Sustained transmission over two decades of a previously unrecognised MPT64 negative Mycobacterium tuberculosis strain in Queensland, Australia: a whole genome sequencing study.

Background: MPT64 is a key protein used for Mycobacterium tuberculosis (MTB) complex strain identification. We describe protracted transmission of an MPT64 negative MTB strain in Queensland, Australia, and explore genomic factors related to its successful spread. Methods: All MPT64 negative strains identified between 2002 and 2022 by the Queensland Mycobacteria Reference Laboratory, and an additional 2 isolates from New South Wales (NSW), were whole genome sequenced. Bayesian modelling and phylogeographical analyses were used to assess their evolutionary history and transmission dynamics. Protein structural modelling to understand the putative functional effects of the mutated gene coding for MPT64 protein was performed. Findings: Forty-three MPT64 negative isolates were sequenced, belonging to a single MTB cluster of Lineage 4.1.1.1 strains. Combined with a UK dataset of the same lineage, molecular dating estimated 1990 (95% HPD 1987-1993) as the likely time of strain introduction into Australia. Although the strain has spread over a wide geographic area and new cases linked to the cluster continue to arise, phylodynamic analysis suggest the outbreak peaked around 2003. All MPT64 negative strains had a frame shift mutation (delAT, p.Val216fs) within the MPT64 gene, which confers two major structural rearrangements at the C-terminus of the protein. Interpretation: This study uncovered the origins of an MPT64 negative MTB outbreak in Australia, providing a richer understanding of its biology and transmission dynamics, as well as guidance for clinical diagnosis and public health action. The potential spread of MPT64 negative strains undermines the diagnostic utility of the MPT64 immunochromatographic test. Funding: This study was funded from an operational budget provided to the Queensland Mycobacterium Reference Laboratory by Pathology Queensland, Queensland Department of Health.

Diagnostic Accuracy of the Abbott SD Bioline MPT64 antigen test for identification of MTB Complex in a U.S. Clinical Mycobacteriology Laboratory.

Identification of the Mycobacterium tuberculosis complex (MTBC) from culture and differentiation from other non-tuberculous mycobacterial species is required for rapid diagnosis and accurate treatment. However, gaps exist in culture-based identification of acid-fast bacilli (AFB) positive cultures for rapid rule-out of MTBC in the United States. The SD Bioline™ MPT64 (Abbott Inc, South Korea) lateral flow assay (LFA) has high sensitivity and specificity for the detection of MTBC in liquid culture but has not been evaluated in a clinical mycobacteriology laboratory in the United States. We conducted a diagnostic accuracy study of this LFA for detection of MTBC versus NTMs on AFB positive cultures. A total of 362 tests were performed, with a sensitivity and specificity of 100 % (362/362) across all tests. The SD Bioline MPT64 assay provides accurate test results with AFB-positive liquid cultures and could fill the current gap for rapid rule-out of MTBC in U.S.-based clinical laboratories.

Diagnosis of Paediatric Extrapulmonary Tuberculosis by the MPT64 Antigen at a Tertiary Care Hospital.

