Practical manual of processing stool samples for diagnosis of childhood TB

Practical manual of processing stool samples for diagnosis of childhood TB

Practical manual of processing stool samples for diagnosis of childhood TB Practical manual of processing stool samples for diagnosis of childhood TB ISBN 978-92-4-004265-0 (electronic version) ISBN 978-92-4-004266-7 (print version) © World Health Organization 2022 Some rights reserved. This work is available under the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 IGO licence (CC BY-NC-SA 3.0 IGO; https://creativecommons.org/licenses/by-nc-sa/3.0/igo). Under the terms of this licence, you may copy, redistribute and adapt the work for non-commercial purposes, provided the work is appropriately cited, as indicated below. In any use of this work, there should be no suggestion that WHO endorses any specific organization, products or services. The use of the WHO logo is not permitted. If you adapt the work, then you must license your work under the same or equivalent Creative Commons licence. 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Design by Inis Communication Contents Acknowledgements iv About this manual v Target audience v Abbreviations and acronyms vi Part A Background 1 A1 Introduction 1 A2 Stool processing methods 1 A3 Comparison of stool processing methods 2 A4 Stool processing methods described in this manual 3 Part B How to perform stool testing 5 B1 Stool collection and storage 5 B2 Stool processing methods 7 Part C Implementation of stool testing 13 C1 Where to place stool testing in the tiered laboratory network 13 C2 Steps and processes for implementation 14 References 19 Annex 1. Overview of publications on stool processing for TB detection until December 2021 23 Annex 2. List of activities for the implementation of stool testing 31 Annex 3. Laboratory tool for assessing stool testing by Xpert MTB/RIF and Xpert MTB/RIF Ultra 32 iii Acknowledgements This guide was developed as a product of the Global Laboratory Initiative (GLI) Core Group. Development was led by Petra de Haas (KNCV Tuberculosis Foundation, The Hague, The Netherlands), Organization for Migration, Geneva, Switzerland), Patricia Hall (Centers for Disease Control and Prevention, Atlanta, GA, United States of America) and Wayne van Gemert (Stop TB Partnership, Geneva, Switzerland), under the coordination of the GLI Working Group Secretariat within the Global TB Programme of the World Health Organization (WHO). The writing of this manual was supported by Pauline Lempens (KNCV Tuberculosis Foundation, The Hague, The Netherlands). Brands, Nazir Ismail, Carl-Michael Nathanson and Sabine Verkuijl (WHO Global TB Programme, Geneva, Switzerland). Special thanks to Pamela Nabeta (Foundation for Innovative New Diagnostics, Geneva, Switzerland), Maryline Bonnet (TB-Speed project, Université de Montpellier, Montpellier, France), Manon Lounnas (TB-Speed project, Université de Montpellier, Montpellier, France) and Martina Casenghi (Elizabeth Glaser Pediatric AIDS Foundation, Geneva, Switzerland) for their valuable feedback and contribution. WHO also appreciates the feedback provided by the following GLI Core Group members and public health specialists: Khalide Azam, Sarabjit Singh Chadha, Fernanda Dockhorn Costa, Sarder Tanzir Hossain, Kristin Kremer, Edine Tiemersma and Andrii Slyzkyi (KNCV Tuberculosis Foundation, The Hague, The Netherlands). GLI is a Working Group of the Stop TB Partnership. Development and publication of this document Development. Practical manual of processing stool samples for diagnosis of childhood TBiv About this manual This practical manual aims to help countries implement stool testing into their tuberculosis (TB) diagnostic and clinical practice. It provides evidence and incorporates recommendations on the use of stool as a sample type for diagnosing TB. It also contains steps for implementation and details on the laboratory process for conducting stool testing using Xpert MTB/RIF and Xpert MTB/ RIF Ultra (Xpert Ultra). Target audience The target audience for this manual is ministries of health, programme managers, clinicians, front- line health workers (especially in paediatric TB), TB testing site managers, supervisory laboratory staff and GeneXpert users at national, state or provincial and testing site level, as well as implementing partners. About this manual v Practical manual of processing stool samples for diagnosis of childhood TB Abbreviations and acronyms CI confidence interval FIND Foundation for Innovative New Diagnostics GA gastric aspirate GLI Global Laboratory Initiative HIV human immunodeficiency virus KNCV KNCV Tuberculosis Foundation MTBC Mycobacterium tuberculosis complex NTP national TB programme OSF optimized sucrose flotation POSEE Paediatric TB Operational and Sustainability Expertise Exchange PPE personal protective equipment SOP standard operating procedure SOS simple one-step SPK stool processing kit SR sample reagent TB tuberculosis WHO World Health Organization Practical manual of processing stool samples for diagnosis of childhood TBvi Part A Background A1 Introduction Each year, 1.1 million children globally fall ill with tuberculosis (TB), of whom only 400 000 are notified; with the case detection gap being highest in children aged under 5 years. In 2020, this gap was 72.5%, whereas for children aged 5–14 years the gap was 55.4% (1). Obtaining bacteriological confirmation of TB is challenging in children because of the frequent paucibacillary presentation of the disease. Diagnostic specimens have a low bacterial load, which decreases diagnostic test sensitivity. Diagnosis is further complicated by the fact that obtaining a sufficient volume of specimen from children can be difficult. Children, especially young children, generally cannot effectively expectorate and produce a sputum sample. Often, invasive procedures such as sputum induction or gastric aspiration are required to obtain a specimen. In many settings, the equipment and consumables required for sputum induction or gastric aspiration are not available, or clinical staff lack the skills to competently perform these methods. Also, parents or other caregivers may be reluctant to have these invasive procedures performed on their children. Stool collection is a non-invasive method. Mycobacterium tuberculosis complex (MTBC) can be detected in stool specimens because sputum is coughed up and subsequently swallowed, and then passes through the gastrointestinal system. Since 2021, the World Health Organization (WHO) has recommended stool as a new specimen type alongside sputum (expectorated or induced), nasopharyngeal aspirate or gastric aspirate (GA) for both Xpert MTB/RIF and Xpert Ultra as the initial diagnostic test for TB and the detection of rifampicin resistance in children aged under 10 years with signs and symptoms of pulmonary TB (2, 3). The recent WHO consolidated guidelines on TB, Module 5, and the accompanying operational handbook, provide recommendations on the management of TB in children and adolescents, including on the use of stool (4, 5). Recent systematic reviews for the detection of MTBC on stool using Xpert MTB/RIF found pooled sensitivities of 50% (95% confidence interval [CI]: 44–56), 57% (95% CI: 40–72), 62% (95% CI: 44–76) and 67% (95% CI: 52–79), and pooled specificities of 99% (95% CI: 98–99), 98% (95% CI: 96–99), 99% (95% CI: 97–99) and 99% (95% CI: 98–99) compared with a microbiological reference standard (4–9). A systematic review and meta-analysis of Xpert Ultra data found a sensitivity of 53% (95% CI: 35–70) and a specificity of 98% (95% CI: 93–99) (10, 11). Given the fact that all specimen types have incomplete sensitivity, using two specimens (preferably of two different specimen types) might increase the chance of obtaining a bacterial diagnosis of TB (12). However, there is no WHO recommendation on this to date. A2 Stool processing methods Various methods to process stool for Xpert MTB/RIF or Xpert Ultra testing have been described (13– 20). The methods vary in terms of technique (or combination of techniques) used to bring stool into suspension (e.g. hand shaking or mechanical shaking using a vortex) and to separate M. tuberculosis Part A Background 1 Practical manual of processing stool samples for diagnosis of childhood TB bacilli from stool debris (e.g. centrifugation, filtration and sedimentation). Consequently, methods vary in complexity, labour intensity, time investment, and the required equipment, supplies and infrastructure. Annex 1 provides a list of relevant publications, giving the stool processing methods used and the reported sensitivities and specificities against various reference standards. Several authors have described relatively simple, centrifuge-free methods for stool processing – for example, Banada et al. (15), Walters et al. (17) and Andriyoko et al. (18). Other simple methods are the optimized sucrose flotation (OSF) method developed by the TB-Speed consortium (19), and the simple one-step (SOS) method developed by the KNCV Tuberculosis Foundation (KNCV) (20). A3 Comparison of stool processing methods Standardized comparison studies are needed owing to the high heterogeneity among stool processing methods and study designs, and consequently among the pooled sensitivity and specificity values from the four systematic reviews (6–9). Jasumback et al. applied four different stool processing methods (15, 17, 20, 21) to stool samples spiked with multiple log concentrations of Mycobacterium bovis bacille Calmette-Guérin (BCG) (22). Walters et al. used a method that included centrifugation (21); this resulted in more frequent detection of BCG at lower concentrations (5/5 replicates at 103 colony forming units [cfu]/mL and 3/5 replicates at 102 cfu/mL) compared with the other three methods – that is, the method used by Banada et al. (15) (3/5 replicates at 103 cfu/mL and 1/5 replicates at 102 cfu/mL), the centrifuge- free swab-based method used by Walters et al. (17) (1/5 replicates at 103 cfu/mL and 0/5 replicates at 102 cfu/mL) and the SOS stool method (20) (3/5 replicates at 103 cfu/mL and 1/5 replicates at 102 cfu/mL). However, numbers were too small to carry out statistical analyses. The SOS stool method was considered to be most suitable for low-resource settings, because of its low error rate, short processing time and minimal requirements regarding biosafety precautions and laboratory equipment (22). In an in vitro study (20), the TB-Speed consortium, in collaboration with KNCV, compared the two-step method described by Andriyoko et al. (18) with the SOS stool processing method (20). The comparison was done using stool samples with different consistency spiked with multiple log concentrations of M. tuberculosis. The SOS stool method was found to have a higher sensitivity than the two-step method; this was attributed to the fact that the two-step method uses an additional dilution step. Three stool processing methods have been assessed in parallel as part of two studies led by the Foundation for Innovative New Diagnostics (FIND) and the TB-Speed consortium. The studies were performed at referral laboratories in Africa and Asia and included the disposable stool processing kit (SPK), which resulted from optimization of the methods described by Banada et al. (15) and Walters et al. (17); the OSF (19); and the SOS (20) stool processing methods. Pooled data from an interim analysis of the FIND and TB-Speed studies suggest similar performance of the three stool processing methods in terms of sensitivity and specificity (23, 24). Briefly, the sensitivity of Xpert Ultra for TB detection in stool was 52.1% (95% CI: 38.3–65.5) for SOS, 48.3% (95% CI: 35.9–60.8) for SPK and 46.8% (95% CI: 33.4–60.8) for OSF, and the specificity was 97.5% (95% CI: 94.9–98.9) for SOS, 97.1% (95% CI: 94.5–98.5) for SPK and 97.8% (95% CI: 95.1–99.1) for OSF (see Table 1). Practical manual of processing stool samples for diagnosis of childhood TB2 The proportion of non-determinate Xpert Ultra results was 8.7% (35/401) for SOS, 11.8% (53/541) for SPK and 10.3% (40/388) for OSF (23). No calculations were planned at this point to determine statistical significance or assess repeat Xpert Ultra test results. A limitation of this interim analysis is the low number of MTBC-positive children included, which resulted in wide confidence intervals around the sensitivity estimates. Table 1. Pooled interim results of the studies by FIND and TB-Speed M et ho d Total number of tests True positive results False positive results False negative results True negative results Sensitivity (95% CI) Specificity (95% CI) SOS 332 25 7 23 277 52.1% (38.3–65.5) 97.5% (94.9–98.9) SPK 368 28 9 30 301 48.3% (35.9–60.8) 97.1% (94.5–98.5) OSF 319 22 6 25 266 46.8% (33.4–60.8) 97.8% (95.1–99.1) CI: confidence interval; FIND: Foundation for Innovative New Diagnostics; OSF: optimized sucrose flotation; SOS: simple one-step; SPK: stool processing kit. The FIND and TB-Speed studies also looked at user acceptability and feasibility of the stool methods, and the findings suggest good acceptability of stool as a sample for TB diagnosis in children (24). All methods were found to be easy to process by laboratory staff at reference level, and all had a high median ease-of-use score. However, most users considered that these methods cannot be performed by non-laboratory staff (e.g. nurses and health care workers) in primary health care settings without access to a laboratory. Overall, the SOS stool method appeared to be the preferred method because it does not require additional equipment or supplies compared with sputum Xpert testing (23, 24). A4 Stool processing methods described in this manual This practical manual focuses on two stool processing methods: the OSF and SOS stool methods. The disposable SPK assessed during the FIND and TB-Speed studies was a prototype kit; although shown to be accurate, the kit offers no additional benefits over the simpler OSF and SOS stool methods and its development and commercialization will therefore not be advanced. Hence, the SPK and the underlying methods described by Banada et al. (15) and Walters et al. (17) are not the focus of this manual. Part A Background 3 Practical manual of processing stool samples for diagnosis of childhood TB Practical manual of processing stool samples for diagnosis of childhood TB4 Part B How to perform stool testing B1 Stool collection and storage Stool collection is usually done by the caregivers or the patients themselves, depending on the age of the child. Ideally, the collection takes place at the health care facility. However, to obtain a specimen on demand is often challenging; therefore, stool is collected at home and the patient or caregiver needs to return to the facility for specimen submission. Following the procedures at the local setting, the patient or caregiver is provided with a stool container. Different types of containers can be used; some of these have a small spoon integrated into the screwcap, as shown in Fig. 1. Importantly, the container should be wide-mouthed to make it easy to add the stool, and it should be able to hold at least 3–5 g of stool. Thus, sputum containers can also be used for stool collection. Fig. 1. Example of a stool container The stool container should be provided in a plastic bag containing absorbent material, to keep the container clean during transport to and from the child’s home. The absorbent material will absorb any substances that may leak out of the container if it is not closed properly. The patient or caregiver should be instructed by the laboratory staff or other health care worker on how to collect the stool sample. The instructions below can be adapted to country-specific needs. A flyer to provide to the patient or caregiver, showing the steps on how to collect the stool sample and explaining the importance of returning the sample to the clinic, can be useful. Part B How to perform stool testing 5 Practical manual of processing stool samples for diagnosis of childhood TB Instructions for the patient or caregiver on how to collect the stool sample: 1. Ideally, collect the stool sample during the first daily bowel movement. If possible, first empty the bladder, to avoid mixing urine with the stool sample. 