Fast, Simple, and Cheap: the Kudoh-Ogawa Swab Method as an Alternative to the Petroff–Lowenstein-Jensen Method for Culturing of Mycobacterium tuberculosis

ABSTRACT The objective of this study was to compare the diagnostic yield of the Kudoh-Ogawa (K-O) swab method for the culturing of Mycobacterium tuberculosis from clinical samples with the standard Petroff–Lowenstein-Jensen (P-LJ) procedure. A total of 2,287 sputum samples and 685 extrapulmonary clinical specimens were processed with both decontamination methods and compared for M. tuberculosis detection rate, recovery of M. tuberculosis colonies, and culture contamination. Overall, 23.9% and 23.5% of the samples, processed with, respectively, the K-O swab method and the P-LJ procedure, yielded M. tuberculosis after 8 weeks of incubation. The K-O swab method and the P-LJ procedure provided comparable diagnostic yields for extrapulmonary clinical specimens (P = 0.688), but the K-O method showed a slightly but statistically significantly higher diagnostic yield for pulmonary samples (P = 0.002). No significant difference for culture contamination or colony recovery was found for either method. The turnaround time for the isolation of M. tuberculosis was significantly shorter for the K-O swab method, with 77% of the M. tuberculosis cultures being positive within 3 weeks of incubation, and only 6.1% positivity for the P-LJ method. Concerning the workload, the K-O swab method needs a minimum sample manipulation and takes less than 4 min per sample, as the samples are not centrifuged in this procedure. The K-O swab method is an efficient and fast (in terms of sample processing and culture growth) alternative for culturing M. tuberculosis from either pulmonary or extrapulmonary clinical specimens. The method is particularly suitable for laboratories with a high workload and for laboratories lacking a special infrastructure.

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A Novel Solid Medium for Culturing Mycobacterium tuberculosis Isolates from Clinical Specimens

Journal of Clinical Microbiology ◽

10.1128/jcm.01149-15 ◽

2015 ◽

Vol 53 (8) ◽

pp. 2566-2569 ◽

Cited By ~ 17

Author(s):

S. Asmar ◽

S. Chatellier ◽

C. Mirande ◽

A. van Belkum ◽

I. Canard ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Solid Medium ◽

Laboratory Diagnosis ◽

Contamination Rate ◽

Content Type ◽

Exact Test ◽

Clinical Specimens ◽

Fisher's Exact Test ◽

Fisher’S Exact Test ◽

Lowenstein Jensen

The laboratory diagnosis of tuberculosis usually relies on culture-based isolation of the causativeMycobacterium tuberculosisbacteria. We developed and evaluated the performance of MOD9, a new blood-free derivative of the MOD4 solid medium on which we previously reported for the isolation and culture of mycobacteria. First, inoculation of Lowenstein-Jensen medium with 21M. tuberculosisisolates at 105, 103, and 10 CFU yielded colonies in 5.7 ± 1.5 days, 7.6 ± 0.8 days, and 10.8 ± 1.7 days versus 1.5 ± 0.4 days, 3.5 ± 0.6 days, and 4.9 ± 1 days in MOD9 (P< 0.05, Student'sttest). Further, the time to detectable growth ofM. tuberculosiswas measured on MOD9 and Lowenstein-Jensen media after duplicate inoculation of 250 clinical specimens decontaminated with 0.7% chlorhexidine. The contamination rate was 1.6% (4/250) on MOD9 versus 4.4% (11/250) on Lowenstein-Jensen medium (P= 0.11, Fisher's exact test). Chlorhexidine-MOD9 yielded 38/250 (15.2%) isolates versus 32/250 (12.8%) isolates for the chlorhexidine-LJ (P= 0.5195, Fisher's exact test). All together, eightM. tuberculosisisolates were cultured solely from chlorhexidine-MOD9, and twoM. tuberculosisisolates were cultured solely from chlorhexidine-LJ. The time to detection was 9.8 ± 3.9 (range, 5 to 18) days for chlorhexidine-MOD9 versus 17.4 ± 5.9 (range, 10 to 35) days for chlorhexidine-LJ (P< 0.05, Student'sttest). These data indicate the superiority of the MOD9 medium over the standard LJ medium following chlorhexidine decontamination for the recovery ofM. tuberculosis.

