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.

The use of rats to detect drug-resistant TB.

Although detection of drug-susceptible TB by Anti-Persoonsmijnen Ontmijnende Product Ontwikkeling-trained African giant pouched rats has been known for more than a decade, the detection of drug-resistant TB (DR-TB) using rats has never been explored before. We present what we believe to be the first report on rifampicin-resistant TB (RR-TB) detected using Xpert® MTB/RIF Ultra, comparably identified by rats sniffing sputum samples from presumptive TB patients: 88% of RR-TB detected using Ultra were identified by the rats. Further evaluation of the usefulness of rats for large-scale DR-TB contact triage testing is needed, especially in low- and middle-income countries, where resources are limited.

Bien que la détection de la TB pharmacosensible par des rats géants de Gambie dressés par APOPO (Anti-Persoonsmijnen Ontmijnende Product Ontwikkeling) soit connue depuis plus d'une décennie, la détection de la TB pharmacorésistante (DR-TB) à l'aide de rats n'a jamais été explorée auparavant. Nous présentons ce que nous pensons être le premier rapport sur la TB résistante à la rifampicine (RR-TB) détectée par test Xpert® MTB/RIF Ultra, identifiée de manière comparable par des rats reniflant des échantillons d'expectorations de patients avec une TB présumée : 88% des RR-TB détectées par test Ultra ont été identifiées par les rats. L'évaluation de l'utilité des rats dans le cadre de tests de triage des contacts de cas de DR-TB à grande échelle doit être poursuivie, en particulier dans les pays à revenu faible ou intermédiaire, où les ressources sont limitées.

CHARACTERIZING TUBERCULOSIS PROGRESSION IN WILD MEERKATS (SURICATA SURICATTA) FROM FECAL SAMPLES AND CLINICAL SIGNS.

Tuberculosis (TB) is an increasing threat to wildlife, yet tracking its spread is challenging because infections often appear to be asymptomatic, and diagnostic tools such as blood tests can be invasive and resource intensive. Our understanding of TB biology in wildlife is therefore limited to a small number of well-studied species. Testing of fecal samples using PCR is a noninvasive method that has been used to detect Mycobacterium bovis shedding amongst badgers, yet its utility more broadly for TB monitoring in wildlife is unclear. We combined observation data of clinical signs with PCR testing of 388 fecal samples to characterize longitudinal dynamics of TB progression in 66 wild meerkats (Suricata suricatta) socially exposed to Mycobacterium suricattae between 2000 and 2018. Our specific objectives were 1) to test whether meerkat fecal samples can be used to monitor TB; 2) to characterize TB progression between three infection states (PCR-negative exposed, PCR-positive asymptomatic, and PCR positive with clinical signs); and 3) estimate individual heterogeneity in TB susceptibility, defined here as the time between TB exposure and detection, and survival after TB detection. We found that the TB detection probability once meerkats developed clinical signs was 13% (95% confidence interval 3-46%). Nevertheless, with an adapted test protocol of 10 PCR replicates per sample we detected hidden TB infections in 59% of meerkats before the onset of clinical signs. Meerkats became PCR positive approximately 14 mo after initial exposure, developed clinical signs approximately 1 yr after becoming PCR positive, and died within 5 mo of developing clinical signs. Individual variation in disease progression was high, with meerkats developing clinical signs from immediately after exposure to 3.4 yr later. Overall, our study generates novel insights into wildlife TB progression, and may help guide adapted management strategies for TB-susceptible wildlife populations.

Tween 80 Improves the Acid-Fast Bacilli Quantification in the Magnetic Nanoparticle-Based Colorimetric Biosensing Assay (NCBA).

Despite its reduced sensitivity, sputum smear microscopy (SSM) remains the main diagnostic test for detecting tuberculosis in many parts of the world. A new diagnostic technique, the magnetic nanoparticle-based colorimetric biosensing assay (NCBA) was optimized by evaluating different concentrations of glycan-functionalized magnetic nanoparticles (GMNP) and Tween 80 to improve the acid-fast bacilli (AFB) count. Comparative analysis was performed on 225 sputum smears: 30 with SSM, 107 with NCBA at different GMNP concentrations, and 88 with NCBA-Tween 80 at various concentrations and incubation times. AFB quantification was performed by adding the total number of AFB in all fields per smear and classified according to standard guidelines (scanty, 1+, 2+ and 3+). Smears by NCBA with low GMNP concentrations (≤1.5 mg/mL) showed higher AFB quantification compared to SSM. Cell enrichment of sputum samples by combining NCBA-GMNP, incubated with Tween 80 (5%) for three minutes, improved capture efficiency and increased AFB detection up to 445% over SSM. NCBA with Tween 80 offers the opportunity to improve TB diagnostics, mainly in paucibacillary cases. As this method provides biosafety with a simple and inexpensive methodology that obtains results in a short time, it might be considered as a point-of-care TB diagnostic method in regions where resources are limited.

