Effectiveness of a Multifaceted Mobile Health Intervention (Multi-Aid-Package) in Medication Adherence and Treatment Outcomes Among Patients With Hypertension in a Low- to Middle-Income Country: Randomized Controlled Trial.

BACKGROUND: The high prevalence of uncontrolled hypertension in Pakistan is predominantly attributed to poor medication adherence. As more than 137 million people in Pakistan use cell phones, a suitable mobile health (mHealth) intervention can be an effective tool to overcome poor medication adherence. OBJECTIVE: We sought to determine whether a novel mHealth intervention is useful in enhancing antihypertensive therapy adherence and treatment outcomes among patients with hypertension in a low- to middle-income country. METHODS: A 6-month parallel, single-blinded, superiority randomized controlled trial recruited 439 patients with hypertension with poor adherence to antihypertensive therapy and access to smartphones. An innovative, multifaceted mHealth intervention (Multi-Aid-Package), based on the Health Belief Model and containing reminders (written, audio, visual), infographics, video clips, educational content, and 24/7 individual support, was developed for the intervention group; the control group received standard care. The primary outcome was self-reported medication adherence measured using the Self-Efficacy for Appropriate Medication Adherence Scale (SEAMS) and pill counting; the secondary outcome was systolic blood pressure (SBP) change. Both outcomes were evaluated at baseline and 6 months. Technology acceptance feedback was also assessed at the end of the study. A generalized estimating equation was used to control the covariates associated with the probability of affecting adherence to antihypertensive medication. RESULTS: Of 439 participants, 423 (96.4%) completed the study. At 6 months post intervention, the median SEAMS score was statistically significantly higher in the intervention group compared to the controls (median 32, IQR 11 vs median 21, IQR 6; U=10,490, P<.001). Within the intervention group, there was an increase in the median SEAMS score by 12.5 points between baseline and 6 months (median 19.5, IQR 5 vs median 32, IQR 11; P<.001). Results of the pill-counting method showed an increase in adherent patients in the intervention group compared to the controls (83/220, 37.2% vs 2/219, 0.9%; P<.001), as well as within the intervention group (difference of n=83, 37.2% of patients, baseline vs 6 months; P<.001). There was a statistically significant difference in the SBP of 7 mmHg between the intervention and control groups (P<.001) at 6 months, a 4 mmHg reduction (P<.001) within the intervention group, and a 3 mmHg increase (P=.314) within the controls. Overall, the number of patients with uncontrolled hypertension decreased by 46 in the intervention group (baseline vs 6 months), but the control group remained unchanged. The variables groups (adjusted odds ratio [AOR] 1.714, 95% CI 2.387-3.825), time (AOR 1.837, 95% CI 1.625-2.754), and age (AOR 1.618, 95% CI 0.225-1.699) significantly contributed (P<.001) to medication adherence. Multi-Aid-Package received a 94.8% acceptability score. CONCLUSIONS: The novel Multi-Aid-Package is an effective mHealth intervention for enhancing medication adherence and treatment outcomes among patients with hypertension in a low- to middle-income country. TRIAL REGISTRATION: ClinicalTrials.gov NCT04577157; https://clinicaltrials.gov/study/NCT04577157.

Effect of pharmaceutical promotion and incentives offered by pharmaceutical companies on the prescribing pattern of medical students: a cross-sectional study from a developing nation Pakistan.

