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.

Corrigendum: Disease specific symptoms indices in patients with celiac disease-A hardly recognised entity.

[This corrects the article DOI: 10.3389/fnut.2022.944449.].

Disease specific symptoms indices in patients with celiac disease-A hardly recognised entity.

Background: Celiac disease (CD) was considered a rare disease before and was perceivably only limited to children but now affects almost 1-2% of the global population. This abrupt increase in prevalence is due to advancements in diagnostic criteria and medical facilities but still many countries lack the basic data that can assess the severity of this health issue. The present study was conducted with the aim to assess the common but rarely diagnosed condition with the identification of its underlying secondary ailments. Materials and methods: Patients visiting public sector hospitals were recruited and tested for clinical symptoms secondary to gluten-containing foods (wheat and barley, etc.), followed by serological testing for immunoglobulin A, tissue transglutaminase A, and anti-endomysial antibodies. Only seropositive candidates were included in the endoscopic and biopsy examination for the features of villous atrophy and intestinal cell damage. The secondary ailments including anemia, growth retardation, and gastrointestinal symptoms were also documented for the tested positive patients. The modified European Society of Pediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) criterion was followed throughout the study. Results: From 647 suspected cases from March 2018 to July 2019, 113 were confirmed with CD while 58% were female children and 42% were male children. The majority of them were from a lower class (75%) and 26% of them had a positive family history of CD. A total of 67% of patients with CD were underweight while wasting was observed in 38%, and 80% were stunted as well. Of the positively tested patients with CD, 49% had moderate anemia with 15% having severe anemia. Approximately 33% had hypoalbuminemia as well. The majority of them had a mild to severe range of gastrointestinal symptoms, such as abdominal pain, diarrhea, flatus, eructation, diarrhea, and steatorrhea. Conclusion: The study finding indicates an increased number of patients diagnosed with CD with an excessive sum of secondary ailments, such as anemia, growth failure, growth retardation, malnutrition, and gastrointestinal symptoms.

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.

Sentinel surveillance for influenza A viruses in Lahore District Pakistan in flu season 2015-2016.

BACKGROUND: Influenza A virus (IAV) remains an important global public health threat with limited epidemiological information available from low-and-middle-income countries. The major objective of this study was to describe the proportions, temporal and spatial distribution, and demographic and clinical characteristics of IAV positive patients with influenza like illness (ILI) and severe acute respiratory illness (SARI) in Lahore, Pakistan. METHODS: Prospective surveillance was established in a sentinel hospital from October 2015 to May 2016. All eligible outpatients and inpatients with ILI or SARI were enrolled in the study. Nasal and/or throat swabs were collected along with clinico-epidemiological data. Samples were tested by real-time RT-PCR (rRT-PCR) to identify IAV and subtype. The descriptive analysis of data was done in R software. RESULTS: Out of 311 enrolled patients, 284 (91.3%) were ILI and 27 (8.7%) were SARI cases. A distinct peak of ILI and SARI activity was observed in February. Fifty individuals (16%) were positive for IAV with peak positivity observed in December. Of 50 IAV, 15 were seasonal H3N2, 14 were H1N1pdm09 and 21 were unable to be typed. The majority of IAV positive cases (98%) presented with current or history of fever, 88% reported cough and 82% reported sore throat. The most common comorbidities in IAV positive cases were hepatitis C (4%), obesity (4%) and tuberculosis (6%). The highest incidence of patients reporting to the hospital was seen three days post symptoms onset (66/311) with 14 of these (14/66) positive for IAV. CONCLUSION: Distinct trends of ILI, SARI and IAV positive cases were observed which can be used to inform public health interventions (vaccinations, hand and respiratory hygiene) at appropriate times among high-risk groups. We suggest sampling from both ILI and SARI patients in routine surveillance as recommended by WHO.

Risk Factors of Influenza-Associated Respiratory Illnesses Reported to a Sentinel Hospital of Lahore, Pakistan: 2015-2016.

