Increased tuberculosis case detection in Tanzanian children and adults using African giant pouched rats.

BACKGROUND: African giant pouched rats, trained by Anti-Persoonsmijnen Ontmijnende Product Ontwikkeling (APOPO), have demonstrated their ability to detect tuberculosis (TB) from sputum. We assessed rat-based case detection and compared the mycobacterium bacillary load (MTB-load) in children versus adults. METHODS: From January-December 2022, samples were collected prospectively from 69 Directly Observed Therapy (DOT) facilities' presumed TB patients. Using an average of five rats, APOPO re-evaluated patients with bacteriologically negative (sputum-smear microscopy or Xpert MTB/RIF) results. Rat-positive samples were tested using concentrated smear light-emitting diode microscopy to confirm TB detection before treatment initiation. The rats' identification of pulmonary TB is based on smelling TB-specific volatile organic compounds (VOCs) in sputum. Using STATA, Chi-square for odds ratio and confidence interval was calculated and evaluated: (1) the yield of rat-based TB detection compared to that of the health facilities; (2) rat-based TB detection in children versus adults; and (3) rats' ability to detect TB across MTB-loads and between children and adults. RESULTS: From 35,766 patients, 5.3% (1900/35,766) were smear-positive and 94.7% (33,866/35,766) were smear or Xpert-negatives at DOTS facility. Of those with negative results, 2029 TB cases were detected using rats, contributing to 52% (2029/3929 of total TB identified), which otherwise would have been missed. Compared to DOT facilities, rats were six-fold more likely to detect TB among Acid Fast Bacilli (AFB) 1+/scanty [90% (1829/2029) versus 60% (1139/1900), odds ratio, OR = 6.11, 95% confidence interval, CI: 5.14-7.26]; twice more likely to identify TB cases among children [71% (91/129) versus 51% (1795/3542), OR = 2.3, 95% CI: 1.59-3.42]; and twice more likely to identify TB cases among children with AFB 1+/scanty than adults with the same MTB-load [5% (86/1703) versus 3% (28/1067), OR = 2.0, 95% CI: 1.28-3.03]. CONCLUSIONS: Rats contributed over half of the TB cases identified in program settings, and children, especially those with a lower MTB-load, were more likely to be diagnosed with TB by rats. The chemical signatures, VOCs, were only available for adults, and further research describing the characteristics of VOCs in children versus adults may pave the way to enhance TB diagnosis in children.

Pediatric tuberculosis detection using trained African giant pouched rats.

BACKGROUND: Tuberculosis (TB) diagnosis in children is a challenge with up to 94% of children with TB treated empirically in TB high-burden countries. Therefore, new diagnostic tests are needed for TB diagnosis. We determined the performance of trained rats in the diagnosis of pediatric TB and whether they can improve detection rate compared to the standard of care. METHODS: Presumptive TB patients in 24 TB clinics in Tanzania were tested. Samples indicated as TB-positive by rats underwent confirmation by concentrated smear microscopy. TB yield of bacteriologically confirmed pediatric TB patients (≤5 years) was compared with yield of standard of care. RESULTS: Sputum samples from 55,148 presumptive TB patients were tested. Nine hundred eighty-two (1.8%) were the children between 1 and 5 years. Clinics detected 34 bacteriologically positive children, whereas rats detected additional 23 children yielding 57 bacteriologically TB-positive children. Rats increased pediatric TB detection by 67.6%. Among 1-14-year-old children, clinics detected 331 bacteriologically positive TB whereas rats found the additional 208 children with TB that were missed by clinics. Relative increase in TB case detection by rats decreased with the increase in age (P<0.0001). CONCLUSION: Trained rats increase pediatric TB detection significantly and could help address the pediatric TB diagnosis challenges. Further determination of accuracy of rats involving other sample types is still needed.

Mycobacterium genotypes in pulmonary tuberculosis infections and their detection by trained African giant pouched rats.

