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1.  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.
doi:10.1371/journal.pone.0095726
PMCID: PMC3994144  PMID: 24752365
2.  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
3.  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.
doi:10.1128/JCM.03432-12
PMCID: PMC3716054  PMID: 23554191
4.  LPS-Binding Protein and IL-6 Mark Paradoxical Tuberculosis Immune Reconstitution Inflammatory Syndrome in HIV Patients 
PLoS ONE  2013;8(11):e81856.
Background
Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) remains a poorly understood complication in HIV-TB co-infected patients initiating antiretroviral therapy (ART). The role of the innate immune system in TB-IRIS is becoming increasingly apparent, however the potential involvement in TB-IRIS of a leaky gut and proteins that interfere with TLR stimulation by binding PAMPs has not been investigated before. Here we aimed to investigate the innate nature of the cytokine response in TB-IRIS and to identify novel potential biomarkers.
Methods
From a large prospective cohort of HIV-TB co-infected patients receiving TB treatment, we compared 40 patients who developed TB-IRIS during the first month of ART with 40 patients matched for age, sex and baseline CD4 count who did not. We analyzed plasma levels of lipopolysaccharide (LPS)-binding protein (LBP), LPS, sCD14, endotoxin-core antibody, intestinal fatty acid-binding protein (I-FABP) and 18 pro-and anti-inflammatory cytokines before and during ART.
Results
We observed lower baseline levels of IL-6 (p = 0.041), GCSF (p = 0.036) and LBP (p = 0.016) in TB-IRIS patients. At IRIS event, we detected higher levels of LBP, IL-1RA, IL-4, IL-6, IL-7, IL-8, G-CSF (p ≤ 0.032) and lower I-FABP levels (p = 0.013) compared to HIV-TB co-infected controls. Only IL-6 showed an independent effect in multivariate models containing significant cytokines from pre-ART (p = 0.039) and during TB-IRIS (p = 0.034).
Conclusion
We report pre-ART IL-6 and LBP levels as well as IL-6, LBP and I-FABP levels during IRIS-event as potential biomarkers in TB-IRIS. Our results show no evidence of the possible contribution of a leaky gut to TB-IRIS and indicate that IL-6 holds a distinct role in the disturbed innate cytokine profile before and during TB-IRIS. Future clinical studies should investigate the importance and clinical relevance of these markers for the diagnosis and treatment of TB-IRIS.
doi:10.1371/journal.pone.0081856
PMCID: PMC3842977  PMID: 24312369
5.  Vitamin D deficiency among adult patients with tuberculosis: a cross sectional study from a national referral hospital in Uganda 
BMC Research Notes  2013;6:293.
Background
Vitamin D deficiency has been reported among patients with tuberculosis in Africa despite abundant sunshine. Vitamin D plays a fundamental role in improving anti tuberculosis immunity, reducing progression and severity of TB in humans.
Methods
In this descriptive cross sectional study, 260 hospitalized adults with a confirmed diagnosis of TB were enrolled into the study from the pulmonology wards of Mulago national referral and teaching hospital, Uganda. The serum concentrations of 25-hydroxyvitamin D or 25 (OH) D were determined by an electrochemilumniscence immunoassay. Vitamin D deficiency, vitamin D insufficiency, severe and very severe vitamin D deficiency were defined as serum 25(OH) D concentrations of ≤ 20 ng/ml, 21–29 ng/ml, < 10 ng/ml and <5 ng/ml respectively.
Results
Majority of the study participants were males (146, 56.2%) and < 35 years (154, 59.2%). The mean age ± SD was 34.7 ± 9.5 years. Two hundred eight (80%) patients were HIV co-infected with a median CD4 count of 68 cells/mm3 (IQR: 17–165). The prevalence of vitamin D deficiency, vitamin D insufficiency, severe and very severe vitamin D deficiency among the hospitalized adult tuberculosis patients was 44.2%, 23.5%, 13.5% and 4.2% respectively. The median (IQR) vitamin D concentration in ng/ml was 22.55 (14.59-33.31).
Vitamin D deficiency was more prevalent in patients with hypoalbuminemia (97.4%), anemia (86.1%), HIV co-infected patients with CD4 count <200cells/mm3 (83.2%) and hypocalcemia corrected for serum albumin levels (67%).
