Original research Pooled testing of sputum with Xpert MTB/ RIF and Xpert Ultra during tuberculosis active case finding campaigns in Lao People’s Democratic Republic Vibol Iem ‍ ‍ ,1,2 Phonenaly Chittamany,2 Sakhone Suthepmany,2 Souvimone Siphanthong,2 Phitsada Siphanthong,2 Silaphet Somphavong,3 Konstantina Kontogianni,1 James Dodd,1 Jahangir AM Khan,4 Jose Dominguez,5 Tom Wingfield,6,7 Jacob Creswell,8 Luis E Cuevas ‍ ‍ 9 To cite: Iem V, Chittamany P, ABSTRACT Suthepmany S, et al. Pooled Key questions Introduction Active case finding (ACF) of individuals with testing of sputum with Xpert tuberculosis (TB) is a key intervention to find the 30% of MTB/RIF and Xpert Ultra What is already known? people missed every year. However, ACF requires screening during tuberculosis active ► Our study adds to the emerging body of evidence large numbers of individuals who have a low probability case finding campaigns in that the pooling methods for testing with molecu- Lao People’s Democratic of positive results, typically <5%, which makes using the lar assays can improve the efficiency of testing for Republic. BMJ Global Health recommended molecular tests expensive. tuberculosis (TB), potentially enabling the screen- 2022;7:e007592. doi:10.1136/ Methods We conducted two ACF surveys (in 2020 and ing and testing of larger numbers of people more bmjgh-2021-007592 2021) in high TB burden areas of Lao PDR. Participants cost-­effectively. were screened for TB symptoms and received a chest X-­ray. Handling editor Alberto L Sputum samples of four consecutive individuals were pooled What are the new findings? Garcia-­Basteiro and tested with Xpert Mycobacterium tuberculosis (MTB)/ ► These findings contribute to recognised gaps in rifampicin (RIF) (Xpert-­MTB/RIF) (2020) or Xpert-­Ultra (2021). funding sources for the procurement of sufficient JC and LEC are joint senior The agreement of the individual and pooled samples was cartridges for testing all individuals with presump- authors. compared and the reasons for discrepant results and potential tive TB, which jeopardises access to high sensitiv- cartridge savings were assessed. ity WHO-­recommended rapid molecular diagnostic Received 2 October 2021 Accepted 1 December 2021 Results Each survey included 436 participants, which tests, such as the GeneXpert Xpert MTB/RIF and were tested in 109 pools. In the Xpert-­MTB/RIF survey, Xpert-­Ultra. 25 (sensitivity 89%, 95% CI 72.8% to 96.3%) of 28 pools containing MTB-­positive samples tested positive and 81 What do the new findings imply? pools containing only MTB-­negative samples tested negative ► The method has not been tested during a real health (specificity 100%, 95% CI 95.5% to 100%). In the Xpert-­Ultra crisis situation, such as the COVID-­19 pandemic. The survey, all 32 (sensitivity 100%, 95% CI 89.3% to 100%) pools study took place at a time laboratory resources were containing MTB-­positive samples tested positive and all 77 being diverted and healthcare workers were repur- (specificity 100%, 95% CI 95.3% to 100%) containing only posed for SARS-­CoV2 testing. The higher efficiency of MTB-­negative samples tested negative. Pooling with Xpert-­ the pooling method can contribute to cope with these MTB/RIF and Xpert-­Ultra saved 52% and 46% (227/436 and challenging times. 