RESUMEN
Eis promoter mutations can confer reduced Mycobacterium tuberculosis kanamycin susceptibility. GenoType MTBDRsl, a widely used assay evaluating this region, wrongly classified 17/410 isolates as eis promoter wild type. Six out of seventeen isolates harbored mutations known to confer kanamycin resistance, and the remainder harbored either novel eis promoter mutations (7/11) or disputed mutations (4/11). GenoType MTBDRsl can miss established and new variants that cause reduced susceptibility. These data highlight the importance of reflex phenotypic kanamycin testing.
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Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Pruebas Diagnósticas de Rutina , Farmacorresistencia Bacteriana Múltiple , Genotipo , Humanos , Kanamicina/farmacología , Resistencia a la Kanamicina/genética , Pruebas de Sensibilidad Microbiana , Mutación/genética , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/genéticaAsunto(s)
Antituberculosos/uso terapéutico , Diarilquinolinas/uso terapéutico , Resistencia a Medicamentos , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Anciano , Quimioterapia Combinada , Humanos , Masculino , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Secuenciación Completa del GenomaRESUMEN
BACKGROUND: Papua New Guinea (PNG) is a high tuberculosis (TB) burden country of the WHO Western Pacific Region, but so far research on drug resistance (DR) and genotypes of Mycobacterium tuberculosis (M. tuberculosis) was only conducted in few provinces in the country. The aim of the present study was to obtain baseline data on the level of drug resistance and the genotypic diversity of circulating M. tuberculosis in additional provinces and to investigate the differences between three selected sites across PNG. RESULTS: Genotyping of 147 M. tuberculosis clinical isolates collected in Goroka, Eastern Highlands Province, in Alotau, Milne Bay Province and in Madang, Madang Province revealed three main lineages of M. tuberculosis: Lineage 4 (European-American lineage), Lineage 2 (East-Asian lineage) and Lineage 1 (Indo-Oceanic lineage). All three lineages were detected in all three sites, but the individual lineage compositions varied significantly between sites. In Madang Lineage 4 was the most prevalent lineage (76.6%), whereas in Goroka and Alotau Lineage 2 was dominating (60.5% and 84.4%, respectively) (p < 0.001). Overall, phenotypic drug susceptibility testing showed 10.8% resistance to at least one of the first-line drugs tested. Of all resistant strains (23/212) 30.4% were Streptomycin mono-resistant, 17.4% were Isoniazid mono-resistant and 13% were Rifampicin mono-resistant. Multi-drug resistant (MDR) TB was found in 2.8% of all tested cases (6/212). The highest amount of MDR TB was found in Alotau in Milne Bay Province (4.6%). CONCLUSION: A large number of drug resistant TB infections are present in the country and MDR TB has already been detected in all three surveyed regions of PNG, highlighting the importance of monitoring drug resistance and making it a high priority for the National Control Program. Due to the high prevalence of Lineage 2 in Milne Bay Province and given the frequent association of this lineage with drug resistance, monitoring of the latter should especially be scaled up in that province.
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Farmacorresistencia Bacteriana , Variación Genética , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/microbiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antituberculosos/farmacología , Femenino , Genotipo , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Tipificación Molecular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Papúa Nueva Guinea/epidemiología , Tuberculosis/epidemiología , Adulto JovenRESUMEN
BACKGROUND: Intermittent preventive treatment in infants (IPTi) has been shown in randomized trials to reduce malaria-related morbidity in African infants living in areas of high Plasmodium falciparum (Pf) transmission. It remains unclear whether IPTi is an appropriate prevention strategy in non-African settings or those co-endemic for P. vivax (Pv). METHODS AND FINDINGS: In this study, 1,121 Papua New Guinean infants were enrolled into a three-arm placebo-controlled randomized trial and assigned to sulfadoxine-pyrimethamine (SP) (25 mg/kg and 1.25 mg/kg) plus amodiaquine (AQ) (10 mg/kg, 3 d, nâ=â374), SP plus artesunate (AS) (4 mg/kg, 3 d, nâ=â374), or placebo (nâ=â373), given at 3, 6, 9 and 12 mo. Both participants and study teams were blinded to treatment allocation. The primary end point was protective efficacy (PE) against all episodes of clinical malaria from 3 to 15 mo of age. Analysis was by modified intention to treat. The PE (compared to placebo) against clinical malaria episodes (caused by all species) was 29% (95% CI, 10-43, p ≤ 0.001) in children receiving SP-AQ and 12% (95% CI, -11 to 30, pâ=â0.12) in those receiving SP-AS. Efficacy was higher against Pf than Pv. In the SP-AQ group, Pf incidence was 35% (95% CI, 9-54, pâ=â0.012) and Pv incidence was 23% (95% CI, 0-41, pâ=â0.048) lower than in the placebo group. IPTi with SP-AS protected only against Pf episodes (PEâ=â31%, 95% CI, 4-51, pâ=â0.027), not against Pv episodes (PEâ=â6%, 95% CI, -24 to 26, pâ=â0.759). Number of observed adverse events/serious adverse events did not differ between treatment arms (p > 0.55). None of the serious adverse events were thought to be treatment-related, and the vomiting rate was low in both treatment groups (1.4%-2.0%). No rebound in malaria morbidity was observed for 6 mo following the intervention. CONCLUSIONS: IPTi using a long half-life drug combination is efficacious for the prevention of malaria and anemia in infants living in a region highly endemic for both Pf and Pv.
