Mubashir Hussain

Cutaneous leishmaniasis in Karak, Pakistan: Report of an outbreak and comparison of diagnostic techniques

A total of 339 patients with clinically suspected cutaneous leishmaniasis (CL) were studied from March to April, 2010 in three villages of Karak, Khyber Pakhtunkhwa, Pakistan where an epidemic of the disease was in question. Using polymerase chain reaction (PCR), 78.17% (265/339) were observed having CL. Microscopically, however, only 43.06% (146/339) were diagnosed with the disease. This study reports and confirms epidemic of CL in both gender of all ages in the area. Females (70.94%) were noted to be predominantly affected as compared to males (29%). Clinically, 12.38% of patients had more than three lesions, 29.20% had two lesions, while 58.40% had only single lesion. Most lesions were found on exposed surfaces of the body (predominantly hands, face and feet). The present study confirms that PCR was more sensitive than microscopic examination. Key words: Epidemic, cutaneous leishmaniasis, polymerase chain reaction (PCR), microscopy, Pakistan.

Quantification of antibodies against poultry haemagglutinating viruses by haemagglutination inhibition test in Lahore

A study was conducted for quantification of antibodies to haemagglutinating viruses such as AIV (H9N2 and H7N3), NDV and IBV in vaccinated poultry flocks. A total of 198 serum samples from different commercial broiler and layer farms in Lahore, Pakistan, were checked for their serological statuses. Haemagglutination inhibition (HI) test was performed to determine the titer of serum antibodies against the above mentioned viruses. It was observed that 25 and 44% samples were positive for H7N3 and H9N2 in broilers, respectively. The seropositivity of H7N3 and H9N2 in serum samples of commercial layers were 66 and 94%, respectively. It was recorded that 96% of the samples in broilers and 100% of those in layers, were found seropositive for antibodies against NDV. Moreover, 69 and 86% were seropositive for IBV in broilers and layers, respectively. Calculated geometric mean titers for groups of 0-2, 2-4 and 4-6 weeks of age were found to be 36.8, 78.8, and 111.4 for H9N2; 48.5, 157.6, 222.9 for NDV; and 97, 84.4, 111.4 for IBV, respectively. It was concluded that the samples were not having protective antibody titers indicating unsuccessful vaccination practiced in these poultry farms which may lead to significant economic losses.

Epidemic outbreak of anthroponotic cutaneous leishmaniasis in Kohat District, Khyber Pakhtunkhwa, Pakistan

An epidemiological and molecular study was carried out for the first time in Kohat District of Khyber Pakhtunkhwa (KP) province, Pakistan from April 2015 to May 2016 to determine the prevalence of Cutaneous Leishmaniasis (CL) in local population and Internally Displaced People (IDPs). In 13 different villages, a total of 1359 (out of 26,250 individuals belonging to local population) and 140 (out of 3615 IDPs residing in these villages) cases were recorded and 300 samples were collected. The total prevalence of CL in local population was 5.17% with active lesions and scar prevalence of 3.91% and 1.26% respectively. Similarly a prevalence of 3.86% for CL was recorded in IDPs. Highest number of IDPs having CL active lesions and scars were recorded in villages Sherkot, Surgul, and Jarma and their presence was positively correlated with CL in local population. Age wise prevalence was highest in young children of age group 1-15 years. The microscopic examination showed 64.33% (193/300) positive samples while kinteoplastic PCR showed 84.66% (254/300) positive. For the first time in KP province, 2/784 sandflies trapped from the study villages was found positive for Leishmania by PCR. Restriction Fragment Length Polymorphism of patients and sandflies samples revealed L. tropica as the prevalent Leishmania species in this district. The results of sequencing and RFLP identified L. tropica in Phlebotomus sergenti. This is the first ever report of molecular identification of L. tropica from sandflies of genus P. sergenti in Khyber Pakhtunkhwa province. This data can be helpful for health authorities in finding out new CL foci and to plan effective strategies for the provision of health facilities to poor people of this area.

