Clinical Sputum Specimens Research Articles

Clonality Analysis of Streptococcus pneumoniae in Clinical Specimens

Pneumococcal pneumonia is a significant cause of illness and death globally, particularly among young children and the elderly. The cpsB gene is involved in the biosynthesis of the capsule polysaccharide, and polymorphisms in the cpsB gene are the basis for sequetyping, a molecular biology-based approach to serotyping. In this study, we attempted the sequetyping of pneumococci directly from clinical sputum specimens collected from adult patients diagnosed with community-acquired pneumonia (CAP). We performed conventional PCR for the cpsB gene, followed by TA cloning and Sanger sequencing of the amplicon. The results showed the status of clonality of pneumococci in each specimen. We also performed real-time PCR targeting pneumococci for each specimen. It revealed a significant association between the Ct value of the real-time PCR and the clonality status of pneumococci among the specimens (p-value 0.0007 by Fisher’s exact test analysis). Specifically, when the Ct value was below 22, there was a high probability that pneumococcus existed as a single clone. Thus, this study demonstrates the possible correlation between pneumococcal clonality and bacterial load in clinical specimens, which might indicate the infection status.

Comparison of targeted next-generation sequencing and the Xpert MTB/RIF assay for detection of Mycobacterium tuberculosis in clinical isolates and sputum specimens.

Targeted next-generation sequencing (tNGS) can be used to perform Mycobacterium tuberculosis (MTB) complex-specific amplification or target capture directly from sputum samples, yielding simultaneous coverage of many genes and DNA regions associated with antimicrobial resistance (AMR). Performance comparisons of tNGS and another molecular testing tool, Xpert MTB/rifampicin (RIF), have been empirical. Here, using a dilution series of a RIF-resistant clinical isolate of MTB, we found that tNGS had a slightly lower limit of bacterial detection (102 CFU/mL) compared with Xpert MTB/RIF (103 CFU/mL) in culture medium. However, the minimum detection limit of the rpoB S450L mutation in this isolate was significantly lower with tNGS (102 CFU/mL) than with Xpert MTB/RIF (106 CFU/mL). Sputum samples collected from 129 suspected pulmonary tuberculosis patients were also prospectively studied with the clinical diagnosis as a reference, revealing that the sensitivity of tNGS (48.6%) was higher than those of culture (46.8%), Xpert MTB/RIF (39.4%), and smear microscopy (34.9%) testing. Notably, AMR analysis of 56 MTB-positive samples as determined by tNGS revealed high mutation frequencies of 96.4%, 35.7%, 26.8%, and 19.6% in the following AMR-associated genes: rrs, rpoB, katG, and pncA, respectively. The findings of this study provide theoretical support for the differential clinical application of tNGS and Xpert MTB/RIF and suggest that tNGS has greater application value in tuberculosis drug resistance monitoring and prevention.IMPORTANCETargeted next-generation sequencing (tNGS) can be used to perform Mycobacterium tuberculosis (MTB) complex-specific amplification or target capture directly from sputum samples, yielding simultaneous coverage of genes and DNA regions associated with antimicrobial resistance (AMR). Performance comparisons of tNGS and Xpert MTB/rifampicin (RIF) have been empirical. The Xpert MTB/RIF assay is a commercial system that uses the nucleic acid amplification detection method for rapid (2 hours) diagnosis of tuberculosis (TB). The cost of the tNGS and Xpert MTB/RIF assays in this study was similar, at USD 98 and USD 70-104 per sample, respectively, but the time required for tNGS (3 days) was much longer than that required for the Xpert MTB/RIF assay. However, tNGS yielded more accurate results and a larger number of AMR-associated gene mutations, which compensated for the extra time and highlighted the greater application value of tNGS in TB drug resistance monitoring and prevention.

Microfluidic Capture of Mycobacterium tuberculosis from Clinical Samples for Culture-Free Whole-Genome Sequencing.

