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1.  Treatment Outcomes of New Tuberculosis Patients Hospitalized in Kampala, Uganda: A Prospective Cohort Study 
PLoS ONE  2014;9(3):e90614.
Background
In most resource limited settings, new tuberculosis (TB) patients are usually treated as outpatients. We sought to investigate the reasons for hospitalisation and the predictors of poor treatment outcomes and mortality in a cohort of hospitalized new TB patients in Kampala, Uganda
Methods and findings
Ninety-six new TB patients hospitalised between 2003 and 2006 were enrolled and followed for two years. Thirty two were HIV-uninfected and 64 were HIV-infected. Among the HIV-uninfected, the commonest reasons for hospitalization were low Karnofsky score (47%) and need for diagnostic evaluation (25%). HIV-infected patients were commonly hospitalized due to low Karnofsky score (72%), concurrent illness (16%) and diagnostic evaluation (14%). Eleven HIV uninfected patients died (mortality rate 19.7 per 100 person-years) while 41 deaths occurred among the HIV-infected patients (mortality rate 46.9 per 100 person years). In all patients an unsuccessful treatment outcome (treatment failure, death during the treatment period or an unknown outcome) was associated with duration of TB symptoms, with the odds of an unsuccessful outcome decreasing with increasing duration. Among HIV-infected patients, an unsuccessful treatment outcome was also associated with male sex (P = 0.004) and age (P = 0.034). Low Karnofsky score (aHR = 8.93, 95% CI 1.88 – 42.40, P = 0.001) was the only factor significantly associated with mortality among the HIV-uninfected. Mortality among the HIV-infected was associated with the composite variable of CD4 and ART use, with patients with baseline CD4 below 200 cells/µL who were not on ART at a greater risk of death than those who were on ART, and low Karnofsky score (aHR = 2.02, 95% CI 1.02 – 4.01, P = 0.045).
Conclusion
Poor health status is a common cause of hospitalisation for new TB patients. Mortality in this study was very high and associated with advanced HIV Disease and no use of ART.
doi:10.1371/journal.pone.0090614
PMCID: PMC3948371  PMID: 24608875
2.  Variability of Infectious Aerosols Produced during Coughing by Patients with Pulmonary Tuberculosis 
Rationale: Mycobacterium tuberculosis is transmitted by infectious aerosols, but assessing infectiousness currently relies on sputum microscopy that does not accurately predict the variability in transmission.
Objectives: To evaluate the feasibility of collecting cough aerosols and the risk factors for infectious aerosol production from patients with pulmonary tuberculosis (TB) in a resource-limited setting.
Methods: We enrolled subjects with suspected TB in Kampala, Uganda and collected clinical, radiographic, and microbiological data in addition to cough aerosol cultures. A subset of 38 subjects was studied on 2 or 3 consecutive days to assess reproducibility.
Measurements and Main Results: M. tuberculosis was cultured from cough aerosols of 28 of 101 (27.7%; 95% confidence interval [CI], 19.9–37.1%) subjects with culture-confirmed TB, with a median 16 aerosol cfu (range, 1–701) in 10 minutes of coughing. Nearly all (96.4%) cultivable particles were 0.65 to 4.7 μm in size. Positive aerosol cultures were associated with higher Karnofsky performance scores (P = 0.016), higher sputum acid-fast bacilli smear microscopy grades (P = 0.007), lower days to positive in liquid culture (P = 0.004), stronger cough (P = 0.016), and fewer days on TB treatment (P = 0.047). In multivariable analyses, cough aerosol cultures were associated with a salivary/mucosalivary (compared with purulent/mucopurulent) appearance of sputum (odds ratio, 4.42; 95% CI, 1.23–21.43) and low days to positive (per 1-d decrease; odds ratio, 1.17; 95% CI, 1.07–1.33). The within-test (kappa, 0.81; 95% CI, 0.68–0.94) and interday test (kappa, 0.62; 95% CI, 0.43–0.82) reproducibility were high.
Conclusions: A minority of patients with TB (28%) produced culturable cough aerosols. Collection of cough aerosol cultures is feasible and reproducible in a resource-limited setting.
doi:10.1164/rccm.201203-0444OC
PMCID: PMC3443801  PMID: 22798319
tuberculosis; cough; air microbiology; infectious disease transmission; infection control
3.  M. tuberculosis microbiologic and clinical treatment outcomes in a randomized trial of immediate vs. CD4 initiated antiretroviral therapy in HIV-infected adults with high CD4 cell counts 
In a prospective randomized, controlled trial of ART during TB treatment versus TB treatment alone in Ugandan patients with CD4 >350 cells/μL, ART did not accelerate microbiologic, radiographic or clinical responses to TB therapy. 18% of participants had positive sputum smears after 5 months of TB therapy despite negative cultures.
doi:10.1086/654799
PMCID: PMC2919368  PMID: 20569064
Tuberculosis; HIV; antiretroviral therapy
4.  Effectiveness of the Standard WHO Recommended Retreatment Regimen (Category II) for Tuberculosis in Kampala, Uganda: A Prospective Cohort Study 
PLoS Medicine  2011;8(3):e1000427.
