Population structure and spatial distribution of Mycobacterium tuberculosis in Ethiopia.

Ethiopia is one of the countries with a high tuberculosis (TB) burden, yet little is known about the spatial distribution of Mycobacterium tuberculosis (Mtb) lineages. This study identifies the spoligotyping of 1735 archived Mtb isolates from the National Drug Resistance Survey, collected between November 2011 and June 2013, to investigate Mtb population structure and spatial distribution. Spoligotype International Types (SITs) and lineages were retrieved from online databases. The distribution of lineages was evaluated using Fisher's exact test and logistic regression models. The Global Moran's Index and Getis-Ord Gi statistic were utilized to identify hotspot areas. Our results showed that spoligotypes could be interpreted and led to 4 lineages and 283 spoligotype patterns in 91% of the isolates, including 4% of those with multidrug/rifampicin resistance (MDR/RR) TB. The identified Mtb lineages were lineage 1 (1.8%), lineage 3 (25.9%), lineage 4 (70.6%) and lineage 7 (1.6%). The proportion of lineages 3 and 4 varied by regions, with lineage 3 being significantly greater than lineage 4 in reports from Gambella (AOR = 4.37, P < 0.001) and Tigray (AOR = 3.44, P = 0.001) and lineage 4 being significantly higher in Southern Nations Nationalities and Peoples Region (AOR = 1.97, P = 0.026) than lineage 3. Hotspots for lineage 1 were located in eastern Ethiopia, while a lineage 7 hotspot was identified in northern and western Ethiopia. The five prevalent spoligotypes, which were SIT149, SIT53, SIT25, SIT37 and SIT26 account for 42.8% of all isolates under investigation, while SIT149, SIT53 and SIT21 account for 52-57.8% of drug-resistant TB cases. TB and drug resistant TB are mainly caused by lineages 3 and 4, and significant proportions of the prevalent spoligotypes also influence drug-resistant TB and the total TB burden. Regional variations in lineages may result from both local and cross-border spread.

[Progress in lipoarabinomannan antigen detection for tuberculosis diagnosis in people living with HIV/AIDS].

Tuberculosis (TB) is the leading cause of death among people living with HIV/AIDS (PLWHA), posing a significant disease burden. Early TB screening in PLWHA is a key intervention to reduce transmission and control disease progression. ​Lipoarabinomannan (LAM) is a glycolipid of Mycobacterium tuberculosis (MTB) that can be detected in the urine of tuberculosis patients. LAM is useful for the rapid and accurate diagnosis of tuberculosis. This article reviews LAM and its application and limitations in the diagnosis of PLWHA, hoping to provide a reference for the diagnosis of tuberculosis in PLWHA.

Granulomatous meningoencephalitis and blindness associated with Mycobacterium tuberculosis complex infection in a senile female chimpanzee (Pan troglodytes).

A 40-year old female chimpanzee (Pan troglodytes) developed hyporexia, weight loss, followed by progressive and complete blindness. Tomography demonstrated an intracranial mass in the rostroventral brain involving the optic chiasm, with a presumptive diagnosis of neoplasm. However, histopathology revealed a granulomatous meningoencephalitis, and tissue samples tested positive for Mycobacterium tuberculosis.