Mycobacterium tuberculosis whole-genome sequencing (WGS) is a powerful tool as it can provide data on population diversity, drug resistance, disease transmission, and mixed infections. Successful WGS is still reliant on high concentrations of DNA obtained through M. tuberculosis culture. Microfluidics technology plays a valuable role in single-cell research but has not yet been assessed as a bacterial enrichment strategy for culture-free WGS of M. tuberculosis. In a proof-of-principle study, we evaluated the use of Capture-XT, a microfluidic lab-on-chip cleanup and pathogen concentration platform to enrich M. tuberculosis bacilli from clinical sputum specimens for downstream DNA extraction and WGS. Three of the four (75%) samples processed by the microfluidics application passed the library preparation quality control, compared to only one of the four (25%) samples not enriched by the microfluidics M. tuberculosis capture application. WGS data were of sufficient quality, with mapping depth of ≥25× and 9 to 27% of reads mapping to the reference genome. These results suggest that microfluidics-based M. tuberculosis cell capture might be a promising method for M. tuberculosis enrichment in clinical sputum samples, which could facilitate culture-free M. tuberculosis WGS. IMPORTANCE Diagnosis of tuberculosis is effective using molecular methods; however, a comprehensive characterization of the resistance profile of Mycobacterium tuberculosis often requires culturing and phenotypic drug susceptibility testing or culturing followed by whole-genome sequencing (WGS). The phenotypic route can take anywhere from 1 to >3 months to result, by which point the patient may have acquired additional drug resistance. The WGS route is a very attractive option; however, culturing is the rate-limiting step. In this original article, we provide proof-of-principle evidence that microfluidics-based cell capture can be used on high-bacillary-load clinical samples for culture-free WGS.

Exploratory study on detection of drug resistance of Mycobacterium tuberculosis in sputum specimens by next-generation sequencing

Objective: To compare the diagnostic performance of next-generation sequencing (NGS) detection methods in sputum samples and Mycobacterium tuberculosis strains, in order to explore the feasibility of the NGS method to detect drug resistance in sputum specimens. Methods: In this retrospective study, the sputum specimens and corresponding clinical isolates of 50 pulmonary tuberculosis patients admitted to Beijing Chest Hospital from January 2017 to December 2017 were collected. The gene mutations of katG, inhA, rpoB, embA, embB, rpsL, rrs, gyrA, gyrB and tlyA in sputum specimens and corresponding clinical isolates were detected by NGS method. The phenotypic drug susceptibility test (DST) of the strains was carried out by the proportion method. Using DST results as a reference, the sensitivity, specificity, positive predictive value and negative predictive value of the NGS method for clinical strains and sputum specimens, as well as the consistency statistic (Kappa) with phenotype DST were calculated respectively. The Chi-square test was used to compare the accuracy of the NGS testing in sputum samples and strain samples. Results: The results showed that rpoB(63.83%, 30/47) and rrs(57.45%, 27/47) were the most common mutated genes, followed by katG(46.81%, 22/47), rpsL(29.79%, 14/47), gyrA(27.66%, 13/47), embB(21.28%, 10/47), tlyA(12.77%, 6/47), gyrB(8.51%, 4/47), and inhA promoter(19.15%, 9/47), embA promoter region (12.77%, 6/47) mutation. when the NGS method was compared with the resistance phenotype of isoniazid, rifampicin, ethambutol, second-line injectable drugs (streptomycin, capreomycin, kanamycin, amikacin), levofloxacin, the sensitivity were 85.71%, 91.67%, 77.78%, 81.82%, 100.00%, 87.50%, 100.00%, 69.23%, and the specificity were 100.00%, 94.12, 87.50%, 89.47%, 97.06%, 96.97%, 94.29%, 89.29% in sputum samples, while in strain samples, the sensitivity were 92.86%, 100.00%, 81.82%, 86.96%, 88.89%, 80.00%, 100.00%, 85.71%. The specificity were 100.00%, 92.86%, 87.10%, 94.74%, 100.00%, 100.00%, 97.14%, 92.86%. Compared with the phenotypic drug susceptibility results, the NGS method has better detection performance for isoniazid, rifampicin, capreomycin, kanamycin, and amikacin in sputum specimens (Kappa≥0.75); while among the strains, the NGS method had a good detection performance for isoniazid, rifampicin, streptomycin, capreomycin, kanamycin, amikacin and levofloxacin (Kappa≥0.75). With the accuracy of the NGS method for detecting strains as a reference, there was no statistically significant difference in the accuracy of all drug resistance detected between strains and sputum specimens. Conclusions: This study showed that the NGS technology was effective in predicting the resistance of isoniazid, rifampicin, and second-line injectable drugs (capreomycin, kanamycin and amikacin) by detecting sputum samples and strain genotypes, suggesting the feasibility and potential of direct detection of sputum samples by the NGS method as an early detection method for drug resistance.

