Mycobacterium Tuberculosis Complex Research Articles

Cytokines Levels and Anti-tuberculosis Drug Resistance among HIV Seropositive Patients in Ibadan, Southwest, Nigeria

Susceptibility to TB disease rises on mutations in cytokine receptors which ultimately lead to defective signaling and alter the immune surveillance and bacterial killing. Therefore, this study was undertaken to investigate the association between cytokines and anti-TB drug resistances. A total of 217 people living with human immunodeficiency virus at University College Hospital, Ibadan were recruited. Venous blood and sputum samples were collected. Sputum samples were cultured for Mycobacterium tuberculosis complex and characterized using standard methods. Anti-TB drug resistance was detected using phenotypic and genotypic methods. Cytokines levels (IL-10 and IFN-Y) were estimated using ELISA method. Association between cytokines and anti-TB drug resistance was determined using linear regression analysis. There were 105(48.4%) males and 112(51.6%) females with a mean age of 42.95 ± 8.286 years and a range of 28-65 years. The prevalence of TB among PLHIV was found to be 19.1% with male having higher rate than female (P<0.05), and highest rate among aged ≥40. There was no significant association between cytokines (IL-10 and IFN-y) and sex of the subjects (P>0.05). However, females had higher IFN-y level of 231.06±61.81 IU/ml when compared to 227.53±57.92 IU/ml obtained from males. The prevalence of multi-drug resistance TB was 11.9% with Rifampicin resistance having the highest rate of 5.5%. There was no significant association between cytokines (IL-10 and IFN-y) and sex, but age group >40 years had higher IL-10 and IFN-y level of 226.53±52.39 IU/ml and 232.41±66.63 IU/ml respectively. There was an association between high cytokines levels and anti-TB drug resistance (P<0.05). Highest cytokines levels; IL-10 (401.00±7.07 IU/ml) and IFN-Y (401.00±7.07 IU/ml) were recorded in Levofloxacin resistance. There was an association between high cytokines levels and genotypic anti- TB drug resistance (P<0.05) with highest cytokines levels; IL-10 (400.12±12.37 IU/ml) and IFN-Y (472.87±31.93 IU/ml) recorded in Rifampicin resistance. This entire study revealed that there is high prevalence of MDR-TB among PLHIV, with Rifampicin monoresistance having the highest resistance rate among the drugs tested, and pro and inflammatory cytokines (IL-10 and IFN-y) play significant role in the development of Multi Drug Resistant-Tuberculosis among people living with HIV.

Clonal Diversity and Spatial Dissemination of Drug-resistant Mycobacterium tuberculosis Complex among HIV Seropositive Patients in Southwest Nigeria

Mycobacterium tuberculosis complex display relatively static genomes and 99.9% nucleotide sequence identity. Studying the evolutionary history of such monomorphic bacteria is a difficult and challenging task. Therefore, this study was undertaken to investigate the clonal diversity and spatial dissemination of drug-resistant M. tuberculosis complex among HIV seropositive clients in Southwest Nigeria. A total of two-hundred and seventeen (217) consented HIV positive (case subjects) and fifty (50) non-HIV positive controls were recruited for this study. Two Sputum samples were collected from each participant in two consecutive days. The samples were divided into two parts: one part was decontaminated using NAOH-NALC method and aseptically cultured for Mycobacteria on Lowenstein-Jensen (LJ) medium with and without pyruvate. Mycobacterial isolates were characterized using conventional and molecular methods. Anti-TB drug sensitivity test was done using 1% proportion conventional method on LJ. MTBC isolates were sequenced using Genetic Analyzer 3130xl sequencer. Genetic relatedness and clonal diversity (phylogenetic analysis) of MTBC isolates were done using Bio- Edit software and MEGA 6. Out of 217 HIV positive participants, 105(48.4%) were male, while 112(51.6%) were female. Twenty-six (26(52.0%)) of the control subjects were male, while 24(48.0%) were females. The overall prevalence of TB among PLHIV was found to be 19.1%. The prevalence of NTM among PLHIV was found to be 2.8% with M. intracellulare being the predominant organism isolated. Participants’ age was found to be significantly associated with TB (P<0.05), however there was no significant association between TB and sex of the participants (P<0.05). The prevalence of culture confirmed drug-resistant TB (DR-TB) among PLHIV was found to be 11.9%, with Rifampicin having the highest monoresistance rate of 5.5%. The overall prevalence of MDR-TB among PLHIV was found to be 9.2%. The phylogenetic analysis of M. tuberculosis strains revealed same clone of all isolates. All PLHIV should be screened for drug-resistant TB especially MDR-TB as the prevalence of primary MDR-TB is high among them. Further study like 3R gene-based studies of adaptation and evolution is needed to facilitate further epidemiological studies of these bacteria.

