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.

Virulence and resistance profiling of Staphylococcus aureus isolated from subclinical bovine mastitis in the Pakistani Pothohar region.

Mastitis is considered one of the most widespread infectious disease of cattle and buffaloes, affecting dairy herds. The current study aimed to characterize the Staphylococcus aureus isolates recovered from subclinical mastitis animals in Pothohar region of the country. A total of 278 milk samples from 17 different dairy farms around two districts of the Pothohar region, Islamabad and Rawalpindi, were collected and screened for sub clinical mastitis using California Mastitis Test. Positive milk samples were processed for isolation of Staphylococcus aureus using mannitol salt agar. The recovered isolates were analyzed for their antimicrobial susceptibility and virulence genes using disc diffusion and PCR respectively. 62.2% samples were positive for subclinical mastitis and in total 70 Staphylococcus aureus isolates were recovered. 21% of these isolates were determined to be methicillin resistant, carrying the mecA gene. S. aureus isolates recovered during the study were resistant to all first line therapeutic antibiotics and in total 52% isolates were multidrug resistant. SCCmec typing revealed MRSA SCCmec types IV and V, indicating potential community-acquired MRSA (CA-MRSA) transmission. Virulence profiling revealed high prevalence of key genes associated with adhesion, toxin production, and immune evasion, such as hla, hlb, clfA, clfB and cap5. Furthermore, the Panton-Valentine leukocidin (PVL) toxin, that is often associated with recurrent skin and soft tissue infections, was present in 5.7% of isolates. In conclusion, the increased prevalence of MRSA in bovine mastitis is highlighted by this study, which also reveals a variety of virulence factors in S. aureus and emphasizes the significance of appropriate antibiotic therapy in combating this economically burdensome disease.

Mutational and expressional association of the PIK3CA gene with the risk of breast cancer in the Pakistani population.

PI3K pathway is a very important pathway that is reported to be involved in breast cancer. Mutation of PI3K and p110 alpha-catalytic subunit of phosphatidylinositol 3-kinase (PIK3CA) is of high predictive and prognostic values in breast cancer. The purpose of the current study was to screen the hotspot mutations of the PIK3CA gene i.e. rs2677760, rs3806685, rs121913273 & rs121913279 along with expressional analysis of PI3K and PIK3CA genes in breast cancer female patients. For mutational analysis, TaqMan assay & Sanger sequencing were performed while for expressional analysis real-time PCR was carried out. Mutant allele C of rs2677760 was observed to be high in postmenopausal patients. The frequency of mutant allele G of rs3806685 was significantly high in breast cancer patients. All diseased and control samples were of wild type for the hotspot rs121913273 and rs121913279 with allele G for rs121913273 and A for rs121913279. Expression of the PI3K was high in breast cancer tissue samples as compared to the adjacent controls. While the expression of the thePIK3CA gene was significantly high in premenopausal breast cancer patients. It was concluded that the mutant allele C of rs2677760 might have some sort of association with the menopausal status and it could be used as a diagnostic marker in post-menopausal women if studied further. Mutant allele G of rs3806685 was also found to be associated with breast cancer. While multiallelic rs121913273 and rs121913279 showed a different trend for the studied population. For expressional analysis, PI3K showed over-expression in the cases while PIK3CA gene expression was observed to be significantly associated with pre-menopausal status.

Identification of transcriptional level variations in microRNA-221 and microRNA-222 as alternate players in the thyroid cancer tumor microenvironment.

Thyroid cancer (TC) is caused by genetic factors and or their cross talk with lifestyle and environment. An important role of miRNA involvement has been identified in different human diseases alongside the cancer. The growing cloud of miRNA discoveries narrates miRNA-221 and miRNA-222 as key elements of ready arsenal in the cancer micro-niches. The aim of present study was to identify the variations of miRNA-221 and miRNA-222 expression in TC tissues and their likely association with TC. miRNA-221 and miRNA-222 were investigated for their expressional alterations in TC tissue samples and healthy thyroid tissue. Expression of miRNA-221 and -222 was analyzed through real time PCR. The relative gene expression of both the miRNA was quantified and statistically evaluated. miRNA-221 and miRNA-222 were found to be highly over expressed when compared with samples of multinodular goiter (MNG) and normal controls. Interestingly, it was also noted that miRNA-221 and miRNA-222 expression is working in a cluster in thyroid cancer patients. So, it can be concluded that the expressional alterations of miRNA-221 and -222 are playing their potential role in the development of thyroid cancer.

Epidemiological survey of bovine gammaherpesvirus 4 (BoHV-4) infection in cattle and buffalo from Pakistan.

