) was inactivated using beta-propiolactone followed by vaccine formulation using Alum adjuvant, keeping the S protein concentration as 5 µg/dose. The vaccine efficacy was evaluated by in vivo immunogenicity testing in laboratory animals and in in vitro microneutralization test. The phylogenetic analysis revealed that all the SARS-CoV-2 isolates reported from Pakistan nested into different clades, representing multiple introductions of the virus into Pakistan. The antisera raised against various isolates from different waves in Pakistan showed a varied level of neutralization titers. However, the antisera produced against a variant (hCoV-19/Pakistan/UHSPK3-UVAS268/2021; fourth wave) efficiently neutralized (1:64-1:512) all the tested SARS-CoV-2 isolates. The inactivated whole virus vaccine of SARS-CoV-2 was safe and it also elicited a protective immune response in rabbits and rhesus macaques on the 35th-day post-vaccination. The activity of neutralizing antibodies of vaccinated animals was found at 1:256-1:1024 at 35 days post-vaccination, indicating the effectiveness of the double-dose regime of the indigenous SARS-CoV-2 vaccine.

Assessment of aflatoxin M1 exposure and associated determinants in children from Lahore, Pakistan.

Aflatoxins are potent carcinogenic and immunomodulatory mycotoxins, and exposure may lead to deleterious effects on human health. This study aimed to detect aflatoxin M1 (AFM1) as biomarker of exposure and determine associated risk factors in children attending a specialized-childcare hospital in Lahore. Urine samples collected from 238 children (1-11 years) during winter (January-mid-March 2020) and hot-humid summer (August-September 2020) were tested for AFM1 presence using ELISA. Data on potential risk factors were also collected. Of 238 samples, 156 (65.5%) were positive for urinary AFM1. Season was significantly associated (OR = 2.64; 95% CI = 1.49-4.79; p = 0.001) with AFM1 positivity; prevalence was higher in hot-humid months (74.6%) than winter (57.3%). The place of living was also significantly associated (OR = 2.21; 95% CI = 1.25-3.97; p = 0.007), and urinary AFM1 positivity was higher in urban children (71.1%) compared to rural (58.3%). Median value for creatinine-adjusted AFM1 was 1.9 ng/mg creatinine (Q1-Q3 = 0.82-6.0 ng/mg creatinine), while non-creatinine-adjusted AFM1 was 0.57 ng/mL (Q1-Q3 = 0.23-1.4 ng/mL). Significantly higher urinary AFM1 levels were detected in children; age ≤2 years (p = 0.037), who consumed more milk (p = 0.048), and who presented to the nutrition clinic (p = 0.003). These findings highlight the need for an effective control program to reduce the AFM1 burden in children.

Assessment of aflatoxin B1-lysine adduct in children and its effect on child growth in Lahore, Pakistan.

Aflatoxin B1 is an important toxic food contaminant and there is very little information available about its exposure and effects on the health of the Pakistani population. Therefore, children (n = 238) aged 1-11 years were recruited in this study to estimate the levels of aflatoxin B1-lysine adduct and to measure its adverse effects on growth. Blood samples were analyzed to detect AFB1-lysine adducts through high-performance liquid chromatography. Socio-demographic information and anthropometry measurements were also obtained. All participants had detectable levels of AFB1-lysine adduct with a median concentration of 10.66 pg/mg albumin (95% CI: 8.6-12.4). Differences in area of residence (p < 0.05) and the father's employment (p < 0.05) were significant predictors for aflatoxin concentration levels in ordinary least square and quantile regression models (residence in 75th quantile and father employment in 90th quantile). Children aged from 5 to 11 years in the 5th and 90th quantiles of the regression model had a significant association with aflatoxin levels. A very high (50.4%, 120/238) prevalence of growth impairment (stunting, wasting, and underweight) was also observed in this study. Although we couldn't establish the effect of aflatoxin on growth impairment, children with low serum albumin levels (OR = 0.18; 95% CI: 0.05-0.56; p = 0.004) were likely to be at risk of wasting. Also, low birth weight was strongly associated with wasting (OR = 3.11; 95% CI: 1.36-7.03; p = 0.006) and underweight (OR = 4.60; 95% CI: 2.21-10.05; p= <0.001), while the mother's school level education had a correlation with child stunting (OR = 1.84; 95% CI: 1.07-3.22; p = 0.029). The high prevalence of growth impairment and high concentration of serum AFB1-lysine adduct levels in study participants demand immediate efforts to mitigate the adverse health outcomes in children in Pakistan.

