Shrikala Baliga

Dual color fluorescence in situ hybridization (FISH) assays for detecting mycobacterium tuberculosis and mycobacterium avium complexes and related pathogens in cultures

Two rapid dual color fluorescence in situ hybridization (FISH) assays were evaluated for detecting M. tuberculosis and related pathogens in cultures. The MN Genus-MTBC FISH assay uses an orange fluorescent probe specific for the Mycobacterium tuberculosis complex (MTBC) and a green fluorescent probe specific for the Mycobacterium and Nocardia genera (MN Genus) to detect and distinguish MTBC from other Mycobacteria and Nocardia. A complementary MTBC-MAC FISH assay uses green and orange fluorescent probes specific for the MTBC and M. avium complex (MAC) respectively to identify and differentiate the two species complexes. The assays are performed on acid-fast staining bacteria from liquid or solid cultures in less than two hours. Forty-three of 44 reference mycobacterial isolates were correctly identified by the MN Genus-specific probe as Mycobacterium species, with six of these correctly identified as MTBC with the MTBC-specific probe and 14 correctly as MAC by the MAC-specific probe. Of the 25 reference isolates of clinically relevant pathogens of other genera tested, only four isolates representing two species of Corynebacterium gave a positive signal with the MN Genus probe. None of these 25 isolates were detected by the MTBC and MAC specific probes. A total of 248 cultures of clinical mycobacterial isolates originating in India, Peru and the USA were also tested by FISH assays. DNA sequence of a part of the 23S ribosomal RNA gene amplified by PCR was obtained from 243 of the 248 clinical isolates. All 243 were confirmed by DNA sequencing as Mycobacterium species, with 157 and 50 of these identified as belonging to the MTBC and the MAC, respectively. The accuracy of the MN Genus-, MTBC-and MAC -specific probes in identifying these 243 cultures in relation to their DNA sequence-based identification was 100%. All ten isolates of Nocardia, (three reference strains and seven clinical isolates) tested were detected by the MN Genus-specific probe but not the MTBC- or MAC-specific probes. The limit of detection for M. tuberculosis was determined to be 5.1x104 cfu per ml and for M. avium 1.5x104 cfu per ml in liquid cultures with the respective MTBC- and MAC-specific probes in both the MN Genus-MTBC and MTBC-MAC FISH assays. The only specialized equipment needed for the FISH assays is a standard light microscope fitted with a LED light source and appropriate filters. The two FISH assays meet an important diagnostic need in peripheral laboratories of resource-limited tuberculosis-endemic countries.

Use of the GeneXpert tuberculosis system for HIV viral load testing in India

www.thelancet.com/lancetgh Vol 5 August 2017 e754 Of the 246 blood samples collected, 21 (9%) were precluded from testing because of insufficient blood volume or breaks in the cold chain. These events were unlikely to be related to the viral loads of the patients. Of the 225 (91%) patient samples remaining, 22 tests generated Xpert error results and 17 tests were invalid, resulting in an Xpert error and invalid rate of 17%. 10 samples were retested and their results were included in subsequent analyses. Ultimately, 196 samples had valid Xpert results. Of these, 89 (45%) patients had viral load values above the lower limit of detection for Xpert Use of the GeneXpert tuberculosis system for HIV viral load testing in India

Performance of the Xpert HIV-1 Viral Load Assay: a Systematic Review and Meta-analysis

ABSTRACT Viral load (VL) is the preferred treatment-monitoring approach for HIV-positive patients. However, more rapid, near-patient, and low-complexity assays are needed to scale up VL testing. The Xpert HIV-1 VL assay (Cepheid, Sunnyvale, CA) is a new, automated molecular test, and it can leverage the GeneXpert systems that are being used widely for tuberculosis diagnosis. We systematically reviewed the evidence on the performance of this new tool in comparison to established reference standards. A total of 12 articles (13 studies) in which HIV patient VLs were compared between Xpert HIV VL assay and a reference standard VL assay were identified. Study quality was generally high, but substantial variability was observed in the number and type of agreement measures reported. Correlation coefficients between Xpert and reference assays were high, with a pooled Pearson correlation (n = 8) of 0.94 (95% confidence interval [CI], 0.89, 0.97) and Spearman correlation (n = 3) of 0.96 (95% CI, 0.86, 0.99). Bland-Altman metrics (n = 11) all were within 0.35 log copies/ml of perfect agreement. Overall, Xpert HIV-1 VL performed well compared to current reference tests. The minimal training and infrastructure requirements for the Xpert HIV-1 VL assay make it attractive for use in resource-constrained settings, where point-of-care VL testing is most needed.