Prospective evaluation of the effectiveness of the WHO-recommended standardized retreatment regimen for tuberculosis by Edward Jones-López and colleagues reveals an unacceptable proportion of unsuccessful outcomes.
Background
Each year, 10%–20% of patients with tuberculosis (TB) in low- and middle-income countries present with previously treated TB and are empirically started on a World Health Organization (WHO)-recommended standardized retreatment regimen. The effectiveness of this retreatment regimen has not been systematically evaluated.
Methods and Findings
From July 2003 to January 2007, we enrolled smear-positive, pulmonary TB patients into a prospective cohort to study treatment outcomes and mortality during and after treatment with the standardized retreatment regimen. Median time of follow-up was 21 months (interquartile range 12–33 months). A total of 29/148 (20%) HIV-uninfected and 37/140 (26%) HIV-infected patients had an unsuccessful treatment outcome. In a multiple logistic regression analysis to adjust for confounding, factors associated with an unsuccessful treatment outcome were poor adherence (adjusted odds ratio [aOR] associated with missing half or more of scheduled doses 2.39; 95% confidence interval (CI) 1.10–5.22), HIV infection (2.16; 1.01–4.61), age (aOR for 10-year increase 1.59; 1.13–2.25), and duration of TB symptoms (aOR for 1-month increase 1.12; 1.04–1.20). All patients with multidrug-resistant TB had an unsuccessful treatment outcome. HIV-infected individuals were more likely to die than HIV-uninfected individuals (p<0.0001). Multidrug-resistant TB at enrolment was the only common risk factor for death during follow-up for both HIV-infected (adjusted hazard ratio [aHR] 17.9; 6.0–53.4) and HIV-uninfected (14.7; 4.1–52.2) individuals. Other risk factors for death during follow-up among HIV-infected patients were CD4<50 cells/ml and no antiretroviral treatment (aHR 7.4, compared to patients with CD4≥200; 3.0–18.8) and Karnofsky score <70 (2.1; 1.1–4.1); and among HIV-uninfected patients were poor adherence (missing half or more of doses) (3.5; 1.1–10.6) and duration of TB symptoms (aHR for a 1-month increase 1.9; 1.0–3.5).
Conclusions
The recommended regimen for retreatment TB in Uganda yields an unacceptable proportion of unsuccessful outcomes. There is a need to evaluate new treatment strategies in these patients.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
One-third of the world's population is currently infected with Mycobacterium tuberculosis, the bacterium that causes tuberculosis (TB), and 5%–10% of HIV-uninfected individuals will go on to develop disease and become infectious. The risk of progression from infection to disease in HIV infected is much higher. If left untreated, each person with active TB may infect 10 to 15 people every year, reinforcing the public health priority of controlling TB through adequate treatment. Patients with a previous history of TB treatment are a major concern for TB programs throughout the world because these patients are at a much higher risk of harboring a form of TB that is resistant to the drugs most frequently used, resulting in poorer treatment outcomes and significantly complicating current management strategies. More then 1 million people in over 90 countries need to be “re-treated” after failing, interrupting, or relapsing from previous TB treatment.
Every year, 10%–20% of people with TB in low- and middle-income countries are started on a standardized five-drug retreatment regimen as recommended by the World Health Organization (WHO). Yet, unlike treatment regimens for newly diagnosed TB patients, the recommended retreatment regimen (also known as the category II regimen) has never been properly evaluated in randomized clinical trials or prospective cohort studies. Rather, this regimen was recommended by experts before the current situation of widespread drug-resistant TB and HIV infection.
Why Was This Study Done?
WHO surveillance data suggest that the retreatment regimen is successful in about 70% of patients, but retrospective studies that have evaluated the regimen's efficacy showed variable treatment responses with success rates ranging from 26% to 92%. However, these studies have generally only assessed outcomes at the completion of the retreatment regimen, and few have examined the risk of TB recurrence, especially in people who are also infected with HIV and so are more likely to experience TB recurrence—an issue of particular concern in sub-Saharan Africa. Therefore, in this study based in Kampala, Uganda, the researchers conducted a prospective cohort study to assess treatment and survival outcomes in patients previously treated for TB and to identify factors associated with poor outcomes. Given the overwhelming contribution of HIV infection to death, the researchers categorized their survival analysis by HIV status.
What Did the Researchers Do and Find?
The researchers recruited consecutive smear-positive TB patients who were admitted to Mulago Hospital, Kampala, Uganda, for the retreatment of TB with the standard retreatment regimen between July 2003 and January 2007. Eligible patients received daily directly observed therapy and after hospital discharge, were seen every month during their 8-month TB-retreatment course. Home health visitors assessed treatment adherence through treatment card review, monthly pill counts, and patient self-report. After the completion of the retreatment regimen, patients were evaluated for TB recurrence every 3 months for a median of 21 months. The researchers then used a statistical model to identify treatment outcomes and mortality HIV-uninfected and HIV-infected patients.