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Antimaláricos/uso terapéutico , Malaria/prevención & control , Plasmodium falciparum/patogenicidad , Plasmodium vivax/patogenicidad , Amodiaquina/uso terapéutico , Combinación de Medicamentos , Femenino , Humanos , Lactante , Recién Nacido , Malaria/parasitología , Masculino , Plasmodium falciparum/efectos de los fármacos , Plasmodium vivax/efectos de los fármacos , Pirimetamina/uso terapéutico , Sulfadoxina/uso terapéuticoRESUMEN
BACKGROUND: Monitoring drug resistance in Mycobacterium tuberculosis is essential to curb the spread of tuberculosis (TB). Unfortunately, drug susceptibility testing is currently not available in Papua New Guinea (PNG) and that impairs TB control in this country. We report for the first time M. tuberculosis mutations associated with resistance to first and second-line anti-TB drugs in Madang, PNG. A molecular cluster analysis was performed to identify M. tuberculosis transmission in that region. RESULTS: Phenotypic drug susceptibility tests showed 15.7% resistance to at least one drug and 5.2% multidrug resistant (MDR) TB. Rifampicin resistant strains had the rpoB mutations D516F, D516Y or S531L; Isoniazid resistant strains had the mutations katG S315T or inhA promoter C15T; Streptomycin resistant strains had the mutations rpsL K43R, K88Q, K88R), rrs A514C or gidB V77G. The molecular cluster analysis indicated evidence for transmission of resistant strain. CONCLUSIONS: We observed a substantial rate of MDR-TB in the Madang area of PNG associated with mutations in specific genes. A close monitoring of drug resistance is therefore urgently required, particularly in the presence of drug-resistant M. tuberculosis transmission. In the absence of phenotypic drug susceptibility testing in PNG, molecular assays for drug resistance monitoring would be of advantage.
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Antituberculosos/farmacología , Farmacorresistencia Bacteriana , Mutación Missense , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Tuberculosis/microbiología , Proteínas Bacterianas/genética , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/aislamiento & purificación , Papúa Nueva Guinea , Proyectos PilotoRESUMEN
Extensively drug-resistant tuberculosis (XDR-TB), defined as resistance to at least isoniazid (INH), rifampicin (RIF), a fluoroquinolone (FQ) and a second-line injectable drug (SLID), is difficult to treat and poses a major threat to TB control. The transmission dynamics and distribution of XDR Mycobacterium tuberculosis (Mtb) strains have not been thoroughly investigated. Using whole genome sequencing data on 461 XDR-Mtb strains, we aimed to investigate the geographical distribution of XDR-Mtb strains in the Western Cape Province of South Africa over a 10 year period (2006-2017) and assess the association between Mtb sub-lineage, age, gender, geographical patient location and membership or size of XDR-TB clusters. First, we identified transmission clusters by excluding drug resistance-conferring mutations and using the 5 SNP cutoff, followed by merging clusters based on their most recent common ancestor. We then consecutively included variants conferring resistance to INH, RIF, ethambutol (EMB), pyrazinamide (PZA), SLIDs and FQs in the cluster definition. Cluster sizes were classified as small (2-4 isolates), medium (5-20 isolates), large (21-100 isolates) or very large (>100 isolates) to reflect the success of individual strains. We found that most XDR-TB strains were clustered and that including variants conferring resistance to INH, RIF, EMB, PZA and SLIDs in the cluster definition did not significantly reduce the proportion of clustered isolates (85.5-82.2â%) but increased the number of patients belonging to small clusters (4.3-12.4â%, P=0.56). Inclusion of FQ resistance-conferring variants had the greatest effect, with 11 clustered isolates reclassified as unique while the number of clusters increased from 17 to 37. Lineage 2 strains (lineage 2.2.1 typical Beijing or lineage 2.2.2 atypical Beijing) showed the large clusters which were spread across all health districts of the Western Cape Province. We identified a significant association between residence in the Cape Town metropole and cluster membership (P=0.016) but no association between gender, age and cluster membership or cluster size (P=0.39). Our data suggest that the XDR-TB epidemic in South Africa probably has its origin in the endemic spread of MDR Mtb and pre-XDR Mtb strains followed by acquisition of FQ resistance, with more limited transmission of XDR Mtb strains. This only became apparent with the inclusion of drug resistance-conferring variants in the definition of a cluster. In addition to the prevention of amplification of resistance, rapid diagnosis of MDR, pre-XDR and XDR-TB and timely initiation of appropriate treatment is needed to reduce transmission of difficult-to-treat TB.
