Use of single-enzyme PCR-restriction digestion barcode targeting the internal transcribed spacers [ITs rDNA] to identify dermatophyte species

Dermatophytes are the most common causative agents of superficial mycoses. Species identification of these fungi is important from therapeutic and epidemiological point of wive. Traditional approaches for identification of dermatophytes at the species level, relying on macroscopic and microscopic features of the colonies, usually are time-consuming and unreliable in many circumstances. Recently a broad varieties of rapid and accurate DNA-based techniques were successfuly utilized for species delineation of dermatophytes. The ITS1-5.8S-ITS2 region of rDNA from various reference strains of dermatophyte species were amplified using the universal fungal primers ITS1 and ITS4.The PCR products were digested by a single restriction enzyme, MvaI. The enzyme was evaluated in both in silico and practical PCR-RFLP assay to find the exact differentiating restriction profiles for each species. To validate the standardized PCR-RFLP system, all tested strains were subjected to sequencing and sequence analysis. The obtained RFLP patterns were specific for many species including T. interdigitale, T. rubrum, T. violaceum, M. persicolor, M. audouinii, M. nanum [A. obtusum] and E. floccosum but were similar for some closely related species such as M. canis / M. ferrugineum. Sequencing of the ITS1-5.8S-ITS2 fragment from all type strains affirmed the RFLP findings. It was practically revealed that the ITS-PCR followed by MvaI-RFLP is a useful and reliable schema for identification and differentiation of several pathogenic species and can be used for rapid screening of even closely related species of dermatophytes in clinical and epidemiological settings

Mycological microscopic and culture examination of 400 bronchoalveolar lavage [BAL] samples

The frequency of invasive opportunistic mycoses has increased significantly over the past decades especially in immunocompromised patients. Invasive aspergillosis [IA] has become a major cause of morbidity and mortality among these patients. As bronchoalveolar lavage [BAL] fluid samples are generally useful specimens in the diagnosis of invasive pulmonary aspergillosis [IPA], this study was designed to evaluate the incidence of fungal elements in at-risk patients by direct microscopy and culture of BAL samples. In a 16-month period, 400 BAL samples were obtained from several groups of different patients with pulmonary and respiratory disorders and examined by using both direct microscopy and culture. Of the 400 samples, 16 [4%] were positive direct examination with branching septate hyphae and 46 [11.5%] were positive culture: 25 [54%] Aspergillus flavus, 6 [13%] A. fumigatus, 5 [10.9%] A. niger, 1 [2.2%] A. terreus, 3 [6.5%] Penicillium spp. and 6 [13%] mixed A. flavus/A. niger. A. flavus was the most common cause of Aspergillus infection or colonization. Bone marrow transplant [BMT] recipients were the most susceptible group to fungal infection and/or colonization. Among Aspergillus species, A. flavus was the most common isolate in both infections and colonization in Iran. More studies are needed to clarify the epidemiological aspect of aspergillosis in Iran

Preliminary identification and typing of pathogenic and toxigenic fusarium species using restriction digestion of ITS1-5.8S rDNA-ITS2 region

Fusarium species are capable of causing a wide range of crop plants infections as well as uncommon human infections. Many species of the genus produce mycotoxins, which are responsible for acute or chronic diseases in animals and humans. Identification of Fusaria to the species level is necessary for biological, epidemiological, pathological, and toxicological purposes. In this study, we undertook a computer-based analysis of ITS1-5.8SrDNA-ITS2 in 192 GenBank sequences from 36 Fusarium species to achieve data for establishing a molecular method for specie-specific identification. Sequence data and 610 restriction enzymes were analyzed for choosing RFLP profiles, and subsequently designed and validated a PCR-restriction enzyme system for identification and typing of species. DNA extracted from 32 reference strains of 16 species were amplified using ITS1 universal primers followed by sequencing and restriction enzyme digestion of PCR products. The following 3 restriction enzymes TasI, ItaI and CfoI provide the best discriminatory power. Using ITS1 and ITS4 primers a product of approximately 550bp was observed for all Fusarium strains, as expected regarding the sequence analyses. After RFLP of the PCR products, some species were definitely identified by the method and some strains had different patterns in same species. Our profile has potential not only for identification of species, but also for genotyping of strains. On the other hand, some Fusarium species were 100% identical in their ITS1-5.8SrDNA-ITS2 sequences, therefore differentiation of these species is impossible regarding this target alone. ITS-PCR-RFLP method might be useful for preliminary differentiation and typing of most common Fusarium species

Evaluation of QIAamp DNA mini kit for removing of inhibitors in detection of Cryptosporidium parvum oocysts in water samples by a nested- PCR assay

In recent years, there has been a dramatic increase in the occurrence of waterborne disease outbreaks caused by the Cryptosporidium parvum, and presence of this protozoan parasite in drinking water is a significant health problem faced by the water industry. A new strategy for detection of Cryptosporidium oocysts in water samples is PCR' based techniques. In this study a nested' PCR assay was designed for the specific amplification of a 199 bp DNA fragment of the gene encoding the heat shock protein [hsp70] of Cryptosporidium parvum oocysts. In order to prevent the inhibition of PCR amplification by substances contained in water samples, three DNA purification methods including QIAamp DNA mini kit, InstaGene Matrix, MagExtractor ' Genome were compared in concentrates of tap water samples spiked with the oocysts. After it was found that the QIAamp is only efficient purification technique, the efficiency of QIAamp and immunomagnetic separation for nested'PCR assay of various water samples was compared. The results show that QIAamp provide a useful and rapid tool for removing of PCR inhibitors. It seems that QIAamp purification- nested PCR assay is a sensitive, rapid and cost effective method for detection of Cryptosporidium parvum oocysts in clean water samples with turbidity < 2 nephelometric turbidity unit [NTU]

nested-PCR assay for detection of cryptosporidium parvum oocysts in water samples

Cryptosporidiosis is a gastroenteric disease caused by the protozoan parasite Cryptosporidium parvum. Water-borne transmission of this organism has become more prevalent in recent years. Current method for detection of C. parvum oocysts in water is immunofluoresence assay [IFA]. The method is time consuming, laborious and particularly not-specific. It cannot determine the infectivity of detected oocysts. We have evaluated a nested- PCR assay for sensitive detection of C. parvum oocysts in water samples. Water sample concentrates were spiked with Cryptosporidium oocysts and after DNA extraction and purification by QIAamp DNA mini kit, detection was achieved by nested PCR amplification of a 200 bp region of hsp70 gene specific for C. Parvum. The method could detect as few as one oocyst in seeded tap water samples. On the basis of these results, PCR could be a useful tool in the monitoring of water samples for the detection of Cryptosporidium oocysts