Putative compensatory mutations in the rpoC gene of rifampin-resistant Mycobacterium tuberculosis are associated with ongoing transmission.

Rifampin resistance in clinical isolates of Mycobacterium tuberculosis arises primarily through the selection of bacterial variants harboring mutations in the 81-bp rifampin resistance-determining region of the rpoB gene. While these mutations were shown to infer a fitness cost in the absence of antibiotic pressure, compensatory mutations in rpoA and rpoC were identified which restore the fitness of rifampin-resistant bacteria carrying mutations in rpoB. To investigate the epidemiological relevance of these compensatory mutations, we analyzed 286 drug-resistant and 54 drug-susceptible clinical M. tuberculosis isolates from the Western Cape, South Africa, a high-incidence setting of multidrug-resistant tuberculosis. Sequencing of a portion of the RpoA-RpoC interaction region of the rpoC gene revealed that 23.5% of all rifampin-resistant isolates tested carried a nonsynonymous mutation in this region. These putative compensatory mutations in rpoC were associated with transmission, as 30.8% of all rifampin-resistant isolates with an IS6110 restriction fragment length polymorphism (RFLP) pattern belonging to a recognized RFLP cluster harbored putative rpoC mutations. Such mutations were present in only 9.4% of rifampin-resistant isolates with unique RFLP patterns (P < 0.01). Moreover, these putative compensatory mutations were associated with specific strain genotypes and the rpoB S531L rifampin resistance mutation. Among isolates harboring this rpoB mutation, 44.1% also harbored rpoC mutations, while only 4.1% of the isolates with other rpoB mutations exhibited mutations in rpoC (P < 0.001). Our study supports a role for rpoC mutations in the transmission of multidrug-resistant tuberculosis and illustrates how epistatic interactions between drug resistance-conferring mutations, compensatory mutations, and different strain genetic backgrounds might influence compensatory evolution in drug-resistant M. tuberculosis.

Genomic determinants of speciation and spread of the Mycobacterium tuberculosis complex.

Models on how bacterial lineages differentiate increase our understanding of early bacterial speciation events and the genetic loci involved. Here, we analyze the population genomics events leading to the emergence of the tuberculosis pathogen. The emergence is characterized by a combination of recombination events involving core pathogenesis functions and purifying selection on early diverging loci. We identify the phoR gene, the sensor kinase of a two-component system involved in virulence, as a key functional player subject to pervasive positive selection after the divergence of the Mycobacterium tuberculosis complex from its ancestor. Previous evidence showed that phoR mutations played a central role in the adaptation of the pathogen to different host species. Now, we show that phoR mutations have been under selection during the early spread of human tuberculosis, during later expansions, and in ongoing transmission events. Our results show that linking pathogen evolution across evolutionary and epidemiological time scales points to past and present virulence determinants.

Prevalence and clinical significance of respiratory viruses and bacteria detected in tuberculosis patients compared to household contact controls in Tanzania: a cohort study.

OBJECTIVES: To describe the prevalence of respiratory pathogens in tuberculosis (TB) patients and in their household contact controls, and to determine the clinical significance of respiratory pathogens in TB patients. METHODS: We studied 489 smear-positive adult TB patients and 305 household contact controls without TB with nasopharyngeal swab samples within an ongoing prospective cohort study in Dar es Salaam, Tanzania, between 2013 and 2015. We used multiplex real-time PCR to detect 16 respiratory viruses and seven bacterial pathogens from nasopharyngeal swabs. RESULTS: The median age of the study participants was 33 years; 61% (484/794) were men, and 21% (168/794) were HIV-positive. TB patients had a higher prevalence of HIV (28.6%; 140/489) than controls (9.2%; 28/305). Overall prevalence of respiratory viral pathogens was 20.4% (160/794; 95%CI 17.7-23.3%) and of bacterial pathogens 38.2% (303/794; 95%CI 34.9-41.6%). TB patients and controls did not differ in the prevalence of respiratory viruses (Odds Ratio [OR] 1.00, 95%CI 0.71-1.44), but respiratory bacteria were less frequently detected in TB patients (OR 0.70, 95%CI 0.53-0.94). TB patients with both respiratory viruses and respiratory bacteria were likely to have more severe disease (adjusted OR [aOR] 1.6, 95%CI 1.1-2.4; p 0.011). TB patients with respiratory viruses tended to have more frequent lung cavitations (aOR 1.6, 95%CI 0.93-2.7; p 0.089). CONCLUSIONS: Respiratory viruses are common for both TB patients and household controls. TB patients may present with more severe TB disease, particularly when they are co-infected with both bacteria and viruses.

