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1.  Impact of GeneXpert MTB/RIF on Patients and Tuberculosis Programs in a Low-Burden Setting. A Hypothetical Trial 
Rationale: Guidelines recommend routine nucleic-acid amplification testing in patients with presumed tuberculosis (TB), but these tests have not been widely adopted. GeneXpert MTB/RIF (Xpert), a novel, semiautomated TB nucleic-acid amplification test, has renewed interest in this technology, but data from low-burden countries are limited.
Objectives: We sought to estimate Xpert’s potential clinical and public health impact on empiric treatment, contact investigation, and housing in patients undergoing TB evaluation.
Methods: We performed a prospective, cross-sectional study with 2-month follow-up comparing Xpert with standard strategies for evaluating outpatients for active pulmonary TB at the San Francisco Department of Public Health TB Clinic between May 2010 and June 2011. We calculated the diagnostic accuracy of standard algorithms for initial empiric TB treatment, contact investigation, and housing in reference to three Mycobacterium tuberculosis sputum cultures, as compared with that of a single sputum Xpert test. We estimated the incremental diagnostic value of Xpert, and the hypothetical reductions in unnecessary treatment, contact investigation, and housing if Xpert were adopted to guide management decisions.
Measurements and Main Results: A total of 156 patients underwent Xpert testing. Fifty-nine (38%) received empiric TB treatment. Thirteen (8%) had culture-positive TB. Xpert-guided management would have hypothetically decreased overtreatment by 94%, eliminating a median of 44 overtreatment days (interquartile range, 43–47) per patient and 2,169 total overtreatment days (95% confidence interval, 1,938–2,400) annually, without reducing early detection of TB patients. We projected similar benefits for contact investigation and housing.
Conclusions: Xpert could greatly reduce the frequency and impact of unnecessary empiric treatment, contact investigation, and housing, providing substantial patient and programmatic benefits if used in management decisions.
PMCID: PMC4226012  PMID: 24869625
tuberculosis; diagnosis; health care quality assurance; operations research; public health
2.  Diagnostic accuracy of same-day microscopy versus standard microscopy for pulmonary tuberculosis: a systematic review and meta-analysis 
The Lancet infectious diseases  2012;13(2):10.1016/S1473-3099(12)70232-3.
Sputum smear microscopy is the most widely available diagnostic test for pulmonary tuberculosis in countries with a high burden of the disease. Improving its accuracy is crucial to achievement of case-detection targets established by the Millennium Development Goals. Unfortunately, many patients are unable to submit all of the specimens needed for examination or to return for treatment because standard sputum collection and reporting requires several clinic visits. To inform policy recommendations by a WHO-convened Expert Group, we aimed to assess the accuracy of sputum smear examination with strategies for obtaining sputum on 1 day compared with strategies for obtaining sputum over 2 days.
We did a systematic review and meta-analysis of research articles comparing the accuracy of front-loaded or same-day microscopy and standard sputum smear microscopy for diagnosis of culture-confirmed pulmonary tuberculosis. We searched Medline, Embase, Biosis, and Web of Science for articles published between Jan 1, 2005, and Feb 14, 2012. Two investigators identified eligible articles and extracted data for individual study sites. We generated pooled summary estimates (95% CIs) for sensitivity and specificity by use of random-effects meta-analysis when four or more studies were available.
We identified eight relevant studies from five articles enrolling 7771 patients with suspected tuberculosis in low-income countries. Compared with the standard approach of examination of two smears with Ziehl-Neelsen light microscopy over 2 days, examination of two smears taken on the same day had much the same sensitivity (64% [95% CI 60 to 69] for standard microscopy vs 63% [58 to 68] for same-day microscopy) and specificity (98% [97 to 99] vs 98% [97 to 99]). We noted similar results for studies employing light-emitting diode fluorescence microscopy and for studies examining three smears, whether they were compared with two-smear strategies or with one another.
Same-day sputum smear microscopy is as accurate as standard smear microscopy. Data from tuberculosis programmes are needed to document the changes required in the health system to successfully implement the strategy and understand its effects.
PMCID: PMC3836432  PMID: 23099183
3.  A Transcriptional Signature for Active TB: Have We Found the Needle in the Haystack? 
PLoS Medicine  2013;10(10):e1001539.
Adithya Cattamanchi and colleagues reflect on recent research by Michael Levin and coworkers into the use of whole blood mRNA expression signatures to detect tuberculosis. The authors highlight challenges faced in getting this promising technology into clinics in low-resource settings.
Please see later in the article for the Editors' Summary
PMCID: PMC3805486  PMID: 24167454
4.  Clinical significance of normal chest radiographs among HIV-seropositive patients with suspected tuberculosis in Uganda 
Respirology (Carlton, Vic.)  2011;16(5):836-841.
Background and objectives
The frequency, aetiologies, and outcomes of normal chest radiographs (CXRs) among HIV-seropositive patients with suspected pulmonary tuberculosis (TB) have been infrequently described.