Epidemiological data about determinants of influenza A virus (IAV) in the Pakistani population is scarce. We aimed to conduct a prospective hospital-based active surveillance study from October 2015 to May 2016 to identify potential risk factors associated with IAV infection among patients with influenza-like illness (ILI) and severe acute respiratory illness (SARI). Surveillance was conducted in Lahore General Hospital, selected as a sentinel site in Lahore District, Pakistan. Nasal/throat samples were collected along with epidemiological and clinical data from enrolled patients. Real-time reverse-transcription polymerase chain reaction (rRT-PCR) was performed to identify IAV and its subtypes (H1N1pdm09, H3N2). Data were analyzed to determine risk factors and risk markers associated with IAV infections. A total of 311 suspected ILI and SARI cases were enrolled in the study, and among these 50 were IAV-positive. Of these 50 confirmed cases of IAV, 14 were subtyped as H1N1pdm09 and 15 were H3N2; the remaining 21 were untyped. A final multivariable model identified four independent risk factors/markers for IAV infection: exposure history to ILI patients within last 7 days and gender being male were identified as risk factors of IAV infection, while use of antibiotics prior to hospital consultation and presence of fever were identified as risk markers. We concluded that adopting nonpharmaceutical interventions like hand hygiene, masks, social distancing, and where possible, avoiding identified risk factors could decrease the risk of IAV infection and may prevent imminent outbreaks of IAV in the community.

Seroprevalence and Associated Risk Factors of Bovine Brucellosis in District Gujranwala, Punjab, Pakistan.

Bovine brucellosis is a contagious zoonotic disease that causes economic losses through abortion and infertility. A cross-sectional study was designed to determine the seroprevalence and associated risk factors of bovine brucellosis in district Gujranwala of Punjab, Pakistan. A total of 220 bovine sera (112 from buffaloes, 108 from cattle) from 46 unvaccinated herds were collected. Parallel testing by the Rose Bengal Plate Test (RBPT) and Indirect Enzyme-linked Immunosorbent Assay (I-ELISA) showed a 58.7% (27/46) herd-level and 22.7% (50/220) animal-level seroprevalence. Seroprevalence was higher (p < 0.001, OR = 7.62) in adult animals (37.2%) compared to younger animals (4.9%). A herd size of >10 animals (p = 0.021, OR = 7.83), less housing space (p = 0.037, OR = 6.39) and history of abortion at the farm (p = 0.023, OR = 5.6) were found as risk factors associated with the seropositivity of brucellosis. There was a substantial agreement between the RBPT and I-ELISA results (Cohen's kappa coefficient (κ) = 64.16, percent agreement = 89.5%). In conclusion, a relatively higher seroprevalence was found compared to the previous reports from the country. Standardization and validation of the advanced diagnostic tests would be needed. Biosecurity, personal protection, quarantine measures and routine screening of animals at the farm level and disease awareness programs and consumption of pasteurized milk in the human population will be helpful in preventing the transmission/zoonosis of the disease.

Assessment of aflatoxin in milk and feed samples and impact of seasonal variations in the Punjab, Pakistan.

The present study was designed to assess the incidence of aflatoxin contamination in animal feed and raw milk samples (total 240 each) collected from dairy farms during the complete year of 2015. These samples were collected through a cluster random sampling technique by dividing the province of Punjab, Pakistan into five clusters (north, south, east, west and central). Factors (environmental & physical) affecting aflatoxin contamination in milk and animal feed at farms were also studied. The AFM1 levels in raw milk & AFB1 levels in feed samples were analyzed by using the ELISA technique. Results demonstrated that overall about 53% raw milk samples from dairy farms were contaminated beyond the US MRL (0.50 µg/L) for AFM1 with than average level of 0.59 µg/L, while the 95% farm feed samples were exceeding the FDA MRL (20 µg/kg) of AFB1 with average level of 43 µg/kg. During winter season, the concentration of AFM1 was higher in all clusters with avg 0.68 µg/L, while the AFB1 contamination was highest in the spring season with avg 54 µg/kg. Market feed prices had negative correlation with AFB1 contamilevels, which were further supported by the positive correlation between quantity of feed at farms with AFM1 and AFB1 contamination. Results exhibited significantly positive impact of environmental factors on milk and feed aflatoxin contamination levels, whereas temperature showed an inverse relationship with AFM1 and AFB1 levels. The study recommends need of synergistic extension work to support dairy farms and highlight the contamination levels for regulatory bodies to introduce strategic policies for control measures.

Occurrence and Seasonal Variations of Aflatoxin M1 in Milk from Punjab, Pakistan.

