Predicting Extensively Drug-Resistant Mycobacterium tuberculosis Phenotypes with Genetic Mutations

Molecular diagnostic methods based on the detection of mutations conferring drug resistance are promising technologies for rapidly detecting multidrug-/extensively drug-resistant tuberculosis (M/XDR TB), but large studies of mutations as markers of resistance are rare. The Global Consortium for Drug-Resistant TB Diagnostics analyzed 417 Mycobacterium tuberculosis isolates from multinational sites with a high prevalence of drug resistance to determine the sensitivities and specificities of mutations associated with M/XDR TB to inform the development of rapid diagnostic methods. We collected M/XDR TB isolates from regions of high TB burden in India, Moldova, the Philippines, and South Africa. The isolates underwent standardized phenotypic drug susceptibility testing (DST) to isoniazid (INH), rifampin (RIF), moxifloxacin (MOX), ofloxacin (OFX), amikacin (AMK), kanamycin (KAN), and capreomycin (CAP) using MGIT 960 and WHO-recommended critical concentrations. Eight genes (katG, inhA, rpoB, gyrA, gyrB, rrs, eis, and tlyA) were sequenced using Sanger sequencing. Three hundred seventy isolates were INH^r, 356 were RIF^r, 292 were MOX^r/OFX^r, 230 were AMK^r, 219 were CAP^r, and 286 were KAN^r. Four single nucleotide polymorphisms (SNPs) in katG/inhA had a combined sensitivity of 96% and specificities of 97 to 100% for the detection of INH^r. Eleven SNPs in rpoB had a combined sensitivity of 98% for RIF^r. Eight SNPs in gyrA codons 88 to 94 had sensitivities of 90% for MOX^r/OFX^r. The rrs 1401/1484 SNPs had 89 to 90% sensitivity for detecting AMK^r/CAP^r but 71% sensitivity for KAN^r. Adding eis promoter SNPs increased the sensitivity to 93% for detecting AMK^r and to 91% for detecting KAN^r. Approximately 30 SNPs in six genes predicted clinically relevant XDR-TB phenotypes with 90 to 98% sensitivity and almost 100% specificity.     

Molecular and phenotypic characterization of multidrug-resistant Mycobacterium tuberculosis isolates resistant to kanamycin,amikacin, and capreomycin in China

Although second-line anti-tuberculosis (TB) injectable drugs have been widely used to improve treatment outcomes of multidrug-resistant TB (MDR-TB), little is known about the prevalence and mechanism of second-line injectable drug resistance among MDR Mycobacterium tuberculosis isolates in China. Here, we found that 12.7 % (20/158) of isolates showed resistance to at least one second-line injectable drug among 158 MDR isolates. At the same time, there were 16 (10.1 %) strains resistant to kanamycin (KAN), 9 (5.7 %) to amikacin (AMK), and 12 (7.6 %) to capreomycin (CAP). In addition, our data revealed no significant difference in the drug resistance patterns for Beijing versus non-Beijing genotype strains (p?>?0.05). The most frequently observed mutation was A-to-G substitution at position 1401 of the rrs gene, conferring high-level resistance to KAN and AMK, but had varying minimum inhibitory concentrations (MICs) for CAP. The mutations in the eis promoter and tlyA gene were responsible for low-level resistance to CAP. 83.3 % of A1401G substitutions in the rrs gene was observed in Beijing genotype strains, while the difference was not significant (p?=?0.157). Our data demonstrated that the hot-spot regions localized in the rrs gene serve as excellent markers for AMK, but is not a sensitive marker for KAN and CAP. In addition, the cross-resistance patterns and MICs differed among different genetic mutation types, which challenge the practice in China of generalizing resistance to AMK and CAP based on the resistance to KAN alone. Our findings suggested that the individualized drug susceptibility to three major second-line injectable drugs is essential in order to generate more effective treatment regimens for MDR patients.     

Production of transmitochondrial mouse cell lines by cybrid rescue of rhodamine-6G pre-treated L-cells

Treatment of mouse LMTK⁻ cells with the toxic mitochondrial dye rhodamine 6G (R-6G) at 2.5 μg/ml for 7 days prevented cell growth while maintaining viability, with less than 10⁻⁶ cells recovering to form colonies. Pre-treatment of LMTK⁻ cells with R-6G was followed by fusion with enucleated mouse 501–1 cells harboring a homoplasmic point mutation in the mitochondrial DNA (mtDNA) 16S rRNA gene conferring chloramphenicol resistance (CAP^R). Cybrids and any surviving unfused LMTK⁻ cells were selected in BrdU with or without CAP and their mtDNAs screened for the presence of the CAP^R marker. Approximately 1 colony per 2×10⁵ LMTK⁻ cells appeared in the fusion plates selected both with and without CAP. Most clones investigated were confirmed to be cybrids by showing the presence of the generally homoplasmic CAP^R mutation, whether or not CAP selection was used. Hence, R-6G pre-treatment permits construction of transmitochondrial cybrid cell lines carrying a variety of mtDNAs, without the need for ρ^o cell lines.     