Drug-resistant Mycobacterium tuberculosis among Nepalese patients at a tuberculosis referral center.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB), characterized by isoniazid and rifampicin resistance, is caused by chromosomal mutations that restrict treatment options and complicate tuberculosis management. This study sought to investigate the prevalence of pre-extensively drug-resistant (pre-XDR) and extensively drug-resistant (XDR) tuberculosis, as well as mutation pattern, in Nepalese patients with MDR/rifampicin-resistant (RR)-TB strains. METHODS: A cross-sectional study was conducted on MDR/RR-TB patients at the German Nepal Tuberculosis Project from June 2017 to June 2018. The MTBDRsl line probe assay identified pre-XDR-TB and XDR-TB. Pre-XDR-TB included MDR/RR-TB with resistance to any fluoroquinolone (FLQ), while XDR-TB included MDR/RR-TB with resistance to any FLQ and at least one additional group A drug. Mutation status was determined by comparing bands on reaction zones [gyrA and gyrB for FLQ resistance, rrs for SILD resistance, and eis for low-level kanamycin resistance, according to the GenoType MTBDRsl VER 2.0, Hain Lifescience GmbH, Nehren, Germany definition of pre-XDR and XDR] to the evaluation sheet. SPSS version 17.0 was used for data analysis. RESULTS: Out of a total of 171 patients with MDR/RR-TB, 160 had (93.57%) had MTBC, of whom 57 (35.63%) had pre-XDR-TB and 10 (6.25%) had XDR-TB. Among the pre-XDR-TB strains, 56 (98.25%) were FLQ resistant, while 1 (1.75%) was SLID resistant. The most frequent mutations were found at codons MUT3C (57.14%, 32/56) and MUT1 (23.21%, 13/56) of the gyrA gene. One patient had SLID resistant genotype at the MUT1 codon of the rrs gene (100%, 1/1). XDR-TB mutation bands were mostly detected on MUT1 (30%, 3/10) of the gyrA and rrs, MUT3C (30%, 3/10) of the gyrA, and MUT1 (30%, 3/10) of the rrs. CONCLUSIONS: Pre-XDR-TB had a significantly higher likelihood than XDR-TB, with different specific mutation bands present in gyrA and rrs genes.

Prevalence and genetic basis of Mycobacterium tuberculosis resistance to pretomanid in China.

BACKGROUND: Pretomanid is a key component of new regimens for the treatment of drug-resistant tuberculosis (TB) which are being rolled out globally. However, there is limited information on the prevalence of pre-existing resistance to the drug. METHODS: To investigate pretomanid resistance rates in China and its underlying genetic basis, as well as to generate additional minimum inhibitory concentration (MIC) data for epidemiological cutoff (ECOFF)/breakpoint setting, we performed MIC determinations in the Mycobacterial Growth Indicator Tube™ (MGIT) system, followed by WGS analysis, on 475 Mycobacterium tuberculosis (MTB) isolated from Chinese TB patients between 2013 and 2020. RESULTS: We observed a pretomanid MIC distribution with a 99% ECOFF equal to 0.5 mg/L. Of the 15 isolates with MIC values > 0.5 mg/L, one (MIC = 1 mg/L) was identified as MTB lineage 1 (L1), a genotype previously reported to be intrinsically less susceptible to pretomanid, two were borderline resistant (MIC = 2-4 mg/L) and the remaining 12 isolates were highly resistant (MIC ≥ 16 mg/L) to the drug. Five resistant isolates did not harbor mutations in the known pretomanid resistant genes. CONCLUSIONS: Our results further support a breakpoint of 0.5 mg/L for a non-L1 MTB population, which is characteristic of China. Further, our data point to an unexpected high (14/475, 3%) pre-existing pretomanid resistance rate in the country, as well as to the existence of yet-to-be-discovered pretomanid resistance genes.

Mycobacterium tuberculosis complex in domestic goats in Southern Spain.

Tuberculosis (TB) is a zoonotic infectious disease caused by bacteria belonging to the Mycobacterium tuberculosis complex (MTC), which can affect a wide variety of domestic and wild animal species. Although the role of goats as a reservoir of MTC bacteria has been evidenced, information about the circulation of MTC strains in this species is still very scarce. The aim of the present study was to determine the seroprevalence, spatial distribution, risk factors and MTC spoligotypes circulating in goats from Andalusia (Southern Spain), the Spanish region with the largest goat census and a hotspot area of TB in both cattle and wild ungulates. A total of 2155 serum samples from 80 goat flocks were analyzed by an in-house ELISA using the P22 protein complex as a coating antigen. Antibodies against MTC were detected in 473 goats (21.9%, 95% CI: 20.2-23.7) and the true seroprevalence was 22.3% (95% CI: 20.6-24.1). Seropositivity was found in 72 (90.0%) of the 80 flocks analyzed. The generalized estimating equation model showed that the management system (higher seroprevalence on intensive and semi-intensive farms), and the presence of hospital pens inside the regular stables, were risk factors potentially associated with MTC exposure in goats in Southern Spain. The spatial analysis identified a significant spatial cluster (p < 0.001) in Eastern Andalusia. A total of 16 different MTC spoligotypes, including five of M. caprae and eleven of M. bovis, were identified in goats between 2015 and 2022 in the study area, with SB0157 as the most frequently isolated. The results obtained indicate widespread and non-homogeneous spatial distribution of MTC in goat herds from Southern Spain. The high individual and herd-level seroprevalence values found suggest that goats could play a significant role in the maintenance and transmission of MTC in the study area. Our results highlight the importance of implementing control measures in this species.