The manifestation of aflatoxins in feed and food is a major issue in the world as its presence leads to some health problems. This study investigates the incidence of aflatoxin M1 (AFM1) contamination in raw milk samples which were collected from Punjab, Pakistan. The Cluster Random Sampling technique was used to collect 960 milk samples from five different regions, and samples were collected every month. The AFM1 level in raw milk was analyzed by the ELISA technique. The findings demonstrate that 70% of samples exceeded the United States permissible maximum residue limits (MRL 0.50 µg/L), with an overall AFM1 level that ranged from 0.3 to 1.0 µg/L. AFM1 contamination varied with the season: The highest average contamination was detected in winter (0.875 µg/L), followed by autumn (0.751 µg/L), spring (0.654 µg/L), and summer (0.455 µg/L). The Eastern region exhibited the highest average AFM1 contamination (0.705 µg/L). Milk samples from the Northern region were found to be widely contaminated, as 86.9% samples exceeded the US MRL, followed by the Eastern region, with 72.3% samples being contaminated with >0.5 µg/L AFM1. The study indicated that the raw milk supply chain was heavily contaminated. Recommendations and remedial measures need to be developed by regulatory authorities to improve the raw milk quality.

RETRACTED: An epidemiological investigation of associated risk factors with equine influenza (H3N8) epidemic 2015-16 in Pakistan.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editors-in-Chief. The article duplicates significant parts of a paper that had already appeared in Preventative Veterinary Medicine, 149, 132-139; https://doi.org/10.1016/j.prevetmed.2017.12.005. One of the conditions of submission of a paper for publication is that authors declare explicitly that the paper has not been previously published and is not under consideration for publication elsewhere. Re-use of any data should be appropriately cited. As such this article represents a misuse of the scientific publishing system. The scientific community takes a very strong view on this matter and apologies are offered to readers of the journal that this was not detected during the submission process.

Occurrence of Campylobacter in retail meat in Lahore, Pakistan.

Campylobacter, one of the emerging zoonotic pathogens, is worldwide in distribution. This thermo-tolerant pathogen is one of the leading causes of diarrhea and gastroenteritis in humans. The main sources of infection are contaminated meat and meat products. A cross-sectional study was conducted to estimate the prevalence of Campylobacter species in retail meat in the Lahore district of Pakistan from September 2014 to January 2015. A total of 600 samples (200 samples each of beef, mutton, and chicken) were collected from retail shops through convenience sampling and preceded for Campylobacter contamination using the ISO 10272-1:2006 (E) method. Campylobacter was present in all three types of meat; the highest prevalence being recorded in chicken meat (29%) followed by mutton (18%) and beef (15.5%). Campylobacters were isolated from 125 (20.8%) samples out of the 600 meat samples. Campylobacter jejuni was more common (74.4%) than C. coli (25.6%). The highest number of Campylobacters were isolated in September (25/125) and November (23/125) while low numbers were isolated in October and December with isolates rate of (17/125) and (19/125), respectively. The highest prevalence was seen in the oldest and overpopulated town of Data Gunj Bakhsh 16% (20/125) while lowest prevalence was seen in a newer and least populated town of Gulburg (7/125). These results indicate that Campylobacter species are circulating in various meat sources in Lahore and that it may pose a threat to public health.

Investigating the epidemiology of EI epidemic spread in the Province of Khyber Pakhtunkhwa, Pakistan in 2015-16.

EI in non-vaccinated population causes disruption and economic losses. To identify the risk factors associated with the EI epidemics in equids in Pakistan, a 1:1 matched case control study was conducted during 2015-2016. Including a total of 197 laboratory confirmed cases and negative controls, matched on the basis of geography, time of sampling, specie and age. A piloted questionnaire was used to collect data regarding risk factors associated with the occurrence of EI in face to face interviews. Conditional logistic regression was performed to analyze the data. A total of 16 out of 23 variables were found associated as risk factors in Univariable conditional logistic regression analysis. Multivariable conditional logistic-regression analysis was also performed. Monthly removal of manure doubles the risk of EI (EI) compared to its daily removal. Due to lack of vaccination; the spread of disease was favored by high equine density. Investigating the index-case it was recorded that infected cases were imported from Afghanistan. Most of these risk factors related to biosecurity and management were due to low awareness level regarding EI amongst the respondents. These findings are in line with the results of many other studies identifying similar risk factors for EI infection in various countries. Adopting protective practices, vaccination and controlling the risk factors identified in the present study could reduce the spread and future outbreaks of EI in Pakistan.

