Samuel Yoo

Sensitivity of direct versus concentrated sputum smear microscopy in HIV-infected patients suspected of having pulmonary tuberculosis.

BackgroundSputum concentration increases the sensitivity of smear microscopy for the diagnosis of tuberculosis (TB), but few studies have investigated this method in human immunodeficiency virus (HIV)-infected individuals.MethodsWe performed a prospective, blinded evaluation of direct and concentrated Ziehl-Neelsen smear microscopy on a single early-morning sputum sample in HIV-infected patients with > 2 weeks of cough hospitalized in Kampala, Uganda. Direct and concentrated smear results were compared with results of Lowenstein-Jensen culture.ResultsOf 279 participants, 170 (61%) had culture-confirmed TB. The sensitivity of direct and concentrated smear microscopy was not significantly different (51% vs. 52%, difference 1%, 95% confidence interval (CI): [-7%, 10%], p = 0.88). However, when results of both direct and concentrated smears were considered together, sensitivity was significantly increased compared with either method alone (64%, 95% CI: [56%, 72%], p < 0.01 for both comparisons) and was similar to that of direct smear results from consecutive (spot and early-morning) specimens (64% vs. 63%, difference 1%, 95% CI: [-6%, 8%], p = 0.85). Among 109 patients with negative cultures, one had a positive direct smear and 12 had positive concentrated smears (specificity 99% vs. 89%, difference 10%, 95% CI: [2%, 18%], p = 0.003). Of these 13 patients, 5 (38%) had improved on TB therapy after two months.ConclusionSputum concentration did not increase the sensitivity of light microscopy for TB diagnosis in this HIV-infected population. Given the resource requirements for sputum concentration, additional studies using maximal blinding, high-quality direct microscopy, and a rigorous gold standard should be conducted before universally recommending this technique.

Clinical and Radiographic Factors Do Not Accurately Diagnose Smear-Negative Tuberculosis in HIV-infected Inpatients in Uganda: A Cross-Sectional Study

Background Although World Health Organization guidelines recommend clinical judgment and chest radiography for diagnosing tuberculosis in HIV-infected adults with unexplained cough and negative sputum smears for acid-fast bacilli, the diagnostic performance of this approach is unknown. Therefore, we sought to assess the accuracy of symptoms, physical signs, and radiographic findings for diagnosing tuberculosis in this population in a low-income country with a high incidence of tuberculosis. Methodology We performed a cross-sectional study enrolling consecutive HIV-infected inpatients with unexplained cough and negative sputum smears for acid-fast bacilli at Mulago Hospital in Kampala, Uganda. Trained medical officers prospectively collected data on standard symptoms and signs of systemic respiratory illness, and two radiologists interpreted chest radiographs in a standardized fashion. We calculated positive- and negative-likelihood ratios of these factors for diagnosing pulmonary tuberculosis (defined when mycobacterial cultures of sputum or bronchoalveolar lavage fluid were positive). We used both conventional and novel regression techniques to develop multivariable prediction models for pulmonary tuberculosis. Principal Findings Among 202 enrolled HIV-infected adults with negative sputum smears for acid-fast bacilli, 72 (36%) had culture-positive pulmonary tuberculosis. No single factor, including respiratory symptoms, physical findings, CD4+ T-cell count, or chest radiographic abnormalities, substantially increased or decreased the likelihood of pulmonary tuberculosis. After exhaustive testing, we were also unable to identify any combination of factors which reliably predicted bacteriologically confirmed tuberculosis. Conclusions and Significance Clinical and radiographic criteria did not help diagnose smear-negative pulmonary tuberculosis among HIV-infected patients with unexplained cough in a low-income setting. Enhanced diagnostic methods for smear-negative tuberculosis are urgently needed.

Integrated Strategies to Optimize Sputum Smear Microscopy: A Prospective Observational Study

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.

Role of interferon-gamma release assays in the diagnosis of pulmonary tuberculosis in patients with advanced HIV infection