Tuberculosis (TB) diagnosis in low-income countries is mainly done by microscopy. Hence, little is known about the diversity of Mycobacterium spp. in TB infections. Different genotypes or lineages of Mycobacterium tuberculosis vary in virulence and induce different inflammatory and immune responses. Trained Cricetomys rats show a potential for rapid diagnosis of TB. They detect over 28 % of smear-negative, culture-positive TB. However, it is unknown whether these rats can equally detect sputa from patients infected with different genotypes of M. tuberculosis. A 4-month prospective study on diversity of Mycobacterium spp. was conducted in Dar es Salaam, Tanzania. 252 sputa from 161 subjects were cultured on Lowenstein-Jensen medium and thereafter tested by rats. Mycobacterial isolates were subjected to molecular identification and multispacer sequence typing (MST) to determine species and genotypes. A total of 34 Mycobacterium spp. isolates consisting of 32 M. tuberculosis, 1 M. avium subsp. hominissuis and 1 M. intracellulare were obtained. MST analyses of 26 M. tuberculosis isolates yielded 10 distinct MST genotypes, including 3 new genotypes with two clusters of related patterns not grouped by geographic areas. Genotype MST-67, shared by one-third of M. tuberculosis isolates, was associated with the Mwananyamala clinic. This study shows that diverse M. tuberculosis genotypes (n = 10) occur in Dar es Salaam and trained rats detect 80 % of the genotypes. Sputa with two M. tuberculosis genotypes (20 %), M. avium hominissuis and M. intracellulare were not detected. Therefore, rats detect sputa with different M. tuberculosis genotypes and can be used to detect TB in resource-poor countries.

Machine Learning for Prediction of Tuberculosis Detection: Case Study of Trained African Giant Pouched Rats.

BACKGROUND: Technological advancement has led to the growth and rapid increase of tuberculosis (TB) medical data generated from different health care areas, including diagnosis. Prioritizing better adoption and acceptance of innovative diagnostic technology to reduce the spread of TB significantly benefits developing countries. Trained TB-detection rats are used in Tanzania and Ethiopia for operational research to complement other TB diagnostic tools. This technology has increased new TB case detection owing to its speed, cost-effectiveness, and sensitivity. OBJECTIVE: During the TB detection process, rats produce vast amounts of data, providing an opportunity to identify interesting patterns that influence TB detection performance. This study aimed to develop models that predict if the rat will hit (indicate the presence of TB within) the sample or not using machine learning (ML) techniques. The goal was to improve the diagnostic accuracy and performance of TB detection involving rats. METHODS: APOPO (Anti-Persoonsmijnen Ontmijnende Product Ontwikkeling) Center in Morogoro provided data for this study from 2012 to 2019, and 366,441 observations were used to build predictive models using ML techniques, including decision tree, random forest, naïve Bayes, support vector machine, and k-nearest neighbor, by incorporating a variety of variables, such as the diagnostic results from partner health clinics using methods endorsed by the World Health Organization (WHO). RESULTS: The support vector machine technique yielded the highest accuracy of 83.39% for prediction compared to other ML techniques used. Furthermore, this study found that the inclusion of variables related to whether the sample contained TB or not increased the performance accuracy of the predictive model. CONCLUSIONS: The inclusion of variables related to the diagnostic results of TB samples may improve the detection performance of the trained rats. The study results may be of importance to TB-detection rat trainers and TB decision-makers as the results may prompt them to take action to maintain the usefulness of the technology and increase the TB detection performance of trained rats.

Diagnosis of tuberculosis by trained African giant pouched rats and confounding impact of pathogens and microflora of the respiratory tract.

Trained African giant-pouched rats (Cricetomys gambianus) can detect Mycobacterium tuberculosis and show potential for the diagnosis of tuberculosis (TB). However, rats' ability to discriminate between clinical sputum containing other Mycobacterium spp. and nonmycobacterial species of the respiratory tract is unknown. It is also unknown whether nonmycobacterial species produce odor similar to M. tuberculosis and thereby cause the detection of smear-negative sputum. Sputum samples from 289 subjects were analyzed by smear microscopy, culture, and rats. Mycobacterium spp. were isolated on Lowenstein-Jensen medium, and nonmycobacterial species were isolated on four different media. The odor from nonmycobacterial species from smear- and M. tuberculosis culture-negative sputa detected by ≥2 rats ("rat positive") was analyzed by gas chromatography-mass spectrometry and compared to the M. tuberculosis odor. Rats detected 45 of 56 confirmed cases of TB, 4 of 5 suspected cases of TB, and 63 of 228 TB-negative subjects (sensitivity, 80.4%; specificity, 72.4%; accuracy, 73.9%; positive predictive value, 41.7%; negative predictive value, 93.8%). A total of 37 (78.7%) of 47 mycobacterial isolates were M. tuberculosis complex, with 75.7% from rat-positive sputa. Ten isolates were nontuberculous mycobacteria, one was M. intracellulare, one was M. avium subsp. hominissuis, and eight were unidentified. Rat-positive sputa with Moraxella catarrhalis, Streptococcus pneumoniae, Staphylococcus spp., and Enterococcus spp. were associated with TB. Rhodococcus, Nocardia, Streptomyces, Staphylococcus, and Candida spp. from rat-positive sputa did not produce M. tuberculosis-specific volatiles (methyl nicotinate, methyl para-anisate, and ortho-phenylanisole). Prevalence of Mycobacterium-related Nocardia and Rhodococcus in smear-negative sputa did not equal that of smear-negative mycobacteria (44.7%), of which 28.6% were rat positive. These findings and the absence of M. tuberculosis-specific volatiles in nonmycobacterial species indicate that rats can be trained to specifically detect M. tuberculosis.

