Construction of an internal amplification control for Mycobacterium tuberculosis polymerase chain reaction (PCR) test

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB) which mostly affects the lungs. The disease causes deaths of many people every year. There are different methods to detect MTB such as skin test, staining, culture and molecular techniques. Polymerase chain reaction (PCR) is a simple and rapid method for the detection of MTB; however, positive and negative false results reduce the efficiency of this technique. The aim of this study was to design an internal amplification control (IAC) and apply it in MTB PCR test. PCR technique for MTB was optimized by using specific primers for IS6110 gene. The sensitivity and specificity of the test were determined. IAC was constructed with competitive strategy by PCR-cloning technique and the limitation range was determined. The PCR products of MTB and IAC were 245 and 660 bp, respectively on electrophoresis gel. The IC used in PCR testing of MTB is the competitive form in which the range was between 10 million and 10 bacteria and the most suitable internal control concentration for the mix was 1,000 plasmids. After making IC and using it in MTB amplification, it was observed that IC might guarantee the correctness of PCR reaction.Keywords: Mycobacterium tuberculosis, polymerase chain reaction (PCR), internal control.African Journal of Biotechnology Vol. 12(27), pp. 4277-428

Low prevalence of antifungal resistant Candida africana, in the C. albicans complex causing vulvovaginal candidiasis

The Candida (C.) albicans complex includes C. albicans, C. dubliniensis, C. stellatoidea, and C. africana, with the last mentioned as an important emerging agent of vulvovaginal candidiasis (VVC). The aim of the study was to identify C. africana and C. dubliniensis and assess their drug susceptibility in vaginitis. One-hundred Candida isolates of the C. albicans complex from women diagnosed with vaginitis and from vaginal samples in the culture collection of a medical mycology laboratory were examined. Species of the C. albicans complex were identified with conventional and molecular methods using polymerase chain reaction (PCR) for amplification and sequencing of the internal transcribed spacer (ITS) region, PCR for partial amplification of hyphal wall protein 1 (HWP1) gene and duplex PCR. The effects of antifungal drugs were evaluated according to standard broth microdilution protocols. Ninety-seven C. albicans (97) and three C. africana (3) isolates were identified. Results of susceptibility testing revealed one isolate of C. africana to be resistant to both clotrimazole and fluconazole, and one showed reduced susceptibility to itraconazole. Identification of Candida species especially C. africana in vaginitis is crucial, there are varying levels of resistance to antifungal drugs. © 2020 Microbiology; Mycology; Microbial genomics; DNA sequencing; Antimicrobial; Women's health; Candida albicans complex; Candida dubliniensis; Candida africana; Drug susceptibility; Vulvovaginal candidiasis; HWP1. © 202

Comparison of polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) for diagnosis of Fusarium solani in human immunodeficiency virus (HIV) positive patients

Fusarium solani is the most virulent Fusarium sp., frequently reported in the fatal disseminated fusariosis in immunocompromised patients. However, it has been always considered, as a very rare or case report infection among HIV positive patients. Anyhow, because of its irreparable consequences, early diagnosis is very important. In this study, loop-mediated isothermal amplification (LAMP) method was developed for rapid and specific detection of F. solani in serum samples of human immunodeficiency virus (HIV) positive patients. Transcription elongation factor (TEF-1α) region was considered as the target gene. The test was carried out in 1 h reaction at 65°C in a heater block. The specificity of the test was 100% and its sensitivity was a copy of genome. Using this method among 45 DNAs samples extracted from HIV positive patients’ serums, 9 (20%) cases were positive for F. solani. All of the samples were rechecked by polymerase chain reaction (PCR) and the results were the same. Considering these results, it was concluded that due to advantages of the LAMP technique, it can be a better alternative for PCR, even in low technology laboratories. In addition, these findings revealed that   the possibility of fatal fusariosis due to F. solani is not so rare in HIV positive patients.Keywords: Fusarium solani, loop-mediated isothermal amplification (LAMP), HIV, polymerase chain reaction (PCR)African Journal of Biotechnology, Vol 13(13), 1496-150

Identification of Yeast Species In the Oral Cavity of Iranian Soldiers By Disk Diffusion Method