The researchers found that 29/148 (20%) of HIV-uninfected and 37/140 (26%) of HIV-infected patients had an unsuccessful treatment outcome. Factors associated with an unsuccessful treatment outcome were poor adherence, HIV infection, increasing age, and duration of TB symptoms. All patients with multidrug resistant TB, a form of TB that is resistant to the two most important drugs used to treat TB, had an unsuccessful treatment outcome. In addition, HIV-infected subjects were more likely to die than HIV-uninfected subjects (p<0.0001), and having multidrug resistant TB at enrollment was the only common risk factor for death during follow-up for both HIV-infected and HIV uninfected patients. Other risk factors for death among HIV-infected patients were CD4<50 cells/ml and no antiretroviral therapy treatment and among HIV-uninfected patients were poor adherence and duration of TB symptoms.
What Do These Findings Mean?
The researchers found that although 70%–80% of patients had a successful treatment outcome on completion of antituberculous therapy (a result that compares well with retrospective studies), the standard retreatment regimen had low treatment response rates and was associated with poor long-term outcomes in certain subgroups of patients, particularly those with multidrug resistant TB and HIV.
These findings indicate that the standard retreatment approach to TB as implemented in low- and middle-income settings is inadequate and stress the importance of a new, more effective, strategies. Improved access to rapid diagnostics for TB drug-resistance, second-line TB treatment, and antiretroviral therapy is urgently needed, along with a strong evidence base to guide clinicians and policy makers on how best to use these tools.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000427.
The World Health Organization has information on TB, TB retreatment, and multidrug-resistant TB
WHO also provides information on TB/HIV coinfection
The Stop TB Partnership provides information on the global plan to stop TB
doi:10.1371/journal.pmed.1000427
PMCID: PMC3058098  PMID: 21423586
5.  Male circumcision and women's risk of incident chlamydial, gonococcal and trichomonal infections 
Sexually transmitted diseases  2008;35(7):689-695.
doi:10.1097/OLQ.0b013e31816b1fcc
PMCID: PMC2978019  PMID: 18418300
male circumcision; women; Chlamydia trachomatis; Neisseria gonorrhoeae; Trichomonas vaginalis
6.  Men's circumcision status and women's risk of HIV acquisition in Zimbabwe and Uganda 
AIDS (London, England)  2007;21(13):1779-1789.
doi:10.1097/QAD.0b013e32827b144c
PMCID: PMC2978032  PMID: 17690577
male circumcision; women; HIV; Zimbabwe; Uganda; misclassification
7.  Evaluation of Time to Detection of Mycobacterium tuberculosis in Broth Culture as a Determinant for End Points in Treatment Trials▿ †  
Journal of Clinical Microbiology  2010;48(12):4370-4376.
Time to detection of Mycobacterium tuberculosis in broth culture was examined for utility as a treatment efficacy end point. Of 146 patients in a phase IIB trial, a decreased mean time to detection was found in 5 with treatment failure. Time to detection in an analysis-of-covariance model was associated with lung cavities, less intensive treatment, and differences in the bactericidal effects of treatment regimens.
doi:10.1128/JCM.00757-10
PMCID: PMC3008491  PMID: 20926712
8.  Shortening Treatment in Adults with Noncavitary Tuberculosis and 2-Month Culture Conversion 
Rationale: Cavitary disease and delayed culture conversion have been associated with relapse. Combining patient characteristics and measures of bacteriologic response might allow treatment shortening with current drugs in some patients.
Objectives: To assess whether treatment could be shortened from 6 to 4 months in patients with noncavitary tuberculosis whose sputum cultures converted to negative after 2 months.
Methods: This study was a randomized, open-label equivalence trial. HIV-uninfected adults with noncavitary tuberculosis were treated daily with isoniazid, rifampin, pyrazinamide, and ethambutol for 2 months, followed by 2 months of isoniazid and rifampin. After 4 months, patients with drug-susceptible TB whose sputum cultures on solid media were negative after 8 weeks of treatment were randomly assigned to continue treatment for 2 more months or to stop treatment. Patients were followed for relapse for 30 months after beginning treatment.
Measurements and Main Results: Enrollment was stopped by the safety monitoring committee after 394 patients were enrolled due to apparent increased risk for relapse in the 4-month arm. A total of 370 patients were eligible for per protocol analysis. Thirteen patients in the 4-month arm relapsed, compared with three subjects in the 6-month arm (7.0 vs. 1.6%; risk difference, 0.054; 95% confidence interval with Hauck-Anderson correction, 0.01–0.10).
Conclusion: Shortening treatment from 6 to 4 months in adults with noncavitary disease and culture conversion after 2 months using current drugs resulted in a greater relapse rate. The combination of noncavitary disease and 2-month culture conversion was insufficient to identify patients with decreased risk for relapse.
doi:10.1164/rccm.200904-0536OC
PMCID: PMC2742745  PMID: 19542476
tuberculosis; antitubercular agents; isoniazid; rifampin
9.  Rate and Amplification of Drug Resistance among Previously-Treated Patients with Tuberculosis in Kampala, Uganda 
Background
Drug-resistant Mycobacterium tuberculosis has emerged as a global threat. In resource-constrained settings, patients with a history of tuberculosis (TB) treatment may have drug-resistant disease and may experience poor outcomes. There is a need to measure the extent of and risk factors for drug resistance in such patients.
