Multidrug-resistant patients receiving treatment in Niger who are infected with M. tuberculosis Cameroon family convert faster in smear and culture than those with M. tuberculosis Ghana family.

In this study, we analyzed the M. tuberculosis complex (MTBc) population structure among multidrug-resistant TB (MDR-TB) patients in Niger and tested whether the Cameroon family displayed a slower response to MDR-TB treatment. We genotyped baseline clinical isolates that had been collected from pulmonary MDR-TB patients recruited consecutively between 2008 and 2016 in Niger. Spoligotyping was used to analyze the genetic diversity of mycobacterial lineages, and Kaplan Meier's analysis to compare treatment outcomes. A total of 222 MTBc isolates were genotyped; 204 (91,9%) were identified as the Euro-American L4 lineage, with the Ghana family (106, 47,4%) and the Cameroon family (63, 28,4%) being predominant. Patients infected by Cameroon family isolates 61(96,8%) showed faster conversion (log-rank p < 0.01) than those infected with Ghana family isolates (91,5%), and were more likely to experience favorable outcome (adjusted odds ratio [aOR] 4.4; 95%CI 1.1-17.9]; p = 0.015). We found no association between MTBc families and second-line drug resistance profiles (p > 0.05). Our findings show that MDR-TB in Niger is caused by major spoligotypes of the Euro-American L4; with more rapid smear and culture conversion in patients infected with the Cameroon family. These first insights may alert clinicians that slow conversion may be associated with the type of infecting strain.

Isoniazid resistance levels of Mycobacterium tuberculosis can largely be predicted by high-confidence resistance-conferring mutations.

The majority of Mycobacterium tuberculosis isolates resistant to isoniazid harbour a mutation in katG. Since these mutations cause a wide range of minimum inhibitory concentrations (MICs), largely below the serum level reached with higher dosing (15 mg/L upon 15-20 mg/kg), the drug might still remain partly active in presence of a katG mutation. We therefore investigated which genetic mutations predict the level of phenotypic isoniazid resistance in clinical M. tuberculosis isolates. To this end, the association between known and unknown isoniazid resistance-conferring mutations in whole genome sequences, and the isoniazid MICs of 176 isolates was examined. We found mostly moderate-level resistance characterized by a mode of 6.4 mg/L for the very common katG Ser315Thr mutation, and always very high MICs (≥19.2 mg/L) for the combination of katG Ser315Thr and inhA c-15t. Contrary to common belief, isolates harbouring inhA c-15t alone, partly also showed moderate-level resistance, particularly when combined with inhA Ser94Ala. No overt association between low-confidence or unknown mutations, except in katG, and isoniazid resistance (level) was found. Except for the rare katG deletion, line probe assay is thus not sufficiently accurate to predict the level of isoniazid resistance for a single mutation in katG or inhA.

The predominance of Ethiopian specific Mycobacterium tuberculosis families and minimal contribution of Mycobacterium bovis in tuberculous lymphadenitis patients in Southwest Ethiopia.

BACKGROUND: Ethiopia has an extremely high rate of extrapulmonary tuberculosis, dominated by tuberculous lymphadenitis (TBLN). However, little is known about Mycobacterium tuberculosis complex (MTBc) lineages responsible for TBLN in Southwest Ethiopia. METHODS: A total of 304 MTBc isolates from TBLN patients in Southwest Ethiopia were genotyped primarily by spoligotyping. Isolates of selected spoligotypes were further analyzed by 15-loci mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) (n=167) and qPCR-based single nucleotide polymorphism (n=38). Isolates were classified into main phylogenetic lineages and families by using the reference strain collections and identification tools available at MIRU-VNTRplus data base. Resistance to rifampicin was determined by Xpert MTB/RIF. RESULTS: The majority of isolates (248; 81.6%) belonged to the Euro-American lineage (Lineage 4), with the ill-defined T and Haarlem as largest families comprising 116 (38.2%) and 43 (14.1%) isolates respectively. Of the T family, 108 isolates were classified as being part of the newly described Ethiopian families, namely Ethiopia_2 (n=44), Ethiopia_3 (n=34) and Ethiopia_H37Rv-like (n=30). Other sub-lineages included URAL (n=18), S (n=17), Uganda I (n=16), LAM (n=13), X (n=5), TUR (n=5), Uganda II (n=4) and unknown (n=19). Lineage 3 (Delhi/CAS) was the second most common lineage comprising 44 (14.5%) isolates. Interestingly, six isolates (2%) were belonged to Lineage 7, unique to Ethiopia. Lineage 1 (East-African Indian) and Lineage 2 (Beijing) were represented by 3 and 1 isolates respectively. M. bovis was identified in only two (0.7%) TBLN cases. The cluster rate was highest for Ethiopia_3 isolates showing clonal similarity with isolates from North Ethiopia. Lineage 3 was significantly associated with rifampicin resistance. CONCLUSIONS: In TBLN in Southwest Ethiopia, the recently described Ethiopia specific Lineage 4 families were predominant, followed by Lineage 3 and Lineage 4-Haarlem. The contribution of M. bovis in TBLN infection is minimal.