Conclusion
Vitamin D deficiency is very common among hospitalized adult tuberculosis patients in Uganda especially in patients with hypoalbuminemia, anemia, HIV co-infected patients with CD4 count <200cells/mm3 and hypocalcemia corrected for serum albumin levels.
doi:10.1186/1756-0500-6-293
PMCID: PMC3737052  PMID: 23886009
Vitamin D deficiency; Tuberculosis; Uganda
6.  Overt diabetes mellitus among newly diagnosed Ugandan tuberculosis patients: a cross sectional study 
BMC Infectious Diseases  2013;13:122.
Background
There is a documented increase of diabetes mellitus in Sub Saharan Africa, a region where tuberculosis is highly endemic. Currently, diabetes mellitus is one of the recognised risk factors of tuberculosis. No study has reported the magnitude of diabetes mellitus among tuberculosis patients in Uganda, one of the countries with a high burden of tuberculosis.
Methods
This was a cross-sectional study conducted among 260 consenting adult patients with a confirmed diagnosis of tuberculosis admitted on the pulmonology wards of Mulago national referral and teaching hospital in Kampala, Uganda to determine the prevalence of diabetes mellitus and associated clinical factors. Laboratory findings as well as the socio-demographic and clinical data collected using a validated questionnaire was obtained. Point of care random blood sugar (RBS) testing was performed on all the patients prior to initiation of anti tuberculosis treatment. Diabetes mellitus was diagnosed if the RBS level was ≥ 200mg/dl in the presence of the classical symptoms of diabetes mellitus.
Results
The prevalence of diabetes mellitus among the admitted patients with tuberculosis was 8.5%. Only 5 (1.9%) patients with TB had a known diagnosis of diabetes mellitus at enrolment. Majority of the study participants with TB-DM co-infection had type 2 diabetes mellitus (n=20, 90.9%).
At bivariate analysis, raised mean ALT concentrations of ≥80 U/L were associated with DM (OR-6.1, 95% CI 1.4-26.36, p=0.032) and paradoxically, HIV co-infection was protective of DM (OR-0.32, 95% CI 0.13-0.79, P=0.016). The relationship between DM and HIV as well as that with ALT remained statistically significant at multivariate analysis (HIV: OR- 0.17 95%CI 0.06-0.51, p=0.002 and ALT: OR-11.42 95%CI 2.15-60.59, p=0.004).
Conclusion
This study demonstrates that diabetes mellitus is common among hospitalized tuberculosis patients in Uganda. The significant clinical predictors associated with diabetes mellitus among tuberculosis patients were HIV co-infection and raised mean serum alanine transaminase concentrations.
doi:10.1186/1471-2334-13-122
PMCID: PMC3599954  PMID: 23497232
7.  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.
doi:10.1016/j.diagmicrobio.2011.10.009
PMCID: PMC3308345  PMID: 22153850
Pneumocystis jirovecii; colonization; pneumonia; AIDS
8.  Serologic Responses to Recombinant Pneumocystis jirovecii Major Surface Glycoprotein among Ugandan Patients with Respiratory Symptoms 
PLoS ONE  2012;7(12):e51545.
Background
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.
Methods
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).
Results
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.
Conclusions
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.
doi:10.1371/journal.pone.0051545
PMCID: PMC3528778  PMID: 23284710
9.  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.
doi:10.1371/journal.pone.0049991
PMCID: PMC3500344  PMID: 23166805
10.  Impact of Xpert MTB/RIF Testing on Tuberculosis Management and Outcomes in Hospitalized Patients in Uganda 
PLoS ONE  2012;7(11):e48599.
Rationale
The clinical impact of Xpert MTB/RIF for tuberculosis (TB) diagnosis in high HIV-prevalence settings is unknown.
Objective
To determine the diagnostic accuracy and impact of Xpert MTB/RIF among high-risk TB suspects.
Methods
We prospectively enrolled consecutive, hospitalized, Ugandan TB suspects in two phases: baseline phase in which Xpert MTB/RIF results were not reported to clinicians and an implementation phase in which results were reported. We determined the diagnostic accuracy of Xpert MTB/RIF in reference to culture (solid and liquid) and compared patient outcomes by study phase.
