ITS1 PCR-RFLP Research Articles

Molecular Identification of Morus ssp. in Duhok Using Nuclear ITS Region and Chloroplast Matk Gene

Since Mulberries (Morus)is a tree species with a considerable plant variety. Molecular techniques are methods used to distinguish between species accurately, easily and quickly. This study examines a Molecular method for distinguishing different Morus species in the Duhok - Kurdistan region/ Iraq. The method is based on the use of four techniques: matK gene, the ITS region, PCR-RFLP, and SRAP markers. Twelve Morus species have been selected for this study from different region of Duhok. The ITS region's PCR result was 700 bp, but the matK gene's PCR produce was 900 bp. The same restriction site was found for all utilized cultivars when the 700bp of ITS fragment was used for PCR-RFLP with two restriction enzymes, RsaI GT/AC and HaeIII GG/CC. This study also used six combinations of SRAP markers to aid in grouping and identifying genetic similarities. The results of PCR-RFLP demonstrated an insufficient link between Morus physical appearance and genetic traits, but differences across studied cultivars could be identified using SRAP markers. Furthermore, this study demonstrated the possibility of DNA barcoding Morus cultivars, as well as additional sequence analysis and the identification of probable SNP between cultivars.

DNA Barcoding of Pomegranate (Punica granatum L.) Cultivars in Duhok Province- Kurdistan Region/ Iraq Using 18S–28S rRNA and ITS Region

Pomegranate is a tree species with a significant plant diversity, therefore molecular methods are necessary to define and verify approaches to recognize rapidly and correctly. This research looks at a genetic strategy for identifying pomegranate varieties in Duhok KRG. The approach is based on application of ITS region, PCR-RFLP, sequencing and SNP identification. For this study, 14 pomegranate accessions were taken from various regions, namely the Center of Duhok, Amedi, Akre, Zaxo, the South of Duhok, and Sulav. The PCR product of the 18S–28S rDNA intergenic spacer was 854bp, and the sequence analysis revealed a 99.94 percent similarity with other accession numbers in NCBI, demonstrating the use of the 18S–28S rDNA intergenic spacer for identifying and barcoding pomegranate cultivars. The PCR product of the ITS region was 700bp. This result was then employed for PCR-RFLP using two restriction enzymes namely RsaI GT/AC and HaeIII GG/CC which helped grouping as well as genetic similarities. This study Further involved sequencing examined genes were compared using the NCBI BLASTn tool and clustalo (Version 1.2.4) to determine gene location and SNP. According to this study, the result of PCR-RFLP revealed poor association between pomegranate physical morphology and genetic features, while SNP identification was identified between studied cultivars. Moreover, this result showed possible DNA barcoding of pomegranate cultivars under the study.

RETRACTED: Clinical evolution of equine leishmaniasis with self-limiting cutaneous disease caused by Leishmania infantum in northeastern Brazil: A case report

Leishmania infantum infections have long been described in humans and dogs worldwide, but characterization of equine cases remains scarce. We describe the clinical evolution of a natural L. infantum infection to contribute to the diagnostic knowledge and epidemiology of equine leishmaniasis (EL). An auction-acquired four-year-old Mangalarga Marchador mare from Pernambuco state, presented a few subcutaneous nodules on the head and neck upon arrival at the purchaser's stud at Bahia state, in November of 2019. They progressed to multiple ulcerated and non-ulcerated nodules and spread to both right limbs in seven weeks. Hematology revealed anemia, lymphocytosis, monocytosis, and elevated plasma fibrinogen. Histopathology of the biopsied nodules identified a granulomatous dermatitis with macrophages containing Leishmania amastigotes. PCR detected Leishmania in skin lesions, but not in blood or spleen aspirate samples; ITS1 PCR-RFLP and DNA sequencing confirmed L. infantum species. A topical antiseptic and insect-repellent therapy and a monthly follow-up were established. All lesions improved progressively, without specific anti-Leishmania treatment, and 14months later there was a consistent resolution. This first description of EL by L. infantum in an endemic area is relevant to emphasize the need for epidemiological studies, and to enhance clinicians' awareness for differential diagnosis.

Domestic dogs carriers of Leishmania infantum, Leishmania tropica and Crithidia fasciculata as potential reservoirs for human visceral leishmaniasis in northeastern Iran.

