Patterns and profiles of drug resistance-conferring mutations in Mycobacterium tuberculosis genotypes isolated from tuberculosis-suspected attendees of spiritual holy water sites in Northwest Ethiopia.

Purpose: This study examined the patterns and frequency of genetic changes responsible for resistance to first-line (rifampicin and isoniazid), fluoroquinolones, and second-line injectable drugs in drug-resistant Mycobacterium tuberculosis (MTB) isolated from culture-positive pulmonary tuberculosis (PTB) symptomatic attendees of spiritual holy water sites (HWSs) in the Amhara region. Patients and methods: From June 2019 to March 2020, a cross-sectional study was carried out. A total of 122 culture-positive MTB isolates from PTB-suspected attendees of HWSs in the Amhara region were evaluated for their drug resistance profiles, and characterized gene mutations conferring resistance to rifampicin (RIF), isoniazid (INH), fluoroquinolones (FLQs), and second-line injectable drugs (SLIDs) using GenoType®MTBDRplus VER2.0 and GenoType®MTBDRsl VER2.0. Drug-resistant MTB isolates were Spoligotyped following the manufacturer's protocol. Results: Genetic changes (mutations) responsible for resistance to RIF, INH, and FLQs were identified in 15/122 (12.3%), 20/122 (16.4%), and 5/20 (25%) of MTB isolates, respectively. In RIF-resistant, rpoB/Ser531Lue (n = 12, 80%) was most frequent followed by His526Tyr (6.7%). Amongst INH-resistant isolates, katG/Ser315Thr1 (n = 19, 95%) was the most frequent. Of 15 MDR-TB, the majority (n = 12, 80%) isolates had mutations at both rpoB/Ser531Leu and katG/Ser315Thr1. All 20 INH and/or RIF-resistant isolates were tested with the MTBDRsl VER 2.0, yielding 5 FLQs-resistant isolates with gene mutations at rpoB/Ser531Lue, katG/Ser315Thr1, and gyrA/Asp94Ala genes. Of 20 Spoligotyped drug-resistant MTB isolates, the majority (n = 11, 55%) and 6 (30%) were SIT149/T3-ETH and SIT21/CAS1-Kili sublineages, respectively; and they were any INH-resistant (mono-hetero/multi-). Of 15 RIF-resistant (RR/MDR-TB) isolates, 7 were SIT149/T3-ETH, while 6 were SIT21/CAS1-Kili sublineages. FLQ resistance was detected in four SIT21/CAS1-Kili lineages. Conclusion: In the current study, the most common gene mutations responsible for resistance to INH, RIF, and FLQs were identified. SIT149/T3-ETH and SIT21/CAS1-Kili constitute the majority of drug-resistant TB (DR-TB) isolates. To further understand the complete spectrum of genetic changes/mutations and related genotypes, a sequencing technology is warranted.

Spiritual Holy Water Sites in Ethiopia: Unrecognized High-Risk Settings for Transmission of Pulmonary Tuberculosis.

Ethiopia is a high-tuberculosis (TB) burden country with 157 new cases per 100,000 people, with 23,800 TB-related deaths in 2020. In Ethiopia, TB patients have different healthcare-seeking behaviors. They frequently visit spiritual places, such as holy water sites (HWSs), to seek treatment for their illness spiritually. This study examined the prevalence of pulmonary TB (PTB) and drug susceptibility profiles of Mycobacterium tuberculosis (MTB) isolates among spiritual HWS attendees in Northwest Ethiopia. A cross-sectional study was conducted from June 2019 to March 2020. Sputum samples were collected, processed, and cultured using Löwenstein-Jensen (LJ) culture medium. Second-generation line probe assays (LPAs), GenoType®MTBDRplus VER2.0 and GenoType®MTBDRsl VER2.0, were used to detect anti-TB drug-resistant isolates. STATA 17 was utilized to perform descriptive statistics, bivariate, and multivariate regression analyses. Of 560 PTB-symptomatic participants, 21.8% ((95% confidence interval (95 CI): 18.4-25.2%)) were culture-positive, resulting in a point prevalence of 1,183/100,000 attendees. Amongst HWS attendees, culture-positive TB occurred most commonly in persons 18-33 years of age (28.5% (95 CI 23.4-34.3%)). Other participant characteristics significantly associated with culture-positive PTB were as follows: rural residents (adjusted odds ratio (aOR) 2.65; 95 CI 1.38-5.10), married participants (aOR 2.43; 95 CI 1.28-4.63), family members >5 per household (aOR 1.84; 95 CI 1.04-3.24), and sharing living space (aOR 10.57; 95 CI 3.60-31.13). Also, among 438 participants followed for 12 months after showing negative TB culture results while at the HWS, 6.8% (95 CI 4.4-9.4%) developed or contracted culture-positive TB post-residency at the HWSs. Of the 122 tested isolates, 20 (16.4%) were isoniazid (INH) and/or rifampicin (RIF) resistant. Multidrug-resistant (MDR) TB was detected in 15 cases (12.3%), five of which were fluoroquinolones (FLQs) resistant. The findings from this study should raise a concern about HWSs as potential high-risk settings for TB transmission. It is recommended that appropriate control measures be instituted that include compulsory TB testing and tightened infection control at HWSs, where an increased risk exists for transmission of TB.