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Determination some Immunological Aspects in Pulmonary Tuberculosis Patients in Qadisiyah Province

International Journal of Pharmaceutical Quality Assurance ◽

10.25258/ijpqa.v9i2.13632 ◽

2018 ◽

Vol 9 (2) ◽

Author(s):

Syoof Khowman Alramahy ◽

Akram Hadi Hamza

Keyword(s):

Mycobacterium Tuberculosis ◽

Pulmonary Tuberculosis ◽

Statistical Difference ◽

Positive Culture ◽

Control Group ◽

Tuberculosis Patients ◽

Significant Difference ◽

Lowenstein Jensen ◽

The Mean ◽

Igg Level

This study was carried out to study of some immunological aspects among the pulmonary Tuberculosis patients infected with causative agent, Mycobacterium tuberculosis. A Total of 200 sputum samples were collected from patients attending the consultant Clinic for Chest and Respiratory disease center, Diwaniya. Control group (No=15) also included. According to acid fast stain of sputum, the patients were classified as positive (No=91,45.5%) and negative (No=109,54.5, Lowenstein Jensen medium used for the cultivation of samples, on which 70% of sputum samples where positive culture for this microorganism. The grown microorganism were identified as M. tuberculosis, based on positive A.F.B, Niacin producers ,negative for catlase at 68c. The mean IgG level was l184.053±76.684 mg/100 ml in tuberculosis group compared with 1016.533 ± 44.882 mg/100ml in control group, rendering the statistical difference significant. For IgA and IgM levels, they were at mean of 315.880±38.552 mg/100 ml and 119.527±8.464 mg/100 ml in control group compared with 396.358±38.776 mg/100 ml and 134.207±11.696 mg/100 ml in patients group respectively with significant difference

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Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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Comparison of In Vitro Activity and MIC Distributions between the Novel Oxazolidinone Delpazolid and Linezolid against Multidrug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis in China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00165-18 ◽

2018 ◽

Vol 62 (8) ◽

Cited By ~ 18

Author(s):

Zhaojing Zong ◽

Wei Jing ◽

Jin Shi ◽

Shu'an Wen ◽

Tingting Zhang ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Novel Mutation ◽

Multidrug Resistant ◽

23S Rrna ◽

Drug Resistant ◽

Content Type ◽

Extensively Drug Resistant ◽

Significant Difference ◽

Mdr Tb

ABSTRACT Oxazolidinones are efficacious in treating mycobacterial infections, including tuberculosis (TB) caused by drug-resistant Mycobacterium tuberculosis. In this study, we compared the in vitro activities and MIC distributions of delpazolid, a novel oxazolidinone, and linezolid against multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) in China. Additionally, genetic mutations in 23S rRNA, rplC, and rplD genes were analyzed to reveal potential mechanisms underlying the observed oxazolidinone resistance. A total of 240 M. tuberculosis isolates were included in this study, including 120 MDR-TB isolates and 120 XDR-TB isolates. Overall, linezolid and delpazolid MIC90 values for M. tuberculosis isolates were 0.25 mg/liter and 0.5 mg/liter, respectively. Based on visual inspection, we tentatively set epidemiological cutoff (ECOFF) values for MIC determinations for linezolid and delpazolid at 1.0 mg/liter and 2.0 mg/liter, respectively. Although no significant difference in resistance rates was observed between linezolid and delpazolid among XDR-TB isolates (P > 0.05), statistical analysis revealed a significantly greater proportion of linezolid-resistant isolates than delpazolid-resistant isolates within the MDR-TB group (P = 0.036). Seven (53.85%) of 13 linezolid-resistant isolates were found to harbor mutations within the three target genes. Additionally, 1 isolate exhibited an amino acid substitution (Arg126His) within the protein encoded by rplD that contributed to high-level resistance to linezolid (MIC of >16 mg/liter), compared to a delpazolid MIC of 0.25. In conclusion, in vitro susceptibility testing revealed that delpazolid antibacterial activity was comparable to that of linezolid. A novel mutation within rplD that endowed M. tuberculosis with linezolid, but not delpazolid, resistance was identified.