Background Tuberculosis (TB) remains a global health concern, with India bearing a substantial burden. Paediatric TB, especially extrapulmonary TB (EPTB), presents unique diagnostic challenges due to its paucibacillary nature and the difficulty in obtaining suitable samples in children. Accurate and timely diagnosis is crucial to initiate appropriate treatment and mitigate disease spread. The MPT64 antigen test has shown promise in diagnosing TB, but its performance in paediatric EPTB remains underexplored. This study aimed to evaluate the diagnostic utility of the MPT64 antigen test in paediatric EPTB cases at a tertiary care hospital in India. Methods We conducted a prospective cross-sectional study at the Indira Gandhi Institute of Medical Sciences (IGIMS), a tertiary care hospital in India. A total of 250 paediatric participants, aged 0-18 years, with clinical suspicion of extrapulmonary tuberculosis (EPTB) were included. Diagnostic samples (e.g., tissue biopsies, pus, cerebrospinal fluid (CSF), and lymph node aspirates) were obtained, and tests including microscopy for acid-fast bacilli (AFB), mycobacterial cultures, GeneXpert MTB/RIF assay, and the TB Antigen MPT64 Rapid ICT Kit were performed. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy of the MPT64 antigen test were calculated using culture and GeneXpert as reference standards. Results Among the 250 participants, 34 (13.6%) were confirmed to have EPTB. The MPT64 antigen test demonstrated a sensitivity of 70.6% and specificity of 92.1% in detecting EPTB cases. Mycobacterial cultures had the highest sensitivity (91.2%) and specificity (97.7%). GeneXpert showed a sensitivity of 70.6% and specificity of 93.9%. Overall diagnostic accuracy ranged from 88.7% for acid-fast bacteria (AFB) staining to 96.9% for mycobacterial cultures. The MPT64 antigen test had an area under the curve (AUC) of 0.814, indicating a good diagnostic accuracy. Conclusion The MPT64 antigen test demonstrates promising sensitivity and specificity for diagnosing paediatric EPTB, making it a valuable diagnostic tool, especially in resource-limited settings. However, mycobacterial cultures maintain the highest accuracy. Combining the MPT64 antigen test with other methods may enhance diagnostic capabilities.

Association of lineage 4.2.2 of Mycobacterium tuberculosis with the 63-bp deletion variant of the mpt64 gene.

IMPORTANCE: To date, rapid diagnostic methods based on the MPT64 antigen assay are increasingly utilized to differentiate between non-tuberculous mycobacteria and TB disease in clinical settings. Furthermore, numerous novel techniques based on the MPT64 release assay are continuously being developed and applied for the identification of both pulmonary and extrapulmonary TB. However, the diagnostic accuracy of the MPT64 antigen assay is influenced by the presence of 63 bp deletion variants within the mpt64 gene. To our knowledge, this is the first report on the association between the 63 bp deletion variant in mpt64 and Mycobacterium tuberculosis L4.2.2 globally, which highlights the need for the cautious utilization of MPT64-based testing in regions where L4.2.2 isolates are prevalent, such as China and Vietnam, and MPT64 negative results should be confirmed with another assay. In addition, further studies on vaccine development and immunology based on MPT64 should consider these isolates with 63 bp deletion variant.

Rapid diagnosis of TB using Aptamer-based assays for Mycobacterium tuberculosis antigens in children and adolescents.

Background: Despite advances establishing microbiological evidence of tuberculosis (TB) is still a concern in children due to the limitation of availability of sample and predominance of extrapulmonary TB, there is unmet need for diagnostic tests which are low cost, rapid and sensitive and specific. Methods: This study evaluated the utility of aptamer-based assay for detecting mycobacterium tuberculosis antigens HspX and MPT 64 in rapid diagnosis of TB in children up to 18 years of age in a tertiary medical college. A total of 100 children were sequentially enrolled with presumptive pulmonary (n = 52 and extrapulmonary n = 48) TB based on clinico-radiological characteristics. The samples were evaluated with ALISA technique for TB antigens and compared with the results of ZN microscopy, GeneXpert and mycobacterial culture MGIT. Results: The enrolled children had mean age (11.7 + 4.4 years) with both pulmonary (n = 52) and extrapulmonary TB (n = 48). Our study results concluded poor results of smears (11% positivity, sensitivity: 17.7%, NPV: 42.7%) and better of GeneXpert (positivity: 42%, sensitivity of 67.4%, NPV: 65.5%) and culture (positivity 57%, sensitivity 91.9%, NPV 88.3%). HspX antigen by ALISA had comparable results (positivity: 49%, sensitivity: 62.9%; NPV: 54.9%). MPT 64 antigen by ALISA also had similar results (positivity: 45%, sensitivity: 58% and NPV 52, 3%). Sensitivity and specificity were higher in pulmonary TB compared to EPTB for both antigens. HspX antigen assay by ALISA and MPT 64 ALISA over existing microbiological diagnostic methods had additional of 13%. Conclusion: ALISA technique for mycobacterium antigens HspX and MPT 64 was rapid, low-cost test (1-3$/test) high sensitivity and specificity and comparable to currently available methods.