Correlation of different phenotypic drug susceptibility testing methods for four fluoroquinolones in Mycobacterium tuberculosis.

BACKGROUND: Molecular resistance testing fails to explain all fluoroquinolone resistance, with a continued need for a suitable rapid phenotypic drug susceptibility testing method. OBJECTIVE: To evaluate the optimal method for phenotypic fluoroquinolone susceptibility testing. METHODS: Using Löwenstein-Jensen medium, Middlebrook 7H11 agar, BACTEC-MGIT 960 and the resazurin microtitre plate assay, we determined susceptibility to fluoroquinolones in Mycobacterium tuberculosis and investigated cross-resistance between ofloxacin, levofloxacin, moxifloxacin and gatifloxacin. We compared MICs of all four fluoroquinolones for 91 strains on Löwenstein-Jensen (as the gold standard) with their MICs in resazurin plates, and with ofloxacin susceptibility at a single concentration in MGIT and on 7H11 agar, in addition to sequencing of the gyrAB genes. RESULTS AND CONCLUSIONS: Applying a cut-off of 2 mg/L ofloxacin, 1 mg/L levofloxacin and 0.5 mg/L moxifloxacin and gatifloxacin in all methods, some discordance between solid medium and MGIT methods was observed, yet this tended to be explained by MICs around the cut-off. The high discordance between Löwenstein-Jensen (LJ) and resazurin plates suggests that the currently applied cut-offs for all fluoroquinolones in the resazurin method should decrease and minor changes in colour (from blue to purple) be considered as meaningful. High-level resistance in all assays to all drugs correlated well with the presence of gyrA mutations, in support of recent findings that fluoroquinolone resistance should be tested at different concentrations, as patients with lower levels of resistance may continue to benefit from high-dose fluoroquinolone-based therapy.

The majority of patients with multidrug-resistant tuberculosis in Sub-Saharan Africa present a concomitant resistance to pyrazinamide.

OBJECTIVE/BACKGROUND: Pyrazinamide (PZA) is an antibacterial used in the first-line regimen against tuberculosis (TB) for its action against dormant bacilli. PZA is also included in the new short regimen to treat multidrug-resistant TB (MDR-TB). However, the prevalence and significance of PZA resistance is not known in Central and West Africa. METHODS: Between 2013 and 2016, we collected samples from MDR-TB patients recruited in an observational study implementing the new short MDR-TB regimen in six countries: Burundi (n=35), Cameroon (n=135), Niger (n=57), Central African Republic (n=35), Democratic Republic of the Congo (n=99), and Rwanda (n=16). Resistance to rifamipicine, isoniazide, injectables, and fluoroquinolones was tested by phenotypic (live strains) or genotypic methods (inactivated strains). Resistance to PZA was analyzed through sequencing of the pncA gene. Relevance of mutations was established based on recent literature. RESULTS: From 377 patients with MDR-TB, 354 (94%) samples were successfully sequenced. Among those, 53% (189) presented a mutation in pncA that confers a reported (121), potential (56), or unclear (12) resistance. Furthermore, six isolates presented a mutation associated with PZA sensitivity. The frequency of resistance per country was 26% in Central African Republic, 39% in Niger, 49% in Cameroon, 66% in Burundi, 68% in Democratic Republic of the Congo, and 87% in Rwanda. Isolates presented 109 different profiles of mutations, including 73 occurring only once. Codon 12 was most frequently affected (15 isolates), including 10 isolates with Asp12Ala. These 10 isolates came from three different countries, and presented different profiles of resistance to other drugs. The two next most frequent mutations, Met175Ile and Gln10Pro (8 isolates and 7 isolates, respectively), each suggest clusters of transmission, with similar geographical and resistance characteristics. Moreover, four isolates presented two simultaneous genetic variations, and 11 patients had a mix of sensitive and resistant bacilli. Preliminary data tend to indicate that patients carrying a PZA-resistant isolate had a higher failure rate on the new short MDR-TB treatment regimen (7% vs. 3%). All isolates resistant to injectables (4) and most (19/21) of those resistant to fluoroquinolones, including two extremely-resistant TB isolates, were also resistant to PZA. CONCLUSION: Similar to other regions in the world, the majority of MDR-TB strains from Sub-Saharan Africa countries are resistant to PZA, albeit with diverse rates between countries. We identified a diverse range of mutations in pncA, with 30% of them not previously reported. The impact of such resistance on the success of the short MDR-TB regimen will require more investigation.