Rapidly training African giant pouched rats (Cricetomys ansorgei) with multiple targets for scent detection.

Since 1997, APOPO, a non-profit organization based in Tanzania, has deployed African giant pouched rats (Cricetomys ansorgei) to detect landmines in post-conflict areas. More recent research suggests the pouched rats can also be trained to detect tuberculosis in human sputum samples. Despite proven success on both tasks, the potential impact of each rat is limited by the required training time and constraint to a single target odor. The aim of this project was to establish a technique to rapidly train pouched rats to detect multiple odor targets. Eight pouched rats were trained to detect five unrelated target odors in Experiment 1. In addition to training duration, we measured maintenance of all odor targets. In Experiment 2, we examined response persistence under conditions of extinction. Experiment 3 investigated whether refresher sessions before tests would maintain detection accuracy. The animals mastered all odor targets in significantly fewer sessions than APOPO's operational rats require to master a single target odor. Importantly, rats demonstrated strong discrimination between targets and non-targets despite the potential for interference and forgetting, suggesting pouched rats can be trained to detect at least five targets simultaneously. These results have the potential to increase the impact of each detection rat by both decreasing training time and expanding operational versatility, e.g., a single rat could be trained to detect multiple diseases.

Behavioral skills training in scent detection research: Interactions between trainer and animal behavior.

Animal trainers working in scent detection programs are responsible for arranging training contingencies as well as for observing and recording animal behavior. We provided behavioral skills training (BST) to animal trainers working with scent detection rats to improve the treatment integrity of scent-detection research sessions. We evaluated the trainers' behavior at baseline and during the sequential introduction of each component of BST (instructions, modeling, and feedback). We observed incremental improvements in treatment integrity with the introduction of each BST component. Posttraining probes revealed that these improvements were sustained at least 3 weeks post-BST. As the trainers' behavior was modified during BST, we observed decrements in measures of rat performance. We discuss the nature of these interactions and their implications for the use of BST in scent detection research and in situations in which intervention with one party produces concomitant effects on the behavior of another.

Tuberculosis detection by pouched rats: Opportunities for reinforcement under low-prevalence conditions.

Giant African pouched rats (Cricetomys ansorgei) have been employed successfully in two operational tuberculosis-detection projects in which they sniff sputum samples from symptomatic individuals who have visited tuberculosis clinics. The prevalence of pulmonary tuberculosis in this population is high, approximately 20% in the regions where the rats have been used. If the rats are to be used to screen individuals from lower-prevalence populations, their performance under such conditions must first be evaluated. In this study, the prevalence of tuberculosis-positive samples presented to eight pouched rats was reduced to approximately 5%, and the percentage of known-positive samples included as opportunities for reinforcement was varied in sequence from 10 to 8, 6, 4, 2, 4, and 2. Liquid food reinforcers were delivered for identification responses to known-positive samples and at no other time. The rats' accuracy was clinically and statistically significantly lower at 2% than at the other values. These results indicate that the rats can perform well in low-prevalence scenarios but, if they are used under the conditions of the present study, at least 4% of the samples presented to them must be opportunities for reinforcement.

Active tuberculosis detection by pouched rats in 2014: More than 2,000 new patients found in two countries.

Tuberculosis (TB) is a major problem in poor countries because sensitive diagnostic tools are unavailable. In 2014, our pouched rats evaluated sputum from 21,600 Tanzanians and 9,048 Mozambicans whose sputum had previously been evaluated by microscopy, the standard diagnostic for TB. Evaluation by the rats revealed 1,412 new patients with active TB in Tanzania and 645 new patients in Mozambique, increases of 39% and 53%, respectively, when compared to detections by microscopy alone. These results provide further support for the applied use of scent-detecting rats.

Effects of schedules of reinforcement on pouched rats' performance in urban search-and-rescue training.

