Hematological Parameters as Potential Markers for Distinguishing Pulmonary Tuberculosis from Genitourinary Tuberculosis.

Mycobacterium tuberculosis complex (MTBC) infection is an important public health concern in Taiwan. In addition to pulmonary tuberculosis (PTB), MTBC can also cause genitourinary tuberculosis (GUTB). This study aimed to examine the role of laboratory data and the values that can be calculated from them for the early detection of GUTB. Patients admitted from 2011 to 2020 were retrospectively recruited to analyze their associated clinical data. Statistical significance was analyzed using the chi-square test and univariate analysis for different variables. A receiver operating characteristic (ROC) curve analysis was used to evaluate the performances of the examined laboratory data and their calculated items, including the neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), neutrophil-to-monocyte-plus-lymphocyte ratio (NMLR), and platelet-to-lymphocyte ratio (PLR), in diagnosing PTB or GUTB. A p-value of <0.05 was considered significant. The ROC curve showed that the discriminative power of the neutrophil count, NLR, and MLR was within the acceptable level between patients with both PTB and GUTB and those with GUTB alone (area under the curve [AUC] values = 0.738, 0.779, and 0.725; p = 0.024, 0.008, and 0.033, respectively). The discriminative power of monocytes and the MLR was within the acceptable level (AUC = 0.782 and 0.778; p = 0.008 and 0.010, respectively). Meanwhile, the neutrophil and lymphocyte counts, NLR, NMLR, and PLR had good discriminative power (AUC = 0.916, 0.896, 0.898, 0.920, and 0.800; p < 0.001, <0.001, <0.001, <0.001, and 0.005, respectively) between patients with GUTB and those with PTB alone. In conclusion, the neutrophil count, lymphocyte count, NLR, NMLR, and PLR can be used as potential markers for distinguishing PTB from GUTB.

Evaluation of an AI-Based TB AFB Smear Screening System for Laboratory Diagnosis on Routine Practice.

The most robust and economical method for laboratory diagnosis of tuberculosis (TB) is to identify mycobacteria acid-fast bacilli (AFB) under acid-fast staining, despite its disadvantages of low sensitivity and labor intensity. In recent years, artificial intelligence (AI) has been used in TB-smear microscopy to assist medical technologists with routine AFB smear microscopy. In this study, we evaluated the performance of a TB automated system consisting of a microscopic scanner and recognition program powered by artificial intelligence and machine learning. This AI-based system can detect AFB and classify the level from 0 to 4+. A total of 5930 smears were evaluated on the performance of this automatic system in identifying AFB in daily lab practice. At the first stage, 120 images were analyzed per smear, and the accuracy, sensitivity, and specificity were 91.3%, 60.0%, and 95.7%, respectively. In the second stage, 200 images were analyzed per smear, and the accuracy, sensitivity, and specificity were increased to 93.7%, 77.4%, and 96.6%. After removing disqualifying smears caused by poor staining quality and smear preparation, the accuracy, sensitivity, and specificity were improved to 95.2%, 85.7%, and 96.9%, respectively. Furthermore, the automated system recovered 85 positive smears initially identified as negative by manual screening. Our results suggested that the automated TB system could achieve higher sensitivity and laboratory efficiency than manual microscopy under the quality control of smear preparation. Automated TB smear screening systems can serve as a screening tool at the first screen before manual microcopy.

High Rates of Antimicrobial Resistance in Rapidly Growing Mycobacterial Infections in Taiwan.