Integrated nucleic acid testing system to enable TB diagnosis in peripheral settings.

To facilitate treatment and limit transmission of tuberculosis (TB), new methods are needed to enable rapid and affordable diagnosis of the disease in high-burden low-resource settings. We have developed a prototype integrated nucleic acid testing device to detect Mycobacterium tuberculosis (M.tb) in sputum. The device consists of a disposable cartridge and compact, inexpensive instrument that automates pathogen lysis, nucleic acid extraction, isothermal DNA amplification and lateral flow detection. A liquefied and disinfected sputum sample is manually injected into the cartridge, and all other steps are automated, with a result provided in <1.5 h. Cell disruption and DNA extraction is executed within a four-port active valve containing a miniature bead blender (based on PureLyse® technology, Claremont BioSolutions LLC). The DNA-containing eluate is combined with dry master-mix reagents and target DNA is isothermally amplified. Amplified master-mix is then pumped into a lateral flow strip chamber for detection. The entire process is performed in a single-use closed-system cartridge to prevent amplicon carryover. For testing of M.tb-spiked sputum the system provided a limit of detection of 5 × 103 colony forming units (CFU) per mL. None of the negative sputum-only controls yielded a false-positive result. Testing of 45 clinical sputum specimens from TB cases and controls relative to a validated manual qPCR-based comparator method revealed a preliminary sensitivity of 90% and specificity of 96%. With further development, the herein described integrated nucleic acid testing device can enable TB diagnosis and treatment initiation in the same clinical encounter in near-patient low-resource settings of high TB burden countries.

Multicenter evaluation of the biochip assay for rapid detection of mycobacterial isolates in smear-positive specimens

ObjectivesThe objective of this study was to conduct a multicentre evaluation of the performance of the biochip assay in the rapid identification of mycobacteria in smear-positive sputum specimens. MethodsA total of 1751 sputum specimens were obtained from 7 cities in Zhejiang, China. All of the specimens were used for the discrimination of Mycobacterium species using the biochip assay, and the results were compared to the golden standard method of culture, hsp65, 16S rRNA and rpoB sequence analysis. ResultsIn the 1751 sputum specimens, 1685 samples were cultured successfully; among these samples, 1361 were Mycobacterium tuberculosis, 323 were NTM and 1 was Nocadia farcinica. Of the 323 NTM, most of them were Mycobacterium intracellulare(52.5%) followed by Mycobacterium abscessus (20.7%), Mycobacterium avium (11.7%), Mycobacterium kansasii (9.6%) and Mycobacterium fortuitum (1.9%). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the biochip assay to differentiate TB and NTM from AFB positive specimens were 99.8%, 99.7%, 99.9%, 99.1%, 98.8%, 1, 1, and 99.7%, respectively. The concordance between the biochip assay and mycobacterial culture for the identification of NTM species was 95.4%. ConclusionsThe biochip assay is a reliable tool for the rapid identification of most mycobacteria in clinical sputum specimens. This assay can be helpful for physicians in the early diagnosis and treatment of mycobacterium infections.

Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens.

Background: When available, nucleic acid tests (NATs) offer powerful tools to strengthen the potential of tuberculosis (TB) diagnosis assays. The sensitivity of molecular assays is critical for detection of Mycobacterium tuberculosis (MTB) in paucibacillary sputum.Materials and Methods: The impact of targeting repetitive IS6110 sequences on the PCR sensitivity was evaluated across mycobacterium strains and reference material. Six lysis-extraction protocols were compared. Next, 92 clinical sputum specimens including 62 culture-positive samples were tested and the results were compared to sputum-smear microscopy, culture, and Xpert MTB/RIF test. Finally, the capacity to detect low MTB DNA concentrations was assessed in 40 samples containing <1.5 × 102 copies/ml ex vivo or after dilution.Results: The lower limit of detection (LOD) using the IS6110 PCR was 107 genome copies/ml (95% CI: 83–130) using MTB H37Rv as a reference strain, versus 741 genome copies/ml (95% CI: 575–1094) using the senX3 PCR. The proportion of recovered MTB DNA after lysis and extraction ranged from 35 to 82%. The Chelex® method appeared as a more efficient protocol among the six different protocols tested. The sensitivity and specificity in clinical sputum samples were 95.1% (95% CI: 90.7–99.6) and 100% (95% CI: 96.2–100.8), respectively. Among 40 samples with low MTB DNA concentration, 75% tested positive for IS6110 PCR, versus 55% using the Xpert MTB/RIF assay (p = 0.03).Conclusion: Laboratory assays based on an efficient MTB lysis and DNA extraction protocols combined with amplification of IS6110 repeat sequences appear as a sensitive diagnostic method to detect MTB DNA in sputum with low bacterial load.

