Mycobacterium Tuberculosis Culture Research Articles

Genital tuberculosis screening at an academic fertility center in the United States

Infertility is a common presentation of female genital tuberculosis in endemic areas. Female genital tuberculosis-related maternal and neonatal complications have increased in recent years after assisted reproductive technology treatments. Despite rising emigration rates to the United States, guidelines to identify those with latent tuberculosis or female genital tuberculosis in fertility centers do not exist. This study aimed to characterize the prevalence of female genital tuberculosis in infertile patients at our academic fertility center. This is a prospective cohort study. All patients presenting for infertility evaluation between January 2014 and January 2017 were assessed for risk factors for latent tuberculosis. Patients at risk for latent tuberculosis underwent screening using QuantiFERON-TB Gold serum assay. QuantiFERON-TB Gold-positive patients underwent further testing for female genital tuberculosis consisting of endometrial biopsy with histopathologic examination by a clinical pathologist, polymerase chain reaction for tuberculosis, and culture for acid-fast Mycobacterium tuberculosis. Twenty-five of 323 infertility patients (7.7%) screened for latent tuberculosis had positive QuantiFERON-TB Gold results. A greater number of patients with a positive test result for QuantiFERON-TB Gold were foreign born than those with a negative test result for QuantiFERON-TB Gold (92% vs 29%; P<.001). Of note, the QuantiFERON-TB Gold-positive population had a higher incidence of both recurrent pregnancy loss (28% vs 7%; P=.003) and Asherman syndrome (8% vs 0.3%; P<.001). Among those with a positive test result for QuantiFERON-TB Gold, chest x-ray was abnormal in only 2 patients (8.0%). Endometrium evaluation revealed abnormalities in 2 patients (8.0%), in whom chest x-ray was normal, with 1 showing evidence of female genital tuberculosis. This was indicated by histology consistent with chronic granulomatous endometritis and positive endometrial testing for tuberculosis by polymerase chain reaction, acid-fast bacilli smear, and culture for Mycobacterium tuberculosis. Although the prevalence of female genital tuberculosis in infertile women in the United States seems to be low, this study indicates that it can be underdiagnosed without utilization of multiple diagnostic modalities including endometrial sampling. Given the potential for serious maternal and neonatal morbidity in affected patients utilizing assisted reproductive technology, we propose that all at-risk women seeking infertility care in the United States be screened for latent tuberculosis. In patients who screen positive, endometrial biopsy should be obtained for evaluation by histology, polymerase chain reaction, and culture for Mycobacterium tuberculosis to rule out female genital tuberculosis before infertility treatments are initiated.

Bronchoalveolar Lavage Fluid Caseating Granulomas in A Case of Pulmonary Tuberculosis

Pulmonary tuberculosis (TB) is a common infectious disease that is considered one of the leading causes of morbidity and mortality worldwide. Special diagnostic studies for TB can be used including smear microscopy and cultures for acid fast bacilli (AFB) and Mycobacterium tuberculosis (MTB) nucleic acid amplification test (NAAT). The specimen is usually taken from the sputum but might also include pleural and bronchoalveolar lavage (BAL) fluids. In addition, BAL cytology and lung biopsy are commonly done if bronchoscopy is performed. Here, we present a case of suspected pulmonary TB in a patient who was complaining of cough and hemoptysis for several months. Chest x-ray showed right upper lung opacity with cystic changes that was confirmed by CT chest. Work up done for this case included three samples of sputum and one BAL fluid specimen for AFB smear microscopy, culture and NAAT in addition to transbronchial lung biopsies and they were all negative. However, Cytologic examination of BAL fluid using Papanicolaou stain surprisingly showed evidence of intact caseating granulomas characteristic for tuberculous infection. Ultimately, the patient was started on the standard anti-TB regimen for 6 months with significant clinical improvement confirming TB diagnosis. To the best of our knowledge, this unusual cytologic finding in culture-negative BAL fluid coupled with unremarkable lung biopsy and persistently negative sputum samples for TB was never reported in the English literature.