Interplay of strain and race/ethnicity in the innate immune response to M. tuberculosis.

BACKGROUND: The roles of host and pathogen factors in determining innate immune responses to M. tuberculosis are not fully understood. In this study, we examined host macrophage immune responses of 3 race/ethnic groups to 3 genetically and geographically diverse M. tuberculosis lineages. METHODS: Monocyte-derived macrophages from healthy Filipinos, Chinese and non-Hispanic White study participants (approximately 45 individuals/group) were challenged with M. tuberculosis whole cell lysates of clinical strains Beijing HN878 (lineage 2), Manila T31 (lineage 1), CDC1551 (lineage 4), the reference strain H37Rv (lineage 4), as well as with Toll-like receptor 2 agonist lipoteichoic acid (TLR2/LTA) and TLR4 agonist lipopolysaccharide (TLR4/LPS). Following overnight incubation, multiplex assays for nine cytokines: IL-1ß, IL-2, IL-6, IL-8, IL-10, IL-12p70, IFNγ, TNFα, and GM-CSF, were batch applied to supernatants. RESULTS: Filipino macrophages produced less IL-1, IL-6, and more IL-8, compared to macrophages from Chinese and Whites. Race/ethnicity had only subtle effects or no impact on the levels of IL-10, IL-12p70, TNFα and GM-CSF. In response to the Toll-like receptor 2 agonist lipoteichoic acid (TLR2/LTA), Filipino macrophages again had lower IL-1 and IL-6 responses and a higher IL-8 response, compared to Chinese and Whites. The TLR2/LTA-stimulated Filipino macrophages also produced lower amounts of IL-10, TNFα and GM-CSF. Race/ethnicity had no impact on IL-12p70 levels released in response to TLR2/LTA. The responses to TLR4 agonist lipopolysaccharide (TLR4/LPS) were similar to the TLR2/LTA responses, for IL-1, IL-6, IL-8, and IL-10. However, TLR4/LPS triggered the release of less IL-12p70 from Filipino macrophages, and less TNFα from White macrophages. CONCLUSIONS: Both host race/ethnicity and pathogen strain influence the innate immune response. Such variation may have implications for the development of new tools across TB therapeutics, immunodiagnostics and vaccines.

Consensus numbering system for the rifampicin resistance-associated rpoB gene mutations in pathogenic mycobacteria.

The rpoB gene codes for the RNA polymerase ß subunit, which is the target of rifampicin, an essential drug in the treatment of tuberculosis and other mycobacterial infections. This gene is present in all bacteria, but its length and nucleotide sequence vary between bacterial species, including mycobacteria. Mutations in the rpoB gene alter the structure of this protein and cause drug resistance. To describe the resistance-associated mutations, the scientific and medical communities have been using, since 1993, a numbering system based on the Escherichia coli sequence annotation. Using E. coli reference for describing mutations in mycobacteria leads to misunderstandings, particularly with the increasing use of whole genome sequencing, which brought an alternative numbering system based on the Mycobacterium tuberculosis rpoB sequence. We propose using a consensus numbering system for the reporting of resistance mutations based on the reference genomes from the species interrogated (such as strain H37Rv for M. tuberculosis). This manuscript provides the necessary figures and tables allowing researchers, microbiologists and clinicians to easily convert other annotation systems into one common language.

Detection and characterization of drug-resistant conferring genes in Mycobacterium tuberculosis complex strains: A prospective study in two distant regions of Ghana.

