Does multiple gastric aspirate collection increase sensitivity of M. tuberculosis detection in children with pulmonary tuberculosis?

In this retrospective study, all children (0-18 years) with the diagnosis of active pulmonary TB -as defined below-, who underwent three GAs, because they did not have spontaneous expectoration, between March 2007 and June 2019 at the study centre were enrolled. Patients who underwent GAs that were finally judged to not have TB were excluded. According to the local ethical board all parents had signed the informed consent for children’s data inclusion in observational studies with anonymised data extraction. Demographic, clinical, radiological and microbiological data were collected from medical charts. The result of microscopy, culture and PCR on each GA was recorded. Moreover, the results of IFN-γ releasing assays (IGRA), tuberculin skin test (TST), chest radiograph (CXR), and chest tomography (CT) closest in time to the first GA were registered. All these records were entered into the study database following international standards for data protection.

A TST was performed in all children, by trained nurses, injecting 5 UI of PPD-S through the Mantoux method. After 48-72 hours, a pediatrician assessed the result. The TST was defined positive in the case of an induration ≥5 mm in children suspected to have TB disease, immunosuppressed or with a close TB contact, ≥10 mm in children <4 years of age, coming/travelling from high TB burden countries, with underlying chronic conditions, and ≥15 mm in children without any risk factors [2]. The IGRAs performed to assess the diagnosis were the QuantiFERON-TB Gold In-Tube from 2007 to 2015 and the QuantiFERON-TB Gold Plus from 2015.

Pulmonary TB was diagnosed according to the American Academy of Pediatrics criteria, i.e: close contact with a confirmed TB case and/or positive IGRA/TST and/or suggestive signs/symptoms and radiological findings of pulmonary TB [2]. The following radiological findings were considered suggestive of TB in children and adolescents: hilar lymphadenopathy, broncopneumonic consolidation, pleural effusion, cavitation, miliary disease, atelectasis and intrabronchial involvement. Active pulmonary TB was defined as “microbiologically confirmed” if at least one microbiological test (microscopy, culture, PCR) was positive in at least one respiratory sample. Otherwise, it was defined as “not microbiologically confirmed” [11, 12].

Findings from CXR and, if available, CT were defined as “complicated TB” according to the radiological classification proposed by Marais et al. [13]. Moreover, for the present study purpose, we extended the definition of “complicated TB” to miliary disease. Therefore, patients with segmental collapse or hyperinflation, miliary disease, cavitation, or intra-bronchial spread with multiple consolidations, were considered “complicated TB” cases [13].

A standardized protocol for GAs was applied. The procedure was performed, by a trained nurse, on three consecutive days with the patient in supine position and on an empty stomach. At least 5-10 ml of gastric fluids were collectedusing a 8 French nasogastric tube. In the case of scarce gastric fluid, the sample was collected after instillating 10 ml of normal saline solution. The sample was refrigerated and sent to the laboratory within 4 hours and no buffering or pH adjustments were applied. For the purposes of the present study, we defined 1^st GA, 2^nd GA and 3^rd GA as the first, the second, and the third GA performed, respectively.

A direct smear fluorescence microscopy was performed, after auramine-rhodamine staining of the samples, Lowenstein-Jensen medium and Mycobacteria Growth Indicator Tube (MGIT) (Becton, Dickinson, and Company) were used for cultures, and samples were incubated for 60 or 42 days, respectively. Mycobacterium species were identified using Line Probe Assay (Hain), and antibiotic susceptibility was assessed through MGIT. PCR was performed to detect the Mycobacterium tuberculosis complex genome in GAs. During the study period, several assays for molecular detection were used (Cobas-Roche, Artus-Qiagen, Gene Xpert MTB/RIF and Gene Xpert MTB/RIF Ultra- Cepheid).

Quantitative data were reported as absolute numbers and percentages or median and interquartile range (IQR), as appropriate. Differences among groups were calculated using the χ2 or Fisher’s test for categorical variables and the Mann–Whitney test for continuous variables. A p value <0.05 was considered significant.

The sensitivity of each microbiological test was calculated for each GA with the correspondent confidence intervals (95% CI). Furthermore, the cumulative sensitivity of microbiological detection was calculated for each GA, for the first two (GA 1-2), and for all the three GAs (GA 1-3). Sensitivity was calculated in relation to children diagnosed with tuberculosis disease according to the abovementioned criteria.

Univariable and multivariable logistic regression analyses were used to identify the variables influencing the sensitivity of microbiological detection on GAs, and risk was expressed as Odds Ratio (OR) with 95% CI.

All statistical analyses were carried out using SPSS package (Statistical Package of Social Science, Chicago, IL), version 27.0.

Overall, 156 children with active pulmonary TB were enrolled. Of those, 46.8% were males, and the median age was 51.5 months (IQR: 25.2–113.2). Figure 1 shows the flow chart of cases selection*.

Among 156 enrolled patients, 143 (92%) were diagnosed with isolated pulmonary TB, whereas 13 patients had both pulmonary and extra-pulmonary TB. All patients were HIVnegative. The demographic and clinical characteristics of the study participants are shown in Table 1. Seventy-four (47.4%) patients had microbiologically confirmed pulmonary TB; the median age of the group was 60.9 months (IQR: 20.7-153.2). , Overall, children with negative GAs (52.6%) were younger, with a median age of 47.4 months (IQR: 25.5-107.4) (p=0.823). However, considering the microbiological confirmation among the different age groups, it was more frequent among children below 1 year (p=0.026) compared to the other groups.

