An unlabelled probe-based real time PCR and modified semi-nested PCR as molecular tools for analysis of chloroquine resistant Plasmodium vivax isolates from Afghanistan.

BACKGROUND: Plasmodium vivax resistance to chloroquine (CQ) has been reported from many endemic regions in the world. Plasmodium vivax is responsible for 95% of malaria cases in Afghanistan and CQ is the first-line treatment given for vivax malaria. The pvmdr-1 and pvcrt-o (K10 insertion) genes are possible markers for CQ-resistance in P. vivax isolates. There have been no studies done on the presence or absence of molecular markers for CQ-resistance P. vivax in Afghanistan. The present work aimed to evaluate the frequency of mutations in the pvmdr-1 and K10 insertion in the pvcrt-o genes of P. vivax. METHODS: Plasmodium vivax isolates were collected from Laghman, Baghlan and Khost provinces. For investigation of polymorphisms of desired regions in pvmdr-1 and pvcrt-o genes, sequencing was applied on the PCR products. A new asymmetric qPCR and melting analysis assay based on unlabelled probe developed for scanning of K10 insertion in pvcrt-o gene. RESULTS: The analysis of sequencing data of the pvmdr-1 gene showed wild type Y976 and K997 and mutant M958 and L1076 in 33 isolates from three provinces. Of the 36 samples evaluated for K10 insertion in pvcrt-o, 2/18(11%), 0/10(0%) and 0/8(0%) isolates from Laghman, Baghlan and Khost province, respectively, possessed K10 insertion, confirmed by either sequencing and unlabelled probes. CONCLUSION: Two samples with K10 insertion and 33 samples with pvmdr1 polymorphism, indicating on the possibility of CQ resistance in P. vivax populations in Afghanistan. Furthermore, unlabelled probes are simple and inexpensive alternative tools for screening of P. vivax mutations.

Evaluation of bacterial co-infections of the respiratory tract in COVID-19 patients admitted to ICU.

BACKGROUND: COVID-19 is known as a new viral infection. Viral-bacterial co-infections are one of the biggest medical concerns, resulting in increased mortality rates. To date, few studies have investigated bacterial superinfections in COVID-19 patients. Hence, we designed the current study on COVID-19 patients admitted to ICUs. METHODS: Nineteen patients admitted to our ICUs were enrolled in this study. To detect COVID-19, reverse transcription real-time polymerase chain reaction was performed. Endotracheal aspirate samples were also collected and cultured on different media to support the growth of the bacteria. After incubation, formed colonies on the media were identified using Gram staining and other biochemical tests. Antimicrobial susceptibility testing was carried out based on the CLSI recommendations. RESULTS: Of nineteen COVID-19 patients, 11 (58%) patients were male and 8 (42%) were female, with a mean age of ~ 67 years old. The average ICU length of stay was ~ 15 days and at the end of the study, 18 cases (95%) expired and only was 1 case (5%) discharged. In total, all patients were found positive for bacterial infections, including seventeen Acinetobacter baumannii (90%) and two Staphylococcus aureus (10%) strains. There was no difference in the bacteria species detected in any of the sampling points. Seventeen of 17 strains of Acinetobacter baumannii were resistant to the evaluated antibiotics. No metallo-beta-lactamases -producing Acinetobacter baumannii strain was found. One of the Staphylococcus aureus isolates was detected as methicillin-resistant Staphylococcus aureus and isolated from the patient who died, while another Staphylococcus aureus strain was susceptible to tested drugs and identified as methicillin-sensitive Staphylococcus aureus. CONCLUSIONS: Our findings emphasize the concern of superinfection in COVID-19 patients due to Acinetobacter baumannii and Staphylococcus aureus. Consequently, it is important to pay attention to bacterial co-infections in critical patients positive for COVID-19.

Application of Nested-qPCR-High Resolution Melting (HRM) Technology on Strongyloides stercoralis Isolates from Iran.