2. Put some clean plastic sheeting on the spot where the stool will be dropped, to ensure the collection of a clean sample. Avoid contamination of the plastic with soil, detergent or disinfectant from the toilet. 3. If the stool sample needs to be collected from a child that uses a diaper (i.e. a nappy), then either collect the stool directly from the diaper as soon as possible after defecation, or put a plastic sheet in the diaper to avoid (prolonged) contact between the stool and the surface of the diaper (diapers may contain substances that inhibit the test). 4. Fill the stool container with the stool sample up to half full, using (for example) the spoon provided with some types of containers, a clean plastic bag, a clean piece of cardboard or a clean spoon. Do not fill the container to the brim. Only a small amount of stool is required for testing (2 g is sufficient for both testing and retesting if the first test is unsuccessful). 5. Close the container tightly, place the container in the plastic bag provided (preferably a self- sealable bag) and close the bag. Leave the absorbent material in the plastic bag so that this material can absorb any substances that may leak out of the container. 6. As soon as the stool sample has been collected, store the plastic bag containing the stool container in a clean, cool place (e.g. in a fridge if possible), avoiding exposure to direct sunlight. Do not freeze the sample. 7. Take the plastic bag containing the stool container to the health care facility, preferably on the same day that you collected the stool sample. For transport and storage of stool specimens, the same conditions apply as for transport and storage of sputum specimens for Xpert testing. Thus, between collection and testing, stool specimens can be kept at a maximum of 35 °C for up to 3 days, followed by a maximum of 7 days at 2–8 °C. Ideally, stool sample containers should be kept at 2–8 °C while being sent to the laboratory and should then be stored in the refrigerator (at 2–8 °C) until testing can be performed. Sample preparation and testing should be started as soon as possible. Research is ongoing on how best to store stool samples (25, 26). Before handling, allow the stool sample to warm up to room temperature. Practical manual of processing stool samples for diagnosis of childhood TB6 B2 Stool processing methods B2.1 OSF stool processing method Principle The TB-Speed OSF method is based on the creation of a sucrose density gradient to support separation of M. tuberculosis from stool debris (19). Biosafety requirements Although the OSF method involves a separation step before inactivation, this step leads to only minimal concentration of the M. tuberculosis bacilli. Therefore, the OSF method can be performed in an open, well-ventilated space with appropriate aerosol reduction practices and appropriate use of personal protective equipment (PPE). Procedure In brief, 0.5 g of stool is transferred into a 50 mL Falcon tube (for solid stool specimens) or an empty stool container (for liquid stool specimens) (Fig. 2). Next, 10 mL of Sheather’s solution (56% sucrose solution) is added to the Falcon tube or stool container, which is then shaken 20 times to homogenize the stool specimen and left to stand for 30 minutes at room temperature, to sediment larger particulate matter. Then 0.5 mL of the resulting supernatant is transferred to another 15 mL Falcon tube, together with 1.8 mL sample reagent (SR) from the Xpert MTB/RIF or Xpert Ultra assay. The mixture is shaken vigorously 20 times then incubated for 15 minutes at room temperature, after which 2 mL of the mixture is transferred to the Xpert MTB/RIF or Xpert Ultra cartridge. The cartridge is then inserted into the GeneXpert instrument. Use of the GeneXpert instrument and interpretation of Xpert results are done according to the manufacturer’s instructions. Detailed information on how to prepare the Sheather’s solution and on how to perform the OSF stool processing method can be found in the standard operating procedure (SOP) on the TB-Speed website (27). TB-Speed is a research project sponsored by Inserm (Institut national de la santé et de la recherche médicale, the French National Institute of Health and Medical Research) and funded by Unitaid and L’Initiative (28), with support from ANRS-MIE (an agency within Inserm) (29). Part B How to perform stool testing 7 Practical manual of processing stool samples for diagnosis of childhood TB x20 Fig. 2. Schematic overview of the SOP for the detection of M. tuberculosis complex and resistance to rifampicin in stool by using the TB-Speed OSF method and the Xpert MTB/RIF or Xpert Ultra assay 2. Sedimentation during 30 minutes 3. Transfer 0.5 mL from the top of the specimen to a 15 mL Falcon tube and add 1.8 mL of Sample Reagent + shake vigorously 20 times 4. Incubation and transfer to the Xpert Ultra cartridge 1. Add 0.5 g of stool in 10 mL of Sheather’s solution + shake 20 times OSF: optimized sucrose flotation; SOP: standard operating procedure. 15 minutes 30 minutes Practical manual of processing stool samples for diagnosis of childhood TB8 B2.2 SOS stool processing method Principle The SOS stool processing method uses one step to release M. tuberculosis from stool. Particulate matter is sedimented by gravity, and it is assumed that this allows TB bacilli to float to the top of the watery solution because of their lipid-containing cell wall (20). Biosafety requirements In the SOS stool method, stool is added directly into the SR bottle provided in the Xpert kit; this results in immediate inactivation of the bacteria. Therefore, the SOS stool method can be performed in an open, well-ventilated space with appropriate aerosol reduction practices and appropriate use of PPE. Procedure Before stool processing, the consistency of the stool specimen is assessed using the Bristol stool scale (30). For stool with the appearance of Bristol type 1 to 5 (formed stool), 0.8 g or a thumbnail size amount of stool (Fig. 3) is directly transferred from the stool container into the SR bottle using a wooden stick or applicator (Fig. 4). For stool with the appearance of Bristol type 6 and 7 (liquid stool), 2 mL SR is removed from the SR bottle, then 2 mL of stool is transferred to the SR bottle using a balloon pipette (Fig. 3). For all types of stools, the SR is shaken vigorously for 30 seconds and then incubated for 10 minutes at room temperature. This step is repeated once. After carefully ensuring that solid particles and debris have settled, 2 mL of the supernatant is then transferred from the SR bottle to the Xpert MTB/RIF or Xpert Ultra cartridge. The cartridge is then inserted into the GeneXpert instrument. Use of the GeneXpert instrument and interpretation of Xpert results is done according to the manufacturer’s instructions. A detailed SOP on how to perform the SOS stool processing method can be found in the KNCV stool toolbox on the KNCV website (31). Part B How to perform stool testing 9 Practical manual of processing stool samples for diagnosis of childhood TB Fig. 3. In the SOS stool processing method, 0.8 g or an amount of stool equal to the size of a thumbnail is used SOS: simple one-step. Fig. 4. Schematic overview of the SOP of the SOS stool processing method and the Xpert MTB/RIF or Xpert Ultra assay for different types of stoolsa SOP: standard operating procedure; SOS: simple one-step; SR: sample reagent. a The upper panel shows the procedure for stool of Bristol type 1–5 (i.e. formed stool) and the lower panel for stool of Bristol type 6 and 7 (i.e. liquid stool). Transfer 0.8 g of stool into the SR* bottle Transfer 2 mL ‘debris free’ supernatant # into Xpert cartridge B Take 2 mL SR* out of the SR* bottle and dispose of it Take 2 mL of stool out of the stool bottle and transfer it into the SR bottle Transfer 2 mL ‘debris free’ supernatant # into Xpert Ultra cartridge Two times: shake 30 seconds and stand 10 minutes Two times: shake 30 seconds and stand 10 minutes * sample reagent buer SOS STOOLBOX7 Figure 1. The Bristol Stool Scale, named after the University of Bristol where it was fi rst described by Lewis and Heaton 21; from type 1, being the most solid, to type 7, being the most liquid. Practical manual of processing stool samples for diagnosis of childhood TB10 B2.3 Characteristics summary of OSF and SOS stool processing methods Table 2 summarizes the characteristics of the OSF and SOS stool processing methods. The table includes additional materials required, preparation time (32), incubation time and biosafety requirements. Table 2. Characteristics of the OSF and SOS stool processing methods SOS OSF Additional suppliesa Applicator to transfer stool (wooden stick), balloon pipettesb Applicator to transfer stool, sample preparation tubes, distilled water, sucrose (Difco), balloon pipets Additional equipmentc, d None Electronic balance, heating magnetic stirrer and bar magnet, screw cap glass bottle (1 L), graduated cylinder (1 L), autoclave Median preparation time (range) 23 (20–30) minutes 56 (45–87) minutes Incubation time Incubation 10 minutes Sedimentation 10 minutes Sedimentation 30 minutes Incubation 15 minutes OSF: optimized sucrose flotation; SOS: simple one-step. a Additional supplies are supplies that are required in addition to what is provided in the Xpert MTB/RIF or Xpert Ultra kit (the kit includes cartridges, sample reagent buffer and balloon pipettes). b Additional balloon pipettes might be required when a high load of liquid stool is processed and the balloon pipets provided in the Xpert kit are not sufficient to cover the work. c Additional equipment is equipment required in addition to the GeneXpert instrument. d Preparation of the Sheather’s solution will be done in batches at central level and distributed to the sites, meaning that the equipment listed is for the central level, not for the GeneXpert site. Part B How to perform stool testing 11 Practical manual of processing stool samples for diagnosis of childhood TB Practical manual of processing stool samples for diagnosis of childhood TB12 Part C Implementation of stool testing This section describes the requirements for the implementation of stool testing. It follows the 10 steps outlined in the chapter on implementation of new diagnostics in the WHO operational handbook on tuberculosis Module 3: Diagnosis (3). C1 Where to place stool testing in the tiered laboratory network Introduction of stool testing for diagnosis of paediatric TB adds a new sample type to an existing diagnostic platform. Currently, WHO recommends that stool be tested using the Xpert MTB/RIF and Xpert Ultra assays. Thus, it is logical to incorporate stool testing in the existing laboratory network for Xpert MTB/RIF or Xpert Ultra testing, and to use the GeneXpert instruments and network already present in-country. When scaling up GeneXpert instruments, the placement strategy needs to consider access for specific key populations benefiting from stool testing (e.g. children) and an effective sample transportation system. Ultimately, countries should be able to perform stool testing at any GeneXpert site; for example, in the same laboratory performing sputum testing for TB. However, when resources are limited, the use of stool should mainly be promoted where it would be of most value, and it is important to consider that stool testing might be most cost-effective in settings with higher prevalence of TB (32). Furthermore, the choice of specimen type (GA, induced sputum or stool) collected for children depends on the acceptability for children, parents and caregivers, health care workers and other stakeholders; on the feasibility of collecting and preparing specimens in the local context; and on local test availability. To help clinicians decide on whether a child should be started on TB treatment, the WHO operational handbook on the management of TB in children and adolescents provides guidance on treatment decision algorithms that integrate a risk assessment for rapid progression of TB disease, bacteriological confirmation where possible and available (including stool testing as relevant), history of contact, clinical signs and symptoms and findings on chest radiography (5). The Pediatric TB Operational and Sustainability Expertise Exchange (POSEE) is a task force under the Child and Adolescent TB Working Group and an entity of the Stop TB Partnership. POSEE has written a position paper that clearly describes the position of the microbiological diagnosis in the diagnostic pathway (12). Part C Implementation of stool testing 13 Practical manual of processing stool samples for diagnosis of childhood TB C2 Steps and processes for implementation C2.1 Policies and planning Globally, countries have adopted either Xpert MTB/RIF or Xpert Ultra (or both) in their national guidelines as an initial test to diagnose TB and detect resistance to rifampicin. However, most countries do not include stool specimens as a sample type for diagnosis of TB among children. Therefore, national guidelines and policies need to be adapted and updated to include stool for the diagnosis of TB in children, in line with the new WHO recommendations (3). The in-country technical working group leads the review of the national guidelines and policies, including the implementation of stool testing. Generally, a situational analysis plan contains a map of the health care centres that have a GeneXpert instrument and linkages to referring and referral laboratories. To prepare a costed operational plan with timelines and milestones for the introduction of stool testing, the working group can use the existing situation analysis if one is available or can undertake a new analysis. The POSEE task force has developed a budget tool for specimen collection for TB diagnosis to support national TB programmes (NTPs) in budget forecasting, and has included stool as one of the possible specimens. The budget tool and other relevant tools are available from the Child and Adolescent TB Working Group page on the Stop TB Partnership website (33). The best approach is a phased implementation, starting with a pilot implementation at a few selected sites that have a high TB notification rate or are actively involved in diagnosis of paediatric TB. These sites can subsequently train other sites, ensuring that all provinces achieve access to Xpert stool testing and have trained staff to perform the tests. To determine which sites to prioritize for the implementation of stool Xpert testing, the number of children seen with signs and symptoms of TB (presumptive TB) can be used as an indicator. Alternatively, the number of children with respiratory infections, pneumonia or malnutrition can be considered. However, it may be difficult to retrieve this information as NTPs often do not systematically collect such data – the number of children clinically or radiographically diagnosed with TB is a good alternative for selecting the sites. C2.2 Regulatory processes No registration specific for stool testing is needed, because stool is a sample type that is already collected in routine settings for other diseases and laboratory tests. Diagnosis of TB from stool samples uses GeneXpert instruments, which are already registered for this purpose. C2.3 Equipment and site preparation For site preparation, equipment additional to the GeneXpert instruments might be needed, depending on the stool processing method to be implemented. Implementation of the SOS stool method requires no additional equipment other than the equipment currently used for Xpert MTB/ RIF or Xpert Ultra sputum testing. Implementation of the OSF method does require some additional equipment, but it is low cost (see Table 2). The introduction of stool testing will increase the use of the GeneXpert platform in each site. Therefore, for each site enrolled in stool testing, the expected workload needs to be estimated and matched with the diagnostic capacity of the available GeneXpert instruments. The number of broken or in other ways non-functional, modules should be considered, because this naturally