In recent years, cases of human visceral leishmaniasis (HVL) have been reported in some districts of Golestan Province, northeastern Iran, particularly in rural areas. Recent epidemiological evidence in Leishmania infantum endemic regions of in Iran indicates approximately 50%-80% of seropositive dogs are asymptomatic for Leishmania infection. The goal in this study was to determine Leishmania species infecting domestic dogs in Golestan Province, Iran. Between 2015 and 2016, blood samples were obtained from 100 domestic dogs in rural regions of Golestan Province, northeastern Iran. All samples were tested for anti-Leishmania antibodies using a direct agglutination test (DAT), and for Leishmania spp. kinetoplast DNA (kDNA) using PCR. Seven (7%) dogs were antibody positive and 25 dogs (25%) were Leishmania spp. DNA positives by PCR positive for leishmaniasis. Four of the seven (71%) antibody-positive dogs and 19 of the 25 (76%) PCR-positive dogs were asymptomatic. The rate of infection detected by PCR was significantly higher in male dogs (21/75, 28%) than that in female dogs (4/25, 16%). The ITS1 PCR-RFLP assay identified the presence of L. infantum, L. tropica or Crithidia spp. in the 25 PCR-positive samples. The high proportion of asymptomatic dogs in the study areas represent they act as potential reservoirs in the transmission cycle of Leishmania spp. and also Crithidia fasciculata as an emerging agent for the first time. Moreover, our data showed that PCR is a more reliable assay than DAT for detecting Leishmania spp. infection among asymptomatic dogs.

Nannizzia incurvata in Hue city - Viet Nam: Molecular identification and antifungal susceptibility testing.

Nannizzia incurvata, a species belonging to the Nannizzia gypsea complex, is considered a neglected pathogen. To detected N. incurvata isolates from dermatophytosis patients in Hue city - Viet Nam, and test the antifungal susceptibility of this species. Moreover, fungal capability to produce hydrolytic enzymes was evaluated. Patients' samples were collected and cultured on Sabouraud-chloramphenicol-cycloheximide medium. Dermatophytes isolates were initially macroscopically and microscopically identified. ITS PCR-RFLP and ITS rDNA sequences were performed to determine and confirm species. An ITS Neighbor-Joining phylogenetic tree evaluated the genetic relationship among isolates. Fungal hydrolytic enzymes were examined, including lipase, phospholipase and protease. Antifungal susceptibility testing was carried out by the disk diffusion method. MICs of itraconazole, voriconazole, and terbinafine against these isolates were determined by the broth microdilution method. Twelve isolates of N. gypsea complex were preliminary morphologically identified. PCR-RFLP and ITS-rDNA sequencing identified and confirmed dermatophytes as N. incurvata strains, respectively. An evident polymorphism among isolates was highlighted in the phylogenetic tree. All isolates showed the activity of lipase, phospholipase, and protease production. Overall, all N. incurvata isolates were susceptible to itraconazole, voriconazole, clotrimazole, miconazole, and terbinafine. Few isolates were susceptible to griseofulvin, and none of them were susceptible to fluconazole. There was a presence of polyclonal N. incurvata isolates in dermatophytosis patients from Hue city, identified by PCR-RLFP and confirmed by ITS sequencing. We confirmed PCR-RLFP as a reliable technique to identify this species. Azole and terbinafine are the optimal choices for N. incurvata treatment except for fluconazole.

Successful Isolation of Leishmania RNA Virus (LRV) from Leishmania major in a Cutaneous Leishmaniasis Focus in Central Iran: An Update on Cases.

PurposeCutaneous leishmaniasis (CL) is a major vector-borne disease that affects people globally, including Iran. Different factors are associated with leishmaniasis pathogenicity; recently, a link of the possible relationship between Leishmania RNA Virus (LRV) and disease severity was proposed, especially in the New World leishmaniasis (NWL). This study was aimed to investigate the presence of LRV2 in Leishmania isolates in Aran o Bidgol, Isfahan province.MethodsSamples were collected from 110 CL-suspected patients referred to the health center. In this study, we aimed to investigate CL cases (parasitologically and clinically), identify Leishmania species (by ITS1-PCR–RFLP), and finally detection of LRV2 (by RdRp-semi-nested PCR).ResultsParasitological methods showed 60 positive cases, based on the HaeIII enzyme restriction profile, 59 cases were caused by L. major and 1 case by L. tropica. Our project is the first study on LRV2 isolation in Aran o Bidgol city and the LRV was successfully detected from a single L. major isolated in a women’s hand lesion. Using BLAST, 94.8–100% similarity was observed in the RdRp sequence of current LRV isolate with those available in GenBank from Iran or overseas.ConclusionL. major was the main cause of CL in Aran o Bidgol, although L. tropica is also present in a much lower proportion in the area. This is the first report on the presence of LRV2 in Aran o Bidgol and the fifth in Iran.