Transmission characteristics in Tuberculosis by WGS: nationwide cross-sectional surveillance in China.

China, with the third largest share of global tuberculosis cases, faces a substantial challenge in its healthcare system as a result of the high burden of multidrug-resistant and rifampicin-resistant tuberculosis (MDR/RR-TB). This study employs a genomic epidemiological approach to assess recent tuberculosis transmissions between individuals, identifying potential risk factors and discerning the role of transmitted resistant isolates in the emergence of drug-resistant tuberculosis in China. We conducted a population-based retrospective study on 5052 Mycobacterium tuberculosis (MTB) isolates from 70 surveillance sites using whole genome sequencing (WGS). Minimum spanning tree analysis identified resistance mutations, while epidemiological data analysis pinpointed transmission risk factors. Of the 5052 isolates, 23% (1160) formed 452 genomic clusters, with 85.6% (387) of the transmissions occurring within the same counties. Individuals with younger age, larger family size, new cases, smear positive, and MDR/RR were at higher odds for recent transmission, while higher education (university and above) and occupation as a non-physical workers emerged as protective factors. At least 61.4% (251/409) of MDR/RR-TB were likely a result of recent transmission of MDR/RR isolates, with previous treatment (crude OR = 2.77), smear-positive (cOR = 2.07) and larger family population (cOR = 1.13) established as risk factors. Our findings highlight that local transmission remains the predominant form of TB transmission in China. Correspondingly, drug-resistant tuberculosis is primarily driven by the transmission of resistant tuberculosis isolates. Targeted interventions for high-risk populations to interrupt transmission within the country will likely provide an opportunity to reduce the prevalence of both tuberculosis and drug-resistant tuberculosis.

Characteristics of TB cases without documented sputum culture in the United States, 2011-2021.

Culture-based diagnostics are the gold standard for diagnosing pulmonary TB (PTB). We characterized culture practices by comparing cases with documented sputum culture to those without.Using multivariable logistic regression, we examined associations between PTB case characteristics and no documented sputum culture reported to the U.S. National TB Surveillance System during 2011-2021.Among 69,538 PTB cases analyzed, no sputum culture attempt was documented for 5,869 (8%). Non-sputum culture specimens were documented for 54%, 80%, and 89% of cases without documented sputum culture attempts among persons aged <15 years, 15-64, and 65+ years, respectively; bronchial fluid and lung tissue were common non-sputum specimens among cases in persons >15 years old. Having no documented sputum culture was associated with age <15 years (aOR 23.84, 99% CI 20.09-28.27) or ≥65 years (aOR 1.22, 99% CI 1.07-1.39), culture of a non-sputum specimen (aOR 6.57, 99% CI 5.93-7.28), residence in a long-term care facility (aOR 1.58, 99% CI 1.23-2.01), and receiving TB care outside of a health department (aOR 1.79, 99% CI 1.61-1.98).Inability to obtain sputum from children and higher diagnostic suspicion for disease processes that require tissue-based diagnostics could explain these findings..

A Portable Nucleic Acid Testing Platform with Photosensitization, a Three-Dimensionally Printed Multipiece Chip, and Digital Color Sensing.

