High yield of culture-based diagnosis in a TB-endemic setting

For years, the World Health Organization (WHO) and the International Union against Tuberculosis and Lung Disease (IUATLD) recommended sputum smear microscopy as the cornerstone to diagnose pulmonary TB, as smear-positive subjects are most contagious [1, 2]. However, since smear microscopy has a detection limit of about 5000–10 000 organisms/mL [3] - compared to 10–100 organisms/mL for culture [4] - specimens with < 5000 bacteria are negative by microscopy.

In settings where microscopy and culture are both done, such as developed countries, approximately half of culture positive specimens have negative smears [5‐8]. Based on this, one anticipates a doubling of case detection if culture methods are introduced into a setting that only does microscopy.

WHO recommends the use of liquid culture in low income settings where feasible [9]. However, other authorities consider the role of culture to be primarily for surveillance and diagnosis of MDR-TB [10]. Little is known on the yield of culture in the context of national TB control programmes in high incidence countries. We have evaluated the relative (RY) and absolute yield (AY) of introducing culture-based diagnosis in a high incidence setting (Cape Town, South Africa).

During the study period, 3742 SE had at least 1 smear and 1 culture done. After excluding the SE which were contaminated, showed the presence of a NTM or lost viability, and after removing the SE with ≥3 smears or cultures, 2537 SE were left for analysis (67.8% of 3742) (Figure 1). These 2537 SE represented 2207 individuals: 62% were male with a median age of 35 years (interquartile range 27–45). Of note, only 1 of the 30 excluded SE with NTM had a mixed infection with M. tuberculosis.

When culture was added to the routine smear-based diagnosis of TB suspects in a high incidence area of South Africa, we found that 29.9% of 2537 SE were culture positive, compared to 20.0% for smear microscopy. The relative yield of culture-based diagnosis was almost double, consistent with the observation that microscopy typically has a sensitivity of about 50% when compared to culture [5‐8]. In this high TB setting, one can obtain an additional TB diagnosis with just 8 cultures, or alternatively by submitting 19 second specimens for microscopy.

Although smear is less sensitive than culture, nevertheless it has excellent PPV in high incidence areas [3]. Our data indicate that while many patients can be detected by microscopy in a high-prevalence setting [22], the addition of culture-based diagnosis can detect many more. Moreover, the present study used the more sensitive fluorescent technique to define a microscopy-positive sample; the incremental yield of culture should be even greater in settings that do only light microscopy. Reviews on the sensitivity of smear compared to culture are based mainly on laboratory-based studies from developed countries, often with no description of the patient population studied [5, 7, 23]. If smears and cultures were performed and read exactly the same way on specimens from clinically similar patients in a low then in high TB prevalence area, we should observe similar sensitivity. However, because of the higher prevalence of positive smear and culture results observed in high endemic areas, the absolute and relative benefit will also increase, while the NND decreases. Paradoxically, countries with the lowest rates of TB typically do both microscopy and culture, while countries with the highest rates of TB often forego culture due to costs and lack of needed infrastructure. Our data supports recent statements by the WHO [9, 24‐27] and the International Standards for TB Care [28] on the use of culture in low income settings, recognizing that for each setting the incremental yield will need to be assessed against the incremental cost of culture-based testing.

In our study, almost 90% of positive cultures were positive on the first specimen, and the IY of the second culture was 10%. A lower yield (77.6%) was observed for the first specimen in smear negative SE compared to smear positive SE. These findings were also reported in studies that performed 3 cultures on patients [23, 29‐31]. However, finding 77% of culture positive patients with the first specimen is still significant and much more informative than a negative smear result. Table 5 compares our results to some other studies that have performed cultures in TB suspects and where results for first and second specimens were reported. The RY of 1C vs 1S was higher (2.7) in a study performed in HIV patients in Thailand and Vietnam [32], most likely because of the lower yield of light microscopy (Ziehl-Nielsen) compared to fluorescent microscopy.

