Specimen pooling as a diagnostic strategy for
microbiologic confirmation in children with pulmonary
(intrathoracic) tuberculosis
9.1 Rationale
Detection of M.tb in children with suspected PTB is improved by increasing the number 123-125 and variety 108,235 of respiratory specimens collected. Furthermore,
collecting specimens over consecutive days may result in a higher cumulative yield than same-day specimen collection 108,109, but it is not feasible in many clinical
settings due to the high cost and the frequent need for hospitalisation. Overnight sputum pooling in adults allows for the collection of higher volume sputum specimens, and has been shown to increase the detection of M.tb by smear microscopy and culture 239-242. This strategy is used mainly in the context of early
bactericidal activity trials of novel antituberculosis drugs and regimens in adults with PTB 240. It is not recommended in routine clinical care due to the higher risk of culture
contamination 237. In young children, pooling together multiple specimens after
collection by health care workers has been suggested as an approach to increase the specimen volume and improve microbiological yield 60, but no studies had evaluated
9.2 Study Aims
This study aimed to compare the diagnostic utility (detection of M.tb as well as proportion of culture contamination) of multiple respiratory specimens of different types collected on one day and pooled, in the laboratory, before testing by Xpert and liquid culture, vs. testing individual respiratory specimens for each individual child enrolled in the study.
9.3 Methods
The study was implemented from May 2014 - March 2017. Children with suspected PTB enrolled in the parent study (See Chapters 3-5), who were unable to expectorate sputum, were included in this pooling sub-study. Specimen collection included a GA, IS, and NPA on each of two consecutive week days. GA, IS, and NPA specimens collected on the second day were pooled for each child individually in the laboratory, after digestion/decontamination. Specimens of different types collected on the same day were pooled, rather than specimens of the same type collected over consecutive days, in order to improve the feasibility of specimen collection in young children. The reconstituted pellets from specimens collected on the second day were combined (pooled) into one centrifuge tube, and vortex-mixed. Individual (day 1) and pooled (day 2) specimens for each child were tested by smear microscopy, Xpert and liquid culture. McNemar’s test was used to compare the paired binary diagnostic outcomes of the pooled specimens by culture or Xpert (day 2) and individual specimens (GA/ IS/ NPA culture or Xpert) (day 1), for each child.
9.4 Results
Three hundred and four children were analysed; the median age was 15.1 (IQR 9.6-27.2) months, with only 4/304 (1.3%) children >5 years of age. Overall, 44 (14.5%) had M.tb confirmed on any of the study specimens. The diagnostic yield for pooled specimens was significantly higher than single IS and single NPA, by culture alone, Xpert alone and by culture and Xpert combined (i.e. any test positive), but it was similar for pooled specimens vs. single GA.
There was no difference in culture contamination between pooled vs. individual specimens of any type. The overall diagnostic yield of pooled specimens vs. all individual specimens combined, was not significantly different, by Xpert, culture, or Xpert/culture combined. Xpert/culture combined detected 35/44 (79.5%) confirmed cases using pooled specimens, vs. 38/44 (86.4%) for all individual day 1 specimens combined. The yield from a single GA was 29/44=65.9%. GA detected 7/44 (15.9%) cases not detected by any other specimen; for pooled specimens, this proportion was 6/44 (13.6%) and for IS, 2/44 (4.5%). NPA specimens did not add any diagnoses that were not already confirmed on other specimen types.
9.5 Conclusions
Although pooled respiratory specimens in this study were not superior to GA for the detection of M.tb by culture or Xpert, given the good performance of GA, pooling two GA could be considered in future studies. “Front-loading” specimen collection (GA, IS, NPA) on one day was feasible and effective for the detection of M.tb,
while a single GA remains a useful specimen for TB testing in young children and as an individual specimen, has a high diagnostic yield.
9.6 Citation
Walters E, van der Zalm MM, Demers AM, Whitelaw A, Palmer M, Bosch C,
Draper HR, Schaaf HS, Goussard P, Lombard CJ, Gie RP, and Hesseling AC. Specimen pooling as a diagnostic strategy for microbiologic confirmation in children with intrathoracic tuberculosis. Pediatr Infec Dis J, in press.
9.7 Candidate’s Contribution
I was responsible for the design, implementation and data collection for this study. I designed the statistical analyses, interpreted the results, drafted and finalised the manuscript after feedback from the other co-authors.
Specimen pooling as a diagnostic strategy for microbiologic confirmation in children with intrathoracic tuberculosis
Elisabetta Walters,1# Marieke M. van der Zalm,1 Anne-Marie Demers,1 Andrew
Whitelaw,2 Megan Palmer,1 Corné Bosch,1 Heather R. Draper,1 H. Simon Schaaf, 1
Pierre Goussard,3 Carl J. Lombard,4,5,6 Robert P. Gie,1 and Anneke C. Hesseling.1 1Desmond Tutu TB Centre, Department of Paediatrics and Child Health, Faculty of
Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa;
2Division of Medical Microbiology, Faculty of Medicine and Health Sciences,
Stellenbosch University and National Health Laboratory Service, Tygerberg Hospital, Cape Town, South Africa; 3Division of Paediatric Pulmonology, Department of
Paediatrics and Child Health, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; 4Division of Epidemiology and Biostatistics,
Department of Global Health, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; 5Biostatistics Unit, South African Medical
Research Council, South Africa; 6School of Public Health and Family Medicine,
University of Cape Town, Cape Town, South Africa
Key words: pulmonary tuberculosis; pediatric; diagnosis; specimen pooling Abbreviated title: Sample pooling to diagnose pediatric pulmonary TB Running title: Diagnosis of pediatric pulmonary TB
#Address correspondence to Elisabetta Walters, [email protected], +27 21 9389764.
Alternative corresponding author: Marieke M. van der Zalm,
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 from that of 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
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
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 (SDC3 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. 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
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). 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.