A high titer antibody response against P22 protein immunocomplex is not correlated with protection in naturally tuberculosis-infected goats

Caprine livestock are significant reservoirs of the Mycobacterium tuberculosis complex (MTBC), contributing to tuberculosis (TB) transmission among animals and humans. The P22 protein immunocomplex (P22PI), derived from bovine tuberculin, shows immunostimulating capacity and is used for TB diagnosis. This study assessed the immunogenicity and protective efficacy of P22PI in two groups of goats: 24 naïve goats (12 immunised, 12 controls) from a TB-free herd, and 24 infected goats (12 immunised, 12 controls), referred to as pre-infected animals, from a M. bovis-infected herd. Both were exposed for 5 months to M. bovis-naturally infected goats. Reactors to single and comparative intradermal tuberculin (SIT and SCIT, respectively) tests and interferon-gamma release assay (IGRA) significantly increased (p < 0.05) in both groups 5 months’ post-exposure, with no significant differences between immunised and control animals. However, immunised animals exhibited a significantly higher (p < 0.05) antibody response against P22PI. Most naïve animals (83.3%) and all pre-infected animals developed TB-compatible lesions, with extensive necrosis in the lungs and associated lymph nodes, compared to 50% and 83.3% of control animals, respectively. These findings suggest that while P22PI stimulates an intense antibody response under the conditions of the present study, it does not confer protection against TB and may exacerbate disease severity.

A simplified pyrazinamidase test for Mycobacterium tuberculosis pyrazinamide antimicrobial susceptibility testing

ABSTRACT Pyrazinamide (PZA) is an important first-line drug for tuberculosis (TB) treatment by eradicating the persisting Mycobacterium tuberculosis complex (MTBC). Due to cost and technical challenges, end TB strategies are hampered by the lack of a simple and reliable culture-based PZA antimicrobial susceptibility testing (AST) for routine use. We initially developed a simplified chromogenic pyrazinamidase (PZase) test in the TB reference laboratory using a training set MTBC isolates with various drug-resistant profiles, and validated its performance using consecutive BACTEC MGIT 960 (MGIT)-culture-positive culture in 10 clinical laboratories. The pncA gene Sanger sequencing results were used as the reference, and compared to the MGIT-PZA AST. Differential diagnosis of Mycobacterium bovis was conducted using patented in-house real-time PCR. Of the 106 training isolates, the PZase test and MGIT-PZA AST showed 100.0% and 99.1% concordance as compared to Sanger sequencing, respectively. We found 32.1% (34/106) isolates harbored pncA mutations, including one isolate with silent mutation S65S. For validation, 1,793 clinical isolates were tested including 150 duplicate isolates from specimens of the same cases and 16 isolates with uncharacterized drug resistance (UDR)-associated mutations. Excluding duplicated and UDR isolates, we identified 2.6% (43/1,627) PZA-resistant isolates, including 1.3% (21/1,627) M . bovis isolates. The kappa values were 0.851–1.000. In addition, the accuracy of the PZase test conducted by 10 laboratories was 98.5%–100.0%. Our simplified PZase test demonstrated high concordance with Sanger sequencing and MGIT-PZA AST. Integrating the PZase test into routine first-line AST is effortless and represents an improvement in laboratory services for ending TB. IMPORTANCE We developed and validated a simple pyrazinamidase (PZase) test for pyrazinamide (PZA) antimicrobial susceptibility testing (AST). Our results demonstrated that the PZase test had high agreement with the pncA gene sequencing and MGIT-PZA AST. Integrating PZase test into routine AST is effortless and represents an improvement in laboratory services for ending TB.