PURPOSE: Strongyloides stercoralis is a parasite with special characteristics presenting it as a unique nematode. Iran is an endemic area for S. stercoralis. In this study, nested-qPCR-high resolution melting (HRM) technology was applied on some human isolates of S. stercoralis from this country by focusing on evolutionary genetics analysis. METHODS: Twelve human isolates of S. stercoralis were collected from four endemic provinces of Iran. Genomic DNA was extracted from a single filariform larva for every isolate. Using specific primers targeting partial regions in cox1 gene, nested-qPCR-HRM was performed and melting-curve profiles were analyzed alongside the evaluation of genetic proximity and phylogenetic analysis using MEGA7 and DnaSP5 software. RESULTS: The melting temperature (Tm) values of the isolates were 77.9 °C-78.3 °C. All isolates from Guilan, Mazandaran, and Khouzestan Provinces shared Tm values of 78.2 °C to 78.3 °C, while the isolates from Hormozgan Province showed Tm values of 77.9 °C, 78.0 °C, and 78.1 °C. The phylogenetic tree illustrated that the sequences of the current study included nine haplotypes. Tajima's D index analyses showed that cox1 gene in S. stercoralis isolates was negative (Tajima's D = - 0.27). CONCLUSION: The isolates were divided into five temperature groups. Although HRM assay compared to PCR sequencing identified more limited genetic changes, it revealed that the mean of Tm of the isolates from Hormozgan Province was lower than those of other provinces and represented specific haplotypes for this geographical region on the phylogenetic tree.

A Comprehensive Comparison of Rapid RNA Extraction Methods for Detection of SARS-CoV-2 as the Infectious Agent of the Upper Respiratory Tract using Direct RT-LAMP Assay.

Background: The current COVID-19 pandemic has highlighted the need for faster and more cost-effective diagnostic methods. The RNA extraction step in current diagnostic methods, such as real-time qPCR, increases the cost and time required for testing. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is a promising technique for developing diagnostic tests with desired sensitivity and specificity without the need for RNA extraction. Materials and Methods: An RT-LAMP assay was developed to detect SARS-CoV-2 with a sensitivity of 0.5 copies of positive control plasmid per microliter in 40 min. Several rapid RNA extraction protocols were evaluated using different reagents, including bovine serum albumin, Triton X-100, Tween 20, proteinase K, guanidine hydrochloride, guanidinium isothiocyanate (GITC), and thermal treatment. Finally, the sensitivity and specificity of the developed direct RT-LAMP were determined using 150 upper respiratory tract samples. Results: Method 10 was selected as the most efficient protocol for the RNA extraction step. The sensitivity and specificity of the developed direct RT-LAMP assay with clinical samples were estimated at 98.4% and 88.8%, respectively. Conclusion: These results suggest that the combination of GITC and Triton X-100 detergent is a highly efficient method for RNA extraction and direct RT-LAMP detection of SARS-CoV-2 in clinical samples, providing a valuable tool for the rapid and cost-effective diagnosis of COVID-19.

Accurate Identification of Leishmania Parasites in Sand Flies by Polymorphism Analysis of Cytochrome Oxidase Subunit 2 Gene Using Polymerase Chain Reaction and Quantitative PCR-High Resolution Melting Techniques in Iranian Border with Iraq.

Background: Firmly identification of Leishmania in Phlebotomus papatasi and understanding of natural transmission cycles of parasites in sand flies are important for treatment and local control. Methods: Modified and developed method of High Resolution Melting (HRM) as a preferable technique was employed to accurate identification of Leishmania in sand flies from Iranian border with Iraq, by targeting cytochrome oxidase II (COII) gene and designing suitable primers. PCR products cloned into pTG19-T vector, then purified plasmid concentration was measured at 260 and 280nm wavelength. The melting curve plots were generated and DNA sequences were analyzed using Sequencher 3.1.1, CLC Main Workbench 5.5, MEGA 6, DnaSP5.10.01 and MedCalc® version 13.3.3 soft wares. Results: Among about 3000 collected sand flies, 89 female Ph. papatasi were identified and two with L. major. In amplified fragment of COII gene among 611bp, 452bp had no genetic variations with low polymorphic sites (P= 0.001) and high synonymous (79.8%) as compare to non-synonymous sites (20.2%). Leishmania major was discriminated in Ph. papatasi with 0.84 °C melting temperature (Tm) and unique curve based on thermodynamic differences was an important criterion using HRM technique. Conclusion: Subsequent war in Iraq made a high risk habitat for parasites transmission. It is important to discover accurate diagnostic procedures for leishmaniasis control.