Fosfomycin to Control Contamination in &amp;lt;i&amp;gt;M. tuberculosis&amp;lt;/i&amp;gt; Culture in BACTEC MGIT 960 System

The BACTEC MGIT 960 system is widely used for recovery of Mycobacterium tuberculosis, but high contamination rates compromise its diagnostic performance. Efforts to control contamination must balance elimination of contaminating organisms while preserving growth of tuberculous bacilli. Fosfomycin has activity against many bacteria responsible for culture contamination yet is inactive against Mycobacterium tuberculosis. Therefore, it holds potential as a selection agent for the culture of M. tuberculosis clinical samples with the MGIT system. In this study, we assess the ability of fosfomycin supplement to MGIT cultures of clinical sputum specimens. Sputum specimens collected at 62 diagnosis and treatment monitoring visits during a randomized controlled trial of high-dose rifampin (ClinicalTrials.gov NCT01408914) were split: one-half was treated as conventional and the other half was treated with 1mg of fosfomycin. The frequencies of contamination and of recovery of M. tuberculosis were compared using McNemar and Wilcoxon signed-rank test, respectively, with a=0.05. In the fosfomycin-treated samples, 2/62 (3.2%) yielded contaminated cultures while 12/62 (19.4%) of untreated cultures were contaminated (p<0.001, McNemar Test). Detection of M. tuberculosis was not significantly altered by the addition of fosfomycin (Time to positivity, p=0.576, Student’s T Test; log colony forming units), nor the Colony Forming Unit Counts (Wilcoxon signed-rank test p=0.671). These results suggest that the use of fosfomycin to control contamination does not affect the natural growth of MTB. Its use could result in a significant improvement in recovery of M. tuberculosis in MGIT.

Performance of the Xpert MTB/RIF assay for the diagnosis of pulmonary tuberculosis and rifampin resistance in a low-incidence, high-resource setting.

Performance of the Xpert MTB/RIF assay, designed to simultaneously detect Mycobacterium tuberculosis complex (MTBC) and rifampin (RIF) resistance, has been well documented in low-resource settings with high TB-incidence. However, few studies have assessed its accuracy in low TB incidence settings. We evaluated the performance of Xpert MTB/RIF using clinical sputum specimens routinely collected from suspect pulmonary TB patients over a 4-year time period in San Diego County, California. Xpert MTB/RIF results were compared to acid-fast bacilli (AFB) smear microscopy, mycobacterial culture, and phenotypic drug susceptibility testing (DST). Of 751 sputum specimens, 134 (17.8%) were MTBC culture-positive and 2 (1.5%) were multidrug-resistant (MDR). For the detection of MTBC, Xpert MTB/RIF sensitivity was 89.6% (97.7% and 74.5% in smear-positive and -negative sputa, respectively) and specificity was 97.2%; while AFB smear sensitivity and specificity were 64.9% and 77.8%, respectively. Xpert MTB/RIF detected 35 of 47 smear-negative culture-positive specimens, and excluded 124 of 137 smear-positive culture-negative specimens. Xpert MTB/RIF also correctly excluded 99.2% (121/122) of nontuberculous mycobacteria (NTM) specimens, including all 33 NTM false-positives by smear microscopy. For the detection of RIF resistance, Xpert MTB/RIF sensitivity and specificity were 100% and 98.3%, respectively. Our findings demonstrate that Xpert MTB/RIF is able to accurately detect MTBC and RIF resistance in routinely collected respiratory specimens in a low TB-incidence setting, with comparable performance to that achieved in high-incidence settings; and suggest that under these conditions the assay has particular utility in detecting smear-negative TB cases, excluding smear-positive patients without MTBC disease, and differentiating MTBC from NTM.