Paradoxical deterioration during treatment of tuberculous meningitis responded to infliximab but not to steroids

Paradoxical deterioration in patients with tuberculous meningitis is a significant diagnostic and treatment challenge. We present the case of a 55-year-old previously healthy male, human immunodeficiency virus (HIV)-negative, presenting with headache, fever, agitation and slurred speech, who was ultimately diagnosed with tuberculous meningoencephalitis confirmed by cerebrospinal fluid polymerase chain reaction positive for Mycobacterium tuberculosis complex. After initiation of a standard antitubercular therapy combined with glucocorticoids, the patient experienced an initial neurological improvement. However, after 3 months of therapy, he had a major stroke with neurological deterioration, clinically and radiologically attributable to a paradoxical deterioration or an immune reconstruction syndrome as described in HIV-positive patients. Due to the persistence of inflammatory symptoms and radiological evidence of worsening tuberculomas despite ongoing antituberculosis therapy, off-label use of the tumour necrosis factor (TNF) inhibitor infliximab was initiated. The patient received three doses of infliximab at 5 mg/kg at monthly intervals, which led to marked clinical and radiological improvement. Follow-up magnetic resonance imaging showed regression of lesions, and the patient was eventually discharged with a significant recovery of motor and speech functions. This case underscores the potential of TNF inhibitors in managing severe cases of paradoxical deterioration in patients with tuberculous meningitis, challenging existing treatment paradigms and suggesting a need for further research into the pathophysiology and treatment of this condition.

Overexpression of outer membrane protein A (OmpA) increases aminoglycoside sensitivity in mycobacteria

BackgroundTuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) complex infection, is a leading cause of death worldwide from a single infectious agent. The emergence of drug resistance Mtb clinical strains makes the situation more serious. The role of Mtb outer membrane protein A (OmpA) in antimicrobial resistance remains unclear. This study aimed to evaluate the effect of OmpA expression on mycobacterial drug resistance. In this study, a Mycobacterium smegmatis (Ms) strain overexpressing OmpA (Ms-OmpA) and a Mycobacterium bovis (Mb) strain overexpressing OmpA (Mb-OmpA) were constructed, and their susceptibility to anti-TB drugs was determined by performing the minimal inhibitory concentrations (MICs), the plate assay and the macrophage infection assays.ResultsThe streptomycin MIC of the overexpressing strain was 2-fold lower than those of the wide-type (Ms) and empty plasmid strains (pMV-261) as well as amikacin and gentamicin. Moreover, both the plate and the macrophage infection assays indicate that overexpression of OmpA increases streptomycin sensitivity in Mycobacteria. The other aminoglycosides like amikacin and gentamicin have the same phenotypes as streptomycin on the plates for the virulent strain Mb-OmpA. The porin inhibitor spermidine can increase streptomycin tolerance in the overexpressing strain, and overexpressing OmpA can increase the intracellular accumulation of hydrophilic ethidium bromide, which indicates that porin protein OmpA contributes to aminoglycosides sensitivity in Mycobacteria.ConclusionsIn this study, we have characterized the contribution of OmpA in the antimicrobial resistance phenotype of Mycobacteria, which may provide valuable insights for understanding antibiotic resistance and designing new strategies for TB treatment.

Population-level frequency of fluoroquinolone resistance by whole-genome sequencing drug predictions in Mycobacterium tuberculosis complex isolates in England from 2017-2023.

Fluoroquinolones are an important component of anti-tuberculosis treatment and identifying fluoroquinolone resistance is essential. We present the first survey of fluoroquinolone resistance in England from sequencing of over 16,000 unselected isolates. Fluoroquinolone resistance was 1.4% overall and 23.9% in multidrug-resistant TB. Routine sequencing allows resistance surveillance and should be widely adopted.

Optimal incubation duration of liquid cultures for assessing culture negative conversion in patients with Mycobacterium avium complex and Mycobacterium abscessus pulmonary diseases.