Background: Pharmaceutical companies continuously pursue healthcare professionals, starting from the medical college level, which can ultimately lead to irrational prescribing of drugs and antibiotics. Therefore, our main aim was to evaluate the opinions and attitudes of medical students toward pharmaceutical promotion. Methods: This study utilized a cross-sectional online survey that applied the snowball sampling technique. Data were collected from three public and three private sector medical colleges in Punjab, Pakistan using snowball sampling. A modified version of a pre-structured questionnaire was used to collect data between October 2020 and January 2021. Medical students from the third year onward were captivated. The tool was made available on Google Forms and students could access it by clicking the link shared. The effect of promotion on prescribing pattern and future prescribing of antibiotics were measured. Descriptive statistics, chi-square, and t-test were used to analyze the data. Results: A total of 1,301 students filled out the survey, but only 1,227 responses were acceptable. The average age was found to be 23.4 ± 1.59 years. Slightly more than half of the respondents were male participants (57.7%), and a significant proportion (84.1%) reported being aware of pharmaceutical promotion. A smaller number (27.7%) felt that physicians who meet medical representatives more frequently tend to prescribe more antibiotics and 46.3% indicated they would be willing to prescribe antibiotics under the promotional influence. Medical students who were male, in senior college years, attended government institutions, and had lower parental income showed significantly higher perception and attitude scores (p < 0.05) which, in turn, may show their inclination to promotional activities. Many students agreed with the view that pharmaceutical promotion (PP) activities may alter prescribing practices and also believed that they contribute to the increased irrational prescribing of drugs and antibiotics. Conclusion: The study revealed that only a small number of students are willing to engage in promotional activities and accept rewards, which influences their choice toward selection of drugs and antibiotics. This study highlighted the necessity of giving proper educational instructions regarding the promotion of drugs to medical students. This study also focused on the educational prerequisites of the students.

Rinderpest: A Disease of the Past, and a Present Threat.

Rinderpest is a highly contagious viral disease that affects ungulates such as cattle, buffalo, yak, and various wildlife species, leading to significant morbidity and mortality. The global eradication of rinderpest was successfully accomplished in 2011 through extensive vaccination efforts. Today, safeguarding against the re-emergence of rinderpest in animal populations is paramount. The Food and Agriculture Organization of the United Nations and the World Organization for Animal Health are entrusted through a series of resolutions with the responsibility to prevent the re-emergence of rinderpest in animals.

Respectful Maternal Care Experience in Low- and Middle-Income Countries: A Systematic Review.

Background and Objectives: Respectful maternity care promotes practices that acknowledge women's preferences and women and newborns' needs. It is an individual-centered strategy founded on ethical and human rights principles. The objective of this systematic review is to identify the impact of income on maternal care and respectful maternity care in low- and middle-income countries. Materials and Methods: Data were searched from Google Scholar, PubMed, Web of Science, NCBI, CINAHL, National Library of Medicine, ResearchGate, MEDLINE, EMBASE database, Scopus, Cochrane Central Register of Controlled Trials (CENTRAL), and Maternity and Infant Care database. This review followed PRISMA guidelines. The initial search for publications comparing low- and middle-income countries with respectful maternity care yielded 6000 papers, from which 700 were selected. The review articles were further analyzed to ensure they were pertinent to the comparative impact of income on maternal care. A total of 24 articles were included, with preference given to those published from 2010 to 2023 during the last fourteen years. Results: Considering this study's findings, respectful maternity care is a crucial component of high-quality care and human rights. It can be estimated that there is a direct association between income and maternity care in LMICs, and maternity care is substandard compared to high-income countries. Moreover, it is determined that the evidence for medical tools that can enhance respectful maternity care is sparse. Conclusions: This review highlights the significance of improving maternal care experiences, emphasizing the importance of promoting respectful practices and addressing disparities in low- and middle-income countries.

Efficacy of mHealth and education-led peer counseling for patients with hypertension and coronary artery disease in Pakistan: study protocol for a double-blinded pragmatic randomized-controlled trial with factorial design.

