Specimen Pooling as a Diagnostic Strategy for Microbiologic Confirmation in Children With Intrathoracic Tuberculosis

Abstract

304 young children with suspected pulmonary tuberculosis had a gastric aspirate, induced sputum and nasopharyngeal aspirate collected on each of two consecutive weekdays. Specimens collected on the second day were pooled in the laboratory for each child individually. The diagnostic yield by Xpert and culture from pooled specimens was not significantly different to a single gastric aspirate.

Background

Intrathoracic (pulmonary) tuberculosis (PTB) in children is largely paucibacillary. In young children who cannot expectorate, specimen collection requires gastric aspiration, sputum induction (with nasopharyngeal suctioning), or nasopharyngeal aspiration. Respiratory specimens typically contain low concentrations of Mycobacterium tuberculosis (M.tb) bacilli, resulting in low sensitivity of currently available molecular tests and culture (1).

In children, increasing the number and variety (2–4) of specimens improves the overall diagnostic yield (detection of M.tb). Specimen collection over consecutive days may have a higher cumulative yield than same-day collection (3, 5). However, it is less practical and more costly if hospitalization is required. In young children, pooling multiple specimens after collection, in order to obtain a higher volume specimen, has been suggested as an approach to improve the bacteriologic yield (6).

In this study of children with suspected PTB, we compared the diagnostic yield and culture contamination of multiple respiratory specimen types pooled for microbiologic testing, with the yield from individual respiratory specimens for an individual child.

Materials and Methods

This analysis was part of a prospective diagnostic cohort study enrolling children with suspected PTB in Cape Town, South Africa. Eligibility criteria and enrolment investigations have been previously described (7). In brief, children <13 years of age presenting to two public referral hospitals, with history and symptoms of suspected PTB, were consecutively enrolled May 2014 to March 2017 (7). We excluded children who had received >1 dose of antituberculosis therapy before the first day of respiratory specimen collection, those with a convincing alternative clinical diagnosis, children with only extrathoracic TB or those who lived remotely. The pooling strategy was assessed in children who could not expectorate sputum, typically children <5 years of age.

Investigations included HIV testing, tuberculin skin test (TST; Mantoux, 2 Tuberculin Units PPD RT-23, Statens Serum Institute, Copenhagen) and a chest radiograph (CR; antero-posterior and lateral), evaluated by two independent experts. During the study, there was a global stock-out of TST, leading to a number of children not having the test. Interferon-gamma release assays were not used.

The attending clinicians were responsible for treatment decisions, with all study-related results made available to them. International consensus clinical case definitions were used to classify participants as “confirmed TB”, “unconfirmed TB”, and “unlikely TB” (8). Categories were assigned retrospectively at the 2-month follow-up, following assessment of treatment response and review of culture results.

Specimen collection and laboratory methods

The study protocol required the collection of one specimen of three different types, on each of two days (SDC1: Figure 1). The standard schedule was an early morning gastric aspirate (GA), a nasopharyngeal aspirate (NPA) and induced sputum (IS). The same order of collection was followed on both days, although specimens for pooling were collected with no time lag between them. On day 1 a minimum of two hours between GA, NPA and IS were observed (SDC2: Document 1_Specimen collection). In November 2015, NPA collection was stopped, due to low yield of M.tb for NPA (SDC3: Table 1) which did not justify the cost of individual testing. NPA was however still collected for the pooled specimen.

Specimens were processed at the National Health Laboratory Service, Tygerberg Hospital following standard protocols. The reconstituted pellets from specimens collected on the second day were combined (pooled) into one centrifuge tube, and vortex-mixed (SDC1: Figure 1). The individual concentrated day 1 specimens and the pooled respiratory specimen were subjected to fluorescent Auramine-O smear microscopy, Xpert MTB/RIF (Xpert: Cepheid, Sunnyvale, CA) and liquid Mycobacteria Growth Indicator Tube (MGIT, Becton Dickinson, Sparks, MD, USA) culture.

GenoType® MTBDRplus line probe assay (LPA: Hain Lifescience, Nehren, Germany) was performed on positive cultures for mycobacterial identification.

Statistical Analysis

The paired binary diagnostic outcomes of the pooled specimens by culture or Xpert (day 2) for each child and individual specimens (GA/ IS/ NPA culture or Xpert) (day 1) for each child were compared for marginal homogeneity using McNemar’s test. The Benjamini-Hochberg method was used to correct for multiple testing (m=34) of the pooled specimens using a false discovery rate of 10%, which corresponded to a p≤0.04. Diagnostic yield was defined as number of children (not specimens) positive for M.tb by Xpert or culture. We compared: a) the diagnostic yield of pooled vs. each individual day 1 specimen type separately (pooled vs. GA, pooled vs. NPA, pooled vs. IS); b) the proportion of contaminated cultures and invalid/error Xpert results for pooled vs. individual specimens separately); c) the total diagnostic yield by Xpert and culture (either positive) of pooled specimens vs. the combined yield from day 1 individual specimens (yield by Xpert or culture from any individual specimen). For a) and b), the pooled specimen had to contain the specimen type to which it was compared (per-protocol approach). For c), both a per-protocol (the number and type of specimens in the pooled specimen being the same as the number and type of day 1 individual specimens) and a pragmatic approach (including all participants) were used.