Consecutive HIV-seropositive adults hospitalized for cough of ≥ 2 weeks duration at Mulago Hospital (Kampala, Uganda), between September 2007 and July 2008, were enrolled. Baseline CXRs were obtained on admission. Patients with sputum smears that were negative for acid-fast bacilli (AFB) were referred for bronchoscopy with bronchoalveolar lavage (BAL). BAL fluid was examined for mycobacteria, Pneumocystis jirovecii, and other fungi. Patients were followed for two months after enrolment.
Of the 334 patients, 54 (16%) had normal CXRs. These patients were younger (median age 30 vs. 34 years, P=0.002), had lower counts of CD4+ T lymphocytes (median 13 vs. 57 cells/μL, P<0.001), and were less likely to be smear positive for AFB (17% vs. 39%, P=0.002) than those with abnormal CXRs. Pulmonary TB was the most frequent diagnosis (44%) among those with normal CXRs, followed by unknown diagnoses, pulmonary aspergillosis, and pulmonary cryptococcosis. The frequency of normal CXRs was 12% among pulmonary TB patients. There was a trend towards increased two-month mortality among patients with normal CXRs compared to those with abnormal CXRs (40% vs. 29%, P=0.15).
Normal CXR findings were common among HIV-seropositive patients with suspected TB, especially those who were young, those with low CD4+ T cell counts, and those with sputum smears that were negative for AFB. Mortality was high among those with normal CXRs. Normal CXR findings should not preclude further diagnostic evaluation in this population.
PMCID: PMC3126910  PMID: 21518124
clinical epidemiology; critical care medicine; immunodeficiency; radiology and other imaging; tuberculosis
5.  SecA1 PCR on Sputum or Oral Wash for the Diagnosis of Pulmonary Tuberculosis* 
Nucleic acid amplification tests are sensitive and specific for identifying Mycobacterium tuberculosis in sputum-smear-positive populations, but less sensitive in sputum-smear-negative populations. Few studies have assessed their performance in patients with HIV, and no studies have assessed their performance using oral-wash specimens, which may be easier to obtain than sputum.
We performed a prospective study of 127 adults from two populations undergoing evaluation for respiratory complaints at Mulago Hospital in Kampala, Uganda. We tested sputum and simultaneously collected oral-wash specimens for Mycobacterium tuberculosis DNA by polymerase chain reaction(PCR) amplification at a novel locus, the secA1 gene. A positive sputum mycobacterial culture defined cases of tuberculosis(TB); we calculated sensitivity and specificity of the PCR assay on sputum or oral wash in reference to this gold standard.
TB(75/127, 59%) and HIV(58/126, 46%) were both common in the study population. Sputum PCR was highly sensitive(99%, 95% confidence interval (CI) 93% to 100%) and specific(88%, 95% CI 77% to 96%) for pulmonary TB, and performed well in patients with HIV and in those with negative sputum smears. Oral-wash PCR was less sensitive(73%, 95% CI 62% to 83%) but also detected a substantial proportion of TB cases.
PCR targeting the secA1 gene was highly sensitive and specific for identifying M. tuberculosis in sputum, independent of smear or HIV status. Oral washes showed promise as an easily obtained respiratory specimen for TB diagnosis. SecA1 PCR on sputum could be a rapid, effective diagnostic tool at tuberculosis referral centers.
PMCID: PMC2657807  PMID: 19207077
HIV/AIDS; PCR; secA1 gene; sensitivity and specificity; tuberculosis
6.  Low Tidal Volume Ventilation Is Associated with Reduced Mortality in HIV-infected Patients with Acute Lung Injury 
Thorax  2008;63(11):988-993.
Respiratory failure remains the leading indication for intensive care unit admission and a leading cause of death for HIV-infected patients in spite of overall improvements in ICU mortality. It is unclear if these improvements are due to combination antiretroviral therapy, low tidal-volume ventilation for acute lung injury, or both.
Our aims were to identify therapies and clinical factors associated with mortality in acute lung injury among HIV-infected patients with respiratory failure in the period 1996–2004. A secondary aim was to compare mortality before and after introduction of a low tidal-volume ventilation protocol in 2000.
We performed a retrospective cohort study of 148 consecutive HIV-infected adults admitted to the ICU at San Francisco General Hospital with acute lung injury requiring mechanical ventilation. We abstracted demographic and clinical information, including data on mechanical ventilation, from medical records, and performed multivariate analysis using logistic regression.
In-hospital mortality was similar before and after introduction of a low tidal-volume ventilation protocol, although the study was not powered to exclude a clinically significant difference (Risk Difference −5.4%, 95% Confidence Interval −21% to 11%, p=0.51). Combination antiretroviral therapy was not clearly associated with mortality, except in patients with Pneumocystis pneumonia. Among all those with acute lung injury, lower tidal volume was associated with decreased mortality (Adjusted Odds Ratio 0.76 per 1 mL/kg decrease, 95% Confidence Interval 0.58 − 0.99, p=0.043), after controlling for Pneumocystis pneumonia, serum albumin, illness severity, gas-exchange impairment, and plateau pressure.
Lower tidal volume ventilation was independently associated with reduced mortality in HIV-infected patients with acute lung injury and respiratory failure.
