Correlation of Xpert MTB/RIF with measures to assess Mycobacterium tuberculosis bacillary burden in high HIV burden areas of Southern Africa

Traditionally, smear microscopy has been used as a point-of-care measure of bacillary burden in tuberculosis patients to inform infection control and contact tracing. Xpert MTB/RIF has the potential to replace smear. However, data to support the use of its quantitative output [cycle threshold (CT)] as an alternate point-of-care measure of bacillary burden are limited. This study assessed the correlation (Spearman’s) between CT, smear, culture time-to-positivity (TTP), and clinical factors in patients with Xpert-positive sputum from Mozambique (n = 238) and South Africa (n = 462). Mean CT and smear grade correlated well (ρ0.72); compared to TTP and smear (ρ0.61); and mean CT and TTP (ρ0.50). In multivariate analyses, lower CT (higher bacillary load) was associated with negative HIV serostatus and low BMI. A smear positivity rule-out (95% sensitivity) CT cut-off of 28.0 was identified, with 54.1% specificity, 2.07 positive likelihood ratio, 0.09 negative likelihood ratio and 79.0% correctly classified. Cut-offs were higher for HIV positive compared to HIV negative individuals for any set sensitivity level. This study suggests Xpert CT values correlate well with smear, both in HIV positive and negative individuals, and that CT cut-offs might be broadly applicable to multiple settings. Studies to directly assess the association of CT with infectiousness are needed.

less starting material than the last, thereby providing a semi-quantitative result of bacillary burden, with higher C T results reflecting lower bacillary burden.
In-line with the WHO recommendation, 15 countries in one of the high-burden lists (TB, TB-HIV or MDR-TB) have now adopted Xpert as the initial diagnostic test for TB 15 . In these settings, there is a need for an alternative measure of bacillary burden that correlates with smear positivity to help predict transmission risk and disease severity. Xpert C T could potentially be used as such a measure until evidence is available that directly assesses the relationship between baseline Xpert C T and outcomes.
A small number of studies have demonstrated a correlation between TTP and C T results in pulmonary samples, suggesting that an assessment of bacillary burden can be made that is more precise than smear microscopy and cheaper and faster than culture 8,[16][17][18][19] . However, Hanrahan et al. demonstrated that the correlation of TTP and C T is less strong among HIV positive individuals comparted to HIV negative individuals 18 . A recent meta-analysis identified cut-of C T values of 27.7 and 31.8 to predict smear positivity with 85% and 95% sensitivity respectively 20 .
In this study, we aimed to further evaluate the correlation between measures of bacillary burden and identify a cut-point for smear positivity using Xpert C T . We have done so through the inclusion of several study sites in Mozambique and South Africa in the context of a high HIV prevalence with a larger sample size than previous studies. Furthermore, we evaluated the effect of clinical features and symptomatology on the different measures of bacillary burden.