Rapidly growing mycobacteria (RGM) has gained increasing clinical importance, and treatment is challenging due to diverse drug resistance. The minimum inhibitory concentrations (MIC) of 13 antimicrobial agents using modified broth microdilution and E-test were determined for 32 clinical isolates of RGM, including Mycobacterium abscessus (22 isolates) and Mycobacterium fortuitum (10 isolates). Our results showed high rates of resistance to available antimicrobial agents. Amikacin remained highly susceptible (87.5%). Clarithromycin was active against the isolates of M. abscessus (95.5%), and M. fortuitum (50%), but 36.4% and 20% had inducible macrolide resistance, respectively. Rates of susceptibility to tigecycline were 68.2-70%, and linezolid 45.5-50%, respectively. The quinolones (ciprofloxacin and moxifloxacin) showed better in vitro activity against M. fortuitum isolates (50% susceptibility) than the M. abscessus isolates (31.8% susceptibility). The susceptibilities to other conventional anti-mycobacterial agents were poor. The MICs of E-test were higher than broth microdilution and may result in reports of false resistance. In conclusion, the implementation of the modified broth microdilution plates into the routine clinical laboratory workflow to provide antimicrobial susceptibility early, allows for the timely selection of appropriate treatment of RGM infections to improve outcome.

Rapid identification of mycobacteria from positive MGIT broths of primary cultures by MALDI-TOF mass spectrometry.

BACKGROUND: Rapid identification of mycobacteria is important for timely treatment and the implementation of public health measures. The MGIT system ensures rapid detection of mycobacteria, but identification is usually delayed by days to weeks due to further subculture on solid medium. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) was demonstrated to effectively identify mycobacteria isolates subcultured from solid or liquid media. Reports of identification directly from MGIT broths of both sterile and non-sterile clinical specimens, omitting the subculture step, were limited and not satisfactory before. Our identification method dramatically shortened delay from detection to identification of mycobacteria. METHODOLOGY: We assessed the performance of the Vitek MS IVD version 3.0 for direct identification of NTM and M.tuberculosis from primary MGIT cultures, and assessed two sample preparation methods. RESULTS: Direct identification of NTM from positive MGIT broths, using MALDI-TOF VITEK MS with IVD v.3.0, generated high rates of acceptable results reaching 96.4% (80/83), and up to 100% (83/83) for sample preparations including a 0.1% SDS washing step. The sensitivity of VITEK MS to identify M.tuberculosis from MGIT tubes was 58/72 (80.6%), when using immunochromatography (ICA) test as gold standard. A characteristic colony clumping, wool-like appearance was observed in 48, and all 58 (100%) were correctly identified as M.tuberculosis using MALDI-TOF. The detection rate of M.tuberculosis complex was low (10/24, 41.6%) in the 24 MGIT tubes that was polymicrobial. Our method significantly reduced both the reagent cost and turnaround time. CONCLUSIONS: Based on a simplified protocol, we showed that MALDI-TOF MS can be used for rapid identification of NTM directly from primary MGIT cultures within the routine clinical laboratory workflow. However, we recommend an initial ICA test to screen for M.tuberculosis complex, due to a low identification rate of M. tuberculosis in the presence of polymicrobial cultures using MALDI-TOF.

Differential diagnosis of tuberculous and malignant pleurisy using pleural fluid adenosine deaminase and interferon gamma in Taiwan.

Accurately differentiating tuberculous pleurisy from lung cancer is important for disease management but difficult using conventional laboratory methods. This study assessed the value of adenosine deaminase (ADA) and interferon gamma (IFN-γ) for differentiating the two conditions in a region of Taiwan with a high prevalence of tuberculosis. The study population comprised patients with lymphocytic exudative pleural effusions: tuberculous (n=24) and malignant (n=42). Mean levels of ADA and IFN-γ in pleural fluid, measured with commercial standardized kits, were significantly higher for tuberculous than for malignant pleurisy (p<0.001 for both). For differentiating the two effusions, results for ADA versus IFN-γ were: sensitivity, 70.8% versus 91.7%; specificity, 95.2% versus 97.6%; positive predictive value, 89.5% versus 96.7%; and negative predictive value, 85.1% versus 95.3%. IFN-γ allows precise diagnosis of pleural tuberculosis, but ADA is easier to use, has a low cost, and results are quickly available. Our study confirms previous studies and extends the usefulness of these diagnostic methods to a wider group of clinical laboratories by showing the reliability of standardized relatively inexpensive commercial kits. We recommend that initial ADA screening be used in conjunction with IFN-γ measurements for differential diagnosis of tuberculous pleurisy.