PMCID: PMC2677080  PMID: 18535118
HIV/AIDS; Respiratory Distress Syndrome; Adult; Intensive Care; Tidal Volume; Antiretroviral Therapy; Highly Active
7.  Respiratory Infection Complicating HIV Infection 
Purpose of review
Respiratory infections remain a major cause of morbidity among HIV-infected persons. Thus, an up-to-date knowledge of recent advances regarding HIV-associated opportunistic pneumonias is crucial to the optimal care of persons with HIV.
Recent findings
Bacterial pneumonia is the most common HIV-associated opportunistic pneumonia in the United States and its incidence remains appreciable. Worldwide, tuberculosis (TB) dominates the clinical picture. The absence of rapid, affordable diagnostics for active and latent TB remains a major obstacle that must be overcome for the global epidemic to be slowed. The specter of extensively-drug-resistant (XDR) TB and its overlap with HIV infection highlight the importance of rapid diagnostics and the need for accessible drug susceptibility testing. Pneumocystis pneumonia (PCP) appears to be a more common etiology among HIV-infected persons residing in developing countries than previously appreciated. Similar to TB, the absence of available diagnostics in developing areas is a major obstacle to clinical care and epidemiologic studies. The critical care of HIV-infected persons is challenging.
Although tremendous advances in our understanding of the management, treatment, and prevention of HIV and its associated respiratory infections have been made, significant gaps remain. Thus, continued epidemiologic, clinical, and bench research is needed.
PMCID: PMC2636795  PMID: 18317044
HIV/AIDS; bacterial pneumonia; tuberculosis (TB); Pneumocystis pneumonia (PCP); critical care; AFB; ART; BAL; CFP-10; ESAT-6; ICU; IDU; IPT; IRIS; MDR; MODS; NNRTI; PCP; PCR; PPV; QFT; TB; TMP-SMZ; TIGRA; TST; USPHS/IDSA; XDR
8.  A Minimum Variance Method for Genome-wide Data-driven Normalization of qRT-PCR Expression Data 
Analytical biochemistry  2014;458:11-13.
Advances in multiplex qRT-PCR have enabled increasingly accurate and robust quantification of RNA, even at lower concentrations, facilitating RNA expression profiling in clinical and environmental samples. Here we describe a data-driven qRT-PCR normalization method, the minimum variance method, and evaluate it on clinically derived Mycobacterium tuberculosis samples with variable transcript detection percentages. For moderate to significant amounts of non-detection (~50%), our minimum variance method consistently produces the lowest false discovery rates compared to commonly used data-driven normalization methods.
PMCID: PMC4100614  PMID: 24780223
Multiplex qRT-PCR; Data-driven normalization
9.  Theory-Informed Interventions to Improve the Quality of Tuberculosis Evaluation at Ugandan Health Centers: A Quasi-Experimental Study 
PLoS ONE  2015;10(7):e0132573.
Tuberculosis (TB) remains under-diagnosed in many countries, in part due to poor evaluation practices at health facilities. Theory-informed strategies are needed to improve implementation of TB evaluation guidelines. We aimed to evaluate the impact of performance feedback and same-day smear microscopy on the quality of TB evaluation at 6 health centers in rural Uganda.
We tested components of a multi-faceted intervention to improve adherence to the International Standards for Tuberculosis Care (ISTC): performance feedback and same-day smear microscopy. The strategies were selected based on a qualitative assessment guided by the Theory of Planned Behavior and the PRECEDE model. We collected patient data 6 months before and after the introduction of each intervention component, and compared ISTC adherence in the pre- and post-intervention periods for adults with cough ≥ 2 weeks’ duration.
The performance feedback evaluation included 1,446 adults; 838 (58%) were evaluated during the pre-intervention period and 608 (42%) during the post-intervention period. Performance feedback resulted in a 15% (95%CI +10% to +20%, p<0.001) increase in the proportion of patients receiving ISTC-adherent care. The same-day microscopy evaluation included 1,950 adults; 907 (47%) were evaluated during the pre-intervention period and 1,043 (53%) during the post-intervention period. Same-day microscopy was associated with a 14% (95%CI +10% to +18%, p<0.001) increase in the proportion of patients receiving ISTC-adherent care.
Performance feedback and same-day microscopy should be considered along with ISTC training as part of a multi-faceted intervention to improve the quality of TB evaluation in other high TB burden countries.
PMCID: PMC4501843  PMID: 26172948
10.  A Clinical Predictor Score for 30-Day Mortality among HIV-Infected Adults Hospitalized with Pneumonia in Uganda 
PLoS ONE  2015;10(5):e0126591.
Pneumonia is a major cause of mortality among HIV-infected patients. Pneumonia severity scores are promising tools to assist clinicians in predicting patients’ 30-day mortality, but existing scores were developed in populations infected with neither HIV nor tuberculosis (TB) and include laboratory data that may not be available in resource-limited settings. The objective of this study was to develop a score to predict mortality in HIV-infected adults with pneumonia in TB-endemic, resource-limited settings.