Household COVID-19 secondary attack rate and associated determinants in Pakistan; A retrospective cohort study.

BACKGROUND: COVID-19 household transmissibility remains unclear in Pakistan. To understand the dynamics of Severe Acute Respiratory Syndrome Coronavirus disease epidemiology, this study estimated Secondary Attack Rate (SAR) among household and close contacts of index cases in Pakistan using a statistical transmission model. METHODOLOGY: A retrospective cohort study was conducted using an inclusive contact tracing dataset from the provinces of Punjab and Khyber-Pakhtunkhwa to estimate SAR. We considered the probability of an infected person transmitting the infection to close contacts regardless of residential addresses. This means that close contacts were identified irrespective of their relationship with the index case. We assessed demographic determinants of COVID-19 infectivity and transmissibility. For this purpose based on evolving evidence, and as CDC recommends fully vaccinated people get tested 5-7 days after close contact with a person with suspected or confirmed COVID-19. Therefore we followed the same procedure in the close contacts for secondary infection. FINDINGS: During the study period from 15th May 2020 to 15th Jan 2021, a total of 339 (33.9%) index cases were studied from 1000 cases initially notified. Among close contact groups (n = 739), households were identified with an assumed mean incubation period of 8.2+4.3 days and a maximum incubation period of 15 days. SAR estimated here is among the household contacts. 117 secondary cases from 739 household contacts, with SAR 11.1% (95% CI 9.0-13.6). All together (240) SAR achieved was 32.48% (95% CI; 29.12-37.87) for symptomatic and confirmed cases. The potential risk factors for SAR identified here included; old age group (>45 years of age), male (gender), household members >5, and residency in urban areas and for index cases high age group. Overall local reproductive number (R) based on the observed household contact frequencies for index/primary cases was 0.9 (95% CI 0.47-1.21) in Khyber Pakhtunkhwa and 1.3 (95% CI 0.73-1.56) in Punjab. CONCLUSIONS: SAR estimated here was high especially in the second phase of the COVID-19 pandemic in Pakistan. The results highlight the need to adopt rigorous preventive measures to cut the chain of viral transmission and prevent another wave of COVID-19.

Outbreak investigation and identification of risk factors associated with the occurrence of foot and mouth disease in Punjab, Pakistan.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of domesticated and wild ruminants and pigs that causes enormous economic losses through morbidity, mortality, and trade restrictions. Although the disease is endemic in Pakistan, seasonal outbreaks occur every year throughout the country. This study aimed to investigate FMD outbreaks and to identify the risk factors associated with FMD outbreaks between January and April 2019 in the Punjab province. We performed outbreak investigations (n = 64) for FMD in nine districts of the Punjab province through Divisional Disease Diagnostic Laboratories, Livestock and Dairy Development Department Punjab. Subsequently, we conducted a case-control study including 128 livestock farms (case to control ratio = 1). The data on various plausibly associated factors were collected using a structured questionnaire from each farm. The FMD outbreaks started during the month of January, culminated in February (n = 36, 56.25%) and stopped during the month of April 2019. The most common serotype was O (45.83%), followed by Asia1 (29.17%) and A (13.89%); however, some case farms had mixed infection with serotypes A and O (9.72%) and serotypes O and Asia1 (1.39%). Multivariable analysis revealed that the history of introducing a new animal with unknown FMD vaccination status in the herd (OR =11.51, 95% CI = 3.28 - 40.47), not practicing regular vaccination against FMD (OR = 20.81, 95% CI = 4.59 - 94.35), history of the visit of an animal broker (OR = 9.06, 95% CI = 2.31 - 35.61), distance of the farm to a nearby livestock farm (OR = 6.13, 95% CI = 1.39 - 27.01) and large herd size (OR = 20.79, 95% CI = 2.45 - 176.27) were significantly associated with the occurrence of FMD outbreaks in Punjab province during 2019. In conclusion, improving biosecurity measures, avoiding the introduction of animals without FMD vaccination history and regular vaccination against FMD can significantly reduce the occurrence of the disease on livestock farms in Pakistan.