Identification of Leishmania tropica from Pediatric Visceral Leishmaniasis in Southern Mediterranean Region of Turkey.

Background and ObjectiveProtozoa of the genus Leishmania are obligate intracellular parasites, and Leishmania species cause a spectrum of species-specific clinical symptoms known as cutaneous, mucocutaneous, and visceral leishmaniasis. For example, Leishmania major and Leishmania tropica cause cutaneous leishmaniasis, while Leishmania infantum and Leishmania donovani cause visceral leishmaniasis (VL). However, molecular studies in recent years have shown that Leishmania species cause different clinical symptoms.ObjectivesOur aim was to evaluate the relationship between the clinical and molecular characterization of leishmania isolates in children with VL defined in Turkey, an intercontinental transitional region.MethodsThe clinical diagnosis of VL was confirmed by detecting amastigotes in the bone marrow aspirate and/or the rK39 test and/or molecular methods (genus-specific PCR, Real-Time PCR, ITS1 PCR-RFLP, DNA sequencing).ResultsMost of the VL patients were referred from the districts of Adana (53.3%) and others from neighboring provinces; Hatay (16.6%), Osmaniye (3%), Gaziantep (3%), Adıyaman (3%), and 20% case were Syrian immigrants A clinical diagnosis of VL was confirmed in 30 patients with different diagnostic methods. 93% was found positive with microscopic examination, 79.1% with rK39 dipstick test, and 60% with genus-specific PCR assay in clinical samples. The Leishmania isolates were identified as L. infantum (40%), L. donovani (26.7%), and L. tropica (23.3%) using Real-Time PCR, ITS1 PCR-RFLP, and DNA sequencing. There was no cutaneous finding in any case in clinical examination.The most common clinical findings were fever (93.3%) and splenomegaly (90%), followed by hepatomegaly (76.6%). The most common laboratory finding was thrombocytopenia (86.6%), followed by anemia (70%). In addition, hemophagocytic lymphohistiocytosis was detected in bone marrow aspiration in two of our patients. Since pentavalent antimony salts treatment initially failed in four patients, it was necessary to switch to Liposomal-Amphotericin B with treatment success.ConclusionsThe presence of L. tropica in VL patients, despite the absence of cutaneous findings in any of the cases, shows that this strain can cause VL, contrary to conventional knowledge. In the Adana province, where this study was carried out, L. infantum from CL cases in previous studies should be taken into account, and visceral spread in CL cases and accompanying cutaneous lesions in VL cases should be investigated in detail.

Typing of Leishmania Species Causing Cutaneous Leishmaniasis by Sybr Green Based ITS-1 Real Time Polymerase Chain Reaction Method