We conducted a secondary analysis of data from a prospective study enrolling HIV-infected adults with cough ≥2 weeks and <6 months and clinically suspected pneumonia admitted to Mulago Hospital in Kampala, Uganda from September 2008 to March 2011. Patients provided two sputum specimens for mycobacteria, and those with Ziehl-Neelsen sputum smears that were negative for mycobacteria underwent bronchoscopy with inspection for Kaposi sarcoma and testing for mycobacteria and fungi, including Pneumocystis jirovecii. A multivariable best subsets regression model was developed, and one point was assigned to each variable in the model to develop a clinical predictor score for 30-day mortality.
Overall, 835 patients were studied (mean age 34 years, 53.4% female, 30-day mortality 18.2%). A four-point clinical predictor score was identified and included heart rate >120 beats/minute, respiratory rate >30 breaths/minute, oxygen saturation <90%, and CD4 cell count <50 cells/mm3. Patients’ 30-day mortality, stratified by score, was: score 0 or 1, 12.6%, score 2 or 3, 23.4%, score 4, 53.9%. For each 1 point change in clinical predictor score, the odds of 30-day mortality increased by 65% (OR 1.65, 95% CI 1.39-1.96, p <0.001).
A simple, four-point scoring system can stratify patients by levels of risk for mortality. Rapid identification of higher risk patients combined with provision of timely and appropriate treatment may improve clinical outcomes. This predictor score should be validated in other resource-limited settings.
PMCID: PMC4427329  PMID: 25962069
11.  Point-of-Care C-Reactive Protein Testing to Facilitate Implementation of Isoniazid Preventive Therapy for People Living with HIV 
Symptom-based tuberculosis screening identifies less than one-third of eligible HIV-infected patients as candidates for isoniazid preventive therapy (IPT). We evaluated whether testing for C-reactive protein (CRP) improves patient selection for IPT.
We measured CRP levels (normal < 10mg/L) using a point-of-care (POC) assay on stored serum samples from HIV-infected Ugandan adults initiating antiretroviral therapy. We assessed diagnostic accuracy in reference to baseline tuberculosis status adjudicated by an expert committee and calculated net reclassification improvement (NRI) to quantify the incremental discriminatory benefit of POC-CRP in determining IPT-eligibility compared to the WHO symptom screen.
Of 201 patients (median CD4 cell-count 137 cells/μL, IQR 83-206), five (2.5%) had tuberculosis. Compared to the WHO symptom screen, POC-CRP had similar sensitivity (100% vs. 80%, p=0.30) but greater specificity (21% vs. 87%, p<0.0001) for tuberculosis. If based on the WHO symptom screen, no patients with tuberculosis but only 42/196 patients without tuberculosis would have been considered IPT-eligible. If POC-CRP were used instead, one patient with tuberculosis (reclassification of cases -20%, p=0.32) and 129 patients without tuberculosis (reclassification of non-cases +66%, p<0.001) would have been reclassified as IPT-eligible, an NRI of 46% (p=0.03). In addition, POC-CRP testing would have reduced the proportion of patients without active tuberculosis requiring confirmatory tuberculosis testing (87% vs. 21%, p<0.0001).
POC-CRP testing increased more than four-fold the proportion of HIV-infected adults immediately identified as IPT-eligible and decreased the proportion of patients requiring referral for further tuberculosis diagnostic testing. POC-CRP testing could substantially improve implementation of tuberculosis screening guidelines.
PMCID: PMC3999220  PMID: 24346636
12.  Procalcitonin Predicts Mortality in HIV-infected Ugandan Adults with Lower Respiratory Tract Infections 
Respirology (Carlton, Vic.)  2014;19(3):382-388.
Background and objective
In low and middle-income countries where HIV infection is prevalent, identifying patients at high risk of dying from lower respiratory tract infections is challenging and validated prognostic models are lacking. Serum procalcitonin may be a useful prognostic tool in these settings. We sought to determine if elevated serum procalcitonin is associated with increased in-hospital mortality and to combine serum procalcitonin with available clinical characteristics to create a clinically useful prognostic model.
We conducted a prospective, nested case-control study of 241 HIV-infected adults admitted to Mulago Hospital in Kampala, Uganda with cough ≥2 weeks in duration. We collected demographic and clinical information, baseline serum for procalcitonin analysis, and followed patients to determine in-hospital mortality.
Serum procalcitonin was a strong and independent predictor of inpatient mortality (aOR=7.69, p=0.01, sensitivity=93%, negative predictive value=97%). Best subset multivariate analysis identified 3 variables that were combined into a prognostic model to risk stratify patients; these variables included respiratory rate ≥30 breaths/minute (aOR=2.07, p=0.11), oxygen saturation <90% (aOR=3.07, p=0.02), and serum procalcitonin >0.5ng/ml (aOR=7.69, p=0.01). The predicted probability of inpatient mortality ranged from 1% when no variables were present, to 42% when all variables were present.
Elevated serum procalcitonin >0.5ng/ml is an independent predictor of in-hospital mortality. Elevated serum procalcitonin, tachypnea, and hypoxemia may be combined into a prognostic model to identify patients at high risk of dying in the hospital. This model may be used to estimate the probability of death and to guide triage and treatment decisions.
