Javed Muhammad

Evidence of Coxiella burnetii in Punjab province, Pakistan.

Coxiella burnetii causes query (Q) fever, an important zoonotic disease with worldwide significance. The role of environment in the ecology of C. burnetti, and its influence on seroconversion in animals has not been elucidated in Pakistan. We carried out a cross-sectional study in Punjab province to (1) determine the prevalence and distribution of C. burnetii in soil using an ISIIII gene-based real time-polymerase chain reaction (RT-PCR) assay, (2) analyze association between the occurrence of C. burnetii in soil and its predictors i.e. soil characteristics (macro- and micro-nutrients) and several likely risk factors including the seroconversion in small ruminants at places where its genome had or had not been detected, and (3) predict homology and genetic diversity of the identified strains using sequences originated from different hosts worldwide. A total of 2425 soil samples from nine districts of Punjab province were processed. C. burnetii DNA was detected in 47 samples (1.94%, 95% CI: ±0.55) originating from 35 villages of studied districts (7.22%, 95% CI: ±2.30). The highest prevalence was found in Attock (7.11%, 95% CI: ±3.36), followed by Lahore (4.83%, 95% CI: ±3.49), Sahiwal (4.70%, 95% CI: ±2.6), Dera Ghazi Khan (2.33%, 95% CI: ±2.02), Faisalabad (1.35%, 95% CI: ±1.18) and Sheikhupura (0.68%, 95% CI: ±0.94). The odds of detecting bacterial DNA in soil was increased with a unit increase in organic matter [2.511 (95% CI: 1.453-4.340), p=0.001] and sodium [1.013 (95% CI: 1.005-1.022), p=0.001], whereas, calcium [0.984 (95% CI: 0.975-0.994), p=0.002] and potassium [0.994 (95% CI: 0.990-0.999), p=0.011] had protective effect where a unit increase in each analyte decreased odds for its occurrence by 1.0% approximately. Likewise, for categorical variables (risk factors), the odds of detecting C. burnetii were higher at locations >500m away from a main road [1.95 (95% CI: 1.06-3.78), p=0.04]. The enzyme-linked immunosorbent assay (ELISA) revealed an increased prevalence of antibodies in sheep (17.9%, 95% CI: ±5.54) compared with goats (16.4%, 95% CI: ±4.34). When determining the association between soil DNA and C. burnetii antibodies in small ruminants, the odds of detecting these antibodies were significant in sheep at the livestock barns [2.81 (95% CI: 1.20-7.37), p=0.02]. The IS1111 gene-based sequence analysis revealed a clustering of the DNA into two distinct groups with much genetic divergence (0.76-68.70%): the first group that contained sequences from Lahore district clustered with human and buffalo origin isolates, whereas the second group that contained the sequences from the remaining study districts clustered with goat-, rodent- and human-origin isolates. This study provides the first evidence of the presence of C. burnetii in the environment in Punjab province, Pakistan. Future studies are needed to ascertain the bacterias molecular epidemiology over a wide geographical area, type the isolates, and evaluates the potential risks to human populations, particularly farmers and veterinarians.

Incidence and Drug Resistance of Zoonotic Mycobacterium bovis Infection in Peshawar, Pakistan

Prevalence of zoonotic Mycobacterium bovis (bTB) disease in human population is underreported from the North of Pakistan. Here, we report on the proportion of human bTB disease among the overall TB patients, drug resistance pattern of bTB isolates, and knowledge, attitude, and practices (KAP)-based analysis of bTB disease. For this purpose, sputum samples from a total of 300 clinically diagnosed TB patients and 100 randomly selected school children suspected of pulmonary TB were processed by culture as well as polymerase chain reaction (PCR) for isolation, identification, and confirmation of Mycobacterium tuberculosis (mTB) and bTB species. Isolates of bTB were processed for drug susceptibility tests. Data on KAP regarding TB were obtained on a pretested questionnaire. Sputum-based PCR results indicated that 288/300 (96%) were confirmed as mTB, while 12/300 (4%) were found as bTB diseases. Interestingly, none of the school child was declared positive for either mTB or bTB. Notably, 274/300 (91.3%) positively cultured samples were identified as mTB, 13/300 (4.3%) as bTB, while 5/300 (1.7%) as mixed containing both. Importantly, except one, all of the bTB isolates were found resistant to pyrazinamide. Surprisingly, most of the bTB isolates (~70%) were found resistant to a broad range of first- and second-line anti-TB drugs. SplitsTree and recombination analysis indicated no evidence of intergenic recombination. Finally, residence, occupation, presence of animals at home, and sleeping alongside animals were found significantly associated with occurrence of bTB disease. To the best of our knowledge, we report for the first time on the high (4%) burden of bTB disease in human TB patients in Peshawar, Pakistan.

