Charles M. Bark

Time to detection of Mycobacterium tuberculosis as an alternative to quantitative cultures

Testing new drugs is critical to improving the treatment of tuberculosis. Quantitative cultures of Mycobacterium tuberculosis on solid media have been used in Phase 1 and 2 trials, but are time and resource intensive. Time to detection (TTD) of growth of M. tuberculosis in automated liquid culture systems is an alternative. TTD has been shown to correlate with CFU in quantitative cultures, and is faster and simpler to perform. We compared TTD in the BACTEC 460 liquid culture system with CFU in a clinical trial that included 110 subjects. Comparing all sputum cultures collected between baseline and 2 months we found a strong negative correlation between log(10) CFU and TTD (rho = -0.91). In addition, when TTD at baseline was compared with 1 and 2 month sputum culture positivity, subjects whose cultures were negative after 1 and 2 months had a significantly longer median baseline TTD compared with subjects whose cultures were positive at 1 and 2 months (5 vs. 3 days and 3 vs. 2 days, respectively). TTD compares closely with CFU and represents a faster, simpler alternative to quantitative cultures.

Comparison of time to positive and colony counting in an early bactericidal activity study of anti-tuberculosis treatment

SETTING Patients with smear-positive, newly diagnosed pulmonary tuberculosis (TB) presenting to the out-patient TB clinic in Kampala, Uganda. OBJECTIVE To compare colony-forming unit (cfu) counting and time to positive (TTP) in Mycobacteria Growth Indicator Tube (MGIT) culture as measures of early bactericidal activity (EBA). DESIGN Patients were enrolled in an EBA feasibility study of standard TB chemotherapy. Sixteen-hour overnight sputum collections were obtained before and on days 2, 4, 7, 10, 12 and 14 of treatment for quantitative culture on selective Middlebrook 7H11 agar media and TTP in the MGIT liquid culture system. RESULTS Log cfu and TTP were correlated over all time points (r(s) = -0.71, P < 0.001). Within-subject (day to day) variation as a percentage of total variation was very similar between the two measures: 25.7% for cfu and 25% for TTP. Mean EBA 0-14, 0-2 and 2-14 measured by TTP were similar to those previously reported. CONCLUSION TTP measured by an automated, standardized, commercially available culture system correlates with cfu determinations. EBA measured by TTP provides similar information to cfu counting, and is reproducible across sites and in different patient populations. These findings support replacing cfu counting with TTP as the primary measurement in EBA studies.

Pretreatment Time to Detection of Mycobacterium tuberculosis in Liquid Culture Is Associated with Relapse after Therapy

The most important outcome of tuberculosis (TB) treatment is nonrelapsing cure. Identification of predictors of relapse is useful for evaluating new drugs and treating patients. Relapse has been associated with a greater pretreatment sputum bacillary load, measured by sputum smear grade and numbers

Disseminated Mycobacterium chelonae Infection in a Patient Receiving an Epidermal Growth Factor Receptor Inhibitor for Advanced Head and Neck Cancer

ABSTRACT We report a case of disseminated cutaneous Mycobacterium chelonae infection in a patient with head and neck cancer on salvage chemotherapy, including the epidermal growth factor receptor inhibitor cetuximab. Mycobacterium chelonae should be considered in the differential diagnosis of cutaneous infections in cancer patients receiving epidermal growth factor receptor inhibitors.

Resistance and Susceptibility to Mycobacterium tuberculosis Infection and Disease in Tuberculosis Households in Kampala, Uganda

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major public health problem. Household contact studies identify children and adults along the spectrum from Mtb exposure to disease. In the Kawempe Community Health Study (conducted in Kampala, Uganda), 872 culture-confirmed pulmonary TB cases and their 2,585 contacts were enrolled during 2002-2012 and followed for up to 2 years each. Risk factors identified by time-to-event analysis for secondary TB differed among children, women, and men. Younger age (P = 0.0061), human immunodeficiency virus (HIV) (P = 0.0002), thinness (P = 0.01), absent bacille Calmette-Guérin vaccination (P = 0.002), and epidemiologic risk score (P < 0.0001) were risks for children. For women, risks were HIV (P < 0.0001), thinness (World Health Organization criteria; P < 0.0001), and epidemiologic risk score (P = 0.003). For men, HIV (P = 0.0007) and low body mass index (P = 0.008) resulted in faster progression to TB. Tuberculin skin testing (TST) identified contacts with Mtb infection and those with persistently negative TST. Risks for faster time to Mtb infection were identified, and included age (P = 0.0007), baseline TST induration (P < 0.0001), and epidemiologic risk score (P < 0.0001) only in children. Those with persistently negative TST comprised 10% of contacts but had no unique epidemiologic characteristics among adults. The burden of Mtb infection and disease is high in TB households, and risk factors for progression from exposure to infection and disease differ among children, women, and men.

Drug-Resistant Tuberculosis: Challenges and Progress.