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Tuberculosis Extensivamente Resistente a Drogas , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Resistencia a Medicamentos , Tuberculosis Extensivamente Resistente a Drogas/epidemiología , Fluoroquinolonas/farmacología , Fluoroquinolonas/uso terapéutico , Humanos , Mutación , Rifampin , Sudáfrica/epidemiologíaRESUMEN
Background: Lineage 1 (L1) and 3 (L3) are two lineages of the Mycobacterium tuberculosis complex (MTBC) causing tuberculosis (TB) in humans. L1 and L3 are prevalent around the rim of the Indian Ocean, the region that accounts for most of the world's new TB cases. Despite their relevance for this region, L1 and L3 remain understudied. Methods: We analyzed 2,938 L1 and 2,030 L3 whole genome sequences originating from 69 countries. We reconstructed the evolutionary history of these two lineages and identified genes under positive selection. Results: We found a strongly asymmetric pattern of migration from South Asia toward neighboring regions, highlighting the historical role of South Asia in the dispersion of L1 and L3. Moreover, we found that several genes were under positive selection, including genes involved in virulence and resistance to antibiotics. For L1 we identified signatures of local adaptation at the esxH locus, a gene coding for a secreted effector that targets the human endosomal sorting complex, and is included in several vaccine candidates. Conclusions: Our study highlights the importance of genetic diversity in the MTBC, and sheds new light on two of the most important MTBC lineages affecting humans.
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Mycobacterium tuberculosis , Genotipo , Humanos , Océano Índico , Mycobacterium tuberculosis/genéticaRESUMEN
BACKGROUND: In order to provide a cost-effective tool to analyse pharmacogenetic markers in malaria treatment, DNA microarray technology was compared with sequencing of polymerase chain reaction (PCR) fragments to detect single nucleotide polymorphisms (SNPs) in a larger number of samples. METHODS: The microarray was developed to affordably generate SNP data of genes encoding the human cytochrome P450 enzyme family (CYP) and N-acetyltransferase-2 (NAT2) involved in anti-malarial drug metabolisms and with known polymorphisms, i.e. CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, and NAT2. RESULTS: For some SNPs, i.e. CYP2A6*2, CYP2B6*5, CYP2C8*3, CYP2C9*3/*5, CYP2C19*3, CYP2D6*4 and NAT2*6/*7/*14, agreement between both techniques ranged from substantial to almost perfect (kappa index between 0.61 and 1.00), whilst for other SNPs a large variability from slight to substantial agreement (kappa index between 0.39 and 1.00) was found, e.g. CYP2D6*17 (2850C>T), CYP3A4*1B and CYP3A5*3. CONCLUSION: The major limit of the microarray technology for this purpose was lack of robustness and with a large number of missing data or with incorrect specificity.