Association of soil chemistry and other factors with spatially distributed Burkholderia mallei DNA in Punjab province, Pakistan

Association of soil chemistry and other risk factors with soil borne Burkholderia mallei (B. mallei) DNA in eight districts (Sheikhupura, Gujranwala, Faisalabad, Sargodha, Chakwal, Attock, Sahiwal, Dera Ghazi Khan) of Punjab province, Pakistan, was studied. A total of 22 soil samples (n=11, each from soil positive and negative for B. mallei DNA) were processed for chemical analysis from B. mallei positive districts (Sheikhupura and Chakwal). The relationship between soil composition, absence or presence of the pathogen was ascertained. In soil samples of Chakwal district, DNA of B. mallei was found to be highly associated with 0.80 to 39.20% range of moisture contents (p=0.008) and 1.74 to 21.75 mg/Kg of P (p=0.050). The association in Sheikhupura district was with sodium (1.90 to 133.59 mg/Kg; p=0.018) and moisture (0.80 to 39.20%; p=0.026). The odds of detecting DNA of B. mallei was 1.4, 6.8, 5.0, 2.8 and 10.6 higher when soil sample sites were < 500 meters from vehicular traffic roads, < 01 km from animal markets, < 100 meters from canal, < 1000 animals and < 300 houses/village, respectively. While the odds of detecting DNA of B. mallei were 0.1, 0.3, 0.4, 0.2 and 0.5 when soil sample sites were > 500 meters from vehicular traffic roads, > 01 km from animal markets, > 100 meters from canal, > 1000 animals and > 300 houses/village. Soilborne B. mallei DNA is more likely to be detected in areas closer to roads having vehicular traffic along with interstate routes and soil containing low levels of moisture.

Seroprevalence and risk factors of glanders in working equines – Findings of a cross-sectional study in Punjab province of Pakistan

Glanders is an infectious and contagious bacterial disease of equines. A little is known about its seroprevalence and risk factors in working equines in countries where the disease is endemic. Also, there are no reports on prevalence of the disease in areas where there is a prior evidence of Burkholderia (B.) mallei detection in soil. A cross-sectional study was conducted in selected districts (n=09) of Punjab province of Pakistan during 2014-2015. A total of 1008 serum samples were screened for detection of antibodies to B. mallei with complement fixation test followed by western blot. The overall seroprevalence was found to be 3.17% (95% CI: 2.25-4.44). The seropositivity was significantly higher from the sampling sites where B. mallei was detected in soil [OR: 10.66 (95% CI: 4.42-31.66), p=0.00]. Other risk factors significantly associated with animal seropositivity were: age group [OR: 1.78 (95% CI: 4.58-15.56), p=0.00], location in urban area [OR: 2.99 (95% CI: 1.46-6.51), p=0.00],body condition [OR: 3.47 (95% CI: 1.64-7.99), p=0.00], presence of farcy lesion[OR: 7.71 (95% CI: 3.47-19.50), p=0.00], proximity to water bodies [OR: 7.71 (95% CI: 3.47-19.50), p=0.00]; domestic animal population [OR: 3.20 (95% CI: 1.24-10.87), p=0.03] and number of households in sampling area [OR: 4.18 (95%CI: 1.82-11.30), p=0.00]. The study provides an estimate of prevalence of glanders and a potential link between animal seropositivity and presence of B. mallei in soil. The risk factors identified in this study can be used in surveillance and disease awareness. The high prevalence of disease in draught horses and contact of infected animals with their care-takers in developing countries signify need to initiate progressive control of the disease using one health approach.