Evaluation of Drug Resistant Genotypes to Fansidar and Chloroquine by Studying Mutation in Pfdhfr and Pfmdr1 Genes in Plasmodium falciparum Isolates from Laghman Province, Afghanistan.

Background: Malaria is one of the major health problems in endemic countries like Afghanistan. Evidence has been reported about reducing the effects of chloroquine against Plasmodium falciparum in many endemic countries. The aim of this study was to investigate the resistance mutations in pfmdr1 and pfdhfr genes of P. falciparum samples detected in blood samples of malaria patients in Laghman Province, Afghanistan. Methods: Samples were taken on DNA retention cards and 3 glass slides (thin and thick spread) from Laghman Province, Afghanistan in 2018. The pfmdr and pfdhfr mutations in 30 P. falciparum positive samples were examined using PCR-RFLP techniques. The PCR product was then sequenced to determine the mutation at the N86Y and D1246Y mutations of the pfmdr1 and N51, C59, I164, S108 and A16 points of pfdhfr genes. Results: In the pfmdr1 gene, all samples were wild-type and no mutation was detected at point 86 and D1246Y. In the pfdhfr gene sequences using CLC main workbench software no mutations were detected at codons 16, 51. However, some mutation was observed at codons 59, 108 and 164. These mutations were L164I, S108N and C59R. Conclusion: Our findings provide evidence of the possible emergence of fansidar-resistant specimens in Laghman. The data of this study provide the basis for future prospective studies in other endemic areas of Afghanistan. The absence of significant mutations in P. falciparum samples of Laghman Province may indicate that this parasite may have switched to chloroquine re-sensitization in this area.

Discrimination of human papillomavirus genotypes using innovative technique nested-high resolution melting.

The prompt detection of human papillomavirus and discrimination of its genotypes by combining conventional methods in new molecular laboratories is essential to achieve the global call of eliminating cervical cancer. After predicting the melting temperature of an approximately 221 bp region of the L1 gene from different HPV genotypes by bioinformatics software, an innovative technique based on the nested- high resolution melting was designed with three approaches and using conventional PCR, qPCR, and diagnostic standards. HPV-positive samples identified by microarray along with diagnostic standards were evaluated by qPCR-HRM and discordant results were subjected to sequencing and analyzed in silico using reference types. In addition to screening for human papillomavirus, nested-qPCR-HRM is one of the modified HRM techniques which can discriminate some genotypes, including 6, 16, 18, 52, 59, 68 and 89. Despite the differences in diagnostic capabilities among HRM, microarray and sequencing, a number of similarities between HRM, and sequencing were diagnostically identified as the gold standard method. However, the bioinformatics analysis and melting temperature studies of the selected region in different HPV genotypes showed that it could be predicted. With numerous HPV genotypes and significant genetic diversity among them, determining the virus genotype is important. Therefore, our goal in this design was to use the specific molecular techniques with several specific primers to increase sensitivity and specificity for discriminating a wide range of HPV genotypes. This approach led to new findings to evaluate the ability of different approaches and procedures in accordance with bioinformatics.

Developing, Modifying, and Validating a TaqMan Real-Time PCR Technique for Accurate Identification of Leishmania Parasites Causing Most Leishmaniasis in Iran.