First insights into circulating Mycobacterium tuberculosis complex lineages and drug resistance in Guinea.

In this study we assessed first-line anti-tuberculosis drug resistance and the genotypic distribution of Mycobacterium tuberculosis complex (MTBC) isolates that had been collected from consecutive new tuberculosis patients enrolled in two clinical trials conducted in Guinea between 2005 and 2010. Among the total 359 MTBC strains that were analyzed in this study, 22.8% were resistant to at least one of the first line anti-tuberculosis drugs, including 2.5% multidrug resistance and 17.5% isoniazid resistance, with or without other drugs. In addition, further characterization of isolates from a subset of the two trials (n = 184) revealed a total of 80 different spoligotype patterns, 29 "orphan" and 51 shared patterns. We identified the six major MTBC lineages of human relevance, with predominance of the Euro-American lineage. In total, 132 (71.7%) of the strains were genotypically clustered, and further analysis (using the DESTUS model) suggesting significantly faster spread of LAM10_CAM family (p = 0.00016). In conclusion, our findings provide a first insight into drug resistance and the population structure of the MTBC in Guinea, with relevance for public health scientists in tuberculosis control programs.

Shifts in Mycobacterial Populations and Emerging Drug-Resistance in West and Central Africa.

In this study, we retrospectively analysed a total of 605 clinical isolates from six West or Central African countries (Benin, Cameroon, Central African Republic, Guinea-Conakry, Niger and Senegal). Besides spoligotyping to assign isolates to ancient and modern mycobacterial lineages, we conducted phenotypic drug-susceptibility-testing for each isolate for the four first-line drugs. We showed that phylogenetically modern Mycobacterium tuberculosis strains are more likely associated with drug resistance than ancient strains and predict that the currently ongoing replacement of the endemic ancient by a modern mycobacterial population in West/Central Africa might result in increased drug resistance in the sub-region.

Pseudo-outbreak of pre-extensively drug-resistant (Pre-XDR) tuberculosis in Kinshasa: collateral damage caused by false detection of fluoroquinolone resistance by GenoType MTBDRsl.

Fluoroquinolones are the core drugs for the management of multidrug-resistant tuberculosis (MDR-TB). Molecular drug susceptibility testing methods provide considerable advantages for scaling up programmatic management and surveillance of drug-resistant TB. We describe here the misidentification of fluoroquinolone resistance by the GenoType MTBDRsl (MTBDRsl) (Hain Lifescience GmbH, Nehren, Germany) line probe assay (LPA) encountered during a feasibility and validation study for the introduction of this rapid drug susceptibility test in Kinshasa, Democratic Republic of Congo. The double gyrA mutation 80Ala and 90Gly represented 57% of all fluoroquinolone mutations identified from MDR-TB patient sputum samples, as confirmed by DNA sequencing. This double mutation was previously found to be associated with susceptibility to fluoroquinolones, yet it leads to absent hybridization of a wild-type band in the MTBDRsl and is thus falsely scored as resistance. Our findings suggest that MTBDRsl results must be interpreted with caution when the interpretation is based solely on the absence of a wild-type band without confirmation by visualization of a mutant band. Performance of the MTBDRsl LPA might be improved by replacing the gyrA wild-type probes by additional probes specific for well-documented gyrA mutations that confer clinically relevant resistance.

