Lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by PCR

<p>Abstract</p> <p>Background</p> <p>Nucleic acid amplification techniques are being used increasingly in diagnosing tuberculosis. In developing countries clinical samples are often stored for subsequent analysis since molecular tests are conducted at only a limited number of laboratories. This study was conducted to assess the speed at which mycobacteria undergo autolysis and free DNA is detected in the supernatant during low-temperature storage.</p> <p>Results</p> <p>Eighty-seven smear positive sputa from tuberculosis patients were analysed immediately and after storage at -20°C. Timelines of 1 and 2 months were selected to assess the maximum extent of DNA loss that occurred during storage. All samples remained PCR- and smear-positive at 1 month and only 1 sample turned negative after 2 months. Bacterial lysis in the specimens was demonstrated by PCR analysis of supernatant fractions; 53% of the freshly analysed samples contained mycobacterial DNA in supernatants. PCR positivity increased significantly during storage (to 69% and 77% after 1 and 2 months of storage, respectively, P < 0.0001). Storage-associated bacterial lysis was accompanied by a decrease in smear grade status in 28 of 87 samples (P < 0.0001 after 2 months of storage) and a significant storage-associated reduction in bacterial numbers in the remaining samples.</p> <p>Conclusion</p> <p>We conclude that (i) freshly isolated sputum contains both intact and lysed mycobacteria, (ii) lysis increased during storage and (iii) supernatant fractions routinely discarded during sample processing contain mycobacterial DNA. We propose that supernatant is a valuable sample for PCR for both fresh and stored specimens, particularly those with a low bacterial load in addition to conventional sediment.</p

Highly Sensitive Detection of Staphylococcus aureus Directly from Patient Blood

Background: Rapid detection of bloodstream infections (BSIs) can be lifesaving. We investigated the sample processing and assay parameters necessary for highly-sensitive detection of bloodstream bacteria, using Staphylococcus aureus as a model pathogen and an automated fluidic sample processing – polymerase chain reaction (PCR) platform as a model diagnostic system. Methodology/Principal Findings: We compared a short 128 bp amplicon hemi-nested PCR and a relatively shorter 79 bp amplicon nested PCR targeting the S. aureus nuc and sodA genes, respectively. The sodA nested assay showed an enhanced limit of detection (LOD) of 5 genomic copies per reaction or 10 colony forming units (CFU) per ml blood over 50 copies per reaction or 50 CFU/ml for the nuc assay. To establish optimal extraction protocols, we investigated the relative abundance of the bacteria in different components of the blood (white blood cells (WBCs), plasma or whole blood), using the above assays. The blood samples were obtained from the patients who were culture positive for S. aureus. Whole blood resulted in maximum PCR positives with sodA assay (90 % positive) as opposed to cell-associated bacteria (in WBCs) (71 % samples positive) or free bacterial DNA in plasma (62.5 % samples positive). Both the assays were further tested for direct detection of S. aureus in patient whole blood samples that were contemporaneous culture positive. S. aureus was detected in 40/45 of culture-positive patients (sensitivity 89%, 95 % CI 0.75–0.96) and 0/59 negative controls with the sodA assay (specificit

Updating the approaches to define susceptibility and resistance to anti-tuberculosis agents: implications for diagnosis and treatment

11 páginas, 2 figuras, 1 tablaInappropriately high breakpoints have resulted in systematic false-susceptible AST results to anti-TB drugs. MIC, PK/PD and clinical outcome data should be combined when setting breakpoints to minimise the emergence and spread of antimicrobial resistance.I. Comas was supported by PID2019-104477RB-I00 from the Spanish Science Ministry and by ERC (CoG 101001038)Peer reviewe

Novel Multipurpose Methodology for Detection of Mycobacteria in Pulmonary and Extrapulmonary Specimens by Smear Microscopy, Culture, and PCR

A novel, robust, reproducible, and multipurpose universal sample processing (USP) methodology for highly sensitive smear microscopy, culturing on solid and liquid media, and inhibition-free PCR which is suitable for the laboratory diagnosis of both pulmonary and extrapulmonary tuberculosis (TB) has been developed. This method exploits the chaotropic properties of guanidinium hydrochloride for sample processing and involves incubating the specimen with USP solution, concentrating bacilli by centrifugation, and using the processed specimen for smear microscopy, culture, and PCR. The detection limit for acid-fast bacilli in spiked sputum by smear microscopy is approximately 300 bacilli per ml of specimen. USP solution-treated specimens are fully compatible with culturing on solid and liquid media. High-quality, PCR-amplifiable mycobacterial DNA can be isolated from all types of clinical specimens processed with USP solution. The method has been extensively validated with both pulmonary and extrapulmonary specimens. Furthermore, the USP method is also compatible with smear microscopy, culture, and PCR of mycobacteria other than tubercle bacilli. In summary, the USP method provides smear microscopy, culture, and nucleic acid amplification technologies with a single sample-processing platform and, to the best of our knowledge, is the only method of its kind described to date. It is expected to be useful for the laboratory diagnosis of TB and other mycobacterial diseases by conventional and modern methods

Utility of Universal Sample Processing Methodology, Combining Smear Microscopy, Culture, and PCR, for Diagnosis of Pulmonary Tuberculosis