Methods
From July 2003 through November 2006, we enrolled 410 previously treated patients with TB in Kampala, Uganda. We measured the prevalence of resistance to first- and second-line drugs and analyzed risk factors associated with baseline and acquired drug resistance.
Results
The prevalence of multidrug-resistant TB was 12.7% (95% confidence interval [95% CI], 9.6%–16.3%). Resistance to second-line drugs was low. Factors associated with multidrug-resistant TB at enrollment included a history of treatment failure (odds ratio, 23.6; 95% CI, 7.7–72.4), multiple previous TB episodes (odds ratio, 15.6; 95% CI, 5.0–49.1), and cavities present on chest radiograph (odds ratio, 5.9; 95% CI, 1.2–29.5). Among a cohort of 250 patients, 5.2% (95% CI, 2.8%–8.7%) were infected with M. tuberculosis that developed additional drug resistance. Amplification of drug resistance was associated with existing drug resistance at baseline (P<.01) and delayed sputum culture conversion (P<.01).
Conclusions
The burden of drug resistance in previously treated patients with TB in Uganda is sizeable, and the risk of generating additional drug resistance is significant. There is an urgent need to improve the treatment for such patients in low-income countries.
doi:10.1086/592252
PMCID: PMC2883442  PMID: 18808360
10.  Impact of pulmonary tuberculosis on survival of HIV-infected adults: a prospective epidemiologic study in Uganda 
AIDS (London, England)  2000;14(9):1219-1228.
Background
Retrospective cohort studies of tuberculosis suggest that active tuberculosis accelerates the progression of HIV infection. The validity of these findings has been questioned because of their retrospective design, diverse study populations, variable compliance with anti-tuberculous therapy and use of anti-retroviral medication. To assess the impact of tuberculosis on survival in HIV infection we performed a prospective study among HIV-infected Ugandan adults with and without tuberculosis.
Methods
In a prospective cohort study, 230 patients with HIV-associated tuberculosis and 442 HIV-infected subjects without tuberculosis were followed for a mean duration of 19 months for survival. To assess changes in viral load over 1 year, 20 pairs of tuberculosis cases and controls were selected and matched according to baseline CD4 lymphocyte count, age, sex and tuberculin skin test status.
Results
During the follow-up period, 63 out of of 230 tuberculosis cases (28%) died compared with 85 out of 442 controls (19%), with a crude risk ratio of 1.4 [95% confidence interval (CI), 1.07–1.87]. Most deaths occurred in patients with CD4 lymphocyte counts < 200 × 106 cells/l at baseline (n = 99) and occurred with similar frequency in the tuberculosis cases (46%) and the controls (44%). When the CD4 lymphocyte count was > 200 × 106 /l, however, the relative risk of death in HIV-associated tuberculosis was 2.1 (95% CI, 1.27–3.62) compared with subjects without tuberculosis. For subjects with a CD4 lymphocyte count > 200 × 106/l, the 1-year survival proportion was slightly lower in the cases than in the controls (0.91 versus 0.96), but by 2 years the survival proportion was significantly lower in the cases than in the controls (0.84 versus 0.91; P < 0.02; log-rank test). For subjects with a CD4 lymphocyte count of 200 × 106 cells/l or fewer, the survival proportion at 1 year for the controls was lower than cases (0.59 versus 0.64), but this difference was not statistically significant (P = 0.53; logrank test). After adjusting for age, sex, tuberculin skin test status, CD4 lymphocyte count, and history of HIV-related infections, the overall relative hazard for death associated with tuberculosis was 1.81 (95% CI, 1.24–2.65). In a nested Cox regression model, the relative hazard for death was 3.0 (95% CI, 1.62–5.63) for subjects with CD4 lymphocyte counts > 200 × 106/l and 1.5 (95% CI, 0.99–2.40) for subjects with a CD4 lymphocyte count of 200 × 106/l or fewer.
Conclusion
The findings from this prospective study indicate that active tuberculosis exerts its greatest effect on survival in the early stages of HIV infection, when there is a reserve capacity of the host immune response. These observations provide a theoretical basis for the treatment of latent tuberculous infection in HIV-infected persons.
PMCID: PMC2869086  PMID: 10894287
HIV-1; tuberculosis; survival; Africa; prospective cohort study
11.  IMMUNE CORRELATES OF ACUTE MYCOBACTERIUM TUBERCULOSIS INFECTION IN HOUSEHOLD CONTACTS IN KAMPALA, UGANDA 
To determine immunologic and epidemiologic correlates of acute Mycobacterium tuberculosis infection in household contacts of infectious tuberculosis cases, we performed a prospective, community-based cohort study of index cases and their household contacts in Kampala, Uganda. Contacts were evaluated for tuberculin skin test (TST) conversion over two years. Interferon-γ expression was measured using a whole blood assay after stimulating with M. tuberculosis culture-filtrate. In 222 contacts with a TST less than 5 mm at baseline, the one-year rate of TST conversion was 27%. The TST conversion was associated with the infectiousness of the index case and proximity of contact. Interferon-γ levels at baseline were greater among TST converters compared with those who did not convert. The risk of TST conversion increased four-fold as the baseline interferon-γ increased 10-fold, but only in contacts with BCG vaccination. In household contacts of tuberculosis, interferon-γ responses to non-specific mycobacterial antigens may be used to make an early diagnosis of tuberculosis infection, especially in resource-limited settings where bacille Calmette-Guérin vaccination is commonly used.
