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

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 responsible for 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 32 L. (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.

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

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.

Pattern of sexually transmitted infections in males in interior Sindh: a 10-year-study.

BACKGROUND: Sexually transmitted infections (STIs) are widespread in Pakistan and have not been fully documented particularly in Sindh Province. The aim of this study is to determine the number and clinical pattern of various types of STIs in general population of Larkana division and its surrounding cities. METHODS: A hospital based prospective study was carried out at Male-STD-Clinic in the Department of Dermatology, Shaheed Muhtarma Benazir Bhutto Medical University Hospital Larkana from January 2000 to December 2009. RESULTS: Among 4,288 patients, 3,947 (92.04%) had the history of extra marital sexual contact and simultaneously had developed the clinical signs of STIs; 341 (7.95%) had history of extra marital sexual contact but did not have the manifestation of STIs. Majority of the patients (3,860, 90.01%) had the history of heterosexual contact with different partners, but only few 171 (3.98%) of them had the history of homosexual contact. According the syndromic diagnosis 1930 (45.00%) patients had genital ulcer (including herpes genitals) with or without skin manifestations, 690 (16.09%) had urethral discharge, 431 (10.05%) had genital warts, 349 (8.14%) had lesions other than STIs related, 304 (7.08%) had more than one syndrome, 193 (4.50%) had scrotal swelling, 46 (1.07%) had inguinal bubo, 3 (0.06%) were human immunodeficiency virus (HIV) positive, and 1 (0.02%) had ophthalmia neonatorum. Based on the clinical and etiological grounds: 2560 (59.70%) had syphilis, 640 (14. 92%) had gonorrhoea, 399 (9.30%) had mixed infections, 40 (0.93%) had chancroid, 431 (10.05%) had genital warts, 40 (0.93%) had lymphogranuloma venerum (LGV) and granuloma inguinale (GI), 3 (0.06%) were HIV positive, 208 (4.85%) had genital herpes, 120 (2.79%) had orchitis, 56 (1.30%) had non gonococcal urethritis (chlamydia were 19), and 1 (0.02%) had ophthalmia neonatorum. CONCLUSIONS: Mode of transmission of STIs in this region is mainly by heterosexual contact and syphilis is the commonest followed by gonorrhoea.

Cutaneous leishmaniasis caused by Leishmania (L.) major infection in Sindh province, Pakistan.

Leishmaniasis is endemic in Pakistan and is wide-spread throughout the country. Polymerase chain reaction (PCR) was performed to identify the Leishmania species present in cutaneous leishmaniasis (CL) patients from new endemic areas of the central part of Sindh province, Pakistan. The PCR primers used were designed for the identification and differentiation of Leishmania (Leishmania) major and Leishmania (Leishmania) tropica species, and PCR bands at 620 and 830 bp of the parasite-specific kinetoplast DNA sequences was identified for L. (L.) major and L. (L.) tropica, respectively. Among a total of 144 DNA samples purified from the skin biopsies of clinically suspected CL patients, 108 (75%) were positive for PCR amplification. Out of the 108 cases, 105 (97.2%) were determined to be positive for L. (L.) major infection, and 3 (2.8%) were positive for L. (L.) tropica infection. It was concluded that CL caused by L. (L.) major is the main source of infection in the central part of Sindh province in Pakistan. This rapid screening technique could be used for the diagnosis of a large number of samples from skin lesions, which commonly contain other bacterial and fungal infections.

The attachment and entry of Leishmania (Leishmania) major into macrophages: observation by scanning electron microscope.

Leishmaniasis, a zoonotic protozoan disease, starts with the inoculation of the Leishmania promastigotes into the skin at the time of blood ingestion by a female sandfly. The infection of leishmaniasis is established when the Leishmania organisms start their own intracellular multiplication after having been phagocytized by the host's macrophages. In the earliest stage of the infection, therefore, the attachment of the promastigates to the macrophages is essential. We incubated a mixed culture of macrophages (JM774-1A) and Leishmania (Leishmania) major for 6 hours in vitro and observed the process of the attachment between the parasite and host cell by scanning electron microscope. We found for the first time that the attachment between the two occurred at the site of the parasite body, in addition to the previously reported sites of the flagellar tip, flagellar base, and aflagellar tip (posterior pole).

Detection of new endemic areas of cutaneous leishmaniasis in Pakistan: a 6-year study.

BACKGROUND: Cutaneous leishmaniasis (CL) is endemic in Pakistan and is widely spreading. Recently, an outbreak of the disease was observed in the region. We report some new endemic areas of CL in the country. METHODS: A total of 1210 cases of CL who visited our department from 1996 to 2001 are reported. Among them, 760 were residents of the Jacobabad, Larkana, and Dadu districts of Sindh province and had never previously traveled to endemic areas. These districts have never been reported/recognized as endemic for CL. Others were residents of endemic areas of Balochistan province. Diagnosis was made on clinical presentation; a giemsa-stained smear test and histopathological results. All the cases were treated with the meglumine antimoniate 600 mg/day (adults) and 15 mg/kg/day (children) intramuscularly for 20 consecutive days. RESULTS: All the patients were aged between 2.5 months and 65 years. Three hundred and ninety-two patients were females and 368 were males. Duration of the disease ranged from 2 to 18 months. Most of the patients had a single lesion on the face and/or extremities. Clinically, the disease was classified as: dry papular type, 407 cases; dry ulcerative type, 335 cases; and wet ulcerative type, 18 cases. No cases of muco-cutaneous or visceral leishmaniasis were found during this period. Smear testing was positive in 845 cases, while 365 cases were histopathologically positive. An ultrastructural study was performed using specimens of a few of the cases. Leishmania parasites were detected in the dermal tissues as well as in the macrophages. CONCLUSIONS: We propose that the Jacobabad, Larkana and Dadu districts could be considered endemic for CL. Wet- and dry-type lesions indicate the presence of both Leishmania tropica and L. major in this tropical region.

