Suspected Cases Of Cutaneous Leishmaniasis Research Articles

Epidemiological survey, molecular profiling and phylogenetic analysis of cutaneous leishmaniasis in Khyber Pakhtunkhwa, Pakistan.

Cutaneous leishmaniasis (CL), an emerging vector-borne ailment in Khyber Pakhtunkhwa (KPK), Pakistan, exhibits diverse spread patterns and outbreaks. To comprehend its epidemiology and identify parasite species, we conducted an active survey on suspected CL cases (n=8845) in KPK. Microscopy and internal transcribed spacer-1 PCR-restriction fragment length polymorphism (RFLP) molecular techniques detected Leishmania spp. in blood samples. Phylogenetic analysis gauged genetic affinities with other areas. District Bannu displayed the highest CL impact (14.58%), while Swat had the lowest impact (4.33%) among cases. Annual blood examination rate, parasite incidence and slide positivity rate were 4.96 per 1000 people, 0.0233 and 0.047%, respectively. CL infections were prevalent in 1- to 20-y-olds, with males (57.17%) more vulnerable than females (42.82%). Single lesions occurred in 43.73% of patients, while 31.2% people had two lesions, 17.31% had three lesions and 7.74% had more than three lesions. Most had sand-fly exposure but lacked preventive measures like repellents and bed nets. Leishmania tropica was confirmed via RFLP analysis in amplified samples. Phylogenetic analysis unveiled genetic parallels between L. tropica of KPK and isolates from China, Iran, Afghanistan, India, Syria and Morocco. Urgent comprehensive control measures are imperative. Early detection, targeted interventions and raising awareness of CL and sand-fly vectors are vital for reducing the disease's impact. International collaboration and monitoring are crucial to tackle Leishmania spp.'s genetic diversity and curtail its cross-border spread.

Evaluation of expression variations in virulence-related genes of Leishmania major after several culture passages compared with Phlebotomus papatasi isolated promastigotes.

Cutaneous leishmaniasis (CL) is a prevalent infectious disease with considerable morbidity annually. Here, we aimed to investigate the likely variations in gene expression of glycoprotein63 (gp63), heat shock protein 70 (HSP70), histone, arginase, cysteine protease B (CPB), Leishmania homologue of receptors for activated C kinase (LACK), small hydrophilic endoplasmic reticulum-associated protein (SHERP) in metacyclic promastigotes of L. major isolated from Phlebotomus papatasi sand flies and promastigotes excessively cultured in culture medium. The parasites were collected from suspected CL cases in Pasteur Institute of Iran, cultured and inoculated into the female BALB/c mice (2×106 promastigotes). Sand flies were trapped in Qom province, fed with the blood of euthanized infected mice and subsequently dissected in order to isolate the midgut including stomodeal valve. The metacyclic promastigotes were isolated from Ph. papatasi (Pro-Ppap) using peanut agglutinin test (PNA), then continuously cultured in RPMI-1640 medium enriched with fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 mg/ml) to reach stationary phase (Pro-Stat). The gene expression was evaluated in both parasitic stages (Pro-Ppap and Pro-Stat) using qRT-PCR. Out results showed a significant increased gene expression at Pro-Ppap stage for gp63 (P = 0.002), SHERP (P = 0.001) and histone (P = 0.026) genes, in comparison with Pro-Stat stage. Noticeably, significant changes were, also, demonstrated in 10th to 15th passages [gp63 (P = 0.041), arginase (P = 0.016), LACK (P = 0.025)] and in 5th to 20th passage (SHERP) (P = 0.029). In conclusion, the findings of the present study seem to be essential in designing Leishmania studies, in particular regarding host-parasite interaction, immunization and infectivity studies.

A Preliminary Study to Compare Recombinase Polymerase Amplification-Lateral Flow and Quantitative PCR in the Detection of Cutaneous Leishmania in Communities from the Volta Region of Ghana.