High Positivity Rate of Urinary Mycobacterium tuberculosis Antigens Cocktail (ESAT-6, CFP-10, and MPT-64) in Active Tuberculosis Patients With and Without Human Immunodeficiency Virus Infection: A Cross-Sectional Study.

Introduction: Human immunodeficiency virus (HIV) infection is a risk factor for the occurrence of a large of Mycobacterium tuberculosis (Mtb) antigen load in the body. The antigens cocktail namely early secretory antigenic target protein 6-kDa (ESAT-6), Culture filtrate protein 10 kDa (CFP-10), and Mycobacterium tuberculosis protein 64 (MPT-64) are secreted by Mtb during replication, hence, their concentration increase in patients with active Tuberculosis (TB). This increased levels facilitates their entry into the systemic circulation, followed by secretion by the glomerulus into the urine. The aim of this study was to determine the positivity rate of the urinary Mtb antigens cocktail between TB patients with and without HIV infection. Methods: This is an observational descriptive comparative study conducted with a cross-sectional design. Random urine samples were collected from patients diagnosed with active TB in Dr. Hasan Sadikin Bandung Hospital in 2021. The subjects were divided into 2 groups, TB-HIV group and TB without HIV group. The samples were tested using the quantitative immunochromatography method. Result: Sixty active TB patients consisting of TB patients with HIV infection (n = 30) and TB patients without HIV infection (n = 30). The positivity in the urinary Mtb antigens cocktail was 93.3% for TB-HIV group and 100% for TB without HIV group (P = .492). The median concentration of urinary Mtb antigens cocktail in TB patients without HIV infection was higher than that of TB patients with HIV infection (137.73 ng/mL vs 96.69 ng/mL, respectively; P = .001). Conclusion: There was no significant difference in the positivity rate, meanwhile, there was a significant difference in concentration of the urinary Mtb antigens cocktail between active TB patients with and without HIV infection. Interestingly, this urinary Mtb antigens cocktail can be found in both groups without being affected by the patient's immune condition, thus becoming a test to assist diagnose active TB.

Quantification of mycobacterial proteins in extrapulmonary tuberculosis cases by nano-based real-time immuno-PCR.

Aim: Diagnosis of extrapulmonary tuberculosis (EPTB) is difficult, and a rapid and dependable diagnostic test is urgently needed. Methods: A nano-based assay, SYBR Green magnetic bead-coupled gold nanoparticle-based real-time immuno-polymerase chain reaction (MB-AuNP-RT-I-PCR) was studied for the quantitative detection of Mycobacterium tuberculosis MPT-64+CFP-10 proteins in clinically suspected EPTB patients. Results: A wide range (270 fg/ml-9.9 ng/ml) of MPT-64+CFP-10 was quantified by MB-AuNP-RT-I-PCR in EPTB cases, whereas magneto-ELISA demonstrated a narrow range (1.8-10 ng/ml). Furthermore, high sensitivity (88.2%) and specificity (100%) were attained by MB-AuNP-RT-I-PCR in EPTB (n = 51) and non-TB control (n = 49) subjects, respectively. Both MB-AuNP-I-PCR/magneto-ELISA exhibited significantly lower (p < 0.05-0.01) sensitivities than MB-AuNP-RT-I-PCR. Conclusion: The MB-AuNP-RT-I-PCR described herein shows good diagnostic accuracy, which may translate into a credible diagnostic kit.

Extrapulmonary tuberculosis (EPTB) is a type of tuberculosis disease caused by the bacteria Mycobacterium tuberculosis (Mtb) that affect other regions of the body, rather than the lungs. Detecting EPTB is difficult, and a fast and reliable test is needed. This study developed a test based on a small particle, known as a nanoparticle, to identify Mtb in people with EPTB. The test shows good accuracy and could be used for routine testing.

Gel Protein Extraction's Impact on Conformational Epitopes of Linear Non-Tagged MPT64 Protein.

