key: cord-0841753-d2vz7e95
authors: Iem, Vibol; Chittamany, Phonenaly; Suthepmany, Sakhone; Siphanthong, Souvimone; Siphanthong, Phitsada; Somphavong, Silaphet; Kontogianni, Konstantina; Dodd, James; Khan, Jahangir AM; Dominguez, Jose; Wingfield, Tom; Creswell, Jacob; Cuevas, Luis E
title: Pooled testing of sputum with Xpert MTB/RIF and Xpert Ultra during tuberculosis active case finding campaigns in Lao People’s Democratic Republic
date: 2022-02-14
journal: BMJ Glob Health
DOI: 10.1136/bmjgh-2021-007592
sha: cb82e5740ea50ef7146c15535d4da5887b2df5b7
doc_id: 841753
cord_uid: d2vz7e95

INTRODUCTION: Active case finding (ACF) of individuals with tuberculosis (TB) is a key intervention to find the 30% of people missed every year. However, ACF requires screening large numbers of individuals who have a low probability of positive results, typically <5%, which makes using the recommended molecular tests expensive. METHODS: We conducted two ACF surveys (in 2020 and 2021) in high TB burden areas of Lao PDR. Participants were screened for TB symptoms and received a chest X-ray. Sputum samples of four consecutive individuals were pooled and tested with Xpert Mycobacterium tuberculosis (MTB)/rifampicin (RIF) (Xpert-MTB/RIF) (2020) or Xpert-Ultra (2021). The agreement of the individual and pooled samples was compared and the reasons for discrepant results and potential cartridge savings were assessed. RESULTS: Each survey included 436 participants, which were tested in 109 pools. In the Xpert-MTB/RIF survey, 25 (sensitivity 89%, 95% CI 72.8% to 96.3%) of 28 pools containing MTB-positive samples tested positive and 81 pools containing only MTB-negative samples tested negative (specificity 100%, 95% CI 95.5% to 100%). In the Xpert-Ultra survey, all 32 (sensitivity 100%, 95% CI 89.3% to 100%) pools containing MTB-positive samples tested positive and all 77 (specificity 100%, 95% CI 95.3% to 100%) containing only MTB-negative samples tested negative. Pooling with Xpert-MTB/RIF and Xpert-Ultra saved 52% and 46% (227/436 and 199/436, respectively) of cartridge costs alone. CONCLUSION: Testing single and pooled specimens had a high level of agreement, with complete concordance when using Xpert-Ultra. Pooling samples could generate significant cartridge savings during ACF campaigns.

Despite being treatable and curable, tuberculosis (TB) remains one of the main infectious killers in the world, as ten million people fall ill and 1.4 million die from the disease each year. 1 Its diagnosis is usually reliant on passive case finding (PCF), in which health services wait for individuals with symptoms of TB to attend a health facility to initiate the diagnostic process. Although PCF identifies most people with TB in locations with adequate access to health services, it misses those unwilling or unable to attend the clinics and is a major reason only seven of the ten million people with TB are diagnosed and notified. 2 Individuals missed by passive approaches often include vulnerable populations such as internally displaced, migrant or rural populations, women, the unemployed and ethnic minorities, 3 4 who may face multiple societal and economic barriers to attend the service, including catastrophic costs. 5 6 It is,

What is already known? ► Our study adds to the emerging body of evidence that the pooling methods for testing with molecular assays can improve the efficiency of testing for tuberculosis (TB) , potentially enabling the screening and testing of larger numbers of people more cost-effectively.

What are the new findings?

► These findings contribute to recognised gaps in funding sources for the procurement of sufficient cartridges for testing all individuals with presumptive TB, which jeopardises access to high sensitivity WHO-recommended rapid molecular diagnostic tests, such as the GeneXpert Xpert MTB/RIF and Xpert-Ultra.

What do the new findings imply?

► The method has not been tested during a real health crisis situation, such as the COVID-19 pandemic. The study took place at a time laboratory resources were being diverted and healthcare workers were repurposed for SARS-CoV2 testing. The higher efficiency of the pooling method can contribute to cope with these challenging times.

thus, recognised that, to be inclusive and reduce the socioeconomic impact of TB, 7 health services need to include active case finding (ACF) approaches that involve proactive interventions to extend the reach of TB services for diagnosis 8 and treatment. 9 Although ACF interventions can be very effective, 10 11 they are less standardised than PCF, as they address the specific barriers of multiple target populations and are more resource and time intensive than PCF. 12 The WHO recommends testing all individuals with presumptive TB with molecular assays, such as the Xpert Mycobacterium tuberculosis/rifampicin (Xpert MTB/ RIF) and Xpert-Ultra (Cepheid Sunnyvale, California), 13 with the latter being preferred given its higher sensitivity. 14 Although the use of these assays is expanding, the assay cartridge unit costs of US$ 9.98 per test 15 remains one of the main hurdles for its wider implementation in low-income and middle-income countries. Diagnostic test costs can limit the expansion of ACF activities, as they require testing large numbers of individuals with relatively lower yields than PCF. 16 Since 2015, the Lao National Tuberculosis Control Center (NTC) has conducted ACF by implementing intensified case finding activities to increase the detection of individuals with TB in high burden districts of the country. These activities include the sensitisation of the population, the local provision of chest X-rays for screening (independently of symptoms) and the identification of individuals with symptoms of TB who have not attended health facilities. Participants with abnormal chest X-rays or symptoms of TB are tested using Xpert MTB/RIF or Xpert-Ultra. 17 The activities have increased case detection, although the cost of the Xpert cartridges is considered high and is the main limiting factor to implement the intervention on a larger scale.