Mycobacterial liquid culturing typically requires six weeks or longer, primarily because of the slow growth rate of Mycobacterium tuberculosis. This study aimed to evaluate the potential of shortening the duration of mycobacterial liquid culturing in healthcare settings with high prevalence rates of non-tuberculous mycobacteria. We retrospectively analyzed the relationship between mycobacterial species and time to positive testing of liquid cultures from sputum samples using the Mycobacteria Growth Indicator Tube system over a 3.5-year period beginning in July 2020 at a university hospital in Japan. We analyzed 15,147 sputum culture samples and found a 1.1% positivity rate for Mycobacterium tuberculosis complex, while the rates for Mycobacterium avium complex and Mycobacterium abscessus were 17.6% and 2.1%, respectively. The median time to positivity was 17 days for Mycobacterium tuberculosis complex, 9 days for Mycobacterium avium complex, and 4 days for Mycobacterium abscessus. Comparing a 4-week culture period with an eight-week period, the positivity rates for Mycobacterium avium complex and Mycobacterium abscessus were 97.0% and 99.4%, respectively. In settings with a high incidence of non-tuberculous mycobacteria, the basic liquid culturing period can be safely shortened to 4 weeks without significantly compromising detection sensitivity, except for the samples that are highly suspected to contain tuberculosis, extremely slow-growing mycobacteria, smear-positive, or nucleic acid amplification testing positive.

A histopathological study in road-killed European badgers (Meles meles) from the English midlands with isolation of novel non-tuberculous atypical mycobacteria

European badgers (Meles meles) play an important role in the epidemiology of bovine tuberculosis (caused by Mycobacterium bovis) in England, but little is known about the prevalence of atypical mycobacteriosis. Badgers are also known to be infected by other infectious agents, and the relationship between mycobacteriosis and concomitant infections needs further investigation. Overall, 88 badger carcasses from the Midlands of England collected between July 2016-August 2017 were selected for histopathological examination based on the degree of autolysis (mild), mycobacterial culture results and a balanced sex ratio. Mycobacteria were cultured from 44 badgers, of which 31 were mycobacteria belonging to the M. tuberculosis complex (MTBC) (based on IS6110 PCR and Hsp64 and/or rRNA PCR and sequencing) and 13 were non-tuberculous atypical mycobacteria (NTM). Mycobacteria were not cultured from the remaining 44 animals. Histologically, the most common findings were silica-laden macrophages (85%), granulomas (53%), sarcocystosis (47%), nephritis (31%), portal/periportal hepatitis (26%), ulcerative dermatitis (18%). Culturable mycobacteriosis was associated with higher prevalence of granulomas (p < 0.001) and lower prevalence of hepatitis (p = 0.003). NTM (M. nonchromogenicum, M. avium complex, M. hassiacum, M. malmoense, M. vaccae.) infections were associated with granulomatous pneumonia, and M. malmoense was associated with pyogranulomatous and ulcerative dermatitis. In conclusion, this study describes, for the first time, histological lesions associated with NTM in badgers, the histomorphology of which was similar to those caused by MTBC. In addition, the negative relationship between mycobacteriosis and periportal hepatitis may indicate a complex relationship between mycobacteriosis and other diseases, as previously observed with tuberculosis.

Evaluation of Multiplex loop-mediated isothermal amplification assay for the detection of Mycobacterium tuberculosis complex from clinically suspected cases of pulmonary tuberculosis