PMCID: PMC4276722  PMID: 24460728
HIV; Pneumonia; Procalcitonin; Prognosis; Tuberculosis
13.  Health worker perspectives on barriers to delivery of routine tuberculosis diagnostic evaluation services in Uganda: a qualitative study to guide clinic-based interventions 
Studies of the quality of tuberculosis (TB) diagnostic evaluation of patients in high burden countries have generally shown poor adherence to international or national guidelines. Health worker perspectives on barriers to improving TB diagnostic evaluation are critical for developing clinic-level interventions to improve guideline implementation.
We conducted structured, in-depth interviews with staff at six district-level health centers in Uganda to elicit their perceptions regarding barriers to TB evaluation. Interviews were transcribed, coded with a standardized framework, and analyzed to identify emergent themes. We used thematic analysis to develop a logic model depicting health system and contextual barriers to recommended TB evaluation practices. To identify possible clinic-level interventions to improve TB evaluation, we categorized findings into predisposing, enabling, and reinforcing factors as described by the PRECEDE model, focusing on potentially modifiable behaviors at the clinic-level.
We interviewed 22 health center staff between February 2010 and November 2011. Participants identified key health system barriers hindering TB evaluation, including: stock-outs of drugs/supplies, inadequate space and infrastructure, lack of training, high workload, low staff motivation, and poor coordination of health center services. Contextual barrier challenges to TB evaluation were also reported, including the time and costs borne by patients to seek and complete TB evaluation, poor health literacy, and stigma against patients with TB. These contextual barriers interacted with health system barriers to contribute to sub-standard TB evaluation. Examples of intervention strategies that could address these barriers and are related to PRECEDE model components include: assigned mentors/peer coaching for new staff (targets predisposing factor of low motivation and need for support to conduct job duties); facilitated workshops to implement same day microscopy (targets enabling factor of patient barriers to completing TB evaluation), and recognition/incentives for good TB screening practices (targets low motivation and self-efficacy).
Our findings suggest that health system and contextual barriers work together to impede TB diagnosis at health centers and, if not addressed, could hinder TB case detection efforts. Qualitative research that improves understanding of the barriers facing TB providers is critical to developing targeted interventions to improve TB care.
Electronic supplementary material
The online version of this article (doi:10.1186/s12913-014-0668-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4307676  PMID: 25609495
Tuberculosis; Qualitative research; Diagnosis; Barriers; Provider behavior; PRECEDE
14.  Assessing the Quality of Tuberculosis Evaluation for Children with Prolonged Cough Presenting to Routine Community Health Care Settings in Rural Uganda 
PLoS ONE  2014;9(8):e105935.
Improving childhood tuberculosis (TB) evaluation and care is a global priority, but data on performance at community health centers in TB endemic regions are sparse.
To describe the current practices and quality of TB evaluation for children with cough ≥2 weeks' duration presenting to community health centers in Uganda.
Cross-sectional analysis of children (<15 years) receiving care at five Level IV community health centers in rural Uganda for any reason between 2009–2012. Quality of TB care was assessed using indicators derived from the International Standards of Tuberculosis Care (ISTC).
From 2009–2012, 1713 of 187,601 (0.9%, 95% CI: 0.4–1.4%) children presenting to community health centers had cough ≥ 2 weeks' duration. Of those children, only 299 (17.5%, 95% CI: 15.7–19.3%) were referred for sputum microscopy, but 251 (84%, 95% CI: 79.8–88.1%) completed sputum examination if referred. The yield of sputum microscopy was only 3.6% (95% CI: 1.3–5.9%), and only 55.6% (95% CI: 21.2–86.3%) of children with acid-fast bacilli positive sputum were started on treatment. Children under age 5 were less likely to be referred for sputum examination and to receive care in accordance with ISTC. The proportion of children evaluated in accordance with ISTC increased over time (4.6% in 2009 to 27.9% in 2012, p = 0.03), though this did not result in increased case-detection.
The quality of TB evaluation was poor for children with cough ≥2 weeks' duration presenting for health care. Referrals for sputum smear microscopy and linkage to TB treatment were key gaps in the TB evaluation process, especially for children under the age of five.
PMCID: PMC4149493  PMID: 25170875
15.  Detection of Mycobacterium tuberculosis Peptides in the Exosomes of Patients with Active and Latent M. tuberculosis Infection Using MRM-MS 
PLoS ONE  2014;9(7):e103811.