Many laboratory methods are used to diagnose leishmaniasis because it is characterized by varied symptoms and caused by different Leishmania species. A quantitative real-time PCR method based on a TaqMan probe was developed and modified for accurate identification of human cutaneous leishmaniasis (caused by Leishmania major or Leishmania tropica) from endemic areas of Iran. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were considered. Six new sets of species-specific primers and probes were designed. A total of 123 samples were examined and employed to evaluate and validate real-time PCR. According to parasitic load of the genesig®Leishmania Advanced Standard Kit, a serial dilution of purified plasmid (2-2×107 copies/reaction) was prepared under the same conditions for both genes. Specific primers and probes were able to detect three and six parasite copies in AAP3 and COII genes, respectively, and were able to detect three copies of parasites for L. major and L. tropica. The sensitivities of the reference kit and our method were 98.7 and 98.1%, respectively, and specificity was 100% for detecting parasite genomes in all assays. Designed primers and probes performed well in terms of efficiency and regression coefficient. For AAP3 and COII genes, respectively, the linear log range was 7 and the correlation coefficient (R2) was 0.749 and 0.996 for the reference kit using the standard generated curve and 0.98 and 0.96 with serial dilutions of parasite DNA. This research detected L. major and L. tropica definitely and opens the horizon for the other scientists in the multiplex reactions in designing and optimization of the conditions in silico and in vivo.

Molecular Evaluation of the Novel Coronavirus Infection of Cockroaches and Flies Collected from Kamkar-Arabnia Hospital in Qom City, Central Iran: With Innovated Internal Control.

Background: Due to the confirmation of the presence of the novel coronavirus in the feces and municipal sewerage system, and the feeding of domestic insects from fecal matter, as well as the ability of these insects to mechanically transmit microbes from the sewerage system. This study was aimed at molecular evaluation of the novel coronavirus infection isolated on cockroaches and flies collected from Kamkar-Arabnia Hospital in Qom City, Iran. Methods: Totally, 18 samples; (12 samples cockroaches and 6 flies) from the external surface of cockroaches and houseflies as well as their digestive system were prepared. After designed and synthetized exogenous heterologous internal control, the RNA was extracted to investigate the contamination of these samples with the novel coronavirus. To detect the virus, the E and RdRp genes were identified. Results: Investigation of coronavirus E gene using the multiplex one-step qPCR technique on the collected samples showed an amplification plot in CT= 35.70 related to the internal surfaces of cockroaches collected from the treatment and sick room of the hospital. Also, the design of internal control to ensure the accuracy of the extraction process was successful. Conclusion: According to the findings of the present study regarding detecting the presence of the coronavirus infection in the digestive system of domestic insects such as American cockroaches and considering their ability to mechanically transmit viruses, it is recommended to control the domestic insects that are in close contact with humans in crowded places such as hospitals and health centers during the Covid-19 pandemic.

Designing and developing of high-resolution melting technique for separating different types of Toxoplasma gondii by analysis of B1 and ROP8 gene regions.

BACKGROUND: Determination of Toxoplasma gondii genotypes plays an important role in the health management and epidemiology of toxoplasmosis. We developed HRM analysis to differentiate genotypes of T. gondii using the B1 and ROP8 genes, through comparing the sensitivity and specificity of both genes and methods used for the detection of T. gondii. METHODS: A total of 96 DNA samples of muscle tissue of livestock and poultry brain tissue with three standard strains RH (type I), PRU (type II) and VEG (type III) were prepared and analyzed. Three methods of nested PCR, PCR-PCR and nested-qPCR-HRM were used. Specific new primers were designed and synthesized for developing HRM. Thirty positive samples obtained from nested-qPCR-HRM were sequenced (18 B1 and 12 ROP8). RESULTS: Overall, 87 infected samples were identified using both genes. Through the B1 gene, we could separate type I (Tm = 84.8 °C) from II/III types (Tm = 84.6 °C). Also, the ROP8 gene could separate type II (Tm = 84.5 °C) from I/III types (Tm = 84.12 °C). Highest sensitivity (100%) and specificity (78.72%) were observed by nested-qPCR-HRM assays of the B1 and ROP8 genes than by other methods, respectively. Thus, the B1 gene can be used to most accurately detect T. gondii, while the ROP8 gene was more appropriate for T. gondii genotyping. PCR-sequencing results were consistent with HRM results in most selected samples. CONCLUSION: HRM analysis is a powerful diagnostic tool for rapid detection and determination of main clonal lineages, and even unusual T. gondii genotypes.

Phylogenetic Analysis and Genetics Polymorphisms Evaluation of ROP8 and B1 Genes of Toxoplasma gondii in Livestock and Poultry Hosts of Yazd, Qom and Golestan Provinces of Iran.