Molecular Epidemiology of a novel re-assorted epidemic strain of equine influenza virus in Pakistan in 2015-16.

BACKGROUND: A widespread epidemic of equine influenza (EI) occurred in nonvaccinated equine population across multiple districts in Khyber Pakhtunkhwa Province of Pakistan during 2015-2016. OBJECTIVES AND METHODS: An epidemiological surveillance study was conducted from Oct 2015 to April 2016 to investigate the outbreak. EI virus strains were isolated in embryonated eggs from suspected equines swab samples and were subjected to genome sequencing using M13 tagged segment specific primers. Phylogenetic analyses of the nucleotide sequences were concluded using Geneious. Haemagglutinin (HA), Neuraminidase (NA), Matrix (M) and nucleoprotein (NP) genes nucleotide and amino acid sequences of the isolated viruses were aligned with those of OIE recommended, FC-1, FC-2, and contemporary isolates of influenza A viruses from other species. RESULTS: HA and NA genes amino acid sequences were very similar to Tennessee/14 and Malaysia/15 of FC-1 and clustered with the contemporary isolates recently reported in the USA. Phylogenetic analysis showed that these viruses were mostly identical (with 99.6% and 97.4% nucleotide homology) to, and were reassortants containing chicken/Pakistan/14 (H7N3) and Canine/Beijing/10 (H3N2) like M and NP genes. Genetic analysis indicated that A/equine/Pakistan/16 viruses were most probably the result of several re-assortments between the co-circulating avian and equine viruses, and were genetically unlike the other equine viruses due to the presence of H7N3 or H3N2 like M and NP genes. CONCLUSION: Epidemiological data analysis indicated the potential chance of mixed, and management such as mixed farming system by keeping equine, canine and backyard poultry together in confined premises as the greater risk factors responsible for the re-assortments. Other factors might have contributed to the spread of the epidemic, including low awareness level, poor control of equine movements, and absence of border control disease strategies.

Diabetes mellitus among tuberculosis patients: a cross sectional study from Pakistan.

BACKGROUND: The co-occurrence of diabetes mellitus (DM) and tuberculosis (TB) is largely associated with high frequency of morbidity. OBJECTIVE: To determine the prevalence of DM among TB patients and describe the socio-demographic and behavioral factors associated with TB-DM co-occurrence. METHODS: We enrolled 500 TB patients from September, 2014 to August 2015 at four major public sector hospitals of Lahore, Pakistan. A questionnaire was used to collect information regarding associated socio-demographic and behavioral factors of the patients. We monitored the fasting blood sugar of each patient by using a semi automated clinical chemistry analyzer followed by an HbA1c level check of all hyperglycemic patients. RESULTS: The prevalence of TB-DM co-occurrence was 14.8%. The prevalence of TB-DM was higher (62.2%) among males. The >57 year age group had the highest proportion of patients (35.1%), with co-existent TB-DM. Most were illiterate (73.0%) and unemployed (48%). Moreover, among the 74 patients positive for TB-DM had a history of smoking. Age and education level were significantly associated with DM-TB while gender, occupation and smoking were not associated. CONCLUSION: The study revealed a 14.8% prevalence of DM among TB patients. This was associated with several socio-demographic factors, including age, unemployment, literacy and polluted environment. Thus, poor and unhealthy lifestyles were the factors associated with DM among immunologically compromised individuals due to TB.

Risk factors associated with Mycobacterium bovis skin positivity in cattle and buffalo in Peshawar, Pakistan.

A cross-sectional study was carried out to determine the prevalence of bovine tuberculosis (bTB) and associated risk factors in cattle and buffalo in Peshawar, Pakistan. Cattle and buffalo, randomly selected from all four towns of District Peshawar, were screened for bovine tuberculosis using comparative cervical intradermal tuberculin test (CCIT). For obtaining data on risk factors, sociodemographic condition, animal characteristics, and management, interviewer administered pretested questionnaire to animal owners. Multivariable logistic regression models were used to measure association between risk factors and comparative cervical intradermal tuberculin reactors. A total of 556 cattle and buffalo were screened for bovine tuberculosis. Out of 556 animals screened, 5.75 % (3.9-8.0 %) were found positive. The prevalence was higher in old animals (p = 0.001) as compared to younger animals. Prevalence also varied with source of animal (either raised on farm or purchased), stay of animals at night (indoor or outdoor), and herd size. Farmer's knowledge about transmission of TB from animals to human as well as signs and symptoms of TB was extremely low. Only 3.6 % farmers correctly stated the combination of three major symptoms of TB. Results of the study call for immediate intervention to control bTB in animals as well as its transmission to human population. Furthermore, it is suggested to emphasize on local epidemiology of bTB and husbandry practices of cattle and buffalo during the control program.