Targeted-Sequencing Workflows for Comprehensive Drug Resistance Profiling of Mycobacterium tuberculosis Cultures Using Two Commercial Sequencing Platforms: Comparison of Analytical and Diagnostic Performance, Turnaround Time, and Cost.

The emergence of Mycobacterium tuberculosis with complex drug resistance profiles necessitates a rapid and comprehensive drug susceptibility test for guidance of patient treatment. We developed two targeted-sequencing workflows based on Illumina MiSeq and Nanopore MinION for the prediction of drug resistance in M. tuberculosis toward 12 antibiotics. A total of 163 M. tuberculosis isolates collected from Hong Kong and Ethiopia were subjected to a multiplex PCR for simultaneous amplification of 19 drug resistance-associated genetic regions. The amplicons were then barcoded and sequenced in parallel on MiSeq and MinION in respective batch sizes of 24 and 12 samples. A web-based bioinformatics pipeline, BacterioChek-TB, was developed to translate the raw datasets into clinician-friendly reports. Both platforms successfully sequenced all samples with mean read depths of 1,127× and 1,649×, respectively. The variant calling by MiSeq and MinION could achieve 100% agreement if variants with an allele frequency of <40% reported by MinION were excluded. Both workflows achieved a mean clinical sensitivity of 94.8% and clinical specificity of 98.0% when compared with phenotypic drug susceptibility test (pDST). Turnaround times for the MiSeq and MinION workflows were 38 and 15 h, facilitating the delivery of treatment guidance at least 17-18 days earlier than pDST, respectively. The higher cost per sample on the MinION platform ($71.56) versus the MiSeq platform ($67.83) was attributed to differences in batching capabilities. Our study demonstrates the interchangeability of MiSeq and MinION platforms for generation of accurate and actionable results for the treatment of tuberculosis.

THUMB TUBERCULOSIS: A CASE REPORT

Tuberculosis is caused by Mycobacterium tuberculosis, occurs in about 2 billion people. Approximately 8 million people/year develop the active form.1,2 Tuberculosis in the hand is manifested as osteomyelitis in carpals, metacarpals and phalanges.1 Musculoskeletal tuberculosis occurs, in most cases, through haematogenous dissemination from the primary focus. In immunosuppression circumstances, it is reactivated.Methods:47 years old lady, who had underlying pulmonary tuberculosis on anti-TB medications since June 2018, presented to us for swelling over right thumb. Associated with tender, erythematous skin and limited range of motion of right thumb.Results:Plain radiograph demonstrated soft tissue swelling, joint space narrowing, mottled lucency of the proximal phalanx and cystic degenerative changes. MRI shows osteomylities proximal phalanx of right thumb. Patient underwent wound debridement of right thumb, culture and sensitivity shows Mycobacterium tuberculosis infections. Post debridement, range of motion of MCP joint of right thumb was improved and anti-TB medications to restart.Discussions:Tuberculosis involvement of the metacarpals and phalanges is a rare presentation of extrapulmonary TB. The radiographic features of osseous tuberculosis are present in conditions such as inflammatory arthritis, pyogenic osteomyelitis, osteopenia, softtissue swelling with minimal periosteal reaction, narrowing of the joint space, cysts in bone adjacent to joints, and subchondral erosions. The gold standard to diagnose is culture of Mycobacterium tuberculosis from bone tissue. Current treatment is a 2 month initial phase of isoniazid, rifampin, pyrazinamide, and ethambutol followed by a 6 to 12 month regimen of isoniazid and rifampin.Conclusion:Finger swelling is a rare presenting sign of disseminated tuberculosis. Early biopsy and appropriate microbiologic testing can avoid diagnostic delay.

The diagnostic utility of pleural markers for tuberculosis pleural effusion.