Results
477 patients were included (baseline phase 287, implementation phase 190). Xpert MTB/RIF had high sensitivity (187/237, 79%, 95% CI: 73–84%) and specificity (190/199, 96%, 95% CI: 92–98%) for culture-positive TB overall, but sensitivity was lower (34/81, 42%, 95% CI: 31–54%) among smear-negative TB cases. Xpert MTB/RIF reduced median days-to-TB detection for all TB cases (1 [IQR 0–26] vs. 0 [IQR 0–1], p<0.001), and for smear-negative TB (35 [IQR 22–55] vs. 22 [IQR 0–33], p = 0.001). However, median days-to-TB treatment was similar for all TB cases (1 [IQR 0–5] vs. 0 [IQR 0–2], p = 0.06) and for smear-negative TB (7 [IQR 3–53] vs. 6 [IQR 1–61], p = 0.78). Two-month mortality was also similar between study phases among 252 TB cases (17% vs. 14%, difference +3%, 95% CI: −21% to +27%, p = 0.80), and among 87 smear-negative TB cases (28% vs. 22%, difference +6%, 95% CI: −34 to +46%, p = 0.77).
Conclusions
Xpert MTB/RIF facilitated more accurate and earlier TB diagnosis, leading to a higher proportion of TB suspects with a confirmed TB diagnosis prior to hospital discharge in a high HIV/low MDR TB prevalence setting. However, our study did not detect a decrease in two-month mortality following implementation of Xpert MTB/RIF possibly because of insufficient powering, differences in empiric TB treatment rates, and disease severity between study phases.
doi:10.1371/journal.pone.0048599
PMCID: PMC3490868  PMID: 23139799
11.  Performance of Frontloading for Smear Microscopy in the Diagnosis of Pulmonary Tuberculosis: A Cross-Sectional Study at a Referral Hospital in Uganda 
PLoS ONE  2012;7(10):e48531.
Objective
To compare the performance of frontloading and the standard WHO method for diagnosis of pulmonary TB at Mulago Hospital in order to validate the technique in this setting.
Methods
This was a cross-sectional study in which 229 adult (≥18 years) TB suspects were consecutively enrolled. Suspects submitted three sputum samples as follows: at initial presentation, one hour after the first sample, and the next morning. The first and next morning samples formed the standard WHO method, while the first and the one hour later samples formed the frontloading method. Sample processing was by the standard N-acetyl L-cystein (NALC)-NaOH method, and fluorescent microscopy was done for both methods, while cultures of the first sample on Lowenstein-Jensen slants acted as a gold standard. The sensitivity, specificity and predictive values for the WHO standard and frontloading methods were compared.
Results
The sensitivity of both the frontloading and standard schemes was 91.1% while their specificities were 86.2% and 91.7% respectively. There was excellent agreement between the diagnostic capacity of the two methods (kappa statistic = 0.87, P<0.0001). The positive predictive value for the frontloading scheme was 87.2% and that for the standard approach was 91.9%, while the negative predictive values were 90.4% and 90.9%, respectively. Among the HIV positive patients, frontloading identified 59/79 (74.7%) culture positive samples while the standard approach identified 55/79 (69.6%). In the HIV sero-negative category, on the other hand, front-loading identified 48/110 (43.6%) culture positive samples compared to 45/110 (40.9%) by the standard approach.
Conclusion
Frontloading based on smear examination of two same-day sputum samples has a similar performance to the current standard method and would not be associated with any significant missed diagnosis. It may therefore be advocated for use in our setting so as to reduce time to completion of diagnosis and patient loss to follow-up.
doi:10.1371/journal.pone.0048531
PMCID: PMC3483226  PMID: 23144768
12.  Evaluation of in-house PCR for diagnosis of smear-negative pulmonary tuberculosis in Kampala, Uganda 
BMC Research Notes  2012;5:487.
Background
Nucleic acid amplification tests (NAATs) have offered hope for rapid diagnosis of tuberculosis (TB). However, their efficiency with smear-negative samples has not been widely studied in low income settings. Here, we evaluated in-house PCR assay for diagnosis of smear-negative TB using Lowenstein-Jensen (LJ) culture as the baseline test. Two hundred and five pulmonary TB (PTB) suspects with smear-negative sputum samples, admitted on a short stay emergency ward at Mulago Hospital in Kampala, Uganda, were enrolled. Two smear-negative sputum samples were obtained from each PTB suspect and processed simultaneously for identification of MTBC using in-house PCR and LJ culture.