Tuberculosis (TB) is the second leading cause of death from a single infectious agent worldwide. Bangladesh ranks 7th among the 30 high TB burdened countries in the world. Accurate detection of Mycobacterium tuberculosis complex (MTBC) is challenging for developing countries as most of the resource poor settings are not suitable to perform molecular techniques.The purpose of the study was to compare the multiplex TB-LAMP assay with MTB/NTM qPCR, culture, Z-N staining, and fluorescence microscopy in order to assess the effectiveness of the LAMP assay for detecting cases of pulmonary tuberculosis.This research work was done from March 2022 to February 2023. Fulfilling the inclusion criteria 130 sputum samples were collected. TB-LAMP assay, qPCR, culture in L-J media, Z-N staining and fluorescence microscopy were performed.Out of 130 samples qPCR detected MTBC in 56.92 % cases and TB-LAMP detected 53.85 %. MTBC was detected by culture 46.15 %, by Fluorescence microscopy 40.77 % and Z-N staining 36.92 %. TB-LAMP detected 16.93 % more cases than Z-N staining and 13.08 % more cases than fluorescence microscopy. The sensitivity, specificity, positive, and negative predictive values of multiplex-LAMP assay were 95 %, 81.4 %, 81.4 % and 95 % respectively considering culture as a gold-standard. MTBC negative culture samples (18.57 %) showed positivity by LAMP assay as well as by qPCR. This study detected 7.69 % non-tuberculous mycobacteria (NTM) by qPCR. All NTM positive samples were negative by TB-LAMP.TB-LAMP is an easy to perform, cost-effective, reliable assay with high sensitivity and specificity. World Health Organization recommended TB-LAMP as a rapid molecular test for rapid detection of tuberculosis and as replacement of microscopy in resource poor settings/hard to reach areas. Bangladesh being a high TB burden country it is essential to implement TB-LAMP to achieve End TB Strategy by 2035.

Challenges in diagnosing bovine tuberculosis through surveillance and characterization of Mycobacterium species in slaughtered cattle in Kolkata

BackgroundTuberculosis in cattle is caused by Mycobacterium tuberculosis complex (MTBC) species. Apart from MTBC, different Nontuberculous Mycobacteria (NTM) species have also been isolated from cattle. The presence of NTM infection in bovines makes the diagnosis of bovine tuberculosis (bTB) a cumbersome task. Therefore, a cross sectional study was conducted to isolate and characterize different Mycobacterium spp. from a slaughterhouse situated in Kolkata, a city in the eastern part of India.ResultsOut of 258 morbid samples, 98 isolates were found to be positive for bacterial growth, and 35% (n = 34) were positive for Mycobacterium. 94% of Mycobacterial cultural isolates were NTM (n = 32), and the rest (n = 2) were found to be MTBC. Species-level identification of the isolates by hsp65 sequencing revealed that out of 32 isolates, 24 were M. fortuitum, three were M. abscessus, two each were M. chelonae and M. parascrofulaceum, and one was M. novocastrense. A phylogenetic tree with partial hsp65 gene sequences was also constructed to determine the relatedness of the unknown isolates to the reference strains.ConclusionBoth NTM species and MTBCs were identified from TB-like lesions in cattle that were slaughtered at the Kolkata abattoir. This discovery may indicate that NTM contributes to the development of lesions in cattle. Also, we recommend implication of more specific diagnostic tests for bTB.

Detection of Mycobacterium bovis in Free-Ranging Sapajus nigritus, Argentina.

Mycobacterium bovis and Mycobacterium tuberculosis are the most relevant among pathogenic mycobacteria, both belonging to the M. tuberculosis complex (MTC). Samples of blood, liver, spleen, kidneys, lungs and caseous tubercles were collected from a free-ranging juvenile black capuchin monkey (Sapajus nigritus) showing non-specific signs of illness. Macroscopic findings included emaciation, a caseous lesion in a tooth and gingiva, disseminated nodules in both lungs and left kidney parenchyma and caseous nodules on the pleura and mesentery. The lesions suggested MTC infection, a diagnosis subsequently supported in the lung by bacilloscopy, immunochromatography and PCR. A multiplex PCR further validated the presence of M. bovis genes. Cases of tuberculosis in platyrrhine primates have only been reported in animals maintained in captivity. We describe for the first time the pathological and molecular findings of M. bovis infection in a free-ranging platyrrhine monkey within an area of intense human-wildlife interaction, which has important implications from a One Health perspective.