199/436, respectively) of cartridge costs alone. Conclusion Testing single and pooled specimens had a high level of agreement, with complete concordance health facility to initiate the diagnostic process. when using Xpert-­Ultra. Pooling samples could generate Although PCF identifies most people with TB in significant cartridge savings during ACF campaigns. locations with adequate access to health services, © Author(s) (or their it misses those unwilling or unable to attend employer(s)) 2022. Re-­use the clinics and is a major reason only seven of permitted under CC BY-­NC. No commercial re-­use. See rights INTRODUCTION the ten million people with TB are diagnosed and permissions. Published by Despite being treatable and curable, tubercu- and notified.2 Individuals missed by passive BMJ. losis (TB) remains one of the main infectious approaches often include vulnerable popula- For numbered affiliations see killers in the world, as ten million people fall ill tions such as internally displaced, migrant or end of article. and 1.4 million die from the disease each year.1 rural populations, women, the unemployed Correspondence to Its diagnosis is usually reliant on passive case and ethnic minorities,3 4 who may face multiple Professor Luis E Cuevas; finding (PCF), in which health services wait for societal and economic barriers to attend the ​luis.​cuevas@​lstmed.​ac.​uk individuals with symptoms of TB to attend a service, including catastrophic costs.5 6 It is, Iem V, et al. BMJ Global Health 2022;7:e007592. doi:10.1136/bmjgh-2021-007592  1 BMJ Global Health thus, recognised that, to be inclusive and reduce the soci- recruitment and testing procedures. The 2020 survey oeconomic impact of TB,7 health services need to include aimed to assess the performance of the pooling method active case finding (ACF) approaches that involve proactive when testing samples with Xpert MTB/RIF, while the interventions to extend the reach of TB services for diag- 2021 survey assessed the method when using Xpert-­Ultra, nosis8 and treatment.9 Although ACF interventions can be after its release for routine use by Lao’s NTC. very effective,10 11 they are less standardised than PCF, as they ACF was conducted in Lao’s high TB burden areas, which address the specific barriers of multiple target populations are programmatically defined as TB incidence ≥100 cases and are more resource and time intensive than PCF.12 per 100 000 population. The 2020 survey was conducted The WHO recommends testing all individuals with in Vientiane Capital, Luang Prabang and Savannakhet presumptive TB with molecular assays, such as the Xpert provinces with estimated populations of 890,129, 468 375 Mycobacterium tuberculosis/rifampicin (Xpert MTB/ and 1 051 675 inhabitants, respectively, and TB notifica- RIF) and Xpert-­Ultra (Cepheid Sunnyvale, California),13 tion rate of 134, 88 and 102 cases per 100 000 population with the latter being preferred given its higher sensi- in 2020, respectively. The 2021 survey was conducted in tivity.14 Although the use of these assays is expanding, the Saravane and Oudomxay, with 430 428 and 333 934 popu- assay cartridge unit costs of US$ 9.98 per test15 remains lation and TB notification rates of 127 and 110 cases per one of the main hurdles for its wider implementation in 100 000 population in 2020, respectively. low-­income and middle-­ income countries. Diagnostic Both surveys were conducted in the same fashion. test costs can limit the expansion of ACF activities, as they Before an ACF activity, the NTC team met the province require testing large numbers of individuals with rela- and district health authorities and conducted prepa- tively lower yields than PCF.16 ration visits with the provincial TB coordinator, district Since 2015, the Lao National Tuberculosis Control TB manager and village authorities, distributed health Center (NTC) has conducted ACF by implementing education materials, obtained the addresses of individ- intensified case finding activities to increase the detec- uals with TB and line listed household contacts. At an tion of individuals with TB in high burden districts of agreed date, the NTC team set up a digital chest X-­ray the country. These activities include the sensitisation of machine and a four-­module GeneXpert platform in the the population, the local provision of chest