Cutaneous leishmaniasis (CL) is an important public health problem, most frequently seen in Şanlıurfa in Turkey. It is important to determine the species in regions where infection occurs with different Leishmania species, as in our province. In this study, it was aimed to genotype 136 samples with suspected Leishmania from Şanlıurfa using the Sybr Green-based ITS-1 real time polymerase chain reaction (Rt-PCR) method and then to compare them with ITS-1 PCR RFLP and direct microscopy methods. Wound fluid samples from patient lesions suspected of leishmaniasis were mounted on a slide, fixed, and stained with Giemsa dye. The preparations were examined under the microscope and evaluated for the presence of amastigote. After the extraction of DNA from Giemsa stained preparations by using the QIAmp DNA Mini Kit (Qiagen, Germany), the samples were studied with the Sybr Green based ITS-1 Rt-PCR method using LITSR and L5.8S primers. As a result of the PCR study, melting curve analysis was determined and the melting curves were compared with the reference strains. Then, PCR was performed in 136 samples for ITS1 region amplification using primers LITSR and L5.8S. PCR products were digested with Hae III restriction enzyme and RFLP process was performed. The products were run on metaphor agarose gel than the gels were stained with ethidium bromide for 15 min and visualized in a UV transilluminator In our study, the results of Sybr Green-based ITS-1 Rt-PCR, ITS-1 PCR-RFLP and direct microscopy methods were compared. The highest positivity rate was determined as 97% (136/132) in ITS-1 Rt-PCR method. With ITS-1 PCR-RFLP method 95.5% (136/130) positivity and with direct microscopy 94.1% (136/128) positivity were obtained, respectively. Of 132 samples, which were studied with the Sybr Green-based ITS-1 Rt-PCR method and found as positive, 121 were genotyped as L.tropica and 11 were genotyped as L.major by melting curve analysis. It was determined that, of 130 samples studied with ITS-1 PCR RFLP method 119 (91.5%) were detected as L.tropica and 11 (8.5%) were detected as L.major. The ITS-1 Rt-PCR method we used in our study was the method that detected the most positivity rate. With this method, Leishmania specimens were typed as L.tropica and L.major. It is thought that this method may be useful for the detection of the presence of Leishmania parasite and in the rapid identification of Leishmania species, as it does not require extra processes such as cutting and staining after PCR and results in a short time, but new studies are needed to observe its effectiveness in detecting other species other than L.tropica and L.major.

Human-type and pig-type Ascaris hybrids found in pigs

The discovery of hybrids between Ascaris lumbricoides and Ascaris suum has complicated our understanding of the relationship between the two species. We examined the same Ascaris specimens (48 from humans and 48 from pigs) using two methods: microsatellite markers combined with Bayesian clustering and PCR-RFLP of the nuclear internal transcribed spacer region. The results obtained by the two methods were inconsistent but showed that hybrid Ascaris identified through both approaches could infect pigs. The results of this study suggest that PCR-RFLP of ITS alone is not suitable for molecular identification of human-type and pig-type Ascaris hybrids. Use of multiple SSR markers combined with Bayesian analysis was the most reliable method in our study. Our results indicate that, in addition to host-specific Ascaris types, there may be some that do not show host specificity. Our results show for the first time that hybrid individuals can infect pigs as well as humans. This study has important theoretical and practical implications, including suggesting the need to re-evaluate long-term ascariasis control strategies.

Molecular epidemiological survey of cutaneous leishmaniasis from Azad Jammu and Kashmir, Pakistan.

Cutaneous leishmaniasis (CL) is an emerging neglected tropical disease in Azad Jammu and Kashmir which is an underdeveloped area. Prevalence and parasite species identification are the key factors to control disease in a particular population, which were the objectives of the present study. Due to a lack of previous data, we performed a district-based active CL surveillance in 2018. The data of CL, suspected (n = 20,000) cases were analyzed statistically and identified the parasite species in microscopic positive cases by ITS1-PCR RFLP and also obtained accession numbers MN891719-28 from gene Bank. The phylogenetic tree was constructed using MEGA6 software. Out of 20,000 CL, suspected cases the highest rate of 4.02% (135/3360) of CL in Mirpur and the lowest 1.58% (8/505) in Neelum was reported. The slide positivity rate, annual parasite incidence rate and annual blood examination rate were 2.27 per 1000 population, 0.08 and 0.34%. The males were more infected 58.12% (297/511) than females 41.88% (214/511) and the age group of 1–20 years were found highly infected 82.78% (423/511) than 21–40 years 13.89% (71/511) and 41–60 years 3.33% (17/511) in the studied population. The patients 56.36% (288/511) had a single lesion whereas 29.35% (150/511) had two, only 10.76% (31/288) and 8% (12/150) were using bed nets. The patients 14.29% (73/511) had three or more lesions were not using bed nets. Only 27.98% (143/511) patients had received treatment, while 72.02% (368/511) didn't. Microscopically positive cases were found to be 2.56% (511/20,000) and ITS1-PCR positive cases were found to be 91.39% (467/511). The RFLP assay confirmed the presence of Leishmania tropica in 467 samples.

Leishmaniasis in Northern Morocco: Predominance of Leishmania infantum Compared to Leishmania tropica.