Expanding the clinical and genetic spectrum of G6PD deficiency: The occurrence of BCGitis and novel missense mutation

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway that ensures sufficient production of coenzyme nicotinamide adenine dinucleotide phosphate (NADPH) by catalyzing the reduction of NADP+ to NADPH. Noteworthy, the latter mediates the production of reactive oxygen species (ROS) by phagocytic cells such as neutrophils and monocytes. Therefore, patients with severe forms of G6PD deficiency may present impaired NADPH oxidase activity and become susceptible to recurrent infections. This fact, highlights the importance to characterize the immunopathologic mechanisms underlying the susceptibility to infections in patients with G6PD deficiency. Here we report the first two cases of G6PD deficiency with Bacille Calmette-Guérin (BCG) adverse effect, besides jaundice, hemolytic anemia and recurrent infections caused by Staphylococcus aureus. The qualitative G6PD screening was performed and followed by oxidative burst analysis using flow cytometry. Genetic and in silico analyses were carried out by Sanger sequencing and mutation pathogenicity predicted using bioinformatics tools, respectively. Activated neutrophils and monocytes from patients displayed impaired oxidative burst. The genetic analysis revealed the novel missense mutation c.1157T>A/p.L386Q in G6PD. In addition, in silico analysis indicated that this mutation is pathogenic, thereby hampering the oxidative burst of neutrophils and monocytes from patients. Our data expand the clinical and genetic spectrum of G6PD deficiency, and suggest that impaired oxidative burst in this severe primary immune deficiency is an underlying immunopathologic mechanism that predisposes to mycobacterial infections.

Epidemiology of Cutaneous Leishmaniasis Outbreak, Waziristan, Pakistan

During 2013–2015, prevalence of cutaneous leishmaniasis in war-affected Waziristan areas was 3.61% by PCR. Youths (1–15 years of age) were more susceptible. Internal transcribed spacer 1 PCR followed by restriction fragment length polymorphism analysis identified Leishmania tropica in 215 samples and Leishmania major in 6 samples.

Molecular detection of Leishmania species in human and animals from cutaneous leishmaniasis endemic areas of Waziristan, Khyber Pakhtunkhwa, Pakistan

Objectives: To detect Leishmania species in human patients, animal reservoirs and Phlebotomus sandflies in Waziristan, Pakistan. Methods: Tissue smears and aspirates from 448 cutaneous leishmaniasis (CL) suspected patients were analyzed. To sort out role of the reservoir hosts, skin scrapings, spleen and liver samples from 104 rodents were collected. Furthermore, buffy coat samples were obtained from 60 domestic animals. Sandflies were also trapped. All human, animals and sandfly samples were tested by microscopy, kinetoplastic PCR and internal transcribed spacer 1 (ITS1) PCR followed by restriction fragment length polymorphism for detection of Leishmania species. Results: An overall prevalence of 3.83% and 5.21% through microscopy and ITS1 PCR respectively was found. However, the statistically non-significant correlation was found between area, gender, and number of lesions. The presence of rodents, sandflies, domestic animals and internally displaced people increased the risk of CL. Using ITS1-PCR-RFLP, Leishmania tropica (L. tropica) was confirmed in 106 samples while 25 of the isolates were diagnosed as Leishmania major (L. major). Similarly, 3/104 rodents were positive for L. major and 14 pools of DNA samples containing Phlebotomus sergenti sandflies were positive for L. tropica. None of samples from domestic animals were positive for leishmaniasis. Conclusions: In the present study, L. tropica and L. major are found to be the main causative agents of CL in study area. Movement of internally displaced people from CL endemic areas presents a risk for nearby CL free areas. To the best of our knowledge, we report for the first time L. major infection in rodents (Rattus rattus) and L. tropica in Phlebotomus sergenti sandflies trapped in Waziristan, Pakistan.