Tuberculosis pleural effusion (TPE) is common in clinical practice, and its diagnosis remains a challenge for clinicians. Ziehl-Neelsen staining, PE Mycobacterium tuberculosis culture, and biopsy are the gold standards for TPE diagnosis; however, they are time-consuming, invasive, observer-dependent, and insensitive. PE markers represent a rapid, low-cost, and non-invasive objective diagnostic tool for TPE. In the past decades, several PE biomarkers have been developed, and their diagnostic accuracy has been evaluated in many studies. Here, we reviewed the literature to summarize the diagnostic accuracy of these biomarkers, especially using the evidence from systematic review and meta-analysis. The current research strongly suggests that adenosine deaminase (ADA), interferon-gamma (IFN-γ), and interleukin 27 (IL-27) have extremely higher diagnostic accuracy for TPE, while the diagnostic accuracy of interferon gamma release assays (IGRAs), tumor necrosis factor-α (TNF-α), and interferon-γ-induced protein 10 kDa (IP-10) is moderate. Although some evidence supports C-X-C motif chemokine ligand 9 (CXCL9), CXCL11, CXCL12, sFas ligand, angiotensin-converting enzyme (ACE), calpain-1, spectrin breakdown products (SBDP), matrix metalloproteinase-1 (MMP-1), soluble CD26 (sCD26), soluble interleukin 2 receptor (sIL-2R) as useful diagnostic markers for TPE, more support is needed to validate their diagnostic accuracy. Finally, nucleic acid amplification tests (NAATs) have extremely high diagnostic specificity, but their sensitivity is low. Taken together, ADA is the preferred marker for TPE because its low cost and suitability for standardization.

Diagnosis of laryngeal tuberculosis in a high TB burden area.

The larynx is the second most commonly affected site in the head and neck region in patients with extrapulmonary tuberculosis (TB). Despite this, the prevalence of laryngeal TB is largely unknown, particularly in areas with a high TB burden. The laboratory diagnosis of TB includes microscopy, culture and molecular testing. The aims of this study were to determine the prevalence of laryngeal TB in patients presenting with laryngeal pathology in a region with a high TB burden and to determine the optimal diagnostic methods for the diagnosis of laryngeal TB. This was a prospective descriptive study of 80 adult patients undergoing direct laryngoscopy and biopsy for laryngeal pathology in the Department of Otorhinolaryngology, Universitas Academic Hospital, Bloemfontein, South Africa over a 1year period. Histopathological and microbiological investigations (microscopy, Xpert MTB/RIF, and TB culture) were performed on all laryngeal biopsies. Five (6.25%) out of 80 patients were diagnosed with laryngeal TB. In one patient, the Xpert MTB/RIF assay was positive on the laryngeal tissue and histology showed granulomas. Two patients had granulomas on histology although the microbiological tests on the tissue were negative. Two patients had only positive tissue cultures for Mycobacterium tuberculosis. None of the biopsies had positive Ziehl-Neelsen stains. The results suggest that the diagnosis of laryngeal TB required a combination of histopathology, culture and PCR and that the Xpert MTB/RIF assay is not a sensitive test for the diagnosis of laryngeal TB.

Imaging and clinical features of breast tuberculosis: a review series of 62 cases

To outline the disease burden of breast tuberculosis (TB) as a quantitative analysis amongst three tertiary hospitals in South Africa, with correlation to their clinical, demographic, and imaging features. A retrospective analysis was undertaken over an 18-month period (01/01/2017-30/06/2018) of all patients undergoing laboratory investigations for breast disease at the mammography departments of these three tertiary centres. The prevalence of breast TB was 2.5% (n=62) of 2,516 patients. The median age of presentation was 38.5 years (interquartile range [IQR] 33-45). HIV status was known in 45 patients, of whom 36 were HIV infected (80%, 95% CI: 0.65-0.90, p<0.0001). Based on the ultrasound and/or mammogram findings, the patients were classified into five categories: TB breast abscess (40.3%), inflammatory/disseminated (24.2%), isolated TB lymphadenitis (22.6%), nodular (11.3%), and sclerosing form (1.6%). Histology demonstrated necrotising granulomatous inflammation in 57 cases (92%). Acid-fast bacilli (AFB) were positive in 8.1% (n=5) of the cytology and 16.1% (n=10) of the histology specimens. Culture for Mycobacterium tuberculosis was positive in 27% (17 cases), and in 12.9% (n=8). AFB were detected histologically using polymerase chain reaction (PCR) testing. Knowledge of the varied clinical and radiological features is necessary to maintain a high degree of suspicion to prevent misdiagnoses, inappropriate management, and complications. Ultrasound-guided core biopsy rather than fine-needle aspiration (FNA) is advocated as the first-line intervention in diagnosing or excluding this disease, as it yields a better tissue sample and more often a positive diagnosis.