In Morocco, Leishmania infantum species is the main causative agents of visceral leishmaniasis (VL). However, cutaneous leishmaniasis (CL) due to L. infantum has been reported sporadically. Moreover, the recent geographical expansion of L. infantum in the Mediterranean subregion leads us to suggest whether the nonsporadic cases of CL due to this species are present. In this context, this review is written to establish a retrospective study of cutaneous and visceral leishmaniasis in northern Morocco between 1997 and 2018 and also to conduct a molecular study to identify the circulating species responsible for the recent cases of leishmaniases in this region. Data concerning leishmaniases cases were collected from the Epidemiology and Disease Control Directorate from 1997 to 2018. Human samples obtained from peripheral laboratories were examined using PCR-ITS1 method. The ITS1 products were subjected to digestion with the restriction endonuclease Mn1-I. Between 1997 and 2018, a total of 1,255 cases of cutaneous and visceral leishmaniasis were recorded in Tangier-Tetouan-Al Hoceima Region, i.e., 1.56% of the reported cases in Morocco (1,255/80,299). Concerning the geographical study covering the period 2007-2018, 79.5% (105/132) of the sectors were affected by leishmaniases. The molecular results showed that Humans were found to be infected with the L. infantum species with a high infection rate compared to L. tropica infection. Moreover, molecular characterization using ITS1 PCR-RFLP showed that the density of L. infantum was significantly higher (n = 68/81; 84%) than that of L. tropica (n = 13/81; 16%) (P-value 9.894e-10). While regarding visceral leishmaniasis, L. infantum was the only species responsible of this form. These findings of this study showed the emergence of L. infantum in Morocco and suggest that this species might be more prevalent than previously thought. Furthermore, the molecular determination of L. infantum will be helpful for control strategies by taking into consideration the reservoir of this species.

Molecular Phylogenetic Variability of Fasciola gigantica in Iran

Fascioliasis is one of important zoonotic disease caused by Fasciola gigantica and F. hepatica. The final hosts of this parasite are ruminants and humans. Iran is one of the endemic areas in the world, about six million people at risk of infection. The aim of this study was to identify and determine the genetic diversity of Fasciola species in cattle after distinguish of their species. One hundred and seventeen liver specimens collected from naturally infected cattle in 5 geographical regions in 2014-2017. Flukes stained with Hematoxylin-Carmine dye to examine for the existence of sperm within seminal vesicles. DNA was extracted from each individual, and ITS1, ND1and CO1 genes were amplified using specific primers. For discrimination of Fasciola species, ITS1 PCR-RFLP was used based on digestion pattern of RsaI enzyme. Genetic analyses and diversity and neutrality indices estimated by Dnasp5 based on NDI. Six nonspermic and 111 spermic flukes were diagnosed. All of nonspermic specimens were F. gigantica and collected from South East, South West and North West of Iran. Genetic haplotype diversity has been observed in F. gigantica based on ND1. Fst value analysis showed that minimum and maximum genetic difference between Iranian F. gigantica with Bangladesh (F st = 0.01414) and Egypt (F st = 0.36653) respectively. It is the first report of existing of nonspermic Fasciola. High haplotype and nucleotide diversity could be due to ecological factors in life cycle, animal migration and coexisting of the final host of this parasite. Haplotype and nucleotide diversity of spermic F. gigantica in Iran and other countries in the world led to creating a variety of haplogroups.

Species distribution and antifungal susceptibility profile of Candida isolates from blood and other normally sterile foci from pediatric ICU patients in Tehran, Iran.