BACKGROUND: Hypertension is a highly relevant public health challenge. Digital interventions may support improving adherence to anti-hypertensive medications and alter health behavior. Therefore, this protocol describes a study that aims to assess the effectiveness of mHealth and educational support through peer counseling (Ed-counselling) to control blood pressure in hypertensive patients when compared to standard care. METHODS: We chose a double-blinded pragmatic randomized-controlled with factorial design for this investigation. The trial is going to recruit 1648 hypertensive patients with coronary artery disease at the age of 21 to 70 years. All participants will already be on anti-hypertensive medication and own a smartphone. They will be randomized into four groups with each having 412 participants. The first group will only receive standard care; while the second group, in addition to standard care, will receive monthly Ed-counselling (educational booklets with animated infographics and peer counseling); the third group will receive daily written and voice reminders and an education-led video once weekly together with standard care; while the fourth one gets both interventions given to second and third groups respectively. All groups will be followed-up for 1 year (0, 6, and 12 months). The primary outcome will be the change in systolic blood pressure while secondary outcomes include health-related quality of life and changes in medication adherence. For measuring changes in systolic blood pressure (SBP) and adherence scores difference at 0, 6, and 12 months between and within the group, parametric (ANOVA/repeated measure ANOVA) and non-parametric tests (Kruskal-Wallis test/Friedman test) will be used. By using the general estimating equation (GEE) with negative binomial regression, at 12 months, the covariates affecting primary and secondary outcomes will be determined and controlled. The analysis will be intention-to-treat. All the outcomes will be analyzed at 0, 6, and 12 months; however, the final analysis will be at 12 months from baseline. DISCUSSION: Besides adding up to existing evidence in the literature on the subject, our designed modules using mHealth technology can help in reducing hypertension-related morbidity and mortality in developing countries.

Preparation and in Vitro Evaluation of Tamoxifen-Conjugated, Eco-Friendly, Agar-Based Hybrid Magnetic Nanoparticles for Their Potential Use in Breast Cancer Treatment.

Tamoxifen is the drug of choice as hormonal therapy for hormone receptor-positive breast cancers and can reduce the risk of breast cancer recurrence. However, oral tamoxifen has a low bioavailability due to liver and intestinal metabolic passes. To overcome this problem and utilize the potential of this drug to its maximum, inorganic nanoparticle carriers have been exploited and tested to increase its bioavailability. Biocompatibility and unique magnetic properties make iron oxide nanoparticles an excellent choice as a drug delivery system. In this study, we developed and tested a "green synthesis" approach to synthesize iron nanoparticles from green tea extract and coated them with agar for longer stability (AG-INPs). Later, these hybrid nanoparticles were conjugated with tamoxifen (TMX). By using this approach, we synthesized stable agar-coated tamoxifen-conjugated iron nanoparticles (TMX-AG-INPs) and characterized them with Fourier-transform infrared (FTIR) spectroscopy. The average particle size of AG-INPs was 26.8 nm, while the average particle size of tamoxifen-loaded iron nanoparticles, TMX-AG-INPs, was 32.1 nm, as measured by transmission and scanning electron microscopy. The entrapment efficiency of TMX-AG-INPs obtained by the drug release profile was 88%, with a drug loading capacity of 43.5%. TMX-AG-INPs were significantly (p < 0.001) efficient in killing breast cancer cells when tested in vitro on the established breast cancer cell line MCF-7 by cell viability assay, indicating their potential to control cell proliferation.

Effectiveness of mHealth Interventions in Medication Adherence among Patients with Cardiovascular Diseases: A Systematic Review.

mHealth interventions have been reported to improve adherence to long-term therapies in chronic conditions. Therefore, this study aimed at determining the effectiveness of mHealth interventions in medication adherence among patients with cardiovascular diseases (CVDs), a leading cause of mortality globally. Relying on our inclusion criteria and the PRISMA recommendations, a literature search was carried out in the PubMed, Medline, and ProQuest databases for primary studies that investigated the impact of mHealth on medication adherence for cardiovascular disease (CVD) between 2000-2021. A total of 23 randomized controlled trials with 34,915 participants matched the selection criteria. The mHealth interventions used included text messages, mobile phone applications, and voice calls, which were used either as a single intervention or combined. Additionally, studies on enhancing drug adherence had contradictory findings: most of the studies elaborated positive results; however, six studies were unable to reveal any significant effect. Finally, a risk bias analysis revealed varying outcomes across all studies. This review, as a whole, supported the notion that mHealth interventions can be effective in improving adherence to CVD medication even though they could not improve adherence to all CVD medications when compared with controls. Further trials with more refined designs integrated with comprehensive interventions are needed to produce better health outcomes.

Functional Gastrointestinal Diseases and Dietary Practices among Pakistani Children-A Schools Based Cross-Sectional Study.