Combination of Xpert MTB/RIF and TBAg/PHA Ratio for Prompt Diagnosis of Active Tuberculosis: A Two-Center Prospective Cohort Study

The prompt diagnosis of active tuberculosis (ATB) is still a challenge in clinical practice, especially in TB-endemic countries. We prospectively enrolled consecutive patients with suspected pulmonary TB from two tertiary hospitals. Acid-fast staining (AFS), Xpert MTB/RIF (Xpert), Mycobacterium tuberculosis culture, and T-SPOT.TB were simultaneously performed. 226 ATB and 348 non-TB patients were diagnosed in Tongji hospital (test cohort), and 86 ATB and 110 non-TB patients were diagnosed in Guangzhou Chest Hospital (validation cohort). Using ATB as patient group and non-TB as control group, for diagnosis of ATB in Tongji Hospital, the sensitivity of AFS was 17.70% (95% CI: 13.08–23.44%). The sensitivity of Xpert and culture were 53.54% (95% CI: 46.81–60.14%) and 46.46% (95% CI: 39.86–53.19%), respectively. The sensitivity of T-SPOT.TB was 81.42% (95% CI: 75.60–86.14%), but the specificity was 71.55% (95% CI: 66.60–76.04%). Calculation of the ratio of TB-specific antigen to phytohaemagglutinin (TBAg/PHA) of T-SPOT.TB assay increased the specificity but with a loss of sensitivity. Combination of Xpert and culture slightly increased the sensitivity compared to using these methods separately. Combination of Xpert and TBAg/PHA ratio (defined as Xpert positive or TBAg/PHA ≥ 0.2) increased diagnostic accuracy, and the sensitivity and specificity of combination of them were 85.84% (95% CI: 80.45–89.98%) and 95.98% (95% CI: 93.36–97.59%), respectively. The diagnostic model was also established based on combination of Xpert and TBAg/PHA ratio. The area under the curve of the diagnostic model was 0.952 (95% CI: 0.932–0.973) for diagnosis of ATB, with a sensitivity of 88.05% (95% CI: 83.10–91.98%) and a specificity of 96.26% (95% CI: 93.70–98.00%) when a cutoff value of 0.44 was used in Wuhan cohort. The performance of combination of Xpert and TBAg/PHA ratio was similar in Guangzhou Chest Hospital. Our data suggest that combination of Xpert and TBAg/PHA ratio may be a good algorithm for prompt diagnosis of ATB in high endemic areas.

Diagnosing Pulmonary Tuberculosis in the Elderly

Pulmonary tuberculosis (TB) remains a diagnostic challenge, particularly in low endemic settings with ageing populations. The impact has both individual and infection control implications. Over the past 20 years, progress has been made in trying to improve TB diagnostics. International recommendations for investigation however still rely heavily on the index of suspicion, imaging and ultimately culturing Mycobacterium tuberculosis which remains the gold standard for diagnosing TB disease. Recent advances include molecular diagnostics with the ability to rapidly identify TB disease and drug resistance and whole-genome sequencing. In this review, we explore the validity of TB diagnostics in the elderly to assist the practicing clinician in the diagnostic workup in this population.

Management of children with tuberculous broncho-esophageal fistulae.