X-­rays for village and invited all residents to complete a question- screening (independently of symptoms) and the iden- naire on signs and symptoms, history and treatment of tification of individuals with symptoms of TB who have TB and offered chest X-­rays for screening, independently not attended health facilities. Participants with abnormal of the presence of symptoms. Individuals with abnormal chest X-­rays or symptoms of TB are tested using Xpert chest X-­rays and those who indicated having cough >2 MTB/RIF or Xpert-­Ultra.17 The activities have increased weeks duration were asked to provide sputum samples for case detection, although the cost of the Xpert cartridges Xpert testing and were managed according to the deci- is considered high and is the main limiting factor to sion tree shown in figure 1. Sputum samples were tested implement the intervention on a larger scale. with Xpert following the manufacturer’s instructions.21 One approach that could increase the affordability of Sputum samples tested individually with Xpert MTB/ Xpert testing is to test several samples together using the RIF or Xpert-­Ultra were processed in the village GeneX- pooling method.18 This procedure combines (or pools) pert platform. Consecutive samples with remnant volumes the sputum of several individuals into one pot and tests ≥0.5 mL were included in the pooling studies and were them together with a single test. If the test is positive, transported to the National TB Reference Laboratory in the pool’s samples are retested individually to identify Vientiane using a cold chain. Samples were transported the positive sample(s) while if the test is negative, all after the sample reagent had been added. Turned around samples in the pool are considered negative, resulting time to testing was <48 hours after the sample reagents in 30%–40% savings in Xpert cartridge costs alone had been added and samples were maintained in a cold depending on the prevalence of TB in the population chain at all times. Sputum samples from four participants tested.19 Therefore, pooling may hold great promise for were pooled together, with a volume of 0.5 mL of sputum ACF, but there are few reports of its performance under each added to a pot, to obtain an aggregated volume of operational conditions.20 2 mL.21 Samples for a pool were selected consecutively Here, we report a prospective study to assess the and staff were blind to the individual Xpert test results sensitivity and specificity of the pooling method using and the pooled specimen was tested using one new Xpert Xpert MTB/RIF and Xpert-­Ultra during intensified case cartridge. finding interventions, and its potential to increase the affordability of Xpert testing in Lao PDR. Statistical analysis Categorical data were summarised using descriptive statis- METHODS tics and χ2 tests were used to test for statistically significant We conducted two independent prospective surveys differences. Individuals unable to produce sputum were embedded within the ACF activities of Lao’s NTC, from excluded from the analysis. The pooled samples were March to April 2020 and from January to March 2021. compared with the four Xpert MTB/RIF and Xpert-­Ultra Both surveys were cross-­ sectional and used the same individual results and their agreement was tested using 2 Iem V, et al. BMJ Global Health 2022;7:e007592. doi:10.1136/bmjgh-2021-007592 BMJ Global Health Figure 1 Flow diagram of the sputum processing. kappa statistics. The CT values and grades (trace, very (2.9%) women, respectively, p=0.014); but women were low, low, medium and high) of individual and pooled tests more likely to be MTB-­positive than men in 2021 (12/222 were compared with describe the effect of combining the (5.4%) vs 12/214 (11.7%), respectively, p<0.008). Each samples. Cost differences were calculated