The production and purification of recombinant proteins are crucial to acquiring pure MPT64 protein. Due to the fact that protein epitopes may undergo conformational changes during purification, this study, therefore, investigated an effective rapid purification method to produce highly intracellular pure MPT64 protein without causing conformational changes in the epitope under denaturing conditions. MPT64 was isolated from E. coli and electrophoresed using gel SDS-PAGE. Then, the desired protein bands were excised and purified with two methods: electroelution and passive elution. The isolated protein was identified via peptide mass fingerprinting using MALDI-TOF MS and reacted with IgG anti-MPT64, and the cross-reactivity of the isolated protein with IgY anti-MPT64 was confirmed using Western blot. The results show that both of these methods produced pure MPT64 protein, and the MPT64 protein was confirmed based on the MALDI-TOF MS results. Neither of these two methods resulted in epitope changes in the MPT64 protein so it could react specifically with both antibodies. The yield of MPT64 protein was higher with electroelution (2030 ± 41 µg/mL) than with passive elution (179.5 ± 7.5 µg/mL). Thus, it can be inferred that the electroelution method is a more effective method of purifying MPT64 protein and maintaining its epitope than the passive elution method.

MPT51 and MPT64-based antigen detection assay for the diagnosis of extrapulmonary tuberculosis from urine samples.

In view of WHO's "End-TB" strategy, we developed a non-invasive, urine-based ELISA, targeting 2 Mycobacterium tuberculosis antigens namely MPT51 and MPT64 for extrapulmonary TB (EPTB) diagnosis. Suspected EPTB patients (n = 137) [Pleural TB, Abdominal TB and Tuberculous meningitis] were categorized in "Definite" EPTB (n = 10) [Xpert-MTB/RIF and/or culture-positive], "Probable" EPTB (n = 77) and "Non-EPTB" (n = 50) groups using defined composite reference standards. ROC-curves were generated using ELISA results of "Definite" EPTB and "Non-EPTB" groups for both antigens independently and cut-off values were selected to provide 86.3% (95%CI:73.3-94.2) specificity for MPT51 and 92% (95%CI:80.8-97.8) for MPT64. The sensitivity of MPT51-ELISA and MPT64-ELISA was 70% (95%CI:34.7-93.3) and 90% (95%CI:55.5-99.7) for "Definite" EPTB group and 32.5% (95%CI:22.2-44.1) and 30.8% (95%CI:20.8-42.2) for "Probable" EPTB group, respectively. Combining the results of both ELISAs showed a 100% (95%CI:69.1-100) sensitivity in "Definite" EPTB group and 41.6% (95%CI:30.4-53.4) in "Probable" EPTB group, with an 80% (95%CI:66.3-89.9) specificity. The results demonstrated the potential of urine-based ELISAs as screening tests for EPTB diagnosis.

Synthesis of plant-based, self-adjuvanted, dual antigen specific to Mycobacterium tuberculosis as a novel tuberculosis subunit vaccine that elicits immunogenicity in rabbit.

OBJECTIVES: The only approved vaccine, Bacillus Calmette Guérin (BCG) used in global tuberculosis (TB) immunization programmes has been very effective in childhood TB but not in adult pulmonary and latent TB. Moreover, the emergence of multi-drug resistance-TB cases demands either to increase efficiency of BCG or replace it with the one with improved efficacy. RESULTS: A novel combination of two most effective secreted protein antigens specific for Mycobacterium tuberculosis (Mtb), ESAT-6 and MPT-64 (but not present in BCG strains) fused with a cholera toxin B subunit (CTB) and tagged with 6xHis was expressed for the first time in Escherichia coli as well as in transgenic cucumber plants developed using Agrobacterium tumefaciens-mediated transformation. The recombinant fusion protein (His6x.CTB-ESAT6-MPT64) expressed in E. coli was purified by a single-step affinity chromatography and used to produce polyclonal antibodies in rabbit. The transgenic cucumber lines were confirmed by polymerase chain reaction (PCR), Southern blot hybridization, reverse transcriptase PCR (RT-PCR), real-time PCR (qRT-PCR) and expression of recombinant fusion protein by western blot analysis and its quantification by enzyme-linked immunosorbent assay (ELISA). A maximum value of the fusion protein, 478 ng.g-1 (0.030% of the total soluble protein) was obtained in a transgenic cucumber line. Rabbit immunized orally showed a significant increase in serum IgG levels against the fusion protein as compared to the non-immunized rabbit. CONCLUSIONS: Stable expression of Mtb antigens with CTB in edible cucumber plants (whose fruits are eaten raw) in sufficient amount possibly would facilitate development of a safe, affordable and orally delivered self-adjuvanted, novel dual antigen based subunit vaccine against TB.