Methods
Study sites and design. The analysis was conducted on anonymised data from participants of larger TB studies conducted in Mozambique and South Africa with sputum samples positive for Xpert MTB/RIF. In Mozambique, consecutive individuals aged 15 or over presenting with possible TB to any of the 10 clinics in the catchment area of Manhiça District Hospital were recruited from August 2013 to August 2014. Manhiça District Hospital is located in a high HIV and TB burden area in Maputo Province, in the south of the country 21 . In South Africa, data from six studies that recruited consecutive patients with presumptive TB from primary care clinics or hospitals were included, four of which have been previously published 17,[22][23][24] . While the district of Manhiça is a semi-rural area in Mozambique, the studies based in South Africa were conducted in the Cape Town metropole, yet both sites are in areas with high TB and HIV prevalence 21,25 .
Sociodemographic and clinical details including HIV status and CD4 count were collected for all participants with TB from standardised questionnaires at the time of diagnosis and clinical records. HIV testing was offered to those without a known result within the last three months.
Informed consent was obtained for all participants. The Mozambique study was approved by the CISM Scientific Committee, the CISM Institutional Review Board and the National Bioethics Committee of Mozambique; the South African studies were approved by the University of Cape Town Faculty of Health Sciences Ethics Committee and the City of Cape Town. All study methods were carried out in accordance with the relevant guidelines and regulations established by the National Bioethics Committees in Mozambique and South Africa.
Data availability statement. The datasets generated during the current study are kept at the data center of CISM. An anonymized version of the dataset can be made available upon request to CISM's Internal Scientific Committee (Email: cci@manhica.net).
Diagnostic tests and measures of bacillary burden. All participants in Mozambique had two samples taken, which were pooled and centrifuged and tested for both smear microscopy (Ziehl Neelsen) and Xpert (MTB/RIF G4 cartridge Cepheid, Sunnyvale, CA). All participants in South Africa had at least one (maximum 3) sample tested using fluorescence microscopy and Xpert. If more than one smear result was available, the highest smear grade was used. Smear microscopy results were graded using the International Union Against Tuberculosis and Lung Disease standard 26 . The mean C T (of positive probes A-E) was recorded for those with positive Xpert results. Liquid culture (BACTEC MGIT 960, BD Diagnostics, Franklin Lakes, NJ) was done, after decontamination with NALC-NaOH, only for those with a positive Xpert result and/or those who had been treated for TB in the past (for a minimum of 4 weeks). TTP (from starting the culture to positive results) was recorded in days.
The sample for this study included only patients with pulmonary samples who had positive Xpert results and an available smear result. Those with rifampicin resistance were excluded as the C T of the probe(s) detecting resistance have values ≥ 4 C T from any other 27 , which could skew the mean C T value of the probes. Those with a C T ≥ 34 for the internal positive control (IPC) -a measure of the presence of inhibitors -were also excluded as this has previously been shown to affect the quantitative ability of Xpert 19 . Data analysis. Data was analysed using Stata 13.1 (StataCorp, College Station, Texas, USA). Descriptive analysis was carried out and differences were assessed between study sites and according to HIV status. The distributions of C T and TTP values were assessed and, as they did not follow a normal distribution, a nonparametric equality of medians test was used to assess differences by HIV status, CD4 count and other clinical factors including symptoms. Differences in smear positivity were assessed using chi-squared. Correlation between TTP, C T and smear grade were assessed using Spearman's ρ with 95% confidence intervals for all participants and by study site and HIV status. In case no statistical differences in the correlation of Xpert C T and smear grade or TTP were observed, estimates for the optimal cut off for smear positivity could be estimated with data from both sites.
Optimum cut-off for smear positivity (considering as all smears scanty to 3+ as positive) was calculated using a receiver operating characteristic (ROC) curve. The cut-off to detect smear positivity with 99%, 95%, 90% and 85% sensitivity (for use as a 'rule-out' cut-off) were derived from the combined data set and evaluated for sensitivity, specificity, negative and positive likelihood ratios and percentage of tests correctly classified overall and according to HIV status. Differing levels of C T cut-offs were also assessed for sensitivity, specificity, negative and SCIENTIFIC REPORTS | (2018) 8:5201 | DOI:10.1038/s41598-018-23066-2 positive likelihood ratios and percentage of tests correctly classified overall and according to HIV status. Rule-in C T cut-offs (95% specificity for smear positivity) were not calculated due to their poor clinical utility -previous studies have demonstrated a high degree of misclassification of smear positives as smear negative 28 . Univariate regression analysis was conducted to identify variables associated with measures of bacillary burden (age, sex, type of sample, history of prior treatment, HIV status, CD4 count, anti-retroviral therapy (ART), body mass index (BMI), presence and duration of constitutional symptoms or cough) and study site. Multivariate regression analysis was conducted including variables with p < 0.10 from univariate analysis. Bidirectional stepwise multivariate regression analysis was also conducted to ensure the validity of this approach.