BACKGROUND: Functional gastrointestinal diseases (FGIDs) are an important yet highly under explored area among public health issues. FGIDs' complex etiology makes them of interest along with their prevalence in children steadily increasing, especially in the developing world. We aimed to determine the burden FGIDs pose on school-going children, and to determine its association with the dietary intake patterns in Pakistani children. METHODOLOGY: The study included 385 school-children from public and private schools in Pakistan through multistage random sampling, from March to August 2022. We used the Food Frequency Questionnaire (FFQ) and Rome IV Criteria for a comprehensive exploration of the issue. Associations between the FGIDs and dietary factors were analyzed using chi-square and Fischer's exact tests in SPSS version 26.0. RESULTS: Females constituted 77.4% (n = 298) of all respondents, while 44.9% (n = 173) of the total reported a family history of gastrointestinal diseases. FFQ analysis showed varying consumption frequencies for different food groups. Functional abdominal pain and irritable bowel syndrome (IBS) were the highest reported FGIDs with a prevalence of 38.7% (n = 149) and 24.9% (n = 96), respectively. Statistical associations were found between different FGIDs and gender, age, household income, family members, and dietary variables such as fruit, vegetable, beverage and pulse consumption. CONCLUSION: FGIDs were found to be associated with a number of socio-demographic and dietary factors which calls for small scale and large scale attention to the issue. Results from the current study and further studies may help develop guidelines to manage these disorders in Pakistan.

The impact of newly synthesized sulfonamides on soil microbial population and respiration in rhizospheric soil of wheat (Triticum aestivum L.).

Antibiotics released into agricultural fields through the manure of grazing animals could exert harmful impacts on soil microbes and plants. Antibiotics exert high impacts on environment than other pharmaceuticals due to their higher biological activity. However, little is known about their impacts on plants, despite indications that antibiotics exert negative effects on soil microorganisms, which ultimately harm the plants. It has been demonstrated that beneficial microorganisms promote plant growth and development under various stresses. This study evaluated the toxicity of four newly derived sulfonamides (SAs), i.e., 2-(phenylsulfonyl) hydrazine carbothioamide (TSBS-1), N, 2-bis phenyl hydrazine carbothioamide (TSBS-2), aminocarbonyl benzene sulfonamide (UBS-1), and N, N'-carbonyl dibenzene sulfonamide (UBS-2) on bacterial growth and soil microbial respiration. Each SA was tested at four different concentrations (i.e., 2.25, 2.5, 3, 4 mg/ml) against five rhizospheric bacterial strains, including AC (Actinobacteria sp.), RS-3a (Bacillus sp.), RS-7a (Bacillus subtilis), RS-4a (Enterobacter sp.), and RS-5a (Enterobacter sp.). Antimicrobial activity was checked by disc diffusion method, which showed that inhibition zone increased with increasing concentration of SAs. The UBS-1 resulted in the highest inhibition zone (11.47 ± 0.90 mm) against RS-4a with the highest concentration (4 mg/ml). Except TSBS-1, all sulfonamide derivatives reduced CO2 respiration rates in soil. Soil respiration values significantly increased till 6th day; however, exposure of sulfonamide derivatives suppressed microbial respiration after 6th day. On the 20th day, poor respiration activity was noted at 0.23, 0.2, and 0.4 (CO2 mg/g dry soil) for TSBS-1, UBS-1, and UBS-2, respectively. Our results demonstrate that sulfonamides, even in small concentrations, significantly affect soil microbial population and respiration. Soil microbial respiration changes mediated by sulfonamides were dependent on length of exposure and concentration. It is recommended that antibiotics should be carefully watched and their impact on plant growth should be tested in the future studies.

Molecular investigation of possible relationships concerning bovine leukemia virus and breast cancer.

Worldwide, breast cancer has an eminent morbidity and mortality rate, as it is a neoplastic disease among females. The query of the prospective danger of bovine leukemia virus (BLV) to humans is an old but exceedingly topical focus of scientific debate. The objective of the current study was to determine the possible relationship between BLV and breast cancer. A total of 2710 formalin-fixed paraffin-embedded (FFPE) breast cancer samples were selected regardless of the age, ethnicity, or municipality origin of the subjects. The presence of BLV in human breast cancer was determined through nested PCR by amplifying tax and gag genes followed by partial sequencing. Homology was confirmed by using the online BLAST Tool. BLV genes were found to be positive in 26.8% (728/2710) of the samples from breast cancer patients and 10% (10/80) of the samples without cancer (negative control). The results indicated a correlation between the presence of the BLV gene and breast cancer (odds ratio = 0.3889; confidence interval = 1,18; p = 0.0029). The current findings suggest a possible link between BLV and human breast carcinoma. Therefore, screening cattle herds and milk products is suggested to reduce the viral transmission risk to humans.

