Mohsen Kalantari

Asymptomatic human carriers of Leishmania infantum : possible reservoirs for Mediterranean visceral leishmaniasis in southern Iran

Abstract Over the last decade, the incidence of visceral leishmaniasis (VL) has increased in many districts of the province of Fars, in southern Iran. Recent epidemiological reports indicate that asymptomatic human infections with Leishmania infantum (the causative agent of VL throughout the Mediterranean basin) occur more frequently in Iran than was previously believed. Between 2004 and 2006, blood samples were collected from 802 apparently healthy subjects from communities, in the north-west and south-east of Fars province, where VL cases had been recorded. Each of these samples was tested for anti-Leishmania antibodies, in direct agglutination tests (DAT), and for L. infantum kinetoplast DNA, in PCR-based assays. Of the 426 subjects from north-western Fars, eight (1.9%) were found seropositive and 68 (16.0%) PCR-positive. The corresponding values for the 376 subjects from south-eastern Fars were lower, with five (1.3%) seropositive and 32 (8.5%) PCR-positive. Of the 100 PCR-positive subjects, 18 (18.0%) each lived in a household in which there had been a case of VL, and six (6.0%) had had VL themselves (in each case, more than a year before the blood sampling for the present study). Although 21 of the PCR-positives have now been followed-up for at least 18 months, none has developed symptomatic VL. Since positivity in the PCR-based assay probably indicated the presence of L. infantum amastigotes in the peripheral blood of 12.5% of the subjects, it is clear that asymptomatic human carriers of L. infantum are quite common in the study areas and probably act as reservoirs in the transmission of the parasite, to humans and to dogs, by sandflies.

Molecular detection of Leishmania major kDNA from wild rodents in a new focus of zoonotic cutaneous leishmaniasis in an oriental region of Iran.

Human cutaneous leishmaniasis is one of the most challenging public health issues in many tropical and subtropical countries of the world, including Iran. More than half (54%) of the new zoonotic cutaneous leishmaniasis (ZCL) cases among the Eastern Mediterranean countries were reported from Iran in 2008. The detection of Leishmania parasites in rodents is essential to incriminate them as probable reservoir hosts of ZCL infection. As a result of the annual detection of about 200-250 clinical ZCL cases in the Jask district of southern Iran, feral rodents were trapped, identified to species level, and examined for Leishmania presence by preparing routine blood smears on microscopic slides from 2007 to 2008. Overall, 27 Tatera indica, 17 Gerbillus nanus, 29 Meriones persicus, 26 M. hurrianae, and 7 M. libycus were identified. Females of T. indica, M. hurrianae, and G. nanus appeared to be naturally infected with the protozoan parasite, L. major. This is the first report of microscopic and molecular detection of this trypanosomatid parasite infecting these three rodents reported from Hormozgan province in southeast Iran. More than three-quarters (82%) of the parasite-infected rodents came from the eastern plain of this province, but none of the other rodents were found to be smear-positive or kinetoplast DNA-positive by PCR. M. hurrianae, G. nanus, and T. indica are therefore incriminated as three potential reservoir hosts of L. major in Oriental parts of Iran.

Molecular and Serological Evaluation of Toxoplasma gondii Infection in Reared Turkeys in Fars Province, Iran.

Background: Toxoplasmosis is a parasitic disease caused by the protozoan Toxoplasma gondii. This parasite infects most of warm-blooded animals, including birds. Turkeys are one of these animals which might be infected by this parasite. Little is known about the prevalence of T. gondii in turkeys in Iran. Objectives: The current study aimed to evaluate the rate of Toxoplasma infection in turkeys in Fars Province, Southern Iran. Materials and Methods: Sera and tissues (brain, neck and tongue) of 54 turkeys were collected from Shiraz slaughterhouse in Fars province. Anti-Toxoplasma antibodies were assessed in the collected sera using modified agglutination test (MAT), while tissues were evaluated by polymerase chain reaction (PCR) and bioassay methods. Results: T. gondii antibodies (MAT titer: ≥ 1:40) were found in 89.8% of turkeys. T. gondii DNA was detected in 61.6% of turkey tissues and brain had the highest rate of infection. Brain tissues from each animal were bioassayed and Toxoplasma tissue cysts were found in 11.5% and Toxoplasma DNA in 62% of inoculated mice. Conclusions: Results of this study validated a relatively high level of Toxoplasma infection in reared turkeys and turkey meat might be considered as an infection sources for human.