One approach that could increase the affordability of Xpert testing is to test several samples together using the pooling method. 18 This procedure combines (or pools) the sputum of several individuals into one pot and tests them together with a single test. If the test is positive, the pool's samples are retested individually to identify the positive sample(s) while if the test is negative, all samples in the pool are considered negative, resulting in 30%-40% savings in Xpert cartridge costs alone depending on the prevalence of TB in the population tested. 19 Therefore, pooling may hold great promise for ACF, but there are few reports of its performance under operational conditions. 20 Here, we report a prospective study to assess the sensitivity and specificity of the pooling method using Xpert MTB/RIF and Xpert-Ultra during intensified case finding interventions, and its potential to increase the affordability of Xpert testing in Lao PDR.

We conducted two independent prospective surveys embedded within the ACF activities of Lao's NTC, from March to April 2020 and from January to March 2021. Both surveys were cross-sectional and used the same recruitment and testing procedures. The 2020 survey aimed to assess the performance of the pooling method when testing samples with Xpert MTB/RIF, while the 2021 survey assessed the method when using Xpert-Ultra, after its release for routine use by Lao's NTC.

ACF was conducted in Lao's high TB burden areas, which are programmatically defined as TB incidence ≥100 cases per 100 000 population. The 2020 survey was conducted in Vientiane Capital, Luang Prabang and Savannakhet provinces with estimated populations of 890,129, 468 375 and 1 051 675 inhabitants, respectively, and TB notification rate of 134, 88 and 102 cases per 100 000 population in 2020, respectively. The 2021 survey was conducted in Saravane and Oudomxay, with 430 428 and 333 934 population and TB notification rates of 127 and 110 cases per 100 000 population in 2020, respectively.

Both surveys were conducted in the same fashion. Before an ACF activity, the NTC team met the province and district health authorities and conducted preparation visits with the provincial TB coordinator, district TB manager and village authorities, distributed health education materials, obtained the addresses of individuals with TB and line listed household contacts. At an agreed date, the NTC team set up a digital chest X-ray machine and a four-module GeneXpert platform in the village and invited all residents to complete a questionnaire on signs and symptoms, history and treatment of TB and offered chest X-rays for screening, independently of the presence of symptoms. Individuals with abnormal chest X-rays and those who indicated having cough >2 weeks duration were asked to provide sputum samples for Xpert testing and were managed according to the decision tree shown in figure 1. Sputum samples were tested with Xpert following the manufacturer's instructions. 21 Sputum samples tested individually with Xpert MTB/ RIF or Xpert-Ultra were processed in the village GeneXpert platform. Consecutive samples with remnant volumes ≥0.5 mL were included in the pooling studies and were transported to the National TB Reference Laboratory in Vientiane using a cold chain. Samples were transported after the sample reagent had been added. Turned around time to testing was <48 hours after the sample reagents had been added and samples were maintained in a cold chain at all times. Sputum samples from four participants were pooled together, with a volume of 0.5 mL of sputum each added to a pot, to obtain an aggregated volume of 2 mL. 21 Samples for a pool were selected consecutively and staff were blind to the individual Xpert test results and the pooled specimen was tested using one new Xpert cartridge.

Categorical data were summarised using descriptive statistics and χ 2 tests were used to test for statistically significant differences. Individuals unable to produce sputum were excluded from the analysis. The pooled samples were compared with the four Xpert MTB/RIF and Xpert-Ultra individual results and their agreement was tested using BMJ Global Health kappa statistics. The CT values and grades (trace, very low, low, medium and high) of individual and pooled tests were compared with describe the effect of combining the samples. Cost differences were calculated on the bases of the number of cartridges required to test all specimens using pooled and individual testing.

Sample size for the surveys was not formally estimated as we were limited by the expected number of participants attending the campaigns before the COVID-19 lockdown, the capacity of staff to conduct additional testing to their routine activities and the number of spare cartridges available for research purposes.

Patient and public involvement It was not appropriate or possible to involve patients or the public in the design, or conduct, or reporting or dissemination plans of our research.

