The emergence of multidrug-resistant tuberculosis (MDR-TB) has complicated the situation due to the decline in potency of second-line anti-tubercular drugs. This limits the treatment option for ...extensively drug-resistant tuberculosis (XDR-TB). The aim of this study was to determine and compare the minimum inhibitory concentration (MIC) by agar dilution and resazurin microtiter assay (REMA) along with the detection of mutations against linezolid and clofazimine in confirmed XDR-TB clinical isolates. A total of 169 isolates were found positive for Mycobacterium tuberculosis complex (MTBC). The MIC was determined by agar dilution and REMA methods. The isolates which showed non-susceptibility were further subjected to mutation detection by targeting rplC gene (linezolid) and Rv0678 gene (clofazimine). The MIC for linezolid ranged from 0.125 microg/ml to > 2 microg/ml and for clofazimine from 0.25 microg/ml to > 4 microg/ml. The MIC.sub.50 and MIC.sub.90 for linezolid were 0.5 microg/ml and 1 microg/ml respectively while for clofazimine both were 1 microg/ml. The essential and categorical agreement for linezolid was 97.63% and 95.26% and for clofazimine, both were 100%. The sequencing result of the rplC gene revealed a point mutation at position 460 bp, where thymine (T) was substituted for cytosine (C) while seven mutations were noted between 46 to 220 bp in Rv0678 gene. REMA method has been found to be more suitable in comparison to the agar dilution method due to lesser turnaround time. Mutations in rplC and Rv0678 genes were reasons for drug resistance against linezolid and clofazimine respectively.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The burden of non-tuberculous mycobacterial (NTM) disease is increasing worldwide but still its diagnosis is delayed and it is mistaken as multidrug-resistant tuberculosis (MDR-TB).The present study ...was performed to develop a multiplex PCR assay for detection and identification of clinically most common NTM to the species level from pulmonary samples.
Out of 50 isolates, 26 were identified as Mycobacterium kansasii (MK), 20 were identified as Mycobacterium abscessus (MA) and 4 were identified as Mycobacterium avium complex (MAC) through multiplex PCR and further confirmed by sequencing.
Our study showed that multiplex PCR assay is a simple, convenient, and reliable technique for detection and differential identification of major NTM species.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Success of India's TB control program relies on rapid case detection, monitoring, care and treatment of drug resistance. Patients on multidrug resistance (MDR) treatment are monitored by follow up ...cultures. Discordant results (culture and smear positive while capilia negative) are usually declared negative Mycobacterium tuberculosis complex (MTBC). This study was designed to understand the possible causes of discordant results.
The capilia kit was evaluated to test its utility among 4737 follow up MDR patients enrolled during a period of 1 year. A total of 889 were liquid culture positive, 3375 were negative and 473 were contaminated. Of the 889 cultures positive, 829 were found positive by ZN smear, capilia test and MTBDR plus assay. The cultures which gave a positive result on Mycobacterium Growth Indicator Tube 960 (MGIT 960) and ZN smear but were negative on capilia test with no growth on Brain Heart Infusion agar (BHI) were included in this study. The conflicting results of capilia were compared with other molecular techniques; MTBDR plus assay and DNA sequence analysis of MPT64 gene.
Out of 889 culture positive, 60 (6.7%) were found positive on liquid culture and ZN smear but were negative on capilia. Of these 60 cultures, 10 (16.7%) were found positive by both MTBDR plus assay and PCR. The sequencing analysis revealed that all of the capilia negative isolates had mutations within the MPT64 gene.
Re-evaluation of culture positive but capilia negative isolates should be done before declaring them as Mycobacterium other than tuberculosis (MOTT) because such cases can act as chronic carriers of TB in the population which can lead to the rise of this lethal disease.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The potential of genetic testing for rapid and accurate diagnosis of drug-resistant Mycobacterium tuberculosis strains is vital for efficient treatment and reduction in dissemination. MTBDR plus ...assays rapidly detect mutations related to drug resistance and wild type sequences allied with susceptibility. Although these methods are promising, the examination of molecular level performance is essential for improved assay result interpretation and continued diagnostic development. Therefore this study aimed to determine novel mutations that were inhibiting wild type probe hybridization in the Line probe assay by DNA sequencing. Using data collected from Line Probe assay (GenoType MTBDRplus assay) the contribution of absent wild type probe hybridization to the detection of rifampicin resistance was assessed via comparison to a reference standard method i.e. DNA sequencing.