We spoligotyped and screened 1490 clinical Mycobacterium tuberculosis complex strains from Northern and Greater Accra regions of Ghana against INH and RIF using the microplate alamar blue phenotypic assay. Specific drug resistance associated genetic elements of drug resistant strains were analyzed for mutations. A total of 111 (7.5%), 10 (0.7%) and 40 (2.6%) were mono-resistant to INH, RIF, and MDR, respectively. We found the Ghana spoligotype to be associated with drug resistance (INH: 22.1%; p = 0.0000, RIF: 6.2%; p = 0.0103, MDR: 4.6%; p = 0.0240) as compared to the Cameroon spoligotype (INH: 6.7%, RIF: 2.4%, MDR: 1.6%). The propensity for an isolate to harbour katG S315T mutation was higher in M. tuberculosis (75.8%) than Mycobacterium africanum (51.7%) (p = 0.0000) whereas the opposite was true for inhApro mutations; MAF (48.3%) compared to MTBSS (26.7%) (p = 0.0419). We identified possible novel compensatory INH resistance mutations in inhA (G204D) and ahpCpro (-88G/A and -142G/A) and a novel ndh mutation K32R. We detected two possible rpoC mutations (G332R and V483G), which occurred independently with rpoB S450L, respectively. The study provides the first evidence that associate the Ghana spoligotype with DR-TB and calls for further genome analyses for proper classification of this spoligotype and to explore for fitness implications and mechanisms underlying this observation.

Clinical implications of molecular drug resistance testing for Mycobacterium tuberculosis: a TBNET/RESIST-TB consensus statement.

The emergence of drug-resistant strains of Mycobacterium tuberculosis is a challenge to global tuberculosis (TB) control. Although culture-based methods have been regarded as the gold standard for drug susceptibility testing (DST), molecular methods provide rapid information on mutations in the M. tuberculosis genome associated with resistance to anti-tuberculosis drugs. We ascertained consensus on the use of the results of molecular DST for clinical treatment decisions in TB patients. This document has been developed by TBNET and RESIST-TB groups to reach a consensus about reporting standards in the clinical use of molecular DST results. Review of the available literature and the search for evidence included hand-searching journals and searching electronic databases. The panel identified single nucleotide mutations in genomic regions of M. tuberculosis coding for katG, inhA, rpoB, embB, rrs, rpsL and gyrA that are likely related to drug resistance in vivo. Identification of any of these mutations in clinical isolates of M. tuberculosis has implications for the management of TB patients, pending the results of in vitro DST. However, false-positive and false-negative results in detecting resistance-associated mutations in drugs for which there is poor or unproven correlation between phenotypic and clinical drug resistance complicate the interpretation. Reports of molecular DST results should therefore include specific information on the mutations identified and provide guidance for clinicians on interpretation and on the choice of the appropriate initial drug regimen.

Survival and sequelae of meningococcal meningitis in Ghana.

BACKGROUND: Meningococcal meningitis epidemics are frequent in the Sahel zone of Africa but there is little information on the frequency of long-term sequelae. We analysed excess mortality in the two years following the 1997 epidemic in northern Ghana and carried out a case-control study to assess sequelae in the survivors. METHODS: Two-year survival of 696 meningitis cases recorded at the War Memorial Hospital, Navrongo, was analysed using data from a demographic surveillance system. A structured questionnaire on disability and on psychiatric, neuropsychological and behavioural problems was administered to 505 of the survivors and 505 age- sex- and location-matched controls as well as to their respective relatives. Cases and controls underwent full neurological and neuropsychological examination and were evaluated for hearing impairment by audiometry. RESULTS: Survival rates after the first month following the attack were similar in cases and controls. Hearing impairment was the major sequela, and was reported in 6 per cent of cases and 2 per cent of controls (odds ratio [OR] = 3.10; 95% CI : 1.48-7.09). Audiometry detected severe and profound hearing loss in the worse affected ear (> or =70 db) in 8/496 (1.6%) survivors but in only one control. Survivors of meningitis were more likely to suffer from feelings of tiredness (OR = 1.47; 95% CI : 1.03-2.11) and were more often reported by relatives to have insomnia (OR = 2.31; 95% CI : 1.17-4.82) and daily alcohol consumption. INTERPRETATION: Meningococcal meningitis annually causes approximately 10 000 cases of deafness in sub-Saharan Africa; there is a need for early detection of affected survivors and promotion of simple hearing devices. There is a sizeable burden of depressive disorders secondary to meningitis which should be identified and looked after appropriately.