As data on pediatric invasive candidiasis (IC) and the antifungal susceptibility pattern of associated isolates are scarce in Iran, this study aimed to determine species distribution and antifungal susceptibility profile of Candida species isolated from pediatric patients with suspected or documented IC. A total of 235 yeast strains recovered from normally sterile body fluids of patients admitted at the intensive care units of Children's Medical Centre, Tehran, Iran, were identified using CHROMagar Candida, molecular methods (ITS PCR-RFLP and sequencing), and MALDI-TOF. Susceptibility to amphotericin B, fluconazole, voriconazole, micafungin, and anidulafungin was determined according to the European on Antimicrobial Susceptibility testing reference microdilution method (EUCAST E.Def 7.3.1). Candida albicans (53.6%), C. parapsilosis (24.7%), and C. tropicalis (8.5%) were the most common species, followed by C. lusitaniae (4.3%), C. glabrata (3.0%), C. guilliermondii and C. orthopsilosis (each 1.7%), C. kefyr (1.3%), C. dubliniensis (0.8%), and C. intermedia (0.4%). Amphotericin B MICs were ≤1mg/l for all Candida isolates. C. albicans isolates were susceptible to all five antifungal agents. All C. parapsilosis isolates categorised as intermediate to micafungin and anidulafungin, except two isolates that had the MICs >2mg/l for micafungin. MIC50, MIC90, and MIC range for fluconazole were 0.25mg/l, 1mg/l, and 0.125 - ≥32mg/l, respectively. Fluconazole and voriconazole showed 100% activity against the most prevalent Candida species. The low resistance rate, favorable safety profile and low cost of fluconazole make it a reasonable choice for treatment of candidemia/invasive candidemia in Iran.

Risk factors and prediction analysis of cutaneous leishmaniasis due to Leishmania tropica in Southwestern Morocco

Cutaneous leishmaniasis is currently a serious public health problem in northern Africa, especially in Morocco. The causative parasite is transmitted to a human host through the bite of infected female sandflies of the genus Phlebotomus. The objective of the present study is to characterize the causative organisms and to predict the risk of cutaneous leishmaniasis (CL) cases in six provinces in southwestern Morocco, based on the spatial distribution of cases in relation to environmental factors and other risk factors such as socio-economic status and demographics. A molecular study was carried out using ITS1 PCR-RFLP method of the ribosomal DNA of Leishmania. An epidemiological study on CL cases was reported between 2000 and 2016 in this current investigation in six provinces in southwestern Morocco. Statistical analysis was performed using a linear regression model to identify the impact as well as the interaction between all predictor variables on the distribution of CL in the studied provinces. The forecast Holt-Winters (HW) method was used to describe the trend and seasonality of CL cases. The ITS1-PCR- RFLP analysis revealed the presence of Leishmania tropica in all studied provinces. The spatial distribution of CL cases documented in all studied provinces during the sixteen years showed a heterogeneous pattern and fluctuation trend with an average prevalence of 9.92 per 100,000 inhabitants. In addition, the forecast HW model predicts continued variability of trend and seasonality of CL cases in the upcoming years. This study confirmed the importance of socioeconomic factors, in particular poverty and the vulnerability rate, on distribution and emergence of CL. This study revealed a relationship between increasing risk of CL occurrence due to Leishmania tropica, as well as the distribution and emergence thereof, and socioeconomic factors in the investigated area.

Molecular Detection of Leishmania DNA in Wild-Caught Phlebotomine Sand Flies (Diptera: Psychodidae) From a Cave in the State of Minas Gerais, Brazil.

Leishmania spp. are distributed throughout the world, and different species are associated with varying degrees of disease severity. In Brazil, Leishmania transmission involves several species of phlebotomine sand flies that are closely associated with different parasites and reservoirs, and thereby giving rise to different transmission cycles. Infection occurs during the bloodmeals of sand flies obtained from a variety of wild and domestic animals, and sometimes from humans. The present study focused on detection of Leishmania DNA in phlebotomine sand flies from a cave in the state of Minas Gerais. Detection of Leishmania in female sand flies was performed with ITS1 PCR-RFLP (internal transcribed spacer 1) using HaeIII enzyme and genetic sequencing for SSUrRNA target. The survey of Leishmania DNA was carried out on 232 pools and the parasite DNA was detected in four: one pool of Lutzomyia cavernicola (Costa Lima, 1932), infected with Le. infantum (ITS1 PCR-RFLP), two pools of Evandromyia sallesi (Galvão & Coutinho, 1939), both infected with Leishmania braziliensis complex (SSUrRNA genetic sequencing analysis), and one pool of Sciopemyia sordellii (Shannon & Del Ponte, 1927), infected with subgenus Leishmania (SSUrRNA genetic sequencing analysis). The present study identified the species for Leishmania DNA detected in four pools of sand flies, all of which were captured inside the cave. These results represent the first molecular detection of Lu cavernicola with Le infantum DNA, Sc sordellii with subgenus Leishmania DNA, and Ev sallesi with Leishmania braziliensis complex DNA. The infection rate in females captured for this study was 0.17%.