This study was conducted to evaluate the prevalence of bovine gammaherpesvirus 4 (BoHV-4) among healthy cattle and buffaloes as well as those associated with different diseases (respiratory tract infection, mastitis and reproductive tract infection) in District Chakwal, Pakistan. Blood, swab and milk samples of cattle and buffaloes were randomly collected from different areas of Chakwal. DNA was isolated from the samples and subjected to nested PCR using thymidine kinase gene primers. Out of 300 samples (200 blood, 50 swab and 50 milk samples) from both species (cattle and buffalo), an overall prevalence of BoHV-4 of 3.33% was obtained. Samples from cattle showed a higher species-specific prevalence (4.16%) than samples from buffalo (2.78%). One sample out of 50 swab samples and 1 out of 50 milk samples were also positive for BoHV-4. DNA sequencing of a positive PCR product from cattle confirmed that the sequence was from the thymidine kinase gene of BoHV-4. Phylogenetic analysis also revealed close similarities with other BOHV-4 thymidine kinase sequences. To detect BoHV-4 antibodies, an indirect ELISA was also performed. Two hundred blood samples were also collected from the same animals in nonanticoagulant-containing tubes for the isolation of serum and were subjected to indirect ELISA. Sixteen samples (8%) were positive for BoHV-4 antibodies. This study will be useful in further diagnoses of BoHV-4 in Pakistan and in devising measures to control the spread of BoHV-4.

Recent Innovations in Non-dairy Prebiotics and Probiotics: Physiological Potential, Applications, and Characterization.

Non-dairy sources of prebiotics and probiotics impart various physiological functions in the prevention and management of chronic metabolic disorders, therefore nutraceuticals emerged as a potential industry. Extraction of prebiotics from non-dairy sources is economical and easily implemented. Waste products during food processing, including fruit peels and fruit skins, can be utilized as a promising source of prebiotics and considered "Generally Recognized As Safe" for human consumption. Prebiotics from non-dairy sources have a significant impact on gut microbiota and reduce the population of pathogenic bacteria. Similarly, next-generation probiotics could also be isolated from non-dairy sources. These sources have considerable potential and can give novel strains of probiotics, which can be the replacement for dairy sources. Such strains isolated from non-dairy sources have good probiotic properties and can be used as therapeutic. This review will elaborate on the potential non-dairy sources of prebiotics and probiotics, their characterization, and significant physiological potential.

Molecular detection and characterization of ovine herpesvirus-2 using heminested PCR in Pakistan.

BACKGROUND: Malignant catarrhal fever (MCF) is a highly fatal lymphoproliferative disease of cattle, deer, bison, water buffalo, and pigs caused by the gamma-herpesviruses alcelaphine herpesvirus-1 (AlHV-1) and ovine herpesvirus-2 (OvHV-2). OBJECTIVES: This study aimed to determine the prevalence of OvHV-2 in sheep, goats, cattle, and buffalo in Rawalpindi and Islamabad, Pakistan, by applying molecular and phylogenetic methods. METHODS: Blood samples were aspirated from sheep (n = 54), goat (n = 50), cattle (n = 46) and buffalo (n= 50) at a slaughterhouse and several farms. The samples were subjected to heminested polymerase chain reaction (PCR), followed by sequencing and phylogenetic analysis of the OvHV-2 POL gene and the OvHV-2 ORF75 tegument protein gene. RESULTS: The highest percentage of MCF positive samples was in sheep (13%), whereas goat, cattle, and buffalo had lower positive percentages, 11%, 9%, and 6.5%, respectively. Four OvHV-2-positive PCR products obtained from sheep samples were sequenced. The sequences obtained were submitted to the NCBI GenBank database (MK852173 for the POL gene; MK840962, MK852171, and MK852172 for the ORF75 tegument protein gene). Phylogenetic analysis revealed a close similarity of study sequences with those of worldwide samples. CONCLUSIONS: This study is the first cross-sectional study on the prevalence and molecular detection of OvHV-2 in apparently healthy cattle and buffalo that could be carrying OvHV-2 acquired from OvHV-2-positive sheep and goats. The results indicate that OvHV-2 is circulating in Pakistan. Further studies are needed to characterize OvHV-2 and elucidate further its prevalence.

Compartmentalized Neuronal Culture for Viral Transport Research.