Asymptomatic domestic dogs are carriers of Leishmania infantum: possible reservoirs host for human visceral leishmaniasis in southern Iran

In the past few years, the incidence of human visceral leishmaniasis (HVL) has increased in many districts of Fars Province, southwest of Iran, particularly, among communities of nomadic tribes. Recent epidemiological reports in Leishmania infantum endemic regions of Iran indicate that more than 50–70% of seropositive dogs are asymptomatic for Leishmania infection. Between 2004 and 2006, blood samples were collected from 110 domestic dogs from nomadic and rural areas. Each of these samples was tested for anti-Leishmania antibodies, in direct agglutination tests (DATs), and for L. infantum kinetoplast deoxyribonucleic acid (kDNA), in polymerase chain reaction (PCR)-based assays. Of the 110 dogs, 5.5% (6/110) were found seropositive and 23% (25/110) PCR-positive. Four of the six seropositive (67%) and 22 of the 25 PCR-positive (88%) were asymptomatic. The rate of infection in dogs from nomadic communities was higher (27%) than dogs from rural areas (18%). Since positivity in the PCR-based assay indicated the presence of L. infantum amastigotes in the peripheral blood of 23% of the subjects, it is clear that these asymptomatic dogs (88%) are quite common in the study areas and probably act as reservoirs in the transmission of Leishmania parasites, to humans and to other dogs, by sandflies. Moreover, our study showed that application of PCR to buffy coat samples gave a better estimate of the real rate of infection in asymptomatic dogs than DAT.

Molecular detection of Leishmania parasites and host blood meal identification in wild sand flies from a new endemic rural region, south of Iran.

Zoonotic Cutaneous Leishmaniosis (ZCL) remains the most crucial vector-borne public health disease particularly in endemic rural parts of Iran. The main aim of this study is to identify wild sand flies (Diptera: Psychodidae), determine their infection rate, and differentiate their host blood meal sources using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Sand fly populations were caught with sticky paper traps from 10 different villages in the county of Darab, Fars province, southern Iran. Following their species identification, they were used in one step PCR to determine their infection with Leishmania spp. parasites. They were then subjected to PCR-RFLP protocol to identify and differentiate their blood meal sources. Two genera of Phlebotomus and Sergentomyia comprising 13 species of sand flies were identified in this region. From a total of 150 parous female sand flies, encompassing 4 different medically important species, 7 specimens (4.7%) including 6 Phlebotomus papatasi and 1 Phlebotomus bergeroti were infected with Leishmania major. Molecular data indicated that about 32% of female sand flies fed on man, while nearly 43% fed on rodent and canine hosts. Molecular detection is an efficient way of differentiating the source of blood meals in female sand flies feeding on different vertebrate hosts. It is suggested that P. papatasi is not highly anthropophagic and appears to be an opportunistic feeder on man. This species is, however, the primary vector of ZCL in this region.

Molecular and Microscopic-Based Characterization of Plasmodium spp. in Fars and Hormozgan Provinces, South of Iran

Despite malaria control programs in recent years, malaria transmission has not been eliminated in Iran. Molecular techniques including PCR, which has proved more sensitive and specific than microscopic examination methods, help to detect infection in low levels of parasitemia and mixed infections. Main our objectives were setting up nested PCR for detection of malaria and evaluating PCR based on plasmodia DNA from blood smears in Fars province, the comparison of this method with traditional microscopy and also evaluate the data in comparison with its neighboring province, Hormozgan. A total of 149 malaria positive samples including 116, 19, and 14 samples from Shiraz, Jask, and Lengeh ports were utilized in this study, respectively. Blood slides were prepared for microscopic observation. DNA from thin smears was extracted and nested PCR was analyzed using rPLU5 and rPLU6 for genus specification, rFAL1, rFAL2, and rVIV1, rVIV2 for P. falciparum and P. vivax detection, respectively. The results showed that 126 (84.6%), 16 (10.7%), and 7 (4.7%) out of 149 cases were positive for P. vivax, P. falciparum, and mixed infections, respectively, by microscopy. The PCR indicated that 95 (63.7%), 15 (10.1%), and 22 (14.8%) cases were infected with P. vivax, P. falciparum, and mixed mentioned species, respectively, and 17 (11.4%) cases were uninfected. Our results confirmed the considerable sensitivity of nested PCR for detection of the mixed infections. Simultaneous application of PCR even based on microscopy slides can facilitate access to the highest level of confidence in malaria researches.

Faunal identification and frequency distribution of wild sand flies infected with Leishmania tropica

Abstract To determine the sand fly composition, its frequency distribution and natural infection with Leishmania parasites to incriminate the likely vectors of cutaneous leishmaniosis in Jiroft, Southeast Iran. Methods Sand flies were caught with sticky paper traps during a 6-month period in 2013. They were morphologically identified to species level using valid taxonomic keys. They were then subjected to nested PCR method and the results were analyzed. Results A total of 3 751 sand flies were identified to belong to 21 species in two genera (8 spp. in the Phlebotomus genus, and 13 spp. in the Sergentomyia genus) most of which were males (65.60%) and exophilic (63.80%). The two most frequent species were Phlebotomus papatasi (39.40%) and Phlebotomus sergenti (17.20%). The latter was confirmed by PCR to be naturally infected with Leishmania tropica (3.33%). Conclusions It was thus concluded that wild-caught Phlebotomus sergenti naturally infected with Leishmania tropica was mainly incriminated by molecular method to be the principal vector of cutaneous leishmaniosis in this endemic focus.