There were several limitations to our study. First, our analysis was done on 68.9% of the 3742 TB suspects with at least 1 smear and 1 culture result. This shows the challenges of obtaining 2 samples per TB suspect in regular programme settings. However, we were able to estimate the proportion of positives for the missing specimens using established formulas [19]. We excluded 15.4% of SE with contaminated culture results from our analysis in order to calculate the yield of first and second specimens accurately . Lower or similar contamination rates have been reported with the MGIT® system: 8.6% [33], 13% [32], 16.9% [34], 16.4% [35]. Despite excluding these contaminated results, we were nevertheless able to analyse a large number of SE. However, in real life, these results have an impact on costs and patient management [34]. Second, the HIV status was available only for treated patients that had consented to HIV testing, or 13.6% of 2537 SE. HIV voluntary counseling and testing (VCT) was offered at the clinic but low rates could be explained by the absence of routinely available antiretroviral therapy at the time of the study. Third, we were not able to compare the yield of the solid vs liquid culture media, as the individual results for these were not available. However, other studies have shown the higher yield of liquid vs solid culture [32‐34]. Finally, some inconsistency rates in smear results were observed. When using all smear positive SE as a denominator, the proportion of SE positive on the first smear was 74.3%, lower than the 85% reported in a meta-analysis [8]. This may be due to the setting of the study in a regular programme. However, the incremental yield of culture over microscopy in this study is not due to false-positive cultures due to laboratory cross-contamination. We have assessed this possibility in another manuscript, using dummy samples, and found that the specificity of culture in this setting is 98.9%, which is comparable to other rates described in the literature [13].

Recently, a novel and simple nucleic-acid amplification test (Xpert® MTB/RIF) has been developed [36] and endorsed by WHO for global implementation [37]. Performing 1 Ziehl–Neelsen smear, 1 Xpert® MTB/RIF and 1 liquid culture on TB suspects yielded 38.4%, 46.2% and 50.1% positive results respectively. Compared to doing 1 smear, the RY, AY and NND would be 1.2, 7.8% and 13 for the Xpert® MTB/RIF and 1.3, 11.7% and 9 for culture [36]. However, as stated by WHO, the Xpert MTB/RIF technology does not eliminate the need for conventional microscopy culture and drug susceptibility testing [37]. The rapidity and simplicity of the method are clear advantages. However, sensitivity in smear negative patients remains an issue, with some reported sensitivities of 43.4% [38], 54.5% [39] and 72.5% [36]. Furthermore, drug susceptibility testing is still required to detect resistance to drugs other than rifampicin [37]. As the Xpert technique is implemented, initial studies show it can potentially be cost-effective compared to sputum smear diagnosis and clinical diagnosis in smear-negative cases [40]. Future studies could evaluate the cost-effectiveness of smear versus Xpert, as compared with smear versus culture, or even the integration of two of these tests. Depending on the prevalence of positive samples by new diagnostic techniques, the NND can be determined for either test, in order to predict the incremental gain in case detection, and ultimately, the impact on patient care.

In conclusion, in a high TB burden setting, the use of culture methods can almost double the number of microbiologically-confirmed cases of TB, with just under 8 cultures required to detect 1 additional case.

Yield calculations (adapted from Rieder et al. [19]).There are 6 possible patterns among those who had a diagnostic examination (Ad) in a setting where 3 specimens are required: Px, NPx, NNP, NN, NN9 and N99, where P is a positive smear, x a subsequent result of no interest, N a negative result, and 9 a missing result. For our study, since we aimed to collect 2 specimens and since less than 10% of SE had ≥ 3 smears or 3 cultures done, we kept 4 patterns (Px, NP, NN and N9) and adapted the formulas accordingly.To calculate the incremental yields, the assumption was made that those with an N9 result had the same probability of being positive on the second smear as those with an NP result. Those (positive) missed by failing to do a second smear examination will be denoted as Md (Table 6).