Javed Hussain Baloch

Bio: Javed Hussain Baloch is an academic researcher from Chandka Medical College. The author has contributed to research in topics: Cutaneous leishmaniasis & Leishmania. The author has an hindex of 4, co-authored 4 publications receiving 85 citations.

Multilocus enzyme electrophoresis and cytochrome b gene sequencing-based identification of leishmania isolates from different foci of cutaneous leishmaniasis in pakistan

Abstract: Seventeen Leishmania stocks isolated from cutaneous lesions of Pakistani patients were studied by multilocus enzyme electrophoresis and by polymerase chain reaction amplification and sequencing of the cytochrome b (Cyt b) gene. Eleven stocks that expressed nine zymodemes were assigned to L. (Leishmania) major. All of them were isolated from patients in the lowlands of Larkana district and Sibi city in Sindh and Balochistan provinces, respectively. The remaining six, distributed in two zymodemes (five and one), isolated from the highland of Quetta city, Balochistan, were identified as L. (L.) tropica. The same result at species level was obtained by the Cyt b sequencing for all the stocks examined. No clear-cut association between the clinical features (wet or dry type lesions) and the Leishmania species involved was found. Leishmania (L.) major was highly polymorphic compared with L. (L.) tropica. This difference may be explained by the fact that humans may act as a sole reservoir of L. (L.) tropica in anthroponotic cycles; however, many wild mammals can be reservoirs of L. (L.) major in zoonotic cycles.

Natural infection of the sand fly Phlebotomus kazeruni by Trypanosoma species in Pakistan.

Abstract: The natural infection of phlebotomine sand flies by Leishmania parasites was surveyed in a desert area of Pakistan where cutaneous leishmaniasis is endemic. Out of 220 female sand flies dissected, one sand fly, Phlebotomus kazeruni, was positive for flagellates in the hindgut. Analyses of cytochrome b (cyt b), glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) and small subunit ribosomal RNA (SSU rRNA) gene sequences identified the parasite as a Trypanosoma species of probably a reptile or amphibian. This is the first report of phlebotomine sand flies naturally infected with a Trypanosoma species in Pakistan. The possible infection of sand flies with Trypanosoma species should be taken into consideration in epidemiological studies of vector species in areas where leishmaniasis is endemic.

Zoophilic feeding behaviour of phlebotomine sand flies in the endemic areas of cutaneous leishmaniasis of Sindh Province, Pakistan.

Abstract: Leishmania (Leishmania) major has been identified as the major causative agent of cutaneous leishmaniasis in Sindh Province of southern Pakistan. To make a rational approach for understanding the pathogen transmission cycles, the sand fly species and their natural blood meals in the endemic areas were examined. Total DNA was individually extracted from sand flies collected in four villages in Sindh Province. PCR–RFLP (restriction fragment length polymorphism) and sequence analysis of the 18S ribosomal RNA gene revealed that female sand flies identified were Sergentomyia clydei/Sergentomyia ghesquierei/Sergentomyia magna (68.6%), Sergentomyia dubia (17.1%), Phlebotomus papatasi (7.4%), Phlebotomus alexandri-like sand flies (3.4%) and Sergentomyia dentata (3.4%). PCR amplification of leishmanial kinetoplast DNA did not result in positive signals, suggesting that all 175 tested female sand flies were not infected with leishmanial parasites or contained undetectable levels of leishmanial DNA. Amplification and sequencing of the vertebrate cytochrome b gene in 28 blood-fed sand flies revealed that P. papatasi fed on cattle and wild rat whereas P. alexandri-like specimens fed on human, cattle, goat and dog. Although Sergentomyia sand flies are generally known to feed on cold-blooded animals, S. clydei, S. dubia and S. ghesquierei preferred humans, cattle, goat, sheep, buffalo, dog, donkey, wild rat and Indian gerbil. The epidemiological significance of the zoophilic feeding on various host species by Phlebotomus and Sergentomyia sand flies in Pakistan is further required to study for better understanding the zoonotic transmission of sand-fly-borne pathogens and for appropriate management of the vectors.