First molecular-based detection of mucocutaneous leishmaniasis caused by Leishmania major in Iran

Mucocutaneous leishmaniasis, which mostly occurs in the New World, is mainly associated with Leishmania braziliensis and to a lesser degree L. panamensis and L. amazonensis infections. Primary mucosal leishmaniasis is very rare in Iran in spite of high prevalence of cutaneous and visceral leishmanisis. A nine-year-old boy had cutaneous leishmaniaisis for five years involving the left side of his face; he then developed swelling and ulceration of the lip and left side buccal mucosa five months before hospital admission. He had severe swelling of the lower lip and there was ulceration and bleeding of the buccal mucosa. Direct smear revealed leishman bodies and nested PCR confirmed the presence of kinetoplast DNA of L. major in the oral mucosal specimen. The patient received amphotericin B deoxycholate 1 mg/kg/day for one month. The lip and face inflammatory reaction disappeared to nearly normal after one month of therapy. The patient was discharged with ketoconazole (5mg/kg/day) for six weeks. To our knowledge, this is the first report of mucocutaneous leishmaniasis caused by L. major in Iran.

Sandflies species composition, activity, and natural infection with Leishmania , parasite identity in lesion isolates of cutaneous leishmaniasis, central Iran

Zoonotic cutaneous leishmaniosis (ZCL) is a crucial public health challenge in Iran. Sandflies feed on reservoir rodents’ blood infected with Leishmania parasite and transmit it to other hosts. This study was conducted to find out the composition and monthly activity of sandflies as well as to identify the protozoan pathogens (Leishmania/Crithidia) by polymerase chain reaction (PCR) in an emerging ZCL focus of Abarkooh, Yazd province, Iran, in 2016. A cross-sectional study was done in rural areas of Abarkooh. From April to November 2016, sticky traps were used indoor and outdoor to capture sandflies once every fortnight. Their composition and monthly activity were recorded. Following identification of sandflies and DNA extraction from them, PCR was used to identify their parasite and match it against samples taken from ZCL confirmed and suspected patients’ lesions. After collection, a total of 2045 sandflies (779 indoor, 1266 outdoor) were identified to species level. Sandfly activity started early April in this area with two active peaks (one late May and the other late August) terminated about mid-November. Seven Phlebotomus species and three Sergentomyia species were identified. The most and the least abundant species were P. papatasi (40.1%) and P. alexandri (0.09%), respectively. Using PCR, only 6% (12:200) of P. papatasi sandflies were infected with Leishmania parasite. No Crithidia was detected in either sandflies or human lesions (176 specimen). Based on the highest abundance both indoor and outdoor of P. papatasi, this sandfly was considered the main vector of ZCL in this area. The capture of P. caucasicus, P. mongolensis, and P. ansarii from rodent burrows showed these species were likely involved in pathogen transmission in reservoir rodents’ burrows.

Co-detection and isolation of Leishmania and Crithidia among naturally infected Tatera indica (Rodentia: Muridae) in Fars province, southern Iran

Objective: To explore the co-detection of natural infection of Trypanosomatidae parasites such as Leishmania and Crithidia in reservoir hosts of leishmaniasis. Methods: Rodent populations were monitored in two endemic foci of cutaneous leishmaniasis of Fars province, southern Iran from March to October 2016. Rodents were trapped alive in several parts of Shiraz and Kharameh cities. Afterwards, their organs were prepared for detection of Leishmania and Crithidia species by molecular, microscopic, and culture methods. Results: Totally, 115 rodents of five species; Tatera indica (T. indica) (85), Rattus rattus (12), Meriones libycus (9), Mus musculus (7), and Rattus norvegicus (2), were trapped alive and their tissue samples were examined using microscopic, cultivation, and molecular assays. Overall, 59 (51.3%) rodents were positive for Leishmania or Crithidia parasites. The highest rate (61.2%; 52/85) of Leishmania infection was related to the T. indica population. The cultivation, and molecular observations showed that two (2.4%; 2/85) of T. indica (foot-pad, and spleen samples) were positive to Crithidia. Conclusions: This is the first report of Crithidia infection in T. indica in Iran. Consequently, more epidemiological and ecological studies are needed to understand the role of Crithidia and Leishmania in T. indica.