Portable nucleic acid testing (NAT) holds great promise for point-of-care disease diagnosis and field-based applications but remains difficult to achieve. Herein, we describe a portable NAT that streamlines loop-mediated isothermal amplification with photosensitization-based color development in a fully sealed 3D-printed multipiece chip. Using a smartphone accessory and an APP, we also introduce a calibration-free quantification approach via digital color sensing and library matching. With these innovative approaches, our detection platform is highly accessible, allowing for rapid and sensitive NAT without requiring sophisticated instruments and well-trained personnel. The field applicability of our NAT platform was demonstrated by detecting tuberculosis infections in clinical sputum samples and food adulteration in commercial salmon meat products.

Whole-genome sequencing of clinical isolates from tuberculosis patients in India: real-world data indicates a high proportion of pre-XDR cases.

Treatment decisions for tuberculosis (TB) in the absence of full drug-susceptibility data can result in amplifying resistance and may compromise treatment outcomes. Genomics of Mycobacterium tuberculosis (M.tb) from clinical samples enables detection of drug resistance to multiple drugs. We performed whole-genome sequencing (WGS) for 600 clinical samples from patients with tuberculosis to identify the drug-resistance profile and mutation spectrum. We documented the reasons reported by clinicians for referral. WGS identified a high proportion (51%) of pre-extensively drug-resistant (pre-XDR) cases followed by multidrug-resistant tuberculosis (MDR-TB) (15.5%). This correlates with the primary reason for referral, as non-response to the first-line treatment (67%) and treatment failure or rifampicin resistance (14%). Multivariate analysis indicated that all young age groups (P < 0.05), male gender (P < 0.05), and Beijing strain (P < 0.01) were significant independent predictors of MDR-TB or MDR-TB+ [pre-extensively drug-resistant tuberculosis (XDR-TB) and XDR-TB]. Ser315Thr (72.5%) in the inhA gene and Ser450Leu in the rpoB gene (65.5%) were the most prevalent mutations, as were resistance-conferring mutations to pyrazinamide (41%) and streptomycin (61.33%). Mutations outside the rifampicin resistance-determining region (RRDR), Ile491Phe and Val170Phe, were seen in 1.3% of cases; disputed mutations in rpoB (Asp435Tyr, His445Asn, His445Leu, and Leu430Pro) were seen in 6% of cases, and mutations to newer drugs such as bedaquiline and linezolid in 1.0% and 7.5% of cases, respectively. This study on clinical samples highlights that there is a high proportion of pre-XDR cases and emerging resistance to newer drugs; ongoing transmission of these strains can cause serious threat to public health; and whole-genome sequencing can effectively identify and support precision medicine for TB. IMPORTANCE: The current study is based on real-world data on the TB drug-resistance profile by whole-genome sequencing of 600 clinical samples from patients with TB in India. This study indicates the clinicians' reasons for sending samples for WGS, which is for difficult-to-treat cases and/or relapse and treatment failure. The study reports a significant proportion of cases with pre-XDR-TB strains that warrant policy makers' attention. It reflects the current iterative nature of the diagnostic tests under programmatic conditions that leads to delays in appropriate diagnosis and empirical treatment. India had an estimated burden of 2.95 million TB cases in 2020 and 135,000 multidrug-resistant cases. However, WGS profiles of M.tb from India remains disproportionately poorly represented. This study adds a significant body of data on the mutation profiles seen in M.tb isolated from patients with TB in India, mutations outside the RRDR, disputed mutations, and resistance-conferring mutations to newer drugs such as bedaquiline and linezolid.

Comparison of targeted next-generation sequencing and the Xpert MTB/RIF assay for detection of Mycobacterium tuberculosis in clinical isolates and sputum specimens.

