This study evaluated the sensitivity of the collection of three GAs in the microbiological confirmation of active pulmonary TB in children in a low prevalence and high-income setting, analysing the diagnostic yield of each sample. The global sensitivity of the combination of microscopy, culture and PCR on three GAs in 156 children with active pulmonary TB was 47.4%. An interesting result of our study is the significant increase in sensitivity by 13.4% determined by the collection of three GAs compared to the first one. In particular, the second and the third aspirate did not significantly increase the sensitivity individually, but cumulatively.
The heterogeneity of the available studies in terms of setting, number of samples collected, and microbiological tests performed allows only a partial comparison with our data. Most studies carried out in high prevalence and low-middle income countries [4, 14, 13] where molecular assays were rarely used, reported a heterogeneous and extremely wide range of sensitivity (1–45%) [16]. Moreover, the majority of the available studies consider the sensitivity of a single GA or the overall sensitivity of three GAs, not distinguishing the diagnostic yield of each sample, and not combining all the microbiological tests available [17–22]. In a previous retrospective study carried out in our centre on 102 children with active pulmonary TB the global sensitivity of the combination of the three GAs was 44.1% [17], resulting comparable to our actual population. A retrospective German study included 454 children who underwent at least two GAs between 2002 and 2010. Culture and PCR were performed, with a global sensitivity of 63%, higher than the sensitivity of 43.6% in our study. This difference might be related to a higher sensitivity of PCR (48% vs. 25.3%) [20]. This difference might be also explained by the age of the German patients, who were mainly below 1 year of age (68% vs. 8% in our population). Children of this age usually have a more aggressive disease leading to a higher probability of microbiological confirmation. In our study, the microbiologically confirmed cases were frequently found among children below one year of age (76.9%) and in those above 13 years (62.1%), who could develop a bacillary disease, similarly to adults. In our population, the collection of three GAs produced a significant increase in sensitivity compared to one GA in the group of children ≤ 4 years but not among children > 4 years, and in the group of patients with uncomplicated TB but not in those with complicated TB [13]. In fact, as the uncomplicated disease is often paucibacillary, it is reasonable to conclude that a higher number of GAs may increase the chance of microbiological confirmation.
In our study, a significant correlation between complicated radiological patterns and microbiological confirmation was found. The association between radiological pattern and global sensitivity of GAs has been also investigated by Kordy et al [19], who reported that miliary disease and hilar lymphadenopathy were prevalent among cases with positive GAs. However, these results are not comparable with our data because of the small sample size of the previous study and the exclusive use of CXR [19].
The sensitivity of culture on each GAs was analysed by a retrospective study in the United States [21]. Of children whose gastric aspirates grew M. tuberculosis, 24/32 (75%) grew it from the first specimen obtained. Second and third GAs increased diagnostic yield of 19% (6/32) and 8% (2/24), respectively [21]. A retrospective study, carried out in Canada from 1999 to 2011, included 202 children who underwent three GAs [19]. The global sensitivity of cultural exams on the three samples was 31.7%, compared to 42.9% of our study. The reason for this difference might rely on the more stringent inclusion criteria used in our study. Of 15 culture-positive patients who underwent exactly 3 GAs, M. tuberculosis was isolated from the first lavage in 10 (67%), only from the second in 3 (20%) and only from the third in 2 (13%) patients. A prospective Spanish pilot study on 17 children reported a global sensitivity of the combination of microscopy and culture of 47%, in agreement with our study [18]. Interestingly, the first GA had a higher sensitivity if compared with our results (41.2% vs. 34.1%), but this difference could not be clearly interpreted due to the small study population [18].
A systematic review of 30 studies, including 11,554 children, found that the sensitivity of culture and Xpert MTB/RIF on different respiratory samples [16] were within the following ranges: 1–30% and 2–17% for IS, 1–45% and 5–51% for GA, and 4–24% and 3–8% for nasopharyngeal aspirate (NPA), respectively. Sampling a second specimen contributed for 6–33% of the cumulative yield and combination of different methods significantly increase the detection yields [16]. A recent meta-analysis on Gene Xpert MTB/RIF Ultra in children confirmed that sensitivity differs by specimen type, with sputum having the highest sensitivity, followed by gastric aspirate [22].
Different type of samples have been recently evaluated, as alternatives to the standard of care. In particular, preliminary data on the diagnostic yield of microbiological analysis on stools seems promising [23, 24]. Moreover, few studies conducted in low-income settings evaluated the possibility to combine different samples type in order to increase the diagnostic yield in young children [25, 26]. In a study conducted in Kenya in 300 children, the average incremental yield for testing up to two of each of GA, NPA or IS specimens by Xpert MTB/RIF or culture resulted to an additional 3 children with positive test results for GA, 2.5 for NPA, and 3 for IS, if comparing to test only one specimen [25]. The combination of minimally invasive techniques such as 2 NPA samples or 1 NPA sample plus 1 stool sample had similar bacteriologic yield to testing 2 GA or 2 IS samples [25]. These data although interesting should be confirmed in larger population. Another option explored in a study conducted in 304 young children in South Africa was the pooling of multiple respiratory specimen types for microbiologic testing [26]. The overall diagnostic yield from pooled GA, NPA, and IS was not different from that of a single GA specimen, suggesting that the GA was probably the main contributor to the diagnostic yield of pooled specimens [26].
The main limitations of our study are the retrospective design and the limited number of children enrolled. However, the highly selective inclusion criteria thus reducing the numerosity of the cohort, allowed to calculate the sensitivity of the three microbiological tests on three GAs in a homogeneous group of children with pulmonary TB. Moreover, the possible limitation of the inter-operator variability in GAs collection is partially overcome by the use of a standardized protocol.
In conclusion, microscopy, culture, and PCR on three sequential GAs represent the gold standard for the microbiological confirmation of paediatric pulmonary TB in a low prevalence setting. The results of this study supports the practice of obtaining three GAs, and this continues to be our institutional practice.
Performing a higher number of GAs might increase the sensitivity, particularly in children ≤ 4 years and in those with an uncomplicated radiological pattern. However, these results need to be confirmed by other studies with a prospective design and with larger population. Similarly, larger studies on the diagnostic yield of different samples combinations are needed in order to find the best association able to reach the highest sensitivity with the less discomfort, especially for younger children.