BackgroundT-cell interferon-gamma release assays (IGRAs) may have a role in the diagnosis of active tuberculosis when evaluating patients for whom standard microbiology has limited sensitivity. Our objective was to examine the accuracy of a commercial IGRA for diagnosis of active tuberculosis in HIV-infected persons.MethodsWe enrolled HIV-infected patients admitted to Mulago Hospital in Kampala, Uganda with cough ≥ 2 weeks. All patients underwent standard medical evaluation. We collected peripheral blood specimens at enrollment and performed a commercial, ELISPOT-based IGRA according to the manufacturers recommendations. IGRA sensitivity and specificity were determined using mycobacterial culture results as the reference standard.ResultsOverall, 236 patients were enrolled. The median CD4+ T-lymphocyte count was 49 cells/μl and 126 (53%) patients were diagnosed with active pulmonary tuberculosis. IGRAs were not performed in 24 (10%) patients due to insufficient mononuclear cell counts. In the remaining 212 patients, results were indeterminate in 54 (25%). IGRAs were positive in 95 of 158 (60%) patients with interpretable results. The proportion of positive test results was similar across CD4+ count strata. IGRA sensitivity was 73% and specificity 54%. IGRA results did not meaningfully alter the probability of active tuberculosis in patients with negative sputum smears.ConclusionsAn ELISPOT-based IGRA detected a high prevalence of latent tuberculosis infection in a hospitalized population of tuberculosis suspects with advanced HIV/AIDS but had limited utility for diagnosis of active tuberculosis in a high prevalence setting. Further research is needed to identify stronger and more specific immune responses in patients with active tuberculosis.

Poor Performance of Universal Sample Processing Method for Diagnosis of Pulmonary Tuberculosis by Smear Microscopy and Culture in Uganda

ABSTRACT Laboratory methods to improve smear microscopy are an urgent priority for global tuberculosis control. The novel universal sample processing (USP) method has been reported to improve conventional diagnostic testing for tuberculosis while also providing inhibitor-free specimens for molecular assays. However, no studies evaluating the method in the field have been conducted. In this study, we compared the performance of the USP method to that of the standard N-acetyl-l-cysteine-NaOH (NALC) method for conventional diagnosis of tuberculosis in 252 adults admitted to Mulago Hospital in Kampala, Uganda, with a clinical suspicion of pneumonia. A single early-morning sputum specimen collected from each patient was divided into two aliquots, each of which was assigned a random identification number. One randomly numbered specimen was processed by the USP method and the other by the NALC method. Mycobacterial cultures were more frequently negative in USP compared to NALC specimen aliquots (58% versus 43%; P < 0.001). There was no difference in the proportion of contaminated mycobacterial cultures (12% versus 11%; P = 0.87). The sensitivity and specificity of smear microscopy for the USP method were 52% and 86%, respectively, and were not significantly different from those for the NALC method (56% and 86%, respectively) using mycobacterial culture results as a reference standard. These results suggest that the USP method did not provide any significant advantage over the standard NALC method for conventional diagnosis of tuberculosis in our setting and illustrate the importance of well-designed, field-level evaluations of novel diagnostic techniques.

Prevalence and outcomes of cryptococcal antigenemia in HIV-seropositive patients hospitalized for suspected tuberculosis in Uganda.

Background:Cryptococcal infection occurs in HIV-seropositive patients and is associated with high mortality. However, limited information is available on the prevalence and outcomes of cryptococcal antigenemia among hospitalized HIV-seropositive patients in sub-Saharan Africa. Objectives:To determine the prevalence of and risk factors for cryptococcal antigenemia among HIV-seropositive patients presenting to Mulago Hospital (Kampala, Uganda) with unexplained cough ≥2 weeks and suspected tuberculosis (TB) and also to determine if antigenemia is associated with an increased mortality. Methods:Between September 2009 and September 2010, we enrolled consecutive HIV-seropositive adults hospitalized at Mulago Hospital with cough ≥2 weeks and suspected TB. Banked serum was tested for cryptococcal antigen. We compared demographic and clinical characteristics, and 2-month mortality in patients with and without cryptococcal antigenemia. Results:Of 563 HIV-seropositive patients, 32 (5.7%) were cryptococcal antigen (CrAg) positive. None had Cryptococcus neoformans detected on fungal culture of bronchoalveolar lavage fluid (n = 116). CrAg-positive patients had a lower median CD4 count compared with CrAg-negative patients (25 vs. 55 cells/&mgr;L, P = 0.02), and a substantial proportion of CrAg-positive patients also had concurrent TB (31%). A positive CrAg test was not associated with increased mortality during the 2-month follow-up period (hazard ratio: 0.99, 95% confidence interval: 0.63 to 1.54, P = 0.95) after adjusting for CD4 count and antiretroviral therapy use at enrollment and/or follow-up. Conclusions:Occult cryptococcal antigenemia occurs commonly among hospitalized HIV-seropositive patients with suspected TB. CrAg testing should be considered in hospitalized HIV-seropositive patients with CD4 count <50 cells/&mgr;L, coupled with longer follow-up to evaluate the diagnostic value of CrAg and therapeutic interventions in patients with asymptomatic cryptococcal antigenemia.