In vitro activities of linezolid against clinical isolates of Mycobacterium tuberculosis complex isolated in Taiwan over 10 years.

Significant increases in the MIC(90)s of linezolid in multidrug-resistant Mycobacterium tuberculosis isolates were seen between the baseline period of 2001 to 2003 (0.5 microg/ml) and 2004 (2 microg/ml). The MICs were 4 microg/ml in three strains. Both fluoroquinolones (except levofloxacin) and kanamycin were found to have statistically significant degrees of concordance with linezolid.

Rapid purity check method for susceptibility testing of M. tuberculosis complex with the MGIT 960 system.

The Bactec MGIT 960 system is a rapid and reliable automated method for drug susceptibility testing of Mycobacterium tuberculosis complex (MTBC) that yields a high percentage of agreement with the standard method. The microscopic cord morphology of M. tuberculosis in liquid medium is characteristic, and readily differentiates MTBC from nontuberculous mycobacteria (NTM). The goals of this study were to describe the microscopic and macroscopic growth morphology of MTBC in antimicrobial-containing MGIT tubes and to evaluate the usefulness of the growth appearance during purity checking. The macroscopic cotton wool-like appearance of MTBC isolates in isoniazid (INH), streptomycin (SM), rifampin (RMP), and ethambutol (EMB)-containing tubes was observed in 97, 90, 93, and 71% of the isolates, respectively. The percentage of typical cord, loose, or frayed rope microscopic features in smears prepared from MTBC-positive cultures of INH, SM, RMP, and EMB-containing tubes was 96, 86, 97, and 71%, respectively. The sensitivity of the macroscopic morphology for predicting the purity of drug-containing MGIT tubes was 93%, while the microscopic morphology predicted the purity with a sensitivity rate of 92%. We found that simply examining the macroscopic morphology of the antimicrobial-containing MGIT tubes of drug-resistant MTBC isolates is useful in preventing false resistant results of susceptibility testing by the MGIT 960 system.

Use of a disposable water filter for prevention of false-positive results due to nontuberculosis mycobacteria in a clinical laboratory performing routine acid-fast staining for tuberculosis.

A point-of-use 0.2-microm filter was evaluated for elimination of nontuberculosis mycobacteria in laboratory water to reduce false-positive acid-fast bacillus staining results. Use of the point-of-use filter can significantly reduce the false-positive rate to 1.2% compared to samples treated with tap water (10.7%) and deionized water (8.7%).

Mycobacterium abscessus cervical lymphadenitis: an immunocompetent child.

Nontuberculous mycobacterium (NTM) is one of the well-known causes of cervicofacial lymphadenopathy in children under 5 years of age. Children often present with a painless cervical mass that fails to respond to conventional antibiotics. They are often referred under the suspicion of a neoplasm or bacterial adenitis rather than NTM cervical lymphadenitis. The lack of systemic symptoms, modest or negative purified protein derivative test and absence of exposure to active tuberculosis are characteristics of NTM lymphadenitis. The diagnosis usually requires the isolation of pathogen or pathologic proof. Complete excision is the choice of treatment by the majority of authors in the literature. This not only enables rapid diagnosis but ensures the lowest recurrence rate. Medical management is sometimes successful when complete resection is impossible or refused. To our knowledge, the incidence of NTM cervical lymphadenitis in children is increasing throughout the world. However, such reports of children in Taiwan is lacking. Clinicians should suspect a possible nontuberculous mycobacterial infection when a cervical lump is found in a child.

Preservation of clinical isolates of Mycobacterium tuberculosis complex directly from MGIT culture tubes.

Preservation of M. tuberculosis complex strains isolated from clinical specimens is important for epidemiological investigations related to tuberculosis. In this study the efficacy of preservation was evaluated by calculating the recovery rate of preserved strains, with various patterns of resistance, after periods of storage and subculture. The recovery rates from strains preserved in enriched solid medium were >90% for storage periods

Trends in fluoroquinolone resistance of Mycobacterium tuberculosis complex in a Taiwanese medical centre: 1995-2003.