The first phylogeographic population structure and analysis of transmission dynamics of M. africanum West African 1--combining molecular data from Benin, Nigeria and Sierra Leone.

Mycobacterium africanum is an important cause of tuberculosis (TB) in West Africa. So far, two lineages called M. africanum West African 1 (MAF1) and M. africanum West African 2 (MAF2) have been defined. Although several molecular studies on MAF2 have been conducted to date, little is known about MAF1. As MAF1 is mainly present in countries around the Gulf of Guinea we aimed to estimate its prevalence in Cotonou, the biggest city in Benin. Between 2005-06 we collected strains in Cotonou/Benin and genotyped them using spoligo- and 12-loci-MIRU-VNTR-typing. Analyzing 194 isolates, we found that 31% and 6% were MAF1 and MAF2, respectively. Therefore Benin is one of the countries with the highest prevalence (37%) of M. africanum in general and MAF1 in particular. Moreover, we combined our data from Benin with publicly available genotyping information from Nigeria and Sierra Leone, and determined the phylogeographic population structure and genotypic clustering of MAF1. Within the MAF1 lineage, we identified an unexpected great genetic variability with the presence of at least 10 sub-lineages. Interestingly, 8 out of 10 of the discovered sub-lineages not only clustered genetically but also geographically. Besides showing a remarkable local restriction to certain regions in Benin and Nigeria, the sub-lineages differed dramatically in their capacity to transmit within the human host population. While identifying Benin as one of the countries with the highest overall prevalence of M. africanum, this study also contains the first detailed description of the transmission dynamics and phylogenetic composition of the MAF1 lineage.

An alternative, sensitive method to detect Helicobacter pylori DNA in feces.

BACKGROUND: Despite the high sensitivity and specificity of PCR, detection of Helicobacter pylori DNA in feces is still challenging. Fecal samples contain inhibitory molecules that can prevent amplification of the target DNA. Even by using specific DNA extraction kits for stools, monitoring of infection by analyzing stool samples remains problematic and endorses the need for improved diagnostic methods. MATERIALS AND METHODS: The newly proposed method uses selective hybridization of target DNA with biotin-labeled probes, followed by DNA isolation with streptavidin-coated magnetic beads. After three washing steps, the purified DNA can be amplified immediately using conventional or quantitative PCR. In order to test this technique on biological samples, Mongolian gerbils were infected with H. pylori ATCC 43504 and fecal samples were analyzed on days 1, 4, and 10 post infection. RESULTS: A detection limit of one bacterial cell per 100 mg stool sample was established, but only after removal of the magnetic beads from the target DNA by heating. This resulted in a 10-fold increase of sensitivity compared to a commercially available stool DNA extraction kit. Analysis of fecal samples from infected gerbils demonstrated the presence of H. pylori DNA on each time point, while the uninfected animal remained negative. CONCLUSIONS: The proposed technique allows detection of very low quantities of H. pylori DNA in biological samples. In laboratory animal models, detailed monitoring of infection and complete clearance of infection can be demonstrated thanks to the low detection limit.

Evaluation of the BD MGIT TBc Identification Test (TBc ID), a rapid chromatographic immunoassay for the detection of Mycobacterium tuberculosis complex from liquid culture.

The BACTEC MGIT 960 system is increasingly used to culture Mycobacterium tuberculosis. We evaluated the performance of the new immunochromatographic assay BD MGIT TBc Identification Test (TBc ID) for the rapid identification of M. tuberculosis complex in clinical samples when performed directly from BACTEC MGIT 960 culture positive for acid-fast bacilli (AFB). Of 92 cultures evaluated, the sensitivity and specificity of the TBc ID test was 98.5% and 100%, respectively compared to sequencing of the 16S rRNA gene. One culture that was TBc ID test negative but that was identified as M. tuberculosis by 16S rRNA sequencing was confirmed to have a mutation in the mpt64 gene. The TBc ID test is an easy and sensitive method for the identification of M. tuberculosis complex in liquid culture medium, does not require a high level of skills, neither any additional specific equipment and gives results in 15 min, which provide a good alternative for the rapid identification of M. tuberculosis complex in liquid medium.