Genetic diversity of Mycobacterium tuberculosis strains isolated from spiritual holy water site attendees in Northwest Ethiopia. A cross-sectional study.

Background: The genetic diversity of Mycobacterium tuberculosis complex (MTBC) strains was characterized among isolates from individuals with pulmonary tuberculosis (PTB) symptoms attended holy water sites (HWSs) in the Amhara region, Ethiopia. Methods: A cross-sectional study was done from June 2019 to March 2020 to describe the genetic diversity and drug-resistance profiles of MTBC isolates. Sputum specimens were collected and cultured in the Löwenstein-Jensen culture medium. Line Probe Assay, MTBDRplus VER 2.0, and MTBDRsl VER 2.0 were used to detect first-and second-line anti-TB drug-resistance patterns. A spoligotyping technique was utilized to characterize the genetic diversity. Statistical analysis was performed using STATA 15. Results: Of 560 PTB-symptomatic participants, 122 (21.8%) were culture-positive cases. Spoligotyping of 116 isolates revealed diverse MTBC sublineages, with four major lineages: Euro-American (EA) (Lineage 4), East-African-Indian (EAI) (Lineage 3), Ethiopian (ETH) (Lineage 7), East Asian (EA) (Lineage 2). The majority (96.6%) of the isolates were EA (lineage 4) and EAI, with proportions of 54.3% and 42.2%, respectively. A total of 31 spoligotype patterns were identified, 26 of which were documented in the SITVIT2 database. Of these, there were 15 unique spoligotypes, while eleven were grouped with 2-17 isolates. SIT149/T3-ETH (n = 17), SIT26/CAS1-DELHI (n = 16), SIT25/CAS1-DELHI (n = 12), and SIT52/T2 (n = 11) spoligotypes were predominant. A rare spoligotype pattern: SIT41/Turkey and SIT1/Beijing, has also been identified in North Shewa. The overall clustering rate of sub-lineages with known SIT was 76.4%.Of the 122 culture-positive isolates tested, 16.4% were resistant to rifampicin (RIF) and/or isoniazid (INH). Multidrug-resistant TB (MDR-TB) was detected in 12.3% of isolates, five of which were fluoroquinolones (FLQs) resistant. SIT149/T3-ETH and SIT21/CAS1-KILI sublineages showed a higher proportion of drug resistance. Conclusions: Diverse MTBC spoligotypes were identified, with the T and CAS families and EA (lineage 4) predominating. A high prevalence of drug-resistant TB, with SIT149/T3-ETH and CAS1-KILI sublineages comprising a greater share, was observed. A study with large sample size and a sequencing method with stronger discriminatory power is warranted to understand better the genetic diversity of circulating MTBC in this cohort of study, which would help to adopt targeted interventions.

Genetic diversity of Mycobacterium tuberculosis isolates from the central, eastern and southeastern Ethiopia.