Results
Seventy two PTB suspects (35%, 72/205) were LJ culture positive while 128 (62.4%, 128/205) were PCR-positive. The sensitivity and specificity of in-house PCR for diagnosis of smear-negative PTB were 75% (95% CI 62.6-85.0) and 35.9% (95% CI 27.2-45.3), respectively. The positive and negative predictive values were 39% (95% CI 30.4-48.2) and 72.4% (95% CI 59.1-83.3), respectively, while the positive and negative likelihood ratios were 1.17 (95% CI 0.96-1.42) and 0.70 (95% CI 0.43-1.14), respectively. One hundred and seventeen LJ culture-negative suspects (75 PCR-positive and 42 PCR-negative) were enrolled for follow-up at 2 months. Of the PCR-positive suspects, 45 (60%, 45/75) were still alive, of whom 29 (64.4%, 29/45) returned for the follow-up visit; 15 (20%, 15/75) suspects died while another 15 (20%, 15/75) were lost to follow-up. Of the 42 PCR-negative suspects, 22 (52.4%, 22/42) were still alive, of whom 16 (72.7%, 16/22) returned for follow-up; 11 (26.2%, 11/42) died while nine (21.4%, 9/42) were lost to follow-up. Overall, more PCR-positive suspects were diagnosed with PTB during follow-up visits but the difference was not statistically significant (27.6%, 8/29 vs. 25%, 4/16, p = 0.9239). Furthermore, mortality was higher for the PCR-negative suspects but the difference was also not statistically significant (26.2% vs. 20% p = 0.7094).
Conclusion
In-house PCR correlates poorly with LJ culture for diagnosis of smear-negative PTB. Therefore, in-house PCR may not be adopted as an alternative to LJ culture.
doi:10.1186/1756-0500-5-487
PMCID: PMC3497582  PMID: 22947399
Pulmonary tuberculosis; Smear-negative TB; HIV-infected; HIV-TB co-infection; CD4 cell counts; Nucleic acid amplification tests; In-house PCR; Lowenstein-Jensen culture; Sensitivity; Specificity; Resource limited settings
13.  Oral Antimicrobial Rinse to Reduce Mycobacterial Culture Contamination among Tuberculosis Suspects in Uganda: A Prospective Study 
PLoS ONE  2012;7(7):e38888.
Rationale
Contamination by bacterial or fungal organisms reduces the effectiveness of mycobacterial culture for diagnosis of pulmonary tuberculosis (TB). We evaluated the effect of an anti-microbial and an anti-fungal oral rinse prior to expectoration on culture-contamination rates.
Methods
We enrolled a consecutive random sample of adults with cough for ≥2 weeks and suspected TB admitted to Mulago Hospital (Kampala, Uganda) between October 2008 and June 2009. We randomly assigned patients to oral rinse (60 seconds with chlorhexidine followed by 60 seconds with nystatin) vs. no oral rinse prior to initial sputum collection. Uganda National Tuberculosis Reference Laboratory technicians blinded to the method of sputum collection (with or without oral rinse) processed all sputum specimens for smear microscopy (direct Ziehl-Neelsen) and mycobacterial culture (Lowenstein-Jensen media).
Results
Of 220 patients enrolled, 177 (80%) were HIV-seropositive (median CD4-count 37 cells/uL, IQR 13–171 cells/uL). Baseline characteristics were similar between patients in the oral-rinse (N = 110) and no oral-rinse (N = 110) groups. The proportion of contaminated cultures was significantly lower in the oral-rinse group compared to the no oral-rinse group (4% vs. 15%, risk difference −11%, 95% CI −18 to −3%, p = 0.005). Oral rinse significantly reduced the proportion of contaminated cultures among HIV-infected patients (3% vs. 18%, risk difference −14%, 95% CI −23 to −6%, p = 0.002) but not HIV-uninfected (6% vs. 4%, risk difference 2%, 95% CI −12 to +15%, p = 0.81) patients. However, the proportion of smear-positive specimens (25% vs. 35%, p = 0.10) and culture-positive specimens (48% vs. 56%, p = 0.24) were lower in the oral-rinse compared to the no oral-rinse group, although the differences were not statistically significant.
Conclusions
Oral rinse prior to sputum expectoration is a promising strategy to reduce mycobacterial culture contamination in areas with high HIV prevalence, if strategies can be devised to reduce the adverse impact of oral rinse on smear- and culture-positivity.
doi:10.1371/journal.pone.0038888
PMCID: PMC3395623  PMID: 22808020
14.  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.
Methods
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.
Results
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).
Conclusions
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.
doi:10.1111/j.1440-1843.2011.01981.x
PMCID: PMC3126910  PMID: 21518124
clinical epidemiology; critical care medicine; immunodeficiency; radiology and other imaging; tuberculosis
15.  Bronchoalveolar Lavage Enzyme-Linked Immunospot for Diagnosis of Smear-Negative Tuberculosis in HIV-Infected Patients 
PLoS ONE  2012;7(6):e39838.