Sequence analysis of the rpoB gene of 47 MTB resistant strains from clinical specimens showed that 37 had a single mutation, 9 had double mutations and one had triple mutations in the ropB gene.
The absence of wild type probe hybridization without mutation probe hybridization was mainly the result of the failure of mutation probe hybridization and the result of the novel or rare mutations. Additional probes are necessary to be included in the Line probe assay to improve the detection of rifampicin-resistant Mycobacterium tuberculosis strains.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Objective
Coagulase-negative staphylococci (CoNS) are being implicated as one of the leading causes of bloodstream infection (BSI). To study the spectrum, prevalence, and antimicrobial ...susceptibility of CoNS causing BSI in neonates.
Materials and Methods
A cross-sectional study was done in level III neonatal intensive care unit (NICU). Blood samples in automated culture bottles were processed as per the standard technique. Previously validated methods were followed for the characterization of CoNS and for AST of standard antibiotics by Kirby Bauer disk diffusion and vancomycin by agar dilution. The prevalence of causative organisms and susceptibility of CoNS were statistically analyzed. Categorical variables were compared by chi-square or Fisher's exact probability tests.
Result
In total, 1,365 blood samples (1,365 neonates) were studied, of which 383 (28.05%) were positive and 982 (71.94%) were negative. Gram-positive organisms (GPC) predominated (
n
= 238; 62.14%) (
p
< 0.001) with 41.77% (160/383)
S. aureus
and 13.83% (53/383) CoNS. CoNS included
S. epidermidis
(19, 38%),
S
.
haemolyticus
(7, 14%),
S. hominis
(6, 12%),
S. simulans
(6,12%),
S. capitis
(5,10%),
S. cohnii
(4, 8%),
S. warneri
(1, 2%), and
S. xylosus
(1, 2%). The susceptibility to netilmicin, linezolid, and vancomycin was 100% (
p
≤ 0.001), and 54% (
n
= 27) had vancomycin MIC of 0.125 μg/mL but methicillin-resistant CoNS (MRCoNS) was 70%. Methicillin-susceptible (MS) CoNS had lower MIC of vancomycin (
p
< 0.05) than MRCoNS.
Conclusion
The spectrum of pathogens causing BSI in neonates is changing with predominance of GPC and among CoNS,
S. epidermidis
. Considerable proportion of MRCoNS with the emergence of MIC creep for vancomycin requires immediate attention.
Drug resistance in tuberculosis is a major public health challenge in developing countries. The limited data available on drug resistance in extra pulmonary tuberculosis stimulated us to design our ...study on anti-tuberculosis drug resistance pattern in cases of extra pulmonary tuberculosis in a tertiary referral hospital of North India. We performed Geno Type MTBDRplus assay in comparison with conventional drug susceptibility testing by proportion method to study the mutation patterns in rpoB, katG and inhA genes.
A total of 510 extra pulmonary samples were included in this study. After the smear microscopy, all the specimens were subjected for culture on Lowenstein Jensen (LJ) media. Phenotypic drug susceptibility testing (DST) was performed on LJ media for all the MTB isolates and compared with the results of Geno Type MTBDRplus assay which was performed with the DNA isolated from the culture by conventional method.
Of 510 specimens cultured, the total culture positivity obtained was 11.8% (60) encompassing 54 (10.6%) Mycobacterium tuberculosis and 6 (1.2%) non-tubercular mycobacteria (NTM). DST results by Geno Type MTBDRplus assay and solid culture methods were compared in 51 MTB isolates excluding the two Rif indeterminate and one invalid test. Geno Type MTBDRplus accurately identified 13 of 14 rifampicin-resistant strains, 14 of 15 isoniazid-resistant strains and 13 of 14 as multi drug resistant tuberculosis (MDR-TB) in comparison with conventional method. Sensitivity and specificity were 92.86% and 97.30% respectively for detection of RIF resistance, 93.33% and 94.44% respectively for detection of INH resistance, 92.86% and 97.30% respectively for detection of MDR-TB, while the overall concordance of Geno Type MTBDRplus assay with conventional DST was 94.11%. The turn-around time for performing Geno Type MTBDRplus assay test was 48 hours.