The universal sample processing (USP) multipurpose methodology was developed for the diagnosis of tuberculosis (TB) and other mycobacterial diseases by using smear microscopy, culture, and PCR (S. Chakravorty and J. S. Tyagi, J. Clin. Microbiol. 43:2697-2702, 2005). Its performance was evaluated in a blinded study of 571 sputa and compared with that of the direct and N-acetyl l-cysteine (NALC)-NaOH methods of smear microscopy and culture. With culture used as the gold standard, USP smear microscopy demonstrated a sensitivity and specificity of 98.2% and 91.4%, respectively, compared to 68.6% and 92.6%, respectively, for the direct method. For a subset of 325 specimens, the USP method recorded a 97.1% sensitivity and 83.2% specificity compared to the NALC-NaOH method, which had a sensitivity and specificity of 80.0% and 89.7%, respectively, with culture used as the gold standard. Thus, the USP method exhibited a highly significant enhancement in sensitivity (P < 0.0001) compared to the direct and NALC-NaOH methods of smear microscopy. The USP culture sensitivity was 50.1% and was not significantly different from that of conventional methods (53.6%). The sensitivity and specificity of IS6110 PCR were 99.1% and 71.2%, respectively, with culture used as the gold standard, and increased to 99.7% and 78.8%, respectively, when compared with USP smear microscopy. Thus, the USP methodology was highly efficacious in diagnosing TB by smear microscopy, culture, and PCR in a clinical setting

Diagnosis of Extrapulmonary Tuberculosis by Smear, Culture, and PCR Using Universal Sample Processing Technology

Definitive and rapid diagnosis of extrapulmonary tuberculosis is challenging since conventional techniques have limitations. We have developed a universal sample processing (USP) technology for detecting mycobacteria in clinical specimens. In this study, this technology was evaluated blindly on 99 extrapulmonary specimens collected from 87 patients. USP-processed specimens were submitted to smear microscopy for detection of acid-fast bacilli (AFB), culture, and two PCR tests targeting devR (Rv3133c) and IS6110 gene sequences. On the basis of clinical characteristics, histology and cytology, conventional microbiology results, and response to antitubercular therapy, 68 patients were diagnosed with tuberculosis. Although USP smear and culture were significantly superior to conventional microbiology, which was not optimized (P < 0.0001), these approaches fell short of PCR tests (P < 0.0001). The low yields by smear and culture are attributed to the paucibacillary load in the specimens. The highest sensitivity in PCR was achieved when devR and IS6110 test results were combined; the sensitivity and specificity values were 83 and 93.8%, 87.5 and 100%, and 66.7 and 75%, respectively, in pleural fluid, needle-biopsied pleural tissue, and lymph node specimens. In conclusion, the application of USP technology, together with clinicopathological characteristics, promises to improve the accuracy and confidence of extrapulmonary tuberculosis diagnosis

Simplified detection of Mycobacterium tuberculosis in sputum using smear microscopy and PCR with molecular beacons

The prompt diagnosis of smear-negative cases is a prerequisite to controlling tuberculosis (TB). Several new laboratory approaches, including nucleic acid amplification (NAA), are being evaluated in various disease settings to meet this challenge. However, NAA needs simplification before it is widely accepted. Furthermore, a supporting smear result improves confidence in and reliability of PCR. In this context, an asymmetric devR PCR assay using two molecular beacon probes for visual or fluorimetric end-point detection of Mycobacterium tuberculosis was developed. The assays reproducibly detected 25 fg M. tuberculosis DNA versus 100 fg by conventional gel electrophoresis (henceforth referred to as gel assay). The devR and IS6110 PCR assays were blindly evaluated on sputum specimens obtained from a directly observed-treatment short-course centre. Universal sample processing (USP) smear microscopy and culture were used as a supportive test and the 'gold' standard, respectively. Among the 148 specimens analysed, 120 were M. tuberculosis culture-positive. Amongst the 122 direct smear-negative samples, 96 were culture-positive, of which 61 were detected by USP smear microscopy. All 35 USP smear-negative samples were positive by three or more PCR methods. devR PCR had a sensitivity of 92.5 % in the fluorimetric assay versus 86.7 % by visual inspection and 90.8 % by the gel method. IS6110 PCR performed at almost equivalent levels. devR visual and fluorimetric assays considered together yielded an increased sensitivity of 95 % without compromising on a specificity of 92.9 %. The results suggest that the USP smear test is useful for diagnosing direct smear-negative TB and judiciously restricting PCR testing to only smear-negative samples. When used together, these tests can provide rapid diagnosis of smear-negative TB in a cost-effective manner

Lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by PCR-6

<p><b>Copyright information:</b></p><p>Taken from "Lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by PCR"</p><p>http://www.biomedcentral.com/1471-2180/7/83</p><p>BMC Microbiology 2007;7():83-83.</p><p>Published online 4 Sep 2007</p><p>PMCID:PMC2008200.</p><p></p>ctions from fresh, 1 month and 2 months stored samples, respectively

PCR amplification of shorter fragments from the devR (Rv3133c) gene significantly increases the sensitivity of tuberculosis diagnosis

This study was designed to assess the vital issue of gene target length and PCR assay performance in relation to the detection of Mycobacterium tuberculosis in clinical specimens. Two PCR assays that amplify fragments of varying lengths from the devR gene of M. tuberculosis were evaluated. Using M. tuberculosis DNA the 'short-length' PCR assay detected 250-500 genome equivalents vs. 500-1000 genome equivalents by the 'long-length' assay. In comparison to a highly sensitive smear microscopy test (universal sample processing smear), the sensitivity of the 'short-length' assay was 97.8% vs. 69.9% of the 'long-length' assay in sputum specimens (n=506) from patients being evaluated for a possible diagnosis of tuberculosis. The 27.9% absolute increase in sensitivity was statistically significant (P&#60;0.001). Our results indicate that in a clinical setting when all other conditions are equal, the amplification of a shorter gene fragment of devR increases the sensitivity and efficiency of the PCR assay in spite of using a single copy gene as target