Population genetics of Leishmania (Leishmania) major DNA isolated from cutaneous leishmaniasis patients in Pakistan based on multilocus microsatellite typing.

Abstract: Background: Cutaneous leishmaniasis (CL) is a major and fast increasing public health problem, both among the local Pakistani populations and the Afghan refugees in camps. Leishmania (Leishmania) major is one of the etiological agents responsibl ef or CL in Pakistan. Genetic variability and population structure have been investigated for 66 DNA samples of L. (L.) major isolated from skin biopsy of CL patients. Methods: Multilocus microsatellite typing (MLMT), employing 10 independent genetic markers specific to L. (L.) major, was used to investigate the genetic polymorphisms and population structures of Pakistani L. (L.) major DNA isolated from CL human cases. Their microsatellite profiles were compared to those of 130 previously typed strains of L. (L.) major from various geographical localities. Results: All the markers were polymorphic and fifty-one MLMT profiles were recognized among the 66 L. (L.) major DNA samples. The data displayed significant microsatellite polymorphisms with rare allelic heterozygosities. A Bayesian model-based approach and phylogenetic analysis inferred two L. (L.) major populations in Pakistan. Thirty-four samples belonged to one population and the remaining 32L. (L.) major samples grouped together into another population. The two Pakistani L. (L.) major populations formed separate clusters, which differ genetically from the populations of L. (L.) major from Central Asia, Iran, Middle East and Africa. Conclusions: The considerable genetic variability of L. (L.) major might be related to the existence of different species of sand fly and/or rodent reservoir host in Sindh province, Pakistan. A comprehensive study of the epidemiology of CL including the situation or spreading of reservoirs and sand fly vectors in these foci is, therefore, warranted.

Leishmaniasis: Prevention, Parasite Detection and Treatment

Abstract: Leishmaniasis remains a public health problem worldwide, affecting approximately 12 million people in 88 countries; 50 000 die of it each year. The disease is caused by Leishmania, obligate intracellular vector-borne parasites. In spite of its huge health impact on the populations in vast areas, leishmaniasis is one of the most neglected diseases. No safe and effective vaccine currently exists against any form of human leishmaniasis. The spectrum and efficacy of available antileishmanial drugs are also limited. First part of this review discusses the approaches used for the vaccination against leishmaniasis that are based on the pathogen and includes virulent or attenuated parasites, parasites of related nonpathogenic species, whole killed parasites, parasites' subunits, DNA vaccines, and vaccines based on the saliva or saliva components of transmitting phlebotomine vector. Second part describes parasite detection and quantification using microscopy assays, cell cultures, immunodetection, and DNA-based methods, and shows a progress in the development and application of these techniques. In the third part, first-line and alternative drugs used to treat leishmaniasis are characterized, and pre-clinical research of a range of natural and synthetic compounds studied for the leishmanicidal activity is described. The review also suggests that the application of novel strategies based on advances in genetics, genomics, advanced delivery systems, and high throughput screenings for leishmanicidal compounds would lead to improvement of prevention and treatment of this disease.

Detection of Leishmania infantum and identification of blood meals in Phlebotomus perniciosus from a focus of human leishmaniasis in Madrid, Spain

Abstract: Since 2010, the number of cases of both human visceral leishmaniasis and cutaneous leishmaniasis in southwestern Madrid region (Spain) and more specifically in the town of Fuenlabrada has increased. Direct xenodiagnosis of leishmaniasis proved that hares (Lepus granatensis) from this focus are able to infect with Leishmania infantum colonized Phlebotomus perniciosus. To a better understanding of this focus of leishmaniasis, we conducted an entomological survey using CDC light traps, at the end of the seasonal transmission period of 2011 before the beginning of control measures of the disease, to study the phlebotomine sand flies species involved. Detection of Leishmania DNA in the sand flies captured was studied by kDNA-PCR and cpb-PCR. In addition, blood fed and gravid female P. perniciosus were analysed by a PCR based in vertebrate cytochrome b (cyt b) gene. Taxonomic identification of captured sand flies (n = 174) as P. perniciosus (n = 171) and Sergentomyia minuta (n = 3) together with the analysis of blood feeding in ten sand flies that shows a high preference for hares (n = 6), followed by humans (n = 3), and cats (n = 1) confirm a strong association between P. perniciosus hares and humans in the focus. Moreover, 79 out of 135 (58.5 %) P. perniciosus were positive to L. infantum by PCR approaches. These data support the increase of human leishmaniasis cases in the area and the existence of an unusual sylvatic cycle alternative to the classical domestic one, where the dog is the main reservoir of L. infantum.