Toxinotyping and antimicrobial susceptibility of enterotoxigenic Clostridium perfringens isolates from mutton, beef and chicken meat.

A total of 300 meat samples comprising mutton, beef, and chicken meat (n = 100) collected from either local butcher shops or large meat outlets situated at various areas of Lahore City located in Punjab province of Pakistan were tested for the isolation of Clostridium perfringens. Prevalence of the organism was highest in the chicken (6 %) followed by mutton (5 %) and beef (1 %). Contamination level was high (10/150) in the samples collected from local butcher shops in comparison to the samples collected from large meat outlets (2/150). All of the raw meat samples were negative for the presence of alpha, beta and epsilon toxins of C. perfringens as detected through ELISA. Out of a total number of 12 isolates only half were capable of producing enterotoxins when cultured in trypticase glucose yeast (TGY) broth. Toxinotyping of the isolates showed that 3 were of type A while one each of the remaining three belonged to type B, C, and D. Antibiotic susceptibility testing of the toxin producing isolates revealed that C. perfringens were susceptible to chloramphenicol, ciprofloxacin, metronidazole, and ceftriaxone. All of the other drugs were relatively less effective with a least activity of amoxicillin against the isolates.

Prevalence and Molecular Diagnosis of Babesia ovis and Theileria ovis in Lohi Sheep at Livestock Experiment Station (LES), Bahadurnagar, Okara, Pakistan.

BACKGROUND: Babesia ovis and Theileria ovis are among the important and main etiological agents causing ovine babesiosis and ovine theileriosis, causing severe economic losses among sheep and goats. The aim of the present study was to determine the prevalence and molecular diagnosis of B. ovis and T. ovis in Lohi sheep at Livestock Experiment Station Bahadurnagar, Okara, Pakistan. METHODS: The prevalence of B. ovis and T. ovis was investigated in 200 Lohi sheep of mixed age and sex by PCR during 2011. The assay was employed using primers Bbo-F & Bbo-R, specific for a 549-bp fragment in B. ovis genomic DNA and primers TSsr 170F & TSsr 670R, specific for a 520-bp fragment in T. ovis genomic DNA. The animals were also screened for both haemoparasites through stained thin blood smears. RESULTS: Thirty two (16%), 48 (24%) and 26 (13%) were the number of animals found positive for B. ovis, T. ovis and for mixed infection with both parasites, respectively, through microscopy. Sixty eight (34%), 73 (37%) and 42 (21%) were the number of animals found positive for B. ovis, T. ovis and for mixed infection with both parasites, respectively, through PCR test. CONCLUSION: The results indicate the high sensitivity of PCR for surveying babesiosis and theileriosis and there is noteworthy prevalence of these diseases in sheep at an experimental station where environmental conditions are relatively controlled as compared to field conditions.

Investigation of equine influenza virus in two geographical regions of Pakistan.

PURPOSE: The present study was an attempt to elucidate the seroprevalence of equine influenza virus (H3N8) in two geographically distinct regions of Pakistan where vaccination is not routinely practiced. METHODS: A total number of 315 animals of family Equidae were included in the survey. Blood samples and nasal swabs were collected from the same animal and analyzed through ELISA and Hemagglutination Inhibition. RESULTS: The seroprevalence for EIV was 10 and 8.39 in districts under study. Out of 29 ELISA positive sera, 7 (24.1 %) showed antibodies against H1 and 22 (75.9 %) showed against H3 of influenza strains. Specie-wise antibody titer against H1 was 7.5 and 9.0 for horses and donkeys, respectively, while mules were negative. Similarly, antibody titer against H3 was 7.12, 6.9, and 6.0 for horses, donkeys, and mules, respectively. No strain was isolated from the nasal swabs despite very high antibody titers of H1 and H3 in the sera of the same animals. CONCLUSIONS: Influenza viruses are circulating in equine species and continuous surveillance is needed to keep check on any future outbreak.