on the bases of survey included 109 pools of four patients. the number of cartridges required to test all specimens using pooled and individual testing. Xpert MTB/RIF survey Sample size for the surveys was not formally estimated In 2020, 28 (25.7%) pools contained one or more Xpert as we were limited by the expected number of partici- MTB/RIF MTB-­positive sample (27 pools with one and pants attending the campaigns before the COVID-­ 19 one pool with two MTB-­positive samples) and 81 (74.3%) lockdown, the capacity of staff to conduct additional pools contained solely MTB-­negative samples (table 2). testing to their routine activities and the number of spare The pool with two MTB-­positive and 24 of 27 pools with cartridges available for research purposes. one MTB-­positive sample tested MTB-­positive and three tested MTB-­ negative, resulting in a sensitivity of 89% Patient and public involvement (25/28, 95% CI 72.8% to 96.3%). All 81 pools containing It was not appropriate or possible to involve patients or solely MTB-­negative samples tested MTB-­negative in the the public in the design, or conduct, or reporting or pooled assay (specificity 100%, 95% CI 95.5% to 100%). dissemination plans of our research. Therefore, the accuracy performance of the 109 pools in correlation to the 436 individual results resulted in RESULTS 97.3% agreement (kappa: 0.925). Among the 27 pools The 2020 survey included 436 participants, 334 (76.6%) containing single MTB-­positive samples, five contained men and 102 (23.4%) women, and 29 (6.7%, 95% CI very low, 15 low, 6 medium and one high MTB grades. The 4.7% to 9.4%) were Xpert MTB/RIF MTB-­positive. The pooled MTB grade was similar to the individual test in 2021 survey also included 436 participants, 222 (50.9%) four (14.8%), one grade lower in 21 (77.8%), two grades men and 214 (49.1%) women, and 37 (8.5%, 95% CI lower in one (3.7 %) and one grade higher in one (3.7%) 6.5% to 11.5%) were Xpert-­Ultra MTB-­positive (p value of the pools. Of the five pools containing very low indi- >0.1, table 1). Men were more likely to be MTB-­positive vidual MTB-­grades, three tested MTB-­not detected and than women in 2020 (26/334 (7.8%) men vs 3/102 two very low MTB-­grade in the pooled assay (table 3). Iem V, et al. BMJ Global Health 2022;7:e007592. doi:10.1136/bmjgh-2021-007592 3 BMJ Global Health Table 1 Baseline characteristics of participants and Xpert MTB/RIF and Xpert Ultra results Xpert MTB/RIF Xpert Ultra Individual Pool Individual Pool n (%) n (%) n (%) n (%) Sex 436 – 436 – Male 334 (76.6) – 222 (50.9) – Female 102 (23.4) – 214 (49.1) – Age 436 – 436 – Mean (SD) (range) 45 (16.1) (12-­89) – 54 (13.7) (10-­90) – <35 131 (30.0) – 41 (9.4) – 35–54 184 (42.2) – 159 (36.5) – ≥55 121 (27.8) – 236 (54.1) – Xpert MTB result 436 109 436 109 Detected/≥1 MTB included 29 (6.7) 25/28 (22.9) 37 (8.5) 32/32 (29.4) Not detected/≥1 MTB included – 3 (2.7) – – Not detected/only MTB-­negative 407 (93.3) 81 (74.3) 399 (91.5) 77 (70.6) Xpert MTB result by sex Male 26/334 (7.8) – 12/222 (5.4) – Female 3/102 (2.9) – 25/214 (11.7) – MTB grade 29 25 37 32 Trace NA NA 6 (16.2) 21 (65.6) Very low 6 (20.7) 15 (60.0) 8 (21.6) 11 (34.4) Low 16 (55.2) 9 (37.5) 15 (40.5) 0 (0.0) Medium 6 (20.7) 1 (4.2) 2 (5.4) 0 (0.0) High 1 (3.4) 0 (0.0) 6 (16.2) 0 (0.0) RIF resistance 29 25 37 32 Detected 0 0 0 0 Not detected 27 (93.1) 24 (96.0) 30 (81.1) 10 (31.2) Indeterminate 2 (6.9) 1 (4.0) 7 (18.9) 22 (68.8) Bold figures are frequencies and do not indicte statistical significance. Xpert MTB/RIF, Xpert Mycobacterium tuberculosis/rifampicin. The CT values for the Xpert MTB/RIF probes for both pooled tests, with an increase in CT values ranging from individual and pooled testing are shown in table 4. The 5.4 to 7.1. median CT values for probes A-­E ranged from 23.4 to Two of the