Background: Leishmaniasis is a parasitic disease that mostly affects populations in tropical and subtropical countries. In Ghana, cutaneous leishmaniasis (CL) is the most common form of the disease affecting communities of the Volta Region. Conventional parasitological method (microscopy) is the commonly used test for CL diagnosis in many endemic countries, but has low sensitivity in chronic cases. Therefore, there is a clear need for a sensitive and easy-to-use point-of-care diagnostic method like an isothermal recombinase polymerase amplification-lateral flow (RPA-LF) test, suitable for use in austere and low-resource settings for the identification of CL cases. This study compared the efficacy of RPA-LF test with quantitative PCR (qPCR) in detecting Leishmania in suspected CL cases from the Volta Region. Methods: Twenty-five participants between 5 and 14 years were enrolled in the study from whom a total of 26 samples were obtained. Lesion samples were collected using FTA® filter papers applied to ulcerated lesions for molecular diagnosis. DNA isolated from filter papers was used for both the RPA-LF test and qPCR. Results: Twenty-two participants (88%) presented with one or two ulcerated active lesions per individual, while the rest of them had plaques or dried lesions. Among the 26 samples, 19/26 (73%) had concordant results when comparing the two diagnostic methods. Conclusion: Data from this study suggest that the RPA-LF test can be used in addition to a conventional parasitological diagnostic test (microscopy) to detect CL cases in communities of the Volta Region.

Highly sensitive nested polymerase chain reaction to improve the detection of Leishmania species in clinical specimens.

Cutaneous leishmaniasis (CL) is one of the most neglected tropical diseases and an important health problem in many countries. It is an endemic disease in most regions of Iraq, while being non-endemic in the Kurdistan Region. The techniques frequently used for detection of CL are not very sensitive. Therefore, this study aimed to identify a sensitive method for diagnosis of CL in clinical samples. The present study was performed in December 2019 to December 2020 in Kalar General Hospital. Clinical samples were collected from 85 suspected CL cases. Sixty-four (75.29%), 71 (83.53%) and 84 (98.82%) cases were detected as positive for CL by microscopy, PCR, and nested PCR, respectively. Of the 84 nested PCR-confirmed CL patients, 46 (54.8%) were female and 38 (45.2%) were male. The most predominate rate of infection was in the 30-39-year age group (29.76%) and the lowest was in the ≥ 60-year group (3.57%). Forty (47.62%) patients had a single lesion. The statistical analysis showed significant differences between age groups and between the number of lesions. The sensitivities of microscopy, conventional PCR, and nested PCR were 80.77%, 86.6% and 100%, respectively, while all three methods showed 100% specificity. Furthermore, PCR-ITS1 followed by a simple restriction fragment length polymorphism (RFLP) analysis using HaeIII endonuclease indicated that Leishmania major was responsible for all CL infections in the study area.

Molecular characterization and genetic diversity of cutaneous leishmaniasis from North Eastern Pakistan

The genetic diversity of Leishmania spp. in North Eastern Pakistan remains undetermined despite increased cases of cutaneous leishmaniasis (CL). This study was designed to decipher the molecular characterization and genetic diversity of Leishmania spp. in North Eastern Pakistan. Out of 13761 CL suspected cases, 567 cases were microscopically positive and confirmed as Leishmania spp. by internal transcribed spacer (ITS) gene amplification through the PCR- RFLP technique. Further, isolates were directly sequenced to conduct phylogenetic analysis for genetic diversity. Among suspected CL cases, Mirpur showed the highest proportion of CL infection with 4.85% (118/2431) of the cases, while the Neelum district showed the lowest percentage at 3.29% (9/273). The slide positivity rate, annual blood examination rate, and annual parasite incidence rate were 3.84, 0.27, and 0.01% respectively, and the incidence of CL in the age group 1–20 years old was higher in males (50.92%) than females (25.75%). The RFLP analysis and sequencing confirmed the occurrence of Leishmania tropica, Leishmania major, and Leishmania infantum. Leishmania tropica (p = 0.02) confirmed significantly higher nucleotides variation than L. major (p = 0.05). Current findings confirmed the prior assumption that anthroponotic CL is the primary CL form present in North Eastern Pakistan. Moreover, this is the first report based on molecular identification of L. major, and L. infantum from North Eastern Pakistan. This remarkable heterogeneity in the Leishmania spp. is the leading cause of treatment failure and emergence of new haplotypes. Therefore more extensive investigations are recommended from all geographical regions of North Eastern Pakistan, especially those using a large sample size.