Introduction: The population structure of Mycobacterium tuberculosis complex (MTBC) in Ethiopia is diverse but dominated by Euro-American (Lineage 4) and East-African-Indian (Lineage 3) lineages. The objective of this study was to describe the genetic diversity of MTBC isolates in Central, Eastern and Southeastern Ethiopia. Methods: A total of 223 MTBC culture isolates obtained from patients referred to Adama and Harar TB reference laboratories were spoligotyped. Demographic and clinical characteristics were collected. Results: Six major lineages: Euro-American (Lineage 4), East-African-Indian (Lineage 3), East Asian (Lineage 2), Indo-Oceanic (Lineage 1), Mycobacterium africanum (Lineage 5 and Lineage 6) and Ethiopian (Lineage 7) were identified. The majority (94.6 %) of the isolates were Euro-American and East-African-Indian, with proportions of 75.3 % and 19.3 %, respectively. Overall, 77 different spoligotype patterns were identified of which 42 were registered in the SITVIT2 database. Of these, 27 spoligotypes were unique, while 15 were clustered with 2-49 isolates. SIT149/T3_ETH (n = 49), SIT53/T1 (n = 33), SIT21/CAS1_Kili (n = 24) and SIT41/Turkey (n = 11) were the dominant spoligotypes. A rare Beijing spoligotype pattern, SIT541, has also been identified in Eastern Ethiopia. The overall clustering rate of sub-lineages with known SIT was 71.3 %. Age group (25-34) was significantly associated with clustering. Conclusion: We found a heterogeneous population structure of MTBC dominated by T and CAS families, and the Euro-American lineage. The identification of the Beijing strain, particularly the rare SIT541 spoligotype in Eastern Ethiopia, warrants a heightened surveillance plan, as little is known about this genotype. A large-scale investigation utilizing a tool with superior discriminatory power, such as whole genome sequencing, is necessary to gain a thorough understanding of the genetic diversity of MTBC in the nation, which would help direct the overall control efforts.

Antimicrobial effects of Medicines for Malaria Venture Pathogen Box compounds on strains of Neisseria gonorrhoeae.

Therapeutic options for Neisseria gonorrhoeae are limited due to emerging global resistance. New agents and treatment options to treat patients with susceptible and multi-extensively drug-resistant N. gonorrhoeae is a high priority. This study used an in vitro approach to explore the antimicrobial potential, as well as synergistic effects of Medicine for Malaria Venture (MMV) Pathogen Box compounds against ATCC and clinical N. gonorrhoeae strains. Microbroth dilution assay was used to determine pathogen-specific minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the Pathogen Box compounds against susceptible and resistant N. gonorrhoeae strains, with modification, by adding PrestoBlue HS Cell Viability Reagent. A checkerboard assay was used to determine synergy between the active compounds and in conjunction with ceftriaxone. Time-kill kinetics was performed to determine if the compounds were either bactericidal or bacteriostatic. The Pathogen Box compounds: MMV676501, MMV002817, MMV688327, MMV688508, MMV024937, MMV687798 (levofloxacin), MMV021013, and MMV688978 (auranofin) showed potent activity against resistant strains of N. gonorrhoeae at an MIC and MBC of ≤10 µM. Besides the eight compounds, MMV676388 and MMV272144 were active against susceptible N. gonorrhoeae strains, also at MIC and MBC of ≤10 µM. All the compounds were bactericidal and were either synergistic or additive with fractional inhibitory concentration index ranging between 0.40 and 1.8. The study identified novel Pathogen Box compounds with potent activity against N. gonorrhoeae strains and has the potential to be further investigated as primary or adjunctive therapy to treat gonococcal infections.

Profile and Frequency of Mutations Conferring Drug-Resistant Tuberculosis in the Central, Southeastern and Eastern Ethiopia.