BACKGROUND: A high correlation is observed between specific clonal lineages and host types in toxoplasmosis. The main objectives of this study were comparing polymorphism and evolutionary analysis of the B1 and ROP8 genes, as well as the evaluation of phylogenic and Toxoplasma gondii isolates obtained from different hosts and regions. METHODS: Overall 96 brain/diaphragm tissue samples of livestock and poultry from three provinces of Iran (cows: 9 from Yazd, 9 from Qom; sheep: 19 from Yazd, 7 from Qom; goats: 7 from Yazd, 4 from Qom; one camel from Yazd and 37 chickens, 2 roosters and one duck from Golestan) were tested during 2018-19. A nested PCR and PCR-PCR methods were developed with the B1 and ROP8 genes. Evaluation of genetic proximity, genetic diversity and evolutionary analysis were done using MEGA-X and DnaSP5 software. Thirty samples of both genes were sequenced (18 B1 and 12 ROP8 genes), and submitted to the GenBank (MN275903-MN275932). RESULTS: Tajima's D index analyses showed that both genes were in the negative direction of evolution. The B1 gene was more sensitive than the ROP8 gene. The ROP8 gene showed better and more acceptable results in terms of the relationship between the host and the genotyping of the samples. CONCLUSION: The B1 gene was only an attractive target for rapid detection of T. gondii parasites, whereas the ROP8 gene due to a high level of polymorphism was able to isolate the three clonal lineages (type I, II and III), intertypes and even atypical strains from different isolates of T. gondii.

Development of an Innovative Method by Optimizing qPCR Technique for Isolating and Determining Oxalobacter Formigenes Microbial Load in the Stool of Patients with Urolithiasis.

INTRODUCTION: Oxalobacter formigenes, as a gram-negative anaerobic bacterium, metabolizes oxalate in the intestine by the enzymes oxalyl-CoA decarboxylase (OXC) and formyl-CoA transferase (FRC). Therefore, not only the presence of the bacterium but also microbial load may affect intestinal absorption and urinary exertion. We evaluated the relationship between Oxalobacter formigenes load and the formation of calcium oxalate urolithiasis using quantitative molecular methods. METHODS: By clinical manifestation and stone analysis, we selected the urine and stool specimens of 73 patients with calcium oxalate urolithiasis. First, the gene regions of the two genes FRC and OXC in Oxalobacter formigenes were selected utilizing bioinformatics and specific primers designed for these regions. Following DNA extraction from stool specimens by specific primers of each gene, PCR was carried out and positive samples were sequenced. Then, qPCR was applied to determine the effective load of Oxalobacter. Also, biochemical tests were performed to measure the excretion rate of oxalate in urine specimens. RESULTS: In addition to oxalobacter identification by PCR, the load of bacteria was quantitatively assessed using qPCR by specific primers for both FRC and OXC gene regions. A significant negative relationship had found between the formation of calcium oxalate urolithiasis and the presence of Oxalobacter formigenes in patients with kidney stone disease. The mean excretion of oxalate and citrate in urolithiasis cases were 22.93 and 552.106 mg/24h, respectively. CONCLUSION: The presence of Oxalobacter formigenes can highly inhibit the generation of calcium oxalate urolithiasis. Furthermore, molecular techniques are more effective than other methods such as culture for the isolation of this bacterium.

Designing and developing a high-resolution melting technique for accurate identification of Leishmania species by targeting amino acid permease 3 and cytochrome oxidase II genes using real-time PCR and in silico genetic evaluation.