OBJECTIVES: Fluoroquinolones are being used more frequently for the treatment of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis complex (MTB). This study was designed to determine the frequency of the emergence of fluoroquinolone-resistant strains in Taiwan and to assess whether this might be due to use of fluoroquinolones for treatment of patients with MDR or because of increased use of fluoroquinolones in the community for treatment of other infections. We also sought to determine whether there might be clonal spread of fluoroquinolone resistance. METHODS: A total of 3497 clinical isolates of M. tuberculosis complex were obtained during 1995-2003, of which 141 were selected. They consisted of 62 isolates fully susceptible to four first-line drugs, 33 isolates resistant to rifampicin and isoniazid (MDR), and 46 isolates with a variety of any drug resistant patterns other than MDR (combination group). The MICs were determined for ciprofloxacin, ofloxacin and levofloxacin. RESULTS: An increase in the MIC90 and rates of resistance to ciprofloxacin, ofloxacin and levofloxacin were noted only in the MDR group. The rates were higher among strains isolated between 1998-2003 compared with those obtained between 1995-1997 (rate of resistance, 20% versus 7.7%; MIC > or = 4 mg/L versus 1-2 mg/L). Among the 10 fluoroquinolone-resistant isolates, five (50%) possessed mutations other than S95T in the gyrA gene. No gyrB mutation was found in any of the clinical isolates. CONCLUSIONS: These findings suggest that fluoroquinolone resistance is the result of treatment of patients with MDR strains rather than from use in the general community in Taiwan. The emergence of fluoroquinolone resistance among MDR strains reinforces the need for routine fluoroquinolone susceptibility testing whenever these drugs might be used.

Discordant molecular characterization results in a Mycobacterium avium complex strain isolated from an AIDS patient.

This report describes an unusual strain of Mycobacterium avium complex isolated from the sputum of an immunocompromised AIDS patient, which did not react with the MAC probe of the BDProbe Tec system, but was identified as Mycobacterium intracellulare by 16S rRNA gene sequencing. Its PCR restriction-enzyme analysis pattern was compatible with an allelic variant of M. avium. It was scotochromogenic, slow-growing and phenotypically identified as Mycobacterium scrofulaceum. Its clinical significance is not certain.

Use of MGIT 960 for rapid quantitative measurement of the susceptibility of Mycobacterium tuberculosis complex to ciprofloxacin and ethionamide.

OBJECTIVES: Tentative standards for testing MICs for Mycobacterium tuberculosis include agar dilution and the BACTEC method. However, the conventional agar dilution method requires 3-5 weeks to complete; whereas BACTEC, although a rapid test, involves the use of radioisotopes. In contrast, the MGIT 960 system uses a fluorescence quenching based oxygen sensor that can be read automatically. This system is not only robust, safe and simple, but has been validated for susceptibility tests of first-line antituberculous agents. METHODS: We evaluated 46 clinical strains of M. tuberculosis isolated from patients admitted to Kaohsiung Veterans General Hospital. Testing of MICs of ciprofloxacin and ethionamide was carried out by MGIT 960 and compared with the agar dilution method. RESULTS: Good agreement was found between MGIT 960 and agar dilution. The greatest concordance between the agar dilution and MGIT assay at +/-1 and +/-2 dilution was 80.4% and 97.8% for ciprofloxacin, and 82.6% and 93.5% for ethionamide, respectively. CONCLUSION: MGIT 960 was found to be comparable to the current NCCLS standard method, agar dilution, and has the advantage of being rapid (obtaining results within 5-17 days, average 8.9 days) and easy to achieve standardization.

Correlation between pyrazinamide activity and pncA mutations in Mycobacterium tuberculosis isolates in Taiwan.

A total of 76 clinical Mycobacterium tuberculosis isolates from Taiwan were tested for pyrazinamidase activity, pyrazinamide susceptibility, and pncA mutations. Frequency of resistance to PZA rose with increases in resistance to first-line drugs. Of 17 pyrazinamide-resistant strains, 7 (3 of which had not been previously described) possessed mutations in the pncA gene.