PMCID: PMC2869089  PMID: 16837709
12.  Vδ2+γδ T Cell Function in Mycobacterium tuberculosis– and HIV-1–Positive Patients in the United States and Uganda: Application of a Whole-Blood Assay 
The Journal of infectious diseases  2005;192(10):1806-1814.
Background
Vγ9+Vδ2+ γδ T cells (Vδ2+ T cells) are activated by Mycobacterium tuberculosis and secrete interferon (IFN)–γ. Vδ2+ T cells recognize phosphoantigens, such as bromohydrin pyrophosphate (BrHPP), and link innate and adaptive immunity.
Methods
A whole-blood assay was developed that used IFN-γ secretion in response to BrHPP as a measurement of Vδ2+ T cell function.
Results
Peak IFN-γ levels were detected after stimulating whole blood with BrHPP for 7–9 days. IFN-γ production in whole blood in response to BrHPP paralleled IFN-γ production and Vδ2+ T cell expansion of peripheral-blood mononuclear cells. The assay was used to evaluate Vδ2+ T cell function in subjects in the United States (n = 24) and Uganda (n = 178) who were or were not infected with M. tuberculosis and/or human immunodeficiency virus (HIV) type 1. When 50 μmol/L BrHPP was used, 100% of healthy subjects produced IFN-γ. The Vδ2+ T cell response was independent of the tuberculin skin test response. In Uganda, Vδ2+ T cell responses were decreased in patients with tuberculosis (n = 73) compared with responses in household contacts (n = 105). HIV-1–positive household contacts had lower responses than did HIV-1–negative household contacts. HIV-1–positive patients with tuberculosis had the lowest Vδ2+ T cell responses.
Conclusions
Tuberculosis and HIV-1 infection are associated with decreased Vδ2+ T cell function. Decreased Vδ2+ T cell function may contribute to increased risk for tuberculosis in HIV-1–positive patients.
doi:10.1086/497146
PMCID: PMC2869092  PMID: 16235181
13.  Effect of Tuberculosis Preventive Therapy on HIV Disease Progression and Survival in HIV-Infected Adults 
HIV clinical trials  2006;7(4):172-183.
Purpose:
To determine whether tuberculosis (TB) preventive therapies alter the rate of disease progression to AIDS or death and to identify significant prognostic factors for HIV disease progression to AIDS.
Method:
In a randomized placebo-controlled trial in Kampala, Uganda, 2,736 purified protein derivative (PPD)-positive and anergic HIV-infected adults were randomly assigned to four and two regimens, respectively. PPD-positive patients were treated with isoniazid (INH) for 6 months (6H; n = 536), INH plus rifampicin for 3 months (3HR; n = 556), INH plus rifampicin plus pyrazinamide for 3 months (3HRZ; n = 462), or placebo for 6 months (n = 464). Anergic participants were treated with 6H (n = 395) or placebo (n = 323).
Results:
During follow-up, 404 cases progressed to AIDS and 577 deaths occurred. The cumulative incidence of the AIDS progression was greater in the anergic cohort compared to the PPD-positive cohort (p < .0001). Among PPD-positive patients, the relative risk of the AIDS progression with INH alone was 0.95 (95% CI 0.68–1.32); with 3HR it was 0.83 (95% CI 0.59–1.17); and with 3HRZ it was 0.76 (95% CI 0.52–1.08), controlling for significant baseline predictors. Among anergic patients, the relative risk of the AIDS progression was 0.81 (95% CI 0.56–1.15). Survival was greater in the PPD-positive cohort compared to the anergic cohort (p = .0001).
Conclusion:
The number of signs or symptoms at baseline and anergic status are associated with increasing morbidity and mortality. Even though the tuberculosis preventive therapies were effective in reducing the incidence of TB for HIV-infected adults, their benefit of delaying HIV disease progression to AIDS was not observed.
doi:10.1310/hct0704-172
PMCID: PMC2860292  PMID: 17065029
disease progression; HIV/AIDS; natural history; prevention; survival; tuberculosis
14.  Tuberculosis Treatment in HIV Infected Ugandans with CD4 Counts >350 Cells/mm3 Reduces Immune Activation with No Effect on HIV Load or CD4 Count 
PLoS ONE  2010;5(2):e9138.
Background
Both HIV and TB cause a state of heightened immune activation. Immune activation in HIV is associated with progression to AIDS. Prior studies, focusing on persons with advanced HIV, have shown no decline in markers of cellular activation in response to TB therapy alone.
