The effect of sputum quality and volume on the yield of bacteriologically-confirmed TB by Xpert MTB/RIF and smear

Introduction The World Health Organization endorsed (2010) the use of Xpert MTB/RIF and countries are shifting from smear microscopy (smear)-based to Xpert MTB/RIF-based tuberculosis (TB) diagnostic algorithms. As with smear, sputum quality may predict the likelihood of obtaining a bacteriologically-confirmed TB when using Xpert MTB/RIF. Methods From 08/12-11/2014, all people living with HIV were recruited at 22 clinics. For patients screened positive using the four TB symptoms their sputa were tested by Xpert MTB/RIF and smear. Laboratorians assessed and recorded sputum appearance and volume. The yield of bacteriologically-positive sputum evaluated using Xpert MTB/RIF and smear, likelihood-ratios were calculated. Results Among 6,041 patients enrolled 2,296 were presumptive TB, 1,305 (56.8%) had > 1 sputa collected and 644/1,305 (49.3%) had both Xpert MTB/RIF and smear results. Since >1 sputa collected from 644 patients 954 sputa were tested by Xpert MTB/RIF and smear. Bacteriologically-positive sputum was two-fold higher with Xpert MTB/RIF 11.4% versus smear 5.3%, p < 0.001. Sputum appearance and quantity were not predictive of bacteriologically-positive results, except volume of 2ml to < 3ml, tested by Xpert MTB/RIF LR+= 1.26 (95% CI, 1.05–1.50). Conclusion Xpert MTB/RIF test yield to bacteriologically-positive sputum was superior to smear. Sputum quality and quantity, however, were not consistently predictive of bacteriologically-positive results by Xpert MTB/RIF or smear.


Introduction
After endorsement by the World Health Organization (WHO) in 2010, over 145 countries implemented the Xpert MTB/RIF assay by 2016 [1]. With such an increased capacity many countries are shifting from a smear microscopy (smear)-to an Xpert MTB/RIF -based tuberculosis (TB) diagnostic algorithm. In 2011, the Botswana Ministry of Health and Wellness adopted WHO guidelines and incorporated Xpert MTB/RIF into the national TB diagnostic algorithm [2]. Optimal performance of Xpert MTB/RIF relies on the quality of the sputum samples submitted for testing [3]. The WHO quality control standard underscores collection of quality sputum, and salivary samples were considered suboptimal and thus prone to rejection by testing laboratories [4]. The minimum required raw sputum sample for smear is 3 -5 ml [5] compared to 1ml for Xpert MTB/RIF per the Cepheid manufacturer (Cepheid, Sunnyvale, CA, USA) recommendation [6,7].

Yoon et al. and Bhat et
al.demonstrated that sputum gross appearance (quality) and volume (quantity) were associated with smear positivity [8,9]. Similar to smear result, sputum quality and quantity may have an impact on the likelihood of obtaining a positive result when using Xpert MTB/RIF [3].
While national TB programs are expanding implementation of Xpert MTB/RIF [1], focus on collection of quality sputum with adequate volume has not been given priority [3]. Data on the effect of sputum quality and quantity on the yield of bacteriologically-confirmed TB using molecular tests such as Xpert MTB/RIF are scarce [3,10]. We

Study population
This is a sub-study of the Xpert MTB/RIF Package Rollout Evaluation  (Table 2).

Association between sputum appearance and TB diagnosis
Of 954 tested sputum samples, 43.3% were classified as salivary. The   [1]. Such investments and their ultimate impact will potentially be compromised if TB diagnostic algorithms do not encompass collection of quality sputum [10]. A wide variability in the proportion of patients with salivary sample (24-70%) among clinics ( Figure 2) suggest an inconsistency in sputum collection practices across the clinics. Bhat et al have shown an association of improved sputum quality and diagnostic yield [9]. It is time that TB screening and diagnostic algorithms include standardized methods of sputum collection and introduce a sputum collection system less prone to variability as suggested by Ho et al. [3].
In some previous reports, improving sputum quality increased TB diagnostic yield [18,[20][21][22][23][24] [24]. In addition, training of health care workers and laboratorians on standardized methods of sputum collection and assessment of adequate good quality sputum can improve sputum quality [9] and measuring a volume in millilitres using a graded reference container can facilitate appropriate volume collection [9].
With these various methods, achieving improved quality and quantity of sputum were possible but well-designed studies are still needed to define a more comprehensive approach and standard.
While endeavouring to standardize sputum collection methods, under current clinical and laboratory practices, at least five reasons stand out about why salivary sputum should not be rejected: (1) a high percentage of salivary sputum are still being collected in clinical practice by health workers [10]; (2) sputum specimen appearance and volume are poor "negative predictors" of MTB in sputum [8,18]; (3) a limited volume (only 1ml) of non-concentrated sputum maybe acceptable for Xpert MTB/RIF testing [6,7]; (4) recent study demonstrate higher sensitivity of Xpert MTB/RIF in salivary samples than mucoid [19] and (5) above all, to minimize any missed opportunities for TB diagnosis [25]. Sputum rejection criteria, that consider salivary sample as unsuitable for testing should be reconsidered, particularly when using Xpert MTB/RIF testing [3].