Broncho-esophageal fistula (BOF) is a rare complication of Mycobacterium tuberculosis (MTB). TB-associated BOF presents either as acute respiratory failure, aspiration pneumonia or as a complication of surgical decompression of thoracic lymph nodes. All children with TB- associated BOF were included. TB was diagnosed if MTB was cultured from respiratory secretions, Ziehl-Neelsen (ZN) smear was positive, GeneXpert MTB/RIF was positive or a chest radiograph revealed radiographic features typical of TB. BOF was diagnosed by a contrast swallow study and/or flexible bronchoscopy. Chest computed tomography (CT) scan was performed, if required. Total of 20 children were diagnosed with TB-associated BOF between 1999 and 2019, with a 75% survival. A total of 85% BOF involved the left main bronchus. A total of 80% of patients were MTB culture or ZN smear-positive. Chest X-ray abnormalities included: extensive parenchymal disease (80%) and lymph gland enlargement (45%). CT features included visualization of the BOF (60%), esophageal air (73%) and pneumomediastinum (40%). BOF closure was achieved by surgical closure (46%), spontaneous closure (26%), fibrin glue (13%), and esophageal stent (13%). Multivariant regression analysis showed that C- reactive protein (CRP), albumin and CRP/albumin ratio were associated with mortality. Most TB-associated BOF are left-sided. It presents either acutely, with respiratory failure, or with chronic respiratory symptoms of aspiration. Children requiring invasive ventilation have high mortality. Most TB-associated BOF requires surgical intervention, although the use of fibrin glue offers an attractive alternative option.

Current Status of Genitourinary Tuberculosis: Presentation, Diagnostic Approach and Management-Single Centre Experience at IGIMS (Ptana, Bihar, India)

Genitourinary tuberculosis (GUTB) is associated with significant morbidity and mortality. As patients with GUTB have no specific symptoms or clinical features, early diagnosis and treatment of GUTB is clinically challenging. The present study assessed the current spectrum of clinical presentation, urinary findings and radiological signs of patients with GUTB. Patients with confirmed GUTB (n???=???53) were analysed for clinical presentation, diagnostic tests and management. Data about demographic details, clinical presentation, urine routine examination, urine acid-fast bacillus (AFB) smear test, urine Mycobacterium tuberculosis culture, routine blood investigations, radiological, cystoscopic and histopathological examinations were recorded for all study patients. Various surgical interventions were performed on the basis of the site involved. After surgical procedure, patients were treated with chemotherapy and followed up for one year to till date. The present study patients were from the age range of 19???75 years with female preponderance. The most common clinical presentation was irritative voiding symptoms (69.81%), haematuria and flank pain (56.60%, each). The most commonly affected organ was kidney (62.26%) following ureter (30.19%), urinary bladder (24.53%), prostate (9.43%) and epididymis (1.89%). The urine AFB smear and urine culture for M. tuberculosis tests were positive in 13.18% and 54.71% of patients, respectively. Surgical interventions were done in majority (75.47%) of patients. Accurate and early diagnosis is vital for the better management of patients with GUTB. Apart from the available course of chemotherapy, surgical intervention is also required for complete management.

The role of mini-bronchoalveolar lavage fluid in the diagnosis of pulmonary tuberculosis in critically ill patients

BackgroundThe detection of Mycobacterium tuberculosis (MTB) in the intensive care unit (ICU) presents several challenges, mainly associated to the clinical state of the patient. The presence of HIV infection further aggravates this scenario, requiring a reliable collection method, with better performance in the microbiological/molecular techniques to be used. We evaluated the performance of two methods for sample collection, mini bronchoalveolar lavage (Mini-BAL) and endotracheal aspirate (ETA), for diagnosis of pulmonary tuberculosis (PTB) in critically ill patients.MethodsThis prospective study involved 26 HIV positive ICU internalized patients, with presumptive PTB who required mechanical ventilation. Two samples were obtained prospectively from 26 HIV ICU patients with presumptive PTB by Mini-BAL and ETA. The samples were processed for smear microscopy, Löwenstein-Jensen medium and the BACTEC Mycobacteria Growth Indicator Tube 960 system®. We define as confirmed PTB patients with positive MTB culture. Furthermore, all samples obtained through the Mini-BAL were analyzed by Xpert® MTB/RIF.ResultsOur results demonstrated that the respiratory samples obtained by Mini-BAL were able to increase MTB detection in critically ill patients with presumptive PTB. The Mini-BAL allowed 30% increased recovery and guaranteed enough sample volume for processing in all methods. In addition, the larger volume of the samples obtained with this technique enabled the Xpert® MTB/RIF molecular test for diagnosis of TB.ConclusionsThe Mini-BAL showed be an acceptable alternative to ETA in this population, since these critically ill and often-immunocompromised patients are more likely to develop complications related to invasive procedures.