Detection and Identification of Leishmania Species from Clinical Specimens by Using a Real-Time PCR Assay and Sequencing of the Cytochrome b Gene

Abstract: Visceral and cutaneous leishmaniases are heterogenous entities. The Leishmania species that a given patient harbors usually cannot be determined clinically, and this identification is essential to prescribe the best species-specific therapeutic regimen. Our diagnosis procedure includes a real-time PCR assay targeted at the 18S rRNA gene, which detects all Leishmania species but which is not specific for a given Leishmania species. We developed a species identification based on sequencing of the cytochrome b (cyt b) gene directly from the DNA extracted from the clinical specimen. The sequences were analyzed using the Sequence Analysis/Seqscape v2.1 software (Applied Biosystems). This software is designed to automatically identify the closest sequences from a reference library after analysis of all known or unknown polymorphic positions. The library was built with the Leishmania cyt b gene sequences available in GenBank. Fifty-three consecutive real-time PCR-positive specimens were studied for species identification. The cyt b gene was amplified in the 53 specimens. Sequencing resulted in the identification of six different species with ≥99% identity with the reference sequences over 872 nucleotides. The identification was obtained in two working days and was in accordance with the multilocus enzyme electrophoresis identification when available. Real-time PCR followed by sequencing of the cyt b gene confirmed the diagnosis of leishmaniasis and rapidly determined the infecting species directly from the clinical specimen without the need for the isolation of parasites. This technique has the potential to significantly accelerate species-adapted therapeutic decisions regarding treatment of leishmaniasis.

Establishment of a Mass Screening Method of Sand Fly Vectors for Leishmania Infection by Molecular Biological Methods

Abstract: Surveillance of the prevalence of Leishmania and its vector, sand fly species, in endemic and surrounding areas is important for prediction of the risk and expansion of leishmaniasis. In this study, a method for the mass screening of sand flies for Leishmania infection was established. This method was applied to 319 field-captured specimens, and 5 positive sand flies were detected. Sand fly species were identified by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) of the18S rRNA gene, and all the positive flies were Lu. hartmanni. Furthermore, cytochrome b (Cyt b) gene sequence analyses identified all the parasites as Endotrypanum species including a probable novel species. Because the method requires minimum effort and can process a large number of samples at once, it will be a powerful tool for studying the epidemiology of leishmaniasis.

The Geographical Distribution of Cutaneous Leishmaniasis Causative Agents in Iran and Its Neighboring Countries, A Review

Abstract: Leishmania tropica and Leishmania major are both the main cause of anthroponotic (ACL) and zoonotic cutaneous leishmaniasis (ZCL), respectively, in the Old World. Leishmania infantum and Leishmania donovani, which are important causes of visceral leishmaniasis, have also occasionally been reported in CL patients. The present study investigates the current distribution of causative species of CL in Iran and neighboring countries in the Middle East. There has been expansion of L. tropica into new urban and rural foci in Iran, with well-documented cases of visceralization, a substantial increase of CL in Syria, and the emergence of new foci and outbreaks in Turkey and Iraq, especially due to L. major. Civil war in Syria and Iraq, population movement, poverty, and climatic change play important roles in the changing CL distribution in this region. Control programs should adopt a multidisciplinary approach based on active surveillance and case finding, especially in vulnerable refugee populations, determination of hazard maps for CL hot points using GIS and other advanced technology, the free distribution of drugs, rodent control, and greater community engagement in poor and marginalized populations. Comprehensive molecular studies that could show the species and strains of Leishmania in different areas of each country can give a better view from the distribution of CL in this region.