Direct detection of rpoB and katG gene mutations of Mycobacterium tuberculosis in clinical samples

Objectives: To study the rpoB and katG gene mutation rate and its markers. Methods: Cross-sectional study methods were used to study Tuberculosis. A total of 45 sputum samples were collected from Annapurna Neurological Institute and Allied sci-ences. Then, acid fast bacilli staining were performed. Positive and negative samples were carried for conventional polymerase chain reaction identification and electrophoresis. Results: Out of 45 samples, 3 were acid fast bacilli positive and the rest were negative. Male participants were more as compare to female participants and the mutation in rpoB and katG gene was found similar i.e. 6.66％among the total samples. Conclusions: We can conclude that genetic mutation in Mycobacterium tuberculosis can be identified directly from the clinical samples. However, we have carried this study in less sample size and to validate research on large number of sample is recommended.

Direct detection of rpoB and katG gene mutations of Mycobacterium tuberculosis in clinical samples

Objectives To study the rpoB and katG gene mutation rate and its markers. Methods Cross-sectional study methods were used to study Tuberculosis. A total of 45 sputum samples were collected from Annapurna Neurological Institute and Allied sciences. Then, acid fast bacilli staining were performed. Positive and negative samples were carried for conventional polymerase chain reaction identification and electrophoresis. Results Out of 45 samples, 3 were acid fast bacilli positive and the rest were negative. Male participants were more as compare to female participants and the mutation in rpoB and katG gene was found similar i.e. 6.66% among the total samples. Conclusions We can conclude that genetic mutation in Mycobacterium tuberculosis can be identified directly from the clinical samples. However, we have carried this study in less sample size and to validate research on large number of sample is recommended.

Direct Detection of Dichlorvos in Honey by Neutral Desorption-Extractive Electrospray Ionization Mass Spectrometry / 分析化学

In this study, a neutral desorption-extractive electrospray ionization mass spectrometry ( ND-EESI-MS) method was developed for the direct and rapid detection of dichlorvos ( DDVP) in honey samples without any sample pretreatment procedure. Under the positive ionization mode, the main characteristic parent ion of DDVP was m/z 223 (MW:222) and daughter ions were m/z 109 and m/z127. Under the optimized working conditions, with the signal intensity of m/z 127 as quantitative index, the quantitative information of DDVP residues in honey was acquired effectively. The results showed that the linear range of DDVP for spiked honey was 5-1000 ng/mL (R2=0. 998) with the limit of detection (LOD) of 1. 0 ng/mL (n=3) and the recoveries for the DDVP spiked honey samples at the concentration levels of 10 , 30 and 400 ng/mL were 93 . 0%-103. 0%, with the relative standard deviations (RSDs, n=6) of less than 4. 4%. Meanwhile, for detection of spiked honey with gas chromatography-flame photometric detector ( GC-FPD ) , the linear range was 5-1000 ng/mL (R2=0. 999) with the LOD of 1. 6 ng/mL(n=3), and the recoveries of DDVP at the spiked honey concentration levels of 10 , 30 and 400 ng/mL were 94 . 9%-110 . 3%, with the RSDs of less than 7. 6%.

Direct Detection of Methicillin-Resistant Staphylococcus aureus from Blood Cultures Using Three Non-Molecular Methods: PBP2a Latex Agglutination, PBP2a Rapid Immunochromatographic Assay and MRSA-Chromogenic Medium / 대한임상미생물학회지

BACKGROUND: This study compared three non-molecular methods for the detection of methicillin-resistance directly from blood cultures containing Staphylococcus aureus: penicillin-binding protein (PBP) 2a latex agglutination (LA), PBP2a immunochromatographic assay (ICA) and MRSA chromogenic medium (CM). METHODS: Fifty methicillin-resistant S. aureus (MRSA) and 50 methicillin-susceptible S. aureus (MSSA) were seeded into blood-culture bottles. When isolates returned a positive signal, 5 mL of culture was added to serum separator tubes and centrifuged at 1,300 g for 10 min. The pellets were then used as the inoculum for the PBP2a LA, MRSA-CM and PBP2a ICA. The pure colony was used for PBP2a LA test, additionally. RESULTS: The respective sensitivities and specificities were 98 and 100% for PBP2a ICA, and 100 and 100% for MRSA-CM in direct detection of MRSA from positive blood culture. The results of PBP2a LA test using pure colony were entirely compatible with those by mecA gene PCR but the PBP2a LA test using the pellets directly isolated from positive blood culture showed sometimes ambiguous agglutination; its sensitivity and specificity were 78 and 100%, if ambiguous results were scored as negative, and were 90 and 92%, if ambiguous results were scored as positive, respectively. CONCLUSION: For direct detection of MRSA in positive blood culture, MRSA-CM and PBP2a ICA provided excellent results. The PBP2a LA test using pure colony also gave excellent results but the PBP2a LA test by the direct method using pellet of positive blood culture was slightly less sensitive than the other two methods.

Nuevos métodos para el diagnóstico de la tuberculosis / New methods for the diagnosis of tuberculosis

La magnitud del problema mundial de la tuberculosis y su potencial de incremento han conducido a la necesidad de mejorar los métodos de diagnóstico como una de las estrategias conducentes al control de la enfermedad. Los nuevos métodos incluyen pruebas moleculares para la detección directa, medios de cultivo rápido y novedosos procedimientos de identificación. Para la detección directa de micobacterias se dispone hoy de pruebas basadas en la amplificación de los ácidos nucleicos, cuya sensibilidad supera a la de la baciloscopia y es cercana a la de los cultivos, pero con mayores costos que los procedimientos tradicionales.La automatización para detectar el crecimiento en medios de cultivo produjo un cambio radical en la micobacteriología, pues con ella se logró disminuir considerablemente el tiempo requerido para aislar el M. tuberculosis (MTB); la combinación de métodos de detección rápida permite en la actualidad mejores tasas de aislamiento y la demostración más precoz del crecimiento. Las nuevas alternativas han logrado aumentar tanto la sensibilidad como la especificidad y la rapidez diagnósticas; sin embargo, es necesario mejorar aún más los métodos de diagnóstico de esta enfermedad de modo que se combinen el mejor desempeño y la simplicidad de los procedimientos con la disminución de los costos.

The magnitude of the global tuberculosis problem and its potential for increase have led to efforts to improve the diagnostic methods as one of the leadingcontrol strategies. New diagnostic tools have been developed, including direct detection tests, rapid culture media and identification methods. For the direct detection of mycobacteria, tests based on nucleic acid amplification have been implemented, with higher sensitivity than the direct smear, close to that of the cultures; however, they are still expensive methods. Availability of the automated cultures resulted in a radical change in mycobacteriology, reducing the time required for the isolation ofMycobacterium tuberculosis. The combination of rapid methods has increased the rates of isolation and decreased the time required for the detection of growth. Different tools for identifying mycobacteria have also been developed, aimed at improving theperformance of traditional biochemical methods and achieving species identification in a few hours. The new diagnostic alternatives have succeeded in improving the sensitivity, specificity and speed in the diagnosis of tuberculosis; however, the challengeremains to reduce their cost and to make them more accessible in countries where the problem with this disease is still serious.