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Evaluation of the efficacy of two methods for direct extraction of DNA from Mycobacterium tuberculosis sputum

The Journal of Infection in Developing Countries ◽

10.3855/jidc.10592 ◽

2018 ◽

Vol 12 (12) ◽

pp. 1067-1072

Author(s):

Victor Ndhlovu ◽

Wilson Mandala ◽

Derek Sloan ◽

Mercy Kamdolozi ◽

Maxine Caws ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Single Molecule ◽

Disease Transmission ◽

Extraction Efficiency ◽

Ease Of Use ◽

Clinical Samples ◽

Direct Extraction ◽

Significant Difference ◽

Ctab Method ◽

Dna Purity

Introduction: Whole genome sequencing (WGS) has shown superiority over other bacterial typing methods and can be used to monitor disease transmission. The long culture period hinders use of WGS as a diagnostic tool for TB. The ideal situation would be to efficiently sequence directly from clinical specimens such as sputum. Attempts to sequence directly from Mtb clinical samples have achieved very low coverage (less than 0.7X). We compared DNA extraction methods for direct extraction from Mycobacterium tuberculosis positive sputum and assessed their suitability for Single Molecule Real Time sequencing. Methodology: We evaluated the extraction efficiency of the PrimeXtract kit and an in-house CTAB method by extracting DNA from Mtb sputum. We evaluated the methods on these parameters: ease of use, efficiency (quantity and purity) and the cost per extraction. Results: The PrimeXtract kit was able to isolate 5.93 µg/mL ± 0.94, (Mean ± SEM) concentration of DNA and a yield of 0.2975 µg ± 0.04723, (Mean ± SEM). Comparatively, the CTAB method isolated 1.88 µg/mL ± 0.38 DNA and a yield of 0.09 µg ± 0.02. Both concentration and yield from the kit were significantly (p = 0.0002) higher than those from CTAB. The PrimeXtract kit had a DNA purity ratio of 1.69 ± 0.09 compared to the CTAB’s 1.73 ± 0.14 and this difference was not statistically different. Conclusion: PrimeXtract kit has a superior extraction efficiency than the CTAB method on Mtb sputum in terms of DNA yield although no significant difference by DNA purity was seen.

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A comparative characterization of nasal and clinical isolates of Staphylococcus aureus from west of Iran

Iranian Journal of Microbiology ◽

10.18502/ijm.v13i6.8086 ◽

2021 ◽

Author(s):

Gholamreza Goudarzi ◽

Yaser Hasanvand ◽

Faranak Rezaei ◽

Somayeh Delfani

Keyword(s):

Staphylococcus Aureus ◽

Antibiotic Resistance ◽

Healthcare Workers ◽

Meca Gene ◽

Staphylococcal Enterotoxin ◽

Healthcare Personnel ◽

Clinical Samples ◽

Resistance Pattern ◽

Clinical Specimens ◽

Significant Difference

Background and Objectives: Recently, the rise of methicillin-resistant Staphylococcus aureus (MRSA) isolated from hos- pital healthcare workers (HCWs) and various infectious samples has become one of the main concerns in hospital settings. Therefore, epidemiological studies are necessary to monitor antibiotic resistance patterns in each region and to study the pathogenesis of this strain to control infections. Materials and Methods: In this cross-sectional study, a total of 100 S. aureus isolates, including 50 isolates obtained from the anterior nares of healthcare workers, as well as 50 other isolates cultured from the various clinical specimens from the referral hospitals in Khorramabad (West of Iran) were tested. All isolates were examined to determine antibiotic resistance pattern, and the presence of staphylococcal enterotoxin A (sea), staphylococcal enterotoxin B (seb) and mecA genes. Results: The mecA gene was found among 36% (18/50) of the clinical S. aureus isolates (CSIs) and 14% (7/50) of nasal S. aureus isolates (NSIs), with statistically significant difference (X2 = 6.53; p = 0.011). The difference between the frequency rate of sea gene among MRSA strains isolated from clinical specimens (46.6%, 7/15) was significant compared to strains isolated from nostrils (14.3%, 1/7) (X2 = 3.85; p = 0.049). Conclusion: The frequency of mecA, sea, and seb genes among the clinical samples was more than strains isolated from the nostrils of healthcare personnel.