Epi-Gene: An R-Package for Easy Pan-Genome Analysis.

The main aim of this study was to develop a set of functions that can analyze the genomic data with less time consumption and memory. Epi-gene is presented as a solution to large sequence file handling and computational time problems. It uses less time and less programming skills in order to work with a large number of genomes. In the current study, some features of the Epi-gene R-package were described and illustrated by using a dataset of the 14 Aeromonas hydrophila genomes. The joining, relabeling, and conversion functions were also included in this package to handle the FASTA formatted sequences. To calculate the subsets of core genes, accessory genes, and unique genes, various Epi-gene functions have been used. Heat maps and phylogenetic genome trees were also constructed. This whole procedure was completed in less than 30 minutes. This package can only work on Windows operating systems. Different functions from other packages such as dplyr and ggtree were also used that were available in R computing environment.

Cutaneous Leishmaniasis in Pakistan: a neglected disease needing one health strategy.

BACKGROUND: Cutaneous Leishmaniasis (CL) is a neglected tropical disease, which mainly affects poor communities. It is one of the major vector-borne disease and endemic in Pakistan. METHODS: A case-control study to evaluate potential risk factors of human-CL was conducted in Khewra region, District Jhelum, Pakistan from January-April 2014. Case data about 90 cases registered during October 2012 to November 2013 was retrieved from Municipal Hospital. Controls were matched (1,1 ratio) on the date of registration with cases from same hospital. Both cases and controls were invited to participate and data was collected in a face-to-face interview. A prospective study of canine leishmaniasis (canine-CL) was also conducted at Civil Veterinary Hospital in the same area. Suspected dogs with skin ulceration signs were included in the study and blood samples were collected. Statistical analyses were conducted to determine association between various parameters and outcome of interest. RESULTS: The ages of cases ranged from 1 to 76 years (median = 15 years) and proved to be protective factor i.e. increase in each year in age reduced the likelihood of being infected with human-CL [Odds Ratio (OR) = 0.4, 95% Confidence Interval (CI) = 0.25-0.76]. People sleeping outsides in an open area were more likely to become a case (OR = 8.7, 95% CI = 2.90-26.37) than a control. Poor sanitary condition inside the house (OR = 3.3, 95% CI 1.03-10.56) and presence of other animals in house (livestock, poultry) (OR = 3.6, 95% CI = 1.07-12.12) also identified as risk factors of high significance. The proportion of positive dogs with canine-CL was 21.05% and was significantly associated with human-CL cases in the same area (p < 0.05). CONCLUSIONS: We concluded that adopting self-protections measures against sand-fly, and maintaining good hygiene may lower the risk of human-CL. One-Health Strategy is suggested to control leishmaniasis in human and dog population.

Molecular epidemiology and genotype distribution of hepatitis C in Pakistan; a multicenter cross-sectional study.