Mitochondrial DNA analysis of mouse-rat hybrid cells: Effect of chloramphenicol selection on the relative amounts of parental mitochondrial DNAs

Several mouse-rat somatic hybrid cell lines were isolated by fusing chloramphenicol-resistant (CAP^R) and CAP-sensitive (CAP^S) parent cells, and propagation of the parent mitochondrial DNA (mtDNA) species in the hybrid cells was studied. The restriction endonucleases EcoRI, HpaII, and HaeIII were used for identification of mtDNA species. Both mouse and rat mtDNAs were propagated in all the hybrid cells examined and maintained during long-term cultivation and repeated cell division. Moreover, in CAP^R mouse-rat hybrid cells, selection and successive cultivation in the presence of CAP did not increase the relative amount of mtDNA species of CAP^R parent cell origin, and when CAP was removed from the culture medium, mtDNA species of CAP^R parent cell origin did not decrease appreciably. The amount of mouse mtDNAs was consistently 1–4 times that of rat mtDNAs in the mouse-rat hybrid cells regardless of the species of parent cells from which the CAP resistance was derived. Thus mouse-rat hybrid cells have a stable mtDNA population in which the amount of mouse mtDNAs is larger than that of rat mtDNAs without any influence of CAP selection.     

Resistance Sniffer: An online tool for prediction of drug resistance patterns of Mycobacterium tuberculosis isolates using next generation sequencing data

The effective control of multidrug resistant tuberculosis (MDR-TB) relies upon the timely diagnosis and correct treatment of all tuberculosis cases. Whole genome sequencing (WGS) has great potential as a method for the rapid diagnosis of drug resistant Mycobacterium tuberculosis (Mtb) isolates. This method overcomes most of the problems that are associated with current phenotypic drug susceptibility testing. However, the application of WGS in the clinical setting has been deterred by data complexities and skill requirements for implementing the technologies as well as clinical interpretation of the next generation sequencing (NGS) data. The proposed diagnostic application was drawn upon recent discoveries of patterns of Mtb clade-specific genetic polymorphisms associated with antibiotic resistance. A catalogue of genetic determinants of resistance to thirteen anti-TB drugs for each phylogenetic clade was created. A computational algorithm for the identification of states of diagnostic polymorphisms was implemented as an online software tool, Resistance Sniffer (http://resistance-sniffer.bi.up.ac.za/), and as a stand-alone software tool to predict drug resistance in Mtb isolates using complete or partial genome datasets in different file formats including raw Illumina fastq read files. The program was validated on sequenced Mtb isolates with data on antibiotic resistance trials available from GMTV database and from the TB Platform of South African Medical Research Council (SAMRC), Pretoria. The program proved to be suitable for probabilistic prediction of drug resistance profiles of individual strains and large sequence data sets.     