Microheterogeneity of serogroup A (subgroup III) neisseria meningitidis during an outbreak in northern ghana

During a meningitis outbreak in the eastern subdistrict of the Kassena-Nankana District of the Upper East Region of Ghana, we analysed cerebrospinal fluid from suspected meningitis cases for the most common causative organisms. In 50 of 92 samples analysed, serogroup A Neisseria meningitidis were detected. The ages of serogroup A N. meningitidis patients ranged from 4 months to 64 years. The case fatality ratio was 20%. Coma or stupor on presentation worsened the prognosis. All serogroup A N. meningitidis isolates recovered revealed the A: 4: P1.9, 20 phenotype characteristic for the subgroup III clonal grouping. No evidence for resistance to penicillin G, chloramphenicol, cefotaxime, ciprofloxacin, rifampicin or tetracycline was found. All strains were resistant to sulphadiazine. Restriction analysis patterns of opa, iga and ingA genes were characteristic for the majority of N. meningitidis serogroup A subgroup III bacteria isolated in Africa after the 1987 epidemic in Mecca. Differences in pulsed-field gel electrophoresis patterns of NheI and SpeI digested DNA revealed microheterogeneity among the Ghanaian isolates.

Risk factors for meningococcal meningitis in northern Ghana.

Meningococcal meningitis is a major cause of morbidity and mortality in the meningitis belt of sub-Saharan Africa where it occurs in epidemics every 8-12 years. Risk factors for the disease in this setting remain largely unknown. We carried out a case-control study to investigate possible risk factors among survivors of a meningitis epidemic occurring in 1997 in northern Ghana. A structured questionnaire on socio-economic factors, housing and household overcrowding, smoking and exposure to smoke and close contact with a case was administered to 505 of the survivors and 505 of age-, sex- and location-matched controls. Cooking in kitchens with firewood stoves (OR 9.00, CI 1.25-395) and sharing a bedroom with a case (OR 2.18 CI 1.43-3.4) were found to be risk factors for disease. Socio-economic factors, overcrowding, smoking and passive exposure to tobacco smoke were not found to be risk factors. Exposure to smoke from cooking fires or close contact with a case puts people at risk of contracting meningococcal meningitis. In the hot dry months, exposure to smoke from cooking fires should be minimized by encouraging alternatives to cooking over wood fires, or cooking outside. If wood-burning stoves cannot be avoided, kitchens should be made larger with improved ventilation. Meningitis cases should be nursed in well-ventilated rooms and the number of people sharing a room with a case kept at a minimum.

[Diarrhea in urban agricultural workers in Nouakchott in Mauritania]. / La diarrhée chez les agriculteurs urbains de Nouakchott en Mauritanie.

Nearly 200 million people in the developing world are dependent or urban gardening for food and income. This practice has been accelerated by the droughts of recent decades which have forced more and more migrants into urban areas. Numerous potential health hazards have been attributed to urban gardening but the exact risks in Sahelian areas remain unclear. The purpose of this cross-sectional study was to evaluate the incidence of diarrhea at the Tel Zatar gardening site in urban Nouakchott, Mauritania. In addition, a case-control study was carried out to identify risk factors for diarrhea in function of gardeners' activity and living conditions. Statistical analysis was performed using univariate and logistical regression methods. The annual incidence of diarrhea ranged from 6.9 (IC95 p. 100 = 5.0-8.8) to 8.5 (IC95 p. 100 = 6.2-10.8) episodes per gardener and year. Multivariate analysis identified four significant risk factors. Two of these factors were unrelated to gardening, i.e., not having spent more than USD 3.50 the previous day (odds ratio (OR = 2.8, IC95 p. 100 = 1.01-7.81) and poor food hygiene (cooking outside (OR = 4.69, IC95 p. 100 = 1.06-20.83). The other two factors were regular consumption of raw vegetables (OR = 25.5, IC95 p. 100 = 2.0-32.0) and use of untreated well water (OR = 3.85, IC95 p. 100 = 1.08-14.29). Unprotected well water was the cause of 59.2 p. 100 of diarrheal episodes reported by gardeners at Tel Zatar. The results of this study confirm that vegetable production in urban gardens such as Tel Zatar is associated with health risks. Public health measures should address not only the garden sites but also domestic hygiene.

Genetic diversity of Mycobacterium tuberculosis in Madang, Papua New Guinea.