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Real-Time Sequencing of Mycobacterium tuberculosis: Are We There Yet?

Journal of Clinical Microbiology ◽

10.1128/jcm.00358-17 ◽

2017 ◽

Vol 55 (5) ◽

pp. 1249-1254 ◽

Cited By ~ 23

Author(s):

Robyn S. Lee ◽

Madhukar Pai

Keyword(s):

Mycobacterium Tuberculosis ◽

Real Time ◽

Phylogenetic Trees ◽

Illumina Miseq ◽

Turnaround Time ◽

Epidemiologic Studies ◽

Content Type ◽

Leading Role ◽

Oxford Nanopore ◽

Oxford Nanopore Technologies

ABSTRACT Whole-genome sequencing has taken a leading role in epidemiologic studies of tuberculosis, but thus far, its real-time clinical utility has been low, in part because of the requirement for culture. In their report in this issue, Votintseva et al. (A. A. Votintseva, P. Bradley, L. Pankhurst, C. del Ojo Elias, M. Loose, K. Nilgiriwala, A. Chatterjee, E. G. Smith, N. Sanderson, T. M. Walker, M. R. Morgan, D. H. Wyllie, A. S. Walker, T. E. A. Peto, D. W. Crook, and Z. Iqbal, J Clin Microbiol 55:1285–1298, 2017, https://doi.org/10.1128/JCM.02483-16 ) present a new method for extracting Mycobacterium tuberculosis DNA directly from smear-positive respiratory samples, making it feasible to generate drug resistance predictions and phylogenetic trees in 44 h with the Illumina MiSeq. They also illustrate the potential for a <24-h turnaround time from DNA extraction to clinically relevant results with Illumina MiniSeq and Oxford Nanopore Technologies MinION. We comment on the promise and limitations of these approaches.

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Evaluation of the Abbott RealTi m e MTB and RealTi m e MTB INH/RIF Assays for Direct Detection of Mycobacterium tuberculosis Complex and Resistance Markers in Respiratory and Extrapulmonary Specimens

Journal of Clinical Microbiology ◽

10.1128/jcm.01144-16 ◽

2016 ◽

Vol 54 (12) ◽

pp. 3022-3027 ◽

Cited By ~ 27

Author(s):

Sabine Hofmann-Thiel ◽

Nikolay Molodtsov ◽

Uladzimir Antonenka ◽

Harald Hoffmann

Keyword(s):

Mycobacterium Tuberculosis ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Nucleic Acid Amplification ◽

Content Type ◽

Clinical Specimens ◽

Different Types ◽

Resistance Markers ◽

Smear Negative

The Abbott RealTi m e MTB (RT MTB) assay is a new automated nucleic acid amplification test for the detection of Mycobacterium tuberculosis complex (MTBC) in clinical specimens. In combination with the RealTi m e MTB INH/RIF (RT MTB INH/RIF) resistance assay, which can be applied to RT MTB-positive specimens as an add-on assay, the tests also indicate the genetic markers of resistance to isoniazid (INH) and rifampin (RIF). We aimed to evaluate the diagnostic sensitivity and specificity of RT MTB using different types of respiratory and extrapulmonary specimens and to compare performance characteristics directly with those of the FluoroType MTB assay. The resistance results obtained by RT MTB INH/RIF were compared to those from the GenoType MTBDR plus and from phenotypic drug susceptibility testing. A total of 715 clinical specimens were analyzed. Compared to culture, the overall sensitivity of RT MTB was 92.1%; the sensitivity rates for smear-positive and smear-negative samples were 100% and 76.2%, respectively. The sensitivities of smear-negative specimens were almost identical for respiratory (76.3%) and extrapulmonary (76%) specimens. Specificity rates were 100% and 95.8% for culture-negative specimens and those that grew nontuberculous mycobacteria, respectively. RT MTB INH/RIF was applied to 233 RT MTB-positive samples and identified resistance markers in 7.7% of samples. Agreement with phenotypic and genotypic drug susceptibility testing was 99.5%. In conclusion, RT MTB and RT MTB INH/RIF allow for the rapid and accurate diagnosis of tuberculosis (TB) in different types of specimens and reliably indicate resistance markers. The strengths of this system are the comparably high sensitivity with paucibacillary specimens, its ability to detect INH and RIF resistance, and its high-throughput capacities.