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Antimaláricos/farmacología , Sistema Enzimático del Citocromo P-450/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Plasmodium/genética , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple/efectos de los fármacos , Antimaláricos/metabolismo , Antimaláricos/uso terapéutico , Arilamina N-Acetiltransferasa , Cambodia , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/farmacología , Variación Genética , Genotipo , Humanos , Malaria/tratamiento farmacológico , Malaria/genética , Malaria/parasitología , Plasmodium/efectos de los fármacos , Plasmodium/aislamiento & purificación , Polimorfismo de Nucleótido Simple/genética , Sensibilidad y Especificidad , TanzaníaRESUMEN
BACKGROUND: Molecular typing of Mycobacterium tuberculosis (M.tb) isolates can inform Tuberculosis (TB) control programs on the relative proportion of transmission driving the TB epidemic. There is limited data on the M. tb genotypes that are circulating in Botswana. The aim of this study was to generate baseline data on the genetic diversity of M.tb isolates circulating in the country. METHODS: A total of 461 M.tb isolates received at the Botswana National Tuberculosis Reference Laboratory between March 2012 and October 2013 were included in this study. Drug susceptibility testing was conducted using the BD BACTEC MGIT 960 System. M.tb strains were genotyped using spoligotyping and spoligotype patterns were compared with existing patterns in the SITVIT Web database. A subset of drug resistant isolates which formed spoligo clusters (n = 65) was additionally genotyped with 12-loci MIRU. Factors associated with drug resistance and clustering were evaluated using logistic regression. RESULTS: Of the 461 isolates genotyped, 458 showed 108 distinct spoligotype patterns. The predominant M.tb lineages were Lineage 4 (81.9%), Lineage 2 (9%) and Lineage 1 (7.2%). The predominant spoligotype families within Lineage 4 were LAM (33%), S (14%), T (16%), X (16%). Three hundred and ninety-two (86%) isolates could be grouped into 44 clusters (2-46 isolates per cluster); giving a clustering rate of 76%. We identified 173 (37.8%) drug resistant isolates, 48 (10.5%) of these were multi-drug resistant. MIRU typing of the drug resistant isolates allowed grouping of 46 isolates into 14 clusters, giving a clustering rate of 49.2%. There was no association between age, sex, treatment category, region and clustering. CONCLUSIONS: This study highlights the complexity of the TB epidemic in Botswana with multiple strains contributing to disease and provides baseline data on the population structure of M.tb strains in Botswana.
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Variación Genética , Genotipo , Tipificación Molecular , Mycobacterium tuberculosis , Filogenia , Tuberculosis , Adolescente , Adulto , Botswana/epidemiología , Estudios Transversales , Femenino , Humanos , Persona de Mediana Edad , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Estudios Retrospectivos , Tuberculosis/epidemiología , Tuberculosis/genética , Tuberculosis/microbiologíaRESUMEN
The emergence and transmission of multidrug resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis (M.tb) strains is a threat to global tuberculosis (TB) control. The early detection of drug resistance is critical for patient management. The aim of this study was to determine the proportion of isolates with additional second-line resistance among rifampicin and isoniazid resistant and MDR-TB isolates. A total of 66 M.tb isolates received at the National Tuberculosis Reference Laboratory between March 2012 and October 2013 with resistance to isoniazid, rifampicin or both were analyzed in this study. The genotypes of the M.tb isolates were determined by spoligotyping and second-line drug susceptibility testing was done using the Hain Genotype MTBDRsl line probe assay version 2.0. The treatment outcomes were defined according to the Botswana national and World Health Organization (WHO) guidelines. Of the 57 isolates analyzed, 33 (58%) were MDR-TB, 4 (7%) were additionally resistant to flouroquinolones and 3 (5%) were resistant to both fluoroquinolones and second-line injectable drugs. The most common fluoroquinolone resistance-conferring mutation detected was gyrA A90V. All XDR-TB cases remained smear or culture positive throughout the treatment. Our study findings indicate the importance of monitoring drug resistant TB cases to ensure rapid detection of second-line drug resistance.
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OBJECTIVES: The characterization of differential gene expression in Giardia lamblia WB C6 strain C4 resistant to metronidazole and nitazoxanide using microarray technology and quantitative real-time PCR. METHODS: In a previous study, we created and characterized the G. lamblia WB C6 clone C4 resistant to nitazoxanide and metronidazole. In this study, using a microarray-based approach, we have identified open-reading frames (ORFs) that were differentially expressed in C4 when compared with its wild-type WB C6. Using quantitative real-time PCR, we have validated the expression patterns of some of those ORFs, focusing on chaperones such as heat-shock proteins in wild-type and C4 trophozoites. In order to induce an antigenic shift, trophozoites of both strains were subjected to a cycle of en- and excystation. Expression of selected genes and resistance to nitazoxanide and metronidazole were investigated after this cycle. RESULTS: Forty of a total of 9115 ORFs were found to be up-regulated and 46 to be down-regulated in C4 when compared with wild-type. After a cycle of en- and excystation, resistance of C4 to nitazoxanide and metronidazole was lost. Resistance formation and en-/excystation were correlated with changes in expression of ORFs encoding for major surface antigens such as the variant surface protein TSA417 or AS7 ('antigenic shift'). Moreover, expression patterns of the cytosolic heat-shock protein HSP70 B2, HSP40, and of the previously identified nitazoxanide-binding proteins nitroreductase and protein disulphide isomerase PDI4 were correlated with resistance and loss of resistance after en-/excystation. C4 trophozoites had a higher thermotolerance level than wild-type trophozoites. After en-/excystation, this tolerance was lost. CONCLUSIONS: These results suggest that resistance formation in Giardia to nitazoxanide and metronidazole is correlated with altered expression of genes involved in stress response such as heat-shock proteins.