Determination of Leishmania species by PCR-RFLP in the smear samples taken from the lesions of cutaneous leishmaniasis cases

The forms of the disease caused by Leishmania species in Turkey as well as in Aegean region are cutaneous and visceral leishmaniasis (CL and VL, respectively), and the agent of CL is commonly L.tropica. However, L.infantum was also reported as being CL agent recently. Direct microscopic examination, serological tests and culture are the conventional methods used for the diagnosis of CL. Since the specificities of these methods are high their sensitivities are variable and identification at species level is not possible. Recently, the use of polymerase chain reaction (PCR)-based molecular methods enabled the rapid and reliable diagnosis and species identification. The aim of this study was to investigate the performance of PCR-restriction fragment length polymorphism (RFLP) method both for the detection and identification of Leishmania species simultaneously in CL patients. A total of 30 smear samples that were positive for Leishmania amastigotes with microscopic examination, obtained from CL-suspected cases admitted to Adnan Menderes University Medical School Hospital, Parasitology Laboratory (located at Aydin, in the Aegean region of Turkey) between 2012-2014 period were included in the study. Ten samples taken from the skin lesions caused by Staphylococcus aureus (n= 5) and Candida albicans (n= 5) were also included as negative controls. DNA extractions from the smears were performed by the use of a commercial kit (Macherey-Nagel NucleoSpin Tissue® Kit, Germany). DNA isolation was also performed from L.major, L.infantum and L.tropica promastigotes that were grown in culture as positive controls. In PCR method LITSR and L5.8S primers targeting to ITS (internal transcribed spacer)-1 region were used. In RFLP method, the amplified PCR products were cleaved by BsuRI (HaeIII) restriction enzyme for the species identification. As a result, restriction profiles of all samples (n= 30) were in accordance with L.tropica restriction profile. No band was observed in the control samples (n= 10). The data of this study showed that the most common CL agent in Aydin is L.tropica. In conclusion, ITS-1 PCR-RFLP method may be used directly as a single routine procedure for both the detection and identification of Leishmania species in the clinical samples of CL patients, in laboratories with adequate facilities.

Genetic diversity of Leishmania tropica strains isolated from clinical forms of cutaneous leishmaniasis in rural districts of Herat province, Western Afghanistan, based on ITS1-rDNA

Despite the high incidence of cutaneous leishmaniasis (CL) in Afghanistan, there is a little information concerning epidemiological status of the disease and phylogenetic relationship and population structure of causative agents. This study was conducted to determine the prevalence and distribution of CL cases and investigate the Leishmania tropica population structure in rural districts of Heart province in the West of Afghanistan in comparison to neighboring foci. Overall, 4189 clinically suspected CL cases from 177 villages (including 12 districts) in Herat province were enrolled in the referral laboratory of WHO sub-office in Herat city from January 2012 to December 2013. 3861 cases were confirmed as CL by microscopic examination of Giemsa-stained slides. ITS1 PCR–RFLP analysis showed dominance of L. tropica (more than 98%) among 127 randomly chosen samples. Analysis of the ITS1 sequences revealed 4 sequence types among the 21 L. tropica isolates. Comparison of sequence types from Herat rural districts with the representatives of L. tropica from Iran, India, and Herat city showed two main population groups (cluster A and B). All isolates from Herat province, India and Southeast, East, and Central Iran were found exclusively in cluster A. The close proximity of West Afghanistan focus and Birjand county as the capital of Southern Khorasan province in East Iran can explain relatively equal to the genetic composition of L. tropica in these two neighboring regions. In addition, two populations were found among L. tropica isolates from Herat rural districts. Main population showed more similarity to some isolates from Birjand county in East Iran while minor population probably originated from the Southeast and East Iranian L. tropica. Recent study provided valuable information concerning the population structure of L. tropica and epidemiology of ACL in the West of Afghanistan, which could be the basis for molecular epidemiology studies in other regions of Afghanistan.