The 2020 survey included 436 participants, 334 (76.6%) men and 102 (23.4%) women, and 29 (6.7%, 95% CI 4.7% to 9.4%) were Xpert MTB/RIF MTB-positive. The 2021 survey also included 436 participants, 222 (50.9%) men and 214 (49.1%) women, and 37 (8.5%, 95% CI 6.5% to 11.5%) were Xpert-Ultra MTB-positive (p value >0.1, table 1). Men were more likely to be MTB-positive than women in 2020 (26/334 (7.8%) men vs 3/102 (2.9%) women, respectively, p=0.014); but women were more likely to be MTB-positive than men in 2021 (12/222 (5.4%) vs 12/214 (11.7%), respectively, p<0.008). Each survey included 109 pools of four patients. 

Our surveys compared pooling with single testing during ACF for TB in a low-income country. Our results confirm that testing individual and pooled samples with the GeneXpert platform can achieve a high level of concordance. Concordance was higher with Xpert-Ultra than with Xpert MTB/RIF, which is in agreement to regional studies evaluating pooling with Xpert-Ultra in Cambodia 20 and Vientiane, Lao PDR (Iem et al, in press). Discrepancies between individual and pooled Xpert MTB/RIF tests only occurred among pauci-bacillary samples with high Xpert CT values, suggesting that some samples with low DNA concentrations fall below the assay's limit of detection and that the better agreement of Xpert-Ultra is due to its higher sensitivity. Consequently, some patients with paucibacillary disease could be missed by pooling, especially if testing is based on Xpert MTB/RIF. The pooling strategy can lead to significant cost savings and facilitate testing of more individuals for a given number of cartridges. In our setting, pooling samples would double the number of people tested with the same number of cartridges. This is higher than in PCF studies, where pooling is reported to save up to 40% of cartridges. 19 Cartridge savings are a function of the proportion of people with MTB-positive results and their distribution within the pools. If the proportion positive is low, a low number of pools would need to be retested, resulting in higher cartridge savings. For example, in a survey in Lao's district clinics, 12% of individuals tested Xpert-positive, and pooling resulted in 38.3% and 41.7% cartridge saving costs with Xpert MTB/RIF and Xpert-Ultra, respectively (Iem et al, in press), while in our survey setting, the proportion of positives was 8.5%, which led BMJ Global Health to higher savings. The proportion of participants with positive tests in ACF is often lower than reported from studies using PCF, typically below 5% depending on the target population, 16 22 and lower to 10%-20% of individuals attending TB clinics in PCF. 23 24 We have, thus, shown that pooling could be highly efficient when testing populations using ACF, and further studies among such populations are warranted. Since the pooling method is a laboratory change, it would not affect the screening algorithm and can be easily instituted without any major modifications. Previous systematic reviews have highlighted that individual and pooled RIF results are often discordant, with pools containing RIF-negative samples often returning RIF-indeterminate pooled results, 19 and our findings are in agreement with these observations. Although samples with pooled RIF results would be routinely confirmed at the time of retesting, the samples of a positive pool to identify the individual MTB-positive samples, it is important to highlight that pooled RIF results are unreliable and should not be used for clinical management.

Further studies could explore ways to further improve the efficiency of pooling when combined with other screening tools, such as C-reactive protein (CRP) 25 and digital chest X-rays with Computer-aided diagnosis (CAD). 26 27 Both tools can identify individuals with and without the traditional symptoms of TB, although their relatively lower specificity requires confirming the diagnosis with more specific molecular assays. Although using tests combinations could increase assay costs, individuals with a positive CRP or abnormal chest X-rays CAD could be confirmed using the pooling method, and its efficiency gains could increase the affordability of tests combinations.

In conclusion, we have shown pooling samples for TB diagnoses during ACF campaigns, which can replicate testing samples with individual tests. The approach can facilitate testing higher numbers of patients with lower cartridge costs, increasing the affordability of testing with molecular assays. The high level of agreement between individual and pooled samples obtained with Xpert-Ultra demonstrates that pooling can be reliable and contribute to achieve the WHO End TB strategy targets in resourcelimited settings.

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Acknowledgements The authors would like to thank the National Tuberculosis Control Center (NTC) of Lao PDR for authorising the study and facilitating samples transportation from the field until the National TB Reference Laboratory (NTRL) in 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 finding team for their engagement, screening and enrolling patients, collecting, and shipping samples from the collection sites and to laboratory technicians of 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 Global Health Trial Unit, LSTM for generating databases and data curation.Contributors The study was designed by LEC, JC, VI, KK and JAMK. Patients screening and enrolling, individual sample testing on Xpert MTB/RIF and Xpert Ultra were conducted by PC, SaS, SoS, PS, and SiS in the field during the ACF campaign. VI performed the pooled testing. Data collection and analysis were conducted by VI, 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 manuscript. LEC is responsible for the overall content and is the guarantor of the study The final version has been read and approved by all named authors. 

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.

Ethics approval The study was approved by the Lao NTC and the Liverpool School of Tropical Medicine Research Ethics Committee, UK (Ethical waiver 20-037), and informed consent waiver was obtained.Provenance and peer review Not commissioned; externally peer reviewed.Data availability statement Data are available upon reasonable request. Not applicable.Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is 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/.

Vibol Iem http://orcid.org/0000-0001-6155-1402 Luis E Cuevas http://orcid.org/0000-0002-6581-0587