Methodology
This prospective cohort study, composed of participants within a larger phase 3 open-label randomized controlled clinical trial, measured the impact of TB treatment on immune activation in persons with non-advanced HIV infection (CD4>350 cells/mm3) and pulmonary TB. HIV load, CD4 count, and markers of immune activation (CD38 and HLA-DR on CD4 and CD8 T cells) were measured prior to starting, during, and for 6 months after completion of standard 6 month anti-tuberculosis (TB) therapy in 38 HIV infected Ugandans with smear and culture confirmed pulmonary TB.
Results
Expression of CD38, and co-expression of CD38 and HLA-DR, on CD8 cells declined significantly within 3 months of starting standard TB therapy in the absence of anti-retroviral therapy, and remained suppressed for 6 months after completion of therapy. In contrast, HIV load and CD4 count remained unchanged throughout the study period.
Conclusion
TB therapy leads to measurable decreases in immune activation in persons with HIV/TB co-infection and CD4 counts >350 cells/mm3.
doi:10.1371/journal.pone.0009138
PMCID: PMC2825253  PMID: 20179751
15.  Rapid Detection of Mycobacterium tuberculosis and Rifampin Resistance by Use of On-Demand, Near-Patient Technology▿ † ‡  
Journal of Clinical Microbiology  2009;48(1):229-237.
Current nucleic acid amplification methods to detect Mycobacterium tuberculosis are complex, labor-intensive, and technically challenging. We developed and performed the first analysis of the Cepheid Gene Xpert System's MTB/RIF assay, an integrated hands-free sputum-processing and real-time PCR system with rapid on-demand, near-patient technology, to simultaneously detect M. tuberculosis and rifampin resistance. Analytic tests of M. tuberculosis DNA demonstrated a limit of detection (LOD) of 4.5 genomes per reaction. Studies using sputum spiked with known numbers of M. tuberculosis CFU predicted a clinical LOD of 131 CFU/ml. Killing studies showed that the assay's buffer decreased M. tuberculosis viability by at least 8 logs, substantially reducing biohazards. Tests of 23 different commonly occurring rifampin resistance mutations demonstrated that all 23 (100%) would be identified as rifampin resistant. An analysis of 20 nontuberculosis mycobacteria species confirmed high assay specificity. A small clinical validation study of 107 clinical sputum samples from suspected tuberculosis cases in Vietnam detected 29/29 (100%) smear-positive culture-positive cases and 33/39 (84.6%) or 38/53 (71.7%) smear-negative culture-positive cases, as determined by growth on solid medium or on both solid and liquid media, respectively. M. tuberculosis was not detected in 25/25 (100%) of the culture-negative samples. A study of 64 smear-positive culture-positive sputa from retreatment tuberculosis cases in Uganda detected 63/64 (98.4%) culture-positive cases and 9/9 (100%) cases of rifampin resistance. Rifampin resistance was excluded in 54/55 (98.2%) susceptible cases. Specificity rose to 100% after correcting for a conventional susceptibility test error. In conclusion, this highly sensitive and simple-to-use system can detect M. tuberculosis directly from sputum in less than 2 h.
doi:10.1128/JCM.01463-09
PMCID: PMC2812290  PMID: 19864480
16.  Comparison of rapid tests for detection of rifampicin-resistant Mycobacterium tuberculosis in Kampala, Uganda 
Background
Drug resistant tuberculosis (TB) is a growing concern worldwide. Rapid detection of resistance expedites appropriate intervention to control the disease. Several technologies have recently been reported to detect rifampicin resistant Mycobacterium tuberculosis directly in sputum samples. These include phenotypic culture based methods, tests for gene mutations and tests based on bacteriophage replication. The aim of the present study was to assess the feasibility of implementing technology for rapid detection of rifampicin resistance in a high disease burden setting in Africa.
Methods
Sputum specimens from re-treatment TB patients presenting to the Mulago Hospital National TB Treatment Centre in Kampala, Uganda, were examined by conventional methods and simultaneously used in one of the four direct susceptibility tests, namely direct BACTEC 460, Etest, "in-house" phage test, and INNO- Rif.TB. The reference method was the BACTEC 460 indirect culture drug susceptibility testing. Test performance, cost and turn around times were assessed.
Results
In comparison with indirect BACTEC 460, the respective sensitivities and specificities for detecting rifampicin resistance were 100% and 100% for direct BACTEC and the Etest, 94% and 95% for the phage test, and 87% and 87% for the Inno-LiPA assay. Turn around times ranged from an average of 3 days for the INNO-LiPA and phage tests, 8 days for the direct BACTEC 460 and 20 days for the Etest. All methods were faster than the indirect BACTEC 460 which had a mean turn around time of 24 days. The cost per test, including labour ranged from $18.60 to $41.92 (USD).
Conclusion
All four rapid technologies were shown capable of detecting rifampicin resistance directly from sputum. The LiPA proved rapid, but was the most expensive. It was noted, however, that the LiPA test allows sterilization of samples prior to testing thereby reducing the risk of accidental laboratory transmission. In contrast the Etest was low cost, but slow and would be of limited assistance when treating patients. The phage test was the least reproducible test studied with failure rate of 27%. The test preferred by the laboratory personnel, direct BACTEC 460, requires further study to determine its accuracy in real-time treatment decisions in Uganda.
doi:10.1186/1471-2334-9-139
PMCID: PMC2744678  PMID: 19709423
17.  Genome Scan of M. tuberculosis Infection and Disease in Ugandans 
PLoS ONE  2008;3(12):e4094.