Spontaneous Latency in a Rabbit Model of Pulmonary Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is an exquisitely adapted human pathogen capable of surviving for decades in the lungs of immune-competent individuals in the absence of disease. The World Health Organization estimates that 2 billion people have latent TB infection (LTBI), defined by a positive immunological response to Mtb antigens, with no clinical signs of disease. A better understanding of host and pathogen determinants of LTBI and subsequent reactivation would benefit TB control efforts. Animal models of LTBI have been hampered generally by an inability to achieve complete bacillary clearance. Herein, we have characterized a rabbit model of LTBI in which, similar to most humans, complete clearance of pulmonary Mtb infection and pathological characteristics occurs spontaneously. The evidence that Mtb-CDC1551–infected rabbits achieve LTBI, rather than sterilization, is based on the ability of the bacilli to be reactivated after immune suppression. These rabbits showed early activation of T cells and macrophages and an early peak in the TNFα level, which decreased in association with clearance of bacilli from the lungs. In the absence of sustained tumor necrosis factor-α production, no necrosis was seen in the evolving lung granulomas. In addition, bacillary control was associated with down-regulation of several metalloprotease genes and an absence of lung fibrosis. This model will be used to characterize molecular markers of protective immunity and reactivation.Tuberculosis (TB) has been declared a global public health emergency, accounting for 8.8 million new cases and 1.1 million deaths among HIV-negative people in 2010.¹ In addition to those with known active disease, the World Health Organization has estimated that >2 billion people are latently infected with the causative agent, Mycobacterium tuberculosis (Mtb).² Individuals with latent TB infection (LTBI) can experience reactivation, leading to active infectious TB later in life, and, thus, pose a huge reservoir of potential new TB cases and additional sources of Mtb infection. These estimates are based on immunological tests, in which LTBI is defined as T-cell recognition of Mtb antigens, determined by either a skin test reaction to Mtb purified protein derivative (PPD) or an in vitro interferon gamma blood assay, in the absence of any clinical signs of disease. However, it is not clear whether all individuals diagnosed as having LTBI actually harbor viable Mtb.³ Autopsy studies have confirmed the presence of viable bacilli in the tissues of individuals who died of other causes, with no known history of TB, but these reports are limited.^4–6 Although individuals with LTBI have only a 5% to 10% lifetime risk of reactivating the infection, the odds are significantly higher with immune compromise, such as that due to HIV infection, diabetes mellitus, or treatment with immune-suppressive drugs. Unfortunately, we presently have no means of identifying which individuals are at greatest risk of reactivating infection. TB control efforts would benefit from a better understanding of the driving forces leading to establishment and maintenance of latency and an ability to predict which individuals are more likely to reactivate LTBI. Recent studies suggest that LTBI is not a single manifestation. Rather, they suggest that TB exists as a spectrum of disease, ranging from the most severe disseminated forms of disease to classic pulmonary TB, then to subclinical active infection, and, finally, to true LTBI, in which there are no clinical signs of active infection or disease.^5–7The outcome of Mtb pulmonary infection is determined by both host and pathogen factors. In addition to host immune compromise, several studies have implicated various genetic polymorphisms in association with resistance or susceptibility to TB in humans.^8–11 On the pathogen side, numerous studies have demonstrated reproducible differences in virulence and/or immunogenicity induced by different clinical Mtb strains.^12–14 Although some studies have compared the host response with large collections of clinical strains, other investigators have taken a more focused approach, using a limited number of strains for more in-depth comparative studies. For example, clinical Mtb strain CDC1551 elicits an early and robust host immune response in mice and in human monocytes, compared with the laboratory strain H37Rv and the clinical strain HN878, a member of the W-Beijing lineage.^15,16 Mtb strain-specific differences in virulence and immunogenicity have been particularly strikingly demonstrated in the rabbit TB infection model.^17–19 By using a rabbit model of TB meningitis, we showed that Mtb CDC1551 infection is more effectively controlled, resulting in lower bacterial colony-forming units (CFUs) in the cerebrospinal fluid and brain, with minimal dissemination to other organs, compared with infection by Mtb HN878, which causes severe pathological characteristics and more dissemination.²⁰ Similarly, we and others have shown significant Mtb strain-specific differences in the ability of rabbits to control bacillary growth and pathological characteristics after pulmonary infection. In general, although Mtb CDC1551 infection appears to be relatively well controlled, the rabbit is more permissive for growth and dissemination of Mtb HN878; Mtb H37Rv infection produces an intermediate response.^17,18,21Several animal models, including mice, guinea pigs, rats, rabbits, and nonhuman primates, have been used to study host and bacterial factors that contribute to the establishment and maintenance of LTBI and reactivation.^22–24 However, none of the published reports from these models has demonstrated reproducible and complete clearance of culturable bacilli from host tissues. Even in the nonhuman primate model, considered the closest to human TB, although approximately 50% of Mtb-infected animals spontaneously develop LTBI, as defined by clinical criteria, it is not possible to predict which animals will control the infection. In addition, subsets of the animals that are defined clinically as latently infected retain many cultivable Mtb in their lungs, lymph nodes, or other tissues.^25,26 This distinction is important because, in most reports of human autopsy studies, Mtb was not directly cultivable from the lungs, but it had to be identified by either an ability to cause disease in guinea pigs or PCR determination of Mtb DNA.²⁷Herein, we have used aerosol infection of rabbits with Mtb CDC1551 to determine whether spontaneous LTBI with complete clearance of cultivable bacilli in the lungs can be established and to evaluate lymphocyte activation and lung pathogenesis during the infection. Furthermore, we have immune suppressed infected rabbits by treatment with corticosteroid to ascertain that the absence of cultivable Mtb in the lungs was not sterilization, but true LTBI.     