Red herrings and zebras and TB, oh my! Unexpected bone marrow presentation of Mycobacterium tuberculosis

Abstract Introduction/Objective Epithelioid granulomas are uncommon and non-specific findings in bone marrow samples, and should prompt additional testing for appropriate diagnosis and treatment, especially in the immunocompromised patient. The differential diagnosis for bone marrow granulomas includes infections, neoplasia, autoimmune disease, sarcoidosis, and drug/therapy reaction. Although bone marrow workup to delineate the cause of granulomas should include staining to rule out both infection and lymphoma, morphologic review is often inconclusive. Final diagnosis requires clinical consultation and additional testing. Methods/Case Report A 76-year-old male was admitted to the hospital from the emergency department for a level 2 trauma after being found on the ground ~3 hours after a fall. The patient reported a recent increasing frequency of falls with associated weakness, as well as an almost 30 lb weight loss over the last 3.5 months and low grade fevers over several weeks. His past medical history was significant for treatment for urothelial carcinoma with transurethral resection and BCG therapy, hypertension, coronary artery disease, and peripheral vascular disease. Imaging showed hepatosplenomgaly, suspicious for early cirrhosis, and mild peripancreatic lymphadenopathy. Initial laboratory abnormalities demonstrated mild anemia, low albumin, and mild increase in ALT, AST, and alkaline phosphotase. Initial Infectious Diseases workup was negative for causative etiology (blood cultures, tick-borne parasites, and respiratory pathogens). Patient symptoms did not improve and cytopenias worsened. A bone marrow biopsy showed multiple non-necrotizing epithelioid granulomas. Despite low clinical concern for infectious etiology, mycobacteria blood culture isolated Mycobacterium tuberculosis complex. Final results were available 112 days after specimen collection. Results (if a Case Study enter NA) NA Conclusion Although tuberculosis is commonly associated with granulomatous pathology, infection is very uncommon in North American patients who have not traveled and alternative diagnoses might initially be favored. A high suspicion for mycobacterial infections is essential for diagnosis even in low-incidence populations with no known exposure or risk factor(s).

APERFEIÇOAMENTO SOBRE O RASTREIO DA TUBERCULOSE PARA OS AGENTES COMUNITÁRIOS DE SAÚDE DA ESTRATÉGIA DE SAÚDE DA FAMÍLIA DO NASCENTE I E II EM ARARIPINA-PE.

Tuberculosis (TB), a disease formerly described as typhus, was known in the 19th century as the white plague, decimating hundreds of thousands of people around the world. TB can be caused by any of the seven species that make up the Mycobacterium tuberculosis complex: M. tuberculosis, M. bovis, M. africanum, M. canetti, M. microti, M. pinnipedi and M. caprae. M. tuberculosis is transmitted by air, from a person with pulmonary or laryngeal TB, who eliminates bacilli in the environment (source case), to another person, by inhaling aerosols from coughing, talking or sneezing. Primary pulmonary TB usually occurs after the individual's first contact with the bacillus and is therefore more common in children. Its main characteristic is a dry or productive cough. Evening fever, without chills, does not usually exceed 38.5ºC. Night sweats and anorexia are common. Tuberculosis is a serious disease, but it is curable in the vast majority of cases, as long as the treatment is followed correctly. In Brazil, treatment is standardized and free of charge, provided by the National Tuberculosis Control Program (PNCT) in Health Units. In Brazil, tuberculosis treatment is standardized and consists of two phases: intensive and maintenance. The basic regimen recommended by the Ministry of Health (BRASIL, 2019) for adults and adolescents aged 10 and over includes rifampicin, isoniazid, pyrazinamide and ethambutol (RHZE). Directly Observed Treatment (DOT) is a fundamental strategy for ensuring patient adherence to tuberculosis treatment and reducing abandonment rates. Adopted by the Ministry of Health (MoH) based on WHO recommendations, DOT consists of direct observation of drug intake by health professionals. Community health workers (CHWs) are essential for tuberculosis control, from diagnosis to the outcome of cases. Their duties include Spreading the word to their community about tuberculosis as an important current public health problem. Educate the population about airborne transmission of tuberculosis and the preventive measures that can be adopted. To be aware of the main symptoms of tuberculosis (cough, fever, weight loss and night sweats) at all meetings with the community, as well as publicizing them, and referring suspected cases to the health unit.