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is an enduring public health problem globally, particularly in sub-Saharan Africa. Several studies have suggested a role for host genetic susceptibility in increased risk for TB but results across studies have been equivocal. As part of a household contact study of Mtb infection and disease in Kampala, Uganda, we have taken a unique approach to the study of genetic susceptibility to TB, by studying three phenotypes. First, we analyzed culture confirmed TB disease compared to latent Mtb infection (LTBI) or lack of Mtb infection. Second, we analyzed resistance to Mtb infection in the face of continuous exposure, defined by a persistently negative tuberculin skin test (PTST-); this outcome was contrasted to LTBI. Third, we analyzed an intermediate phenotype, tumor necrosis factor-alpha (TNFα) expression in response to soluble Mtb ligands enriched with molecules secreted from Mtb (culture filtrate). We conducted a full microsatellite genome scan, using genotypes generated by the Center for Medical Genetics at Marshfield. Multipoint model-free linkage analysis was conducted using an extension of the Haseman-Elston regression model that includes half sibling pairs, and HIV status was included as a covariate in the model. The analysis included 803 individuals from 193 pedigrees, comprising 258 full sibling pairs and 175 half sibling pairs. Suggestive linkage (p<10−3) was observed on chromosomes 2q21-2q24 and 5p13-5q22 for PTST-, and on chromosome 7p22-7p21 for TB; these findings for PTST- are novel and the chromosome 7 region contains the IL6 gene. In addition, we replicated recent linkage findings on chromosome 20q13 for TB (p = 0.002). We also observed linkage at the nominal α = 0.05 threshold to a number of promising candidate genes, SLC11A1 (PTST- p = 0.02), IL-1 complex (TB p = 0.01), IL12BR2 (TNFα p = 0.006), IL12A (TB p = 0.02) and IFNGR2 (TNFα p = 0.002). These results confirm not only that genetic factors influence the interaction between humans and Mtb but more importantly that they differ according to the outcome of that interaction: exposure but no infection, infection without progression to disease, or progression of infection to disease. Many of the genetic factors for each of these stages are part of the innate immune system.
doi:10.1371/journal.pone.0004094
PMCID: PMC2605555  PMID: 19116662
18.  Whole Blood Interferon-Gamma Responses to Mycobacterium tuberculosis Antigens in Young Household Contacts of Persons with Tuberculosis in Uganda 
PLoS ONE  2008;3(10):e3407.
Background
Due to immunologic immaturity, IFN-γ-producing T cell responses may be decreased in young children compared to adults, thus we hypothesized that IFN-γ responses to mycobacterial antigens in household contacts exposed to Mycobacterium tuberculosis (Mtb) would be impaired in young children relative to adults. The objective of this study was to compare whole blood IFN-γ production in response to mycobacterial antigens between children and adults in Uganda.
Methodology/Principal Findings
We studied household contacts of persons with culture-positive pulmonary tuberculosis (TB) enrolled in a cohort study conducted in Kampala, Uganda. Whole blood IFN-γ production in response to Mtb culture-filtrate antigens was measured by ELISA and compared between infants (<2 years old, n = 80), young children (2 <5 years old, n = 216), older children (5 <15 years old, n = 443) and adults (≥15 years old, n = 528). We evaluated the relationship between IFN-γ responses and the tuberculin skin test (TST), and between IFN-γ responses and epidemiologic factors that reflect exposure to Mtb, and the effect of prior BCG vaccination on IFN-γ responses. Young household contacts demonstrated robust IFN-γ responses comparable to those of adults that were associated with TST and known risk factors for infection. There was no effect of prior BCG immunization on the IFN-γ response.
Conclusions/Significance
Young children in a TB endemic setting can mount robust IFN-γ responses generally comparable to those of adults, and as in adults, these responses correlated with the TST and known epidemiologic risk factors for Mtb infection.
doi:10.1371/journal.pone.0003407
PMCID: PMC2560997  PMID: 18923705
19.  Low-cost rapid detection of rifampicin resistant tuberculosis using bacteriophage in Kampala, Uganda 
Background
Resistance to anti-tuberculosis drugs is a serious public health problem. Multi-drug resistant tuberculosis (MDR-TB), defined as resistance to at least rifampicin and isoniazid, has been reported in all regions of the world. Current phenotypic methods of assessing drug susceptibility of M. tuberculosis are slow. Rapid molecular methods to detect resistance to rifampicin have been developed but they are not affordable in some high prevalence countries such as those in sub Saharan Africa. A simple multi-well plate assay using mycobacteriophage D29 has been developed to test M. tuberculosis isolates for resistance to rifampicin. The purpose of this study was to investigate the performance of this technology in Kampala, Uganda.