Rapid screening of Mycobacterium tuberculosis complex (MTBC) in clinical samples by a modular portable biosensor

Mycobacterium tuberculosis complex (MTBC) is the well-known causative agents of tuberculosis (TB). Transmission of bacteria via airborne routes makes infection control difficult. Hence, rapid and accurate identification of TB from clinical sputum samples is essential for minimizing the spread of the disease. However, traditional bacterial identification methods for TB are time consuming and labor intensive. The aim of this study is to propose a straightforward and rapid screening platform for MTBC using a portable organic light-emitting diode- (OLED-) based surface plasmon resonance (SPR) biosensor integrated with a nested PCR technique. Experimentally, the limit of detection (LOD) for MTBC PCR products measurement was estimated to be around 63pg/mL. In addition, the biosensor can successfully differentiate MTBC from other nontuberculous mycobacterium strains. In 600 clinical sputum specimens, the sensitivity and specificity were 96.6% (86/89) and 98.4% (503/511), respectively. These results indicate that the portable OLED-based SPR biosensor may be taken into account as a suitable and convenient tool employed in the remote area and developing country for diagnosing TB diseases.

Early detection of multidrug- and pre-extensively drug-resistant tuberculosis from smear-positive sputum by direct sequencing

BackgroundEmergence of multidrug- and extensively drug-resistant tuberculosis (M/XDR-TB) is a major hurdle for TB control programs especially in developing countries like China. Resistance to fluoroquinolones is high among MDR-TB patients. Early diagnosis of MDR/pre-XDR-TB is essential for lowering transmission of drug-resistant TB and adjusting the treatment regimen.MethodsSmear-positive sputum specimens (n = 186) were collected from Wuhan Institute for Tuberculosis Control. The DNA was extracted from the specimens and run through a Sanger sequencing assay to detect mutations associated with MDR/pre-XDR-TB including the rpoB core region for rifampicin (RIF) resistance; katG and inhA promoter for isoniazid (INH) resistance; and gyrA for fluoroquinolone (FQ) resistance. Sequencing data were compared to phenotypic Lowenstein-Jensen (L-J) proportion method drug susceptibility testing (DST) results for performance analysis.ResultsBy comparing the mutation data with phenotypic results, the detection rates of MDR-TB and pre-XDR-TB were 84.31% (43/51) and 83.33% (20/24), respectively. The sequencing assay illustrated good sensitivity for the detection of resistance to RIF (96.92%), INH (86.89%), FQ (77.50%). The specificities of the assay were 98.35% for RIF, 99.20% for INH, and 97.26% for FQ.ConclusionsThe sequencing assay is an efficient, accurate method for detection of MDR-TB and pre-XDR-TB from clinical smear-positive sputum specimens, should be considered as a supplemental method for obtaining early DST results before the availability of phenotypic DST results. This could be of benefit to early diagnosis, adjusting the treatment regimen and controlling transmission of drug-resistant TB.

Multi-Fluorescence Real-Time PCR Assay for Detection of RIF and INH Resistance of M. tuberculosis.

Background: Failure to early detect multidrug-resistant tuberculosis (MDR-TB) results in treatment failure and poor clinical outcomes, and highlights the need to rapidly detect resistance to rifampicin (RIF) and isoniazid (INH).Methods: In Multi-Fluorescence quantitative Real-Time PCR (MF-qRT-PCR) assay, 10 probes labeled with four kinds of fluorophores were designed to detect the mutations in regions of rpoB, katG, mabA-inhA, oxyR-ahpC, and rrs.The efficiency of MF-qRT-PCR assay was tested using 261 bacterial isolates and 33 clinical sputum specimens. Among these samples, 227 Mycobacterium tuberculosis isolates were analyzed using drug susceptibility testing (DST), DNA sequencing and MF-qRT-PCR assay.Results: Compared with DST, MF-qRT-PCR sensitivity and specificity for RIF-resistance were 94.6 and 100%, respectively. And the detection sensitivity and specificity for INH-resistance were 85.9 and 95.3%, respectively. Compared with DNA sequencing, the sensitivity and specificity of our assay were 97.2 and 100% for RIF-resistance and 97.9 and 96.4% for INH-resistance. Compared with Phenotypic strain identification, MF-qRT-PCR can distinguish 227 M. tuberculosis complexes (MTC) from 34 Non-tuberculous mycobacteria (NTM) isolates with 100% accuracy rate.Conclusions: MF-qRT-PCR assay was an efficient, accurate, reliable, and easy-operated method for detection of RIF and INH-resistance, and distinction of MTC and NTM of clinical isolates.