Purpose: Advances in molecular tools that assess genes harboring drug resistance mutations have greatly improved the detection and treatment of drug-resistant tuberculosis (DR-TB). This study was conducted to determine the frequency and type of mutations that are responsible for resistance to rifampicin (RIF), isoniazid (INH), fluoroquinolones (FLQs) and second-line injectable drugs (SLIDs) in Mycobacterium tuberculosis (MTB) isolates obtained from culture-positive pulmonary tuberculosis (TB) patients in the central, southeastern and eastern Ethiopia. Patients and Methods: In total, 224 stored culture-positive MTB isolates from pulmonary TB patients referred to Adama and Harar regional TB laboratories between August 2018 and January 2019 were assessed for mutations conferring RIF, INH, FLQs and SLIDs resistance using GenoType®MTBDRplus (MTBDRplus) and GenoType®MTBDRsl (MTBDRsl). Results: RIF, INH, FLQs and SLIDs resistance-conferring mutations were identified in 88/224 (39.3%), 85/224 (38.0%), 7/77 (9.1%), and 3/77% (3.9%) of MTB isolates, respectively. Mutation codons rpoB S531L (59.1%) for RIF, katG S315T (96.5%) for INH, gyrA A90V (42.1%) for FLQs and WT1 rrs (100%) for SLIDs were observed in the majority of the isolates tested. Over a 10th of rpoB mutations detected in the current study were unknown. Conclusion: In this study, the most common mutations conferring drug resistance to RIF, INH, FLQs were identified. However, a significant proportion of RIF-resistant isolates manifested unknown rpoB mutations. Similarly, although few in number, all SLID-resistant isolates had unknown rrs mutations. To further elucidate the entire spectrum of mutations, tool such as whole-genome sequencing is imperative. Furthermore, the expansion of molecular drug susceptibility testing services is critical for tailoring patient treatment and preventing disease transmission.

Mycobacterium tuberculosis Drug Resistance in Ethiopia: An Updated Systematic Review and Meta-Analysis.

Background: Tuberculosis (TB) remains a significant global public health issue, despite advances in diagnostic technologies, substantial global efforts, and the availability of effective chemotherapies. Mycobacterium tuberculosis, a species of pathogenic bacteria resistant to currently available anti-TB drugs, is on the rise, threatening national and international TB-control efforts. This systematic review and meta-analysis aims to estimate the pooled prevalence of drug-resistant TB (DR-TB) in Ethiopia. Materialsand Methods: A systematic literature search was undertaken using PubMed/MEDLINE, HINARI, the Web of Science, ScienceDirect electronic databases, and Google Scholar (1 January 2011 to 30 November 2020). After cleaning and sorting the records, the data were analyzed using STATA 11. The study outcomes revealed the weighted pooled prevalence of any anti-tuberculosis drug resistance, any isoniazid (INH) and rifampicin (RIF) resistance, monoresistance to INH and RIF, and multidrug-resistant TB (MDR-TB) in newly diagnosed and previously treated patients with TB. Results: A total of 24 studies with 18,908 patients with TB were included in the final analysis. The weighted pooled prevalence of any anti-TB drug resistance was 14.25% (95% confidence interval (CI): 7.05-21.44%)), whereas the pooled prevalence of any INH and RIF resistance was found in 15.62% (95%CI: 6.77-24.47%) and 9.75% (95%CI: 4.69-14.82%) of patients with TB, respectively. The pooled prevalence for INH and RIF-monoresistance was 6.23% (95%CI: 4.44-8.02%) and 2.33% (95%CI: 1.00-3.66%), respectively. MDR-TB was detected in 2.64% (95%CI: 1.46-3.82%) of newly diagnosed cases and 11.54% (95%CI: 2.12-20.96%) of retreated patients with TB, while the overall pooled prevalence of MDR-TB was 10.78% (95%CI: 4.74-16.83%). Conclusions: In Ethiopia, anti-tuberculosis drug resistance is widespread. The estimated pooled prevalence of INH and RIF-monoresistance rates were significantly higher in this review than in previous reports. Moreover, MDR-TB in newly diagnosed cases remained strong. Thus, early detection of TB cases, drug-resistance testing, proper and timely treatment, and diligent follow-up of TB patients all contribute to the improvement of DR-TB management and prevention. Besides this, we urge that a robust, routine laboratory-based drug-resistance surveillance system be implemented in the country.

Prevalence of drug resistance-conferring mutations associated with isoniazid- and rifampicin-resistant Mycobacterium tuberculosis in Ethiopia: a systematic review and meta-analysis.