Results
Baseline characteristics. Seven hundred Xpert positive participants were included -238 (34.0%) from Mozambique and 462 from South Africa. Participant characteristics according to each site are shown in Table 1. Median age was 35 years (IQR 28-44) and 404 (58.1%) were men. A prior history of TB treatment was found in 29.1% of patients. Co-infection with HIV was common (57.2%) with significant immunosuppression -median CD4 was 173 (IQR 69-336).
Symptoms were common: cough was present in 614/697 (88.1%) participants with documented history; fever in 347/592 (58.6%); weight loss in 609/700 (87.0%); and night sweats in 574/700 (82.0%). There were significant variations in symptomatology between study sites (Supplementary Table S1). There were also differences between HIV positive and negative participants. Participants with HIV coinfection were more likely to have productive cough (85.0% vs 76.6%, p 0.010); weight loss (90.5% vs 82.9%, p 0.003); and fatigue (73.7% vs 61.1, p < 0.001); but less likely to have chest pain (45.5% vs 62.3%, p < 0.001).   Table S2). In univariate regression analysis only age, HIV and BMI were significantly associated with smear status, but symptoms of cough, productive cough, fever, weight loss, fatigue and chest pain as well as HIV, CD4, age, a prior history of TB and low BMI were variably associated with C T and TTP. In multivariate regression analyses ( Correlation of measures of bacillary burden. Figure (Table 3).

Measures of bacillary burden.
CT cut-off for smear positivity. Figure 2 shows the ROC curve analysis of C T threshold for smear positivity for overall data (area under the curve of 0.87). From the ROC curve analysis, a C T cut-off value of 28.0 was identified with 95% sensitivity for smear positivity. Analysis according to HIV status found a lower cut-off for HIV negative individuals and higher cut-off for HIV positive individuals. Cut-offs with differing levels of sensitivity are reported in Table 4 (overall and according to HIV status) with associated, specificity, positive and negative likelihood ratios and percentage of correctly classified (with positive and negative predictive values in Supplementary Table S3. These are also presented according to different CT cut-offs between 25 and 30 in Table 5 and Supplementary Table S4. Cut-offs of 31.8 and 27.7 (sensitivity of 95%, specificity of 35% and sensitivity 85%, specificity 67%) identified by the recent meta-analysis of C T predictors of smear positivity 20 , resulted in sensitivity of 98.8%, specificity 22.0% with a cut-off of 31.8 and sensitivity of 94.2% and specificity of 58.3% in this study.

Discussion
This study has added to the evidence that Xpert MTB/RIF C T can be used as a measure of bacillary burden that correlates well with smear grade and moderately with TTP in liquid culture. Given the current recommendation to replace smear with Xpert as the initial diagnostic test for TB and the lack of access to (and delay in obtaining) results from culture in LICs, Xpert may provide the only means for assessing bacillary burden for many countries. This study identified a cut-off of 28 to rule-out smear positivity from studies in high HIV burden settings in Southern Africa. In studies reported by Hanrahan et al. 18 , Theron et al. 28,29 and meta-analysis by Lange et al. 20 , variation of C T values occurs within smear grades and the C T cut-point to rule-out smear positivity varied from 27.7 to 31.8. This may be attributable to a number of factors such as variation in HIV prevalence and differences in type of smear microscopy performed. The sensitivity of any particular cut-off value of mean C T varied according to HIV status, with lower sensitivity for HIV positive individuals. This may be due to the lower bacillary burden in HIV positive individuals as reflected by higher rates of smear negativity, and higher mean C T and TTP values.   Whilst the majority of transmission occurs from individuals with higher smear grades, on which the basis of isolation and contact tracing for smear positive cases has been founded, some transmission does occur from smear negative individuals 30,31 . Factors contributing to this include intensity and duration of exposure, host susceptibility and discordance between sputum and cough aerosol bacillary burden 32 Table 4. Cut-points of mean C T to rule-out smear positivity (with varying degrees of sensitivity). Sensitivity, specificity, positive likelihood ratio (LR), negative LR and percentage of results correctly classified reported according to overall results (n = 700), HIV negative participants (n = 293) and HIV positive participants (n = 391).  categorization of smear grades may also contribute to this and a more precise measure of bacillary burden by Xpert C T could therefore prove useful in more accurately identifying those with the highest risk of transmission at the time of diagnosis to prioritise infection control measures. Until that time, rule-out C T cut-offs have a greater utility than rule-in tests from a public health perspective: while rule-in cut-offs have a high specificity, they have poor sensitivity and therefore misclassify potentially infectious smear positive cases as smear negative 28 . Baseline TTP is a better determinant than smear status of the risk of transmission 9 and response to treatment 4,10,11 but its cost and need for specialist laboratories and training are prohibitive and it is therefore used infrequently in many settings. Exemplifying this is the fact that among the 22 high TB burden countries, there are only 1.8 laboratories able to perform TB culture per 5 million population 12 . Using C T as a same-day measure of bacillary burden that correlates with TTP could provide a useful and more timely surrogate. Unfortunately, C T has been found to have variable correlation with TTP. In some studies, this correlation has been adversely affected by HIV coinfection 18 , likely due to the lower bacillary burden in these individuals. In this study, the correlation between C T and TTP was stronger in HIV positive compared to HIV negative individuals. This could possibly be due to a difference in viable bacilli according to immunosuppression, with potentially a greater number of live bacilli in HIV positive individuals. C T likely correlates more closely with smear given that both methods detect both viable and non-viable organisms compared to culture which only detects viable organisms. This is reflected in the finding that Xpert remains positive beyond culture conversion 33 and patients with previously treated TB are more likely to have Xpert-positive, culture-negative results 34 . Ultimately, studies to directly assess outcomes and transmission based on CT results would be helpful in understanding its utility in clinical practice.