The problem of MDR in extra pulmonary tuberculosis (EPTB) cannot be overlooked and due attention on patients should be given. Routine use of Geno Type MTBDRplus assay for the diagnosis of MDR-EPTB can substantially reduce the time between diagnosis and drug therapy. Culture along with Geno Type MTBDRplus assay could be a solution for rapid and accurate diagnosis of MDR-TB in low bacillary non sputum specimens.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Molecular epidemiological studies of Mycobacterium tuberculosis (MTB) are the core of current research to find out the association of the M. tuberculosis genotypes with its outbreak and transmission. ...The high prevalence of the Beijing genotype strain among multidrug resistance (MDR) TB has already been reported in various studies around India. The overall objective of this study was to detect the prevalence of Beijing genotype strains of MDR M. tuberculosis and their association with the clinical characteristics of TB patients. In this study 381 M. tuberculosis clinical isolates were obtained from sputum samples from 2008 to 2014. The multiplex-PCR and Spoligotyping (n = 131) methods were used to investigate the prevalence of the Beijing genotype strain by targeting the Rv2820 gene and their association with drug resistance and clinical characteristics of TB patients. The drug susceptibility testing of first-line anti-TB drugs was performed by using the proportion method and MGIT960. A collection of isolates having Beijing and non-Beijing strains were also characterized to see if Beijing genotype strains had a higher rate of mutations at codons 516, 526 and 531 of the 81-bp region of the rpoB gene, codon 315 of the katG gene, and codon 306 of the embB gene. The sensitivities and specificities of multiplex-PCR assay compared to that of standard Spoligotyping was detected to be 100%. Further, we observe that the multi drug-resistance was significantly associated with Beijing genotype strains (p = 0.03) and a strong correlation between Beijing genotype strains and specific resistance mutations at the katG315, rpoB531, and embB306 codons (p = < 0.0001, < 0.0001 & 0.0014 respectively) was also found. This rapid, simple, and cost-effective multiplex PCR assay can effectively be used for monitoring the prevalence of Beijing genotype strains in low resource settings. Findings of this study may provide a scientific basis for the development of new diagnostic tools for detection and effective management of DR-TB in countries with a higher incidence rate of Beijing genotype strains.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Detection of drug resistance in Mycobacterium tuberculosis by conventional phenotypic drug susceptibility testing methods requires several weeks. Therefore, molecular diagnostic tests for rapid ...detection of multidrug resistance tuberculosis (MDR-TB) are urgently needed. Early diagnosis helps in initiating optimal treatment which would not only enable cure of an individual patient but also will curb the transmission of drug resistance in the community. Line probe assay (LPA) has shown great promises in the diagnosis of MDR-TB. All MDR suspect patients from ten-linked districts were asked to deposit sputum samples at peripheral designated microscopy centers. The district TB officers facilitated the transport of samples collected during February 2014-December 2014 to our laboratory. The detection of rpoB gene mutations for rifampicin (RIF) and katG and inhA genes for isoniazid (INH), respectively, was performed on 663 samples by LPA. A total of 663 sputum samples from MDR suspects were received of which 321 (50.8%) were found to be MDR. Missing of WT8 along with mutation in codon S531 L was the most common pattern for RIF-resistant isolates (80.8%) and missing WT along with mutation in codon S315T1 of k atG gene was the most common pattern for INH-resistant isolates (91.3%).The MDR-TB in Eastern Uttar Pradesh, India, was found to be 50.8%. The common mutations obtained for RIF and INH in the region was mostly similar to those reported earlier.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Retropharyngeal abscess is a rare manifestation in spinal tuberculosis. Early clinical diagnosis followed by microbiological confirmation and effective treatment is crucial to avoid ...irreversible damage to the spine. Here, we report a case of disseminated tuberculosis in an immunocompetent adolescent male who presented with retropharyngeal abscess, multifocal involvement of the spine, and skin tuberculids. Xpert MTB/RIF assay in this patient facilitated early lifesaving treatment by detecting rifampicin-resistant
Mycobacterium tuberculosis
(MTB) in the clinical specimen.
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