MTB-­ positive samples were RIF-­ 24.8 for the individual tests and from 30.6 to 33.6 for the indeterminate and 27 RIF-­negative. Of the 28 pools with Table 2 Number of pools with 0, 1, 2, 3, 4 positive results Individual Xpert results included in a pool All negative One positive Two positive n (%) n (%) n (%) Three positive n (%) Four positive n (%) All Pooled Xpert MTB/RIF 81 27 1 0 0 109 Detected 0 24 (89%) 1 (100%) 0 0 25 (23%) Not detected 81 (100%) 3 (11%) 0 0 0 84 (77%) Pooled Xpert Ultra 77 27 5 0 0 109 Detected 0 27 (100%) 5 (100%) 0 0 32 (29%) Not detected 77 (100%) 0 0 0 0 77 (71%) Bold figures are frequencies and do not indicte statistical significance. Xpert MTB/RIF, Xpert Mycobacterium tuberculosis/rifampicin. 4 Iem V, et al. BMJ Global Health 2022;7:e007592. doi:10.1136/bmjgh-2021-007592 BMJ Global Health Table 3 Correlation of Individual and pooled Xpert MTB grades (positive pools only include those with only one positive Xpert) Individual Xpert grade included in pool Not detected Trace Very low Low Medium High n (%) n (%) n (%) n (%) n (%) n (%) Pooled Xpert MTB/RIF 81 NA 5 15 6 1 Not detected 81 (100%) NA 3 (60%) 0 0 0 Very low 0 NA 2 (40%) 12 (80%) 0 0 Low 0 NA 0 2 (13.3%) 6 (100%) 1 (100%) Medium 0 NA 0 1 (6.7%) 0 0 High 0 NA 0 0 0 0 Pooled Xpert Ultra 77 2 5 13 1 6 Not detected 77 (100 %) 0 0 0 0 0 Trace 0 2 (100%) 4 (80%) 7 (54%) 1 (100%) 2 (33%) Very low 0 0 1 (20%) 6 (46%) 0 4 (67%) Low 0 0 0 0 0 0 Medium 0 0 0 0 0 0 High 0 0 0 0 0 0 Bold values are frequencies and do not indicte statistical significance. Xpert MTB/RIF, Xpert Mycobacterium tuberculosis/rifampicin. MTB-­ positive samples, 25 pools contained one MTB-­ Xpert-Ultra survey positive RIF-­negative sample, one had two MTB-­positive In 2021, 32 (29.4%) pools contained MTB-­positive samples RIF-­negative samples and two had one MTB-­ positive and 77 (70.6%) solely MTB-­ negative samples. Twenty-­ RIF-­indeterminate samples. Of the 25 MTB-­ positive seven of the 32 MTB-­positive pools contained one and five RIF-­negative pools, three tested MTB-­negative and did contained two MTB-­positive samples and all tested positive in not report RIF results and 22 tested RIF-­negative. The pool containing two MTB-­positive RIF-­negative samples the pooled assay (sensitivity 100%, 95% CI 89.3% to 100%). tested RIF-­negative and the two pools containing RIF-­ All 77 pools containing only MTB-­negative samples tested indeterminate samples tested RIF-­negative in one and MTB-­negative (specificity 100%, 95% CI 95.3% to 100%), RIF-­indeterminate in the other. resulting in 100% agreement (Kappa: 1). Among the 27 Table 4 Median CT values of individual and pooled Xpert MTB/RIF and Xpert Ultra probe results Xpert MTB RIF Individual results n=29 Pooled results n=25 Probe CT median IQ range Min–max CT median IQ range Min–max ΔCT Probe D 24.6 (22.7–27.3) 19.1–35.2 32.3 (28.5–34.2) 21.0–38.2 7.1 Probe C 23.7 (22.0–26.7) 18.9–34.7 30.3 (27.4–31.8) 20.0–35.7 6.5 Probe E 24.8 (23.0–28.1) 20.5–36.2 33.9 (29.3–34.9) 21.2–39.3 7.1 Probe B 24.6 (22.9–27.3) 20.1–33.8 30.1 (27.3–32.5) 20.9–34.7 5.4 Probe A 23.3 (22.0–26.0) 20.5–34.1 31.1 (27.1–32.9) 19.7–34.6 6.6 Xpert Ultra Individual results n=37 Pooled results n=32 Probe CT median IQ range Min–max CT median IQ range Min–max ΔCT Probe IS1081 19.6 (17.1–22.6) 16.0–32.0 24.9 (22.2–26.5) 19.9–29.3 4.6 Probe rpoB1 21.6 (17.9–24.9) 0–32.0 0 (0–30.2) 0–34.9 NA Probe rpoB2 21.3 (17.9–25.8) 0–32.1 0 (0–29.8) 0–35.3 NA Probe rpoB3 23.3 (19.2–27.0) 0–33.7 0 (0–32.9) 0–39.8 NA Probe rpoB4 25.7 (21.2–29.7) 0–35.7 0 (0–33.5b) 0–37.7 NA Xpert MTB/RIF, Xpert Mycobacterium tuberculosis/rifampicin. Iem V, et al. BMJ Global Health 2022;7:e007592. doi:10.1136/bmjgh-2021-007592 5 BMJ Global Health Table 5 Costs and savings to screen consecutive patients using the pooling method and number