OBJECTIVES: Globally, the incidence and mortality of tuberculosis (TB) are declining; however, low detection of drug-resistant disease threatens to reverse current progress toward global TB control. Multiple rapid molecular diagnostic tests have recently been developed to detect genetic mutations in Mycobacterium tuberculosis (Mtb) known to confer drug resistance. However, their utility depends on the frequency and distribution of resistance-associated mutations in the pathogen population. This review aimed to assess the prevalence of gene mutations associated with rifampicin (RIF)- and isoniazid (INH)-resistant Mtb in Ethiopia. METHODS: We searched the literature in PubMed/MEDLINE, Web of Science, Scopus and Cochrane Library. Data analysis was conducted in Stata 11. RESULTS: Totally, 909 (95.8%) of 949 INH-resistant Mtb isolates had detectable gene mutations: 95.8% in katG315 and 5.9% in the inhA promoter region. Meta-analysis resulted in an estimated pooled prevalence of katGMUT1(S315T1) of 89.2% (95% CI 81.94-96.43%) and a pooled prevalence of inhAMUT1(C15T) of 77.5% (95% CI 57.84-97.13%). Moreover, 769 (90.8%) of 847 RIF-resistant strains had detectable rpoB gene mutations. Meta-analysis resulted in a pooled prevalence of rpoBMUT3(S531L) of 74.2% (95% CI 66.39-82.00%). CONCLUSION: RIF-resistant Mtb were widespread, particularly those harbouring rpoB(S531L) mutation. Similarly, INH-resistant Mtb with katG(S315T1) and inhA(C15T) mutations were common. Tracking S531L, S315T1 and C15T mutations among RIF- and INH-resistant isolates, respectively, would be diagnostically and epidemiologically valuable. Rapid diagnosis of RIF- and INH-resistant Mtb would expedite modification of TB treatment regimens, and proper timely infection control interventions could reduce the risk of development and transmission of multidrug-resistant TB.

Molecular detection of Mycobacterium tuberculosis in poor-quality cough specimens.

Clinical specimens unfit for laboratory processing represent missed opportunities for diagnosing tuberculosis. Poor-quality cough specimens (n=61) from presumptive tuberculosis cases were cultured and GeneXpert MTB/RIF (Xpert) successfully performed on samples transferred by flocked swab into PrimeStore molecular transport medium (PS-MTM). Mycobacterium tuberculosis was grown in culture from 13 (21.3 %) and Xpert reported 15 (24.2 %) positive, of which 10 concordant. RT-PCR of PS-MTM samples showed enhanced sensitivity; three positives were missed by Xpert, five by culture and three more detected for a total of 21 positives (34.4 %).

Erratum to "Opsonic monoclonal antibodies enhance phagocytic killing activity and clearance of Mycobacterium tuberculosis from blood in a quantitative qPCR mouse model" [Heliyon 5 (9), (September 2019), e02260].

[This corrects the article DOI: 10.1016/j.heliyon.2019.e02260.].

Opsonic monoclonal antibodies enhance phagocytic killing activity and clearance of Mycobacterium tuberculosis from blood in a quantitative qPCR mouse model.

BACKGROUND: Patients with impaired immunity often have rapid progression of tuberculosis (TB) which can lead to highly lethal Mycobacterium tuberculosis (MTB) sepsis. Opsonic monoclonal antibodies (MABs) directed against MTB that enhance phagocytic killing activity and clearance of MTB from blood may be useful to enhance TB immunity. METHODS: BALB/c mice were immunized with ethanol-killed MTB (EK-MTB) and MABs were produced and screened by ELISA for binding to killed and live Mycobacterium smegmatis (SMEG) and MTB. MAB opsonophagocytic killing activity (OPKA) was examined using SMEG with HL60 and U-937 cells and MTB with U-937 cells. Clearance of MTB from blood was evaluated in Institute of Cancer Research (ICR) mice given opsonic anti-MTB MABs or saline (control) 24 h prior to intravenous infusion with 108 CFUs gamma-irradiated MTB (HN878). MTB levels in murine blood collected 0.25, 4 and 24 h post-challenge were assessed by qPCR. MAB binding to peptidoglycan (PGN) was examined by ELISA using PGN cell wall mixture and ultra-pure PGN. RESULTS: Two MABs (GG9 and JG7) bound to killed and live SMEG and MTB (susceptible and resistant), and promoted OPKA with live MTB. MAB JG7 significantly enhanced OPKA of MTB. Both MABs significantly enhanced clearance of killed MTB from murine blood at 4 and 24 h as measured by qPCR. These opsonic MABs bound to PGN, a major cell wall constituent. CONCLUSIONS: Anti-MTB MABs that promote bactericidal phagocytic activity of MTB and enhance clearance of killed MTB from the blood, may offer an immunotherapeutic approach for treatment of MTB bacteremia or sepsis, and augment treatment of multi-drug resistant (MDR) or extensively drug resistant (XDR) TB.