reduces the Practical manual of processing stool samples for diagnosis of childhood TB14 capacity per site. The costed operational plan for the site preparation will include provision of stool collection and processing, workload analysis, staff mapping, training of staff and other logistical needs. Annex 2 provides an example of what is needed to start stool testing; it can be adapted to country specifics and used to check the readiness of the sites to implement stool testing. C2.4 Supply chain For implementation of stool testing, a few additional supplies might be needed on top of those that are routinely used for sputum Xpert testing (see Table 2). Most important for supply management is the increase in the number of Xpert cartridges used. For each site, the incremental workload needs to be estimated and matched with the number of cartridges to be supplied. Owing to its higher sensitivity, Xpert Ultra is preferable to the Xpert MTB/RIF assay for the detection of TB in children. Another requirement is stool containers, which can vary in type, as discussed in Section B1. Transportation of stool samples should use the same transport network that is used for sputum Xpert testing; ideally, a cold chain should be maintained, with samples kept at low temperature until testing. Each stool container should be packed in a ziplocked plastic bag containing absorbent material with a biohazard label on the outside, as for sputum transportation. If such bags are not available, filled stool containers should at least be wrapped in toilet paper and placed separately in a simple plastic bag, to avoid any cross contamination should leakage occur during transport to the laboratory. C2.5 Procedures The most up to date SOPs of the OSF stool processing method can be found on the TB-Speed website (27), and a detailed SOP for the SOS stool method can be found on the KNCV website (31). The SOPs should be customized to the country requirements, and translated into local languages where necessary. C2.6 Digital data When stool testing is implemented, instructions need to be provided to the laboratory staff on what relevant details should be added when collecting and analysing the data. For example, when starting the test run in the GeneXpert instrument, “stool” should be entered within the field for sample type. This allows the test runs of stool samples to be separated from test runs of other sample types when measuring the positivity rate or other quality indicators. If the GeneXpert instrument is linked to a connectivity system (e.g. GXAlert or DataToCare) that allows additional data to be entered, it is useful to add information about the stool sample or patients (34). Such information could include, for example, the consistency of the stool sample (e.g. as listed in the Bristol chart, or grouped as “formed”, “semi formed” or “liquid”) or the age or other characteristics of the child patient – capturing this type of information can be useful for operational research purposes. Part C Implementation of stool testing 15 Practical manual of processing stool samples for diagnosis of childhood TB C2.7 Quality assurance control and assessment Targets of quality indicators (e.g. error and invalid rates) that are set for sputum Xpert testing might need to be adjusted when performing Xpert testing on stool. Current studies suggest that the initial non-determinate rate for stool is slightly higher than for sputum Xpert testing. In comparison with sputum, stool naturally contains a high load of solid particles, which may lead to errors linked to issues with the sample transfer through the microfluid filter in the cartridge (e.g. if the supernatant containing the bacteria is not well separated from the debris). Current studies on stool testing show that the most common error code obtained is “error 2008”, which is linked to this phenomenon. Also, stool contains other organic substances not present in sputum that might inhibit the polymerase chain reaction, resulting in invalid Xpert results. Repeating the test once using the same sample decreases the rate of non-determinate Xpert results (35). More studies are needed to provide better insights on these aspects. C2.8 Recording and reporting For the implementation of stool testing, the recording and reporting tools need to be carefully reviewed and adapted to include stool as a sample type. For analysis of both the number of notified cases and quality indicators, it is important that the outcome of the test can be stratified by sample type, including stool. C2.9 Training and competency assessment The most cost-effective approach is to first train staff already competent with sputum Xpert testing on how to perform the specific steps for stool processing, using either the OSF or SOS processing method; these staff are already familiar with the use of the GeneXpert instrument. For staff who are not competent with sputum Xpert testing, a more extensive training is required. As observed for sputum Xpert testing, it takes time for the laboratory staff to become familiar with the use of stool samples and with the processing method. Therefore, it might be that, when initially implementing the use of stool, there will be slightly more non-determinate Xpert results; however, this would be expected to decrease as staff gain more experience. This situation should be considered when ordering Xpert cartridges. In parallel with the training on stool processing for the laboratory staff, all involved health care providers should be trained to perform the stool collection procedure and to explain the procedure to parents and caregivers. Generally, at most health care centres, stool is routinely collected for other diseases (e.g. parasitology). Thus, health care staff are likely to already have the knowledge and the tools to collect this specimen type. However, staff working in TB facilities may be less familiar with stool collection, and thus may need specific training on stool collection and on the development and distribution of a stool collection flyer. Furthermore, all health care providers involved in stool sample collection should be trained on the treatment decision algorithms that include stool as a primary specimen. Practical manual of processing stool samples for diagnosis of childhood TB16 C2.10 Monitoring and evaluation Once staff have been trained and have started using stool as a primary specimen to diagnose TB in children, it is essential to closely monitor and supervise their stool-related work on a weekly or fortnightly basis, at least for the first 2 months. Troubleshooting should address the rate of non-determinate results, as discussed in Section C2.7. For stool testing, additional indicators need to be added to the standard laboratory monitoring and supervision checklist for Xpert MTB/RIF and Xpert Ultra testing (36). Annex 3 provides a list of the stool-specific indicators to consider. These indicators can be added to the standard list of indicators used for supervision and monitoring of sputum Xpert testing. 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Geneva: Global Laboratory Initiative; 2016 (https://stoptb.org/wg/gli/assets/documents/gli_connectivity_guide.pdf, accessed January 2022). 35. Tiemersma E. Accuracy of the simple one-step stool method with Xpert MTB/RIF Ultra assay for the diagnosis of M. tuberculosis in children. 2021. 36. Tuberculosis technical scorecard Xpert MTB/RIF. Geneva: Stop TB Partnership; 2020 (https:// stoptb.org/wg/gli/assets/documents/5%20Find-TB-Scorecard-Xpert-Low-Res.pdf, accessed January 2022). References 21 Practical manual of processing stool samples for diagnosis of childhood TB Practical manual of processing stool samples for diagnosis of childhood TB22 A n n ex 1 . O ve rv ie w o f p u b lic at io n s o n s to o l p ro ce ss in g fo r T B d et ec ti o n u n ti l D ec em b er 2 02 1 Fo r e ac h pu bl ica tio n, th e fo llo w in g ar e pr ov id ed if a pp lic ab le a nd if re po rt ed : p at ie nt a ge , n um be r o f p at ie nt s in clu de d in th e an al ys is/ a ll pa tie nt s el ig ib le , st ud y po pu la tio n, s to ol p ro ce ss in g m et ho d us ed a nd s en si tiv iti es a nd s pe ci fic iti es a ga in st v ar io us r ef er en ce s ta nd ar ds . St ud y, ye ar / co un try (re fe re nc e) Ag e (y ea rs ) ra ng e, m ed ia n [IQ R] In clu de d in an al ys is/ al l el ig ib le Po pu la tio n Am ou nt of st oo l St oo l p ro ce ss in g m et ho d in clu de s: No . m icr o- bi ol og ica lly co nfi rm ed (% ) No . cli ni ca lly co nfi rm ed (% ) Re fe re nc e sta nd ar d St oo l X pe rt pe rfo rm an ce dilution in vortexing centrifugation filtration Se ns iti vit y (9 5% CI ) Sp ec ifi ci ty (9 5% CI ) Ai na n, 20 21 / Ta nz an ia (1 ) NR , 2 .1 7 [1 .1 6– 5. 19 ] 22 5/ 25 8 Ch ild re n w ith pr es um pt iv e TB in 6 he al th fa cil iti es in D ar e s S al aa m 2 cm 3 Di st ill ed w at er an d SR Ye s No No 8 (3 .6 ) 42 (1 8. 7) Xp er t a nd /o r so lid cu ltu re o n sp ut um o r G A 62 .5 % (2 5– 92 % ) 10 0% (9 8– 10 0% ) An dr iy ok o, 20 19 / In do ne sia (2 ) 0– 14 , 1 .4 [0 .4 –6 .5 ] 36 /N R La bo ra to ry st ud y in 1 h os pi ta l; co ns ec ut iv e st oo l s pe cim en s su bm itt ed fo r T B di ag no sis 0. 8– 1 g PB S an d SR No No No 6 (1 7) – Xp er t o n GA or in du ce d sp ut um 10 0% 87 .5 % Ba na da , 20 16 / S ou th Af ric a (3 ) 0– 15 , N R 37 /4 0 20 M TB + an d 20 M TB o n in du ce d sp ut um o r G A Xp er t 0. 6 g Co m m er cia l bu ff er Ye s, w ith gl as s be ad s No Ye s 20 (5 4) – Xp er t o n GA or in du ce d sp ut um 85 % (6 2– 97 % ) 10 0% (9 8– 10 0% ) Ch ip in du ro , 20 17 / Zi m ba bw e (4 ) 5– 16 , 10 .6 [8 –1 3] 21 8/ 21 8 Pr es en tin g w ith pr es um pt iv e TB in 8 P HC s: TB sy m pt om s o r hi st or y o f c lo se co nt ac t w ith T B pa tie nt 0. 15 g PB S an d SR Ye s Ye s No 19 (8 .7 ) – LJ cu ltu re /X pe rt on in du ce d sp ut um 68 % (4 3– 87 % ) 98 % (9 5– 99 % ) Annex 1. Overview of publications on stool processing for TB detection until December 2021 23 St ud y, ye ar / co un try (re fe re nc e) Ag e (y ea rs ) ra ng e, m ed ia n [IQ R] In clu de d in an al ys is/ al l el ig ib le Po pu la tio n Am ou nt of st oo l St oo l p ro ce ss in g m et ho d in clu de s: No . m icr o- bi ol og ica lly co nfi rm ed (% ) No . cli ni ca lly co nfi rm ed (% ) Re fe re nc e sta nd ar d St oo l X pe rt pe rfo rm an ce dilution in vortexing centrifugation filtration Se ns iti vit y (9 5% CI ) Sp ec ifi ci ty (9 5% CI ) Ch ip in du ro , 20 17 / Zi m ba bw e (4 ) 5– 16 , 10 .6 [8 –1 3] 32 /2 18 Pr es en tin g w ith sy m pt om s o f TB in 8 P HC s: TB sy m pt om s o r hi st or y o f c lo se co nt ac t w ith T B pa tie nt 0. 15 g PB S an d SR Ye s Ye s No – 32 Cl in ica l 53 % (3 5– 71 % ) NR de H aa s, 20 21 / Et hi op ia (5 ) NR 12 3/ 14 7 Ch ild re n pr es en tin g w ith pr es um pt iv e TB in se le ct ed he al th fa cil iti es , fo r w ho m ro ut in e na so GA w as re qu es te d up on cl in ica l ex am in at io n 0. 8– 1 g SR No No No 9 (7 .3 ) – Xp er t a nd /o r L J cu ltu re a nd /o r M GI T cu ltu re on n as oG A 78 % – Ha nr ah an , 20 19 / S ou th Af ric a (6 ) <1 0, 2 1. 4 m on th s [1 2. 3– 42 .9 ] 11 9 Ch ild re n w ith sig ns a nd sy m pt om s o f T B pr es en tin g at a pr im ar y- ca re cl in ic NR PB S, N AL C- Na OH a nd SR NR Ye s No 4 (3 ) – Sm ea r, cu ltu re or X pe rt o n an y o ne o f th e sa m pl es co lle ct ed 0/ 4 m icr ob io lo gi ca lly co nfi rm ed T B pa tie nt s h ad a po sit iv e te st o n st oo l 0/ 15 p at ie nt s un lik el y t o ha ve TB h ad a p os iti ve te st o n st oo l Ha nr ah an , 20 19 / S ou th Af ric a (6 ) <1 0, 2 1. 4 m on th s [1 2. 3– 42 .9 ] 11 9 Ch ild re n w ith sig ns a nd sy m pt om s o f T B pr es en tin g at a pr im ar y- ca re cl in ic N R PB S, N AL C- Na OH a nd SR NR Ye s No – 10 0 (8 4) At le as t 2 o f t he fo llo w in g: C XR co ns ist en t w ith TB , p os iti ve cli ni c r es po ns e to a nt i-T B tre at m en t, do cu m en te d ex po su re to T B or a p os iti ve TS T 0/ 10 0 cli ni ca lly co nfi rm ed T B pa tie nt s h ad a po sit iv e te st o n st oo l 0/ 15 p at ie nt s un lik el y t o ha ve TB h ad a p os iti ve te st o n st oo l Practical manual of processing stool samples for diagnosis of childhood TB24 St ud y, ye ar / co un try (re fe re nc e) Ag e (y ea rs ) ra ng e, m ed ia n [IQ R] In clu de d in an al ys is/ al l el ig ib le Po pu la tio n Am ou nt of st oo l St oo l p ro ce ss in g m et ho d in clu de s: No . m icr o- bi ol og ica lly co nfi rm ed (% ) No . cli ni ca lly co nfi rm ed (% ) Re fe re nc e sta nd ar d St oo l X pe rt pe rfo rm an ce dilution in vortexing centrifugation filtration Se ns iti vit y (9 5% CI ) Sp ec ifi ci ty (9 5% CI ) Ha sa n, 20 17 / Pa ki st an (7 ) 0– 15 , 6 .8 [2 –9 ] 49 /5 0 of 6 4 ch ild re n w ith cli ni ca l sy m pt om s Pr es en tin g w ith sy m pt om s o f pu lm on ar y T B in 2 te rt ia ry h os pi ta ls 0. 15 g PB S an d SR Ye s Ye s No 11 (2 2) – LJ cu ltu re / Xp er t o n GA o r sp ut um 82 % (4 8– 98 % ) 95 % (8 2– 99 % ) Ha sa n, 20 17 / Pa ki st an (7 ) 0– 15 , 6 .8 [2 –9 ] 49 /5 0 of 6 4 ch ild re n w ith cli ni ca l sy m pt om s Pr es en tin g w ith sy m pt om s o f pu lm on ar y T B in 2 te rt ia ry h os pi ta ls 0. 15 g PB S an d SR Ye s Ye s No – 17 (3 5) Cl in ica l 59 % (3 3– 82 % ) 10 0% (8 9– 10 0% ) La Co ur se , 20 18 / K en ya (8 ) 0– 12 , 2 [1 .1 –4 .8 ] 14 7/ 16 5 HI V- po sit iv e ch ild re n el ig ib le fo r A RT , ho sp ita liz ed fo r ac ut e ill ne ss in 4 ho sp ita ls NR PB S, N AL C- Na OH , S R No Ye s No 11 (7 .5 ) – M GI T cu ltu re / Xp er t o n sp ut um o r G A 70 % (3 5– 93 % ) 10 0% (9 7– 10 0% ) La Co ur se , 20 18 / K en ya (8 ) 0– 12 , 2 [1 .1 –4 .8 ] 16 5/ 16 5 HI V- po sit iv e ch ild re n el ig ib le fo r A RT , ho sp ita liz ed fo r ac ut e ill ne ss in 4 ho sp ita ls NR PB S, N AL C- Na OH , S R No Ye s No – 85 (5 2) Cl in ica l 9% (4 –1 9% ) 10 0% (9 5– 10 0% ) Lo un na s, 20 20 / Fr an ce (9 ) NA . NA . NA . 0. 5 g su cr os e so lu tio n an d SR No No No NA . NA . NA . NA . NA . M ar cy , 20 16 / Bu rk in a Fa so , Ca m bo di a, Ca m er oo n, Vi et N am (1 0) 0– 13 , 7 .2 (4 .1 –7 .2 ) 27 2/ 27 2 HI V- po sit iv e ch ild re n pr es en tin g w ith pr es um pt iv e pu lm on ar y T B in 8 te rt ia ry /p ae di at ric ho sp ita ls 0. 5 g Su cr os e so lu tio n an d SR No Ye s Ye s 27 (1 0) – M GI T/ LJ cu ltu re on G A, in du ce d sp ut um , N GA , st rin g sa m pl e 67 % (4 6– 83 % ) 10 0% (9 8– 10 0% ) Annex 1. Overview of publications on stool processing for TB detection until December 2021 25 St ud y, ye ar / co un try (re fe re nc e) Ag e (y ea rs ) ra ng e, m ed ia n [IQ R] In clu de d in an al ys is/ al l el ig ib le Po pu la tio n Am ou nt of st oo l St oo l p ro ce ss in g m et ho d in clu de s: No . m icr o- bi ol og ica lly co nfi rm ed (% ) No . cli ni ca lly co nfi rm ed (% ) Re fe re nc e sta nd ar d St oo l X pe rt pe rfo rm an ce dilution in vortexing centrifugation filtration Se ns iti vit y (9 5% CI ) Sp ec ifi ci ty (9 5% CI ) M ar cy , 20 16 / Bu rk in a Fa so , Ca m bo di a, Ca m er oo n, Vi et N am (1 0) 0– 13 , 7 .2 (4 .1 –7 .2 ) 27 2/ 27 2 HI V- po sit iv e ch ild re n pr es en tin g w ith pr es um pt iv e pu lm on ar y T B in 8 te rt ia ry or p ae di at ric ho sp ita ls 0. 5 g su cr os e so lu tio n an d SR No Ye s Ye s – 24 5 (9 0) Cl in ica l 11 % (8 –1 6% ) 96 % (8 1– 10 0% ) M em on , 20 18 / I nd ia (1 1) 0. 5– 15 , 11 10 0/ 10 0 Ch ild re n at te nd in g th e pa ed ia tr ic TB cli ni c o f a te rt ia ry ca re h os pi ta l 0. 2 g PB S, N AL C- Na OH , S R No Ye s No 26 (2 6) – M GI T cu ltu re on in du ce d sp ut um o r G A 11 .5 % (2 .4 –3 0. 1% ) 98 .6 % (9 2. 7– 99 .9 % ) M ou ss a, 20 16 / E gy pt (1 2) >1 to < 15 , NR 11 5/ 11 5 Pr es en tin g w ith cli ni ca l s ig ns o f pu lm on ar y T B in a te rt ia ry ca re ho sp ita l 2 g di st ill ed w at er , PB S, N AL C- Na OH , S R No Ye s No 36 (3 1) – LJ cu ltu re on sp ut um or in du ce d sp ut um 83 % (6 7– 94 % ) 99 % (9 3– 10 0% ) Ng ad ay a, 20 20 / Ta nz an ia (1 3) 1– 95 , 3 5 [2 1– 47 ] 59 0/ NR Pr es um pt iv e TB pa tie nt s > 1 ye ar ol d, 7 p rim ar y he al th fa cil iti es an d 5 te rt ia ry he al th fa cil iti es , Xp er t o n st oo l co nd uc te d at CT RL 2 cm 3 Di st ill ed w at er a nd SR Ye s Ye s No 75 (1 2. 