Human cell variants resistant to methylglyoxal-bis(guanylhydrazone) display increased sensitivity to chloramphenicol

Four variants of the cultured human cell line VA₂ have been isolated which are resistant to the antiproliferative and antimitochondrial effects of methylglyoxal-bis(guanylhydrazone) (MGBG). Each of the four variants is two-to fivefold more sensitive to the mitochondrial protein synthesis inhibitor chloramphenicol (CAP) than wild type when grown in the absence of MGBG, and five-to tenfold more sensitive to CAP when grown in the presence of MGBG. Uptake studies demonstrate that each MGBG-resistant variant cell line is freely permeable to CAP. The in vivo rates of mitochondrial protein synthesis are significantly reduced in each of the variants whether pregrown and labeled in the presence or absence of MGBG. When cytoplasts from a cytoplasmically inherited CAP-resistant mutant are fused to an MGBG-resistant recipient cell line, cybrid clones can be isolated which are functionally resistant to low levels of CAP. With continued growth, the levels of resistance to CAP do not, however, approach the levels of resistance of the CAP-resistant donor cell line. When CAP resistance is subsequently transferred from a CAP/MGBG-resistant cybrid by enucleation and fusion to other human cell lines, then CAP-resistant cybrids can be readily selected in high levels of CAP. It is possible that the substantial decrease in mitochondrial protein synthesis observed in the variants fully accounts for their increased sensitivity to CAP, although the basis for this decreased rate of mitochondrial protein synthesis is not understood.     

Induction of supermelanin synthesis and morphological changes in interspecific reconstituted cells and its reversal by tumor promoter

Chloramphenicol-resistant (CAP _r) reconstituted cells and cybrids were isolated by fusion of karyoplasts (or intact cells) of mouse amelanotic melanoma B₁₆ cells with cytoplasts of hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient, CAP_r rat myoblastic cells, L₆TG.CAP_r, and double selection in HAT medium containing CAP. Reconstituted cells or cybrids exhibited unique cellular arrangement, and about one third of the isolated clones expressed high tyrosinase activity and marked melanin synthesis, although the parental mouse cells expressed low tyrosinase activity and the parental rat cells did not express tyrosinase activity. These phenotypic changes have been stable for more than a year. The phenotypic reversions of these clonal cells were induced by treatment with a tumor promoter. There were changes in the morphology of the treated cells to that of the mouse B₁₆ cells and extinction of tyrosinase activity and melanin synthesis in pigmented clonal cells. These phenotypic changes and reversions induced by a promoter were repeatedly reversible.     

Mitotic segregation of cytoplasmic determinants for chloramphenicol resistance in mammalian cells I: Fusions with mouse cell lines

The segregation of cytoplasmically inherited chloramphenicol (CAP) resistance in mouse cells was investigated in fusions between CAP-resistant cells or cytoplasts (enucleated cells) and CAP-sensitive cells of varying tissue origin. All hybrids formed in cell-cell fusions were initially CAP-resistant, indicating that CAP resistance is dominant. Hybrids from fusions of cells of the same tissue origin (homologous) were stably CAP-resistant, whereas the hybrid population from fusions of different origins (heterologous) showed a rapid diminution of average CAP resistance. Individual hybrid clones from these heterologous fusions also showed an overall loss of CAP resistance, and a wide variation in CAP resistance which is consistent with a large number of genetic determinants (possibly mitochondrial DNA molecules) contributing to the CAP phenotype. Similar results were obtained from cytoplast-cell fusions, so the observed CAP segregation is not the result of nuclear-nuclear interactions. This segregation of CAP resistance constitutes a second criterion of cytoplasmic inheritance in mammalian cells.     

Genomic Epidemiology and Molecular Resistance Mechanisms of Azithromycin-Resistant Neisseria gonorrhoeae in Canada from 1997 to 2014

The emergence of Neisseria gonorrhoeae strains with decreased susceptibility to cephalosporins and azithromycin (AZM) resistance (AZM^r) represents a public health threat of untreatable gonorrhea infections. Genomic epidemiology through whole-genome sequencing was used to describe the emergence, dissemination, and spread of AZM^r strains. The genomes of 213 AZM^r and 23 AZM-susceptible N. gonorrhoeae isolates collected in Canada from 1989 to 2014 were sequenced. Core single nucleotide polymorphism (SNP) phylogenomic analysis resolved 246 isolates into 13 lineages. High-level AZM^r (MICs ≥ 256 μg/ml) was found in 5 phylogenetically diverse isolates, all of which possessed the A2059G mutation (Escherichia coli numbering) in all four 23S rRNA alleles. One isolate with high-level AZM^r collected in 2009 concurrently had decreased susceptibility to ceftriaxone (MIC = 0.125 μg/ml). An increase in the number of 23S rRNA alleles with the C2611T mutations (E. coli numbering) conferred low to moderate levels of AZM^r (MICs = 2 to 4 and 8 to 32 μg/ml, respectively). Low-level AZM^r was also associated with mtrR promoter mutations, including the −35A deletion and the presence of Neisseria meningitidis-like sequences. Geographic and temporal phylogenetic clustering indicates that emergent AZM^r strains arise independently and can then rapidly expand clonally in a region through local sexual networks.     