Direct identification and discernment of Mycobacterium avium and Mycobacterium intracellulare using a real-time RNA isothermal amplification and detection method

The purpose of this work was to establish a real-time simultaneous amplification and testing method for identification and discernment of Mycobacterium avium and Mycobacterium intracellulare (SAT-MAC assay) and to evaluate the efficiency with which this method can detect isolated strains and clinical sputum specimens. The specific 16S rRNA sequences of M.avium and M.intracellulare were used as targets to design RNA probes and a reverse transcription primer containing T7 promoter. RNA isothermal amplification and real-time fluorescence detection were performed at 42°C. SAT-MAC assay, culture tests on Lowenstein-Jensen (L-J) culture medium and PCR-sequencing were used to test the clinical isolated strains and sputum specimens. The limit of detection (LOD) of M.avium and M.intracellulare by SAT-MAC was found to be 30CFU/mL and 20CFU/mL. SAT-MAC showed high specificity in 21 species of mycobacteria standard strains and 5 species of non-mycobacteria bacteria. Using PCR-sequencing as the reference method, both rates of SAT-MAC assay for identifying M.avium and M.intracellulare from clinical isolates were 100% (259/259). Consistent with the results of L-J culture combined PCR-sequencing, the coincidence rate of SAT-MAC assay in clinical sputum specimens was 100% (369/369) for M.avium and 99.19% (366/369) for Mycobacterium intracellular. The SAT-MAC assay can identify and distinguish M.avium and M.intracellulare rapidly and accurately. It may be suitable for use in clinical microbiology laboratories.

Laboratory evaluation of a specimen transport medium for downstream molecular processing of sputum samples to detect Mycobacterium tuberculosis

BackgroundModern molecular-based approaches for the detection of Mycobacterium tuberculosis in sputum samples promise quicker and more accurate detection of cases. However, processing sputum samples at central diagnostic facilities provides a diagnostic approach, but requires a safe and efficient system that is not affected by transport delays and ambient temperature to be feasible. We evaluated the technical properties of PrimeStore®-Molecular Transport Medium (PS-MTM) for its ability to inactivate mycobacteria, ensuring stability of DNA over time at ambient temperatures and to assess the compatibility of the transport medium with DNA extraction systems. MethodsAssessment of the transport medium for application of sputum samples processed for the detection of M. tuberculosis included the inactivation of M. tuberculosis in spiked sputum samples, compatibility of the medium with three commercial nucleic extraction systems and stability of DNA in the medium at ambient temperature over 28days. We further performed a clinical laboratory evaluation on 256 sputum specimens sent for tuberculosis investigation. ResultsComplete inactivation of M. tuberculosis occurred within 30min of exposure at a ratio of 1:3 for sputum to PS-MTM. Sputum specimen in PS-MTM showed very good compatibility with automated bead-based extraction systems, producing high DNA output (estimated lower limits of detection: ~170CFU/ml). Furthermore, PS-MTM samples remained stable over 28days at ambient temperature displaying no significant change over time in Ct-values (<5% on a mean starting value of 22.47). Of the 256 clinical sputum specimens, 10.2% were culture positive and 11.0% were positive by real-time PCR of PS-MTM samples. ConclusionsCollecting and transporting sputum from TB suspects in PS-MTM offer safe transport at ambient temperature, DNA stability for extended periods without cooling and specimens directly suitable for molecular testing. This novel approach may support introduction and further scale-up of molecular diagnostics for TB in resource-limited settings.

Development and evaluation of a rapid multiplex-PCR based system for Mycobacterium tuberculosis diagnosis using sputum samples

Global tuberculosis (TB) control and eradication is hampered by the unavailability of simple, rapid and affordable diagnostic tests deployable at low infrastructure microscopy centers. We have developed and evaluated the performance of a nucleic acid amplification test for detection of Mycobacterium tuberculosis (MTB), the NWU-TB test, in clinical sputum specimens from 306 patients with suspected pulmonary tuberculosis. The test involves sputum sample processing using a Lyser device within 7min, followed by rapid multiplex-PCR on a fast thermal cycler within 25min, and amplicon resolution on agarose gel electrophoresis. Samples were also examined for presence of MTB using smear microscopy, GeneXpert and MGIT culture. Results were assessed in comparison to a MGIT culture as gold standard. Of the 306 patients, 174 had a previous TB history or already on treatment, and 132 were TB naïve cases. The NWU-TB system was found to have an overall sensitivity and specificity of 80.8% (95% CI: 75–85.7) and 75.6% (95% CI: 64.9–84.4) respectively, in comparison to 85.3% (95% CI: 79.9–89.6) and 73.2% (95% CI: 62.2–82.4) respectively for GeneXpert; and 62.1% (95% CI: 55.3–68.4) and 56.1% (95% CI: 44.7–67) respectively for smear microscopy. The study has shown that the NWU-TB system allows detection of TB in less than two hours and can be utilized at low infrastructure sites to provide quick and accurate diagnosis at a very low cost.