Clinical and epidemiological observations of cutaneous tuberculosis in Larkana, Pakistan.

BACKGROUND: Cutaneous tuberculosis is widespread in Pakistan but has not been fully documented. This study was conducted to determine the clinical pattern, nature and existence of the disease in Larkana, Sindh province, Pakistan. METHODS: We are reporting 153 cases of patients with cutaneous tuberculosis who visited our department from 1996 to 1999. All cases were diagnosed at the clinic, and the biopsies were examined for histopathological evidence. The patients received three antituberculous treatments during a 9 month course. RESULTS: Clinically, 63 (41.2%) cases of lupus vulgaris, 54 (35.3%) of scrofuloderma, 29 (19.59%) of lupus verrucosa cutis, six (3.92%) of tuberculosis cutis orificialis and one (0.64%) case of disseminated cutaneous tuberculosis were observed in our department from 1996 to 1999. All patients were aged between 3 and 50 years and had experienced the present complaints for 1 to 12 years. Sixty-nine (45.1%) cases were children aged under 10 years, 50 cases (37.25%) were aged between 10 and 20 years, and 27 cases (17.65%) were aged over 20 years. There was no considerable ratio difference of the disease between male and female patients. Histopathologically, all the specimens showed chronic granulomatous changes; the majority was infiltrated with epitheloid cells, langhans giant cells, plasma cells and other inflammatory cells, such as lymphocytes, eosinophils and neutrophils in ulcerated lesions. Increased numbers of mast cells were seen in upper and lower dermis in two-thirds of the specimens. Caseating necrosis was visible in half of the specimens while Ziehl-Neelsen stain was negative in all the sections. CONCLUSIONS: The observed number of patients was moderately large, thus indicating a high incidence of cutaneous tuberculosis in Larkana. Lupus vulgaris, a form of cutaneous tuberculosis, was widespread in this area and prevalent in adults, while scrofuloderma was prevalent in children. Moreover, the existing rate of the disease was higher in children aged under 10 years and lower in adults. This indicates that children are more prone to this disease than adults.

Comparación histopatológica y estructural de leishmaniasis animal experimental causada por diferentes cepas de Leishmania (Leishmania) mexicana aislada de pacientes con lesiones cutáneas localizadas y difusas

Con la finalidad de investigar algunos factores relacionados a diferentes formas clínicas, causadas por cepas o especies de Leishmania, se realizaron comparaciones histopatológicas y ultraestructurales. Con este propósito, se infectaron hámsters con promastigotes de Leishmania (Leishmania) mexicana, aislados de pacientes con dos tipos de formas clínicas, leishmaniasis cutáneo-difusa (LCD) y leishmaniasis cutánea lozalizada (LCL). No se encontraron aspectos histopatológicos ni ultraestructurales que indicasen un clara diferenciación entre las cepas de LCD y LCL. Los animales de experimentación utilizados fueron dividos en dos grupos como sigue: los hámsters en el grupo A fueron infectados con L (L) mexicana, aislada...

Enfermedad de Carrión: hallazgos histopatológicos de los núdulos verrugosos cutáneos de un paciente ecuatoriano

Se han estudiado aquí los nódulos verrugosos cutáneos, tomados de un paciente con la enfermedad de Carrión. Histológicamente, los nódulos verrugosos eran cosistentes lesiones granulomatosas, que mostraron infiltración con varios tipos de células, junto con la proliferación de capilares. Los cortes estuvieron predominantemente infiltrados con neutrófilos y células endoteliales, mientras que los histiocitos, las células plasmáticas, los linfocitos y células mastoideas fueron visibles en cierto grado. Las venas estaban dilatadas con muchas células endoteliales redondeadas localizadas periféricamente, mientras un gran número de neutrófilos invadían las venas. A la microscopía electrónica, se encontraron numerosos organismos...

Observaciones histológicas de hámsters dorados infectados con una cepa ecuatoriana de Leishmania mexiacana

Se desarrolló un estudio experimental para investigar el efecto de la infección de hámsters dorados con Leishmania mexicana. Los animales fueron infectados con L. mexicana de Ecuador. En la autopsia, seis meses después de la inoculación, los sitios de infección se habían tornado en lesiones ulcerativas exudativas inflamatorias cubiertas con gruesas costras. No se observó metástasis cutáneas en otras partes expuestas del cuerpo. Histológicamente las muestras de la nariz y las plantas de los pies mortraban gran número de amastigotes, con infiltración extensiva de histiocitos, linfocitos, y alguna cantidad de neutrófilos, eosinófilos y células plasmáticas. Un importante número de células mastoideas era predominante...

Estudios de microscopía electrónica de la leishmaniasis cutánea en el Ecuador

Se realizaron observaciones de la ultraestructura de las lesiones cutáneas de 3 pacientes con leishmaniasis (LC) en el Ecuador. Los parásitos localizados extra o intracelularmente, estaban en el segundo caso, a su vez, dentro o fuera de las vacuolas. El diámetro promedio de los parásitos fue de 2.62 um (0.17s.d.) y 2.18 um (0.18s.d.) de largo y ancho respectivamente. Los parásitos mostraron degeneración en las vacuolas, ya sea dentro del macrófago o fuera d ela célula. Los linfocitos estaban en estrecho contacto con los macrófagos parasitados, al igual que directamente unidos con los parásitos. Más aún, se confirmaron amstigotes en la epidermis donde linfocitos y otras células mononucleares se encontraban cerca de ellos...