High level of cross-resistance between kanamycin, amikacin, and capreomycin among Mycobacterium tuberculosis isolates from Georgia and a close relation with mutations in the rrs gene.

The aminoglycosides kanamycin and amikacin and the macrocyclic peptide capreomycin are key drugs for the treatment of multidrug-resistant tuberculosis (MDR-TB). The increasing rates of resistance to these drugs and the possible cross-resistance between them are concerns for MDR-TB therapy. Mutations in the 16S rRNA gene (rrs) have been associated with resistance to each of the drugs, and mutations of the tlyA gene, which encodes a putative rRNA methyltransferase, are thought to confer capreomycin resistance in Mycobacterium tuberculosis bacteria. Studies of possible cross-resistance have shown variable results. In this study, the MICs of these drugs for 145 clinical isolates from Georgia and the sequences of the rrs and tlyA genes of the isolates were determined. Of 78 kanamycin-resistant strains, 9 (11.5%) were susceptible to amikacin and 16 (20.5%) were susceptible to capreomycin. Four strains were resistant to capreomycin but were susceptible to the other drugs, whereas all amikacin-resistant isolates were resistant to kanamycin. Sequencing revealed six types of mutations in the rrs gene (A514C, C517T, A1401G, C1402T, C1443G, T1521C) but no mutations in the tlyA gene. The A514C, C517T, C1443G, and T1521C mutations showed no association with resistance to any of the drugs. The A1401G and C1402T mutations were observed in 65 kanamycin-resistant isolates and the 4 capreomycin-resistant isolates, respectively, whereas none of the susceptible isolates showed either of those mutations. The four mutants with the C1402T mutations showed high levels of resistance to capreomycin but no resistance to kanamycin and amikacin. Detection of the A1401G mutation appeared to be 100% specific for the detection of resistance to kanamycin and amikacin, while the sensitivities reached 85.9% and 94.2%, respectively.

First cultivation and characterization of Mycobacterium ulcerans from the environment.

BACKGROUND: Mycobacterium ulcerans disease, or Buruli ulcer (BU), is an indolent, necrotizing infection of skin, subcutaneous tissue and, occasionally, bones. It is the third most common human mycobacteriosis worldwide, after tuberculosis and leprosy. There is evidence that M. ulcerans is an environmental pathogen transmitted to humans from aquatic niches; however, well-characterized pure cultures of M. ulcerans from the environment have never been reported. Here we present details of the isolation and characterization of an M. ulcerans strain (00-1441) obtained from an aquatic Hemiptera (common name Water Strider, Gerris sp.) from Benin. METHODOLOGY/PRINCIPAL FINDINGS: One culture from a homogenate of a Gerris sp. in BACTEC became positive for IS2404, an insertion sequence with more than 200 copies in M. ulcerans. A pure culture of M. ulcerans 00-1441 was obtained on Löwenstein-Jensen medium after inoculation of BACTEC culture in mouse footpads followed by two other mouse footpad passages. The phenotypic characteristics of 00-1441 were identical to those of African M. ulcerans, including production of mycolactone A/B. The nucleotide sequence of the 5' end of 16S rRNA gene of 00-1441 was 100% identical to M. ulcerans and M. marinum, and the sequence of the 3' end was identical to that of the African type except for a single nucleotide substitution at position 1317. This mutation in M. ulcerans was recently discovered in BU patients living in the same geographic area. Various genotyping methods confirmed that strain 00-1441 has a profile identical to that of the predominant African type. Strain 00-1441 produced severe progressive infection and disease in mouse footpads with involvement of bone. CONCLUSION: Strain 00-1441 represents the first genetically and phenotypically identified strain of M. ulcerans isolated in pure culture from the environment. This isolation supports the concept that the agent of BU is a human pathogen with an environmental niche.

Application of the hsp65 PRA method for the rapid identification of mycobacteria isolated from clinical samples in Belgium.