Development of sandwich-form biosensor to detect Mycobacterium tuberculosis complex in clinical sputum specimens

Mycobacterium tuberculosis, the causing agent of tuberculosis, comes second only after HIV on the list of infectious agents slaughtering many worldwide. Due to the limitations behind the conventional detection methods, it is therefore critical to develop new sensitive sensing systems capable of quick detection of the infectious agent. In the present study, the surface modified cadmium-telluride quantum dots and gold nanoparticles conjunct with two specific oligonucleotides against early secretory antigenic target 6 were used to develop a sandwich-form fluorescence resonance energy transfer-based biosensor to detect M. tuberculosis complex and differentiate M. tuberculosis and M. bovis Bacille Calmette–Guerin simultaneously. The sensitivity and specificity of the newly developed biosensor were 94.2% and 86.6%, respectively, while the sensitivity and specificity of polymerase chain reaction and nested polymerase chain reaction were considerably lower, 74.2%, 73.3% and 82.8%, 80%, respectively. The detection limits of the sandwich-form fluorescence resonance energy transfer-based biosensor were far lower (10fg) than those of the polymerase chain reaction and nested polymerase chain reaction (100fg). Although the cost of the developed nanobiosensor was slightly higher than those of the polymerase chain reaction-based techniques, its unique advantages in terms of turnaround time, higher sensitivity and specificity, as well as a 10-fold lower detection limit would clearly recommend this test as a more appropriate and cost-effective tool for large scale operations.

A rapid fluorescence polarization-based method for genotypic detection of drug resistance in Mycobacterium tuberculosis

Rapid detection of drug-resistant Mycobacterium tuberculosis is critical to the effective early treatment and prevention of the transmission of tuberculosis. However, conventional drug susceptibility tests for M. tuberculosis require up to several weeks. In the present study, the One Label Extension genotyping method was adapted for rapid detection of drug resistance-associated sequence variations in six genes of M. tuberculosis, viz. rpoB, rpsL, rrs, embB, katG, or inhA. The method utilizes polymerase chain reaction amplified fragments of the drug resistant genes as reaction templates, and proceeds with template-directed primer extension incorporating a fluorescence-labeled nucleotide, which is then measured by fluorescence polarization. A total of 121 M. tuberculosis isolates from clinical sputum specimens were examined by this genotyping method and verified by direct sequencing of polymerase chain reaction amplicons harboring previously reported mutational sites associated with M. tuberculosis drug resistance. Based on phenotyping results obtained from microbiology-based drug susceptibility tests, the sensitivity, specificity, and test efficiency estimated for One Label Extension assays were respectively 83.9%, 95.5%, and 92.4% with ropB in rifampin resistance, 67.3%, 97.1%, and 84.3% with rpsL and rrs in streptomycin resistance, 60.0%, 96.0%, and 91.4% with embB in ethambutol resistance, 68.4%, 94.9%, and 86.3% with inhA and katG in isoniazid resistance, and 74.1%, 98.9%, and 93.2% in multiple drug resistance defined as resistance to at least both isoniazid and rifampin. In conclusion, examination of clinical sputum specimens by One Label Extension based genotyping provides a valid method for the rapid molecular detection of drug-resistant M. tuberculosis.

Application of Hyperbranched Rolling Circle Amplification for Direct Detection of Mycobacterium Tuberculosis in Clinical Sputum Specimens