Evaluation of the Safety and Immunogenicity of Two Antigen Concentrations of the Mtb72F/AS02A Candidate Tuberculosis Vaccine in Purified Protein Derivative-Negative Adults

Tuberculosis (TB) remains a major cause of illness and death worldwide, making a new TB vaccine an urgent public health priority. Purified protein derivative (PPD)-negative adults (n = 50) were equally randomized to receive 3 doses at 1-month intervals (at 0, 1, and 2 months) of one of the following vaccines: Mtb72F/AS02_A (10 or 40 μg antigen), Mtb72F/saline (10 or 40 μg antigen), or AS02_A. Mtb72F/AS02_A recipients received an additional dose 1 year after the first dose to evaluate if the elicited immune response could be boosted. Mtb72F/AS02_A vaccines were locally reactogenic but clinically well tolerated, with transient adverse events (usually lasting between 1 and 4 days) that resolved without sequelae being observed. No vaccine-related serious adverse events were reported. Vaccination with Mtb72F/AS02_A induced a strong Mtb72F-specific humoral response and a robust Mtb72F-specific CD4⁺ T-cell response, both of which persisted at 9 months after primary immunization and for 1 year after the booster immunization. There was no significant difference between the magnitude of the CD4⁺ T-cell response induced by the 10-μg and 40-μg Mtb72F/AS02_A vaccines. The Mtb72F-specific CD4⁺ T cells predominantly expressed CD40L; CD40L and interleukin-2 (IL-2); CD40L and tumor necrosis factor alpha (TNF-α); CD40L, IL-2, and TNF-α; and CD40L, IL-2, TNF-α, and gamma interferon (IFN-γ). Serum IFN-γ, but not TNF-α, was detected 1 day after doses 2 and 3 for the Mtb72F/AS02_A vaccine but did not persist. Vaccine-induced CD8⁺ T-cell responses were not detected, and no immune responses were elicited with AS02_A alone. In conclusion, Mtb72F/AS02_A is clinically well tolerated and is highly immunogenic in TB-naïve adults. The 10- and 40-μg Mtb72F/AS02_A vaccines show comparable safety and immunogenicity profiles.Tuberculosis (TB) is a major cause of illness and death worldwide, causing approximately 1.7 million deaths a year (43). Despite global efforts to control or eradicate the disease, the WHO estimates that in 2008 an estimated 8.9 million to 9.9 million people became infected with Mycobacterium tuberculosis. The situation is compounded by the emergence of multidrug-resistant TB. Since one-third of the world''s population is estimated to be latently infected with M. tuberculosis and at possible risk of disease, TB prevention remains one of today''s greatest public health challenges. An efficacious vaccination strategy is an essential tool to control TB.M. bovis bacillus Calmette-Guérin (BCG), consisting of attenuated strains of M. bovis, is the only TB vaccine currently available. It effectively prevents meningeal and miliary TB in young children (8) but appears to be ineffective in preventing adult-onset TB (protection rates, 0 to 80%) (10) and pulmonary TB in children.GlaxoSmithKline (GSK) Biologicals'' candidate TB vaccine antigen, Mtb72F, is composed of a fusion protein derived from two highly immunogenic M. tuberculosis antigens: Mtb39A (Rv0125 encoding PepA) and Mtb32A (Rv1196 encoding PPE18) (34, 35). Mtb72F, formulated with GSK Biologicals'' proprietary AS02_A adjuvant system, was shown to be well tolerated in animal models and protected against M. tuberculosis challenge in nonhuman primates, where Mtb72F/AS02_A was shown to be capable of inducing long-term protection against tuberculosis, as determined by protection against severe disease and death and by other clinical and histopathological parameters (6, 30, 34, 39). A first-time-in-human study evaluated Mtb72F/AS02_A (10 μg) in purified protein derivative (PPD)-negative TB-naïve, healthy adults in the United States given according to a 0-, 1-, and 2-month schedule and was found to be clinically well tolerated and highly immunogenic (42).This study assessed whether a larger amount of the Mtb72F/AS02_A vaccine antigen (40 μg) could improve the elicited immune response compared with the response elicited by the previously tested 10-μg antigen dose (42). In addition, a fourth vaccine dose was given to the vaccine recipients to evaluate whether the immune response could be boosted approximately 1 year after the primary vaccination course.     