7) – LJ cu ltu re o n sp ut um 84 % (8 1. 0– 87 .0 % ) 93 .4 % (9 8. 5– 99 .9 % ) Ng ad ay a, 20 20 / Ta nz an ia (1 3) 1– 95 , 3 5 [2 1– 47 ] 59 0/ NR Pr es um pt iv e TB pa tie nt s > 1 ye ar ol d, 7 p rim ar y he al th fa cil iti es an d 5 te rt ia ry he al th fa cil iti es , Xp er t o n st oo l co nd uc te d at p er ip he ra l la bo ra to ri es 2 cm 3 Di st ill ed w at er a nd SR Ye s Ye s No 75 (1 2. 7) – LJ cu ltu re o n sp ut um 63 .0 % (4 7. 8– 76 .1 % ) 76 .7 % (7 2. 1– 81 .4 % ) Practical manual of processing stool samples for diagnosis of childhood TB26 St ud y, ye ar / co un try (re fe re nc e) Ag e (y ea rs ) ra ng e, m ed ia n [IQ R] In clu de d in an al ys is/ al l el ig ib le Po pu la tio n Am ou nt of st oo l St oo l p ro ce ss in g m et ho d in clu de s: No . m icr o- bi ol og ica lly co nfi rm ed (% ) No . cli ni ca lly co nfi rm ed (% ) Re fe re nc e sta nd ar d St oo l X pe rt pe rfo rm an ce dilution in vortexing centrifugation filtration Se ns iti vit y (9 5% CI ) Sp ec ifi ci ty (9 5% CI ) Ni co l, 20 13 / So ut h Af ric a (1 4) 1- <1 5, 2 .6 [1 .6 –4 .8 ] 11 5/ 11 5 PH C an d te rt ia ry pe di at ric h os pi ta l 0. 15 g (F LO Q sw ab s1 ) PB S an d SR No Ye s No 17 (1 5) – M GI T cu ltu re on in du ce d sp ut um 47 % (2 3– 72 % ) 99 % (9 4– 10 0% ) Or ik iri za , 20 18 / Ug an da (1 5) 1 M –1 4, NR 34 9/ 35 7 Pa tie nt s s ta rt in g on T B tre at m en t i n a re gi on al re fe rr al ho sp ita l NR Sa lin e so lu tio n, NA LC - Na OH , PB S an d un sp ec ifi ed bu ff er No Ye s No 9 (2 .6 ) – LJ & M GI T Cu ltu re o n sp ut um o r in du ce d sp ut um 56 % (2 1– 86 % ) 98 % (9 0– 10 0% ) W al te rs , 20 12 / S ou th Af ric a (1 6) 0 to <1 4, 1 7 m on th s [N R] 23 /2 8 (1 4 w ith b ot h GA a nd st oo l, 6 on ly G A, 3 on ly st oo l) Pr es en tin g w ith pr es um pt iv e pu lm on ar y T B in 2 ho sp ita ls NR Sa lin e so lu tio n, NA LC - Na OH , P BS an d SR No Ye s No 4 (1 7) – M GI T cu ltu re / Xp er t o n GA NR (c om bi na tio n of st oo l a nd G A Xp er t v er su s M GI T cu ltu re : 75 % ) NR W al te rs , 20 12 / S ou th Af ric a (1 6) 0 to <1 4, 1 7 m on th s [N R] 23 /2 8 (1 4 w ith b ot h GA a nd st oo l) Pr es en tin g w ith pr es um pt iv e pu lm on ar y T B in 2 ho sp ita ls NR sa lin e so lu tio n, NA LC - Na OH , P BS an d SR No Ye s No – 12 (5 2) Cl in ica l NR (c om bi na tio n of st oo l a nd G A Xp er t v er su s cli ni ca l D x: 2 5% ) NR W al te rs , 20 17 / S ou th Af ric a (1 7) 0 to <1 3, 1 .3 [0 .8 –2 .4 ] 37 9/ 37 9 Pr es en tin g w ith pr es um pt iv e pu lm on ar y T B in 2 re fe rr al h os pi ta ls <5 g a nd 1– 4 g PB S, N AL C- Na OH , S R No Ye s No 72 (1 9) – M GI T cu ltu re on G A, in du ce d sp ut um , N GA , st rin g sa m pl e 32 % (2 1– 44 % ) 10 0% (9 8– 10 0% ) W al te rs , 20 17 / S ou th Af ric a (1 7) 0– 13 , 1 .3 [0 .8 –2 .4 ] 35 1/ 37 9 Pr es en tin g w ith pr es um pt iv e pu lm on ar y T B in 2 re fe rr al h os pi ta ls <5 g a nd 1– 4 g PB S, N AL C- Na OH , S R No Ye s No – 24 2 (6 9) Cl in ica l 10 % (6 –1 4% ) 10 0% (9 7– 10 0% ) 1 F or m or e in fo , p le as e se e FL O Q Sw ab s, C O PA N D ia gn os tic s In c. Annex 1. Overview of publications on stool processing for TB detection until December 2021 27 St ud y, ye ar / co un try (re fe re nc e) Ag e (y ea rs ) ra ng e, m ed ia n [IQ R] In clu de d in an al ys is/ al l el ig ib le Po pu la tio n Am ou nt of st oo l St oo l p ro ce ss in g m et ho d in clu de s: No . m icr o- bi ol og ica lly co nfi rm ed (% ) No . cli ni ca lly co nfi rm ed (% ) Re fe re nc e sta nd ar d St oo l X pe rt pe rfo rm an ce dilution in vortexing centrifugation filtration Se ns iti vit y (9 5% CI ) Sp ec ifi ci ty (9 5% CI ) W al te rs , 20 18 / S ou th Af ric a [1 7] 1. 3 [0 .9 –2 .4 ] 28 0/ 30 2 Pr es en tin g w ith pr es um pt iv e pu lm on ar y T B in 2 re fe rr al h os pi ta ls 0. 6 g an d sw ab PB S an d SR Ye s, w ith gl as s be ad s No Ye s 23 (8 .3 ) – M GI T/ Xp er t o n ex pe ct or at ed or in du ce d sp ut um a nd G A if <5 y (s ub se t o nl y) 44 .4 % (1 3. 7– 78 .8 % ) Xp er t a s R S 25 .0 % (7 .3 –5 2. 4% ) cu ltu re a s R S 99 .1 % (9 6. 8– 99 .9 % ) Xp er t a s R S 99 .5 % (9 7. 5– 10 0% ) cu ltu re a s R S W al te rs , 20 18 / S ou th Af ric a (1 8) NR , 1 .3 [0 .9 –2 .4 ] 28 0/ 30 2 Pr es en tin g w ith pr es um pt iv e pu lm on ar y T B in 2 re fe rr al h os pi ta ls 0. 6 g an d sw ab PB S an d SR Y e s, w ith gl as s be ad s No Ye s – 88 (3 1) Cl in ica l 44 .4 % (1 3. 7– 78 .8 % ) Xp er t a s R S 25 .0 % (7 .3 –5 2. 4% ) cu ltu re a s R S 99 .1 % (9 6. 8– 99 .9 % ) Xp er t a s R S 99 .5 % (9 7. 5– 10 0% ) cu ltu re a s R S W el da y, 20 14 / K en ya (1 9) 0 to < 15 , NR 53 /9 1 La bo ra to ry st ud y in 2 h os pi ta ls in clu di ng ch ild re n re fe rr ed fo r TB te st in g by a cli ni cia n 0. 15 g PB S on ly ve rs us P BS an d SR No No ve rs us ye s No 6 (1 1. 3) – ZN sp ut um sm ea r m icr os co py 10 0% . D ire ct m et ho d di d yi el d m or e ca se s t ha n in di re ct m et ho d 89 % AR T: a nt ire tr ov ira l t he ra py ; C I: co nfi de nc e in te rv al ; C TR L: c en tr al T B r ef er en ce la bo ra to ry ; C XR : c he st X -r ay ; G A: g as tr ic a sp ira te ; H IV : h um an im m un od efi ci en cy v ir us ; I Q R: in te rq ua rt ile ra ng e; L J: Lö w en st ei n– Je ns en ; M G IT : m yc ob ac te ri a gr ow th in di ca to r t ub e; M TB : M yc ob ac te ri um tu be rc ul os is ; N A: n ot a pp lic ab le ; N AL C- N aO H : N -a ce ty l-L -c ys te in e– so di um c itr at e- so di um h yd ro xi de ; N G A: n as op ha ry ng ea l a sp ira te ; N R: n ot r ep or te d; P B S: p ho sp ha te -b uff er ed s al in e; P H C: p ri m ar y he al th c en tr e; R S: r ef er en ce s ta nd ar d; S R: Xp er t s am pl e re ag en t; TB : t ub er cu lo si s; T ST : t ub er cu lin s ki n te st ; Z N : Z ie hl –N ee ls en Practical manual of processing stool samples for diagnosis of childhood TB28 References for Annex 1 1. Ainan S, Furia FF, Mhimbira F, Mnyambwa NP, Mgina N, Zumla A et al. Xpert® MTB/RIF assay testing on stool for the diagnosis of paediatric pulmonary TB in Tanzania. Public Health Action. 2021;11(2):75–9. 2. Andriyoko B, Janiar H, Kusumadewi R, Klinkenberg E, de Haas P, Tiemersma E. Simple stool processing method for the diagnosis of pulmonary tuberculosis using GeneXpert MTB/RIF. Eur Respir J. 2019;53(3):1801832. 3. Banada PP, Naidoo U, Deshpande S, Karim F, Flynn JL, O’Malley M et al. A novel sample processing method for rapid detection of tuberculosis in the stool of pediatric patients using the Xpert MTB/ RIF assay. PLoS One. 2016;11(3):1–13. 4. Chipinduro M, Mateveke K, Makamure B, Ferrand RA, Gomo E. Stool Xpert® MTB/RIF test for the diagnosis of childhood pulmonary tuberculosis at primary clinics in Zimbabwe. Int J Tuberc Lung Dis. 2017;21(2):161–6. 5. de Haas P, Yenew B, Mengesha E, Slyzkyi A, Gashu Z, Lounnas M et al. The simple one-step (SOS) stool processing method for use with the Xpert MTB/RIF assay for a child-friendly diagnosis of tuberculosis closer to the point of care. J Clin Microbiol. 2021;59(8):e0040621. 6. Hanrahan CF, Dansey H, Mutunga L, France H, Omar SV, Ismail N et al. Diagnostic strategies for childhood tuberculosis in the context of primary care in a high burden setting: the value of alternative sampling methods. Paediatr Int Child Health. 2019;39(2):88–94. 7. Hasan Z, Shakoor S, Arif F, Mehnaz A, Akber A, Haider M et al. Evaluation of Xpert MTB/RIF testing for rapid diagnosis of childhood pulmonary tuberculosis in children by Xpert MTB/RIF testing of stool samples in a low resource setting. BMC Res Notes. 2017;10(1):473. 8. LaCourse SM, Pavlinac PB, Cranmer LM, Njuguna IN, Mugo C, Gatimu J et al. Stool Xpert MTB/RIF and urine lipoarabinomannan for the diagnosis of tuberculosis in hospitalized HIV-infected children. AIDS (London, England). 2018;32(1):69–78. 9. Lounnas M, Diack A, Nicol MP, Eyangoh S, Wobudeya E, Marcy O et al. Laboratory development of a simple stool sample processing method diagnosis of pediatric tuberculosis using Xpert Ultra. Tuberculosis (Edinb). 2020;125:102002. 10. Marcy O, Ung V, Goyet S, Borand L, Msellati P, Tejiokem M et al. Performance of Xpert MTB/RIF and alternative specimen collection methods for the diagnosis of tuberculosis in HIV-infected children. Clin Infect Dis. 2016;62(9):1161–8. 11. Memon SS, Sinha S, Sharma SK, Kabra SK, Lodha R, Soneja M. Diagnostic accuracy of Xpert MTB/ RIF assay in stool samples in intrathoracic childhood tuberculosis. Journal of Tuberculosis and Therapeutics. 2018;3(2). 12. Moussa HS, Bayoumi FS, Mohamed AMA. Gene Xpert for direct detection of Mycobacterium tuberculosis in stool specimens from children with presumptive pulmonary tuberculosis. Ann Clin Lab Sci. 2016;46(2):198–203. 13. Ngadaya E, Kimaro G, Sandi E, Mnyambwa NP, Wilfred A, Lubinza C et al. Evaluation of stool GeneXpert MTB/RIF for the diagnosis of pulmonary tuberculosis among presumptive patients in Tanzania. J Clin Tuberc Other Mycobact Dis. 2020;21:100195. Annex 1. Overview of publications on stool processing for TB detection until December 2021 29 14. Nicol MP, Spiers K, Workman L, Isaacs W, Munro J, Black F et al. Xpert MTB/RIF testing of stool samples for the diagnosis of pulmonary tuberculosis in children. Clin Infect Dis. 2013;57(3):18–21. 15. Orikiriza P, Nansumba M, Nyehangane D, Bastard M, Mugisha IT, Nansera D et al. Xpert MTB/RIF diagnosis of childhood tuberculosis from sputum and stool samples in a high TB-HIV-prevalent setting. Eur J Clin Microbiol Infect Dis. 2018;37(8):1465–73. 16. Walters E, Gie RP, Hesseling AC, Friedrich SO, Diacon AH, Gie RP. Rapid diagnosis of pediatric intrathoracic tuberculosis from stool samples using the Xpert MTB/RIF Assay: a pilot study. Pediatr Infect Dis J. 2012;31(12):1316. 17. Walters E, van der Zalm MM, Palmer M, Bosch C, Demers A-M, Draper H et al. Xpert MTB/RIF on stool is useful for the rapid diagnosis of tuberculosis in young children with severe pulmonary disease. Pediatr Infect Dis J. 2017;36(9):837–43. 18. Walters E, Scott L, Nabeta P, Demers A-M, Reubenson G, Bosch C et al. Molecular detection of Mycobacterium tuberculosis from stools in young children by use of a novel centrifugation-free processing method. J Clin Microbiol. 2018;56(9):e00781–18. 19. Welday SH, Kimang’a AN, Kabera BM, Mburu JW, Mwachari C, Mungai E et al. Stool as appropriate sample for the diagnosis of Mycobacterium tuberculosis by Gene Xpert test. Open J Respir Dis. 2014;04:83–9. Practical manual of processing stool samples for diagnosis of childhood TB30 Annex 2. List of activities for the implementation of stool testing No. Activity Condition (yes/no/in progress) 1 Adapt national policies and guidance to include stool as a primary sample for the diagnosis of TB in children with signs and symptoms of TB 2 Train health care providers on the new national guidance including the diagnostic algorithms incorporating stool 3 Train health care providers on treatment initiation using stool Xpert results 4 Train health care providers on the collection of stools 5 Train laboratory staff on the stool processing method(s) 6 Assign and train the focal point (site coordinator) 7 Adapt the reporting and registration tools to include a provision for stool 8 Adapt the digital data collection and connectivity tools to include a provision for stool 9 Add indicators specific for stool testing to the standard monitoring and supervision list for Xpert MTB/RIF and Xpert Ultra testing 10 Ensure availability of supplies needed for stool collection (e.g. stool container and stool collection flyer) 11 Ensure availability of supplies to transport stool samples (e.g plastic bag and cooler box) and arrange that stool can be transported through the routine sample referral network 12 Ensure availability of the additional supplies for the stool processing method using Xpert MTB/RIF or Xpert Ultra 13 Ensure availability of the SOPs and bench aids for stool testing 14 Ensure availability of enough Xpert MTB/RIF or Xpert Ultra cartridges for stool testing SOP: standard operating procedure; TB: tuberculosis. Annex 2. List of activities for the implementation of stool testing 31 Annex 3. Laboratory tool for assessing stool testing by Xpert MTB/RIF and Xpert MTB/RIF Ultra Laboratory stool testing assessment The laboratory indicators listed in this tool should be integrated into the standard monitoring and supervision tool for Xpert MTB/RIF and Xpert Ultra testing already used by the country (1). Name of institution District Date of visit Name and contact details of staff met Name: Designation: Tel/email: Name: Designation: Tel/email: Name: Designation: Tel/email: Names of assessors Practical manual of processing stool samples for diagnosis of childhood TB32 Number or Yes/no/partial Comments 1. Stool samples obtained Number of children with signs and symptoms of TB for whom a stool sample has been collected per month 2. Quality of the collected stool sample Are stool samples kept between 2 °C and 8 °C during transport? Are stool samples kept between 2 °C and 8 °C before testing? Are correct collection pots used? Are lids properly closed? Number of stool pots with stool found on the outside of the pot Are pots properly filled (minimum bottom of container covered, maximum ½ of pot)? Number of stool samples rejected by the laboratory Indicate rejection criteria: 3. Quality of sample processing Number of stool samples processed per month Indicate the type of stool received: Formed Semi formed Liquid Number of samples processed within 3 days of stool submission Number of samples processed 3 or more days after stool submission Maximum number of days Is the correct amount of stool used for stool testing? Is the processing performed according to the SOP for stool testing by Xpert MTB/RIF or Xpert Ultra? Number of stool samples with an Xpert MTB positive result (total per month): MTB detected high MTB detected medium MTB detected low MTB detected very low MTB trace detected Number of stool samples with MTB not detected Number of stool samples with an error code Error codes: Code x no. of tests Annex 3. Laboratory tool for assessing stool testing by Xpert MTB/RIF and Xpert MTB/RIF Ultra 33 Number or Yes/no/partial Comments Number of stool samples with “Invalid” Number of stool samples with “No result” Number of stool samples not tested Reason for not testing: Number of samples with rifampicin resistance Number of samples with rifampicin indeterminate Yes/no/partial Comments 4. Supply management Are Xpert Ultra cartridges always available for stool testing? Reason if not: Does the monthly consumption of Xpert Ultra cartridge consider stool testing? Number of cartridges/ months: Any stockout of containers for stool collection in the last quarter? Any stockout of applicator to support transfer of stool to SR buffer in the last quarter? Any stock out of supplies required for stool processing in the last quarter? Any stock out of reagents for stool processing in the last quarter? Yes/no/partial Comments 5. Registration and reporting tools Does request form have a provision to request stool testing? Does laboratory register have a provision to add stool as sample type? Is “stool” indicated within the sample type field of the GeneXpert? Are stool testing results entered into the laboratory information system? MTB: Mycobacterium tuberculosis; SOP: standard operating procedure; TB: tuberculosis. Additional comments __________________________________________________________________________________________________________________ _______________________________________________________ __________________________________________________________________________________________________________________ _______________________________________________________ Reference for Annex 3 1. Tuberculosis technical scorecard Xpert MTB/RIF. Geneva: Stop TB Partnership; 2020 (https://stoptb.org/ wg/gli/assets/documents/5%20Find-TB-Scorecard-Xpert-Low-Res.pdf, accessed January 2022). Practical manual of processing stool samples for diagnosis of childhood TB34

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Practical manual of processing stool samples for diagnosis of childhood TB

