Molecular Detection of a Pathogenic Entamoeba among Symptomatic Children in Eastern Kurdistan of Iraq.

Entamoeba histolytica infects the large intestine of humans, causing a spectrum of clinical appearances ranging from asymptomatic colonization to severe intestinal and extra-intestinal disease. The parasite is identical microscopically to commensal nonpathogenic amoeba. To detect the pathogenic Entamoeba and estimate the precise prevalence of the parasite among the symptomatic pediatric population using molecular techniques. 323 fecal samples were collected from symptomatic children admitted to Sulaimani Pediatric Teaching Hospital, Sulaimaniyah Province, Iraq, from June to October 2021. A structured, validated questionnaire was prepared and used to report participants' gender, residency, and drinking water source. Then, stool samples were microscopically examined, and the positive samples were submitted to molecular analysis by amplifying the 18s rRNA gene using nested PCR to differentiate E. histolytica from other nonpathogenic Entamoeba. Finally, gene sequences were done to confirm the species. Microscopically, 58 positive samples represented Entamoeba species infection rate of 18% among symptomatic patients. However, only 18 samples were positive for E. histolytica based on molecular methods, which accounts for 31% of the positive by microscopy and 5.6% among the 323 symptomatic populations. NCBI, available in their database, gives the gene sequence and accession number. Patients' sociodemographic data and water sources were directly related to the infection rate. Classical microscopic examination provides a misleading profile about the prevalence of E. histolytica in an endemic region that might lead to unnecessary treatments and a lack of appropriate management for patients.

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-based assay for genotyping Leishmania spp. from clinically suspected cutaneous leishmaniasis lesions in the Garmian area, Kurdistan Region of Iraq.

Cutaneous leishmaniasis (CL) is highly prevalent in southern Iraq and neighboring countries, but is non-endemic to the Kurdistan Region of Iraq, particularly in the Garmian area. This study aimed to investigate the causative agent of CL at the molecular level by amplifying the small subunit (18S) rRNA and internal transcribed spacer 1 (ITS1) region. The present study was conducted from December 2019 to December 2020 at Kalar General Hospital, Kalar, Kurdistan Region, Iraq. Eighty-five clinical specimens were collected selectively from patients with suspected CL lesions via fine needle aspiration. After parasitic genomic DNA was extracted from the removed fluid, PCR and DNA sequencing targeting the 18S rRNA and ITS1 region were performed for molecular detection and species identification. Additionally, for 14 samples, the target bands of amplified DNA fragments for both 18S rRNA and ITS1 were extracted and sequenced via Sanger method using both the directional primers employed in the PCR. Seventy-one (83.53%) of the 85 suspected patients had CL, based on amplification of 18S rRNA and ITS1 via PCR. The sequence analysis revealed that all samples were Leishmania major. Phylogenetic analysis based on ITS1 was also performed. Our study revealed that our molecular method was an efficient technique for detecting CL and a valuable method for identifying Leishmania species in clinical samples. Sequence analysis indicated that the causative agent of CL in the Garmian area was L. major and the disease was rural in origin.