Background:The disk diffusion method for identification of yeasts species was performed based on different but distinct susceptibilities of yeasts spp.to chemicals:janus green, ethidium bromide,2,3,5-triphenyltetrazolium chloride, brilliant green, cycloheximide and rhodamine 6G. Methods: Atotal of 568 Iranian soldiers went under study for isolation and identification of Yeast species from their oral cavity. Asterile swab was used for each individual and specimens were collected from the nasopharynx region, then inoculated to petri dishes containing Sabouraud Dextrose Agar and incubated for 48 hrs at 37 °C. All colonies were counted and stocked in distilled water and stored in a refrigerator for further analysis. The yeasts were identified by the “disk diffusion test” [6,8]. This is a simple, rapid, accurate, and inexpensive technique presented by Sobczak [8]. By this method we identified yeast species within 24-48 hrs. Results: 51.4% of petri dishes were positive for yeast species and 318 strains were identified. Candida albicans, Candida kefyr, Candida tropicalis and Candida guilliermondii were the most common yeast species isolated from the oral cavity of soldiers. Conclusion: We used this method because of its simplicity and other beneficial characteristics for rapid identification of large and numerous isolates and the results were compared with other morphological characters such as chlamydospore and germ tube production. In addition,we used some type strains (Candida parapsilosis: PTCC 5089,Candida tropicalis: PTCC 5028,Saccharomyces cerevisiae:PTCC 5052,Candida lipolytica: PTCC 5063,Candida lipolytica:PTCC 5064),and the results were acceptable

Application of PCR method in specific identification of Fusarium solani in HIV positive patients

Background and Objective: Fusarium solani is the common etiological agent of fungemia and disseminated fusariosis, which is associated with high incidence of mortality in immune-compromised host. Due to high level of resistance of antifungals in Fusarium solani, rapid and specific identification of organism is essential. This study was done to evaluate the PCR method for rapid and specific diagnosis of Fusarium solani in serum samples of HIV positive patients. Methods: In this descriptive study, the PCR test based on mitochondrial cytochrome b gene as the target gene with 330 bp product was optimized. PCR was applied on 45 serum samples of HIV positive patients after evaluation of sensitivity and specificity of the test. Results: In the optimized PCR test, the 330 bp product was amplified. The sensitivity of the test was a copy of Fusarium solani genome, and its specificity was 100%. Among 45 serum samples, 9 cases (20%) were positive for Fusarium solani. Conclusion: The PCR method has functional capabilities for direct, rapid and specific clinical diagnosis of Fusarium solani in HIV positive patients

A study of hydraulic fracturing clean-up efficiency in unconventional gas reservoirs using statistical approaches

Hydraulic fracturing is widely used to improve well productivity especially in unconventional reservoirs. This costly operation, however, sometimes underperforms. One of the main reasons for this poor performance is poor clean-up efficiency of injected fracturing fluid (FF). In this work, a parametric study of FF clean-up efficiency of hydraulic fractured vertical wells was performed with 49152 simulations (in 12 sets) consisting of injection, soaking and production periods. Due to the large number of required simulations, that were conducted using a commercial reservoir simulator, a developed computer code was used to automatically read input data, run simulations and creates output data. In each set (consisting of 4096 runs), simultaneous impacts of 12 parameters (fracture permeability, matrix permeability and capillary pressure, end points and exponents of Corey gas and FF relative permeability curve in both matrix and fracture) were studied. To sample the variables domain and analyse results, two-level full factorial experimental design and linear surface model describing dependency of gas production loss (GPL), compared to 100 clean-up, to pertinent parameters at three production periods (10, 30 and 365 days) were considered and supported by the tornado charts of fitted equations, frequency of simulations with given GPL and FF saturation maps. Results indicate that generally parameters controlling FF mobility within fracture had greatest impact on GPL reduction. However in sets with very low matrix permeability especially when applied pressure drop during production is low, the effect of fluid mobility in the matrix on GPL is more pronounced, in other words, it is important how gas and FF flow within matrix rather than how fast fracture is cleaned. In tighter gas formations, generally more GPL and slower clean-up was observed. The effect of matrix capillary pressure on GPL reduction was more pronounced when drawdown was very low and/or soaking time was extended. This observation was more profound in tighter formations, i.e. for these formations, the effect of a change in drawdown and/or soaking time on matrix capillary pressure and GPL was more pronounced. These findings can be used to make better decisions on the performance and optimised design of hydraulic fracturing, which is a costly but widely used stimulation technique for unconventional low permeability gas reservoirs