Impact of COVID-19 on the Mental Health of Healthcare Workers: A Cross-Sectional Study From Pakistan.

Background: The novel coronavirus (COVID-19) pandemic has significantly increased the rate of mortality and morbidity worldwide due to its rapid transmission rate. The mental health status of individuals could have a negative impact attributed to this global situation. Therefore, this study was intended to explore the symptoms of depression and anxiety among healthcare workers (HCWs) of Pakistan during the ongoing COVID-19 pandemic. Methods: A cross-sectional survey was undertaken by administering a web-based questionnaire between May and June 2020. Two tools, including the Patient Health Questionnaire (PHQ9) and Generalized Anxiety Disorder-7 (GAD-7), were employed to measure anxiety and depression symptoms among HCWs. The data analyses were carried out using descriptive statistics, Man Whitney, and Kruskal Wallis tests. Results: Of 1094 HCWs who participated in this online survey, 742 (67.8%) were physicians, followed by nurses (n = 277, 25.3%) and pharmacists (n = 75, 6.9%). The survey respondents had a median depression and anxiety score of 5.00 (7.00-3.00) and 8.00 (11.00-5.00), respectively. A considerable number of HCWs (82.2%) utilized online psychological resources to deal with their psychological distress. Female HCWs, nurses, frontline HCWs, and HCWs aged 30-49 years were more likely to suffer from depression and anxiety (p < 0.05). Conclusion: During the recent ongoing pandemic of COVID-19, there is a mild level of symptoms of depression and anxiety among HCWs. Our findings call for urgent psychological interventions for vulnerable groups of Pakistani HCWs.

Perceptions, Expectations, and Experience of Physicians About Pharmacists and Pharmaceutical Care Services in Pakistan: Findings and Implications.

Background: Optimal collaboration between pharmacists and other healthcare professionals such as physicians is integral in implementing pharmaceutical care. However, there are concerns regarding the role of pharmacists, especially among low- and middle-income countries. This study explored the perceptions, expectations, and experience of physicians working in various hospital settings of Punjab, Pakistan, about pharmacists and their roles. Methods: A self-administered questionnaire consisting of four sections was administered from October to December 2020. Descriptive and inferential statistics such as Kruskal-Wallis and Mann-Whitney tests were used for data analysis using SPSS. Results: Six hundred and seventy-eight physicians participated in this study with a response rate of 77.9%. Most of the physicians reported minimal to no interaction with pharmacists (n = 521, 76.8%). However, more than three-quarters of physicians (n = 660, 97.3%) accepted pharmacists as evidence-based sources of drug information. In addition, many physicians (n = 574, 84.7%) strongly agreed that pharmacists should attend patient care rounds to respond promptly to questions related to patient medication. A limited number of physicians (n = 124, 18.3%) assumed that pharmacists were advising their patients regarding the judicial use of their drugs. Median expectation and experience score had a significant association with age, experience, and education of physicians (P < 0.05). Conclusions: The perception of physicians was positive toward certain roles of pharmacists, coupled with high expectations. However, their experience was low, with most of the activities of pharmacists due to inadequate interprofessional coordination.

The Escherichia coli Sequence Type 131 Harboring Extended-Spectrum Beta-Lactamases and Carbapenemases Genes from Poultry Birds.