Rapid Pyrazinamide Drug Susceptibility Testing using a Closed-Tube PCR Assay of the Entire pncA gene

The continued use of pyrazinamide in the treatment of tuberculosis in the absence of a rapid, accurate and standardized pyrazinamide drug susceptibility assays is of great concern. While whole genome sequencing holds promise, it is not yet feasible option in low resource settings as it requires expensive instruments and bioinformatic analysis. We investigated the diagnostic performance of a closed-tube Linear-After-The-Exponential (LATE)-PCR assay for pyrazinamide susceptibility in Mycobacterium tuberculosis. Based on a set of 654 clinical Mycobacterium tuberculosis culture isolates with known mutations throughout the pncA gene as determined by Sanger sequencing, the assay displays excellent sensitivity of 96.9% (95% CI: 95.2–98.6) and specificity of 97.9% (95% CI: 96.1–99.7). In a subset of 384 isolates with phenotypic drug susceptibility testing, we also observed high sensitivity of 98.9% (95% CI: 97.5–100) but lower specificity of 91.8% (95% CI: 87.9–95.8) when compared to phenotypic drug susceptibility testing. We conclude that the LATE PCR assay offers both a rapid and accurate prediction of pyrazinamide susceptibility.

Integrative proteomic and glycoproteomic profiling of Mycobacterium tuberculosis culture filtrate.

Despite being the subject of intensive research, tuberculosis, caused by Mycobacterium tuberculosis, remains at present the leading cause of death from an infectious agent. Secreted and cell wall proteins interact with the host and play important roles in pathogenicity. These proteins are explored as candidate diagnostic markers, potential drug targets or vaccine antigens, and more recently special attention is being given to the role of their post-translational modifications. With the purpose of contributing to the proteomic and glycoproteomic characterization of this important pathogen, we performed a shotgun analysis of culture filtrate proteins of M. tuberculosis based on a liquid nano-HPLC tandem mass spectrometry and a label-free spectral counting normalization approach for protein quantification. We identified 1314 M. tuberculosis proteins in culture filtrate and found that the most abundant proteins belong to the extracellular region or cell wall compartment, and that the functional categories with higher protein abundance factor were virulence, detoxification and adaptation, and cell wall and cell processes. We could identify a group of proteins consistently detected in previous studies, most of which were highly abundant proteins. In culture filtrate, 140 proteins were predicted to contain one of the three types of bacterial N-terminal signal peptides. Besides, various proteins belonging to the ESX secretion systems, and to the PE and PPE families, secreted by the type VII secretion system using nonclassical secretion signals, were also identified. O-glycosylation was identified in 46 proteins, many of them lipoproteins and cell wall associated proteins. Finally, we provide proteomic evidence for 33 novel O-glycosylated proteins, aiding to the glycoproteomic characterization of relevant antigenic membrane and exported proteins. These findings are expected to collaborate with the research on pathogen derived biomarkers, virulence factors and vaccine candidates, and to provide clues to the understanding of the pathogenesis and survival strategies adopted by M. tuberculosis.

Accuracy and consequences of using trial-of-antibiotics for TB diagnosis (ACT-TB study): protocol for a randomised controlled clinical trial

IntroductionOver 40% of global tuberculosis case notifications are diagnosed clinically without mycobacteriological confirmation. Standard diagnostic algorithms include ‘trial-of-antibiotics’—empirical antibiotic treatment given to mycobacteriology-negative individuals to treat infectious causes of symptoms...