Multi- and Extensively Drug Resistant Mycobacterium tuberculosis in South Africa: a Molecular Analysis of Historical Isolates.

Modern advances in genomics provide an opportunity to reinterpret historical bacterial culture collections. In this study, genotypic antibiotic resistance profiles of Mycobacterium tuberculosis isolates from a historical 20-year-old multidrug-resistant tuberculosis (MDR-TB) culture collection in South Africa are described. DNA samples extracted from the phenotypically MDR-TB isolates (n = 240) were assayed by Hain line probe assay (LPA) for the confirmation of MDR-TB and by Illumina Miseq whole-genome sequencing (WGS) for the characterization of mutations in eight genes (rpoB, katG, inhA, rpsL, pncA, embB, gyrA, and rrs) that are known to code for resistance to commonly used anti-TB agents. LPA identified 71.3% of the TB isolates as MDR-TB, 18.3% as rifampin (RIF) monoresistant, 2% as isoniazid (INH) monoresistant, and 8.3% as susceptible to both RIF and INH (RIF+INH). In a subset of 42 randomly selected isolates designated as RIF+INH resistant by Löwenstein-Jensen (LJ) culture in 1993, LPA and WGS results confirmed MDR-TB. In all five INH-monoresistant isolates by LPA and in all but one (the wild type) of the 34 successfully sequenced RIF-monoresistant isolates, WGS revealed matching mutations. Only 26% of isolates designated as susceptible by LPA, however, were found to be wild type by WGS. Novel mutations were found in the rpoB (Thr480Ala, Gln253Arg, Val249Met, Val251Tyr, Val251Phe), katG (Trp477STOP, Gln88STOP, Trp198STOP, Trp412STOP), embB (Thr11Xaa, Gln59Pro), and pncA (Thr100Ile, Thr159Ala, Ala134Arg, Val163Ala, Thr153Ile, DelGpos7, Phe106Ser) genes. Three MDR-TB isolates showed mutations in both the gyrA and rrs genes, suggesting that extensively drug-resistant tuberculosis existed in South Africa well before its formal recognition in 2006.

Field evaluation of a novel preservation medium to transport sputum specimens for molecular detection of Mycobacterium tuberculosis in a rural African setting.

OBJECTIVES: To assess the performance of an innovative method of transporting sputum to centralised facilities for molecular detection of Mycobacterium tuberculosis: using a swab to inoculate sputum in a transport medium, PrimeStore(®) Molecular Transport Medium (PS-MTM). METHODS: Two sputum specimens were obtained from suspected patients with tuberculosis (TB) at rural healthcare facilities in South Africa. A swab was taken from each specimen and placed into PS-MTM, prior to it being processed by either liquid culture or Xpert MTB/RIF assay (Xpert). RESULTS: A total of 141 patients (including 47 with laboratory-confirmed TB) were included in this analysis. M. tuberculosis was detected at 29% by culture and 29% by Xpert, whereas 31% tested positive by IS6110 real-time PCR of PS-MTM from the culture and 36% from the Xpert-paired specimen. Concordance between the method under evaluation with culture was 82% (McNemar, P = 0.55) and 84% (McNemar, P = 0.05) for Xpert. Stratified by culture result, the detection rate by IS6110 real-time PCR of PS-MTM was similar to Xpert for patients with positive culture (P = 0.32), but significantly higher if culture was negative (P = 0.008). CONCLUSIONS: These results suggest that swab collection of sputum into PS-MTM for transport is a promising method for diagnosis of TB in rural healthcare settings, thereby potentially improving the options available for molecular diagnosis of TB in countries incapable of applying decentralised high-tech molecular testing.