BACKGROUND AND AIM: The extended-spectrum beta-lactamases (ESBLs), as well as carbapenemases, are considered as the foremost resistance determinants throughout the world. However, the relevant data especially related to the sequence types of ESBL and carbapenemases producing Escherichia coli from the poultry is limited from Pakistan. Here, we present the data on the genetic diversity of E. coli strains isolated from the poultry birds from the poultry farms located in Islamabad, Pakistan, and the underlying resistance mechanisms to beta-lactam agents. METHODS: Of 250 broilers from 25 different farms (10 birds from each farm), the cecal samples were obtained and analyzed for the presence of ESBLs producing E. coli (ESBL-Ec) as well as carbapenemases producing E. coli (CPEc) strains using selective agar for ESBL and carbapenemases screening. The susceptibility profiling of the ESBL-Ec and CPEc isolates was evaluated followed by multi-locus sequence typing. RESULTS: A total of 119 strains were positive for ESBL production whereas 37 strains were found positive to produce carbapenemases in addition to ESBLs. The MLST analysis has shown a diversity of isolates as the E. coli isolates from poultry birds correspond to a total of 16 sequence types (STs). The ST131 (22/48, 46%) followed by ST8051 (10/48, 21%) were the main STs in this study. The blaCTX-M gene was detected in all the poultry E. coli strains whereas the blaTEM was found in 45.5% of strains. The blaVIM was found in all 37 CPEc isolates whereas the blaNDM and blaIMP were found in 31/37 (83.8%) and 16/37 (43.2%) CPEc isolates respectively. CONCLUSION: The overall results have shown the prevalence of diverse genotypes among the ESBL-Ec and carbapenemase-producing E. coli (CPEC) from poultry. Furthermore, the study documents poultry birds as a persisting reservoir of extensively antimicrobial-resistant E. coli ST131 in Pakistan, suggesting a potential threat to public health.

View of Pakistani Residents toward Coronavirus Disease (COVID-19) during a Rapid Outbreak: A Rapid Online Survey.

BACKGROUND: Coronavirus disease (COVID-19) is a deadly disease that is affecting most of the countries worldwide. Public understanding, including knowledge about signs and symptoms, mode of transmission, and hygiene of COVID-19, is vital for designing effective control strategies during a public health crisis. The current study is aimed at investigating the public's perspective about COVID-19, including their knowledge, attitude, and practices. METHODS: A rapid online survey comprising 22 items was administered during the rapid outbreak of COVID-19 in Pakistan. Questions were focused on the prevention, transmission, clinical features, and control of COVID-19. In addition, the attitudes and practices of the participants were explored. Descriptive statistics, Mann-Whitney tests, Kruskal-Wallis tests, and regression analysis were carried out during data analysis. RESULTS: A total of 1257 respondents participated in this study. Most of the respondents had good knowledge (good = 64.8%, average = 30.5%, poor = 4.7%) of COVID-19. Gender, marital status, education, and residence were observed to have a significant association with the knowledge score. A vast majority of the survey respondents (77.0%) believed that COVID-19 would be controlled successfully in Pakistan. The practices of wearing a mask (85.8%) and handwashing (88.1%) were common among the participants. CONCLUSION: The participants demonstrated good knowledge and reasonable attitudes and practices toward most aspects of the COVID-19 outbreak. Improvements in certain areas could be made by mass-level education.

Evaluating the Feasibility of Five Candidate DNA Barcoding Loci for Philippine Lasianthus Jack (Lasiantheae: Rubiaceae).

INTRODUCTION: The pantropical genus Lasianthus Jack is identified for high phenotypic plasticity making traditional taxonomic identification difficult. Having some members with important medicinal properties, a precise complimentary identification through DNA barcoding is needed for species delineation. MATERIALS AND METHODS: In this study, 12 samples representing six Philippine Lasianthus species were used to determine the most efficient barcoding loci among the cpDNA markers (matK, rbcL, rps16, and trnT-F) and nrDNA (ITS) based on the criteria of universality, discriminatory power, and resolution of species. RESULTS: The results revealed that ITS has the recommended primer universality, greatest interspecific divergences, and average resolution of species. Among the cpDNA markers, matK and rbcL are recommended but with minimal resolution of species. While trnT-F showed moderate interspecific variations and resolution of Lasianthus species, rps16 has the lowest interspecific divergence and resolution of species. CONCLUSION: Consequently, ITS is the potential ideal DNA barcode for Lasianthus species. SUMMARY: ITS, matK, and rps16 markers have the excellent amplification and sequence qualityITS marker has the highest interspecific divergence with the maximum values, followed by matK, rbcL, trnT-F, and rps16, respectivelyAll markers except rps16 yielded average resolution to Lasianthus speciesITS marker is the most ideal locus in terms of excellent universality, high interspecific discriminatory ability, and average species resolution. Abbreviations used: ITS: Internal Transcribe Spacer, matK: maturase K, rbcL: ribulose-1,5-biphospahte-carboxylase, rps16: ribosomal protein 16 small subunit gene.