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Antiprotozoarios/farmacología , Resistencia a Medicamentos/genética , Perfilación de la Expresión Génica , Giardia lamblia/efectos de los fármacos , Giardia lamblia/fisiología , Metronidazol/farmacología , Tiazoles/farmacología , Animales , Antígenos de Protozoos/genética , Antígenos de Superficie/genética , Giardia lamblia/genética , Proteínas de Choque Térmico/genética , Mutación , Nitrocompuestos , Nitrorreductasas/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteína Disulfuro Isomerasas/genética , Proteínas Protozoarias/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Chlamydia species have recently been recognized as emerging pathogens in snakes. However, isolation of novel snake chlamydiae is critical and their growth characteristics are largely unknown. In this study, two novel chlamydial species are described: Chlamydia serpentis and Chlamydia poikilothermis, isolated after attempts on 23 cloacal and choanal swabs from 18 PCR-positive captive snakes originating from different Swiss snake collections. Isolation success, growth curve and infectivity rates over a 48-hour time period were dependent on temperature (37 °C for C. serpentis, 28 °C for C. poikilothermis). C. serpentis and C. poikilothermis were sensitive to tetracycline and moxifloxacin during evaluation by in vitro antibiotic susceptibility assay but intermediate to resistant (2-4 µg/ml) to azithromycin. Whole genome sequencing of the isolates provided proof of the novel species status, and gives insights into the evolution of these branches of genus Chlamydia.
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Antibacterianos/uso terapéutico , Azitromicina/uso terapéutico , Infecciones por Chlamydia/veterinaria , Chlamydia/efectos de los fármacos , Farmacorresistencia Bacteriana , Serpientes/microbiología , Temperatura , Animales , Chlamydia/clasificación , Chlamydia/genética , Infecciones por Chlamydia/tratamiento farmacológico , Infecciones por Chlamydia/microbiología , Genoma Bacteriano , Metagenómica , Filogenia , Secuenciación Completa del GenomaRESUMEN
Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.
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ADN Bacteriano/análisis , Genómica/métodos , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , Polimorfismo Genético/genética , Tuberculosis/microbiología , Genotipo , Salud Global , Humanos , Mycobacterium tuberculosis/aislamiento & purificación , Filogeografía , Tuberculosis/genéticaRESUMEN
OBJECTIVE: To determine the proportion of non-tuberculous mycobacteria (NTM) in samples of pulmonary tuberculosis (TB) cases from Papua New Guinea who were diagnosed using acid-fast microscopy. METHODS: As part of a case detection study for TB, conducted in three provincial hospitals in Papua New Guinea, sputum samples of suspected tuberculous cases aged 15 years or older were collected from November 2010 to July 2012. Mycobacterial species isolated from sputum and grown in culture were examined to distinguish between NTM and the Mycobacterium tuberculosis complex (MTBC). RESULTS: NTM were detected in 4% (9/225) of sputum samples grown in culture. Five (2.2%) of them were identified as NTM only and four (1.8%) were identified as mixed cultures containing both MTBC and NTM. Four different NTM species were identified; M. fortuitum, M. intracellulare, M. terrae and M. avium. DISCUSSION: This is the first report from Papua New Guinea identifying NTM in three different locations. As NTM cannot be distinguished from M. tuberculosis through smear microscopy, the presence of NTM can lead to a false-positive diagnosis of tuberculosis. The prevalence of NTM should be determined and a diagnostic algorithm developed to confirm acid-fast bacilli in a smear as M. tuberculosis.
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Infecciones por Mycobacterium no Tuberculosas/epidemiología , Micobacterias no Tuberculosas/aislamiento & purificación , Tuberculosis Pulmonar/microbiología , Adolescente , Adulto , Femenino , Humanos , Masculino , Microscopía/métodos , Persona de Mediana Edad , Papúa Nueva Guinea/epidemiología , Prevalencia , Tuberculosis Pulmonar/epidemiología , Adulto JovenRESUMEN
Little is known about the situation of tuberculosis in Papua New Guinea despite its high TB burden, emerging drug resistance and rising HIV co-infection. This review gives an overview on the current situation of TB in PNG and identifies knowledge gaps that should urgently be addressed in the future.