Practical manual of processing stool samples for diagnosis of childhood TB Practical manual of processing stool samples for diagnosis of childhood TB ISBN 978-92-4-004265-0 (electronic version) ISBN 978-92-4-004266-7 (print version) © World Health Organization 2022 Some rights reserved. This work is available under the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 IGO licence (CC BY-NC-SA 3.0 IGO; https://creativecommons.org/licenses/by-nc-sa/3.0/igo). Under the terms of this licence, you may copy, redistribute and adapt the work for non-commercial purposes, provided the work is appropriately cited, as indicated below. In any use of this work, there should be no suggestion that WHO endorses any specific organization, products or services. The use of the WHO logo is not permitted. If you adapt the work, then you must license your work under the same or equivalent Creative Commons licence. 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Design by Inis Communication Contents Acknowledgements iv About this manual v Target audience v Abbreviations and acronyms vi Part A Background 1 A1 Introduction 1 A2 Stool processing methods 1 A3 Comparison of stool processing methods 2 A4 Stool processing methods described in this manual 3 Part B How to perform stool testing 5 B1 Stool collection and storage 5 B2 Stool processing methods 7 Part C Implementation of stool testing 13 C1 Where to place stool testing in the tiered laboratory network 13 C2 Steps and processes for implementation 14 References 19 Annex 1. Overview of publications on stool processing for TB detection until December 2021 23 Annex 2. List of activities for the implementation of stool testing 31 Annex 3. Laboratory tool for assessing stool testing by Xpert MTB/RIF and Xpert MTB/RIF Ultra 32 iii Acknowledgements This guide was developed as a product of the Global Laboratory Initiative (GLI) Core Group. Development was led by Petra de Haas (KNCV Tuberculosis Foundation, The Hague, The Netherlands), Organization for Migration, Geneva, Switzerland), Patricia Hall (Centers for Disease Control and Prevention, Atlanta, GA, United States of America) and Wayne van Gemert (Stop TB Partnership, Geneva, Switzerland), under the coordination of the GLI Working Group Secretariat within the Global TB Programme of the World Health Organization (WHO). The writing of this manual was supported by Pauline Lempens (KNCV Tuberculosis Foundation, The Hague, The Netherlands). Brands, Nazir Ismail, Carl-Michael Nathanson and Sabine Verkuijl (WHO Global TB Programme, Geneva, Switzerland). Special thanks to Pamela Nabeta (Foundation for Innovative New Diagnostics, Geneva, Switzerland), Maryline Bonnet (TB-Speed project, Université de Montpellier, Montpellier, France), Manon Lounnas (TB-Speed project, Université de Montpellier, Montpellier, France) and Martina Casenghi (Elizabeth Glaser Pediatric AIDS Foundation, Geneva, Switzerland) for their valuable feedback and contribution. WHO also appreciates the feedback provided by the following GLI Core Group members and public health specialists: Khalide Azam, Sarabjit Singh Chadha, Fernanda Dockhorn Costa, Sarder Tanzir Hossain, Kristin Kremer, Edine Tiemersma and Andrii Slyzkyi (KNCV Tuberculosis Foundation, The Hague, The Netherlands). GLI is a Working Group of the Stop TB Partnership. Development and publication of this document Development. Practical manual of processing stool samples for diagnosis of childhood TBiv About this manual This practical manual aims to help countries implement stool testing into their tuberculosis (TB) diagnostic and clinical practice. It provides evidence and incorporates recommendations on the use of stool as a sample type for diagnosing TB. It also contains steps for implementation and details on the laboratory process for conducting stool testing using Xpert MTB/RIF and Xpert MTB/ RIF Ultra (Xpert Ultra). Target audience The target audience for this manual is ministries of health, programme managers, clinicians, front- line health workers (especially in paediatric TB), TB testing site managers, supervisory laboratory staff and GeneXpert users at national, state or provincial and testing site level, as well as implementing partners. About this manual v Practical manual of processing stool samples for diagnosis of childhood TB Abbreviations and acronyms CI confidence interval FIND Foundation for Innovative New Diagnostics GA gastric aspirate GLI Global Laboratory Initiative HIV human immunodeficiency virus KNCV KNCV Tuberculosis Foundation MTBC Mycobacterium tuberculosis complex NTP national TB programme OSF optimized sucrose flotation POSEE Paediatric TB Operational and Sustainability Expertise Exchange PPE personal protective equipment SOP standard operating procedure SOS simple one-step SPK stool processing kit SR sample reagent TB tuberculosis WHO World Health Organization Practical manual of processing stool samples for diagnosis of childhood TBvi Part A Background A1 Introduction Each year, 1.1 million children globally fall ill with tuberculosis (TB), of whom only 400 000 are notified; with the case detection gap being highest in children aged under 5 years. In 2020, this gap was 72.5%, whereas for children aged 5–14 years the gap was 55.4% (1). Obtaining bacteriological confirmation of TB is challenging in children because of the frequent paucibacillary presentation of the disease. Diagnostic specimens have a low bacterial load, which decreases diagnostic test sensitivity. Diagnosis is further complicated by the fact that obtaining a sufficient volume of specimen from children can be difficult. Children, especially young children, generally cannot effectively expectorate and produce a sputum sample. Often, invasive procedures such as sputum induction or gastric aspiration are required to obtain a specimen. In many settings, the equipment and consumables required for sputum induction or gastric aspiration are not available, or clinical staff lack the skills to competently perform these methods. Also, parents or other caregivers may be reluctant to have these invasive procedures performed on their children. Stool collection is a non-invasive method. Mycobacterium tuberculosis complex (MTBC) can be detected in stool specimens because sputum is coughed up and subsequently swallowed, and then passes through the gastrointestinal system. Since 2021, the World Health Organization (WHO) has recommended stool as a new specimen type alongside sputum (expectorated or induced), nasopharyngeal aspirate or gastric aspirate (GA) for both Xpert MTB/RIF and Xpert Ultra as the initial diagnostic test for TB and the detection of rifampicin resistance in children aged under 10 years with signs and symptoms of pulmonary TB (2, 3). The recent WHO consolidated guidelines on TB, Module 5, and the accompanying operational handbook, provide recommendations on the management of TB in children and adolescents, including on the use of stool (4, 5). Recent systematic reviews for the detection of MTBC on stool using Xpert MTB/RIF found pooled sensitivities of 50% (95% confidence interval [CI]: 44–56), 57% (95% CI: 40–72), 62% (95% CI: 44–76) and 67% (95% CI: 52–79), and pooled specificities of 99% (95% CI: 98–99), 98% (95% CI: 96–99), 99% (95% CI: 97–99) and 99% (95% CI: 98–99) compared with a microbiological reference standard (4–9). A systematic review and meta-analysis of Xpert Ultra data found a sensitivity of 53% (95% CI: 35–70) and a specificity of 98% (95% CI: 93–99) (10, 11). Given the fact that all specimen types have incomplete sensitivity, using two specimens (preferably of two different specimen types) might increase the chance of obtaining a bacterial diagnosis of TB (12). However, there is no WHO recommendation on this to date. A2 Stool processing methods Various methods to process stool for Xpert MTB/RIF or Xpert Ultra testing have been described (13– 20). The methods vary in terms of technique (or combination of techniques) used to bring stool into suspension (e.g. hand shaking or mechanical shaking using a vortex) and to separate M. tuberculosis Part A Background 1 Practical manual of processing stool samples for diagnosis of childhood TB bacilli from stool debris (e.g. centrifugation, filtration and sedimentation). Consequently, methods vary in complexity, labour intensity, time investment, and the required equipment, supplies and infrastructure. Annex 1 provides a list of relevant publications, giving the stool processing methods used and the reported sensitivities and specificities against various reference standards. Several authors have described relatively simple, centrifuge-free methods for stool processing – for example, Banada et al. (15), Walters et al. (17) and Andriyoko et al. (18). Other simple methods are the optimized sucrose flotation (OSF) method developed by the TB-Speed consortium (19), and the simple one-step (SOS) method developed by the KNCV Tuberculosis Foundation (KNCV) (20). A3 Comparison of stool processing methods Standardized comparison studies are needed owing to the high heterogeneity among stool processing methods and study designs, and consequently among the pooled sensitivity and specificity values from the four systematic reviews (6–9). Jasumback et al. applied four different stool processing methods (15, 17, 20, 21) to stool samples spiked with multiple log concentrations of Mycobacterium bovis bacille Calmette-Guérin (BCG) (22). Walters et al. used a method that included centrifugation (21); this resulted in more frequent detection of BCG at lower concentrations (5/5 replicates at 103 colony forming units [cfu]/mL and 3/5 replicates at 102 cfu/mL) compared with the other three methods – that is, the method used by Banada et al. (15) (3/5 replicates at 103 cfu/mL and 1/5 replicates at 102 cfu/mL), the centrifuge- free swab-based method used by Walters et al. (17) (1/5 replicates at 103 cfu/mL and 0/5 replicates at 102 cfu/mL) and the SOS stool method (20) (3/5 replicates at 103 cfu/mL and 1/5 replicates at 102 cfu/mL). However, numbers were too small to carry out statistical analyses. The SOS stool method was considered to be most suitable for low-resource settings, because of its low error rate, short processing time and minimal requirements regarding biosafety precautions and laboratory equipment (22). In an in vitro study (20), the TB-Speed consortium, in collaboration with KNCV, compared the two-step method described by Andriyoko et al. (18) with the SOS stool processing method (20). The comparison was done using stool samples with different consistency spiked with multiple log concentrations of M. tuberculosis. The SOS stool method was found to have a higher sensitivity than the two-step method; this was attributed to the fact that the two-step method uses an additional dilution step. Three stool processing methods have been assessed in parallel as part of two studies led by the Foundation for Innovative New Diagnostics (FIND) and the TB-Speed consortium. The studies were performed at referral laboratories in Africa and Asia and included the disposable stool processing kit (SPK), which resulted from optimization of the methods described by Banada et al. (15) and Walters et al. (17); the OSF (19); and the SOS (20) stool processing methods. Pooled data from an interim analysis of the FIND and TB-Speed studies suggest similar performance of the three stool processing methods in terms of sensitivity and specificity (23, 24). Briefly, the sensitivity of Xpert Ultra for TB detection in stool was 52.1% (95% CI: 38.3–65.5) for SOS, 48.3% (95% CI: 35.9–60.8) for SPK and 46.8% (95% CI: 33.4–60.8) for OSF, and the specificity was 97.5% (95% CI: 94.9–98.9) for SOS, 97.1% (95% CI: 94.5–98.5) for SPK and 97.8% (95% CI: 95.1–99.1) for OSF (see Table 1). Practical manual of processing stool samples for diagnosis of childhood TB2 The proportion of non-determinate Xpert Ultra results was 8.7% (35/401) for SOS, 11.8% (53/541) for SPK and 10.3% (40/388) for OSF (23). No calculations were planned at this point to determine statistical significance or assess repeat Xpert Ultra test results. A limitation of this interim analysis is the low number of MTBC-positive children included, which resulted in wide confidence intervals around the sensitivity estimates. Table 1. Pooled interim results of the studies by FIND and TB-Speed M et ho d Total number of tests True positive results False positive results False negative results True negative results Sensitivity (95% CI) Specificity (95% CI) SOS 332 25 7 23 277 52.1% (38.3–65.5) 97.5% (94.9–98.9) SPK 368 28 9 30 301 48.3% (35.9–60.8) 97.1% (94.5–98.5) OSF 319 22 6 25 266 46.8% (33.4–60.8) 97.8% (95.1–99.1) CI: confidence interval; FIND: Foundation for Innovative New Diagnostics; OSF: optimized sucrose flotation; SOS: simple one-step; SPK: stool processing kit. The FIND and TB-Speed studies also looked at user acceptability and feasibility of the stool methods, and the findings suggest good acceptability of stool as a sample for TB diagnosis in children (24). All methods were found to be easy to process by laboratory staff at reference level, and all had a high median ease-of-use score. However, most users considered that these methods cannot be performed by non-laboratory staff (e.g. nurses and health care workers) in primary health care settings without access to a laboratory. Overall, the SOS stool method appeared to be the preferred method because it does not require additional equipment or supplies compared with sputum Xpert testing (23, 24). A4 Stool processing methods described in this manual This practical manual focuses on two stool processing methods: the OSF and SOS stool methods. The disposable SPK assessed during the FIND and TB-Speed studies was a prototype kit; although shown to be accurate, the kit offers no additional benefits over the simpler OSF and SOS stool methods and its development and commercialization will therefore not be advanced. Hence, the SPK and the underlying methods described by Banada et al. (15) and Walters et al. (17) are not the focus of this manual. Part A Background 3 Practical manual of processing stool samples for diagnosis of childhood TB Practical manual of processing stool samples for diagnosis of childhood TB4 Part B How to perform stool testing B1 Stool collection and storage Stool collection is usually done by the caregivers or the patients themselves, depending on the age of the child. Ideally, the collection takes place at the health care facility. However, to obtain a specimen on demand is often challenging; therefore, stool is collected at home and the patient or caregiver needs to return to the facility for specimen submission. Following the procedures at the local setting, the patient or caregiver is provided with a stool container. Different types of containers can be used; some of these have a small spoon integrated into the screwcap, as shown in Fig. 1. Importantly, the container should be wide-mouthed to make it easy to add the stool, and it should be able to hold at least 3–5 g of stool. Thus, sputum containers can also be used for stool collection. Fig. 1. Example of a stool container The stool container should be provided in a plastic bag containing absorbent material, to keep the container clean during transport to and from the child’s home. The absorbent material will absorb any substances that may leak out of the container if it is not closed properly. The patient or caregiver should be instructed by the laboratory staff or other health care worker on how to collect the stool sample. The instructions below can be adapted to country-specific needs. A flyer to provide to the patient or caregiver, showing the steps on how to collect the stool sample and explaining the importance of returning the sample to the clinic, can be useful. Part B How to perform stool testing 5 Practical manual of processing stool samples for diagnosis of childhood TB Instructions for the patient or caregiver on how to collect the stool sample: 1. Ideally, collect the stool sample during the first daily bowel movement. If possible, first empty the bladder, to avoid mixing urine with the stool sample. 