Targeted next-generation sequencing (tNGS) can be used to perform Mycobacterium tuberculosis (MTB) complex-specific amplification or target capture directly from sputum samples, yielding simultaneous coverage of many genes and DNA regions associated with antimicrobial resistance (AMR). Performance comparisons of tNGS and another molecular testing tool, Xpert MTB/rifampicin (RIF), have been empirical. Here, using a dilution series of a RIF-resistant clinical isolate of MTB, we found that tNGS had a slightly lower limit of bacterial detection (102 CFU/mL) compared with Xpert MTB/RIF (103 CFU/mL) in culture medium. However, the minimum detection limit of the rpoB S450L mutation in this isolate was significantly lower with tNGS (102 CFU/mL) than with Xpert MTB/RIF (106 CFU/mL). Sputum samples collected from 129 suspected pulmonary tuberculosis patients were also prospectively studied with the clinical diagnosis as a reference, revealing that the sensitivity of tNGS (48.6%) was higher than those of culture (46.8%), Xpert MTB/RIF (39.4%), and smear microscopy (34.9%) testing. Notably, AMR analysis of 56 MTB-positive samples as determined by tNGS revealed high mutation frequencies of 96.4%, 35.7%, 26.8%, and 19.6% in the following AMR-associated genes: rrs, rpoB, katG, and pncA, respectively. The findings of this study provide theoretical support for the differential clinical application of tNGS and Xpert MTB/RIF and suggest that tNGS has greater application value in tuberculosis drug resistance monitoring and prevention.IMPORTANCETargeted next-generation sequencing (tNGS) can be used to perform Mycobacterium tuberculosis (MTB) complex-specific amplification or target capture directly from sputum samples, yielding simultaneous coverage of genes and DNA regions associated with antimicrobial resistance (AMR). Performance comparisons of tNGS and Xpert MTB/rifampicin (RIF) have been empirical. The Xpert MTB/RIF assay is a commercial system that uses the nucleic acid amplification detection method for rapid (2 hours) diagnosis of tuberculosis (TB). The cost of the tNGS and Xpert MTB/RIF assays in this study was similar, at USD 98 and USD 70-104 per sample, respectively, but the time required for tNGS (3 days) was much longer than that required for the Xpert MTB/RIF assay. However, tNGS yielded more accurate results and a larger number of AMR-associated gene mutations, which compensated for the extra time and highlighted the greater application value of tNGS in TB drug resistance monitoring and prevention.

Droplet based whole genome amplification for sequencing minute amounts of purified Mycobacterium tuberculosis DNA.

Implementation of whole genome sequencing (WGS) for patient care is hindered by limited Mycobacterium tuberculosis (Mtb) in clinical specimens and slow Mtb growth. We evaluated droplet multiple displacement amplification (dMDA) for amplification of minute amounts of Mtb DNA to enable WGS as an alternative to other Mtb enrichment methods. Purified genomic Mtb-DNA (0.1, 0.5, 1, and 5 pg) was encapsulated and amplified using the Samplix Xdrop-instrument and sequenced alongside a control sample using standard Illumina protocols followed by MAGMA-analysis. The control and 5 pg input dMDA samples underwent nanopore sequencing followed by Nanoseq and TB-profiler analysis. dMDA generated 105-2400 ng DNA from the 0.1-5 pg input DNA, respectively. Followed by Illumina WGS, dMDA raised mean sequencing depth from 7 × for 0.1 pg input DNA to ≥ 60 × for 5 pg input and the control sample. Bioinformatic analysis revealed a high number of false positive and false negative variants when amplifying ≤ 0.5 pg input DNA. Nanopore sequencing of the 5 pg dMDA sample presented excellent coverage depth, breadth, and accurate strain characterization, albeit elevated false positive and false negative variants compared to Illumina-sequenced dMDA sample with identical Mtb DNA input. dMDA coupled with Illumina WGS for samples with ≥ 5 pg purified Mtb DNA, equating to approximately 1000 copies of the Mtb genome, offers precision for drug resistance, phylogeny, and transmission insights.

Diagnostic performance of Xpert MTB/RIF ultra in detecting Mycobacterium tuberculosis and Rifampicin Resistance in AFB Smear-negative Pulmonary and Extrapulmonary Tuberculosis samples in Malaysia.