Standard operating procedures have been developed to train Cricetomys to locate humans in collapsed structures and return to the release point on command. The present study demonstrated that the schedule of reinforcement for target location influences the rats' performance. Rats required more time to locate targets when no reinforcement was arranged for target location but less time to return to the release point. These findings suggest that training conditions should be based on the priority assigned to target location and return in an operational scenario.

Giant African pouched rats (Cricetomys gambianus) that work on tilled soil accurately detect land mines.

Pouched rats were employed as mine-detection animals in a quality-control application where they searched for mines in areas previously processed by a mechanical tiller. The rats located 58 mines and fragments in this 28,050-m(2) area with a false indication rate of 0.4 responses per 100 m(2) . Humans with metal detectors found no mines that were not located by the rats. These findings indicate that pouched rats can accurately detect land mines in disturbed soil and suggest that they can play multiple roles in humanitarian demining.

Using giant African pouched rats to detect human tuberculosis: a review.

Despite its characteristically low sensitivity, sputum smear microscopy remains the standard for diagnosing tuberculosis (TB) in resource-poor countries. In an attempt to develop an alternative or adjunct to microscopy, researchers have recently examined the ability of pouched rats to detect TB-positive human sputum samples and the microbiological variables that affect their detection. Ten published studies, reviewed herein, suggest that the rats are able to detect the specific odor of Mycobacterium tuberculosis, which causes TB, and can substantially increase new-case detections when used for second-line TB screening following microscopy. Further research is needed to ascertain the rats' ability to detect TB in children and in HIV-positive patients, to detect TB when used for first-line screening, and to be useful in broad-scale applications where cost-effectiveness is a major consideration.

Training pouched rats to find people.

Giant African pouched rats equipped with video cameras may be a tenable option for locating living humans trapped under debris from collapsed structures. In the present study, 5 pouched rats were trained to contact human targets in a simulated collapsed building and to return to the release point after hearing a signal to do so. During test sessions, each rat located human targets more often than it located similar-sized inanimate targets on which it had not previously been trained and spent more time within 1 m of the human target than within 1 m of the other targets. Overall, the rats found humans, plastic bags containing clothes, and plastic bags without clothes on 83%, 37%, and 11% of trials, respectively. These findings suggest that using pouched rats to search for survivors in collapsed structures merits further attention.

Evaluation of Giant African Pouched Rats for Detection of Pulmonary Tuberculosis in Patients from a High-Endemic Setting.

BACKGROUND: This study established evidence about the diagnostic performance of trained giant African pouched rats for detecting Mycobacterium tuberculosis in sputum of well-characterised patients with presumptive tuberculosis (TB) in a high-burden setting. METHODS: The TB detection rats were evaluated using sputum samples of patients with presumptive TB enrolled in two prospective cohort studies in Bagamoyo, Tanzania. The patients were characterised by sputum smear microscopy and culture, including subsequent antigen or molecular confirmation of Mycobacterium tuberculosis, and by clinical data at enrolment and for at least 5-months of follow-up to determine the reference standard. Seven trained giant African pouched rats were used for the detection of TB in the sputum samples after shipment to the APOPO project in Morogoro, Tanzania. RESULTS: Of 469 eligible patients, 109 (23.2%) were culture-positive for Mycobacterium tuberculosis and 128 (27.3%) were non-TB controls with sustained recovery after 5 months without anti-TB treatment. The HIV prevalence was 46%. The area under the receiver operating characteristic curve of the seven rats for the detection of culture-positive pulmonary tuberculosis was 0.72 (95% CI 0.66-0.78). An optimal threshold could be defined at ≥ 2 indications by rats in either sample with a corresponding sensitivity of 56.9% (95% CI 47.0-66.3), specificity of 80.5% (95% CI 72.5-86.9), positive and negative predictive value of 71.3% (95% CI 60.6-80.5) and 68.7% (95% CI 60.6-76.0), and an accuracy for TB diagnosis of 69.6%. The diagnostic performance was negatively influenced by low burden of bacilli, and independent of the HIV status. CONCLUSION: Giant African pouched rats have potential for detection of tuberculosis in sputum samples. However, the diagnostic performance characteristics of TB detection rats do not currently meet the requirements for high-priority, rapid sputum-based TB diagnostics as defined by the World Health Organization.

Landmine-detection rats: an evaluation of reinforcement procedures under simulated operational conditions.