Stellenbosch University Health Research Ethics Committee (N11/09/282) and local health authorities approved the study.

Results

In 304 enrolled children (SDC 4: Figure 2), the median age was 15.1 (IQR 9.6–27.2) months (SDC 5: Table 2). Fifty-one of 304 (16.8%) children had confirmed TB: 44 were confirmed by study specimens and 7 on other specimens. Therefore, the total diagnostic yield for the pooling study was 44 children with confirmed TB. Antituberculosis treatment was initiated in 51 (100%) children with confirmed TB, 77/97 (79.4%) with unconfirmed TB and 6/156 (3.8%) with unlikely TB.

Pooled specimens vs. individual specimen types (SDC3 Table 1 for overall specimen results and SDC6 Figure 3 for specimen flow).

When comparing pooled specimens to each individual specimen type (Table 1), the proportion difference in diagnostic yield for pooled vs. single IS and single NPA was significantly higher for pooled specimens, by culture alone, Xpert alone and culture and Xpert combined (any test positive), but not for pooled vs. single GA.

Table 1.

Paired comparisons of culture, Xpert and smear microscopy results for Pooled vs gastric aspirate (GA), Pooled vs induced sputum (IS) and Pooled vs nasopharyngeal aspirate (NPA)¹, and paired comparisons of culture, Xpert and smear microscopy results for Pooled vs the combined yield of GA, IS and NPA by per-protocol and pragmatic approaches

		Day 1 GA Culture (n=294)		Day 1 IS Culture (n=298)		Day 1 NPA Culture (n=242)		Day 1 Combined cultures, per-protocol (n=2342)		Day 1 Combined cultures, pragmatic (n=304)
Type of test	Outcome	M.tb+	M.tb−3	M.tb+	M.tb−	M.tb+	M.tb−	M.tb+	M.tb−	M.tb+	M.tb−
Pooled Culture	M.tb +	20	8	18	13	9	15	17	5	25	6
	M.tb −	8	258	2	265	0	218	7	205	10	263
	Difference (95% CI)	0.0 (−0.03, 0.03)		0.04 (0.01, 0.07)		0.06 (0.03, 0.10)		−0.01 (−0.04, 0.02)		−0.01 (−0.04, 0.02)
	McNemar’s p-value	1,000		0,0053		<0.0014		0,564		0,317
		Day 1 GA Xpert (n=281)		Day 1 IS Xpert (n=296)		Day 1 NPA Xpert (n=242)		Day 1 Combined Xpert, per-protocol (n=2232)		Day 1 Combined Xpert, Pragmatic (n=304)
		M.tb+	M.tb−	M.tb+	M.tb−	M.tb+	M.tb−	M.tb+	M.tb−	M.tb+	M.tb−
Pooled Xpert	M.tb+	12	8	14	9	8	11	13	3	19	4
	M.tb−	4	257	1	272	1	222	5	202	5	276
	Difference (95% CI)	0.01 (−0.01, 0.04)		0.03 (0.00, 0.05)		0.04 (0.01, 0.07)		0.00 (−0.04, 0.02)		0.00 (−0.03, 0.02)
	McNemar’s p-value	0,388		0,0114		0,0044		0,478		0,739
		Day 1 GA Culture/Xpert combined (n=294)		Day 1 IS Culture/Xpert Combined (n=298)		Day 1 NPA Culture/Xpert Combined (n=242)		Day 1 Combined culture/Xpert,per-protocol (n=2342)		Day 1 Combined culture/Xpert, pragmatic (n=304)
		M.tb+	M.tb−	M.tb+	M.tb−	M.tb+	M.tb−	M.tb+	M.tb−	M.tb+	M.tb−
Pooled Culture/Xpert combined	M.tb+	22	10	21	14	11	16	20	5	29	6
	M.tb−	7	255	2	261	0	215	6	203	9	260
	Difference (95% CI)	0.01 (−0.02, 0.04)		0.04 (0.01, 0.07)		0.07 (0.03, 0.10)		0.00 (−0.04, 0.03)		−0.01 (−0.04, 0.02)
	McNemar’s p-value	0,467		0,0034		<0.0014		0,763		0,439
		Day 1 GA Smear (n=294)		Day 1 IS Smear (n=298)		Day 1 NPA Smear (n=242)		NA		NA
		Positive	Negative	Positive	Negative	Positive	Negative
Pooled Smear	Positive	3	3	2	4	1	2
	Negative	1	287	1	291	0	239
	Difference (95% CI)	0.01 (−0.01, 0.02)		0.01 (−0.01, 0.03)		0.01 (−0.01, 0.02)
	McNemar’s p-value	0,317		0,180		0,157
		Day 1 GA Culture (n=294)		Day 1 IS Culture (n=298)		Day 1 NPA Culture (n=242)		Day 1 Combined cultures, per-protocol (n=2342)		Day 1 Combined cultures, Pragmatic (n=304)
		Contaminated	Not Contaminated	Contaminated	Not Contaminated	Contaminated	Not Contaminated	Contaminated	Not Contaminated	Contaminated	Not Contaminated
Pooled Culture	Contaminated	3	18	1	20	2	12	2	13	5	16
	Not Contaminated	16	257	9	268	5	223	23	196	27	256
	Difference (95% CI)	0.01 (−0.04, 0.05)		0.04 (0.00, 0.08)		0.03 (−0.01, 0.07)		−0.04 (−0.10, 0.01)		−0.04 (−0.08, 0.01)
	McNemar’s p-value	0,732		0,0414		0,090		0,096		0,093
		Day 1 GA Xpert (n=281)		Day 1 IS Xpert (n=296)		Day 1 NPA Xpert (n=242)		Day 1 Combined Xpert, per- Protocol (n=2232)		Day 1 Combined Xpert, Pragmatic (n=304)
		Error	Not Error	Error	Not Error	Error	Not Error	Error	Not Error	Error	Not Error
Pooled Xpert	Error	0	3	0	3	0	3	0	3	0	3
	Not error	0	278	2	291	0	239	2	218	2	299
	Difference (95% CI)	0.01 (0.00, 0.03)		0.00 (−0.01, 0.02)		0.01 (−0.01, 0.03)		0.00 (−0.02, 0.03)		0.00 (−0.01, 0.02)
	McNemar’s p-value	0,083		0,655		0,083		0,655		0,655