The identification of easily measured, accurate diagnostic biomarkers for active tuberculosis (TB) will have a significant impact on global TB control efforts. Because of the host and pathogen complexities involved in TB pathogenesis, identifying a single biomarker that is adequately sensitive and specific continues to be a major hurdle. Our previous studies in models of TB demonstrated that exosomes, such as those released from infected macrophages, contain mycobacterial products, including many Mtb proteins. In this report, we describe the development of targeted proteomics assays employing multiplexed multiple reaction monitoring mass spectrometry (MRM-MS) in order to allow us to follow those proteins previously identified by western blot or shotgun mass spectrometry, and enhance biomarker discovery to include detection of Mtb proteins in human serum exosomes. Targeted MRM-MS assays were applied to exosomes isolated from human serum samples obtained from culture-confirmed active TB patients to detect 76 peptides representing 33 unique Mtb proteins. Our studies revealed the first identification of bacteria-derived biomarker candidates of active TB in exosomes from human serum. Twenty of the 33 proteins targeted for detection were found in the exosomes of TB patients, and included multiple peptides from 8 proteins (Antigen 85B, Antigen 85C, Apa, BfrB, GlcB, HspX, KatG, and Mpt64). Interestingly, all of these proteins are known mycobacterial adhesins and/or proteins that contribute to the intracellular survival of Mtb. These proteins will be included as target analytes in future validation studies as they may serve as markers for persistent active and latent Mtb infection. In summary, this work is the first step in identifying a unique and specific panel of Mtb peptide biomarkers encapsulated in exosomes and reveals complex biomarker patterns across a spectrum of TB disease states.
PMCID: PMC4117584  PMID: 25080351
17.  The Lung Microbiome of Ugandan HIV-Infected Pneumonia Patients Is Compositionally and Functionally Distinct from That of San Franciscan Patients 
PLoS ONE  2014;9(4):e95726.
Sub-Saharan Africa represents 69% of the total number of individuals living with HIV infection worldwide and 72% of AIDS deaths globally. Pulmonary infection is a common and frequently fatal complication, though little is known regarding the lower airway microbiome composition of this population. Our objectives were to characterize the lower airway microbiome of Ugandan HIV-infected patients with pneumonia, to determine relationships with demographic, clinical, immunological, and microbiological variables and to compare the composition and predicted metagenome of these communities to a comparable cohort of patients in the US (San Francisco). Bronchoalveolar lavage samples from a cohort of 60 Ugandan HIV-infected patients with acute pneumonia were collected. Amplified 16S ribosomal RNA was profiled and aforementioned relationships examined. Ugandan airway microbiome composition and predicted metagenomic function were compared to US HIV-infected pneumonia patients. Among the most common bacterial pulmonary pathogens, Pseudomonas aeruginosa was most prevalent in the Ugandan cohort. Patients with a richer and more diverse airway microbiome exhibited lower bacterial burden, enrichment of members of the Lachnospiraceae and sulfur-reducing bacteria and reduced expression of TNF-alpha and matrix metalloproteinase-9. Compared to San Franciscan patients, Ugandan airway microbiome was significantly richer, and compositionally distinct with predicted metagenomes that encoded a multitude of distinct pathogenic pathways e.g secretion systems. Ugandan pneumonia-associated airway microbiome is compositionally and functionally distinct from those detected in comparable patients in developed countries, a feature which may contribute to adverse outcomes in this population.
PMCID: PMC3994144  PMID: 24752365
18.  Mobile Digital Fluorescence Microscopy for Diagnosis of Tuberculosis 
Journal of Clinical Microbiology  2013;51(6):1774-1778.
Access to sputum smear microscopy in high-tuberculosis (TB)-burden regions is limited by a scarcity of microscopes and experienced technicians. We evaluated the accuracy of CellScope, a novel digital fluorescence microscope that may expand access to microscopy. The study utilized smear microscopy slides prepared from sputum specimens submitted by consecutive adults with ≥2 weeks of cough who were admitted to Mulago Hospital (Kampala, Uganda). Conventional light-emitting diode (LED) fluorescence microscopy (FM) and mycobacterial culture were performed by experienced technicians. Two U.S.-based postgraduate researchers without prior microscopy experience restained, imaged, and interpreted the slides using CellScope. We assessed whether sensitivity and specificity of CellScope-based LED FM was noninferior to conventional LED FM by using a preselected margin of inferiority of 15%. Of 525 patients included, 72% were HIV seropositive and 39% had culture-confirmed TB. The proportions of positive results were similar with CellScope and conventional LED FM (34% versus 32%, respectively; P = 0.32), and agreement was substantial. CellScope accuracy was within the noninferiority margin for both sensitivity (63% versus 70%; difference, −7%; 95% confidence interval [CI], −13% to −1%) and specificity (85% versus 92%; difference, −7%; 95% CI, −12% to −3%). A subanalysis of 43 slides evaluated by each CellScope reader found substantial interreader reliability (custom-weighted kappa, 0.65) and variable intrareader reliability (custom-weighted kappa, 0.11 versus 0.48). CellScope offers promise for expanding microscopy services. Future studies should evaluate the device when operated by health workers in low-resource settings, the feasibility of image transmission and analysis by experienced microscopists, and the accuracy of automated image analysis algorithms.
PMCID: PMC3716054  PMID: 23554191
19.  Rapid Molecular Testing for TB to Guide Respiratory Isolation in the U.S.: A Cost-Benefit Analysis 
PLoS ONE  2013;8(11):e79669.