Rapid and highly accurate diagnostic tools are critically needed to diagnose Mycobacterium tuberculosis and rifampicin resistance in AFB smear-negative samples. In this study, we evaluated the diagnostic performance of Xpert MTB/RIF Ultra (Ultra) as a rapid test to diagnose tuberculosis in smear-negative cases in Malaysia. A retrospective study of 1960 smear-negative pulmonary and extrapulmonary samples obtained from patients was conducted. Culture was used as the reference standard for the study. The overall sensitivity and specificity of Ultra on the tested samples were 88.7 % and 77.2 %, respectively, while the PPV was 32.3 % and the NPV was 98.2 %. Ultra showed slightly higher sensitivity in pulmonary (89.9 %) compared to extrapulmonary samples (86.1 %). The overall accuracy of Ultra was 78.5 % (kappa=0.37; 95 %CI: 0.32,0.42). Ultra showed good diagnostic accuracy for detecting MTB and rifampicin resistance in various AFB smear-negative samples. Ultra also had excellent capability in rifampicin resistance detection.

Community Voices and Whole-Genome Sequencing for Tuberculosis: Storytelling and the Importance of Listening.

One of the primary public health functions of a tuberculosis (TB) program is to arrest the spread of infection. Traditionally, TB programs have relied on epidemiological information, gathered through contact tracing, to infer that transmission has occurred between people. The ability of drawing such inferences is extensively context dependent. Where epidemiological information has been strong, such as 2 cases of TB occurring sequentially within a single household, confidence in such inferences is high; conversely, public health authorities have been less certain about the significance of TB cases merely occurring in the same wider social group or geographic area. Many current laboratory tests for TB used globally may be sufficient to confirm a diagnosis and guide appropriate therapy but still be insufficiently precise for distinguishing two strains reliably. In short, drawing inferences regarding a chain of transmissions has always been as much art as science.

Performance of a stool-based quantitative PCR assay for the diagnosis of tuberculosis in adolescents and adults: a multinational, prospective diagnostic accuracy study.

BACKGROUND: Despite increasing availability of rapid molecular tests for the diagnosis of tuberculosis in high-burden settings, many people with tuberculosis are undiagnosed. Reliance on sputum as the primary specimen for tuberculosis diagnostics contributes to this diagnostic gap. We evaluated the diagnostic accuracy and additive yield of a novel stool quantitative PCR (qPCR) assay for the diagnosis of tuberculosis in three countries in Africa with high tuberculosis burdens. METHODS: We undertook a prospective diagnostic accuracy study in Eswatini, Mozambique, and Tanzania from Sept 21, 2020, to Feb 2, 2023, to compare the diagnostic accuracy for tuberculosis of a novel stool qPCR test with the current diagnostic standard for Mycobacterium tuberculosis DNA detection from sputum and stool, Xpert-MTB/RIF Ultra (Xpert Ultra). Sputum, stool, and urine samples were provided by a cohort of participants, aged 10 years or older, diagnosed with tuberculosis. Participants with tuberculosis (cases) were enrolled within 72 h of treatment initiation for tuberculosis diagnosed clinically or following laboratory confirmation. Participants without tuberculosis (controls) consisted of household contacts of the cases who did not develop tuberculosis during a 6-month follow-up. The performance was compared with a robust composite microbiological reference standard (CMRS). FINDINGS: The cohort of adolescents and adults (n=408) included 268 participants with confirmed or clinical tuberculosis (cases), 147 (55%) of whom were living with HIV, and 140 participants (controls) without tuberculosis. The sensitivity of the novel stool qPCR was 93·7% (95% CI 87·4-97·4) compared with participants with detectable growth on M tuberculosis culture, and 88·1% (81·3-93·0) compared with sputum Xpert Ultra. The stool qPCR had an equivalent sensitivity as sputum Xpert Ultra (94·8%, 89·1-98·1) compared with culture. Compared with the CMRS, the sensitivity of the stool qPCR was higher than the current standard for tuberculosis diagnostics on stool, Xpert Ultra (80·4%, 73·4-86·2 vs 73·5%, 66·0-80·1; p=0·025 on paired comparison). The qPCR also identified 17-21% additional tuberculosis cases compared to sputum Xpert Ultra or sputum culture. In controls without tuberculosis, the specificity of the stool qPCR was 96·9% (92·2-99·1). INTERPRETATION: In this study, a novel qPCR for the diagnosis of tuberculosis from stool specimens had a higher accuracy in adolescents and adults than the current diagnostic PCR gold standard on stool, Xpert-MTB/RIF Ultra, and equivalent sensitivity to Xpert-MTB/RIF Ultra on sputum. FUNDING: National Institutes of Health (NIH) Allergy and Infectious Diseases, and NIH Fogarty International Center.