GA: gastric aspirate; IS: induced sputum; NPA: nasopharyngeal aspirate; M.tb: Mycobacterium tuberculosis; CI: confidence interval.

¹For an explanation for the different denominators used in these comparisons, see SDC4: Figure 1

²For the per-protocol analysis, 234/304 (77.0%) children had the same number and type of specimens collected on day 1 and for pooling. Of these, all 234 (100%) had culture on all specimens tested by culture, while 223 (95.3%) had Xpert on all specimens tested by Xpert.

³M. tb− includes all non-positive results (for culture: contaminated, NTM and negative; for Xpert: error/invalid or negative);

⁴Using the Benjamini-Hochberg method, P≤0.04 was defined as statistically significant (24).

The proportion difference in contaminated cultures was not significant for pooled vs. any individual specimen type (Table 1).

The diagnostic yield of pooled specimens vs. the overall yield from individual specimens

For the per-protocol analysis, 234/304 (77.0%) children who were included had culture on all specimens, while 223 (73.4%) had Xpert on all specimens. For both the per-protocol and pragmatic analyses, the proportion difference in diagnostic yield, culture contamination and Xpert error rate of pooled specimens vs. all individual specimens combined was not significant (Table 1).

Of the 134 children treated for TB, 16 (11.9%) started treatment before (median 1.5 days) collection of specimens for pooling. Only one had a pooled specimen with negative culture and an individual positive GA culture, while two cases had culture-positive pooled specimens with negative individual specimens.

Discussion

We found that the overall diagnostic yield from pooled specimens (pooled GA, NPA, and IS) was not different from that of a single GA specimen, but was significantly higher than the yield from single IS or single NPA specimen. Pooled specimens and individual GA also had similar incremental yield and proportions of culture contamination, suggesting that the GA contained within the pooled specimen was probably the main contributor to both the diagnostic yield and the contamination rate of pooled specimens.

Pooling may result in high specimen volume, and also allows for sampling of the respiratory tract at different time points. Early studies demonstrated that shedding of M.tb bacilli into respiratory secretions may be intermittent (9) and pooling may increase the chance of collecting respiratory secretions which contain higher concentrations of bacilli. GA may be viewed as a naturally “pooled” specimen, as it consists of multiple expectoration and swallowing cycles, collected within the stomach. In contrast, both IS and NPA reflect a single instance of sampling the respiratory tract.

We elected to pool (in the laboratory) specimens of different types collected consecutively in a standard manner, to avoid prolonged periods of fasting, while allowing for collection on a single day. A same-day collection strategy avoids longer hospital stay, and potential loss to follow-up associated with consecutive-day collection in ambulatory settings.

Although we may have compromised some of the diagnostic potential of pooled specimens by collecting specimens consecutively for pooling with no time interval between specimens, our focus was to reduce cost of laboratory testing and improve feasibility, and long waiting times would not be practicable in routine clinical settings. A small proportion of children started antituberculosis therapy before collection of pooled specimens. However, our data suggest that this did not impact negatively on M.tb detection.

As expected in pediatric TB, collecting multiple specimens from children optimizes M.tb detection (SDC7: Figure 4; SDC8: footnote). The yield from multiple specimens on a single day was substantial (38/44=86.4%; Table 1). Previous studies have also shown that “front-loading” specimen collection on one day was comparable to consecutive-day collection (3, 10). However, pooling specimens did not have the expected increase in diagnostic yield. Given the good performance of GA, pooling two GA could be considered in future studies.