An appraisal on the occurrence of foot-and-mouth disease virus serotypes in cattle and buffaloes, Pakistan.

Foot-and-mouth disease (FMD) is a livestock disease that has serious economic repercussions. Here, we report the laboratory results for samples from suspected outbreaks that were sent for confirmation of FMD in Pakistan. From January 2010 to December 2011, 132 suspected outbreaks were investigated, and samples from 58 out of the 76 outbreaks sent to the National Veterinary Laboratory (NVL) were positive. The highest proportion of positives were of serotype O (65.52 %), followed by serotype A (24.14 %) and serotype Asia-1 (10.35 %), whereas amongst the samples sent to the World Reference Laboratory, Pirbright, UK (WRL), samples from 48 out of 56 outbreaks were confirmed to be FMD positive, with the following serotypes identified: O (56.25 %), Asia-1 (37.50 %) and A (6.25 %). The outbreaks affected cattle, buffalo and mixed (cattle and buffalo) herds at rates of 91, 70 and 76 %, respectively. The trend of positive outbreaks was higher in the months of winter and late spring (November to April). Although the serotype O isolates and some of the serotype A isolates from the field samples resembled the vaccine strains (r-value ≥ 0.3), this was not the case for the Asia-1 isolates. These results help to improve our understanding of the occurrence and distribution of FMD in cattle and buffaloes in Pakistan and to provide baseline information for the FMD progressive control program in the country.

Spatial trend of Foot and Mouth Disease virus (FMDV) serotypes in cattle and buffaloes, Pakistan.

The present study describes the frequency of Foot and Mouth Disease (FMD) virus serotypes (O, A and Asia-1) in major regions (all provinces) of Pakistan using Indirect Sandwich ELISA. Also, spatial distribution of various FMD serotypes and their comparison is discussed. A total of 590 samples (Epithelial tissue) have been analyzed during a period of five years (2005-2009). Out of 590 samples, 180 were found positive, giving an overall confirmation of FMDV about 33.2 %. Of the prevalent serotypes, FMDV 'O' serotype caused most outbreaks (20.7 %), followed by serotype A (6.6 %) and serotype Asia-1 (4.6 %) while there was no positive case of type 'C'. The study clearly showed that the disease was more frequent in the agro-climatic zones than in hilly areas. Based on the data of 590 samples (>50 outbreaks), the overall prevalence of FMDV in cattle and buffaloes in Pakistan was 33.2 %, while in cattle alone, it was 37.1 %, higher than in buffalo (28.7 %). There were eight cases of mixed serotypes infection, indicating the presence of endemic state of disease. Another significant feature was the change over time. In phase-I (2005-2007), there was an overall prevalence of 29.4 %, while the occurrence of the serotype O, A and Asia-1 was 20.4 %, 2.9 % and 4.7 %, respectively. During phase-II (2008-2009), the overall prevalence was 59.21 %, while those of serotype O, A and Asia-1 were 22.4 %, 31.6 % and 4.0 %, respectively. This clearly indicated a shift from serotype O to A, which may help to explain the occurrence of more severe outbreaks, despite vaccination.

Natural infection with peste des petits ruminants virus: a pre and post vaccinal assessment following an outbreak scenario.