2. Put some clean plastic sheeting on the spot where the stool will be dropped, to ensure the collection of a clean sample. Avoid contamination of the plastic with soil, detergent or disinfectant from the toilet. 3. If the stool sample needs to be collected from a child that uses a diaper (i.e. a nappy), then either collect the stool directly from the diaper as soon as possible after defecation, or put a plastic sheet in the diaper to avoid (prolonged) contact between the stool and the surface of the diaper (diapers may contain substances that inhibit the test). 4. Fill the stool container with the stool sample up to half full, using (for example) the spoon provided with some types of containers, a clean plastic bag, a clean piece of cardboard or a clean spoon. Do not fill the container to the brim. Only a small amount of stool is required for testing (2 g is sufficient for both testing and retesting if the first test is unsuccessful). 5. Close the container tightly, place the container in the plastic bag provided (preferably a self- sealable bag) and close the bag. Leave the absorbent material in the plastic bag so that this material can absorb any substances that may leak out of the container. 6. As soon as the stool sample has been collected, store the plastic bag containing the stool container in a clean, cool place (e.g. in a fridge if possible), avoiding exposure to direct sunlight. Do not freeze the sample. 7. Take the plastic bag containing the stool container to the health care facility, preferably on the same day that you collected the stool sample. For transport and storage of stool specimens, the same conditions apply as for transport and storage of sputum specimens for Xpert testing. Thus, between collection and testing, stool specimens can be kept at a maximum of 35 °C for up to 3 days, followed by a maximum of 7 days at 2–8 °C. Ideally, stool sample containers should be kept at 2–8 °C while being sent to the laboratory and should then be stored in the refrigerator (at 2–8 °C) until testing can be performed. Sample preparation and testing should be started as soon as possible. Research is ongoing on how best to store stool samples (25, 26). Before handling, allow the stool sample to warm up to room temperature. Practical manual of processing stool samples for diagnosis of childhood TB6 B2 Stool processing methods B2.1 OSF stool processing method Principle The TB-Speed OSF method is based on the creation of a sucrose density gradient to support separation of M. tuberculosis from stool debris (19). Biosafety requirements Although the OSF method involves a separation step before inactivation, this step leads to only minimal concentration of the M. tuberculosis bacilli. Therefore, the OSF method can be performed in an open, well-ventilated space with appropriate aerosol reduction practices and appropriate use of personal protective equipment (PPE). Procedure In brief, 0.5 g of stool is transferred into a 50 mL Falcon tube (for solid stool specimens) or an empty stool container (for liquid stool specimens) (Fig. 2). Next, 10 mL of Sheather’s solution (56% sucrose solution) is added to the Falcon tube or stool container, which is then shaken 20 times to homogenize the stool specimen and left to stand for 30 minutes at room temperature, to sediment larger particulate matter. Then 0.5 mL of the resulting supernatant is transferred to another 15 mL Falcon tube, together with 1.8 mL sample reagent (SR) from the Xpert MTB/RIF or Xpert Ultra assay. The mixture is shaken vigorously 20 times then incubated for 15 minutes at room temperature, after which 2 mL of the mixture is transferred to the Xpert MTB/RIF or Xpert Ultra cartridge. The cartridge is then inserted into the GeneXpert instrument. Use of the GeneXpert instrument and interpretation of Xpert results are done according to the manufacturer’s instructions. Detailed information on how to prepare the Sheather’s solution and on how to perform the OSF stool processing method can be found in the standard operating procedure (SOP) on the TB-Speed website (27). TB-Speed is a research project sponsored by Inserm (Institut national de la santé et de la recherche médicale, the French National Institute of Health and Medical Research) and funded by Unitaid and L’Initiative (28), with support from ANRS-MIE (an agency within Inserm) (29). Part B How to perform stool testing 7 Practical manual of processing stool samples for diagnosis of childhood TB x20 Fig. 2. Schematic overview of the SOP for the detection of M. tuberculosis complex and resistance to rifampicin in stool by using the TB-Speed OSF method and the Xpert MTB/RIF or Xpert Ultra assay 2. Sedimentation during 30 minutes 3. Transfer 0.5 mL from the top of the specimen to a 15 mL Falcon tube and add 1.8 mL of Sample Reagent + shake vigorously 20 times 4. Incubation and transfer to the Xpert Ultra cartridge 1. Add 0.5 g of stool in 10 mL of Sheather’s solution + shake 20 times OSF: optimized sucrose flotation; SOP: standard operating procedure. 15 minutes 30 minutes Practical manual of processing stool samples for diagnosis of childhood TB8 B2.2 SOS stool processing method Principle The SOS stool processing method uses one step to release M. tuberculosis from stool. Particulate matter is sedimented by gravity, and it is assumed that this allows TB bacilli to float to the top of the watery solution because of their lipid-containing cell wall (20). Biosafety requirements In the SOS stool method, stool is added directly into the SR bottle provided in the Xpert kit; this results in immediate inactivation of the bacteria. Therefore, the SOS stool method can be performed in an open, well-ventilated space with appropriate aerosol reduction practices and appropriate use of PPE. Procedure Before stool processing, the consistency of the stool specimen is assessed using the Bristol stool scale (30). For stool with the appearance of Bristol type 1 to 5 (formed stool), 0.8 g or a thumbnail size amount of stool (Fig. 3) is directly transferred from the stool container into the SR bottle using a wooden stick or applicator (Fig. 4). For stool with the appearance of Bristol type 6 and 7 (liquid stool), 2 mL SR is removed from the SR bottle, then 2 mL of stool is transferred to the SR bottle using a balloon pipette (Fig. 3). For all types of stools, the SR is shaken vigorously for 30 seconds and then incubated for 10 minutes at room temperature. This step is repeated once. After carefully ensuring that solid particles and debris have settled, 2 mL of the supernatant is then transferred from the SR bottle to the Xpert MTB/RIF or Xpert Ultra cartridge. The cartridge is then inserted into the GeneXpert instrument. Use of the GeneXpert instrument and interpretation of Xpert results is done according to the manufacturer’s instructions. A detailed SOP on how to perform the SOS stool processing method can be found in the KNCV stool toolbox on the KNCV website (31). Part B How to perform stool testing 9 Practical manual of processing stool samples for diagnosis of childhood TB Fig. 3. In the SOS stool processing method, 0.8 g or an amount of stool equal to the size of a thumbnail is used SOS: simple one-step. Fig. 4. Schematic overview of the SOP of the SOS stool processing method and the Xpert MTB/RIF or Xpert Ultra assay for different types of stoolsa SOP: standard operating procedure; SOS: simple one-step; SR: sample reagent. a The upper panel shows the procedure for stool of Bristol type 1–5 (i.e. formed stool) and the lower panel for stool of Bristol type 6 and 7 (i.e. liquid stool). Transfer 0.8 g of stool into the SR* bottle Transfer 2 mL ‘debris free’ supernatant # into Xpert cartridge B Take 2 mL SR* out of the SR* bottle and dispose of it Take 2 mL of stool out of the stool bottle and transfer it into the SR bottle Transfer 2 mL ‘debris free’ supernatant # into Xpert Ultra cartridge Two times: shake 30 seconds and stand 10 minutes Two times: shake 30 seconds and stand 10 minutes * sample reagent buer SOS STOOLBOX7 Figure 1. The Bristol Stool Scale, named after the University of Bristol where it was fi rst described by Lewis and Heaton 21; from type 1, being the most solid, to type 7, being the most liquid. Practical manual of processing stool samples for diagnosis of childhood TB10 B2.3 Characteristics summary of OSF and SOS stool processing methods Table 2 summarizes the characteristics of the OSF and SOS stool processing methods. The table includes additional materials required, preparation time (32), incubation time and biosafety requirements. Table 2. Characteristics of the OSF and SOS stool processing methods SOS OSF Additional suppliesa Applicator to transfer stool (wooden stick), balloon pipettesb Applicator to transfer stool, sample preparation tubes, distilled water, sucrose (Difco), balloon pipets Additional equipmentc, d None Electronic balance, heating magnetic stirrer and bar magnet, screw cap glass bottle (1 L), graduated cylinder (1 L), autoclave Median preparation time (range) 23 (20–30) minutes 56 (45–87) minutes Incubation time Incubation 10 minutes Sedimentation 10 minutes Sedimentation 30 minutes Incubation 15 minutes OSF: optimized sucrose flotation; SOS: simple one-step. a Additional supplies are supplies that are required in addition to what is provided in the Xpert MTB/RIF or Xpert Ultra kit (the kit includes cartridges, sample reagent buffer and balloon pipettes). b Additional balloon pipettes might be required when a high load of liquid stool is processed and the balloon pipets provided in the Xpert kit are not sufficient to cover the work. c Additional equipment is equipment required in addition to the GeneXpert instrument. d Preparation of the Sheather’s solution will be done in batches at central level and distributed to the sites, meaning that the equipment listed is for the central level, not for the GeneXpert site. Part B How to perform stool testing 11 Practical manual of processing stool samples for diagnosis of childhood TB Practical manual of processing stool samples for diagnosis of childhood TB12 Part C Implementation of stool testing This section describes the requirements for the implementation of stool testing. It follows the 10 steps outlined in the chapter on implementation of new diagnostics in the WHO operational handbook on tuberculosis Module 3: Diagnosis (3). C1 Where to place stool testing in the tiered laboratory network Introduction of stool testing for diagnosis of paediatric TB adds a new sample type to an existing diagnostic platform. Currently, WHO recommends that stool be tested using the Xpert MTB/RIF and Xpert Ultra assays. Thus, it is logical to incorporate stool testing in the existing laboratory network for Xpert MTB/RIF or Xpert Ultra testing, and to use the GeneXpert instruments and network already present in-country. When scaling up GeneXpert instruments, the placement strategy needs to consider access for specific key populations benefiting from stool testing (e.g. children) and an effective sample transportation system. Ultimately, countries should be able to perform stool testing at any GeneXpert site; for example, in the same laboratory performing sputum testing for TB. However, when resources are limited, the use of stool should mainly be promoted where it would be of most value, and it is important to consider that stool testing might be most cost-effective in settings with higher prevalence of TB (32). Furthermore, the choice of specimen type (GA, induced sputum or stool) collected for children depends on the acceptability for children, parents and caregivers, health care workers and other stakeholders; on the feasibility of collecting and preparing specimens in the local context; and on local test availability. To help clinicians decide on whether a child should be started on TB treatment, the WHO operational handbook on the management of TB in children and adolescents provides guidance on treatment decision algorithms that integrate a risk assessment for rapid progression of TB disease, bacteriological confirmation where possible and available (including stool testing as relevant), history of contact, clinical signs and symptoms and findings on chest radiography (5). The Pediatric TB Operational and Sustainability Expertise Exchange (POSEE) is a task force under the Child and Adolescent TB Working Group and an entity of the Stop TB Partnership. POSEE has written a position paper that clearly describes the position of the microbiological diagnosis in the diagnostic pathway (12). Part C Implementation of stool testing 13 Practical manual of processing stool samples for diagnosis of childhood TB C2 Steps and processes for implementation C2.1 Policies and planning Globally, countries have adopted either Xpert MTB/RIF or Xpert Ultra (or both) in their national guidelines as an initial test to diagnose TB and detect resistance to rifampicin. However, most countries do not include stool specimens as a sample type for diagnosis of TB among children. Therefore, national guidelines and policies need to be adapted and updated to include stool for the diagnosis of TB in children, in line with the new WHO recommendations (3). The in-country technical working group leads the review of the national guidelines and policies, including the implementation of stool testing. Generally, a situational analysis plan contains a map of the health care centres that have a GeneXpert instrument and linkages to referring and referral laboratories. To prepare a costed operational plan with timelines and milestones for the introduction of stool testing, the working group can use the existing situation analysis if one is available or can undertake a new analysis. The POSEE task force has developed a budget tool for specimen collection for TB diagnosis to support national TB programmes (NTPs) in budget forecasting, and has included stool as one of the possible specimens. The budget tool and other relevant tools are available from the Child and Adolescent TB Working Group page on the Stop TB Partnership website (33). The best approach is a phased implementation, starting with a pilot implementation at a few selected sites that have a high TB notification rate or are actively involved in diagnosis of paediatric TB. These sites can subsequently train other sites, ensuring that all provinces achieve access to Xpert stool testing and have trained staff to perform the tests. To determine which sites to prioritize for the implementation of stool Xpert testing, the number of children seen with signs and symptoms of TB (presumptive TB) can be used as an indicator. Alternatively, the number of children with respiratory infections, pneumonia or malnutrition can be considered. However, it may be difficult to retrieve this information as NTPs often do not systematically collect such data – the number of children clinically or radiographically diagnosed with TB is a good alternative for selecting the sites. C2.2 Regulatory processes No registration specific for stool testing is needed, because stool is a sample type that is already collected in routine settings for other diseases and laboratory tests. Diagnosis of TB from stool samples uses GeneXpert instruments, which are already registered for this purpose. C2.3 Equipment and site preparation For site preparation, equipment additional to the GeneXpert instruments might be needed, depending on the stool processing method to be implemented. Implementation of the SOS stool method requires no additional equipment other than the equipment currently used for Xpert MTB/ RIF or Xpert Ultra sputum testing. Implementation of the OSF method does require some additional equipment, but it is low cost (see Table 2). The introduction of stool testing will increase the use of the GeneXpert platform in each site. Therefore, for each site enrolled in stool testing, the expected workload needs to be estimated and matched with the diagnostic capacity of the available GeneXpert instruments. The number of broken or in other ways non-functional, modules should be considered, because this naturally reduces the Practical manual of processing stool samples for diagnosis of childhood TB14 