Because the location of landmines is initially unknown, it is impossible to arrange differential reinforcement for accurate detection of landmines by pouched rats working on actual minefields. Therefore, provision must be made for maintenance of accurate responses by an alternative reinforcement strategy. The present experiment evaluated a procedure in which a plastic bag containing 2,4,6-trinitrotoluene (TNT), the active ingredient in most landmines, was placed in contact with the ground in a disturbed area, then removed, to establish opportunities for reinforcement. Each of five rats continued to accurately detect landmines when extinction was arranged for landmine-detection responses and detections of TNT-contaminated locations were reinforced under a fixed-ratio 1 schedule. The results of this translational research study suggest that the TNT-contamination procedure is a viable option for arranging reinforcement opportunities for rats engaged in actual landmine-detection activities and the viability of this procedure is currently being evaluated on minefields in Angola and Mozambique.

Ability of Cricetomys rats to detect Mycobacterium tuberculosis and discriminate it from other microorganisms.

Trained African giant pouched rats (Cricetomys gambianus) have potential for diagnosis of tuberculosis (TB). These rats target volatile compounds of Mycobacterium tuberculosis (Mtb) that cause TB. Mtb and nontuberculous mycobacteria (NTM) species are related to Nocardia and Rhodococcus spp., which are also acid-fast bacilli and can be misdiagnosed as Mtb in smear microscopy. Diagnostic performance of C. gambianus on in vitro-cultured mycobacterial and related pulmonary microbes is unknown. This study reports on the response of TB detection rats to cultures of reference Mtb, clinical Mtb, NTM, Nocardia; Rhodococcus; Streptomyces; Bacillus; and yeasts. Trained rats significantly discriminated Mtb from other microbes (p < 0.008, Fisher's exact test). Detection of Mtb cultures was age-related, with exponential and early stationary phase detected more frequently than early log phase and late stationary phase (p < 0.001, Fisher's test) (sensitivity = 83.33%, specificity = 94.4%, accuracy = 94%). The detection of naturally TB-infected sputum exceeded that of negative sputum mixed with Mtb, indicating that C. gambianus are conditioned to detect odours of TB-positive sputum better than spiked sputum. Although further studies on volatiles from detectable growth phases of Mtb are vital for identification of Mtb-specific volatiles detected by rats, our study underline the potential of C. gambianus for TB diagnosis.

Pouched rats' detection of tuberculosis in human sputum: comparison to culturing and polymerase chain reaction.

Setting. Tanzania. Objective. To compare microscopy as conducted in direct observation of treatment, short course centers to pouched rats as detectors of Mycobacterium tuberculosis. Design. Ten pouched rats were trained to detect tuberculosis in sputum using operant conditioning techniques. The rats evaluated 910 samples previously evaluated by smear microscopy. All samples were also evaluated through culturing and multiplex polymerase chain reaction was performed on culture growths to classify the bacteria. Results. The patientwise sensitivity of microscopy was 58.0%, and the patient-wise specificity was 97.3%. Used as a group of 10 with a cutoff (defined as the number of rat indications to classify a sample as positive for Mycobacterium tuberculosis) of 1, the rats increased new case detection by 46.8% relative to microscopy alone. The average samplewise sensitivity of the individual rats was 68.4% (range 61.1-73.8%), and the mean specificity was 87.3% (range 84.7-90.3%). Conclusion. These results suggest that pouched rats are a valuable adjunct to, and may be a viable substitute for, sputum smear microscopy as a tuberculosis diagnostic in resource-poor countries.

Mycobacterium tuberculosis volatiles for diagnosis of tuberculosis by Cricetomys rats.

Tuberculosis (TB) diagnosis in regions with limited resources depends on microscopy with insufficient sensitivity. Rapid diagnostic tests of low cost but high sensitivity and specificity are needed for better point-of-care management of TB. Trained African giant pouched rats (Cricetomys sp.) can diagnose pulmonary TB in sputum but the relevant Mycobacterium tuberculosis (Mtb)-specific volatile compounds remain unknown. We investigated the odour volatiles of Mtb detected by rats in reference Mtb, nontuberculous mycobacteria, Nocardia sp., Streptomyces sp., Rhodococcus sp., and other respiratory tract microorganisms spiked into Mtb-negative sputum. Thirteen compounds were specific to Mtb and 13 were shared with other microorganisms. Rats discriminated a blend of Mtb-specific volatiles from individual, and blends of shared, compounds (P = 0.001). The rats' sensitivity for typical TB-positive sputa was 99.15% with 92.23% specificity and 93.14% accuracy. These findings underline the potential of trained Cricetomys rats for rapid TB diagnosis in resource-limited settings, particularly in Africa where Cricetomys rats occur widely and the burden of TB is high.