SETTING: Madang and surroundings, Papua New Guinea (PNG). OBJECTIVE: To characterise the genetic diversity and drug susceptibility of Mycobacterium tuberculosis isolates collected in Madang and surroundings. DESIGN: M. tuberculosis was isolated from sputum samples from active pulmonary tuberculosis cases. Drug resistance profiles were obtained by drug susceptibility testing. M. tuberculosis lineages were identified by single nucleotide polymorphisms and sub-typing was performed by spoligotyping. Spoligotyping and 24 locus mycobacterial interspersed repetitive units-variable number of tandem repeats were combined to identify clustered isolates. RESULTS: The 173 M. tuberculosis isolates collected belonged predominantly to the Euro-American lineage (Lineage 4) and the East-Asian lineage (Lineage 2). Multidrug-resistant M. tuberculosis were observed in 5.2% of isolates. Lineage 2 M. tuberculosis, which includes the 'Beijing' genotype, was significantly associated with any drug resistance (OR 5.2, 95%CI 1.8-15.1). Cluster analyses showed 44% molecularly clustered isolates, suggesting transmission of M. tuberculosis in the community, including transmission of primary drug-resistant M. tuberculosis. CONCLUSION: These data provide the first insight into the molecular characteristics of M. tuberculosis in the Madang area of PNG, and indicate substantial drug resistance with evidence of ongoing transmission.

Strain diversity, epistasis and the evolution of drug resistance in Mycobacterium tuberculosis.

Mycobacterium tuberculosis harbours little DNA sequence diversity compared with other bacteria. However, there is mounting evidence that strain-to-strain variation in this organism has been underestimated. We review our current understanding of the genetic diversity among M. tuberculosis clinical strains and discuss the relevance of this diversity for the ongoing global epidemics of drug-resistant tuberculosis. Based on findings in other bacteria, we propose that epistatic interactions between pre-existing differences in strain genetic background, acquired drug-resistance-conferring mutations and compensatory changes could play a role in the emergence and spread of drug-resistant M. tuberculosis.

Strain classification of Mycobacterium tuberculosis: congruence between large sequence polymorphisms and spoligotypes.

Spoligotyping is used in molecular epidemiological studies, and signature patterns have identified strain families. However, homoplasy occurs in the markers used for spoligotyping, which could lead to identical spoligotypes in phylogenetically unrelated strains. We determined the accuracy of strain classification based on spoligotyping using the six large sequence and single nucleotide polymorphisms-defined lineages as a gold standard. Of 919 Mycobacterium tuberculosis isolates, 870 (95%) were classified into a spoligotype family. Strains from a particular spoligotype family belonged to the same lineage. We did not find convergence to the same spoligotype. Spoligotype families appear to be sub-lineages within the main lineages.

Fitness cost of drug resistance in Mycobacterium tuberculosis.

Multidrug-resistant (MDR)--and extensively drug-resistant (XDR)--forms of tuberculosis are growing public health problems. Mathematical models predict that the future of the MDR and XDR tuberculosis epidemics depends in part on the competitive fitness of drug-resistant strains. Here, recent experimental and molecular epidemiological data that illustrate how heterogeneity among drug-resistant strains of Mycobacterium tuberculosis can influence the relative fitness and transmission of this pathogen are reviewed.

Infectiousness, reproductive fitness and evolution of drug-resistant Mycobacterium tuberculosis.

Mathematical models predict that the future of the multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) epidemic will depend to a large extent on the transmission efficiency or relative fitness of drug-resistant Mycobacterium tuberculosis compared to drug-susceptible strains. Molecular epidemiological studies comparing the spread of drug-resistant to that of drug-susceptible strains have yielded conflicting results: MDR strains can be up to 10 times more or 10 times less transmissible than pan-susceptible strains. Experimental work performed with model organisms has highlighted a level of complexity in the biology of bacterial drug resistance that is generally not considered during standard epidemiological studies of TB transmission. Recent experimental studies in M. tuberculosis indicate that drug resistance in this organism could be equally complex. For example, the relative fitness of drug-resistant strains of M. tuberculosis can be influenced by the specific drug resistance-conferring mutation and strain genetic background. Furthermore, compensatory evolution, which has been shown to mitigate the fitness defects associated with drug resistance in other bacteria, could be an important factor in the emergence and spread of drug-resistant M. tuberculosis. However, much more work is needed to understand the detailed molecular mechanisms and evolutionary forces that drive drug resistance in this pathogen. Such increased knowledge will allow for better epidemiological predictions and assist in the development of new tools and strategies to fight drug-resistant TB.