capacity per site. The costed operational plan for the site preparation will include provision of stool collection and processing, workload analysis, staff mapping, training of staff and other logistical needs. Annex 2 provides an example of what is needed to start stool testing; it can be adapted to country specifics and used to check the readiness of the sites to implement stool testing. C2.4 Supply chain For implementation of stool testing, a few additional supplies might be needed on top of those that are routinely used for sputum Xpert testing (see Table 2). Most important for supply management is the increase in the number of Xpert cartridges used. For each site, the incremental workload needs to be estimated and matched with the number of cartridges to be supplied. Owing to its higher sensitivity, Xpert Ultra is preferable to the Xpert MTB/RIF assay for the detection of TB in children. Another requirement is stool containers, which can vary in type, as discussed in Section B1. Transportation of stool samples should use the same transport network that is used for sputum Xpert testing; ideally, a cold chain should be maintained, with samples kept at low temperature until testing. Each stool container should be packed in a ziplocked plastic bag containing absorbent material with a biohazard label on the outside, as for sputum transportation. If such bags are not available, filled stool containers should at least be wrapped in toilet paper and placed separately in a simple plastic bag, to avoid any cross contamination should leakage occur during transport to the laboratory. C2.5 Procedures The most up to date SOPs of the OSF stool processing method can be found on the TB-Speed website (27), and a detailed SOP for the SOS stool method can be found on the KNCV website (31). The SOPs should be customized to the country requirements, and translated into local languages where necessary. C2.6 Digital data When stool testing is implemented, instructions need to be provided to the laboratory staff on what relevant details should be added when collecting and analysing the data. For example, when starting the test run in the GeneXpert instrument, “stool” should be entered within the field for sample type. This allows the test runs of stool samples to be separated from test runs of other sample types when measuring the positivity rate or other quality indicators. If the GeneXpert instrument is linked to a connectivity system (e.g. GXAlert or DataToCare) that allows additional data to be entered, it is useful to add information about the stool sample or patients (34). Such information could include, for example, the consistency of the stool sample (e.g. as listed in the Bristol chart, or grouped as “formed”, “semi formed” or “liquid”) or the age or other characteristics of the child patient – capturing this type of information can be useful for operational research purposes. Part C Implementation of stool testing 15 Practical manual of processing stool samples for diagnosis of childhood TB C2.7 Quality assurance control and assessment Targets of quality indicators (e.g. error and invalid rates) that are set for sputum Xpert testing might need to be adjusted when performing Xpert testing on stool. Current studies suggest that the initial non-determinate rate for stool is slightly higher than for sputum Xpert testing. In comparison with sputum, stool naturally contains a high load of solid particles, which may lead to errors linked to issues with the sample transfer through the microfluid filter in the cartridge (e.g. if the supernatant containing the bacteria is not well separated from the debris). Current studies on stool testing show that the most common error code obtained is “error 2008”, which is linked to this phenomenon. Also, stool contains other organic substances not present in sputum that might inhibit the polymerase chain reaction, resulting in invalid Xpert results. Repeating the test once using the same sample decreases the rate of non-determinate Xpert results (35). More studies are needed to provide better insights on these aspects. C2.8 Recording and reporting For the implementation of stool testing, the recording and reporting tools need to be carefully reviewed and adapted to include stool as a sample type. For analysis of both the number of notified cases and quality indicators, it is important that the outcome of the test can be stratified by sample type, including stool. C2.9 Training and competency assessment The most cost-effective approach is to first train staff already competent with sputum Xpert testing on how to perform the specific steps for stool processing, using either the OSF or SOS processing method; these staff are already familiar with the use of the GeneXpert instrument. For staff who are not competent with sputum Xpert testing, a more extensive training is required. As observed for sputum Xpert testing, it takes time for the laboratory staff to become familiar with the use of stool samples and with the processing method. Therefore, it might be that, when initially implementing the use of stool, there will be slightly more non-determinate Xpert results; however, this would be expected to decrease as staff gain more experience. This situation should be considered when ordering Xpert cartridges. In parallel with the training on stool processing for the laboratory staff, all involved health care providers should be trained to perform the stool collection procedure and to explain the procedure to parents and caregivers. Generally, at most health care centres, stool is routinely collected for other diseases (e.g. parasitology). Thus, health care staff are likely to already have the knowledge and the tools to collect this specimen type. However, staff working in TB facilities may be less familiar with stool collection, and thus may need specific training on stool collection and on the development and distribution of a stool collection flyer. Furthermore, all health care providers involved in stool sample collection should be trained on the treatment decision algorithms that include stool as a primary specimen. Practical manual of processing stool samples for diagnosis of childhood TB16 C2.10 Monitoring and evaluation Once staff have been trained and have started using stool as a primary specimen to diagnose TB in children, it is essential to closely monitor and supervise their stool-related work on a weekly or fortnightly basis, at least for the first 2 months. Troubleshooting should address the rate of non-determinate results, as discussed in Section C2.7. For stool testing, additional indicators need to be added to the standard laboratory monitoring and supervision checklist for Xpert MTB/RIF and Xpert Ultra testing (36). Annex 3 provides a list of the stool-specific indicators to consider. These indicators can be added to the standard list of indicators used for supervision and monitoring of sputum Xpert testing. 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Geneva: Global Laboratory Initiative; 2016 (https://stoptb.org/wg/gli/assets/documents/gli_connectivity_guide.pdf, accessed January 2022). 35. Tiemersma E. Accuracy of the simple one-step stool method with Xpert MTB/RIF Ultra assay for the diagnosis of M. tuberculosis in children. 2021. 36. Tuberculosis technical scorecard Xpert MTB/RIF. Geneva: Stop TB Partnership; 2020 (https:// stoptb.org/wg/gli/assets/documents/5%20Find-TB-Scorecard-Xpert-Low-Res.pdf, accessed January 2022). References 21 Practical manual of processing stool samples for diagnosis of childhood TB Practical manual of processing stool samples for diagnosis of childhood TB22 A n n ex 1 . O ve rv ie w o f p u b lic at io n s o n s to o l p ro ce ss in g fo r T B d et ec ti o n u n ti l D ec em b er 2 02 1 Fo r e ac h pu bl ica tio n, th e fo llo w in g ar e pr ov id ed if a pp lic ab le a nd if re po rt ed : p at ie nt a ge , n um be r o f p at ie nt s in clu de d in th e an al ys is/ a ll pa tie nt s el ig ib le , st ud y po pu la tio n, s to ol p ro ce ss in g m et ho d us ed a nd s en si tiv iti es a nd s pe ci fic iti es a ga in st v ar io us r ef er en ce s ta nd ar ds . St ud y, ye ar / co un try (re fe re nc e) Ag e (y ea rs ) ra ng e, m ed ia n [IQ R] In clu de d in an al ys is/ al l el ig ib le Po pu la tio n Am ou nt of st oo l St oo l p ro ce ss in g m et ho d in clu de s: No . m icr o- bi ol og ica lly co nfi rm ed (% ) No . cli ni ca lly co nfi rm ed (% ) Re fe re nc e sta nd ar d St oo l X pe rt pe rfo rm an ce dilution in vortexing centrifugation filtration Se ns iti vit y (9 5% CI ) Sp ec ifi ci ty (9 5% CI ) Ai na n, 20 21 / Ta nz an ia (1 ) NR , 2 .1 7 [1 .1 6– 5. 19 ] 22 5/ 25 8 Ch ild re n w ith pr es um pt iv e TB in 6 he al th fa cil iti es in D ar e s S al aa m 2 cm 3 Di st ill ed w at er an d SR Ye s No No 8 (3 .6 ) 42 (1 8. 7) Xp er t a nd /o r so lid cu ltu re o n sp ut um o r G A 62 .5 % (2 5– 92 % ) 10 0% (9 8– 10 0% ) An dr iy ok o, 20 19 / In do ne sia (2 ) 0– 14 , 1 .4 [0 .4 –6 .5 ] 36 /N R La bo ra to ry st ud y in 1 h os pi ta l; co ns ec ut iv e st oo l s pe cim en s su bm itt ed fo r T B di ag no sis 0. 8– 1 g PB S an d SR No No No 6 (1 7) – Xp er t o n GA or in du ce d sp ut um 10 0% 87 .5 % Ba na da , 20 16 / S ou th Af ric a (3 ) 0– 15 , N R 37 /4 0 20 M TB + an d 20 M TB o n in du ce d sp ut um o r G A Xp er t 0. 6 g Co m m er cia l bu ff er Ye s, w ith gl as s be ad s No Ye s 20 (5 4) – Xp er t o n GA or in du ce d sp ut um 85 % (6 2– 97 % ) 10 0% (9 8– 10 0% ) Ch ip in du ro , 20 17 / Zi m ba bw e (4 ) 5– 16 , 10 .6 [8 –1 3] 21 8/ 21 8 Pr es en tin g w ith pr es um pt iv e TB in 8 P HC s: TB sy m pt om s o r hi st or y o f c lo se co nt ac t w ith T B pa tie nt 0. 15 g PB S an d SR Ye s Ye s No 19 (8 .7 ) – LJ cu ltu re /X pe rt on in du ce d sp ut um 68 % (4 3– 87 % ) 98 % (9 5– 99 % ) Annex 1. Overview of publications on stool processing for TB detection until December 2021 23 St ud y, ye ar / co un try (re fe re nc e) Ag e (y ea rs ) ra ng e, m ed ia n [IQ R] In clu de d in an al ys is/ al l el ig ib le Po pu la tio n Am ou nt of st oo l St oo l p ro ce ss in g m et ho d in clu de s: No . m icr o- bi ol og ica lly co nfi rm ed (% ) No . cli ni ca lly co nfi rm ed (% ) Re fe re nc e sta nd ar d St oo l X pe rt pe rfo rm an ce dilution in vortexing centrifugation filtration Se ns iti vit y (9 5% CI ) Sp ec ifi ci ty (9 5% CI ) Ch ip in du ro , 20 17 / Zi m ba bw e (4 ) 5– 16 , 10 .6 [8 –1 3] 32 /2 18 Pr es en tin g w ith sy m pt om s o f TB in 8 P HC s: TB sy m pt om s o r hi st or y o f c lo se co nt ac t w ith T B pa tie nt 0. 15 g PB S an d SR Ye s Ye s No – 32 Cl in ica l 53 % (3 5– 71 % ) NR de H aa s, 20 21 / Et hi op ia (5 ) NR 12 3/ 14 7 Ch ild re n pr es en tin g w ith pr es um pt iv e TB in se le ct ed he al th fa cil iti es , fo r w ho m ro ut in e na so GA w as re qu es te d up on cl in ica l ex am in at io n 0. 8– 1 g SR No No No 9 (7 .3 ) – Xp er t a nd /o r L J cu ltu re a nd /o r M GI T cu ltu re on n as oG A 78 % – Ha nr ah an , 20 19 / S ou th Af ric a (6 ) <1 0, 2 1. 4 m on th s [1 2. 3– 42 .9 ] 11 9 Ch ild re n w ith sig ns a nd sy m pt om s o f T B pr es en tin g at a pr im ar y- ca re cl in ic NR PB S, N AL C- Na OH a nd SR NR Ye s No 4 (3 ) – Sm ea r, cu ltu re or X pe rt o n an y o ne o f th e sa m pl es co lle ct ed 0/ 4 m icr ob io lo gi ca lly co nfi rm ed T B pa tie nt s h ad a po sit iv e te st o n st oo l 0/ 15 p at ie nt s un lik el y t o ha ve TB h ad a p os iti ve te st o n st oo l Ha nr ah an , 20 19 / S ou th Af ric a (6 ) <1 0, 2 1. 4 m on th s [1 2. 3– 42 .9 ] 11 9 Ch ild re n w ith sig ns a nd sy m pt om s o f T B pr es en tin g at a pr im ar y- ca re cl in ic N R PB S, N AL C- Na OH a nd SR NR Ye s No – 10 0 (8 4) At le as t 2 o f t he fo llo w in g: C XR co ns ist en t w ith TB , p os iti ve cli ni c r es po ns e to a nt i-T B tre at m en t, do cu m en te d ex po su re to T B or a p os iti ve TS T 0/ 10 0 cli ni ca lly co nfi rm ed T B pa tie nt s h ad a po sit iv e te st o n st oo l 0/ 15 p at ie nt s un lik el y t o ha ve TB h ad a p os iti ve te st o n st oo l Practical manual of processing stool samples for diagnosis of childhood TB24 St ud y, ye ar / co un try (re fe re nc e) Ag e (y ea rs ) ra ng e, m ed ia n [IQ R] In clu de d in an al ys is/ al l el ig ib le Po pu la tio n Am ou nt of st oo l St oo l p ro ce ss in g m et ho d in clu de s: No . m icr o- bi ol og ica lly co nfi rm ed (% ) No . cli ni ca lly co nfi rm ed (% ) Re fe re nc e sta nd ar d St oo l X pe rt pe rfo rm an ce dilution in vortexing centrifugation filtration Se ns iti vit y (9 5% CI ) Sp ec ifi ci ty (9 5% CI ) Ha sa n, 20 17 / Pa ki st an (7 ) 0– 15 , 6 .8 [2 –9 ] 49 /5 0 of 6 4 ch ild re n w ith cli ni ca l sy m pt om s Pr es en tin g w ith sy m pt om s o f pu lm on ar y T B in 2 te rt ia ry h os pi ta ls 0. 15 g PB S an d SR Ye s Ye s No 11 (2 2) – LJ cu ltu re / Xp er t o n GA o r sp ut um 82 % (4 8– 98 % ) 95 % (8 2– 99 % ) Ha sa n, 20 17 / Pa ki st an (7 ) 0– 15 , 6 .8 [2 –9 ] 49 /5 0 of 6 4 ch ild re n w ith cli ni ca l sy m pt om s Pr es en tin g w ith sy m pt om s o f pu lm on ar y T B in 2 te rt ia ry h os pi ta ls 0. 15 g PB S an d SR Ye s Ye s No – 17 (3 5) Cl in ica l 59 % (3 3– 82 % ) 10 0% (8 9– 10 0% ) La Co ur se , 20 18 / K en ya (8 ) 0– 12 , 2 [1 .1 –4 .8 ] 14 7/ 16 5 HI V- po sit iv e ch ild re n el ig ib le fo r A RT , ho sp ita liz ed fo r ac ut e ill ne ss in 4 ho sp ita ls NR PB S, N AL C- Na OH , S R No Ye s No 11 (7 .5 ) – M GI T cu ltu re / Xp er t o n sp ut um o r G A 70 % (3 5– 93 % ) 10 0% (9 7– 10 0% ) La Co ur se , 20 18 / K en ya (8 ) 0– 12 , 2 [1 .1 –4 .8 ] 16 5/ 16 5 HI V- po sit iv e ch ild re n el ig ib le fo r A RT , ho sp ita liz ed fo r ac ut e ill ne ss in 4 ho sp ita ls NR PB S, N AL C- Na OH , S R No Ye s No – 85 (5 2) Cl in ica l 9% (4 –1 9% ) 10 0% (9 5– 10 0% ) Lo un na s, 20 20 / Fr an ce (9 ) NA . NA . NA . 0. 5 g su cr os e so lu tio n an d SR No No No NA . NA . NA . NA . NA . M ar cy , 20 16 / Bu rk in a Fa so , Ca m bo di a, Ca m er oo n, Vi et N am (1 0) 0– 13 , 7 .2 (4 .1 –7 .2 ) 27 2/ 27 2 HI V- po sit iv e ch ild re n pr es en tin g w ith pr es um pt iv e pu lm on ar y T B in 8 te rt ia ry /p ae di at ric ho sp ita ls 0. 5 g Su cr os e so lu tio n an d SR No Ye s Ye s 27 (1 0) – M GI T/ LJ cu ltu re on G A, in du ce d sp ut um , N GA , st rin g sa m pl e 67 % (4 6– 83 % ) 10 0% (9 8– 10 0% ) Annex 1. Overview of publications on stool processing for TB detection until December 2021 25 St ud y, ye ar / co un try (re fe re nc e) Ag e (y ea rs ) ra ng e, m ed ia n [IQ R] In clu de d in an al ys is/ al l el ig ib le Po pu la tio n Am ou nt of st oo l St oo l p ro ce ss in g m et ho d in clu de s: No . m icr o- bi ol og ica lly co nfi rm ed (% ) No . cli ni ca lly co nfi rm ed (% ) Re fe re nc e sta nd ar d St oo l X pe rt pe rfo rm an ce dilution in vortexing centrifugation filtration Se ns iti vit y (9 5% CI ) Sp ec ifi ci ty (9 5% CI ) M ar cy , 20 16 / Bu rk in a Fa so , Ca m bo di a, Ca m er oo n, Vi et N am (1 0) 0– 13 , 7 .2 (4 .1 –7 .2 ) 27 2/ 27 2 HI V- po sit iv e ch ild re n pr es en tin g w ith pr es um pt iv e pu lm on ar y T B in 8 te rt ia ry or p ae di at ric ho sp ita ls 0. 5 g su cr os e so lu tio n an d SR No Ye s Ye s – 24 5 (9 0) Cl in ica l 11 % (8 –1 6% ) 96 % (8 1– 10 0% ) M em on , 20 18 / I nd ia (1 1) 0. 5– 15 , 11 10 0/ 10 0 Ch ild re n at te nd in g th e pa ed ia tr ic TB cli ni c o f a te rt ia ry ca re h os pi ta l 0. 2 g PB S, N AL C- Na OH , S R No Ye s No 26 (2 6) – M GI T cu ltu re on in du ce d sp ut um o r G A 11 .5 % (2 .4 –3 0. 1% ) 98 .6 % (9 2. 7– 99 .9 % ) M ou ss a, 20 16 / E gy pt (1 2) >1 to < 15 , NR 11 5/ 11 5 Pr es en tin g w ith cli ni ca l s ig ns o f pu lm on ar y T B in a te rt ia ry ca re ho sp ita l 2 g di st ill ed w at er , PB S, N AL C- Na OH , S R No Ye s No 36 (3 1) – LJ cu ltu re on sp ut um or in du ce d sp ut um 83 % (6 7– 94 % ) 99 % (9 3– 10 0% ) Ng ad ay a, 20 20 / Ta nz an ia (1 3) 1– 95 , 3 5 [2 1– 47 ] 59 0/ NR Pr es um pt iv e TB pa tie nt s > 1 ye ar ol d, 7 p rim ar y he al th fa cil iti es an d 5 te rt ia ry he al th fa cil iti es , Xp er t o n st oo l co nd uc te d at CT RL 2 cm 3 Di st ill ed w at er a nd SR Ye s Ye s No 75 (1 2. 