Geographical Differences Associated with Single-Nucleotide Polymorphisms (SNPs) in Nine Gene Targets among Resistant Clinical Isolates of Mycobacterium tuberculosis

Alternative diagnostic methods, such as sequence-based techniques, are necessary for increasing the proportion of tuberculosis cases tested for drug resistance. Despite the abundance of data on drug resistance, isolates can display phenotypic resistance but lack any distinguishable markers. Furthermore, because resistance-conferring mutations develop under antibiotic pressure, different drug regimens could favor unique single-nucleotide polymorphisms (SNPs) in different geographical regions. A total of 407 isolates were collected from four geographical regions with a high prevalence of drug-resistant tuberculosis (India, Moldova, the Philippines, and South Africa). The “hot spot” or promoter sequences of nine genes (rpoB, gyrA, gyrB, katG, inhA promoter, ahpC promoter, eis promoter, rrs, and tlyA) associated with resistance to four types of antibiotics (rifampin, isoniazid, fluoroquinolones, and aminoglycosides) were analyzed for markers. Four genes contributed largely to resistance (rpoB, gyrA, rrs, and katG), two genes contributed moderately to resistance (the eis and inhA promoters), and three genes contributed little or no resistance (gyrB, tlyA, and the ahpC promoter) in clinical isolates. Several geographical differences were found, including a double mutation in rpoB found in 37.1% of isolates from South Africa, the C→T mutation at position −12 of the eis promoter found exclusively in 60.6% of isolates from Moldova, and the G→A mutation at position −46 of the ahpC promoter found only in India. These differences in polymorphism frequencies emphasize the uniqueness of isolates found in different geographical regions. The inclusion of several genes provided a moderate increase in sensitivity, and elimination of the examination of other genes might increase efficiency.     

Detecting mutation pattern of drug-resistant Mycobacterium tuberculosis isolates in Himachal Pradesh using GenoType® MTBDRplus assay

Context: Tuberculosis (TB) is a major public health problem in India and a principal cause of death in adults, especially among the economically productive age group. India accounts for one-fifth of the global burden of TB. It is estimated that about 40% of Indian population is infected with TB bacillus. The GenoType® MTBDRplus molecular method allows rapid diagnosis of the clinical samples and detection of the most common mutations in the genes associated with rifampicin (R) and isoniazid (H) resistance. Aims: To study the drug resistance and mutational patterns in multidrug-resistant (MDR) suspects clinical strains using GenoType® MTBDRplus assay. Subjects and Methods: A total of 770 sputum samples of the MDR-TB suspects were included in this study, which were received at Intermediate Reference Laboratory, Government TB Sanatorium, Dharampur, Solan, Himachal Pradesh from the Designated Microscopy Centres of Himachal Pradesh for the culture and susceptibility testing. All the 521 Mycobacterium tuberculosis complex (MTBC) strains were subjected to GenoType® MTBDRplus (HAIN Lifescience) assay to detect molecular resistance pattern to first line anti-tubercular drugs (isoniazid and rifampicin). Results: Of 770 samples, 556 (72.20%) were from male and 214 (27.80%) were from female. Among the 521 MTBC strains, 19.76% were found to be MDR and mono-resistance to isoniazid and rifampicin was detected in 8.63% and 6.14% strains respectively. About 74.81%, 76.35% and 5.40% strains harboured known mutation in rpoB, katG and inhA genes respectively. Conclusions: In rpoB gene, the most common mutation is associated with S531 L region. The GenoType® MTBDRplus assay is a rapid test for the detection of the most common mutations in MDR-TB strains. In our study, unknown rpoB gene mutations were found in 25.18% strains that may further be detected by gene sequencing.     

Expression of cytoplasmically inherited genes for chloramphenicol resistance in interspecific somatic cell hybrids and cybrids

Interspecies cybrids and hybrids were prepared in which one parent contributed the cytoplasmically inherited mutation for mitochondrial chloramphenicol (CAP) resistance. The transfer of CAP-resistant (R) mitochondria in human × mouse cybrids proved to be extremely difficult. Transfer from mouse to hamster cells occurred, but the cybrids had limited proliferative capacity. Hybrids between CAP-R mouse and CAP-sensitive (S) hamster cells could be selected in HAT + CAP at a very low frequency but comparable hybrids could not be isolated in CAP-R human × CAP-S mouse fusions unless ouabain was substituted for HAT. Hybrids selected directly in CAP retained the chromosomes of the resistant parent and segregated those of the sensitive. CAP-R hybrids (CAP-R human × CAP-S mouse) which segregate human chromosomes could be isolated only by sequential selection in HAT and then CAP. These results suggest that the mitochondrial genes of mammalian species are rapidly diverging from each other.     