Respiratory isolation of inpatients during evaluation for TB is a slow and costly process in low-burden settings. Xpert MTB/RIF (Xpert) is a novel molecular test for tuberculosis (TB) that is faster and more sensitive but substantially more expensive than smear microscopy. No previous studies have examined the costs of molecular testing as a replacement for smear microscopy in this setting.
We conducted an incremental cost–benefit analysis comparing the use of a single negative Xpert versus two negative sputum smears to release consecutive adult inpatients with presumed TB from respiratory isolation at an urban public hospital in the United States. We estimated all health-system costs and patient outcomes related to Xpert implementation, diagnostic evaluation, isolation, hospitalization, and treatment. We performed sensitivity and probabilistic uncertainty analyses to determine at what threshold the Xpert strategy would become cost-saving.
Among a hypothetical cohort of 234 individuals undergoing evaluation for presumed active TB annually, 6.4% had culture-positive TB. Compared to smear microscopy, Xpert reduced isolation bed utilization from an average of 2.7 to 1.4 days per patient, leading to a 48% reduction in total annual isolation bed usage from 632 to 328 bed-days. Xpert saved an average of $2,278 (95% uncertainty range $1582–4570) per admission, or $533,520 per year, compared with smear microscopy.
Molecular testing for TB could provide substantial savings to hospitals in high-income countries by reducing respiratory isolation usage and overall length of stay.
PMCID: PMC3835836  PMID: 24278155
20.  Test and Treat: A New Standard for Smear-Positive Tuberculosis 
Optimizing sputum smear microscopy, the principal method of tuberculosis (TB) diagnosis in high-burden settings around the world, is a critical priority for global TB control. To improve rates of testing, completion, and reporting, the World Health Organization (WHO) recently endorsed a policy of same-day diagnosis of TB by microscopy. Unfortunately, the implementation of this policy has emphasized same-day sputum collection alone, with patients required to return on a subsequent day to collect results. We use a simple decision analysis to demonstrate that the timing of results reporting has a greater impact on treatment initiation for smear-positive TB cases than the timing of specimen collection. Same-day diagnosis of smear-positive TB, including sputum collection and reporting of smear results, should be the new global standard.
PMCID: PMC3427531  PMID: 22918128
Tuberculosis; modeling; decision analysis; smear microscopy; same-day diagnosis; same-day treatment
21.  Population-Level Impact of Same-Day Microscopy and Xpert MTB/RIF for Tuberculosis Diagnosis in Africa 
PLoS ONE  2013;8(8):e70485.
To compare the population-level impact of two World Health Organization-endorsed strategies for improving the diagnosis of tuberculosis (TB): same-day microscopy and Xpert MTB/RIF (Cepheid, USA).
We created a compartmental transmission model of TB in a representative African community, fit to the regional incidence and mortality of TB and HIV. We compared the population-level reduction in TB burden over ten years achievable with implementation over two years of same-day microscopy, Xpert MTB/RIF testing, and the combination of both approaches.
Same-day microscopy averted an estimated 11.0% of TB incidence over ten years (95% uncertainty range, UR: 3.3%–22.5%), and prevented 11.8% of all TB deaths (95% UR: 7.7%–27.1%). Scaling up Xpert MTB/RIF to all centralized laboratories to achieve 75% population coverage had similar impact on incidence (9.3% reduction, 95% UR: 1.9%–21.5%) and greater effect on mortality (23.8% reduction, 95% UR: 8.6%–33.4%). Combining the two strategies (i.e., same-day microscopy plus Xpert MTB/RIF) generated synergistic effects: an 18.7% reduction in incidence (95% UR: 5.6%–39.2%) and 33.1% reduction in TB mortality (95% UR: 18.1%–50.2%). By the end of year ten, combining same-day microscopy and Xpert MTB/RIF could reduce annual TB mortality by 44% relative to the current standard of care.
Scaling up novel diagnostic tests for TB and optimizing existing ones are complementary strategies that, when combined, may have substantial impact on TB epidemics in Africa.
PMCID: PMC3741313  PMID: 23950942
22.  Automated Tuberculosis Diagnosis Using Fluorescence Images from a Mobile Microscope 
In low-resource areas, the most common method of tuberculosis (TB) diagnosis is visual identification of rod-shaped TB bacilli in microscopic images of sputum smears. We present an algorithm for automated TB detection using images from digital microscopes such as CellScope [2], a novel, portable device capable of brightfield and fluorescence microscopy. Automated processing on such platforms could save lives by bringing healthcare to rural areas with limited access to laboratory-based diagnostics. Our algorithm applies morphological operations and template matching with a Gaussian kernel to identify candidate TB-objects. We characterize these objects using Hu moments, geometric and photometric features, and histograms of oriented gradients and then perform support vector machine classification. We test our algorithm on a large set of CellScope images (594 images corresponding to 290 patients) from sputum smears collected at clinics in Uganda. Our object-level classification performance is highly accurate, with Average Precision of 89.2% ± 2.1%. For slide-level classification, our algorithm performs at the level of human readers, demonstrating the potential for making a significant impact on global healthcare.