Evaluation of the BD MAX™ MDR-TB assay in a real-world setting for the diagnosis of pulmonary and extra-pulmonary TB

Tuberculosis in London occurs at a rate of 19 cases per 100,000 population, with a significant proportion diagnosed as extra-pulmonary infection. At Barts Health NHS Trust, our TB rates are much higher than the London average and approximately 60% cases are extra-pulmonary in nature. We evaluated the BD MAX™ MDR-TB assay as a molecular tool for rapid diagnosis of TB. One hundred twenty-eight specimens, encompassing pulmonary (70) and extra-pulmonary (58) infection, were tested using the BD MAX™ MDR-TB assay and compared with smear and liquid culture results, to determine PCR performance. The BD MAX™ MDR-TB assay was also compared with the Xpert MTB/RIF assay, where applicable. TB was successfully detected in 50/66 Mycobacterium tuberculosis culture positive specimens, with additional detections in 2 of the culture negative specimens. The BD MAX™ MDR-TB assay demonstrated higher sensitivity with the pulmonary samples (92%) compared with the extra-pulmonary samples (52%), although the performance with fluids and biopsies demonstrated greater potential than the remaining extra-pulmonary samples. Rifampicin and/or isoniazid resistance was successfully detected by the BD MAX™ in 2/3 samples, where WGS susceptibility results were available. The BD MAX™ MDR-TB assay was comparable with the performance of the Xpert MTB/RIF assay. TB can successfully be diagnosed, in both pulmonary and extra-pulmonary samples, using the BD MAX™ MDR-TB assay.

4-month moxifloxacin containing regimens in the treatment of patients with sputum-positive pulmonary tuberculosis in South India - a randomised clinical trial.

Shortening tuberculosis (TB) treatment duration is a research priority. We tested the efficacy and safety of 3- and 4-month regimens containing moxifloxacin in a randomised clinical trial in pulmonary TB (PTB) patients in South India. New, sputum-positive, adult, HIV-negative, non-diabetic PTB patients were randomised to 3- or 4-month moxifloxacin regimens [moxifloxacin (M), isoniazid (H), rifampicin (R), pyrazinamide (Z) and ethambutol (E)] or to a control regimen (2H3 R3 Z3 E3 /4R3 H3 ) [C]. The 4 test regimens were 3R7 H7 Z7 E7 M7 [M3], 2R7 H7 Z7 E7 M7 /2R7 H7 M7 [M4], 2R7 H7 Z7 E7 M7 /2R3 H3 M3 [M4-I] or 2R7 H7 Z7 E7 M7 /2R3 H3 E3 M3 [M4-IE]. Treatment was directly observed. Clinical and bacteriological assessments were done monthly during treatment and for 24months post-treatment. The primary end point was TB recurrence post-treatment. Of 1371 patients, randomised, modified intention-to-treat (ITT) analysis was done in 1329 and per-protocol (PP) analysis in 1223 patients. Regimen M3 was terminated due to high TB recurrence rates. 'Favourable' response at end of treatment was 96-100% in the moxifloxacin regimensand 93% in the control regimen. Among these, the TB recurrence occurred in 4.1% in the M4 regimen and in 4.5% in the control regimen and demonstrated equivalence within a 5% margin (95% CI -3.68, 4.55). Similar findings were observed in modified ITT analysis. The TB recurrence rates in the M4-I and M4-IE regimens did not show equivalence with the control regimen. Sixteen (1.4%) of 1087 patients in the moxifloxacin regimens required treatment modification. The 4-month daily moxifloxacin regimen [M4] was found to be equivalent and as safe as the 6-month thrice-weekly control regimen.

Decision-making in the diagnosis of tuberculous meningitis.

Tuberculous meningitis (TBM) is the most devastating form of tuberculosis (TB) but diagnosis is difficult and delays in initiating therapy increase mortality. All currently available tests are imperfect; culture of Mycobacterium tuberculosis from the cerebrospinal fluid (CSF) is considered the most accurate test but is often negative, even when disease is present, and takes too long to be useful for immediate decision making. Rapid tests that are frequently used are conventional Ziehl-Neelsen staining and nucleic acid amplification tests such as Xpert MTB/RIF and Xpert MTB/RIF Ultra. While positive results will often confirm the diagnosis, negative tests frequently provide insufficient evidence to withhold therapy. The conventional diagnostic approach is to determine the probability of TBM using experience and intuition, based on prevalence of TB, history, examination, analysis of basic blood and CSF parameters, imaging, and rapid test results. Treatment decisions may therefore be both variable and inaccurate, depend on the experience of the clinician, and requests for tests may be inappropriate. In this article we discuss the use of Bayes' theorem and the threshold model of decision making as ways to improve testing and treatment decisions in TBM. Bayes' theorem describes the process of converting the pre-test probability of disease to the post-test probability based on test results and the threshold model guides clinicians to make rational test and treatment decisions. We discuss the advantages and limitations of using these methods and suggest that new diagnostic strategies should ultimately be tested in randomised trials.