Impaired dendritic cell differentiation of CD16‐positive monocytes in tuberculosis: Role of p38 MAPK

Tuberculosis (TB) is one of the world's most pernicious diseases mainly due to immune evasion strategies displayed by its causative agent Mycobacterium tuberculosis (Mtb). Blood monocytes (Mos) represent an important source of DCs during chronic infections; consequently, the alteration of their differentiation constitutes an escape mechanism leading to mycobacterial persistence. We evaluated whether the CD16⁺/CD16^? Mo ratio could be associated with the impaired Mo differentiation into DCs found in TB patients. The phenotype and ability to stimulate Mtb‐specific memory clones DCs from isolated Mo subsets were assessed. We found that CD16^? Mos differentiated into CD1a⁺DC‐SIGN^high cells achieving an efficient recall response, while CD16⁺ Mos differentiated into a CD1a^?DC‐SIGN^low population characterized by a poor mycobacterial Ag‐presenting capacity. The high and sustained phosphorylated p38 expression observed in CD16⁺ Mos was involved in the altered DC profile given that its blockage restored DC phenotype and its activation impaired CD16^? Mo differentiation. Furthermore, depletion of CD16⁺ Mos indeed improved the differentiation of Mos from TB patients toward CD1a⁺DC‐SIGN^high DCs. Therefore, Mos from TB patients are less prone to differentiate into DCs due to their increased proportion of CD16⁺ Mos, suggesting that during Mtb infection Mo subsets may have different fates after entering the lungs.     

New tricks for old dogs: countering antibiotic resistance in tuberculosis with host-directed therapeutics

Despite the availability of Mycobacterium tuberculosis (Mtb) drugs for over 50 years, tuberculosis (TB) remains at pandemic levels. New drugs are urgently needed for resistant strains, shortening duration of treatment, and targeting different stages of the disease, especially for treatment during human immunodeficiency virus co-infection. One solution to the conundrum that antibiotics kill the bacillus yet select for resistance is to target the host rather than the pathogen. Here, we discuss recent progress in so-called ‘host-directed therapeutics’ (HDTs), focusing on two general mechanistic strategies: (i) HDTs that disrupt Mtb pathogenesis in macrophages and (ii) immunomodulatory HDTs that facilitate protective immune responses that kill Mtb or reduce deleterious responses that exacerbate disease. HDTs hold significant promise as adjunctive therapies in that they are less likely to engender resistance, will likely have efficacy against antibiotic-resistant strains, and may have activity against non-replicating Mtb. However, TB is a complex and variegated disease, and human populations exhibit significant diversity in their immune responses to it, which presents a complicated landscape for HDTs to navigate. Nevertheless, we suggest that a detailed mechanistic understanding of drug action, together with careful selection of disease stage targets and dosing strategies may overcome such limitations and allow the development of HDTs as effective adjunctive treatment options for TB.     

Interleukin-17-dependent CXCL13 mediates mucosal vaccine–induced immunity against tuberculosis

The variable efficacy of tuberculosis (TB) vaccines and the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) emphasize the urgency for not only generating new and more effective vaccines against TB but also understanding the underlying mechanisms that mediate vaccine-induced protection. We demonstrate that mucosal adjuvants, such as type II heat labile enterotoxin (LT-IIb), delivered through the mucosal route induce pulmonary Mtb-specific T helper type 17 (Th17) responses and provide vaccine-induced protection against Mtb infection. Importantly, protection is interferon-γ (IFNγ)-independent but interleukin-17 (IL-17)-dependent. Our data show that IL-17 mediates C-X-C motif chemokine ligand 13 (CXCL13) induction in the lung for strategic localization of proinflammatory cytokine-producing CXCR5⁺ (C-X-C motif chemokine receptor 5-positive) T cells within lymphoid structures, thereby promoting early and efficient macrophage activation and the control of Mtb. Our studies highlight the potential value of targeting the IL-17–CXCL13 pathway rather than the IFNγ pathway as a new strategy to improve mucosal vaccines against TB.     

Sputum microbiota as a potential diagnostic marker for multidrug-resistant tuberculosis