Modelling the number of dermal lesions in anthroponotic cutaneous leishmaniasis and its associated factors in Herat province, western Afghanistan, during 2012–2013

Cutaneous leishmaniasis (CL), caused by Leishmania tropica, is the main causative agent of CL in the Herat province, western Afghanistan. In CL patients, one of the most important indicators for reducing the amount of body damage and the choice of treatment is the number of dermal lesions, but there is no strong evidence in this regard. Therefore, using count regression models, the main objective of this historical cohort study was to identify the distribution of the number of dermal lesions and determine the associated factors among CL patients referring to leishmaniasis reference laboratory in Herat province, from January 2012 to December 2013. In a total of 4,127 clinically suspected CL cases, 50.20% were female and 49.80% male. Based on the results of ZINB model, which is the best model suggested by goodness-of-fit criteria, age variables (IRR=1.007), duration of lesion (6-12weeks compared to<6weeks with IRR=1.36 and >12weeks with IRR=1.39 compared to <6weeks), type of lesion(both nodule and ulcer compared to papule with IRR=2.03), location of lesion (trunk with IRR=1.90, upper with IRR=1.66, lower with IRR=1.61 and mix with IRR=10.35 compared to head/neck/ears) and type of lesion (ulcer compared to papule with IRR=0.50, nodule and lupoid, respectively, as IRR=0.72 and IRR=0.51 compared to papule) had a significant effect on the mean number of dermal lesions (p<.05). Also, our results showed that among four models used, the ZINB model represented a better performance to determine the associated probable factors about the number of dermal lesions in ACL patients. Moreover, there was a direct association between age and number of dermal lesions; in addition, duration of lesion, type of lesion and location of lesion had a significant effect on the number of dermal lesions. Therefore, a comprehensive planning is necessary for controlling and reducing the number of dermal lesions.

Investigation of Polymerase Chain Reaction Method in Patients with Suspected Chronic Cutaneous Leishmania of Negative Microscopy

Leishmaniasis is a parasitic disease that is transmitted to humans by the bites of infected female phlebotomine sandflies. In the diagnosis of cutaneous leishmaniasis (CL), in the smear samples, the demonstration of the parasite by microscope remains a gold standard method. However, it becomes difficult to diagnose the parasite since the number of amastigotes in chronic cases with a lesion of one year or longer is very low. Due to many factor such as patients primarily do not to take any notice these lesions in their bodies, do not apply to health institutions or late applied, receive wrong treatment; the diagnosis and treatment are delayed. In addition, it is been worse prognosis by add secondary infection to lesions and wounds become chronic. For this reason, molecular methods are used in addition to microscopic examination in chronic suspected CL cases. It was aimed to reveal of the molecular diagnostic value in chronic suspected CL cases by polymerase chain reaction (PCR) in the smear belonging to Turkish patients that reported to be evaluated clinically because it can not be seen Leishmania amastigotes in microscopic examination. Smear of 50 Turkish patients who were clinically reported of the evaluation of chronic CL were selected. These samples were smears belonging to suspected CL patients that applied Hatay Mustafa Kemal University, Faculty of Medicine, Parasitology laboratory from different polyclinics and were decided to be evaluated clinically as a result of microscopic examination because they came from endemic regions (such as Hassa, Altınözü, Yayladağı). DNA was isolated from selected samples and PCR was performed using 13A, 13B primers targeting the kinetoplastid DNA (kDNA) region. The samples found positive by PCR were typed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using LITSR and L5.8S primers targeting internal transcribed spacer (ITS-1) region. Of the 50 smear samples, 17 (34%) were determined positive with 13A, 13B primers targeting the kinetoplastid DNA (kDNA) region. Positive samples were also found to be positive with LITSR and L5.8S primers targeting ITS-1 region. The PCR products obtained from PCR with ITS-1 gene region were digested with the restriction endonucleases BsuRI (HaeIII). As a result of PCR-RFLP analysis, it was determined that 11 of Leishmania tropica, one of Leishmania major and five of Leishmania infantum/donovani out of 17 samples. Chronic CL can be confused with skin diseases such as sarcoidosis, tuberculosis, malignant tumors. In particular, chronic CL cases can be escaped the attention for many reasons such as failure to diagnose correctly, insufficient microscope experience, fail to see due to low number of parasites. For this reason, it was concluded that PCR, which is a molecular method, should be used in chronic suspected CL samples which are negative for the parasite by microscopic examination.

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.

Molecular and parasitological study of cutaneous leishmaniasis in Bushehr province, southwest of the Islamic Republic of Iran: a cross-sectional study during 2009-2012.