Methods
In a blinded study 149 M. tuberculosis isolates were tested for resistance to rifampicin by the phage assay and results compared to those from routine phenotypic testing in BACTEC 460. Three concentrations of drug were used 2, 4 and 10 μg/ml. Isolates found resistant by either assay were subjected to sequence analysis of a 81 bp fragment of the rpoB gene to identify mutations predictive of resistance. Four isolates with discrepant phage and BACTEC results were tested in a second phenotypic assay to determine minimal inhibitory concentrations.
Results
Initial analysis suggested a sensitivity and specificity of 100% and 96.5% respectively for the phage assay used at 4 and 10 μg/ml when compared to the BACTEC 460. However, further analysis revealed 4 false negative results from the BACTEC 460 and the phage assay proved the more sensitive and specific of the two tests. Of the 39 isolates found resistant by the phage assay 38 (97.4%) were found to have mutations predictive of resistance in the 81 bp region of the rpoB gene. When used at 2 μg/ml false resistant results were observed from the phage assay. The cost of reagents for testing each isolate was estimated to be 1.3US$ when testing a batch of 20 isolates on a single 96 well plate. Results were obtained in 48 hours.
Conclusion
The phage assay can be used for screening of isolates for resistance to rifampicin, with high sensitivity and specificity in Uganda. The test may be useful in poorly resourced laboratories as a rapid screen to differentiate between rifampicin susceptible and potential MDR-TB cases.
doi:10.1186/1476-0711-6-1
PMCID: PMC1779803  PMID: 17212825
20.  Gender and HIV-associated pulmonary tuberculosis: presentation and outcome at one year after beginning antituberculosis treatment in Uganda 
Background
Tuberculosis is responsible for more female deaths around the earth than any other infectious disease. Reports have suggested that responses to tuberculosis may differ between men and women. We investigated gender related differences in the presentation and one year outcomes of HIV-infected adults with initial episodes of pulmonary tuberculosis in Uganda.
Methods
We enrolled and followed up a cohort of 105 male and 109 female HIV-infected adults on treatment for initial episodes of culture-confirmed pulmonary tuberculosis between March 1993 and March 1995. A favorable outcome was defined as being cured and alive at one year while an unfavorable outcome was not being cured or dead. Subjects were followed-up by serial medical examinations, complete blood counts, serum β2 microglobulin, CD4+ cell counts, sputum examinations, and chest x-rays.
Results
Male patients were older, had higher body mass indices, and lower serum β2 microglobulin levels than female patients at presentation. At one year, there was no difference between male and female patients in the likelihood of experiencing a favorable outcome (RR 1.02, 95% CI 0.89–1.17). This effect persisted after controlling for symptoms, serum β2 microglobulin, CD4+ cell count, and severity of disease on chest x-ray (OR 1.07, 95% CI 0.54–2.13) with a repeated measures model.
Conclusions
While differences existed between males and females with HIV-associated pulmonary tuberculosis at presentation, the outcomes at one year after the initiation of tuberculosis treatment were similar in Uganda. Women in areas with a high HIV and tuberculosis prevalence should be encouraged to present for screening at the first sign of tuberculosis symptoms.
doi:10.1186/1471-2466-2-4
PMCID: PMC129982  PMID: 12223113
Tuberculosis; Gender; HIV infection
22.  Drug Tolerance in Mycobacterium tuberculosis 
Antimicrobial Agents and Chemotherapy  1999;43(11):2600-2606.
Although Mycobacterium tuberculosis is eradicated rapidly during therapy in some patients with pulmonary tuberculosis, it can persist for many months in others. This study examined the relationship between mycobacterial drug tolerance (delayed killing in vitro), persistence, and relapse. It was performed with 39 fully drug-susceptible isolates from a prospective trial of standard short-course antituberculous therapy with sputum smear-positive, human immunodeficiency virus-uninfected subjects with pulmonary tuberculosis in Brazil and Uganda. The rate of killing in vitro was determined by monitoring the growth index (GI) in BACTEC 12B medium after addition of drug to established cultures and was measured as the number of days required for 99% sterilization. Drugs differed significantly in bactericidal activity, in the following order from greatest to least, rifampin > isoniazid-ethambutol > ethambutol (P < 0.001). Isolates from subjects who had relapses (n = 2) or in whom persistence was prolonged (n = 1) were significantly more tolerant of isoniazid-ethambutol and rifampin than isolates from other subjects (P < 0.01). More generally, the duration of persistence during therapy was predicted by strain tolerance to isoniazid and rifampin (P = 0.012 and 0.026, respectively). Tolerance to isoniazid-ethambutol and tolerance to rifampin were highly correlated (P < 0.001). Tolerant isolates did not differ from others with respect to the MIC of isoniazid; the rate of killing of a tolerant isolate by isoniazid-ethambutol was not increased at higher drug concentrations. These observations suggest that tolerance may not be due to drug-specific mechanisms. Tolerance was of the phenotypic type, although increased tolerance appeared to emerge after prolonged drug exposure in vivo. This study suggests that drug tolerance may be an important determinant of the outcome of therapy for tuberculosis.
PMCID: PMC89531  PMID: 10543735

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