Antimicrobial resistance is a natural evolutionary process, which in the case of Mycobacterium tuberculosis is based on spontaneous chromosomal mutations, meaning that well-designed combination drug regimens provided under supervised therapy will prevent the emergence of drug-resistant strains. Unfortunately, limited resources, poverty, and neglect have led to the emergence of drug-resistant tuberculosis throughout the world. The international community has responded with financial and scientific support, leading to new rapid diagnostics, new drugs and regimens in advanced clinical development, and an increasingly sophisticated understanding of resistance mechanisms and their application to all aspects of TB control and treatment.

Incubation time of Mycobacterium tuberculosis complex sputum cultures in BACTEC MGIT 960: 4weeks of negative culture is enough for physicians to consider alternative diagnoses.

We retrospectively analyzed time to detection of 3747 positive MGIT sputum cultures at a laboratory in a country with heavy burden of tuberculosis. Ninety-nine percent of diagnostic cultures turned positive within 28days, suggesting that physicians may consider alternative diagnoses if sputum cultures remain negative after 4weeks of incubation.

Identification of Host Proteins Predictive of Early Stage Mycobacterium tuberculosis Infection

The objective of this study was to identify blood-based protein biomarkers of early stage Mycobacterium tuberculosis (Mtb) infection. We utilized plasma and serum specimens from TB patients and their contacts (age ≥ 12) enrolled in a household contact study in Uganda. In the discovery phase cross-sectional samples from 104 HIV-uninfected persons classified as either active TB, latent Mtb infection (LTBI), tuberculin skin test (TST) converters, or persistent TST-negative were analyzed. Two hundred eighty-nine statistically significant (false discovery rate corrected p < 0.05) differentially expressed proteins were identified across all comparisons. Proteins associated with cellular immunity and lipid metabolism were induced early after Mtb infection. One hundred and fifty-nine proteins were selected for a targeted mass spectrometry assay. A set of longitudinal samples from 52 TST-negative subjects who converted to TST-positive or remained TST-negative were analyzed, and multivariate logistic regression was used to identify unique protein panels able to predict TST conversion with cross-validated AUC > 0.85. Panel performance was confirmed with an independent validation set of longitudinal samples from 16 subjects. These candidate protein biomarkers may allow for the identification of recently Mtb infected individuals at highest risk for developing active TB and most likely to benefit from preventive therapy.

Differences in EBA in the first two days of standard anti-tuberculosis treatment in different geographic regions

In a recent article entitled, “Comparison of time to positive and colony counting in an early bactericidal activity study of anti-tuberculosis treatment” we found a relatively lower early bactericidal activity (EBA) of combination anti-TB treatment with isoniazid (INH), rifampicin, ethambutol and pyrazinamide between days 0 and 2 in HIV-uninfected Ugandan adults with newly-diagnosed, initial episodes of smear-positive pulmonary TB.1 The mean EBA 0-2, measured in colony forming units (CFU), from 40 patients receiving directly observed therapy (DOT) was 0.26 ± 0.24 log10 CFU/ml/day. This result was similar to an EBA 0-2 value of 0.28 ± 0.42 log10 CFU/ml/day we calculated using data from an earlier 2003 clinical trial conducted at the same site.2 In comparison, previous studies done mainly in South Africa have demonstrated a higher EBA 0-2 for standard 4-drug therapy of approximately 0.5 log10 CFU/ml/day, while the EBA 0-14 was similar to that found in our study.3,4 The reason for the difference in EBA during the first 2 days of therapy is not clear. Patients in our study received DOT on an inpatient basis with Good Manufacturing Practice grade medications. Medications were administered as loose tablets, and all patients received 300 mg of INH. The mean dose of INH was 5.8 ± 0.8 mg/kg (range 3.7 to 7.9), which should yield maximum EBA 0-2.5 CFU counts were done on 16-hour pooled overnight sputum collections using standardized methods. The mean baseline sputum colony count of 6.57 ± 0.58 log10 CFU/ml was similar to previous EBA studies. Baseline sputum colony count has been associated with EBA 0-2 in previous studies,5,6 and we did find a correlation in this study (rs = 0.34, P = 0.03), however the relatively high mean baseline count in our patients would again predict maximal EBA 0-2. Finally, recent exposure to INH was excluded by urine INH metabolite testing (BBL Taxo INH Test Strips, BD, Sparks, MD, USA) during screening and at the time of enrollment to the ward to begin the study. All Mycobacterium tuberculosis (MTB) isolates from the study subjects were susceptible to INH, rifampicin, ethambutol and pyrazinamide, and sputum collection and processing methods in our study were similar to those used in the earlier studies.

Sulfamethoxazole Susceptibility of Mycobacterium tuberculosis Isolates from HIV-Infected Ugandan Adults with Tuberculosis Taking Trimethoprim-Sulfamethoxazole Prophylaxis

ABSTRACT Additional drugs are needed for the treatment of multidrug-resistant tuberculosis (TB). Sulfamethoxazole has been shown to have in vitro activity against Mycobacterium tuberculosis; however, there is concern about resistance given the widespread use of trimethoprim-sulfamethoxazole prophylaxis among HIV-infected patients in sub-Saharan Africa. Thirty-eight of 40 Mycobacterium tuberculosis isolates (95%) from pretreatment sputum samples from Ugandan adults with pulmonary TB, including HIV-infected patients taking trimethoprim-sulfamethoxazole prophylaxis, were susceptible with MICs of ≤38.4 μg/ml.