Peste des petits ruminants virus (PPRV) infection was confirmed in a herd of goats (n = 55) at an organised farm in Islamabad, Pakistan. PPRV infection was confirmed using both antigen- and antibody-based detection methods, haemagglutination (HA) tests and molecular methods. Animals that survived natural infection developed a typical serological response and virus antigen was detected in fecal matter. Following determination of serological response to infection animals were grouped and either vaccinated or left unvaccinated: group 1 animals succumbed to infection (n = 5) and samples were analysed for PPRV antigen; group 2 animals developed clinical disease (n = 10) and were divided into 2 groups, half being vaccinated (group 2a) whilst the remainder were unvaccinated (group 2b); group 3 (n = 15) animals included those that developed only very mild clinical disease or no clinical disease; group 4 animals (n = 5) were negative for clinical disease and were housed as a negative control group. A variable antibody response was detected following resolution of the initial outbreak. Excretion of virus antigen was assessed at different time points following vaccination. Importantly, animals that were vaccinated (group 2a) excreted antigen in fecal matter for 1 month following vaccination whilst unvaccinated animals (group 2b) continued to shed virus antigen for 2 months. The potential for virus excretion in fecal matter and effects of vaccination upon virus infection are discussed. We postulate that excretion in fecal material may represent a mechanism of virus transmission following natural infection and that this mechanism may demonstrate a potential method by which PPRV outbreaks occur spontaneously in areas not previously known to have circulating virus.

Comparison of RNA extraction methods to augment the sensitivity for the differentiation of vesicular stomatitis virus Indiana1 and New Jersey.

Two methods for the extraction of RNA of vesicular stomatitis virus (VSV) Indiana1 and New Jersey and their simultaneous amplification by one-step polymerase chain reaction using reverse transcriptase were evaluated. A guanidine-thiocyanate-based RNA extraction (Qiagen RNeasy Mini Kit, Qiagen, Valencia, CA ) followed by column-based purification coupled with one-step RT-PCR proved to be a simple, safe, practicable, and reliable tool for rapid, highly sensitive, and specific differential diagnosis of both types of VSV in cell lysate and spiked tissue samples as compared with the tri-phasic extraction method (Tri-reagent method). When RNA was extracted either from VSV cell culture stock or from VSV spiked bovine lymph nodes by using Qiagen RNeasy Mini Kit, the detection limit in the multiplex RT-PCR was as low as 0.505 to 2.84 TCID(50) for VSV-IND and VSV-NJ, respectively. The multiplex RT-PCR consistently detected VSV-IND and NJ RNA in as little as 0.1-1.0 fg of total RNA from spiked BHK-21 cell suspension when Qiagen RNeasy mini kit was used. The multiplex RT-PCR assay was capable of detecting both types of VSV in a one-step reaction tube. The minimum sensitivity of this assay in various experiments was 0.1683 TCID(50) (IND), 0.0946 TCID(50) (NJ), and 0.057 fg (IND and NJ) per 2 µl PCR sample, which is significantly more sensitive than reported previously (0.28-2.8 TCID50/1 µl). So the present study improved the sensitivity of previously reported multiplex RT-PCR for the detection and differentiation of VSV-IND and VSV-NJ in a single assay.

Evidence of peste des petits ruminants virus (PPRV) infection in Sindh Ibex (Capra aegagrus blythi) in Pakistan as confirmed by detection of antigen and antibody.

An outbreak resulting in mortality in Sindh Ibex (Capra aegagrus blythi) was investigated. There was a history of about 36 deaths (both young and adult) during the period of 1 month. Disease appeared in a generalized form, affecting the respiratory and digestive systems. Major lesions were respiratory distress, pustules on and in the mouth, ocular-nasal discharges, and severe diarrhea. The most significant lesion was the oculonasal discharges and diarrhea. Deaths were mainly due to blindness, anorexia, diarrhea, and respiratory arrest. Both adult (mortality = 21) and young (mortality = 15) animals were affected with the disease. Peste des petits ruminants virus (PPRV) antigen was detected in the spleen, lung, lymph node, and swab samples by immunocapture enzyme-linked immunosorbent assay. Spleen and lung samples were also tested and found positive for the presence of F-gene of PPRV by polymerase chain reaction. Thirteen of 20 serum samples from nearby sheep and goats were found positive for antibodies to PPRV. The disease threatened the huge population of ibex in the wild life park, which was spread over a large area, but vaccination of the domestic population of sheep and goats in the surrounding villages appeared to control the disease.