Aluminum Foil vs. Gold Film: Cost-Effective Substrate in Sandwich SERS Immunoassays of Biomarkers Reveals Potential for Selectivity Improvement.

The first application of aluminum foil (Al F) as a low-cost/high-availability substrate for sandwich immunoassay using surface-enhanced Raman spectroscopy (SERS) is reported. Untreated and unmodified Al F and gold film are used as substrates for sandwich SERS immunoassay to detect tuberculosis biomarker MPT64 and human immunoglobulin (hIgG) in less than 24 h. The limits of detection (LODs) for tuberculosis (TB) biomarker MPT64 on Al foil, obtained with commercial antibodies, are about 1.8-1.9 ng/mL, which is comparable to the best LOD (2.1 ng/mL) reported in the literature for sandwich ELISA, made with fresh in-house antibodies. Not only is Al foil competitive with traditional SERS substrate gold for the sandwich SERS immunoassay in terms of LOD, which is in the range 18-30 pM or less than 1 pmol of human IgG, but it also has a large cost/availability advantage over gold film. Moreover, human IgG assays on Al foil and Si showed better selectivity (by about 30-70% on Al foil and at least eightfold on Si) and a nonspecific response to rat or rabbit IgG, in comparison to the selectivity in assays using gold film.

Detection of Mycobacterium tuberculosis Complex in Sputum Samples Using Droplet Digital PCR Targeting mpt64.

Tuberculosis (TB) is one of the top 10 causes of death worldwide. It is challenging to find methods of diagnosis of active pulmonary TB that are sensitive enough to detect cases for proper treatment before unintentional transmission. Droplet digital PCR (ddPCR) is a highly sensitive method to detect genetic material of pathogens, but it has rarely been used for diagnosis of TB. This study compared the sensitivity of ddPCR with that of GeneXpert and AFB smear microscopy in 180 leftover sputum samples from patients suspected of having TB on the basis of clinical symptoms and radiography. Absolute quantification of copy numbers of MTB-specific genes was possible using ddPCR targeting the mpt64 gene. Among the 180 samples, 41.1% were diagnosed as having TB using ddPCR. The sensitivities of AFB smear microscopy, GeneXpert and ddPCR were 41.9%, 82.4% and 100%, respectively. AFB smear microscopy and GeneXpert both had a specificity of 100%, and the specificity of ddPCR was 95.3%. The accuracy of ddPCR (97.2%) is higher than that of GeneXpert (92.7%). This robust ddPCR system could potentially be used as a method for early diagnosis of TB.

Novel Mutations in MPT64 Secretory Protein of Mycobacterium tuberculosis Complex.

Tuberculosis (TB) is a global health problem caused by the Mycobacterium tuberculosis complex (MTBC). These bacteria secrete various proteins involved in the pathogenesis and persistence of MTBC. Among the secretory proteins, MPT64 (Rv1980C) is highly conserved and is also known as a major culture filtrate that is used in rapid diagnosis of MTBC. In the current study, we aimed to find the mutation in this highly conserved protein in isolates from the Pashtun-dominant province of Pakistan. We analyzed 470 M. tuberculosis whole-genome sequences of Khyber Pakhtunkhwa Province. Mutations in the MPT64 gene were screened through TB-Profiler and BioEdit software tools. The DynaMut web server was used to analyze the impact of the mutation on protein dynamics and stability. Among 470 MTB genomes, three non-synonymous mutations were detected in nine isolates, and one synonymous mutation (G208A) was found in four isolates. Mutation G211T (F159L), which was detected at the C-terminal domain of the protein in six isolates, was the most prominent. The second novel mutation, T480C (I70V), was detected in two isolates at the C-terminal side of the protein structure. The third novel mutation, A491C (L66R), was detected in a single isolate at the N-terminal side of the MPT64 protein. The effect of these three mutations was destabilizing on the protein structure. The molecular flexibility of the first two mutations increased, and the last one decreased. MPT64 is a highly conserved secretory protein, harboring only a few mutations. This study provides useful information for better managing the diagnosis of MTB isolates in high TB-burden countries.