Population pharmacokinetic drug-drug interaction pooled analysis of existing data for rifabutin and HIV PIs.

OBJECTIVES: Extensive but fragmented data from existing studies were used to describe the drug-drug interaction between rifabutin and HIV PIs and predict doses achieving recommended therapeutic exposure for rifabutin in patients with HIV-associated TB, with concurrently administered PIs. METHODS: Individual-level data from 13 published studies were pooled and a population analysis approach was used to develop a pharmacokinetic model for rifabutin, its main active metabolite 25-O-desacetyl rifabutin (des-rifabutin) and drug-drug interaction with PIs in healthy volunteers and patients who had HIV and TB (TB/HIV). RESULTS: Key parameters of rifabutin affected by drug-drug interaction in TB/HIV were clearance to routes other than des-rifabutin (reduced by 76%-100%), formation of the metabolite (increased by 224% in patients), volume of distribution (increased by 606%) and distribution to the peripheral compartment (reduced by 47%). For des-rifabutin, clearance was reduced by 35%-76% and volume of distribution increased by 67%-240% in TB/HIV. These changes resulted in overall increased exposure to rifabutin in TB/HIV patients by 210% because of the effects of PIs and 280% with ritonavir-boosted PIs. CONCLUSIONS: Given together with non-boosted or ritonavir-boosted PIs, rifabutin at 150 mg once daily results in similar or higher exposure compared with rifabutin at 300 mg once daily without concomitant PIs and may achieve peak concentrations within an acceptable therapeutic range. Although 300 mg of rifabutin every 3 days with boosted PI achieves an average equivalent exposure, intermittent doses of rifamycins are not supported by current guidelines.

Improved Detection by Next-Generation Sequencing of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates.

The technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising the pncA coding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolates from an archived series collected in 2001. These 88 isolates were previously Sanger sequenced, with 55 (61%) designated as carrying the wild-type pncA gene and 33 (37%) showing mutations. PZA susceptibility of the isolates was also determined using the Bactec 460 TB system and the Wayne test. In this study, isolates were recultured and susceptibility testing was performed in Bactec 960 MGIT. Concordance between NGS and MGIT results was 93% (n = 88), and concordance values between the Bactec 460, the Wayne test, or pncA gene Sanger sequencing and NGS results were 82% (n = 88), 83% (n = 88), and 89% (n = 88), respectively. NGS confirmed the majority of pncA mutations detected by Sanger sequencing but revealed several new and mixed-strain mutations that resolved discordancy in other phenotypic results. Importantly, in 53% (18/34) of these isolates, pncA mutations were located in the 151 to 360 region and warrant further exploration. In these isolates, with their known resistance to rifampin, NGS of pncA improved PZA resistance detection sensitivity to 97% and specificity to 94% using NGS as the gold standard and helped to resolve discordant results from conventional methodologies.

Laboratory evaluation of a specimen transport medium for downstream molecular processing of sputum samples to detect Mycobacterium tuberculosis.

BACKGROUND: Modern molecular-based approaches for the detection of Mycobacterium tuberculosis in sputum samples promise quicker and more accurate detection of cases. However, processing sputum samples at central diagnostic facilities provides a diagnostic approach, but requires a safe and efficient system that is not affected by transport delays and ambient temperature to be feasible. We evaluated the technical properties of PrimeStore®-Molecular Transport Medium (PS-MTM) for its ability to inactivate mycobacteria, ensuring stability of DNA over time at ambient temperatures and to assess the compatibility of the transport medium with DNA extraction systems. METHODS: Assessment of the transport medium for application of sputum samples processed for the detection of M. tuberculosis included the inactivation of M. tuberculosis in spiked sputum samples, compatibility of the medium with three commercial nucleic extraction systems and stability of DNA in the medium at ambient temperature over 28 days. We further performed a clinical laboratory evaluation on 256 sputum specimens sent for tuberculosis investigation. RESULTS: Complete inactivation of M. tuberculosis occurred within 30 min of exposure at a ratio of 1:3 for sputum to PS-MTM. Sputum specimen in PS-MTM showed very good compatibility with automated bead-based extraction systems, producing high DNA output (estimated lower limits of detection: ~170 CFU/ml). Furthermore, PS-MTM samples remained stable over 28 days at ambient temperature displaying no significant change over time in Ct-values (<5% on a mean starting value of 22.47). Of the 256 clinical sputum specimens, 10.2% were culture positive and 11.0% were positive by real-time PCR of PS-MTM samples. CONCLUSIONS: Collecting and transporting sputum from TB suspects in PS-MTM offer safe transport at ambient temperature, DNA stability for extended periods without cooling and specimens directly suitable for molecular testing. This novel approach may support introduction and further scale-up of molecular diagnostics for TB in resource-limited settings.