BackgroundGlobal tuberculosis (TB) control is encumbered by the lack of a rapid and simple detection method for diagnosis, especially in low-resource areas. An isothermal amplification method, hyperbranched rolling circle amplification (HRCA), was optimized to detect Mycobacterium tuberculosis (Mtb) in clinical sputum specimens.MethodsA clinical validation study was performed to assess the diagnostic accuracy of HRCA. In order to analyze the detection limit of HRCA under optimal conditions, the method was initially used to detect purified H37Rv strain DNA and culture suspensions. Next, three strains of Mycobacterium tuberculosis complex (MTC) and eight strains of non-tuberculosis mycobacterium (NTM) were analyzed in order to evaluate specificity. Sputum specimens from 136 patients with diagnosed pulmonary TB, 38 lung cancer patients, and 34 healthy donors were tested by HRCA to validate the clinical application of HRCA for the rapid detection of Mtb.ResultsThe detection limit of HRCA for purified H37Rv DNA and culture suspensions was 740 aM and 200cfu/ml, respectively. The results of all MTC strains were positive in contrast to the NTM specimens which were all negative. The detection sensitivity for the 136 sputum specimens from TB patients was 77.2% (105/136), which was slightly lower than that of quantitative real-time PCR(79.4%, 108/136) and culture (80.9%,110/136). The sensitivity of all three methods was statistically higher than smear microscopy (44.9%, 61/136). The overall specificity of HRCA was 98.6% (71/72) which was similar to that of quantitative real-time PCR (qRT-PCR) and smear/culture methods (100%, 72/72).ConclusionsUse of the HRCA assay for detection of Mtb within clinical sputum specimens was demonstrated to be highly sensitive and specific. Moreover, the performance of HRCA is simple and cost-effective compared with qRT-PCR and is less time consuming than culture. Therefore, HRCA is a promising TB diagnostic tool that can be used routinely in low-resource clinical settings.

Rapid Diagnosis of Mycobacterium tuberculosis with Truenat MTB: A Near-Care Approach

BackgroundControl of the global Tuberculosis (TB) burden is hindered by the lack of a simple and effective diagnostic test that can be utilized in resource-limited settings.MethodsWe evaluated the performance of Truenat MTB™, a chip-based nucleic acid amplification test in the detection of Mycobacterium tuberculosis (MTB) in clinical sputum specimens from 226 patients with suspected pulmonary tuberculosis (TB). The test involved sputum processing using Trueprep-MAG™ (nanoparticle-based protocol run on a battery-operated device) and real-time PCR performed on the Truelab Uno™ analyzer (handheld, battery-operated thermal cycler). Specimens were also examined for presence of MTB using smear microscopy, liquid culture and an in-house nested PCR protocol. Results were assessed in comparison to a composite reference standard (CRS) consisting of smear and culture results, clinical treatment and follow-up, and radiology findings.ResultsBased on the CRS, 191 patients had “Clinical-TB” (Definite and Probable-TB). Of which 154 patients are already on treatment, and 37 were treatment naïve cases. Remaining 35 were confirmed “Non-TB” cases which are treatment naïve cases. The Truenat MTB test was found to have sensitivity and specificity of 91.1% (CI: 86.1–94.7) and 100% (CI: 90.0–100) respectively, in comparison to 90.58% (CI: 85.5–94.3) and 91.43% (CI: 76.9–98.2) respectively for the in-house nested PCR protocol.ConclusionThis preliminary study shows that the Truenat MTB test allows detection of TB in approximately one hour and can be utilized in near-care settings to provide quick and accurate diagnosis.

TRICORE, a novel bead-based specimen concentration method for the culturing of Mycobacterium tuberculosis

Centrifugation is a necessary concentrating step for the detection of Mycobacterium tuberculosis in a liquid culture. However, centrifugation is biologically hazardous and presents an obstacle in the development of an automated culture system. A bead-based bacterial concentration method, TRICORE, was recently developed by Genetein Co., Ltd. We compared the efficacy of TRICORE and conventional centrifugation for concentrating M. tuberculosis in clinical sputum specimens by using liquid and solid culture systems. Among 90 pretreated clinical sputum specimens, 51 (57.3%) and 55 (61.8%) M. tuberculosis isolates were recovered by the MGIT culture system by using the centrifugation and TRICORE methods, respectively (chi‐square test, p=0.5413). The detection time for the centrifugation method was 359.3±117.0h, while that for the bead-based concentration method was 377.6±162.3h (p=0.5637). However, the number of colonies recovered on solid media were significantly higher with the TRICORE method (p=0.003). In particular, among the smear-negative specimens, culture positivity of the TRICORE method was 39.6%, while that of the centrifugation method was 15.1%. The TRICORE bead-based concentration method was considered equivalent to centrifugation and enabled efficient collection of paucibacillary specimens in solution. Thus, the new noncentrifugation concentration method could yield more positive culture results.