Edward C. Jones-López

Rapid Detection of Mycobacterium tuberculosis and Rifampin Resistance by Use of On-Demand, Near-Patient Technology

ABSTRACT 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 Systems 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 assays 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.

Immunoadjuvant Prednisolone Therapy for HIV-Associated Tuberculosis: A Phase 2 Clinical Trial in Uganda

Background. Human immunodeficiency virus (HIV)-infected patients with tuberculosis (TB) respond to effective antituberculous therapy, but their prognosis remains poor. Mounting evidence from clinical studies supports the concept of copathogenesis in which immune activation that is triggered by TB and mediated by cytokines stimulates viral replication and worsens HIV infection, especially when immune function is preserved.Methods. We performed a phase 2, randomized, double-blind, placebo-controlled clinical trial in Kampala, Uganda, to determine whether immunoadjuvant prednisolone therapy in HIV-infected patients with TB who have CD4(+) T cell counts >/=200 cells/ mu L is safe and effective at increasing CD4(+) T cell counts.Results. Short-term prednisolone therapy reduced levels of immune activation and tended to produce higher CD4(+) T cell counts. Although prednisolone therapy was associated with a more rapid clearance of Mycobacterium tuberculosis from the sputum, it was also associated with a transient increase in HIV RNA levels, which receded when prednisolone therapy was discontinued. The intervention worsened underlying hypertension and caused fluid retention and hyperglycemia.Conclusion. The benefits of prednisolone therapy on immune activation and CD4(+) T cell counts do not outweigh the risks of adverse events in HIV-infected patients with TB and preserved immune function.

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.

Cough Aerosols of Mycobacterium tuberculosis Predict New Infection. A Household Contact Study

RATIONALE Airborne transmission of Mycobacterium tuberculosis results from incompletely characterized host, bacterial, and environmental factors. Sputum smear microscopy is associated with considerable variability in transmission. OBJECTIVES To evaluate the use of cough-generated aerosols of M. tuberculosis to predict recent transmission. METHODS Patients with pulmonary tuberculosis (TB) underwent a standard evaluation and collection of cough aerosol cultures of M. tuberculosis. We assessed household contacts for new M. tuberculosis infection. We used multivariable logistic regression analysis with cluster adjustment to analyze predictors of new infection. MEASUREMENTS AND MAIN RESULTS From May 2009 to January 2011, we enrolled 96 sputum culture-positive index TB cases and their 442 contacts. Only 43 (45%) patients with TB yielded M. tuberculosis in aerosols. Contacts of patients with TB who produced high aerosols (≥10 CFU) were more likely to have a new infection compared with contacts from low-aerosol (1-9 CFU) and aerosol-negative cases (69%, 25%, and 30%, respectively; P = 0.009). A high-aerosol patient with TB was the only predictor of new M. tuberculosis infection in unadjusted (odds ratio, 5.18; 95% confidence interval, 1.52-17.61) and adjusted analyses (odds ratio, 4.81; 95% confidence interval, 1.20-19.23). Contacts of patients with TB with no aerosols versus low and high aerosols had differential tuberculin skin test and interferon-γ release assay responses. CONCLUSIONS Cough aerosols of M. tuberculosis are produced by a minority of patients with TB but predict transmission better than sputum smear microscopy or culture. Cough aerosols may help identify the most infectious patients with TB and thus improve the cost-effectiveness of TB control programs.

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.

Low-cost rapid detection of rifampicin resistant tuberculosis using bacteriophage in Kampala, Uganda.

BackgroundResistance 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.MethodsIn 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.ResultsInitial 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

Latent tuberculosis infection--Revisiting and revising concepts.

when testing a batch of 20 isolates on a single 96 well plate. Results were obtained in 48 hours.ConclusionThe 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.

Effectiveness of the Standard WHO Recommended Retreatment Regimen (Category II) for Tuberculosis in Kampala, Uganda: A Prospective Cohort Study

Host- and pathogen-specific factors interplay with the environment in a complex fashion to determine the outcome of infection with Mycobacterium tuberculosis (Mtb), resulting in one of three possible outcomes: cure, latency or active disease. Although much remains unknown about its pathophysiology, latent tuberculosis infection (LTBI) defined by immunologic evidence of Mtb infection is a continuum between self-cure and asymptomatic, yet active tuberculosis (TB) disease. Strain virulence, intensity of exposure to the index case, size of the bacterial inoculum, and host factors such as age and co-morbidities, each contribute to where one settles on the continuum. Currently, the diagnosis of LTBI is based on reactive tuberculin skin testing (TST) and/or a positive interferon-gamma release assay (IGRA). Neither diagnostic test reflects the activity of the infectious focus or the risk of progression to active TB. This is a critical shortcoming, as accurate and efficient detection of those with LTBI at higher risk of progression to TB disease would allow for provision of targeted preventive therapy to those most likely to benefit. Host biomarkers may prove of value in stratifying risk of development of TB. New guidelines are required for interpretation of discordance between TST and IGRA, which may be due in part to a lack of stability (that is reproducibility) of IGRA or TST results or to a delay in conversion of IGRA to positivity compared to TST. In this review, the authors elaborate on the definition, diagnosis, pathophysiology and natural history of LTBI, as well as promising methods for better stratifying risk of progression to TB. The review is centered on the human host and the clinical and epidemiologic features of LTBI that are relevant to the development of new and improved diagnostic tools.

Elucidating emergence and transmission of multidrug-resistant tuberculosis in treatment experienced patients by whole genome sequencing

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.

Mutations in extensively drug-resistant Mycobacterium tuberculosis that do not code for known drug-resistance mechanisms.

Background Understanding the emergence and spread of multidrug-resistant tuberculosis (MDR-TB) is crucial for its control. MDR-TB in previously treated patients is generally attributed to the selection of drug resistant mutants during inadequate therapy rather than transmission of a resistant strain. Traditional genotyping methods are not sufficient to distinguish strains in populations with a high burden of tuberculosis and it has previously been difficult to assess the degree of transmission in these settings. We have used whole genome analysis to investigate M. tuberculosis strains isolated from treatment experienced patients with MDR-TB in Uganda over a period of four years. Methods and Findings We used high throughput genome sequencing technology to investigate small polymorphisms and large deletions in 51 Mycobacterium tuberculosis samples from 41 treatment-experienced TB patients attending a TB referral and treatment clinic in Kampala. This was a convenience sample representing 69% of MDR-TB cases identified over the four year period. Low polymorphism was observed in longitudinal samples from individual patients (2-15 SNPs). Clusters of samples with less than 50 SNPs variation were examined. Three clusters comprising a total of 8 patients were found with almost identical genetic profiles, including mutations predictive for resistance to rifampicin and isoniazid, suggesting transmission of MDR-TB. Two patients with previous drug susceptible disease were found to have acquired MDR strains, one of which shared its genotype with an isolate from another patient in the cohort. Conclusions Whole genome sequence analysis identified MDR-TB strains that were shared by more than one patient. The transmission of multidrug-resistant disease in this cohort of retreatment patients emphasises the importance of early detection and need for infection control. Consideration should be given to rapid testing for drug resistance in patients undergoing treatment to monitor the emergence of resistance and permit early intervention to avoid onward transmission.