Background
Peripheral blood interferon-gamma release assays (IGRAs) have sub-optimal sensitivity and specificity for diagnosis of active pulmonary tuberculosis (TB). However, assessment of local immune responses has been reported to improve the accuracy of TB diagnosis.
Methods
We enrolled HIV-infected adults with cough ≥2 weeks’ duration admitted to Mulago Hospital in Kampala, Uganda and referred for bronchoscopy following two negative sputum acid-fast bacillus smears. We performed an ELISPOT-based IGRA (T-SPOT.TB®, Oxford Immunotec, Oxford, UK) using peripheral blood and bronchoalveolar lavage (BAL) fluid mononuclear cells, and determined the accuracy of IGRAs using mycobacterial culture results as a reference standard.
Results
94 HIV-infected patients with paired peripheral blood and BAL IGRA results were included. The study population was young (median age 34 years [IQR 28–40 years]) and had advanced HIV/AIDS (median CD4+ T-lymphocyte count 60 cells/µl [IQR 22–200 cells/µl]). The proportion of indeterminate IGRA results was higher in BAL fluid than in peripheral blood specimens (34% vs. 14%, difference 20%, 95% CI 7–33%, p = 0.002). BAL IGRA had moderate sensitivity (73%, 95% CI 50–89%) but poor specificity (48%, 95% CI 32–64%) for TB diagnosis. Sensitivity was similar (75%, 95% CI 57–89%) and specificity was higher (78%, 95% CI 63–88%) when IGRA was performed on peripheral blood.
Conclusions
BAL IGRA performed poorly for the diagnosis of smear-negative TB in a high HIV/TB burden setting. Further studies are needed to examine reasons for the large proportion of indeterminate results and low specificity of BAL IGRA for active TB in high HIV/TB burden settings.
doi:10.1371/journal.pone.0039838
PMCID: PMC3383728  PMID: 22745833
16.  HIV-Associated Pneumocystis Pneumonia 
During the past 30 years, major advances have been made in our understanding of HIV/AIDS and Pneumocystis pneumonia (PCP), but significant gaps remain. Pneumocystis is classified as a fungus and is host-species specific, but an understanding of its reservoir, mode of transmission, and pathogenesis is incomplete. PCP remains a frequent AIDS-defining diagnosis and is a frequent opportunistic pneumonia in the United States and in Europe, but comparable epidemiologic data from other areas of the world that are burdened with HIV/AIDS are limited. Pneumocystis cannot be cultured, and bronchoscopy with bronchoalveolar lavage is the gold standard procedure to diagnose PCP, but noninvasive diagnostic tests and biomarkers show promise that must be validated. Trimethoprim-sulfamethoxazole is the recommended first-line treatment and prophylaxis regimen, but putative trimethoprim-sulfamethoxazole drug resistance is an emerging concern. The International HIV-associated Opportunistic Pneumonias (IHOP) study was established to address these knowledge gaps. This review describes recent advances in the pathogenesis, epidemiology, diagnosis, and management of HIV-associated PCP and ongoing areas of clinical and translational research that are part of the IHOP study and the Longitudinal Studies of HIV-associated Lung Infections and Complications (Lung HIV).
doi:10.1513/pats.201009-062WR
PMCID: PMC3132788  PMID: 21653531
acquired immune deficiency syndrome; HIV; Pneumocystis; Pneumocystis pneumonia; dihydropteroate synthase
17.  Integrated Strategies to Optimize Sputum Smear Microscopy 
Rationale: Smear-positive tuberculosis (TB) case detection rates are far below targets in most low-income countries. The standard approach to smear microscopy involves sputum collection over multiple days and examination of sputum smears by light microscopy (LM), an insensitive and time-consuming technique.
Objective: To determine whether two alternative approaches can increase smear-positive case detection by increasing the efficiency (single-specimen microscopy) or sensitivity (light-emitting diode [LED] fluorescence microscopy [FM]) of TB suspect evaluation.
Methods: We enrolled patients with cough of 2 weeks or more admitted to Mulago Hospital in Kampala, Uganda and collected spot and early morning sputum specimens. We compared the diagnostic accuracy of four prespecified strategies based on the number of sputum specimens collected (one specimen versus two specimens) and the type of microscopy (LM versus LED FM) using mycobacterial culture as a reference standard.