Neuron-invading viruses usually enter via the peripheral organs/tissues of their mammalian hosts and are transported to the neurons. Virus trafficking is critical for transport or spread within the nervous system. Primary culture of neurons is a valuable and indispensable method for neurobiological research, allowing researchers to investigate basic mechanisms of diverse neuronal functions as well as retrograde and anterograde virus transport in neuronal axons. Primary ganglion sensory neurons from mice can be cultured in a compartmentalized culture device, which allows spatial fluidic separation of cell bodies and distal axons. These neurons serve as an important model for investigating the transport of viruses between the neuronal soma and distal axons. Alphaherpesviruses are fascinating and important human and animal pathogens, they replicate and establish lifelong latent infection in the peripheral nervous system, the mechanism of the viral transport along the axon is the key to understand the virus spread in the nervous system. In this review, we briefly introduce and evaluate the most frequently used compartmentalization tools in viral transport research, with particular emphasis on alphaherpesviruses.

First detection and molecular characterization of avian polyomavirus in young parrots in Pakistan.

Avian polyomavirus (APV) infection, also called as budgerigar fledgling disease (BFD) causes various health problems in many psittacine species which may cause untimely death. The aims of this study were to investigate, for the first time, the detection, molecular characterization and phylogenetic analysis of avian polyomavirus (APV) in Pakistani psittacine birds. In an aviary a disease similar to APV was found and 90% of the nestlings died within a few weeks. Seven to ten-day-old parrot nestlings (n = 3) from the aviary were presented with feather abnormalities, plumage defect and were clinically depressed. Birds died at 11th, 14th and 16th day of age. Samples of hearts, livers, spleen, feathers and kidneys were collected from the dead birds. Samples were analyzed for the presence of APV DNA by using PCR. APV VP1 gene was partially sequenced, and phylogenetic analysis was performed. The APV strain was similar to those previously reported in other areas of the world. The results of this investigation indicate presence of a high frequency of APV infections in psittacine birds in Pakistan.

Prospective study of avian influenza H9 infection in commercial poultry farms of Punjab Province and Islamabad Capital Territory, Pakistan.

A prospective study was conducted from November 2013 to February 2014 to estimate the spatial clustering; cumulative incidence and risk factors associated with avian influenza (AI) subtype H9 infection on commercial poultry farms of Pakistan. A total of 400 farms were enrolled and followed during the study period. Among these, 109 farms submitted samples suspected for AI to the laboratory, and only 47 farms were confirmed positive by hemagglutinin inhibition (HI) test. Data was collected from these 109 farms about their demography, management, and biosecurity practices. The cumulative incidence of H9N2 was 11.75 % (95 % confidence interval (CI) 8.76-15.23). The highest number of cases (40.42 %) was reported in January. One most likely cluster (p = 0.009, radius = 4.61 km) occurred in the Kasur district. Multivariable logistic regression analyses showed that the presence of wild birds on the farms (odds ratio (OR) = 16.18; 95 % CI 3.94-66.45) was independently associated with H9N2 infection. Cleaning of cages before delivery on farm (OR = 0.16; 95 % CI = 0.06-0.47), presence of a footbath at the entrance of farm (OR = 0.24; 95 % CI 0.08-0.79), and changing of gloves (OR = 0.33; 95 % CI 0.11-0.99) were protective factors against H9N2 infection. Reducing the exposure to risk factors and adapting biosecurity measures may reduce the risk of AI H9N2 infection on commercial poultry farms in Pakistan.

Ovine herpesvirus-2-encoded microRNAs target virus genes involved in virus latency.

Herpesviruses encode microRNAs (miRNAs) that target both virus and host genes; however, their role in herpesvirus biology is understood poorly. We identified previously eight miRNAs encoded by ovine herpesvirus-2 (OvHV-2), the causative agent of malignant catarrhal fever (MCF), and have now investigated the role of these miRNAs in regulating expression of OvHV-2 genes that play important roles in virus biology. ORF20 (cell cycle inhibition), ORF50 (reactivation) and ORF73 (latency maintenance) each contain predicted targets for several OvHV-2 miRNAs. Co-transfection of miRNA mimics with luciferase reporter constructs containing the predicted targets showed the 5' UTRs of ORF20 and ORF73 contain functional targets for ovhv-miR-2 and ovhv2-miR-8, respectively, and the 3' UTR of ORF50 contains a functional target for ovhv2-miR-5. Transfection of BJ1035 cells (an OvHV-2-infected bovine T-cell line) with the relevant miRNA mimic resulted in a significant decrease in ORF50 and a smaller but non-significant decrease in ORF20. However, we were unable to demonstrate a decrease in ORF73. MCF is a disease of dysregulated lymphocyte proliferation; miRNA inhibition of ORF20 expression may play a role in this aberrant lymphocyte proliferation. The proteins encoded by ORF50 and ORF73 play opposing roles in latency. It has been hypothesized that miRNA-induced inhibition of virus genes acts to ensure that fluctuations in virus mRNA levels do not result in reactivation under conditions that are unfavourable for viral replication and our data supported this hypothesis.