of patients that could be tested with Xpert MTB/RIF and Xpert Ultra cartridges Individual Xpert Pooled Xpert MTB/RIF Ultra MTB/RIF Ultra Number of individuals tested 436 436 436 436  Sensitivity Reference Reference 89%* 100%*  Specificity Reference Reference 100%* 100%*  Proportion positive 6.7% 8.5% 22.9% 29.4%  Bacteriologically confirmed 29 37 26 37  Cartridges required 436 436 209 237  Cartridge costs (USD) 4351.28 4351.28 2085.82 2365.26  Cartridge savings (USD) NA NA 2265.46(52%) 1986.02(46%) Numbers tested with 436 cartridges  Number tested 436 436 909 802  Cartridge cost per patient (USD) 9.98 9.98 4.78 5.42 *Assumes pools of 1:4; proportion positive taken from the surveys’ findings. pools with single MTB-­positive samples, two contained trace, RIF and Xpert-­Ultra, respectively—an effective test per 5 very low, 13 low, 1 medium and 6 high MTB grades. The patient cost of U ​ S$​4.​78.​and 5.42, respectively (table 5). pooled MTB grades were the same as the individual grades in three (11%), one grade lower in 10 (42%), two grades lower in seven (29%), three grades lower in five (21%) and DISCUSSION four grades lower in two (8%) of the pooled assays (table 3). Our surveys compared pooling with single testing during The Xpert-­Ultra probes CT values are shown in . Probe ACF for TB in a low-­income country. Our results confirm IS1081/IS6110 had median CT of 19.6 for individual and that testing individual and pooled samples with the 24.9 for pooled results, with a median increase of 4.6. Probes GeneXpert platform can achieve a high level of concord- rpoB1-­B4 median CT values ranging from 19.6 to 25.7 for ance. Concordance was higher with Xpert-­ Ultra than the individual tests, but CT values were not available for with Xpert MTB/RIF, which is in agreement to regional the pools. Among the 37 MTB-­positive samples, 30 (81%) studies evaluating pooling with Xpert-­Ultra in Cambodia20 were RIF-­negative and 7 (18.9%) were RIF-­indeterminate and Vientiane, Lao PDR (Iem et al, in press). Discrepan- and were distributed in 32 pools. Twenty-­ five of the 32 cies between individual and pooled Xpert MTB/RIF tests pools contained only RIF-­negative and 7 contained RIF-­ only occurred among pauci-­bacillary samples with high indeterminate samples. Fifteen of the 25 pools containing Xpert CT values, suggesting that some samples with low only RIF-­negative samples tested RIF-­ indeterminate and DNA concentrations fall below the assay’s limit of detec- 10 RIF-­ negative, while all seven pools containing RIF-­ tion and that the better agreement of Xpert-­Ultra is due indeterminate samples tested pooled RIF-­indeterminate. to its higher sensitivity. Consequently, some patients with paucibacillary disease could be missed by pooling, espe- Xpert MTB/RIF and Xpert Ultra costs cially if testing is based on Xpert MTB/RIF. The cartridges cost for testing individually the 436 partic- The pooling strategy can lead to significant cost savings ipants with Xpert at US$9.98 per test was US$4351.28 for and facilitate testing of more individuals for a given each survey, as shown in table 5. The pooling method in number of cartridges. In our setting, pooling samples 2020 required 109 Xpert MTB/RIF cartridges to test 109 would double the number of people tested with the pools and 100 cartridges to test individual samples of 25 same number of cartridges. This is higher than in PCF MTB-­positive pools. The total of 209 (109+100) cartridges studies, where pooling is reported to save up to 40% for pool testing would cost US$2085.82, resulting in of cartridges.19 Cartridge savings are a function of the US$2265.46 (52%) saving in cartridge costs. Similarly, proportion of people with MTB-­positive results and their testing 109 pools with Xpert-­Ultra in 2021 required 109 distribution within the pools. If