Sputum concentration did not increase the sensitivity of light microscopy for TB diagnosis in this prospective, blinded evaluation of 279 hospitalized, HIV-infected adults in Kampala, Uganda. Moreover, sputum concentration decreased specificity, though this difference was attenuated when clinical response to TB therapy was considered in the gold standard. The performance of concentrated smear microscopy requires further evaluation before recommending universal implementation of this technique.
Our finding that sputum concentration prior to Ziehl-Neelsen staining did not significantly increase sensitivity for TB over direct smear microscopy differs from the results of five prior studies that found higher sensitivity in HIV-endemic populations after sputum concentration [31–35]. This discrepancy may reflect differences in study site (e.g., research versus field setting), patient population (e.g., specially-selected outpatients versus consecutive hospitalized patients), gold standard (e.g., multiple cultures and follow-up data not routinely used versus used), or study methodology (e.g., sodium hypochlorite and sedimentation versus NALC-NaOH and centrifugation). In addition, although many studies employed some form of blinding, it is difficult to blind readers to processing method (direct vs. concentrated) due to the different appearance of unprocessed versus processed slides. This imperfect blinding is known to influence the results of sputum smear microscopy  and would be expected to inflate measured sensitivity for concentrated smear. Although we cannot exclude random chance or weaknesses in our study design as the explanation for our negative results, a number of additional considerations speak to the veracity of our findings. First, our reported 52% sensitivity of concentrated smear is similar to that in previous studies of HIV-infected individuals (i.e., 50% , 52% , and 54% ), suggesting that the discrepancy in findings may reflect higher sensitivity of direct microscopy in the present study, rather than higher sensitivity of concentrated microscopy in other studies. In addition, our study incorporated several features that strengthen its internal validity – consecutive enrollment of a relevant population, clear description of included and excluded patients, blinded reading of index and reference tests by trained non-research staff, and a gold standard which incorporated multiple culture results and follow-up assessment [40, 41]. Thus, while our findings differ from those of previous studies, the present study provides sound evidence that sputum concentration does not universally increase smear sensitivity among HIV-infected TB suspects, and it suggests that provision of high-quality direct microscopy may diminish the benefit of sputum concentration.
In this study, direct and concentrated sputum smear had surprisingly low concordance among patients with culture-confirmed TB (kappa = 0.51), even though both techniques were performed on the same sputum specimen. Although direct and concentrated smear microscopy had similar sensitivities, they detected different patients: 39% (42/109) of all smear-positive patients with culture-confirmed TB were only positive by a single method (Table 2). Although most prior studies [42, 43] have found moderate-to-high concordance between direct and concentrated smear, one earlier study  – also performed using high-speed centrifugation in an African reference lab – found results similar to those presented here (sensitivity of 43% for direct smear, 44% for concentrated smear, and 55% for direct plus concentrated smear). Collection of a single sputum specimen would reduce patient burden in terms of number of visits to health care centers, and could also decrease the delay between clinical presentation and initiation of treatment. Given the similar sensitivity of direct and concentrated smear microscopy also observed in our study, future studies should investigate whether performing multiple direct smears on a single specimen can substantially increase sensitivity for TB, avoiding the need for multiple patient visits.
As with any evaluation of smear microscopy, this study has certain limitations. First, our study was designed to measure a clinically relevant difference between direct and concentrated microscopy and was not powered to demonstrate equivalence. However, our 95% confidence intervals exclude more than a 10% absolute difference in sensitivity between the two methods. Second, this study was conducted in a national reference laboratory using NALC-NaOH on early-morning specimens collected from a population of HIV-infected, hospitalized patients. Thus, our results may not fully generalize to other settings (e.g., peripheral laboratories, laboratories using alternative sputum processing methods, non-HIV populations, and healthier outpatient populations). Estimates of diagnostic performance are known to vary between ambulatory and hospital settings. However, the choice of study population is less likely to impact a comparison between two diagnostic techniques. In addition, given the rigorous training required of microscopists at the Uganda NTRL, it is unlikely that laboratory inexperience explains the results of the present study. Finally, in order to better replicate actual test conditions, internal quality assurance was not performed during the study period. Though we would not expect reliability to differentially affect direct versus concentrated sputum smear results, we were unable to quantify inter-reader and intra-reader agreement.