Detection, molecular typing and phylogenetic analysis of Leishmania isolated from cases of leishmaniasis among Syrian refugees in Lebanon

Leishmania is a parasitic protozoan with more than two-dozen species causing the disease leishmaniasis. It is transmitted to humans through the bite of an infected female phlebotomine sand-fly vector. In the past two years the incidence of leishmaniasis has been drastically increasing in Lebanon. This was in parallel with the deterioration of the security in Syria forcing thousands to flee and seek shelter in poorly maintained refugee camps and collective shelters. Cutaneous leishmaniasis (CL) is now considered a public health problem, but its epidemiology has not been fully elucidated. To our knowledge, this is the first study comparing two different molecular methods for the detection and identification of Leishmania tropica in Lebanon.Two molecular typing methods of 39 FFPE Leishmania isolates were used: the ITS1-PCR RFLP and the nested ITS1-5.8S rDNA gene amplification followed by sequencing and phylogenetic analysis. The efficiency of these two techniques in Leishmania identification was compared and the phylogenetic relationships among these isolates were illustrated based on the neighbor-joining (NJ) method. The results were statistically correlated with the parasitic index (PI). The DNA storage in formalin-fixed paraffin embedded (FFPE) tissues was assessed as well. The parasites identified were all L. tropica as determined by both techniques. ITS1-5.8S rDNA gene based typing proved to be more sensitive in the detection of parasites (positive in 69.2% of the isolates) as opposed to the ITS1-PCR RFLP method that was successful in identifying L. tropica in only 43.6% of the isolates. Sequencing and phylogenetic analysis revealed high levels of heterogeneity. A statistically significant correlation was observed between PI and the results of the nested ITS1-5.8S rDNA gene PCR. Genotyping at the species level is essential for monitoring the relative frequency of CL in the Mediterranean area that is correlated to three different Leishmania species (Leishmania infantum, Leishmania major and L. tropica), each characterized by distinct epidemiological features. The obtained results highlight the need to find a universally accepted diagnostic tool for Leishmania typing.

Phylogenetic structure of Leishmania tropica in the new endemic focus Birjand in East Iran in comparison to other Iranian endemic regions

Iran has been identified being among the countries with the highest number of cutaneous leishmaniasis (CL) cases. South Khorasan province in East Iran is an emerging focus of CL. Species identification of sixty clinical samples by ITS1 PCR-RFLP presented evidence for the dominance of Leishmania tropica (90%) in this region. Analysis of the ITS1 sequence of 19 L. tropica isolates revealed seven closely related sequence types. In addition, ITS1 sequences available in GenBank from other Iranian regions were compiled for comparison with the studied isolates. Iranian L. tropica was distributed in two main clusters. All East Iranian sequence types were grouped with strains from foci from Southeast and Central regions in cluster A, showing highly similar sequences. The highest similarity was observed between most L. tropica from East and all isolates from Southeast regions and from Savojbolagh county in Central Iran. Southwest L. tropica was shown to be paraphyletic as the isolates were distributed in both clusters A and B. All Northeastern L. tropica were part of cluster B, however they showed significant heterogeneity and were distributed in different subclusters. Distribution of L. tropica populations was to some extent congruent with genetic lineages of Phlebotomus sergenti in Iran and may be an evidence for parasite-vector co-evolution. Southeast-East L. tropica was also similar to strains from Herat province in Afghanistan at the East border of Iran. This is the first comprehensive study on population structure of L. tropica in Iran that provides a guideline for appropriate sampling for further molecular based epidemiological studies.

Molecular detection of Leishmania spp. isolated from cutaneous lesions of patients referred to Herat regional hospital, Afghanistan.

Cutaneous leishmaniasis is one of the main public health problems in Afghanistan, particularly in Herat. To identify Leishmania spp., molecular techniques were applied to samples from 64 cutaneous leishmaniasis patients referred to Herat regional hospital during 2013. Polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the ribosomal RNA gene internal transcribed spacer-1 (ITS1) was used. Most of the patients demonstrated dry type single lesions on the head. The results of direct microscopy detection using Giemsastained skin scrapings were compared with that of ITS PCR-RFLP for the diagnosis of cutaneous leishmaniasis. Light microscopy examination showed 37/64 positive cases (58%). PCR revealed 50 positive cases (78%), from which ITS PCR-RFLP identified 48 cases (96%) as L. tropica and 2 cases (4%) as L. major. Cutaneous leishmaniasis in Herat appears to be endemic and of the clinically dry type, caused mainly by L. tropica and occasionally by L. major.