the proportion positive cartridges to test the pools and 128 cartridges to test indi- is low, a low number of pools would need to be retested, vidually the 32 positive pools. The total of 237 cartridges resulting in higher cartridge savings. For example, in a would cost US$2365.26, resulting in US$1986.02 (46%) survey in Lao’s district clinics, 12% of individuals tested savings in cartridge costs. If the number of cartridges is Xpert-­positive, and pooling resulted in 38.3% and 41.7% kept fixed, the pooling method could test more patients cartridge saving costs with Xpert MTB/RIF and Xpert-­ than testing samples individually, as 436 cartridges would Ultra, respectively (Iem et al, in press), while in our survey allow testing 909 and 802 individuals with Xpert MTB/ setting, the proportion of positives was 8.5%, which led 6 Iem V, et al. BMJ Global Health 2022;7:e007592. doi:10.1136/bmjgh-2021-007592 BMJ Global Health 9 to higher savings. The proportion of participants with Clinical Sciences and Recsearch Centre for Drugs and Diagnostics, Liverpool positive tests in ACF is often lower than reported from School of Tropical Medicine, Liverpool, UK studies using PCF, typically below 5% depending on the Twitter Jacob Creswell @Jacob_Creswell target population,16 22 and lower to 10%–20% of indi- Acknowledgements The authors would like to thank the National Tuberculosis viduals attending TB clinics in PCF.23 24 We have, thus, Control Center (NTC) of Lao PDR for authorising the study and facilitating samples shown that pooling could be highly efficient when testing transportation from the field until the National TB Reference Laboratory (NTRL) in populations using ACF, and further studies among such Vientiane. We are also grateful to the local health authorities that allowed access to the study sites and for their coordination locally. We are thankful to the active case populations are warranted. Since the pooling method finding team for their engagement, screening and enrolling patients, collecting, is a laboratory change, it would not affect the screening and shipping samples from the collection sites and to laboratory technicians of algorithm and can be easily instituted without any major the NTRL for their dedication to carefully perform these additional laboratory procedures at the time of the COVID-­19 pandemic. We also thank Jim Read and the modifications. Global Health Trial Unit, LSTM for generating databases and data curation. Previous systematic reviews have highlighted that indi- Contributors The study was designed by LEC, JC, VI, KK and JAMK. Patients vidual and pooled RIF results are often discordant, with screening and enrolling, individual sample testing on Xpert MTB/RIF and Xpert Ultra pools containing RIF-­negative samples often returning were conducted by PC, SaS, SoS, PS, and SiS in the field during the ACF campaign. RIF-­indeterminate pooled results,19 and our findings are VI performed the pooled testing. Data collection and analysis were conducted by VI, in agreement with these observations. Although samples KK, LEC, JD and JC. VI, JD, TW, JC and LEC wrote the first draft of the manuscript. All authors made substantial contributions to the writing and editing of the with pooled RIF results would be routinely confirmed manuscript. LEC is responsible for the overall content and is the guarantor of the at the time of retesting, the samples of a positive pool study The final version has been read and approved by all named authors. to identify the individual MTB-­ positive samples, it is Funding This research was funded in part by a TB REACH, Stop TB Partnership important to highlight that pooled RIF results are unre- grant supported by Global Affairs Canada (STBP/TBREACH/GSA/2020-­04, awarded liable and should not be used for clinical management. to LEC), the