PMCID: PMC3565532  PMID: 23286149
23.  Low prevalence of Pneumocystis jirovecii lung colonization in Ugandan HIV-infected patients hospitalized with non-Pneumocystis pneumonia 
Pneumocystis jirovecii is an important opportunistic infection in HIV-infected patients. In the developed world, P. jirovecii epidemiology is marked by frequent colonization in immunosuppressed patients, but data on the prevalence of colonization is very limited in sub-Saharan Africa, where the majority of persons living with HIV reside. Our objective was to describe the epidemiology of P. jirovecii colonization among HIV-positive patients in a cross-sectional, hospital-based study of patients admitted with suspected pneumonia in Kampala, Uganda. P. jirovecii was detectable in bronchoalveolar lavage fluid from 7 of 124 (6%) consecutive patients with non-Pneumocystis pneumonia. Colonization was not associated with patient demographic or clinical information. This prevalence is substantially lower than in published studies in the developed world, and suggests that there is a limited reservoir of organisms for clinical infections in this Ugandan population. These findings may partially explain the low incidence of Pneumocystis pneumonia in Uganda and other sub-Saharan African countries.
PMCID: PMC3308345  PMID: 22153850
Pneumocystis jirovecii; colonization; pneumonia; AIDS
24.  Serologic Responses to Recombinant Pneumocystis jirovecii Major Surface Glycoprotein among Ugandan Patients with Respiratory Symptoms 
PLoS ONE  2012;7(12):e51545.
Little is known about the serologic responses to Pneumocystis jirovecii major surface glycoprotein (Msg) antigen in African cohorts, or the IgM responses to Msg in HIV-positive and HIV-negative persons with respiratory symptoms.
We conducted a prospective study of 550 patients, both HIV-positive (n = 467) and HIV-negative (n = 83), hospitalized with cough ≥2 weeks in Kampala, Uganda, to evaluate the association between HIV status, CD4 cell count, and other clinical predictors and antibody responses to P. jirovecii. We utilized ELISA to measure the IgM and IgG serologic responses to three overlapping recombinant fragments that span the P. jirovecii major surface glycoprotein: MsgA (amino terminus), MsgB (middle portion) and MsgC1 (carboxyl terminus), and to three variations of MsgC1 (MsgC3, MsgC8 and MsgC9).
HIV-positive patients demonstrated significantly lower IgM antibody responses to MsgC1, MsgC3, MsgC8 and MsgC9 compared to HIV-negative patients. We found the same pattern of low IgM antibody responses to MsgC1, MsgC3, MsgC8 and MsgC9 among HIV-positive patients with a CD4 cell count <200 cells/µl compared to those with a CD4 cell count ≥200 cells/µl. HIV-positive patients on PCP prophylaxis had significantly lower IgM responses to MsgC3 and MsgC9, and lower IgG responses to MsgA, MsgC1, MsgC3, and MsgC8. In contrast, cigarette smoking was associated with increased IgM antibody responses to MsgC1 and MsgC3 but was not associated with IgG responses. We evaluated IgM and IgG as predictors of mortality. Lower IgM responses to MsgC3 and MsgC8 were both associated with increased in-hospital mortality.
HIV infection and degree of immunosuppression are associated with reduced IgM responses to Msg. In addition, low IgM responses to MsgC3 and MsgC8 are associated with increased mortality.
PMCID: PMC3528778  PMID: 23284710
25.  Low Prevalence of Pneumocystis pneumonia (PCP) but High Prevalence of Pneumocystis dihydropteroate synthase (dhps) Gene Mutations in HIV-Infected Persons in Uganda 
PLoS ONE  2012;7(11):e49991.
Pneumocystis jirovecii pneumonia (PCP) is an important opportunistic infection in patients infected with HIV, but its burden is incompletely characterized in those areas of sub-Saharan Africa where HIV is prevalent. We explored the prevalence of both PCP in HIV-infected adults admitted with pneumonia to a tertiary-care hospital in Uganda and of putative P. jirovecii drug resistance by mutations in fungal dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr). In 129 consecutive patients with sputum smears negative for mycobacteria, 5 (3.9%) were diagnosed with PCP by microscopic examination of Giemsa-stained bronchoalveolar lavage fluid. Concordance was 100% between Giemsa stain and PCR (dhps and dhfr). PCP was more prevalent in patients newly-diagnosed with HIV (11.4%) than in patients with known HIV (1.1%; p = 0.007). Mortality at 2 months after discharge was 29% overall: 28% among PCP-negative patients, and 60% (3 of 5) among PCP-positive patients. In these 5 fungal isolates and an additional 8 from consecutive cases of PCP, all strains harbored mutant dhps haplotypes; all 13 isolates harbored the P57S mutation in dhps, and 3 (23%) also harbored the T55A mutation. No non-synonymous dhfr mutations were detected. PCP is an important cause of pneumonia in patients newly-diagnosed with HIV in Uganda, is associated with high mortality, and putative molecular evidence of drug resistance is prevalent. Given the reliability of field diagnosis in our cohort, future studies in sub-Saharan Africa can investigate the clinical impact of these genotypes.
PMCID: PMC3500344  PMID: 23166805

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