7) – LJ cu ltu re o n sp ut um 84 % (8 1. 0– 87 .0 % ) 93 .4 % (9 8. 5– 99 .9 % ) Ng ad ay a, 20 20 / Ta nz an ia (1 3) 1– 95 , 3 5 [2 1– 47 ] 59 0/ NR Pr es um pt iv e TB pa tie nt s > 1 ye ar ol d, 7 p rim ar y he al th fa cil iti es an d 5 te rt ia ry he al th fa cil iti es , Xp er t o n st oo l co nd uc te d at p er ip he ra l la bo ra to ri es 2 cm 3 Di st ill ed w at er a nd SR Ye s Ye s No 75 (1 2. 7) – LJ cu ltu re o n sp ut um 63 .0 % (4 7. 8– 76 .1 % ) 76 .7 % (7 2. 1– 81 .4 % ) Practical manual of processing stool samples for diagnosis of childhood TB26 St ud y, ye ar / co un try (re fe re nc e) Ag e (y ea rs ) ra ng e, m ed ia n [IQ R] In clu de d in an al ys is/ al l el ig ib le Po pu la tio n Am ou nt of st oo l St oo l p ro ce ss in g m et ho d in clu de s: No . m icr o- bi ol og ica lly co nfi rm ed (% ) No . cli ni ca lly co nfi rm ed (% ) Re fe re nc e sta nd ar d St oo l X pe rt pe rfo rm an ce dilution in vortexing centrifugation filtration Se ns iti vit y (9 5% CI ) Sp ec ifi ci ty (9 5% CI ) Ni co l, 20 13 / So ut h Af ric a (1 4) 1- <1 5, 2 .6 [1 .6 –4 .8 ] 11 5/ 11 5 PH C an d te rt ia ry pe di at ric h os pi ta l 0. 15 g (F LO Q sw ab s1 ) PB S an d SR No Ye s No 17 (1 5) – M GI T cu ltu re on in du ce d sp ut um 47 % (2 3– 72 % ) 99 % (9 4– 10 0% ) Or ik iri za , 20 18 / Ug an da (1 5) 1 M –1 4, NR 34 9/ 35 7 Pa tie nt s s ta rt in g on T B tre at m en t i n a re gi on al re fe rr al ho sp ita l NR Sa lin e so lu tio n, NA LC - Na OH , PB S an d un sp ec ifi ed bu ff er No Ye s No 9 (2 .6 ) – LJ & M GI T Cu ltu re o n sp ut um o r in du ce d sp ut um 56 % (2 1– 86 % ) 98 % (9 0– 10 0% ) W al te rs , 20 12 / S ou th Af ric a (1 6) 0 to <1 4, 1 7 m on th s [N R] 23 /2 8 (1 4 w ith b ot h GA a nd st oo l, 6 on ly G A, 3 on ly st oo l) Pr es en tin g w ith pr es um pt iv e pu lm on ar y T B in 2 ho sp ita ls NR Sa lin e so lu tio n, NA LC - Na OH , P BS an d SR No Ye s No 4 (1 7) – M GI T cu ltu re / Xp er t o n GA NR (c om bi na tio n of st oo l a nd G A Xp er t v er su s M GI T cu ltu re : 75 % ) NR W al te rs , 20 12 / S ou th Af ric a (1 6) 0 to <1 4, 1 7 m on th s [N R] 23 /2 8 (1 4 w ith b ot h GA a nd st oo l) Pr es en tin g w ith pr es um pt iv e pu lm on ar y T B in 2 ho sp ita ls NR sa lin e so lu tio n, NA LC - Na OH , P BS an d SR No Ye s No – 12 (5 2) Cl in ica l NR (c om bi na tio n of st oo l a nd G A Xp er t v er su s cli ni ca l D x: 2 5% ) NR W al te rs , 20 17 / S ou th Af ric a (1 7) 0 to <1 3, 1 .3 [0 .8 –2 .4 ] 37 9/ 37 9 Pr es en tin g w ith pr es um pt iv e pu lm on ar y T B in 2 re fe rr al h os pi ta ls <5 g a nd 1– 4 g PB S, N AL C- Na OH , S R No Ye s No 72 (1 9) – M GI T cu ltu re on G A, in du ce d sp ut um , N GA , st rin g sa m pl e 32 % (2 1– 44 % ) 10 0% (9 8– 10 0% ) W al te rs , 20 17 / S ou th Af ric a (1 7) 0– 13 , 1 .3 [0 .8 –2 .4 ] 35 1/ 37 9 Pr es en tin g w ith pr es um pt iv e pu lm on ar y T B in 2 re fe rr al h os pi ta ls <5 g a nd 1– 4 g PB S, N AL C- Na OH , S R No Ye s No – 24 2 (6 9) Cl in ica l 10 % (6 –1 4% ) 10 0% (9 7– 10 0% ) 1 F or m or e in fo , p le as e se e FL O Q Sw ab s, C O PA N D ia gn os tic s In c. Annex 1. Overview of publications on stool processing for TB detection until December 2021 27 St ud y, ye ar / co un try (re fe re nc e) Ag e (y ea rs ) ra ng e, m ed ia n [IQ R] In clu de d in an al ys is/ al l el ig ib le Po pu la tio n Am ou nt of st oo l St oo l p ro ce ss in g m et ho d in clu de s: No . m icr o- bi ol og ica lly co nfi rm ed (% ) No . cli ni ca lly co nfi rm ed (% ) Re fe re nc e sta nd ar d St oo l X pe rt pe rfo rm an ce dilution in vortexing centrifugation filtration Se ns iti vit y (9 5% CI ) Sp ec ifi ci ty (9 5% CI ) W al te rs , 20 18 / S ou th Af ric a [1 7] 1. 3 [0 .9 –2 .4 ] 28 0/ 30 2 Pr es en tin g w ith pr es um pt iv e pu lm on ar y T B in 2 re fe rr al h os pi ta ls 0. 6 g an d sw ab PB S an d SR Ye s, w ith gl as s be ad s No Ye s 23 (8 .3 ) – M GI T/ Xp er t o n ex pe ct or at ed or in du ce d sp ut um a nd G A if <5 y (s ub se t o nl y) 44 .4 % (1 3. 7– 78 .8 % ) Xp er t a s R S 25 .0 % (7 .3 –5 2. 4% ) cu ltu re a s R S 99 .1 % (9 6. 8– 99 .9 % ) Xp er t a s R S 99 .5 % (9 7. 5– 10 0% ) cu ltu re a s R S W al te rs , 20 18 / S ou th Af ric a (1 8) NR , 1 .3 [0 .9 –2 .4 ] 28 0/ 30 2 Pr es en tin g w ith pr es um pt iv e pu lm on ar y T B in 2 re fe rr al h os pi ta ls 0. 6 g an d sw ab PB S an d SR Y e s, w ith gl as s be ad s No Ye s – 88 (3 1) Cl in ica l 44 .4 % (1 3. 7– 78 .8 % ) Xp er t a s R S 25 .0 % (7 .3 –5 2. 4% ) cu ltu re a s R S 99 .1 % (9 6. 8– 99 .9 % ) Xp er t a s R S 99 .5 % (9 7. 5– 10 0% ) cu ltu re a s R S W el da y, 20 14 / K en ya (1 9) 0 to < 15 , NR 53 /9 1 La bo ra to ry st ud y in 2 h os pi ta ls in clu di ng ch ild re n re fe rr ed fo r TB te st in g by a cli ni cia n 0. 15 g PB S on ly ve rs us P BS an d SR No No ve rs us ye s No 6 (1 1. 3) – ZN sp ut um sm ea r m icr os co py 10 0% . D ire ct m et ho d di d yi el d m or e ca se s t ha n in di re ct m et ho d 89 % AR T: a nt ire tr ov ira l t he ra py ; C I: co nfi de nc e in te rv al ; C TR L: c en tr al T B r ef er en ce la bo ra to ry ; C XR : c he st X -r ay ; G A: g as tr ic a sp ira te ; H IV : h um an im m un od efi ci en cy v ir us ; I Q R: in te rq ua rt ile ra ng e; L J: Lö w en st ei n– Je ns en ; M G IT : m yc ob ac te ri a gr ow th in di ca to r t ub e; M TB : M yc ob ac te ri um tu be rc ul os is ; N A: n ot a pp lic ab le ; N AL C- N aO H : N -a ce ty l-L -c ys te in e– so di um c itr at e- so di um h yd ro xi de ; N G A: n as op ha ry ng ea l a sp ira te ; N R: n ot r ep or te d; P B S: p ho sp ha te -b uff er ed s al in e; P H C: p ri m ar y he al th c en tr e; R S: r ef er en ce s ta nd ar d; S R: Xp er t s am pl e re ag en t; TB : t ub er cu lo si s; T ST : t ub er cu lin s ki n te st ; Z N : Z ie hl –N ee ls en Practical manual of processing stool samples for diagnosis of childhood TB28 References for Annex 1 1. Ainan S, Furia FF, Mhimbira F, Mnyambwa NP, Mgina N, Zumla A et al. Xpert® MTB/RIF assay testing on stool for the diagnosis of paediatric pulmonary TB in Tanzania. Public Health Action. 2021;11(2):75–9. 2. Andriyoko B, Janiar H, Kusumadewi R, Klinkenberg E, de Haas P, Tiemersma E. Simple stool processing method for the diagnosis of pulmonary tuberculosis using GeneXpert MTB/RIF. Eur Respir J. 2019;53(3):1801832. 3. Banada PP, Naidoo U, Deshpande S, Karim F, Flynn JL, O’Malley M et al. A novel sample processing method for rapid detection of tuberculosis in the stool of pediatric patients using the Xpert MTB/ RIF assay. PLoS One. 2016;11(3):1–13. 4. Chipinduro M, Mateveke K, Makamure B, Ferrand RA, Gomo E. Stool Xpert® MTB/RIF test for the diagnosis of childhood pulmonary tuberculosis at primary clinics in Zimbabwe. Int J Tuberc Lung Dis. 2017;21(2):161–6. 5. de Haas P, Yenew B, Mengesha E, Slyzkyi A, Gashu Z, Lounnas M et al. The simple one-step (SOS) stool processing method for use with the Xpert MTB/RIF assay for a child-friendly diagnosis of tuberculosis closer to the point of care. J Clin Microbiol. 2021;59(8):e0040621. 6. Hanrahan CF, Dansey H, Mutunga L, France H, Omar SV, Ismail N et al. Diagnostic strategies for childhood tuberculosis in the context of primary care in a high burden setting: the value of alternative sampling methods. Paediatr Int Child Health. 2019;39(2):88–94. 7. Hasan Z, Shakoor S, Arif F, Mehnaz A, Akber A, Haider M et al. Evaluation of Xpert MTB/RIF testing for rapid diagnosis of childhood pulmonary tuberculosis in children by Xpert MTB/RIF testing of stool samples in a low resource setting. BMC Res Notes. 2017;10(1):473. 8. LaCourse SM, Pavlinac PB, Cranmer LM, Njuguna IN, Mugo C, Gatimu J et al. Stool Xpert MTB/RIF and urine lipoarabinomannan for the diagnosis of tuberculosis in hospitalized HIV-infected children. AIDS (London, England). 2018;32(1):69–78. 9. Lounnas M, Diack A, Nicol MP, Eyangoh S, Wobudeya E, Marcy O et al. Laboratory development of a simple stool sample processing method diagnosis of pediatric tuberculosis using Xpert Ultra. Tuberculosis (Edinb). 2020;125:102002. 10. Marcy O, Ung V, Goyet S, Borand L, Msellati P, Tejiokem M et al. Performance of Xpert MTB/RIF and alternative specimen collection methods for the diagnosis of tuberculosis in HIV-infected children. Clin Infect Dis. 2016;62(9):1161–8. 11. Memon SS, Sinha S, Sharma SK, Kabra SK, Lodha R, Soneja M. Diagnostic accuracy of Xpert MTB/ RIF assay in stool samples in intrathoracic childhood tuberculosis. Journal of Tuberculosis and Therapeutics. 2018;3(2). 12. Moussa HS, Bayoumi FS, Mohamed AMA. Gene Xpert for direct detection of Mycobacterium tuberculosis in stool specimens from children with presumptive pulmonary tuberculosis. Ann Clin Lab Sci. 2016;46(2):198–203. 13. Ngadaya E, Kimaro G, Sandi E, Mnyambwa NP, Wilfred A, Lubinza C et al. Evaluation of stool GeneXpert MTB/RIF for the diagnosis of pulmonary tuberculosis among presumptive patients in Tanzania. J Clin Tuberc Other Mycobact Dis. 2020;21:100195. Annex 1. Overview of publications on stool processing for TB detection until December 2021 29 14. Nicol MP, Spiers K, Workman L, Isaacs W, Munro J, Black F et al. Xpert MTB/RIF testing of stool samples for the diagnosis of pulmonary tuberculosis in children. Clin Infect Dis. 2013;57(3):18–21. 15. Orikiriza P, Nansumba M, Nyehangane D, Bastard M, Mugisha IT, Nansera D et al. Xpert MTB/RIF diagnosis of childhood tuberculosis from sputum and stool samples in a high TB-HIV-prevalent setting. Eur J Clin Microbiol Infect Dis. 2018;37(8):1465–73. 16. Walters E, Gie RP, Hesseling AC, Friedrich SO, Diacon AH, Gie RP. Rapid diagnosis of pediatric intrathoracic tuberculosis from stool samples using the Xpert MTB/RIF Assay: a pilot study. Pediatr Infect Dis J. 2012;31(12):1316. 17. Walters E, van der Zalm MM, Palmer M, Bosch C, Demers A-M, Draper H et al. Xpert MTB/RIF on stool is useful for the rapid diagnosis of tuberculosis in young children with severe pulmonary disease. Pediatr Infect Dis J. 2017;36(9):837–43. 18. Walters E, Scott L, Nabeta P, Demers A-M, Reubenson G, Bosch C et al. Molecular detection of Mycobacterium tuberculosis from stools in young children by use of a novel centrifugation-free processing method. J Clin Microbiol. 2018;56(9):e00781–18. 19. Welday SH, Kimang’a AN, Kabera BM, Mburu JW, Mwachari C, Mungai E et al. Stool as appropriate sample for the diagnosis of Mycobacterium tuberculosis by Gene Xpert test. Open J Respir Dis. 2014;04:83–9. Practical manual of processing stool samples for diagnosis of childhood TB30 Annex 2. List of activities for the implementation of stool testing No. Activity Condition (yes/no/in progress) 1 Adapt national policies and guidance to include stool as a primary sample for the diagnosis of TB in children with signs and symptoms of TB 2 Train health care providers on the new national guidance including the diagnostic algorithms incorporating stool 3 Train health care providers on treatment initiation using stool Xpert results 4 Train health care providers on the collection of stools 5 Train laboratory staff on the stool processing method(s) 6 Assign and train the focal point (site coordinator) 7 Adapt the reporting and registration tools to include a provision for stool 8 Adapt the digital data collection and connectivity tools to include a provision for stool 9 Add indicators specific for stool testing to the standard monitoring and supervision list for Xpert MTB/RIF and Xpert Ultra testing 10 Ensure availability of supplies needed for stool collection (e.g. stool container and stool collection flyer) 11 Ensure availability of supplies to transport stool samples (e.g plastic bag and cooler box) and arrange that stool can be transported through the routine sample referral network 12 Ensure availability of the additional supplies for the stool processing method using Xpert MTB/RIF or Xpert Ultra 13 Ensure availability of the SOPs and bench aids for stool testing 14 Ensure availability of enough Xpert MTB/RIF or Xpert Ultra cartridges for stool testing SOP: standard operating procedure; TB: tuberculosis. Annex 2. List of activities for the implementation of stool testing 31 Annex 3. Laboratory tool for assessing stool testing by Xpert MTB/RIF and Xpert MTB/RIF Ultra Laboratory stool testing assessment The laboratory indicators listed in this tool should be integrated into the standard monitoring and supervision tool for Xpert MTB/RIF and Xpert Ultra testing already used by the country (1). Name of institution District Date of visit Name and contact details of staff met Name: Designation: Tel/email: Name: Designation: Tel/email: Name: Designation: Tel/email: Names of assessors Practical manual of processing stool samples for diagnosis of childhood TB32 Number or Yes/no/partial Comments 1. Stool samples obtained Number of children with signs and symptoms of TB for whom a stool sample has been collected per month 2. Quality of the collected stool sample Are stool samples kept between 2 °C and 8 °C during transport? Are stool samples kept between 2 °C and 8 °C before testing? Are correct collection pots used? Are lids properly closed? Number of stool pots with stool found on the outside of the pot Are pots properly filled (minimum bottom of container covered, maximum ½ of pot)? Number of stool samples rejected by the laboratory Indicate rejection criteria: 3. Quality of sample processing Number of stool samples processed per month Indicate the type of stool received: Formed Semi formed Liquid Number of samples processed within 3 days of stool submission Number of samples processed 3 or more days after stool submission Maximum number of days Is the correct amount of stool used for stool testing? Is the processing performed according to the SOP for stool testing by Xpert MTB/RIF or Xpert Ultra? Number of stool samples with an Xpert MTB positive result (total per month): MTB detected high MTB detected medium MTB detected low MTB detected very low MTB trace detected Number of stool samples with MTB not detected Number of stool samples with an error code Error codes: Code x no. of tests Annex 3. Laboratory tool for assessing stool testing by Xpert MTB/RIF and Xpert MTB/RIF Ultra 33 Number or Yes/no/partial Comments Number of stool samples with “Invalid” Number of stool samples with “No result” Number of stool samples not tested Reason for not testing: Number of samples with rifampicin resistance Number of samples with rifampicin indeterminate Yes/no/partial Comments 4. Supply management Are Xpert Ultra cartridges always available for stool testing? Reason if not: Does the monthly consumption of Xpert Ultra cartridge consider stool testing? Number of cartridges/ months: Any stockout of containers for stool collection in the last quarter? Any stockout of applicator to support transfer of stool to SR buffer in the last quarter? Any stock out of supplies required for stool processing in the last quarter? Any stock out of reagents for stool processing in the last quarter? Yes/no/partial Comments 5. Registration and reporting tools Does request form have a provision to request stool testing? Does laboratory register have a provision to add stool as sample type? Is “stool” indicated within the sample type field of the GeneXpert? Are stool testing results entered into the laboratory information system? MTB: Mycobacterium tuberculosis; SOP: standard operating procedure; TB: tuberculosis. Additional comments __________________________________________________________________________________________________________________ _______________________________________________________ __________________________________________________________________________________________________________________ _______________________________________________________ Reference for Annex 3 1. Tuberculosis technical scorecard Xpert MTB/RIF. Geneva: Stop TB Partnership; 2020 (https://stoptb.org/ wg/gli/assets/documents/5%20Find-TB-Scorecard-Xpert-Low-Res.pdf, accessed January 2022). Practical manual of processing stool samples for diagnosis of childhood TB34

For further information, please contact: Global TB Programme World Health Organization 20, Avenue Appia CH-1211 Geneva 27 Switzerland Web site: www.who.int/tb