An Outbreak of Burkholderia cepacia Bacteremia in a Neonatal Intensive Care Unit

ObjectivesTo identify the source of infection, to study the clinical profile and outcomes of neonates with Burkholderia septicemia and to determine the antimicrobial susceptibility patterns of the isolates.MethodsThe authors describe a 3xa0mo outbreak of nosocomial Burkholderia cepacia bacteremia involving 12 neonates. During the outbreak, ventilator humidifier water, intravenous solutions and other possible sources were taken from the concerned neonatal intensive care units (NICUs); cultured and isolates identified by standard microbiological techniques and VITEK system. Clinical details of affected babies were also obtained to ascertain the clinical significance of the isolates.ResultsAll neonates had clinical and biochemical evidence of sepsis and the source could be tracked to intravenous solutions of 5xa0% dextrose, normal saline (opened bottles) and continuous positive airway pressure humidifier water. Strain relatedness of the environmental isolates with the clinical isolates is likely as antibiotic susceptibility patterns were similar.ConclusionsThe investigations revealed the source of the nosocomial outbreak which is crucial for initiating appropriate control measures.

Antibacterial action of Tropical honey on various bacteria obtained from diabetic foot ulcer

The purpose of study was to evaluate the antibacterial action of Indian honey on bacteria obtained from diabetic foot ulcer. Time kill assay was performed with varying concentration of honey. Broth macro dilution test was used to determine MIC and MB

Rapid method for detecting and differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in sputum by fluorescence in situ hybridization with DNA probes

OBJECTIVEnIn resource-limited tuberculosis-endemic countries, Mycobacterium tuberculosis in sputum is mainly detected by acid-fast bacillus (AFB) staining and the identification of sputum-derived cultures. PCR techniques are practical only in well-resourced laboratories. This study investigated the application of a rapid, simple, and inexpensive fluorescence in situ hybridization (FISH) assay to identify and differentiate M. tuberculosis complex (MTBC) from non-tuberculous mycobacteria (NTM) in sputum.nnnMETHODSnThe Mycobacterium/Nocardia Genus (MN Genus)-MTBC FISH assay performed in this study utilizes two different DNA probes labeled with different fluorescent molecules that hybridize respectively with 16S rRNA of the genus Mycobacterium and 23S rRNA of MTBC. The assay was tested on 202 patient sputum samples in Mangaluru, Karnataka State, India. Sputa were first liquefied and bacteria concentrated before performing the FISH assay and parallel culturing and AFB staining. The identities of cultured bacteria from DNA sequencing were compared with FISH assay findings from corresponding sputa.nnnRESULTSnOf the 202 sputum samples tested, 67 reacted with both MN Genus-specific and MTBC-specific probes, none reacted only with the MTBC-specific probe, and 22 reacted only with the MN Genus-specific probe. The FISH assay yielded results in 2h and had a limit of detection of 2.2×104CFU/ml in sputum spiked with cultured M. tuberculosis. The diagnostic sensitivity, specificity, and positive and negative predictive values of the FISH assay for MTBC in patient sputa were 89.7%, 95.5%, 88.0%, and 92.6%, respectively. NTM were a significant cause of tuberculosis-like infections in Mangaluru.nnnCONCLUSIONSnThe MN Genus-MTBC dual probe fluorescence FISH assay previously applied to cultures can also be utilized in resource-limited tuberculosis-endemic countries for rapidly identifying and differentiating MTBC and NTM in sputum samples.

Assessment of the level of knowledge and awareness of dental health care workers about hand Hygiene -A questionnaire based study

Hand hygiene is considered as an important measure to prevent cross-transmission of microorganisms and to reduce the incidence of health care–associated infections. Published literature reveals poor compliance with standard precautions among healthca