Discrimination, accurate identification, and reliable techniques are required for accurate identification of Leishmania parasites. High-resolution melting (HRM) is recognized as an authentic and exact method. The main objective of this research was optimizing HRM analysis for detecting and screening Leishmania major, Leishmania tropica and mix infections. Thirty-six DNA samples of Leishmania parasite were prepared and analyzed. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were targeted and six pairs of specific new primers were designed. Bioinformatics analysis was employed to predict DNA temperature resolution for each species and compared with in-vitro results. The genetic diversity of the selected gene regions was analyzed using PCR-sequencing method and DnaSP 5.10.01 software. They were submitted in GenBank (KU680818- KU680821 and KY041643- KY041649). The haplotype diversity for both AAP3 and COII genes was 96% and 87%, respectively. Tajima's D index was 0.65 for AAP3 and 0.36 for COII. CLC Genomics Workbench 11 software predictions were significant and close to these findings. The designed primers could be able to identify at least two Leishmania species. Temperature variations in HRM technique separated Iranian Leishmania parasites of L. major, L. tropica and mix infections. The target genes and our modified HRM method proved this technique could be useful in both clinical and experimental settings. Also, it can be effective for detecting Leishmania parasites in different hosts such as humans, reservoir hosts and vectors. Indeed, HRM can be used as a technique in Leishmania identification as well as for ecological and epidemiological research.

Assessing genetic evolution and detecting human papillomavirus by matching two complementary highly sensitive approaches, nested-qPCR and sequencing.

Becoming armed with an appropriate strategy to isolate the minimum number of human papillomaviruses (HPV), regardless of DNA extraction method, can be a huge step in preventing false negative; it has a significant effect on the management and control of HPV infection among women's population. This study was conducted in Qom province, considering the risk factors associated with HPV. It was able to analyze genetic evolution in its genotypes and evaluated the limit of detection by a new diagnostic approach. Totally, 486 Pap smear samples were tested; then, the HPV DNA was developed by a semi-nested quantification PCR. Positive samples were sequenced and submitted to the GenBank (MG825048-MG825061). After alignment, phylogenetic and polymorphism analyses were performed on the sequenced samples with a number of GenBank sequences. The overall HPV prevalence among all women in Qom was 11.7%. HPV6 (43.24%) and HPV16 (6.75%) were the most frequent LR and HR genotypes, respectively. Although the Tajima's D of all genotypes was positive, it was negative individually. The position of genotypes 6, 11, and 73 was controversial on phylogenetic trees. Limit of detection (LOD) was obtained as about 10-100 copies per reaction in various genotypes of HPV by semi-nested qPCR. The nature of HPV could be preserved during natural selection. This research, through innovative usage of the primers, could detect different genotypes of the HPV, and inform the women society of the probable risk through its prevalence determination.

Critical diagnosis of complicated strongyloidosis with nested-PCR and High Resolution Melting analysis (HRM)

Diagnosis of strongyloidosis is sometimes problematic and requires novel techniques. Here, critical diagnosis of a complicated case of strongyloidosis using molecular methods is reported. A young woman referred to the Diagnostic Laboratory of Strongyloidiasis in School of Public Health, Tehran University of Medical Sciences. She had taken albendazole before referring to the laboratory. She had cerebral edema, behavior disorders, hypereosinophilia and titer of IgE >2000 IU/mL. The patient had history of intestinal and skin disorders and steroid therapy. For detection of Strongyloides stercoralis infection, parasitological techniques and novel methods of nested-PCR and HRM analysis were applied on stool samples upon admission and during the following up. On the samples provided upon first admission, parasitology showed negative results, while both molecular methods revealed infection with S. stercoralis. After specific treatment, during the following up, the patient general health was much improved and the results of all parasitological and molecular tests were negative for strongyloidosis. Application of novel sensitive diagnostic methods for detection of S. stercoralis is necessary, especially once parasitological techniques have lack of sensitivity.

Assessment of nuclear and mitochondrial genes in precise identification and analysis of genetic polymorphisms for the evaluation of Leishmania parasites.