Bronchoscopy is useful for diagnosing smear-negative tuberculosis in HIV-infected patients

To the Editors: Tuberculosis (TB) is the leading cause of morbidity and mortality in HIV-infected patients in sub-Saharan Africa 1, in part because limited availability of diagnostic tests hinders early, directed treatment. Studies have demonstrated a substantial yield of bronchoscopy for diagnosing HIV-associated opportunistic pulmonary diseases, but few studies have explicitly considered whether bronchoscopy adds to the sensitivity of sputum culture in identifying Mycobacterium tuberculosis , or whether bronchoscopy shortens the time needed to diagnose TB. Although bronchoscopy is unavailable in many HIV and TB endemic settings, where it is available its usefulness for TB diagnosis is uncertain. Thus, we examined the performance of bronchoscopy to diagnose TB and other pulmonary diseases in HIV-infected inpatients with cough in Kampala, Uganda. We performed a prospective cross-sectional study enrolling consecutive HIV-infected patients aged ≥18 yrs hospitalised at Mulago Hospital with cough of ≥2 weeks but <6 months duration. After providing informed consent, patients underwent a standard evaluation including chest radiography, sputum acid-fast bacillus (AFB) microscopy and bronchoscopy with bronchoalveolar lavage (BAL) if they were AFB smear-negative, according to previously described protocols 2. Trained technicians examined BAL by smear and/or culture for mycobacteria, Pneumocystis jirovecii , and other fungi. Specific pneumonia treatment was recorded. Patients were seen at a 2-month follow-up visit, after which a pulmonologist and a medical officer assigned final diagnoses based on all diagnostic information and according to a standardised protocol. A final diagnosis of pulmonary TB was based on a positive sputum mycobacterial culture (using Lowenstein–Jensen media), positive BAL AFB smear or BAL mycobacterial culture, or a clinical response to TB treatment at the 2-month follow-up visit. Between September 2007 and July 2008, 107 (55%) out of 193 patients successfully underwent bronchoscopy with BAL. Among the 86 patients who did not undergo bronchoscopy, 24 patients had an …

Performance of Modified Universal Sample Processing Method in a Field Study in Uganda

We developed the universal sample processing (USP) methodology and demonstrated its excellent performance in several validation studies using blinded samples (1-4). An article titled “Poor performance of Universal Sample Processing Method for Diagnosis of Pulmonary Tuberculosis by Smear Microscopy and Culture in Uganda” was published recently in the Journal of Clinical Microbiology (5). In this study, smear sensitivities and specificities were not significantly different in a modified USP protocol that was used and the standard N-acetyl-l-cysteine-NaOH (NALC-NaOH) method. The authors also noted that mycobacterial cultures were more frequently negative in the former method. This in itself is not entirely surprising since it is well known that even established diagnostic techniques, not to mention new and novel methodologies, vary widely in performance from very poor to excellent depending on the study settings. It is most important that new diagnostic techniques are evaluated in well-designed studies, and we thank Cattamanchi and coworkers for carrying out an independent assessment of USP methodology in Uganda (1). However, we wish to highlight some concerns that we have about the misrepresentation of “USP methodology.” While referring to it as the “USP method,” the authors have actually used a modified version of the USP protocol. Based on a careful examination of their findings, we believe that the “poor” performance of the USP method is likely to be a consequence of using a modified USP protocol. Critical aspects of the method were altered. First, bacteria were sedimented at a lower centrifugation speed (3,000 × g instead of the recommended 5,000 to 6,000 × g). This is likely to be a crucial modification since the USP protocol advocates a higher centrifugation speed. Second, the authors do not mention how much of the processed sample was applied; while the NALC-NaOH method advocates the use of only two loopfuls, the USP method prescribes the application of 10% of the processed sample to the slide (2-5). Third, the use of fluorescence microscopy (which is not advocated by USP methodology) likely increased the sensitivity of the NALC-NaOH method without enhancing the detection of acid-fast bacilli on USP slides that are remarkably free of background interference. Fourth, culture sensitivity could be compromised by inefficient bacterial sedimentation, incomplete removal of guanidinium hydrochloride (GuHCl), or the use of 4 to 6 M GuHCl as opposed to the 4 M GuHCl recommended for optimal culture sensitivity. Finally, our previous validation studies were carried out under laboratory conditions, where the finer technicalities of the USP method were strictly adhered to. These stringent procedures might not have been possible in this first field application of the “modified USP” method. The authors mention that the results of the USP smear method were not significantly different from those of the NALC-NaOH method but contradict this statement by defining its performance as “poor.” They also noted that compared to the NALC-NaOH method, the USP method yielded a greater proportion of scanty positive results in support of its stated efficacy in detecting paucibacillary specimens. On this basis and in light of the modifications in the protocol, the title “poor performance” is not justified and the authors should refer to it as “modified USP method.”