Rifampicin-resistant Mycobacterium tuberculosis complex in Niger: A 5-year retrospective analysis (2017–2022)

Rifampicin-resistant Mycobacterium tuberculosis complex in Niger: A 5-year retrospective analysis (2017–2022)

Mycobacterium tuberculosis complex sample processing by mechanical lysis, an essential step for reliable whole genome sequencing

Mycobacterium tuberculosis complex (MTBC) whole genome sequencing (WGS) turnaround time and WGS success rates are highly influenced by DNA extraction protocols even from cultures. Efficient mycobacterial lysis is crucial for obtaining sufficient DNA from cultures to facilitate reliable genomic drug susceptibility prediction and accurate genotyping with WGS. We compared four DNA extraction protocols from BD BACTEC™ Mycobacterial Growth Indicator Tubes (MGIT) for WGS with a focus on the lysis step: protocol A) column-based protocol without mechanical lysis; protocol B) an adapted protocol including a bead beating step; protocol C) DNA extraction from primary received cultures using bead beating: and protocol D) DNA extraction from pre MGIT-positive (enriched) cultures. Protocol B increased DNA yield approximately 60-fold, and significantly improved the sequencing success rate. The increased yield also allowed DNA extraction from primary cultures with high success rates (protocol C). Additionally, by using pre-positive enriched MGIT cultures, we demonstrated that bead beating opens the possibility of reliable WGS up to five days before a MGIT tube would be flagged positive (protocol D). The most optimal bead beating-based DNA extraction was also evaluated for Nanopore sequencing. Shortening bead beating duration to 15 s resulted in longer read lengths (N50 from 1.4 kb to 2.6 kb) while still providing efficient lysis. Furthermore, AmpureXP bead beating-based DNA capture / purification proved to be as efficient as Qiagen column-based DNA extraction, further simplifying and shortening the DNA extraction protocol. Adding a mechanical lysis step to our routine MTBC DNA extraction protocol has allowed us to reduce the turnaround time while maintaining DNA quality sequencing success rates.

Bovine tuberculosis in Central Ethiopian slaughterhouses and the identification of causative mycobacteria by multiplex real-time PCR

BackgroundBovine tuberculosis (bTB) is a chronic disease caused by members of the Mycobacterium tuberculosis complex (MTBC) that ultimately leads to the development of progressive granulomatous lesions. Although the disease is widespread, especially in crossbred cattle in Ethiopia, routine investigations and surveillance are lacking. Thus, the aim of this study was to determine the prevalence, associated risk factors, and species of mycobacteria causing bTB in slaughtered cattle at four slaughterhouses in Central Ethiopia.MethodsPostmortem examination of 7,640 cattle was conducted using a cross-sectional slaughterhouse survey. A total of 388 tuberculous-like lesions (TBLs) were collected from 173 animals and cultured. Six target genes were used to differentiate mycobacterial species using multiplex real-time PCR (mRT-PCR). Multivariate logistic regression analyses and related odds ratios (ORs) were used to gauge the strength of the associations between risk factors, TBL incidence and culture growth.ResultsThe prevalence of TBL was 2.3% (95% CI = 2.0-2.6). Logistic regression analysis indicated an increased risk of TBL in crossbred cattle (OR = 11.8, 95% CI: 6.4, 21.2, p < 0.001). Animals slaughtered at Adama (OR = 3.2, 95% CI: 1.2, 7.3, p = 0.009) or Burayu (OR = 5.8, 95% CI: 3.9, 8.9, p < 0.001) had a greater risk of TBL than those slaughtered at Sululta. There were significantly more TBL-positive lesions in the lungs and lymph nodes related to the lung (OR = 7.1; 95% CI: 2.7, 24.5, p < 0.001) and the head lymph node (OR = 5.6; 95% CI: 1.8, 21.7; p = 0.006) compared to gut associated lymph nodes. Among the 173 TBL-positive animals, 36% (95% CI = 28.8, 43.2), and among the 388 TBL-positive tissues, 24.2% (95% CI = 20, 29) were culture and mRT-PCR positive. All the culture-generated isolates were positive for M. bovis in mRT-PCR. Among them, two animals had mixed infections including one zebu cattle tested positive for both M. caprae and M. bovis, and a crossbred cow tested positive for both M. tuberculosis and M. bovis in mRT-PCR. This suggests persistent transmission within the cattle population, posing a substantial public health threat.ConclusionThis study revealed an eleven-fold greater risk of bTB-related lesions in crossbred cattle compared to local zebu cattle. This finding highlights the necessity for targeted interventions, continuous vigilance, and thorough carcass inspection to mitigate public health risks.