The proportion of microbiologically confirmed GAs according to the radiological features is shown in Table 2. Overall, 80.1% (125/156) of the children showed an uncomplicated radiological picture. No statistically significant difference in microbiological confirmation was found in complicated vs. uncomplicated cases (p=0.09). Analysing all radiological patterns, parenchymal cavitation was more frequently associated with microbiologically confirmed cases when compared to negative ones (86.7% vs. 13.3%, p=0.001). All patients with miliary TB were microbiologically confirmed (p=0.022). Microbiological confirmation was more likely in children with parenchymal cavitation (p=0.001) and miliary disease (p=0.022). On the contrary, children with hilar lymphadenopathy were more likely to have microbiologically negative GAs (p=0.041), as shown in Table 2.

Microbiological tests showed a sensitivity of 34% (95%CI 26.7, 42) for 1^stGA, 32.1% (95%CI 24.9, 40.1) for 2^ndGA and 35.9% (95%CI 28.5, 44) for 3^rdGA (1^stGA vs. 2^ndGA p=0.722; 1^stGA vs. 3^rdGA p=0.725; 2^ndGA vs. 3^rdGA p=0.479). The cumulative sensitivity of GA (1-2) was 40.4% (95%CI 32.7, 48.5), while that of GA (1-3) was 47.4% (95%CI 39.8, 55.2).

Therefore, the collection of the 2^ndGA increased the sensitivity of the 1^stGA by 6.4% (95%CI -4.3, 16.9) (p=0.243), and the 3^rdGA led to a further increase in sensitivity by 7% (95%CI -3.9, 17.7) (p=0.195), obtaining a microbiological confirmation in other 10 and 11 patients, respectively. The collection of both the second and third GAs led to a significant increase in sensitivity of 13.4% (95%CI 2.8, 24.1; p=0.014) compared to the first GA only (Fig. 2).

The sensitivity of each microbiological investigation (microscopy, PCR, culture) for each GA is reported in Table 3. Microscopy and culture were obtained in all the included patients, whereas PCR was performed in 150/156 children (96.1%). No statistically significant difference was found when comparing the sensitivity of each microbiological investigation among each gastric GA (Table 3).

The increase in sensitivity of each test related to the number of performed GAs is shown in Fig. 3. No significant difference in microscopy sensitivity was found between 1stGA vs. GA (1-2) (p=0.382), GA (1-2) vs. GA (1-3) (p=0.544), and 1stGA vs. all the three GAs (p=0.144). The sensitivity of PCR was significantly higher considering the three GAs compared to the first one (p=0.020), whereas no significant differences were found between 1stGA vs. GA (1-2) (p=0.634) and GA (1-2) vs. GA (1-3) (p=0.649).

The culture showed the highest sensitivity compared to the other tests, which increased significantly if performed on three GAs compared to the first one (p=0.027). On the contrary, the difference was not statistically significant when comparing 1^stGA vs. GA (1-2) (p=0.297) and GA (1-2) vs. GA (1-3) (p=0.258).

Positive results of each microbiological test, considering overall the three GAs, are shown in Fig. 4.

Sensitivity of GAs in children > and ≤ 4 years of age and with uncomplicated/complicated radiological pattern, according to the number of GAs performed, is reported in Table 4. Considering the age groups, in children younger than 4 years the microbiological confirmation obtained with GA(1-3) lead to a significant increase in sensitivity of 19.7% (95%CI 4.5, 33.6; p=0.0011) as compared to 1^stGA, whereas there was not a significant difference in the proportion of microbiological confirmation on GA(1-2) vs. 1^stGA (10.5%, 95%CI -4.1, 24.5; p=0.160) and GA(1-3) vs. GA(1-2) (9.2%, 95%CI -6.3, 24.1; p=0.248). In the group of children older than 4 years a significant increase in sensitivity was not observed in any case: GA(1-2) vs. 1^stGA (2.5%, 95%CI -12.6, 17.4; p=0.751); GA(1-3) vs. 1^st GA (7.5%, 95%CI -7.8, 22.3; p=0.311); GA (1-3) vs. GA (1-2) (5%, 95%CI -10.3, 19.9; p=0.528). In children with uncomplicated TB, performing three GAs increased the sensitivity by 16% when compared to the 1^st GA (95%CI 4.2, 27.2; p=0.008). On the contrary, the increases in sensitivity in GA(1-2) vs. 1^st GA by an additional 7.2% (95%CI -4.1, 18.3; p=0.215), and between GA(1-3) vs. GA(1-2) by an additional 8.8% (95%CI -3.2, 20.4; p=0.152) were not statistically significant. In the multivariate analysis evaluating the role of the radiological classification and age on the overall microbiological performance, only the radiological classification resulted as an independent variable affecting the sensitivity of three GAs (radiological classification: OR 2.8, 95%CI 1.2, 6.5, p=0.013; age: OR 1.23; 95%CI 0.66, 2.32, p=0.511).