Novel licensure pathways for expeditious introduction of new tuberculosis vaccines: a discussion of the adaptive licensure concept.

The ultimate goal of vaccine development is licensure of a safe and efficacious product that has a well-defined manufacturing process resulting in a high quality product. In general, clinical development and regulatory approval occurs in a linear, sequential manner: Phase 1 - safety, immunogenicity; Phase 2 - immunogenicity, safety, dose ranging and preliminary efficacy; Phase 3 - definitive efficacy, safety, lot consistency; and, following regulatory approval, Phase 4 - post-marketing safety and effectiveness. For candidate TB vaccines, where correlates of protection are not yet identified, phase 2 and 3 efficacy of disease prevention trials are, by necessity, very large. Each trial would span 2-5 years, with full licensure expected only after 1 or even 2 decades of development. Given the urgent unmet need for a new TB vaccine, a satellite discussion was held at the International African Vaccinology Conference in Cape Town, South Africa in November 2012, to explore the possibility of expediting licensure by use of an "adaptive licensure" process, based on a risk/benefit assessment that is specific to regional needs informed by epidemiology. This may be appropriate for diseases such as TB, where high rates of morbidity, mortality, particularly in high disease burden countries, impose an urgent need for disease prevention. The discussion focused on two contexts: licensure within the South African regulatory environment - a high burden country where TB vaccine efficacy trials are on-going, and licensure by the United States FDA --a well-resourced regulatory agency where approval could facilitate global licensure of a novel TB vaccine.

Persister populations of Mycobacterium tuberculosis in sputum that grow in liquid but not on solid culture media.

OBJECTIVES: Can the characteristics of persisters in cultures of Mycobacterium tuberculosis also be found in bacilli from the sputum of pulmonary tuberculosis patients? The objective of this study was to explore whether the ability of persisters to grow in liquid but not on solid culture media, as in 100 day static cultures, can also be found in bacilli in sputum. METHODS: Serial dilutions of homogenized sputum obtained from patients before or during the first week of treatment were inoculated into broths to estimate the probable number of organisms and onto plates to give colony counts. RESULTS: Cultures in broths grew slowly to reach a maximal count at 12 weeks of probable numbers about 10-fold higher than the colony counts on plates, which did not grow beyond the initial count at 3-4 weeks. No such excess growth in liquid medium was found with control log-phase cultures. CONCLUSIONS: About 90% of the bacilli in sputum are persisters that can grow in liquid media but not on solid plates.

Dry powder antibiotic aerosol product development: inhaled therapy for tuberculosis.

Inhaled therapies offer a unique approach to the treatment of tuberculosis (TB) using a relevant target organ system as a route of administration. The number of research reports on this topic has been increasing exponentially in the last decade but studies of clinical efficacy have been rare in recent times. The challenge is to take many research findings and translate them into a strategy for product development. Dry powder inhalers are the dominant drug product under consideration by those interested in the inhaled therapy for TB. A range of factors including candidate drug, formulation, device selection, drug product testing for proof of concept, and preclinical and clinical purposes all demand different considerations. The following review is intended to raise awareness of a growing body of evidence, suggesting that inhaled therapy for TB is possible and desirable. In addition, it is intended to outline key elements of the product-development activity for this particular application that has not been discussed elsewhere in the literature. Hopefully, this will encourage those with development expertise to seriously contemplate the steps required to bring such products forward.