Cutaneous leishmaniasis (CL) is one of the most important parasitic disease in Iran. CL is distributed among more than half of 31 provinces of Iran. Studies on epidemiological aspects of the disease and Leishmania species identification among infected humans are necessary for providing a comprehensive prevention and control program thus; this descriptive cross-sectional study was conducted on all CL suspected patients who referred to Health Centers of Bushehr province from 2009 to 2012. Physical examinations were carried out in suspected individuals and CL cases were confirmed by microscopical examinations. Prepared slides from suspicious cases of CL were fixed with absolute methanol and stained by Giemsa 10%. All the Giemsa-stained slides examined under a light microscope with high magnification (1,000×) and classified them based on grading of Leishmania parasites. DNA from each slide was extracted, separately. The ribosomal internal transcribed spacer 1 was amplified with specific primers and PCR products were digested by restrict enzymes (HaeIII), run them in 3% gel agarose for electrophoresis and visualized on a UV transilluminator after staining with ethidium bromide. SPSS version 21 was used for data analyses. A total of 726 suspected CL cases were referred to Health Centers of Bushehr province from 2009 to 2012 and samples were only prepared from 188 of the patients whereas 43 (5.9%) of them were microscopy positive. The most frequent of CL was observed in November (14%) and December (12%). The most distribution of CL lesions were observed on hands (32%), feet (26%), and face (21%), respectively. The highest frequency of CL was observed in 1-9years old (30%). Altogether, 50% of the patients showed one skin lesion and 2-10 skin lesions were occurred in the remained CL patients. Totally, 27 out of 43 (63%) of the Giemsa stained slides were positive by PCR-RFLP assay because all the PCR-RFLP negative slides were prepared 3-4years ago and kept without cover slip, and also observed scarce amastigotes during microscopy observations. Leishmania species were identified in 21 desirable slides which 14 of them were L. major and 7 of the remained isolates were identified L. tropica using PCR-RFLP.

ITS-1 DNA SEQUENCE CONFIRMATION OF LEISHMANIA MAJOR AS A CAUSE OF CUTANEOUS LEISHMANIASIS FROM AN OUTBREAK FOCUS IN THE HO DISTRICT, SOUTHEASTERN GHANA

We report first-time definitive evidence of human cutaneous leishmaniasis (CL) resulting from L. major infections in Ghana. The evidence is in the form of PCR testing and DNA sequence analysis of skin sample biopsies from residents of the Ho District in the forested Volta Region of southeastern Ghana (Figure 1). The moist semi-deciduous forest of this region of Ghana is atypical for CL. Although two sand fly species with known or suspected potential to transmit L. major were present in communities where human cases of CL originated, they were found in such small numbers that their role in the transmission of CL is uncertain. Twelve million people worldwide are believed to be infected with the parasites that cause leishmaniasis, and 88 different nations report this disease. 1 It is estimated that up to 75% of the 1.5 million new cases of leishmaniasis that occur each year are CL with L. major the responsible etiologic agent of this cutaneous disease in Africa and throughout Eurasia. Over this vast range, CL transmission is typically associated with arid desert and savannah conditions where the parasites are harbored in rodents and vectored by night-biting sand flies of the genus Phlebotomus. For many years, leishmaniasis in Africa and Western Asia has been grossly underestimated; it now appears that the disease is much more prevalent than previously suspected. 1 In Ghana, CL was considered a parasitic disease risk that was limited to the arid sahel-savannah region in the north, but no infections or evidence of transmission had been actually described. Thousands of man-hours of mosquito surveillance have been conducted in northern Ghana 2 for the purpose of quantifying man-biting malaria vectors, but there is no record or report of sand flies in these collections. However, since 1999, an increasing number of suspected CL cases have been reported from the southern Volta Region of Ghana in a moist forest ecosystem where such skin lesions had not been previously reported. The assumption of CL was based subjectively on local microscopy, which identified Leishmania amastigotes in skin lesion biopsies taken from a cluster of local patients. Between 1999 and 2002, the Ghana Health Service recorded 2,426 suspected cases of CL in the Ho, Hohoe, and Kpando Districts (2,348, 2, and 76 cases, respectively). In 2003, the number of suspected cases rose to 6,450 (6,185, 174, and 91 in the same respective districts) with 116 villages affected (Ghana Ministry of Health (MoH), Annual Report, 2003). A limited survey of towns in the Ho district during 2002 identified suspected CL lesions in 12.2–32.3% of local school children. In an effort to determine the cause and extent of the outbreak, the Ghana MoH initiated an epidemiologic study in Ho district with assistance from the U.S. Naval Medical Research Unit No. 3 and the Noguchi Memorial Institute of Medical Research. Initial efforts of our study intended to verify the presence of Leishmania parasites by applying newly developed PCR methods to 5 previously collected, archived biopsy samples