Non-amplification nucleic acid detection with thio-NAD cycling.

The PCR technique is indispensable in biology and medicine, but some difficulties are associated with its use, including false positive or false negative amplifications. To avoid these issues, a non-amplification nucleic acid detection protocol is needed. In the present study, we propose a method in which nucleic-acid probe hybridization is combined with thio-NAD cycling to detect nucleic acids without amplification. We report our application of this method for the detection of the gene of MPT64 in Mycobacterium tuberculosis. Two different cDNA probes targeted the mpt64 gene: the first probe was used to immobilize the mpt64 gene, and the second probe, linked with alkaline phosphatase (ALP), was hybridized to a target sequence in the mpt64 gene. A substrate was then hydrolyzed by ALP, and a cycling reaction was conducted by a dehydrogenase with its co-factors (thio-NAD and NADH). The single-stranded DNA, double-stranded DNA, plasmid DNA for the mpt64 gene, and whole genome of M. tuberculosis var. BCG were detected at the level of 105-106 copies/assay, whereas the non-tuberculosis mycobacteria (e.g., M. avium, M. intracellulare, M. kansasii, and M. abscessus) were below the limits of detection. The present method enables us to avoid the errors inherent in nucleic acid amplification methods.

Ultrasensitive biosensing platform for Mycobacterium tuberculosis detection based on functionalized graphene devices.

Tuberculosis (TB) has high morbidity as a chronic infectious disease transmitted mainly through the respiratory tract. However, the conventional diagnosis methods for TB are time-consuming and require specialists, making the diagnosis of TB with point-of-care (POC) detection difficult. Here, we developed a graphene-based field-effect transistor (GFET) biosensor for detecting the MPT64 protein of Mycobacterium tuberculosis with high sensitivity as a POC detection platform for TB. For effective conjugation of antibodies, the graphene channels of the GFET were functionalized by immobilizing 1,5-diaminonaphthalene (1,5-DAN) and glutaraldehyde linker molecules onto the graphene surface. The successful immobilization of linker molecules with spatial uniformity on the graphene surface and subsequent antibody conjugation were confirmed by Raman spectroscopy and X-ray photoelectron spectroscopy. The GFET functionalized with MPT64 antibodies showed MPT64 detection with a detection limit of 1 fg/mL in real-time, indicating that the GFET biosensor is highly sensitive. Compared to rapid detection tests (RDT) and enzyme-linked immunosorbent assays, the GFET biosensor platform developed in this study showed much higher sensitivity but much smaller dynamic range. Due to its high sensitivity, the GFET biosensor platform can bridge the gap between time-consuming molecular diagnostics and low-sensitivity RDT, potentially aiding in early detection or management of relapses in infectious diseases.

Enhanced Serum IgG Detection Potential Using 38KD-MPT32-MPT64, CFP10-Mtb81-EspC Fusion Protein and Lipoarabinomannan (LAM) for Human Tuberculosis.

For the rapid, reliable, and cost-effective methods of tuberculosis (TB) auxiliary diagnosis, antibody (Ab) detection to multiple antigens of Mycobacterium tuberculosis (Mtb) has great potential; however, this methodology requires optimization. We constructed 38KD-MPT32-MPT64, CFP10-Mtb81-EspC, and Ag85B-HBHA fusion proteins and evaluated the serum Ab response to these fusion proteins and to lipoarabinomannan (LAM) by ELISA in 50 TB patients and 17 non-TB subjects. IgG responses to the three fusion proteins and to LAM were significantly higher in TB patients, especially in Xpert Mtb-positive TB patients (TB-Xpert+), than in non-TB subjects. Only the anti-38KD-MPT32-MPT64 Ab showed higher levels in the Xpert Mtb-negative TB patients (TB-Xpert-) than in the non-TB, and only the anti-LAM Ab showed higher levels in the TB-Xpert+ group than in the TB-Xpert- group. Anti-Ag85B-HBHA Ab-positive samples could be accurately identified using 38KD-MPT32-MPT64. The combination of 38KD-MPT32-MPT64, CFP10-Mtb81-EspC, and LAM conferred definite complementarity for the serum IgG detection of TB, with relatively high sensitivity (74.0%) and specificity (88.2%). These data suggest that the combination of 38KD-MPT32-MPT64, CFP10-Mtb81-EspC, and LAM antigens provided a basis for IgG detection and for evaluation of the humoral immune response in patients with TB.