Polymerization-Based Amplification for Target-Specific Colorimetric Detection of Amplified Mycobacterium tuberculosis DNA on Cellulose

Loop-mediated isothermal amplification (LAMP) is an appealing method for low-cost, point-of-care nucleic acid diagnostic assays due to high sensitivity, minimal equipment requirements, and compatibility with user-friendly colorimetric detection methods. The enhanced sensitivity LAMP offers comes with vulnerability to cross-contamination, where negative samples are exposed to minute amounts of nucleic acids from positive samples. These amounts are insignificant in less sensitive amplification methods, but visible when LAMP is paired with common colorimetric methods. Here, we examined the use of eosin photopolymerization, a tunable reaction, for colorimetric detection of LAMP products to reduce this false positive risk. Using eosin and biotin end-labeled primers, we successfully amplified target regions of the Mycobacterium tuberculosis (MTB) genome using PCR and LAMP, captured amplicons on streptavidin-coated cellulose, and detected DNA targets via eosin photopolymerization, producing a bright pink color only if MTB DNA was present in the sample. Consistent with previous reports, the LAMP-based method exhibited high background signal, but tuning the illumination time for the photopolymerization reaction allowed readouts from samples with no added MTB DNA to remain blank and visually distinct from pink positives. This method yielded limits of detection of 30 and 300 copies/μL for LAMP and PCR amplification, respectively. When confronted with boiled MTB culture samples, this method gave clear positive readouts, compared to negligible signal from other Mycobacterium boiled culture samples. This new method of LAMP colorimetric detection has the potential to increase the utility of LAMP as a nucleic acid assay technique by mitigating sensitivity to cross-contamination.

False Positive Tuberculosis Cases (Xpert MTB/RIF Assay) Among People Living With HIV Attending Bahati Comprehensive Care Centre Nairobi, Kenya

The introduction of GeneXpert MTB/RIF assay has impacted positively in tuberculosis diagnosis, providing a rapid way of identifying tuberculosis patients in high burden, low income countries. However Mycobacterium tuberculosis (MTB) detection in previously treated patients, which may be due to old deoxyribonucleic acid or active disease, still remains a diagnostic dilemma for diagnosis of tuberculosis. A retrospective cohort study was conducted among consenting patients with signs and symptoms of tuberculosis attending Bahati comprehensive care centers. A total of three hundred and forty six patients were sampled and their sputa collected, subsequently laboratory analysis was carried out for detection and culture of Mycobacterium tuberculosis. Seventy seven (22%) sputa had Mycobacterium tuberculosis detected on Xpert MTB/RIF assay sputa from these patients with bacteriologically confirmed pulmonary tuberculosis were subjected to culture on Mycobacterium Growth Indicator Tube (MGIT) media. Detection of Mycobacterium tuberculosis on Xpert MTB/RIF assay with no isolation of growth on culture indicated a false positive tuberculosis diagnosis. Out of 77 isolates subjected for culture a total of 0(0%) and 5(7.5%); P=0.484, isoniazid preventive therapy and non- isoniazid preventive therapy patients had false positive tuberculosis cases, while 0(0%) and 5(25%); P=0.001 new and retreatment patient’s had false positive tuberculosis. Our study concluded that there was no significant association between isoniazid preventive therapy and tuberculosis false positivity but there was significant association between patient treatment status and tuberculosis false positivity. Previously treated tuberculosis patients were significantly associated with false positivity, this call for clinicians to exercise caution when interpreting results from previously treated tuberculosis patients.