Pakistan has second highest burden of hepatitis C virus (HCV) infected patients in the World. Little is known about the molecular epidemiology and risk factors for prevailing HCV genotypes in Pakistan. Considering this a multicenter cross-sectional study was conducted at 23different viral hepatitis control and prevention centers. A total of 175,897 patients were registered and screened for HCV, out of which 73,180 (41.6%) were found positive on Architect screening test. The screened positive patients were sequentially tested on RT-PCR; where 41,241 (56.35%) were detected positive. Molecular characterization results showed genotype 3 (73.9%) as the most prevalent type, followed by genotype 1 (9.7%), and genotype 4 (0.3%) was isolated for the first time in Pakistan. On regression analysis; risk factors associated with genotype 3 and 1 included; age group of 30-50 years, rural residence, exposure to >10 injections, barber shaving, circumcision by barbers, and low literacy rate. Phylogenetic analysis based on genotypes identified in this study and sequences isolated from Pakistan in last 10 years demonstrated that genotype 3 and 1 are endemic locally in Punjab province. The high prevalence rate of HCV is a threat for a generalized epidemic and genetic recombination with such variability of genotypes identified here is an alarming condition. More focused attention and resources should be spent in awareness of the population to prevent the spread of HCV among high risk population.

Iron and prebiotic fortified flour improves the immune function of iron deficient women of childbearing age.

Micronutrient deficiencies (MNDs) are common worldwide, in both developing as well as developed countries. MNDs such as Iron Deficiency not only compromise the nutritional status of individuals but can also put them at an increased risk of developing various other diseases by negatively affecting their immunity. The objective of the current research was to determine the effects of prebiotics and iron fortificants on various immunoglobulins among iron deficient women belonging to childbearing age. To serve the purpose, a total of seventy five iron deficient women were selected and randomly divided into one control and four treatment groups. Accordingly, different types of fortified wheat flour were prepared, based on varying dosage of prebiotics and iron fortificants, to be fed to anemic women on daily basis for three months. Two iron salts (FeSO4 and NaFeEDTA) and two prebiotics (Galacto oligosaccharides and Inulin) were used to fortify wheat flour during the trials. Overnight fasted women were asked to give blood samples on monthly basis, up to three months. Four types of Immunoglobulins including IgA, IgE, IgG and IgM were determined at baseline, 30th, 60th and 90th day of trials using their respective protocols. The results of the study indicated that a statistically significant declining trend for IgA, IgE, IgG and IgM was present among the treatment groups (P-value < 0.05), compared to the control group. The study concluded that provision of iron and prebiotic fortified flour improved the immune function of iron deficient women.

Incidence of Animal-Bite Injuries Registered in Public Hospitals of Post-Conflict Swat District, Pakistan in 2014.

The Swat district, a conflict-hit territory due to Taliban militancy, had a damaged local health infrastructure. Animal-bite injuries leading to rabies is one of the major health concerns in developing countries, especially within conflict zones. The current prospective epidemiological study was conducted to estimate the cumulative incidence of animal-bite injuries, to summarize characteristics of bite victims and biting animals, and to collect information about post-exposure prophylaxis (PEP) practices in Swat district, Pakistan, during 2014. A questionnaire was designed to collect data about the sociodemography of the patients, bite incident situation (provoked or unprovoked), injury pattern, animal type, PEP, and vaccination. Descriptive analyses were conducted using bar graphs, frequency tables, and chi-square tests were used to determine associations. The cumulative incidence of animal-bite injuries was 39 per 100,000 people during the study period (May-August 2014). The majority of incidents were reported from rural regions (77.7%) and were males (76.6%) younger than 10 years (37.3%). Dogs were the most frequent biting animal (86.8%) followed by rats (4.7%). About 77.7% patients washed their wound before arrival at hospital. After an eclipse phase of 10 days, 44 (10.1%) animals developed sign of rabies. The current study has highlighted a topic of interest for health, education, veterinary, and local government policy makers regarding prevention of animal bites, benefits of PEP, vaccination of human and domestic animals, control of stray dogs, and eradication of rabies in developing countries with damaged healthcare structures.