Rapid identification of Mycobacterium tuberculosis in cultures by molecular and immunological methods / Identificação rápida de Mycobacterium tuberculosis em culturas por método molecular e método imunológico

Objective: The study aimed to evaluate molecular and immunological methods and to propose a workflow using them for tuberculosis (TB) diagnosis routine. Methods: A cross-sectional retrospective study was performed, including 121 liquid cultures from a TB laboratory located in the extreme south of Brazil. All cultures were positive for Mycobacterium tuberculosis complex (MTBC) by in-house Polymerase Chain Reaction (PCR) using DNA extracted by the CTAB method (PCR-CTAB) for IS6110 detection. These cultures were subjected to faster tests than this one, the immunological MPT64 assay and the PCR using DNA extracted by thermal lysis method (PCR-TL), and these were evaluated for MTBC identification using PCR-CTAB as a reference method. Results: The sensitivity of MPT64 assay and PCR-TL to identify MTBC in positive cultures by PCR-CTAB were 73.6% (89/121) and 98.3% (119/121), respectively. We proposed a workflow based on the use of MPT64 assay in liquid cultures suggestive of MTBC, and in case of a negative result, we suggest the performance of PCR-TL. The PCR-CTAB is suggested only if faster tests are negative. Conclusions: Methods capable of confirming MTBC in cultures should continue to be standardized, tested, and optimized to meet the ideal requirements of simplicity, quickness, and effectiveness. The molecular and immunological methods evaluated have differences in the execution and detection of MTBC in cultures, but they are rapid tools for laboratory TB diagnosi

Objetivos: O estudo objetivou avaliar métodos molecular e imunológico e propor um fluxo de trabalho utilizando-os para a rotina de diagnóstico da tuberculose (TB). Métodos: Foi realizado um estudo transversal retrospectivo, incluindo 121 culturas líquidas de um laboratório de TB localizado no extremo sul do Brasil. Todas as culturas foram positivas para o complexo Mycobacterium tuberculosis (CMTB) por Reação em Cadeia da Polimerase (PCR) in-house para detecção do IS6110, usando DNA extraído pelo método CTAB (PCR-CTAB). Essas culturas foram submetidas a testes mais rápidos que este, o ensaio imunológico MPT64 e a PCR com DNA extraído pelo método de lise térmica (PCR-LT), e estas foram avaliadas para identificação de CMTB usando PCR-CTAB como método de referência. Resultados: A sensibilidade do ensaio MPT64 e da PCR-LT para identificar o CMTB em culturas positivas pela PCRCTAB foi de 73,6% (89/121) e 98,3% (119/121), respectivamente. Propusemos um fluxo de trabalho baseado no uso do ensaio MPT64 em culturas líquidas sugestivas de CMTB e, em caso de resultado negativo, sugerimos a realização de PCR-LT. Sugere-se a PCR-CTAB apenas se os testes mais rápidos forem negativos. Conclusões: Os métodos capazes de confirmar o CMTB em culturas devem continuar sendo padronizados, testados e otimizados para atender aos requisitos ideais de simplicidade, rapidez e eficácia. Os métodos molecular e imunológico avaliados apresentam diferenças na execução e detecção do CMTB em culturas, mas são ferramentas rápidas para o diagnóstico laboratorial da TB.

Simultaneous Amperometric Aptasensor Based on Diazonium Grafted Screen-Printed Carbon Electrode for Detection of CFP10 and MPT64 Biomarkers for Early Tuberculosis Diagnosis.