The polymorphism and genetic diversity of Leishmania genus has status under discussion depending on many items such as nuclear and/or mitochondrial genes, molecular tools, Leishmania species, geographical origin, condition of micro-environment of Leishmania parasites and isolation of Leishmania from clinical samples, reservoir host and vectors. The genetic variation of Leishmania species (L. major, L. tropica, L. tarentolae, L. mexicana, L. infantum) were analyzed and compared using mitochondrial (COII and Cyt b) and nuclear (nagt, ITS-rDNA and HSP70) genes. The role of each enzymatic (COII, Cyt b and nagt) or housekeeping (ITS-rDNA, HSP70) gene was employed for accurate identification of Leishmania parasites. After DNA extractions and amplifying of native, natural and reference strains of Leishmania parasites, polymerase chain reaction (PCR) products were sequenced and evaluation of genetic proximity and phylogenetic analysis were performed using MEGA6 and DnaSP5 software. Among the 72 sequences of the five genes, the number of polymorphic sites was significantly lower as compared to the monomorphic sites. Of the 72 sequences, 54 new haplotypes (five genes) of Leishmania species were submitted in GenBank (Access number: KU680818 - KU680871). Four genes had a remarkable number of informative sites (P=0.00), except HSP70 maybe because of its microsatellite regions. The non-synonymous (dN) variants of nagt gene were more than that of other expression genes (47.4%). The synonymous (dS)/dN ratio in three expression genes showed a significant variation between five Leishmania species (P=0.001). The highest and lowest levels of haplotype diversity were observed in L. tropica (81.35%) and L. major (28.38%) populations, respectively. Tajima's D index analyses showed that Cyt b gene in L. tropica species was significantly negative (Tajima's D=-2.2, P<0.01), while COII and nagt genes were produced through evolutionary processes for both L. tropica and L. major (Tajima's D=2.85 & 2.91, P<0.01). More different clinical lesions with extensive phylogenetic and evolutionary analyses should be employed to avoid confusion in the diagnosis of leishmaniasis and development of vaccines for eradicating Leishmania parasites.

Design and Validation of Real-Time PCR: Quantitative Diagnosis of Common Leishmania Species in Iran.

OBJECTIVE: Design and validation of Real-time PCR on the protected gene region ITS2 to quantify the parasite load in common leishmania (L) species. MATERIALS AND METHODS: Probe and primer were designed from the ITS2 region between the rRNA genes with minimum gene variation in three common leishmania species followed by a Real-time PCR using the Taq man probe method in the form of absolute quantification. A series of different concentrations of leishmania were analyzed. After the purified PCR product was successfully placed in a PTG19-T plasmid vector, specialized ITS2 region was cloned in this plasmid. In the last phase, the cloned gene was transferred to the Ecoli.Top10F bacteria. The standard plasmid was provided in 10(7) to 10(1) copies/rxn concentrations. The specification and clinical sensitivity of the data was analyzed using inter and intra scales. RESULTS: The probe and primer were designed using three species, including L. infantum, L. major, and L.tropica. Seven concentrations of purified parasite in culture media showed that the selected region for quantifying the parasite is suitable. Clinical and analytical specificity and sensitivity were both 100%, respectively. CONCLUSION: The Taq man method for the ITS2 region in leishmania is one the most sensitive diagnostic test for identifying the parasite load and is suggested as a tool for fast identification and quantification of species.

Molecular pathology and histopathological findings in localized leishmania lymphadenitis.

BACKGROUND: A rare variant of Leishmaniasis is Localized Leishmania Lymphadenitis which has been occasionally reported from south-eastern parts of Iran. So far, no molecular assay has been performed for diagnosing this variety of Leishmaniasis. METHODS: Nineteen lymph node paraffin blocks were collected from 1994 to 2007. Parasite load count and histopathological patterns reported on Hematoxylin-Eosin and Giemsa stained slides.DNA extraction was carried out just on the remaining available 7 lymph node paraffin blocks according to QIAamp DNA FFPE kit instructions. A pair of primers and a probe were designed for rRNA ITS region with Allele ID 6.0 software, followed by real time PCR amplification. RESULT: The most common histopathological pattern was necrotizing granuloma with few Leishman bodies. Parasite load was the highest in submental lymph node (3 ± 1.41 per oil field) which was significantly higher compared to cervical and inguinal nodes (P < 0.05). Absolute load of parasite DNA was detectable in all 7 cases. The positive cases revealed a 201 bpamplicon after electrophoresis of end product which was confirmative for Leishmania tropica. CONCLUSION: Real time PCR revealed Leishmania tropica as the etiologic agent of Localized Leishmania Lymphadenitis. Although this molecular method is a sensitive diagnostic tool, histopathological findings are still important.