Measurements and Main Results: Two hundred thirty-three of 464 (50%) patients had culture-positive TB. There was no difference in sensitivity between single-specimen and two-specimen strategies when smears were examined with LM (55 vs. 56%; difference, −1%; 95% confidence interval [CI], −5 to +2%) or LED FM (61 vs. 64%; difference, −3%; 95% CI, −7 to +1%). LED FM was more sensitive than LM with both the single-specimen (61 vs. 55%; difference, 6%; 95% CI, 2–10%) and two-specimen strategies (64 vs. 56%; difference, 8%; 95% CI, 3–12%). Findings were similar among the HIV-infected patient subset (n = 321 patients).
Conclusions: In low-income, high TB burden settings, single-specimen microscopy and LED FM, either alone or in combination, could considerably increase identification of smear-positive TB cases.
doi:10.1164/rccm.201008-1207OC
PMCID: PMC3056227  PMID: 20851925
tuberculosis; diagnosis; smear-positive case detection
18.  Causes of Early Mortality in HIV-Infected TB Suspects in an East African Referral Hospital 
Background
Respiratory infections are a leading cause of death in Africa, especially among HIV-infected patients. Data on the etiology of fatal respiratory diseases are largely based on autopsy studies. We evaluated causes of pneumonia associated with early mortality among hospitalized HIV-infected patients in Kampala, Uganda.
Methods
Prospective cohort study of HIV-infected patients admitted to Mulago Hospital, Kampala, with at least 2 weeks of cough. Consecutively enrolled patients with negative Ziehl Neelsen sputum smears for acid-fast bacilli underwent bronchoscopy with bronchoalveolar lavage and examination for mycobacteria (smear, solid culture), Pneumocystis jirovecii (Giemsa stain), and fungi (KOH mount, India ink stain, Sabouraud culture). Early mortality was defined as death before the 2-month follow-up visit.
Results
Follow-up data were available for 353 (87%) of 407 patients enrolled. Of participants with follow-up data, 112 (32%) died within 2 months. Among patients with early mortality, a diagnosis was confirmed in 74 (66%), including tuberculosis (TB) (56%), cryptococcal pneumonia (1%), Pneumocystis pneumonia (3%), pulmonary Kaposi sarcoma (4%), and pneumonia caused by 2 or more disease processes (3%).
Conclusions
Mortality in HIV-infected TB suspects is high, with TB associated with the largest proportion of deaths. A significant proportion of patients die without a confirmed diagnosis.
PMCID: PMC3249444  PMID: 21105258
HIV; tuberculosis; mortality; hospital admission; Africa
19.  The Role of Speciation in Positive Lowenstein-Jensen Culture Isolates from a High Tuberculosis Burden Country 
PLoS ONE  2011;6(11):e27017.
Objective
To determine the need for routine speciation of positive Lowenstein-Jensen mycobacterial cultures in HIV-infected patients suspected of having pulmonary tuberculosis at Mulago Hospital in Kampala, Uganda.
Methods
Sputum and bronchoalveolar lavage Lowenstein-Jensen mycobacterial culture isolates from consecutive, HIV-infected patients admitted to Mulago Hospital with 2 weeks or more of cough were subjected to IS6110 PCR and rpoB genetic analysis to determine the presence of Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM).
Results
Eighty (100%) mycobacterial cultures from 65 patients were confirmed to be members of MTBC. Subsequent analysis of the cultures from 54 patients by PCR and sequence analyses to identify co-infection with NTM confirmed the presence of MTBC as well as the presence of Micrococcus luteus (n = 4), Janibacter spp. (n = 1) and six cultures had organisms that could not be identified.
Conclusions
Presumptive diagnosis of tuberculosis on the basis of a positive Lowenstein-Jensen culture is sufficient in HIV-infected Ugandans suspected of having tuberculosis. Routine molecular confirmation of positive Lowenstein-Jensen cultures is unnecessary in this low resource setting.
doi:10.1371/journal.pone.0027017
PMCID: PMC3208568  PMID: 22073241
21.  The prevalence and clinical course of HIV-associated pulmonary cryptococcosis in Uganda 
Background
The prevalence and clinical course of pulmonary cryptococcosis in Sub-Saharan Africa are not well-described.
Methods
Consecutive HIV-infected adults hospitalized at Mulago Hospital (Kampala, Uganda) between September 2007 and July 2008 with cough ≥ 2 weeks were enrolled. Patients with negative sputum smears for acid-fast bacilli were referred for bronchoscopy with bronchoalveolar lavage (BAL). BAL fluid was examined for mycobacteria, Pneumocystis jirovecii, and fungi. Patients were followed two and six months after hospital discharge.