The prevalence of drug-resistant Mycobacterium tuberculosis (Mtb) strains makes disease control more complicated, which is the main cause of death in tuberculosis (TB) patients. Early detection and timely standard treatment are the key to current prevention and control of drug-resistant TB. In recent years, despite the continuous advancement in drug-resistant TB diagnostic technology, the needs for clinical rapid and accurate diagnosis are still not fully met. With the development of sequencing technology, the research of human microecology has been intensified. This study aims to use 16 rRNA sequencing technology to detect and analyze upper respiratory flora of TB patients with anti-TB drug sensitivity (DS, n = 55), monoresistance isoniazide (MR-INH, n = 33), monoresistance rifampin (MR-RFP, n = 12), multidrug resistance (MDR, n = 26) and polyresistance (PR, n = 39) in southern China. Potential microbial diagnostic markers for different types of TB drug resistance are searched by screening differential flora, which provides certain guiding significance for drug resistance diagnosis and clinical drug use of TB. The results showed that the pulmonary microenvironment of TB patients was more susceptible to infection by external pathogens, and the infection of different drug-resistant Mtb leads to changes in different flora. Importantly, seven novel microorganisms (Leptotrichia, Granulicatella, Campylobacter, Delfitia, Kingella, Chlamydophila, Bordetella) were identified by 16S rRNA sequencing as diagnostic markers for different drug resistance types of TB. Leptotrichia, Granulicatella, Campylobacter were potential diagnostic marker for TB patients with INH single-resistance. Delftia was a potential diagnostic marker for TB patients with RFP single drug-resistance. Kingella and Chlamydophila can be used as diagnostic markers for TB patients with PR. Bordetella can be used as a potential diagnostic marker for identification of TB patients with MDR.     

Unequal distribution of resistance-conferring mutations among Mycobacterium tuberculosis and Mycobacterium africanum strains from Ghana

Isoniazid (INH) and rifampicin (RMP) resistance in Mycobacterium tuberculosis complex (MTC) isolates are mainly based on mutations in a limited number of genes. However, mutation frequencies vary in different mycobacterial populations. In this work, we analyzed the distribution of resistance-associated mutations in M. tuberculosis and M. africanum strains from Ghana, West Africa. The distribution of mutations in katG, fabG1-inhA, ahpC, and rpoB was determined by DNA sequencing in 217 INH-resistant (INH^r) and 45 multidrug-resistant (MDR) MTC strains isolated in Ghana from 2001 to 2004. A total of 247 out of 262 strains investigated (94.3%) carried a mutation in katG (72.5%), fabG1-inhA (25.1%), or ahpC (6.5%), respectively. M. tuberculosis strains mainly had katG 315 mutations (80.1%), whereas this proportion was significantly lower in M. africanum West-African 1 (WA1) strains (43.1%; p < 0.05). In contrast, WA1 strains showed more mutations in the fabG1-inhA region (39.2%, p < 0.05) compared to M. tuberculosis strains (20.9%). In 44 of 45 MDR strains (97.8%) mutations in the 81-bp core region of the rpoB gene could be verified. Additionally, DNA sequencing revealed that 5 RMP-susceptible strains also showed mutations in the rpoB hotspot region. In conclusion, although principally the same genes were affected in INH^rM. tuberculosis and M. africanum strains, disequilibrium in the distribution of mutations conferring resistance was verified that might influence the efficiency of molecular tests for determination of resistance.     

Magnitude of Gene Mutations Conferring Drug Resistance in Mycobacterium Tuberculosis Isolates from Lymph Node Aspirates in Ethiopia

Objective: Resistance to drugs is due to particular genomic mutations in the specific genes of Mycobacterium tuberculosis. Timely genetic characterization will allow identification of resistance mutations that will optimize an effective antibiotic treatment regimen. We determine the magnitude of gene mutations conferring resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among tuberculosis (TB) lymphadenitis patients.Methods: A cross sectional prospective study was conducted among 226 M.tuberculosis isolates from culture positive lymph node aspirates collected from TB lymphadenitis patients between April 2012 and May 2012. Detection of mutations conferring resistance to drugs was carried out using GenoType^® MTBDRplus and GenoType® MTBDRsl assay.Results: Out of the 226 strains, mutations conferring resistance to INH, RMP, multidrug resistance tuberculosis (MDR-TB) and EMB were 8, 3, 2 and 2 isolates, respectively. There was no isolated strain that showed mutation in the inhA promoter region gene. All INH resistant strains had mutations in the katG gene at codon 315 with amino acid change of S315T1. Among rifampicin resistant strains, two isolates displayed mutations at codon 531 in the rpoB gene with amino acid change of S531L and one isolate was by omission of wild type probes at Q513L. According to mutations associated with ethambutol resistance, all of the isolates had mutations in the embB gene with aminoacid change of M306I. All isolates resistant to INH, RMP and MDR using BacT/AlerT 3D system were correctly identified by GenoType® MTBDRplus assay.Conclusion: We observed mutations conferring resistance to INH at S315T1 of the katG gene, RMP at S531L and Q513L in the rpoB genes and EMB at M306I of the embB gene. In the absence of conventional drug susceptibility testing, the effort to develop easy, rapid and cost effective molecular assays for drug resistance TB monitoring is definitely desirable and the GenoType® MTBDRplus assay was found to be a useful method for diagnosis of resistance to INH, RMP and MDR from lymph node aspirates. Further molecular cluster analysis to determine transmission dynamics of mutated strain is required.