Early diagnosis is highly crucial for life-saving and transmission management of tuberculosis (TB). Despite the low sensitivity and time-consuming issues, TB antigen detection still relies on conventional smear microscopy and culture techniques. To address this limitation, we report the development of the first amperometric dual aptasensor for the simultaneous detection of Mycobacterium tuberculosis secreted antigens CFP10 and MPT64 for better diagnosis and control of TB. The developed sensor was based on the aptamers-antibodies sandwich assay and detected by chronoamperometry through the electrocatalytic reaction between peroxidase-conjugated antibodies, H2O2, and hydroquinone. The CFP10 and MPT64 aptamers were immobilized via carbodiimide covalent chemistry over the disposable dual screen-printed carbon electrodes modified with a 4-carboxyphenyl diazonium salt. Under optimized conditions, the aptasensor achieved a detection limit of 1.68 ng mL-1 and 1.82 ng mL-1 for CFP10 and MPT64 antigens, respectively. The developed assay requires a small sample amount (5 µL) and can be easily performed within 2.5 h. Finally, the dual aptasensor was successfully applied to clinical sputum samples with the obtained diagnostic sensitivity (n = 24) and specificity (n = 13) of 100%, respectively, suggesting the readiness of the developed assay to be used for TB clinical application.

Improving of pelB-Secreted MPT64 protein released by Escherichia coli BL21 (DE3) using Triton X-100 and Tween-80.

pelB has been known as a successful signal peptide to translocate the protein target extracellularly in the Escherichia coli system. However, in our previous study, the yield of MPT64 protein extracellular recovery was still low and plenty of this protein was remain trapped in cytoplasm and periplasm. Recently, nonionic surfactants were efficiently reported to secrete recombinant protein extracellularly. Nonetheless, it must be clarified whether the surfactant supplementation can improve the yield of MPT64 extracellular protein significantly without giving impact on the structure of isolated MPT64 protein and can minimized the cell lysis effect. MPT64 protein secretion was carried out by comparing the effects of surfactants Tween 80 and Triton × 100 at various concentrations. Triton × 100 was able to increase the extracellular MPT64 protein gain up to 3 times higher than Tween 80 and it was in line with the greater level ratio of cell leakage of Triton × 100 compared to that of Tween 80. Similarly, the viable cell of the cultures decreased dramatically. However, both surfactants did not interfere the structure of MPT64 protein. In conclusion, Triton × 100 can be chosen as the supporting surfactant to assist the act of peptide signal in improving the resulting of MPT64 extracellular protein.

Doubled Nontuberculous Mycobacteria Isolation as a Consequence of Changes in the Diagnosis Algorithm.

Background: Nontuberculous mycobacteria (NTM) and their associated diseases remain neglected. Through minor modifications in our diagnostic algorithm, we observed an unexpected higher number of cultivable NTM isolates. Therefore, a retrospective study was performed thoroughly to investigate the effect of changed laboratory procedures on NTM isolation in a specialized tuberculosis hospital. Methods: NTM isolation rates and composition of NTM species were compared for the two diagnostic algorithms: (1) by using traditional p-nitrobenzoic acid (PNB) selective medium as a preliminary test to identify NTM isolates among the positive cultures (procedure I) and (2) by using the MPT64 antigen detection method to distinguish between Mycobacterium tuberculosis complex (MTBC) isolates and possible NTM isolates after a positive MGIT960 liquid culture (procedure II). Results: The NTM isolation rate in procedure II was significantly higher than the procedure I (18.08% vs 9.71%; P<0.001). A noticeable increase in the ratio of NTM isolates among the identified mycobacteria was observed over the studied years (ie, from 58.18% in 2019 to 72.93% in 2021), which indicated a more precise prescription of species identification test after prompt information was provided in procedure II. In addition, the consistency of the identified species using multiple specimens from the same patient did not present a significant difference between the procedures. Conclusion: According to our study, NTM infection might be far more underestimated than it is. A diagnostic procedure combining MGIT960 culture and MPT64 antigen detection could timely and easily identify clues of NTM isolates and improve the diagnosis of NTM infections.