Results
Of 407 patients enrolled, 132 (32%) underwent bronchoscopy. Of 132 BAL fungal cultures, 15 (11%) grew Cryptococcus neoformans. None of the patients were suspected to have pulmonary cryptococcosis on admission. The median CD4 count among those with pulmonary cryptococcosis was 23 cells/µL (IQR 7–51). Of 13 patients who completed six-month follow-up, four died and nine were improved, including five who had started antiretroviral therapy (ART) but had not received antifungal medication.
Conclusions
Pulmonary cryptococcosis is common in HIV-infected TB suspects in Uganda. Early initiation of ART in those with isolated pulmonary infection may improve outcomes, even without anti-fungal therapy. This finding suggests that some HIV-infected patients with C. neoformans isolated from respiratory samples may have colonization or localized infection.
doi:10.1097/QAI.0b013e3181ce6b19
PMCID: PMC2892206  PMID: 20150818
Pulmonary Cryptococcosis; HIV/AIDS; Bronchoscopy
22.  Modified Giemsa method for confirmation of Pneumocystis pneumonia in low-income countries 
BMJ Case Reports  2009;2009:bcr02.2009.1580.
An HIV-seropositive man who presented with cough, low-grade fever and difficulty in breathing was admitted to the pulmonology unit at Mulago Hospital in Kampala, Uganda. He was initially diagnosed with bacterial pneumonia and treated with ceftriaxone without significant improvement. Induced sputum and bronchoalveolar lavage specimens were collected and stained using a modified Giemsa stain (Diff-Quik). With this technique, it was possible to demonstrate cystic and trophic forms of Pneumocystis jirovecii and confirm the diagnosis of Pneumocystis pneumonia in this patient.
doi:10.1136/bcr.02.2009.1580
PMCID: PMC3028160  PMID: 21691388
23.  Feasibility, diagnostic accuracy, and effectiveness of decentralised use of the Xpert MTB/RIF test for diagnosis of tuberculosis and multidrug resistance: a multicentre implementation study 
Lancet  2011;377(9776):1495-1505.
Summary
Background
The Xpert MTB/RIF test (Cepheid, Sunnyvale, CA, USA) can detect tuberculosis and its multidrug-resistant form with very high sensitivity and specificity in controlled studies, but no performance data exist from district and subdistrict health facilities in tuberculosis-endemic countries. We aimed to assess operational feasibility, accuracy, and effectiveness of implementation in such settings.
Methods
We assessed adults (≥18 years) with suspected tuberculosis or multidrug-resistant tuberculosis consecutively presenting with cough lasting at least 2 weeks to urban health centres in South Africa, Peru, and India, drug-resistance screening facilities in Azerbaijan and the Philippines, and an emergency room in Uganda. Patients were excluded from the main analyses if their second sputum sample was collected more than 1 week after the first sample, or if no valid reference standard or MTB/RIF test was available. We compared one-off direct MTB/RIF testing in nine microscopy laboratories adjacent to study sites with 2–3 sputum smears and 1–3 cultures, dependent on site, and drug-susceptibility testing. We assessed indicators of robustness including indeterminate rate and between-site performance, and compared time to detection, reporting, and treatment, and patient dropouts for the techniques used.
Findings
We enrolled 6648 participants between Aug 11, 2009, and June 26, 2010. One-off MTB/RIF testing detected 933 (90·3%) of 1033 culture-confirmed cases of tuberculosis, compared with 699 (67·1%) of 1041 for microscopy. MTB/RIF test sensitivity was 76·9% in smear-negative, culture-positive patients (296 of 385 samples), and 99·0% specific (2846 of 2876 non-tuberculosis samples). MTB/RIF test sensitivity for rifampicin resistance was 94·4% (236 of 250) and specificity was 98·3% (796 of 810). Unlike microscopy, MTB/RIF test sensitivity was not significantly lower in patients with HIV co-infection. Median time to detection of tuberculosis for the MTB/RIF test was 0 days (IQR 0–1), compared with 1 day (0–1) for microscopy, 30 days (23–43) for solid culture, and 16 days (13–21) for liquid culture. Median time to detection of resistance was 20 days (10–26) for line-probe assay and 106 days (30–124) for conventional drug-susceptibility testing. Use of the MTB/RIF test reduced median time to treatment for smear-negative tuberculosis from 56 days (39–81) to 5 days (2–8). The indeterminate rate of MTB/RIF testing was 2·4% (126 of 5321 samples) compared with 4·6% (441 of 9690) for cultures.
