[Molecular identification of corynebacterium minutissimum in skin lesions suspicious for erythrasma by the PCR method]

Erythrasma is a chronic superficial infection of the intertriginous areas. Most laboratories use methylene blue stain and 10% KOH smear to identify Corynebacterium minutissimum [C. minutissimum] by direct observation of filamentous bacilli. Occasionally atypical forms can be seen that create problems in diagnosis. This study aims to use the polymerase chain reaction [PCR] method in order to definitively identify C. minutissimum as an agent of erythrasma. This research was performed during 2013 on 100 skin scrapings suspicious for erythrasma which were obtained from various medical mycology laboratories in Tehran. Samples were tested by three methods - direct examination, culture and PCR. DNA was extracted by the modified phenol-chloroform method after which PCR was performed using designed primers. We sequenced some of the PCR products. The sensitivity and specificity of the PCR method was compared to the direct and culture examinations. Of the 100 samples, there were 25 positive samples according to PCR analysis, 13 positive by direct examination and 23 that cultured positive. DNA sequencing results showed the presence of C. minutissimum. The PCR method in comparison with direct examination had a sensitivity of 100% and a specificity of 86.2%. The study also showed that the PCR method in comparison with culture had a sensitivity of 100% and a specificity of 97.4%. This study showed that the PCR method in comparison with the direct method and culture had a higher sensitivity in the detection of C. minutissimum. The present PCR method confirmed all typical and some of the atypical forms of C. minutissimum which indicated the importance of this method in the definitive diagnosis of erythrasma

[Isolation and identification of fusarium species from maize and wheat and assessment of their ability to produce fumonisin B[1]]

Fusarium species are prevalent contaminants of foodstuffs and agricultural crops. They produce fumonisins, which are carcinogenic mycotoxins. The present study has evaluated maize and wheat samples from ten provinces in Iran that were contaminated with Fusarium species. Special attention was paid to the ability of the isolates to produce fumonisin B[1] [FB[1]] as a public health hazard. We collected 32 maize and 15 wheat samples from ten provinces that were major cultivation areas. Samples surface disinfected with a 1% sodium hypochlorite solution for 2 minutes. Fusarium species were isolated by the flotation method on malachite green agar. Pure cultures on potato dextrose agar [PDA] were identified using a combination of macroscopic and microscopic morphological criteria. The ability of the isolates to produce FB[1] was evaluated by thin layer chromatography [TLC] and the amounts of fumonisin B[1] produced were assessed by high performance liquid chromatography [HPLC]. A total of 55 Fusarium isolates that belonged to five species were isolated. There were 27 of the 32 maize samples [84.4%] and 11 of 15 wheat samples [73.3%] that were contaminated with Fusarium species. Species consisted of F. verticillioides [23 isolates], F. proliferatum [22 isolates], F. subglutinans [5 isolates], F. nygamai [4 isolates] and F. redolens [1 isolate] based on morphological criteria. Twenty-two of the 55 [40%] Fusarium isolates produced FB[1] in a total range from 230.4 to 9565 microg/ml. The highest amounts of FB[1] production were related to toxigenic isolates of F. verticillioides and F. proliferatum. Results of the present work indicates a high degree of contamination of maize and wheat with Fusarium strains that belong to the Gibberella fujikuroi species complex, particularly F. verticillioides and F. proliferatum. This contamination is a potential public health threat due to food spoilage and subsequent production of high levels of carcinogenic FB[1]

A duplex PCR for rapid and simultaneous detection of Brucella spp. in human blood samples

OBJECTIVE@#To design a duplex PCR for rapid and simultaneous detection of Brucella species. in human blood samples.@*METHODS@#Fifty-two peripheral bloods samples were collected from suspicious patients with brucellosis. Following DNA extraction, PCR assay were performed, using three primers that could simultaneously identify and differentiate three major species of pathogenic Brucella in humans and animals.@*RESULTS@#Of the 52 peripheral bloods samples tested, 25 sample (48%) showed positive reactions in PCR. Twelve samples were positive for Brucella abortus 39 (B. abortus 39) (23%), 13 for Brucella melitensis 39 (B. melitensis 39) (25%) and 0 for Brucella ovis 39 (B. ovis 39) (0%).@*CONCLUSIONS@#This work demonstrates that in case where specific primers were utilized, duplex PCR has proved to be a simple, fast, and relatively inexpensive method for simultaneous detection of important species of Brucella in clinical samples.

Rapid detection of trichophyton rubrum in clinical samples from tinea unguium using PCR

Dermatophytosis is one of the most common pandemic fungal infections that is a major health problem in cities and villages. This study aims to evaluate PCR sensitivity and accuracy in the detection of nail dermatophytosis compared to conventional direct and culture detection methods, and performs an assessment of Trichophyton rubrum in patients suspected of having nail dermatophytosis. This experiment was a descriptive-experimental study carried out on 71 nail samples obtained from patients with suspected nail dermatophytosis. All clinical samples of nails or chips were divided into three sections and each section underwent direct examination, culture and molecular tests. In the molecular test, we used fungal rRNA universal primers [ITS1 and ITS4] and Trichophyton rubrum-specific primers. In this study, for the first time in Iran and based on a modified protocol, DNA was directly extracted from tissues of infected nails in less than five hours. Additionally a comparison of the results obtained from routine laboratory methods such as direct examination and culture with PCR verified the high sensitivity and accuracy of PCR compared to the other studied methods. PCR, as a rapid, accurate method, can be a good replacement for conventional culture and direct examination

Simultaneous detection and differentiates of Brucella abortus and Brucella melitensis by combinatorial PCR

OBJECTIVE@#To evaluate simultaneous detection and differentiates of Brucella abortus (B. abortus) and Brucella melitensis (B. melitensis) through the combinatorial PCR method.@*METHODS@#This study was designed using three primers that could simultaneously identify and differentiate two major species of pathogenic Brucella in humans and animals. Identification and differentiation of each species using the size of the PCR product were determined. To determine the specificity of the method, bacteria close to the genus Brucella were used. Finally, to confirm PCR products, In addition to the products sequence, RFLP was performed on PCR products using restriction enzymes.@*RESULTS@#The method of optimized combinatorial PCR in this study could simultaneously detect and differentiate B. abortus and B. melitensis with high specificity and sensitivity in clinical samples. Differentiation of species is based on the resulting bands; therefore, the band 494 bp for B. abortus and 733 bp for B. melitensis were obtained. RFLP and sequencing results confirmed PCR results.@*CONCLUSIONS@#The results of this study shows that without routine diagnostic methods such as culture and serology tests, using the molecular method of combinatorial PCR, important species of Brucella can be simultaneously identified and differentiated in clinical samples.