Microheterogeneity of serogroup A (subgroup III) Neisseria meningitidis during an outbreak in northern Ghana.

During a meningitis outbreak in the eastern subdistrict of the Kassena-Nankana District of the Upper East Region of Ghana, we analysed cerebrospinal fluid from suspected meningitis cases for the most common causative organisms. In 50 of 92 samples analysed, serogroup A Neisseria meningitidis were detected. The ages of serogroup A N. meningitidis patients ranged from 4 months to 64 years. The case fatality ratio was 20%. Coma or stupor on presentation worsened the prognosis. All serogroup A N. meningitidis isolates recovered revealed the A: 4: P1.9, 20 phenotype characteristic for the subgroup III clonal grouping. No evidence for resistance to penicillin G, chloramphenicol, cefotaxime, ciprofloxacin, rifampicin or tetracycline was found. All strains were resistant to sulphadiazine. Restriction analysis patterns of opa, iga and ingA genes were characteristic for the majority of N. meningitidis serogroup A subgroup III bacteria isolated in Africa after the 1987 epidemic in Mecca. Differences in pulsed-field gel electrophoresis patterns of NheI and SpeI digested DNA revealed microheterogeneity among the Ghanaian isolates.

Factors in the dialysis regimen which contribute to alterations in the abnormalities of uremia.

While hemodialysis therapy in its present form is capable of sustaining life, dialysis patients are not metabolically normal and we are unable to say what technical factors contribute adequate therapy. Recent efforts to resolve these problems have led to the assumption that substances in the molecular weight range of 800 to 3000 daltons may be pathogenic in uremia and these may not be effectively removed by dialysis. Accordingly, four groups of patients (ten each) underwent changes in their routine which were theoretically designed to alter independently the concentration of small (urea) and "middle" molecules in the blood. In two groups, the concentration of urea was theoretically increased or decreased while the concentration of so-called middle molecules was maintained unchanged. In the remaining two groups, middle molecule concentration was theoretically increased or decreased while small molecule concentration was unchanged. Patients were evaluated prior to and after completing altered dialysis therapy. The results suggest three related conclusions. First, the uremic syndrome may be viewed as a constellation of abnormalities which can be subgrouped by association so that azotemia may be correlated with neuropathic disease and hypertension with weight gain or body size, for example. Second, those physiologic variables which changed after altered dialysis tended to deteriorate with increasing concentration of small molecules in the blood and remained independent of theoretical changes in middle molecules. Finally, when patients are relatively under-dialyzed, they may spontaneously modulate the reduced removal of metabolites such as urea by decreasing the dietary intake of nutrients.

The effects of varying dialysis regimens on lymphocyte stimulation.

A protocol was designed to alter mean time average serum concentrations of "middle" (B12) and "small" (urea) molecules. Serum before and after studies was tested for its effect on the blastogenic response to PHA. From this study we have concluded that: 1. Serum of patients on hemodialysis inhibits the mitogenic response of normal lymphocytes to PHA when compared to normal sera. 2. This phenomenon is not responsive to pertubations in dialysis designed to alter the concentration of ""middle'' and ""small'' molecules. 3. The inverse correlation between PHA response and serum PO4 and likewise the inverse correlation with changes in bone X-rays suggest that PTH, fragments thereof, or other factors related to bone and calcium metabolism, could conceivably be involved. This could mediated via cyclic AMP.

Fit genotypes and escape variants of subgroup III Neisseria meningitidis during three pandemics of epidemic meningitis.

The genetic variability at six polymorphic loci was examined within a global collection of 502 isolates of subgroup III, serogroup A Neisseria meningitidis. Nine "genoclouds" were identified, consisting of genotypes that were isolated repeatedly plus 48 descendent genotypes that were isolated rarely. These genoclouds have caused three pandemic waves of disease since the mid-1960s, the most recent of which was imported from East Asia to Europe and Africa in the mid-1990s. Many of the genotypes are escape variants, resulting from positive selection that we attribute to herd immunity. Despite positive selection, most escape variants are less fit than their parents and are lost because of competition and bottlenecks during spread from country to country. Competition between fit genotypes results in dramatic changes in population composition over short time periods.