Interpretation
The MTB/RIF test can effectively be used in low-resource settings to simplify patients' access to early and accurate diagnosis, thereby potentially decreasing morbidity associated with diagnostic delay, dropout and mistreatment.
Funding
Foundation for Innovative New Diagnostics, Bill & Melinda Gates Foundation, European and Developing Countries Clinical Trials Partnership (TA2007.40200.009), Wellcome Trust (085251/B/08/Z), and UK Department for International Development.
doi:10.1016/S0140-6736(11)60438-8
PMCID: PMC3085933  PMID: 21507477
24.  Nucleic Acid Amplification Tests for Diagnosis of Smear-Negative TB in a High HIV-Prevalence Setting: A Prospective Cohort Study 
PLoS ONE  2011;6(1):e16321.
Background
Nucleic acid amplification tests are sensitive for identifying Mycobacterium tuberculosis in populations with positive sputum smears for acid-fast bacilli, but less sensitive in sputum-smear-negative populations. Few studies have evaluated the clinical impact of these tests in low-income countries with high burdens of TB and HIV.
Methods
We prospectively enrolled 211 consecutive adults with cough ≥2 weeks and negative sputum smears at Mulago Hospital in Kampala, Uganda. We tested a single early-morning sputum specimen for Mycobacterium tuberculosis DNA using two nucleic acid amplification tests: a novel in-house polymerase chain reaction targeting the mycobacterial secA1 gene, and the commercial Amplified® Mycobacterium tuberculosis Direct (MTD) test (Gen-Probe Inc, San Diego, CA). We calculated the diagnostic accuracy of these index tests in reference to a primary microbiologic gold standard (positive mycobacterial culture of sputum or bronchoalveolar lavage fluid), and measured their likely clinical impact on additional tuberculosis cases detected among those not prescribed initial TB treatment.
Results
Of 211 patients enrolled, 170 (81%) were HIV-seropositive, with median CD4+ T-cell count 78 cells/µL (interquartile range 29-203). Among HIV-seropositive patients, 94 (55%) reported taking co-trimoxazole prophylaxis and 29 (17%) reported taking antiretroviral therapy. Seventy-five patients (36%) had culture-confirmed TB. Sensitivity of MTD was 39% (95% CI 28–51) and that of secA1 was 24% (95% CI 15–35). Both tests had specificities of 95% (95% CI 90–98). The MTD test correctly identified 18 (24%) TB patients not treated at discharge and led to a 72% relative increase in the smear-negative case detection rate.
Conclusions
The secA1 and MTD nucleic acid amplification tests had moderate sensitivity and high specificity for TB in a predominantly HIV-seropositive population with negative sputum smears. Although newer, more sensitive nucleic acid assays may enhance detection of Mycobacterium tuberculosis in sputum, even currently available tests can provide substantial clinical impact in smear-negative populations.
doi:10.1371/journal.pone.0016321
PMCID: PMC3029312  PMID: 21298001
25.  Antiretroviral Treatment-Associated Tuberculosis in a Prospective Cohort of HIV-Infected Patients Starting ART 
Commencement of antiretroviral treatment (ART) in severely immunosuppressed HIV-infected persons is associated with unmasking of subclinical disease. The subset of patients that are diagnosed with tuberculosis (TB) disease while on ART have been classified as ART-associated TB. Few studies have reported the incidence of ART-associated TB and unmasking TB-IRIS according to the International Network for the Study of HIV-Associated IRIS (INSHI) consensus definition. To determine the incidence and predictors of ART-associated TB, we screened 219 patients commencing ART at the Infectious Diseases Clinic in Kampala, Uganda for TB by symptoms, sputum microscopy, and chest X-rays and followed them for one year. Fourteen (6.4%) patients were diagnosed with TB during followup. Eight (3.8%) patients had ART-associated TB (incidence rate of 4.3 per 100 person years); of these, three patients fulfilled INSHI criteria for unmasking TB-associated IRIS (incidence rate of 1.6 per 100 person years). A body mass index of less than 18.5 kg/m2 BMI (HR 5.85 95% CI 1.24–27.46, P = .025) and a C-reactive protein greater than 5 mg/L (HR 8.23 95% CI 1.36–38.33, P = .020) were risk factors for ART-associated TB at multivariate analysis. In conclusion, with systematic TB screening (including culture and chest X-ray), the incidence of ART-associated TB is relatively low in settings with high HIV and TB prevalence.
doi:10.1155/2011/758350
PMCID: PMC3004409  PMID: 21197091

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