Diagnostics of Tuberculosis with Single-Walled Carbon Nanotube-Based Field-Effect Transistors.

Tuberculosis (TB) is still threatening millions of people's lives, especially in developing countries. One of the major factors contributing to the ongoing epidemic of TB is the lack of a fast, efficient, and inexpensive diagnostic strategy. In this work, we developed a semiconducting single-walled carbon nanotube (SWCNT)-based field-effect transistor (FET) device functionalized with anti-Mycobacterium tuberculosis antigen 85B antibody (Ab85B) to detect the major M. tuberculosis-secreted antigen 85B (Ag85B). Through optimizing the device fabrication process by evaluating the mass of the antibody and the concentration of the gating electrolyte, our Ab85B-SWCNT FET devices achieved the detection of the Ag85B spiked in phosphate-buffered saline (calibration samples) with a limit of detection (LOD) of 0.05 fg/mL. This SWCNT FET biosensor also showed good sensing performance in biological matrices including artificial sputum and can identify Ag85B in serum after introducing bovine serum albumin (BSA) into the blocking layer. Furthermore, our BSA-blocked Ab85B-SWCNT FET devices can distinguish between TB-positive and -negative clinical samples, promising the application of SWCNT FET devices in point-of-care TB diagnostics. Moreover, the robustness of this SWCNT-based biosensor to the TB diagnosis in blood serum was enhanced by blocking SWCNT devices directly with a glutaraldehyde cross-linked BSA layer, enabling future applications of these SWCNT-based biosensors in clinical testing.

New Manual Quantitative Polymerase Chain Reaction Assay Validated on Tongue Swabs Collected and Processed in Uganda Shows Sensitivity That Rivals Sputum-based Molecular Tuberculosis Diagnostics.

BACKGROUND: Sputum-based testing is a barrier to increasing access to molecular diagnostics for tuberculosis (TB). Many people with TB are unable to produce sputum, and sputum processing increases assay complexity and cost. Tongue swabs are emerging as an alternative to sputum, but performance limits are uncertain. METHODS: From June 2022 to July 2023, we enrolled 397 consecutive adults with cough >2 weeks at 2 health centers in Kampala, Uganda. We collected demographic and clinical information, sputum for TB testing (Xpert MTB/RIF Ultra and 2 liquid cultures), and tongue swabs for same-day quantitative polymerase chain reaction (qPCR) testing. We evaluated tongue swab qPCR diagnostic accuracy versus sputum TB test results, quantified TB targets per swab, assessed the impact of serial swabbing, and compared 2 swab types (Copan FLOQSWAB and Steripack spun polyester). RESULTS: Among 397 participants, 43.1% were female, median age was 33 years, 23.5% were diagnosed with human immunodeficiency virus, and 32.0% had confirmed TB. Sputum Xpert Ultra and tongue swab qPCR results were concordant for 98.2% (95% confidence interval [CI]: 96.2-99.1) of participants. Tongue swab qPCR sensitivity was 92.6% (95% CI: 86.5 to 96.0) and specificity was 99.1% (95% CI: 96.9 to 99.8) versus microbiological reference standard. A single tongue swab recovered a 7-log range of TB copies, with a decreasing recovery trend among 4 serial swabs. Swab types performed equivalently. CONCLUSIONS: Tongue swabs are a promising alternative to sputum for molecular diagnosis of TB, with sensitivity approaching sputum-based molecular tests. Our results provide valuable insights for developing successful tongue swab-based TB diagnostics.