Microscopic morphology in smears prepared from MGIT broth medium for rapid presumptive identification of Mycobacterium tuberculosis complex, Mycobacterium avium complex and Mycobacterium kansasii.

Mycobacterium species has a specific morphology when grown in liquid medium. Mycobacterium tuberculosis complex (MTB) often exhibits serpentine cording, which is different from the dot and cross-barring morphology observed in Mycobacterium avium complex (MAC) and Mycobacterium kansasii (MK), respectively. These characteristic morphologies can be used as a cost-effective method for rapid, presumptive identification of mycobacterial isolates cultured from the MGIT 960 system. By using Kinyoun acid-fast stain, serpentine cording was found in 840 of 904 (92.1%) samples positive for MTB; dot or loose aggregation was observed in 112 of 136 (82.3%) samples positive for MAC; and the cross-barring, ladder-like, morphology was observed in 45 of 56 (80.5%) samples positive for MK. The sensitivity and specificity were 92.9% and 96.4% for MTB; 82.4% and 94.5% for MAC; and 80.4% and 94.6% for MK, respectively. Using growth rate selection to exclude rapid growers, the positive and negative predictive values were 98% and 87.6% for MTB; 78.3% and 98% for MAC; and 78.9% and 99.1% for MK, respectively. Twenty-eight (93.3%) of 30 strains with ball morphology were rapid growers. Microscopic morphology can be used for rapid, presumptive identification of M. tuberculosis complex, M. kansasii, and M. avium complex and act as a guide for appropriate selection of initial probes to reduce costs.

Rapid detection of pulmonary tuberculosis using the BDProbeTEC ET Mycobacterium tuberculosis Complex Direct Detection Assay (DTB).

The ability to rapidly detect tubercle bacilli in respiratory secretions was determined for the BDProbeTEC ET Mycobacterium tuberculosis Complex Direct Detection Assay in comparison with the acid-fast smear (AFS). A total of 267 respiratory specimens obtained from 89 patients were evaluated. The DTB assay was positive in 70 of 78 culture positive specimens (89.7%) and 12 of 177 culture negative specimens (6.8%). The AFS was positive in 33 of 78 culture positive specimens (42.3%) and 3 of 186 culture negative specimens (1.6%). The sensitivity, specificity, positive predictive value, and negative predictive value of DTB assay were 89.7%, 93.7%, 85.4%, and 95.7%, respectively. The sensitivity of a single DBT (74.4%) was 2.1-times greater than three AFS (35.9%). The greater cost of the DTB assay compared to the AFS was compensated by its valuable information for the diagnosis and control of tuberculosis. These results demonstrated the clinical usefulness of the DTB assay for the rapid diagnosis of tuberculosis in respiratory specimens.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis to first-line drugs: comparisons of the MGIT 960 and BACTEC 460 systems.

The reliability of the Mycobacteria Growth Indicator Tube (MGIT) 960 system for rapid antimicrobial susceptibility testing (AST) of Mycobacterium tuberculosis was evaluated. Forty-seven isolates, including 10 fully susceptible and 37 resistant strains, were tested for susceptibility to the critical concentrations of streptomycin (STR), isoniazid (INH), rifampin (RMP), and ethambutol (EMB), as recommended by the manufacturer. Strains resistant to the critical concentrations were tested with higher concentrations. The results were compared to those obtained by a radiometric method (BACTEC 460TB) and by a conventional agar dilution method, which served as the reference method. Based on these data, we suggest that the following antibiotic concentrations give satisfactory results with the MGIT 960 system: STR, 4.0 microg/ml; INH, 0.1 microg/ml; RMP, 1.0 microg/ml; and EMB, 5.0 microg/ml. The time required to obtain susceptibility results averaged 6.9 days by the MGIT 960 system and 5.4 days by the BACTEC 460TB system; these intervals were not significantly different. This study shows that the MGIT 960 system is a reliable, rapid, automated method for testing the susceptibility of M. tuberculosis isolates to first-line drugs.