Co-infection with Mycobacterium and 20% acid-fast Nocardia in an immunocompetent patient – A case report

Nocardiosis is an opportunistic infectious disease in immunocompromised individuals, which may be fatal and in people with healthy immune systems, which is reported as a case. Mixed infections with Nocardia and Mycobacterium tuberculosis complex (MTBC) are rare. We are reporting a case of co-infection with Nocardia spp. and MTBC in an immunocompetent patient presenting with neck swelling. Interestingly, in this case, the primary identification of Nocardia was done using 20% H2SO4 as decolorizer for Ziehl–Neelsen staining. The patient was given first-line anti-tubercular treatment for the first 2 months as an intensive phase, followed by isoniazid and rifampicin for an additional 4 months, with a total of 6 months treatment, along with linezolid (600 mg twice daily for 15 days). The patient subsequently improved.

Assessing infectiousness and the impact of effective treatment to guide isolation recommendations for people with pulmonary tuberculosis.

Determining the extent and duration of infectiousness of individuals with pulmonary tuberculosis (TB) is critical for various aspects of TB care, including decisions regarding isolation. Studies suggest considerable heterogeneity in infectiousness of people with pulmonary TB. Pre-treatment, measures of bacillary burden including sputum smear microscopy, culture time-to-positivity, and Xpert MTB/RIF cycle threshold (Ct) value, predict the risk of transmission to contacts. Index patients with smear negative disease pose lower infectious risk than those who have smear-positive disease, and household contact infection is more likely with index patients who have lower Xpert Ct values. Newer tools that enable detection of Mycobacterium tuberculosis complex (Mtb complex) from cough aerosol sampling and face mask sampling may be better predictors of contact infection risk. Clinical factors such as cough strength and frequency, and presence of cavitation on chest imaging, may also assist with risk prediction. Post-treatment, smear and culture status are poor predictors of infectiousness. While the exact duration of infectiousness post treatment initiation remains uncertain, data from human-to-guinea pig transmission studies and clinical studies suggest effective treatment results in a rapid decline in infectiousness, irrespective of smear or culture conversion. This is largely supported by early bactericidal activity and transcriptomic studies, and cough aerosol sampling studies, although a subset of patients may have persistent cough aerosol positivity. These findings can enable a more nuanced approach to isolation decision making, while further research studies are awaited.

Quantification of MDR-TB drug JBD0131 and its metabolite in plasma via UPLC-MS/MS: application in first-in-human study.

Aim: JBD0131, a novel anti-multidrug-resistant tuberculosis (MDR-TB) drug, can target and inhibit the synthesis of mycolic acids, which are crucial components of the cell wall of the Mycobacterium tuberculosis complex. To support the results of this clinical trial in healthy subjects, development of a specific and accurate quantification method for detecting JBD0131 and its metabolite DM131 in human plasma is needed.Materials & methods: Samples with prior added stabilizer were pretreated by protein precipitation method and the extracts weresubjected to ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The m/z transitions for the precursor/product ion pairs were 402.1/273 for JBD0131, 333.1/273 for DM131 and 386.1/257 for the internal standard (IS).Results: This method showed good linearity from 1 to 2000ng/ml for JBD0131 and 0.25 to 500ng/ml for DM131 and was validated in terms of selectivity, linearity, accuracy, precision, matrix effect, recovery of pretreament and stability.Conclusion: This method was sensitive and specific for measuring the plasma concentrations of JBD0131 and its metabolites. And it was applied for the investigation of the pharmacokinetics of JBD0131 and DM131 in a clinical trial.