Increased Sensitivity in Diagnosis of Tuberculosis in HIV-Positive Patients through the Small-Membrane-Filter Method of Microscopy

Abstract

The sensitivity of microscopy for the diagnosis of tuberculosis (TB) is around 50% but decreases by about 15% in patients with suspected TB who are coinfected with HIV. Here, we compared the accuracies of three microscopy methods for processing sputum smears (concentration by centrifugation with or without N-acetyl-L-cysteine [NALC] and concentration by filtration on a polycarbonate membrane) to that of culture on Ogawa-Kudoh medium as the gold standard method. Sputum samples were obtained from 432 patients with suspected pulmonary TB, of whom 60% were infected with HIV. Analysis was performed using the first specimen. Compared to the gold standard culture, the small-membrane-filter (SMF) method was the most sensitive microscopic method. In HIV-infected TB patients, the sensitivity of the SMF method was significantly higher than those for centrifugation of sputum samples with or without NALC treatment (61.9%, 47.6%, and 45.2%, respectively; P ‫؍‬ 0.001). Similarly, in TB patients without HIV infection, the sensitivity of the SMF method was significantly higher than those for centrifugation of sputum samples with or without NALC treatment (81.8%, 63.6%, and 57.5%, respectively; P ‫؍‬ 0.001). In the two study groups, TB patients with or without HIV, no significant differences between the specificities of the three methods were observed. Handling of the second sputum sample similarly by centrifugation with or without NALC and by the SMF method increased positivities by 13%, 11%, and 4%, respectively. The overall agreement between microscopy and culture was above 90% for all groups. Microscopic evaluation of the sputum samples treated with NALC compared to those not treated with NALC did not show any increase in sensitivity. Altogether, the sensitivity of the SMF method is higher than those of the other two microscopic methods studied without a loss of specificity. Diagnosing tuberculosis (TB) in HIV-infected patients still presents a major challenge since clinical and radiological findings are often atypical (1, 2), and many cases of smear-negative pulmonary TB are undiagnosed (3). Sputum smear microscopy, a low-cost method, is important in resource-limited scenarios, where cultures are not available, but it provides a lower sensitivity in HIV-infected patients with suspected TB (4-6).Filtration on a polycarbonate membrane, the small-membrane-filter (SMF) method, was proved to have greater sensitivity and specificity than direct microscopy, even after centrifugation of sputum samples (7,8), but the HIV status of the populations was not reported in those studies.Under the routine conditions of a bacteriology laboratory, we evaluated this method in patients with suspected TB, most of whom were infected with HIV, along with bacilloscopy after centrifugation with or without decontamination. The three methods were compared to culture on solid medium (Ogawa-Kudoh [OK]), considered the gold standard for TB diagnosis in our setting, and their sensitivities, specificities, and Cohen's kappa coefficients were calculated. MAT...

Prospective Cross-Sectional Evaluation of the Small Membrane Filtration Method for Diagnosis of Pulmonary Tuberculosis

“…Our results suggest that the new laboratory SMF protocol, which was developed to minimize the filtration failure rate by diluting the clinical sample, may have compromised the performance of the method in specimens with a low bacterial load (or low volume) where the concentrating capacity of the SMF method would be most useful. Prior studies in both HIV-uninfected and HIV-infected patients with pulmonary TB had shown promising results with previous versions of the SMF method (8,9). In a study of 313 HIVuninfected TB suspects using solid Ogawa culture as the reference standard, the mean sensitivity of the SMF method on the first sputum specimen using either light microscopy (LM) or fluorescence microscopy (FM) was 89% (95% CI, 80 to 94), compared to sensitivities of 60% (95% CI, 49 to 70) for centrifuged smears and 56.1% (95% CI, 40 to 71) for direct smears.…”

“…When samples are ready for processing, vacuum is applied to draw a sample through the metal holders containing a nylon net prefilter (pore size, 30 m; Millipore) and a smaller Isopore membrane filter (pore size, 0.8 m; Millipore). The version of the manifold prototype (version 5.0) evaluated in this study had several design and technical improvements over the manifold used (version 4.0) in a recent study in Brazil (9), including (i) a modification of the neck holder in order to fit the screw caps of 50-ml polypropylene tubes (to minimize crosscontamination), (ii) a modification to the tube holder to allow membrane staining, (iii) replacement of O rings, and (iv) fine adjustment of the rotation key to turn or adjust the holder's set screws.…”

“…The SMF method reduces biohazard potential through the use of bleach (sodium hypochlorite), which kills M. tuberculosis bacilli (7), and a sealed processing system that limits sample manipulation, and thus, the method might be amenable for use in the existing global network of facilities that already use smear microscopy. Two prior studies using earlier, more rudimentary (full manual operation) versions of the vacuum manifold showed promising results for both HIV-uninfected (8) and HIV-infected (9) patients, but the processing failure rates were high, since 5 to 20% of samples failed to fully filter through the membrane.…”

Temporal distribution of tuberculosis in the State of Amazonas, Brazil

“…However, the sensitivity of sputum smear microscopy in HIV patients is low; moreover, additional and time-consuming tests are needed for the early diagnosis of drug resistance. Quincó et al (18) showed that the addition of a polycarbonate membrane filtration technique increased the sensitivity of sputum smear microscopy in HIV-infected patients at the FMT-HVD, achieving 61.9% in the HIV-positive group and 81.8% in the HIV-negative cases.…”

Evaluation of a Modified Small Membrane Filtration Method

“…Variability in these reagents might alter their chemical characteristics, which in turn might affect staining characteristics. No coverslips or antiquenching solution were used in the recent study, unlike the prior two (2,3). Three prior studies have suggested that membrane filtration can substantially improve microscopy diagnosis of TB with simple, low-cost materials.…”