C T cutoff
In seeking to develop understanding of the effect of different clinical factors on bacillary burden by these different methods, this study found that many symptoms of TB were associated with higher bacillary burden, however many of their effects on bacillary burden were not present in multivariate analysis suggesting that other factors contribute to this association, including HIV. In multivariate analysis, the only factors remaining significant for higher bacillary burden were HIV (negative), fever, cough, low BMI and study site. This likely reflects the increased severity of pulmonary disease and therefore a higher sputum bacillary load. Given that fever and BMI were associated with higher bacillary burden as measured by TTP, it may be valuable to consider them as clinical indicators of increased disease severity and transmission. Association of bacillary burden with study site may represent differences in the patient population not accounted for in the analysis (e.g. differences in disease severity or presence of pulmonary cavities. Although in general the diagnostic procedures were the same, there might still be subtle differences in laboratory processing techniques, samples chosen for culture/Xpert or different expertise of lab technologists involved which may also contribute to the findings. As Xpert mean C T was not associated with study site, which may reflect the lower inter-operator variability compared to smear and TTP. Correlations between measures of bacillary burden may also be affected by calibration, laboratory processing techniques or differences in the frequency of non-culturable bacilli between populations due to factors leading to immune system containment such as HIV or TB treatment.
Beyond the potential differences by study site in the diagnostic procedures specified previously, this analysis had several limitations. Both sites have high HIV prevalence and are within Southern Africa, therefore it is possible that the findings are not applicable to other settings. Only patients with positive Xpert results were included; culture was not performed on all individuals in the larger study of patients with suspected TB, a reflection of real-world practice in this context. It was therefore not possible to evaluate the TTP for those who were Xpert negative, however these patients are likely to have lower bacillary burden and therefore present a lower transmission risk. However Xpert negative patients have a lower risk of adverse outcomes 35,36 . The study population therefore reflected a population with higher risk of adverse outcomes, in whom measurements of bacillary burden are likely to be more important.
Another limitation of the study was the exclusion of extra-pulmonary samples due to their very small number in the larger study population. Few studies to date have assessed this and found the correlation to be lower in extra-pulmonary samples, but further larger studies are warranted to assess whether C T is reliable as a measure of bacillary burden in specific types of extrapulmonary sample 16,29 . In addition, transmission is almost exclusively from TB in sputum, reinforcing the greater need to understand bacillary burden in pulmonary samples.
With the introduction of Xpert Ultra recently recommended by WHO 37 , further studies are needed to evaluate the correlation of Xpert Ultra's C T with TTP and smear. However, whilst programmes continue to use Xpert MTB/ RIF these findings remain applicable.

Conclusion
This study suggests that Xpert C T can be used as a measure of bacillary burden and a surrogate for smear status, with the recommended cut-off of 28 to rule-out smear positivity. However there is variation in the recommended cut-off between studies which may reflect differences in clinical features of the population. The exact cut-off point that individual national TB programmes that have replaced smear with Xpert will also depend on the specific desire for sensitivity and specificity. However, until studies have been conducted that directly assess the value of C T with outcomes and transmission of TB, C T values could be viewed as a measure of bacillary burden, with lower C T values representing a higher transmission risk, for programmes that no longer utilise smear microscopy.