Magnetic Nanoparticle-Based Biosensing Assay Quantitatively Enhances Acid-Fast Bacilli Count in Paucibacillary Pulmonary Tuberculosis.

A new method using a magnetic nanoparticle-based colorimetric biosensing assay (NCBA) was compared with sputum smear microscopy (SSM) for the detection of pulmonary tuberculosis (PTB) in sputum samples. Studies were made to compare the NCBA against SSM using sputum samples collected from PTB patients prior to receiving treatment. Experiments were also conducted to determine the appropriate concentration of glycan-functionalized magnetic nanoparticles (GMNP) used in the NCBA and to evaluate the optimal digestion/decontamination solution to increase the extraction, concentration and detection of acid-fast bacilli (AFB). The optimized NCBA consisted of a 1:1 mixture of 0.4% NaOH and 4% N-acetyl-L-cysteine (NALC) to homogenize the sputum sample. Additionally, 10 mg/mL of GMNP was added to isolate and concentrate the AFB. All TB positive sputum samples were identified with an increased AFB count of 47% compared to SSM, demonstrating GMNP's ability to extract and concentrate AFB. Results showed that NCBA increased AFB count compared to SSM, improving the grade from "1+" (in SSM) to "2+". Extending the finding to paucibacillary cases, there is the likelihood of a "scant" grade to become "1+". The assay uses a simple magnet and only costs $0.10/test. NCBA has great potential application in TB control programs.

MDR-TB Antibody Response (Western Blot) to Fractions of Isoniazid and Rifampicin Resistant Antigens of Mycobacterium tuberculosis.

Drug-resistant TB poses a major threat to control of TB worldwide. Despite progress in the detection of Multidrug-resistant TB (MDR-TB) cases, a major diagnostic gap remains: 55% of reported TB patients estimated to have MDR-TB were not detected in 2013. MDR-TB antigens were conjugated to CNBr-activated Sepharose 4B. Specific polyclonal antibodies against MDR-TB Ags were prepared in rabbits using two boosted injections of the MDR-TB antigen. The antibodies were purified and treated with susceptible TB to remove any non-specific and cross-reactive antibodies. In the present study, comparative analysis of electrophoretic pattern of different antigens of INH/RIF-resistant TB were studied for identifying protein profiles. A RIF-resistant TB antigen was shown here to have different protein profiles from INH-resistant TB isolate. The results of Western blotting analysis showed that in the RIF- and INH-resistant antigenic fractions some bands of 14.4 and 45 kDa as immunogenic were common. Moreover, four bands of RIF-resistant TB antigen fractions (16, 19, 21, and 45 KDa) and one band of INH-resistant TB (about 26 KDa) were detected as diagnostic antigens. This study suggests that the Western blot is an accurate test to survey INH- and RIF-resistant TB antigens of M. tuberculosis infection. These findings indicate that MDR-TB diagnosis (based on Ag detection) could be useful in the identification of disease stages that precede symptomatic and microbiologically positive TB, such as subclinical and incipient TB.

Evaluation of antigen detection test (chromatographic immunoassay): potential to replace the antibody assay using purified 45-kDa protein for rapid diagnosis of tuberculosis.

BACKGROUND: The current strategy for combating tuberculosis (TB) is based on the early detection and treatment of patients to halt transmission. The present study was conducted to evaluate the diagnostic potential of three Mycobacterium tuberculosis antigens, 45-kDa, A60, and sonicated MTB antigen (SmTB-Ag), as antibody/antigen detection methods for the rapid and accurate diagnosis of TB. METHODS: The SmTB-Ag and 45-kDa antigens were purified and A60 antigen was supplied by Anda-Biologicals, France. The 45-kDa and A60 antigens (for antibody detection procedures) and SmTB-Ag (for antigen detection test) were tested in the same study subjects. ELISA and immunochromatographic (rapid) test were performed on 201 sputum and serum samples. Ninety-eight samples from TB patients and 103 samples from control individuals were studied. RESULTS: The mean absorbance value of antibodies against 45-kDa antigen in the TB patients were (1.17 ± 0.44, CI 1.09-1.26), significantly higher than in the non-TB group, (0.8 ± 0.28, CI 0.74-0.85, P < 0.05). The sensitivities of tests using two antigens, 84% for the 45-kDa antigen and 65% for the A60 antigen, were lower than SmTB-Ag(93%). The rapid test yielded 93% sensitivity and 92% specificity. CONCLUSION: Findings highlighted the importance of antigen detection as a diagnostic tool. The rapid test evaluated in this study may be useful for diagnosis of TB.