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HIV-Associated Mycobacterium tuberculosis Bloodstream Infection Is Underdiagnosed by Single Blood Culture

Journal of Clinical Microbiology ◽

10.1128/jcm.01914-17 ◽

2018 ◽

Vol 56 (5) ◽

Cited By ~ 5

Author(s):

David A. Barr ◽

Andrew D. Kerkhoff ◽

Charlotte Schutz ◽

Amy M. Ward ◽

Gerry R. Davies ◽

...

Keyword(s):

South Africa ◽

Mycobacterium Tuberculosis ◽

Blood Culture ◽

Confidence Interval ◽

Bloodstream Infection ◽

Diagnostic Yield ◽

Blood Cultures ◽

Prospective Evaluation ◽

Content Type ◽

Time Point

ABSTRACT We assessed the additional diagnostic yield for Mycobacterium tuberculosis bloodstream infection (BSI) by doing more than one tuberculosis (TB) blood culture from HIV-infected inpatients. In a retrospective analysis of two cohorts based in Cape Town, South Africa, 72/99 (73%) patients with M. tuberculosis BSI were identified by the first of two blood cultures during the same admission, with 27/99 (27%; 95% confidence interval [CI], 18 to 36%) testing negative on the first culture but positive on the second. In a prospective evaluation of up to 6 blood cultures over 24 h, 9 of 14 (65%) patients with M. tuberculosis BSI had M. tuberculosis grow on their first blood culture; 3 more patients (21%) were identified by a second independent blood culture at the same time point, and the remaining 2 were diagnosed only on the 4th and 6th blood cultures. Additional blood cultures increase the yield for M. tuberculosis BSI, similar to what is reported for nonmycobacterial BSI.

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Wild-Type and Non-Wild-Type Mycobacterium tuberculosis MIC Distributions for the Novel Fluoroquinolone Antofloxacin Compared with Those for Ofloxacin, Levofloxacin, and Moxifloxacin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00393-16 ◽

2016 ◽

Vol 60 (9) ◽

pp. 5232-5237 ◽

Cited By ~ 9

Author(s):

Xia Yu ◽

Guirong Wang ◽

Suting Chen ◽

Guomei Wei ◽

Yuanyuan Shang ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Critical Concentration ◽

Bacterial Species ◽

Drug Susceptibility Testing ◽

World Health ◽

Wild Type ◽

Resistance Mutations ◽

Content Type ◽

Lowenstein Jensen

ABSTRACTAntofloxacin (AFX) is a novel fluoroquinolone that has been approved in China for the treatment of infections caused by a variety of bacterial species. We investigated whether it could be repurposed for the treatment of tuberculosis by studying itsin vitroactivity. We determined the wild-type and non-wild-type MIC ranges for AFX as well as ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MFX), using the microplate alamarBlue assay, of 126 clinicalMycobacterium tuberculosisstrains from Beijing, China, of which 48 were OFX resistant on the basis of drug susceptibility testing on Löwenstein-Jensen medium. The MIC distributions were correlated with mutations in the quinolone resistance-determining regions ofgyrA(Rv0006) andgyrB(Rv0005). Pharmacokinetic/pharmacodynamic (PK/PD) data for AFX were retrieved from the literature. AFX showed lower MIC levels than OFX but higher MIC levels than LFX and MFX on the basis of the tentative epidemiological cutoff values (ECOFFs) determined in this study. All strains with non-wild-type MICs for AFX harbored known resistance mutations that also resulted in non-wild-type MICs for LFX and MFX. Moreover, our data suggested that the current critical concentration of OFX for Löwenstein-Jensen medium that was recently revised by the World Health Organization might be too high, resulting in the misclassification of phenotypically non-wild-type strains with known resistance mutations as wild type. On the basis of our exploratory PK/PD calculations, the current dose of AFX is unlikely to be optimal for the treatment of tuberculosis, but higher doses could be effective.

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