Global Fund to Fight AIDS, Tuberculosis and Malaria (LAO-­T-­GFMOH grant) and the National Tuberculosis Control Program budget from the Ministry Further studies could explore ways to further improve of Health of Lao PDR. TW is supported by grants from: the Wellcome Trust, UK the efficiency of pooling when combined with other (209075/Z/17/Z); the Medical Research Council, Department for International screening tools, such as C-­ reactive protein (CRP)25 Development, and Wellcome Trust, UK (Joint Global Health Trials, MR/V004832/1), and digital chest X-­rays with Computer-­aided diagnosis the Medical Research Council, UK (MR/V028618/1); the Academy of Medical Sciences, UK; and the Swedish Health Research Council, Sweden. (CAD).26 27 Both tools can identify individuals with and without the traditional symptoms of TB, although their Competing interests The authors have no conflicts of interest to declare. relatively lower specificity requires confirming the diag- Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research. nosis with more specific molecular assays. Although using tests combinations could increase assay costs, individ- Patient consent for publication Not applicable. uals with a positive CRP or abnormal chest X-­rays CAD Ethics approval The study was approved by the Lao NTC and the Liverpool School could be confirmed using the pooling method, and its of Tropical Medicine Research Ethics Committee, UK (Ethical waiver 20–037), and informed consent waiver was obtained. efficiency gains could increase the affordability of tests Provenance and peer review Not commissioned; externally peer reviewed. combinations. In conclusion, we have shown pooling samples for TB Data availability statement Data are available upon reasonable request. Not applicable. diagnoses during ACF campaigns, which can replicate Open access This is an open access article distributed in accordance with the testing samples with individual tests. The approach can Creative Commons Attribution Non Commercial (CC BY-­NC 4.0) license, which facilitate testing higher numbers of patients with lower permits others to distribute, remix, adapt, build upon this work non-­commercially, cartridge costs, increasing the affordability of testing with and license their derivative works on different terms, provided the original work is molecular assays. The high level of agreement between properly cited, appropriate credit is given, any changes made indicated, and the use is non-­commercial. See: http://creativecommons.org/licenses/by-nc/4.0/. individual and pooled samples obtained with Xpert-­Ultra demonstrates that pooling can be reliable and contribute ORCID iDs to achieve the WHO End TB strategy targets in resource-­ Vibol Iem http://orcid.org/0000-0001-6155-1402 Luis E Cuevas http://orcid.org/0000-0002-6581-0587 limited settings. Author affiliations 1 Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, UK 2 National Tuberculosis Control Center, XJ7F+P5F, Vientiane, Lao People's REFERENCES Democratic Republic 1 World Health Organization. Global tuberculosis report; 2020. 2020. 3 Center of Infectology Lao Christophe Merieux, Vientiane, Lao People's Democratic 2 Shewade HD, Gupta V, Satyanarayana S, et al. Patient Republic characteristics, health seeking and delays among new sputum 4 smear positive TB patients identified through active case finding Health Economics and Policy Unit, School of Public Health and Community when compared to passive case finding in India. PLoS One Medicine, University of Gothenburg, Gothenburg, Sweden 2019;14:e0213345. 5 Institut d'Investigació Germans Trias i Pujol, CIBER Enfermedades Respiratorias, 3 World Health Organization. Who consolidated guidelines on and Universitat Autònoma de Barcelona, Barcelona, Spain tuberculosis. 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