Biochemical identification of mycobacteria is slow and many times fail to produce correct results. We compared PCR-restriction fragment length polymorphism analysis (PRA) of hsp65 and biochemical methods for the identification of mycobacteria from human samples in Belgium. PRA was found useful in the identification of mycobacteria and simple to implement as a quick method in the laboratory.

Implementation validation performed in Rwanda to determine whether the INNO-LiPA Rif.TB line probe assay can be used for detection of multidrug-resistant Mycobacterium tuberculosis in low-resource countries.

We validated the implementation of the INNO-LiPA Rif.TB line probe assay, a diagnostic test for rapid detection of multidrug-resistant tuberculosis (MDR-TB), in Rwanda. No substantial difference was found between results obtained in Rwanda and results obtained in Belgium with the same samples. This rapid diagnostic test for MDR-TB can therefore be reliably implemented in a resource-poor setting.

Endogenous reactivation and true treatment failure as causes of recurrent tuberculosis in a high incidence setting with a low HIV infection.

OBJECTIVE: To determine the relative frequencies of reinfection vs. reactivation or treatment failure in patients from a high tuberculosis incidence setting with a low prevalence of HIV infection. METHOD: We performed DNA fingerprinting on serial isolates from one and multiple TB episodes from 97 retreatment patients; 35 patients had been previously cured, whereas 62 had not. RESULTS: DNA fingerprinting patterns of recurrence Mycobacterium tuberculosis isolates of 5 of the 35 previously cured patients did not match with those of the corresponding initial isolates, indicating reinfection. We did not document reinfection during treatment. Isolates from each of the remaining 30 previously cured patients had identical DNA fingerprinting results, indicating reactivation. DNA fingerprinting patterns of isolates from the 62 patients with persistently positive sputum smears were identical, suggesting treatment failure. CONCLUSION: These findings suggest that reinfection is not a common cause of relapse and treatment failure in this rural predominantly HIV-free population despite the high incidence of TB.

A DNA sequence capture extraction method for detection of Mycobacterium avium subspecies paratuberculosis in feces and tissue samples.

Culturing of Mycobacterium avium subspecies paratuberculosis (Map) remains difficult and is time consuming. An alternative for the rapid detection of Map in samples is PCR. We have developed a sensitive DNA-extraction method based on sequence capture for the rapid detection of M. avium subspecies paratuberculosis by PCR in fecal and tissue samples. The method detected 10(2)Map/g feces using spiked samples, and reached a diagnostic sensitivity of 33,7% compared to 22% for culture. Analysis of tissue samples gave 65 polymerase chain reaction (PCR)-positive (42.2%) and 49 culture-positive samples (31.8%). Therefore, the detection limit of the DNA-extraction is the same as previously reported for culture, the PCR assay could detect more positive samples than the culture method.

Newly developed primers for comprehensive amplification of the rpoB gene and detection of rifampin resistance in Mycobacterium tuberculosis.

New rpoB gene primers for detecting Rif(r) in Mycobacterium tuberculosis complex bacteria achieved 100% specificity and 88% (fresh sputa) and 92% (ethanol-preserved sputa) diagnostic sensitivity and detected up to 4 CFU/sample. Of the 99 Rif(r) isolates examined, 97% had mutations within cluster I, 2% at codon 176, and 1% at codon 497.

Newly developed primers for the detection of Mycobacterium avium subspecies paratuberculosis.

Recent publications reported the existence of IS900 like sequences in mycobacteria different from Mycobacterium avium subspecies paratuberculosis (Map). The primers used for IS900 detection of Map have amplified these sequences causing false positive results. In this study, we have developed two new PCR assays for the detection of Map. The first assay is based on the IS900 sequence using primers different from the ones previously reported, the second assay on the f57 sequence. The specificity of the tests was checked by analysis of 190 mycobacterial isolates (74 Map and 116 non-Map isolates). All Map strains were positive and all non-Map strains were negative. Serial dilutions of Map bacteria were used to assess the sensitivity of the assays. We achieved a sensitivity of 1CFU per PCR for both assays. In addition, a PCR-simulating computer programme was used to evaluate the specificity of the new IS900 primers. The combination of the two PCR assays has proven to be useful for the identification of Map but validation on a large range of clinical samples still needs to be done.