Background Nigeria has the tenth highest burden of tuberculosis (TB) among the 22 TB high-burden countries in the world. This study describes the biodiversity and epidemiology of drug-susceptible and drug-resistant TB in Ibadan, Nnewi and Abuja, using 409 DNAs extracted from culture positive TB isolates. Methodology/Principal Findings DNAs extracted from clinical isolates of Mycobacterium tuberculosis complex were studied by spoligotyping and 24 VNTR typing. The Cameroon clade (CAM) was predominant followed by the M. africanum (West African 1) and T (mainly T2) clades. By using a smooth definition of clusters, 32 likely epi-linked clusters related to the Cameroon genotype family and 15 likely epi-linked clusters related to other “modern” genotypes were detected. Eight clusters concerned M. africanum West African 1. The recent transmission rate of TB was 38%. This large study shows that the recent transmission of TB in Nigeria is high, without major regional differences, with MDR-TB clusters. Improvement in the TB control programme is imperative to address the TB control problem in Nigeria.

e As a follow-up of the "spoligoriftyping" development, we present here an extension of this technique which includes the detection of isoniazid resistance-associated mutations in a new 59-plex assay, i.e., tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT), running on microbead-based multiplexed systems. This assay improves the synergy between clinical microbiology and epidemiology by providing (i) mutation-based prediction of drug resistance profiles for patient treatment and (ii) genotyping data for tuberculosis (TB) surveillance. This third-generation microbead-based high-throughput assay for TB runs on the Luminex 200 system and on the recently launched MagPix system (Luminex, Austin, TX). Spoligotyping patterns obtained by the TB-SPRINT method were 100% (n ‫؍‬ 85 isolates; 3,655/3,655 spoligotype data points) concordant with those obtained by microbead-based and membrane-based spoligotyping. Genetic drug susceptibility typing provided by the TB-SPRINT method was 100% concordant with resistance locus sequencing (n ‫؍‬ 162 for rpoB gene sequencing and n ‫؍‬ 76 for katG and inhA sequencing). Considering phenotypic drug susceptibility testing (DST) as the reference method, the sensitivity and specificity of TB-SPRINT regarding Mycobacterium tuberculosis complex (n ‫؍‬ 162 isolates) rifampin resistance were both 100%, and those for isoniazid resistance were 90.4% (95% confidence interval, 85 to 95%) and 100%, respectively. Used routinely in national TB reference and specialized laboratories, the TB-SPRINT assay should simultaneously improve personalized medicine and epidemiological surveillance of multidrug-resistant (MDR) TB. This assay is expected to play an emerging role in public health in countries with heavy burdens of MDR TB and/or HIV/TB coinfection. Application of this assay directly to biological samples, as well as development for extensively drug-resistant (XDR) TB detection by inclusion of second-line antituberculosis drug-associated mutations, is under development. With bioinformatical methods and data mining to reduce the number of targets to the most informative ones, locally adapted formats of this technique can easily be developed everywhere.

Two-step electrochemical patterning methods have been employed to elaborate composite nanomaterials formed with multiwalled carbon nanotubes (MWCNTs) coated with polypyrrole (PPy) and redox PAMAM dendrimers. The nanomaterial has been demonstrated as a molecular transducer for electrochemical DNA detection. The nanocomposite MWCNTs-PPy has been formed by wrapping the PPy film on MWCNTs during electrochemical polymerization of pyrrole on the gold electrode. The MWCNTs-PPy layer was modified with PAMAM dendrimers of fourth generation (PAMAM G4) with covalent bonding by electro-oxidation method. Ferrocenyl groups were then attached to the surface as a redox marker. The electrochemical properties of the nanomaterial (MWCNTs-PPy-PAMAM-Fc) were studied using both square wave voltammetry and cyclic voltammetry to demonstrate efficient electron transfer. The nanomaterial shows high performance in the electrochemical detection of DNA hybridization leading to a variation in the electrochemical signal of ferrocene with a detection limit of 0.3 fM. Furthermore, the biosensor demonstrates ability for sensing DNA of rpoB gene of Mycobacterium tuberculosis in real PCR samples. Developed biosensor was suitable for detection of sequences with a single nucleotide polymorphism (SNP) T (TCG/TTG), responsible for resistance of M. tuberculosis to rifampicin drug, and discriminating them from wild-type samples without such mutation. This shows potential of such systems for further application in pathogens diagnostic and therapeutic purpose.

Rio de Janeiro is endemic for tuberculosis (TB) and presents the second largest prevalence of the disease in Brazil. Here, we present the bacterial population structure of 218 isolates of Mycobacterium tuberculosis, derived from 186 patients that were diagnosed between January 2008 and December 2009. Genotypes were generated by means of spoligotyping, 24 MIRU-VNTR typing and presence of fbpC103, RDRio and RD174. The results confirmed earlier data that predominant genotypes in Rio de Janeiro are those of the Euro American Lineages (99%). However, we observed differences between the classification by spoligotyping when comparing to that of 24 MIRU-VNTR typing, being respectively 43.6% vs. 62.4% of LAM, 34.9% vs. 9.6% of T and 18.3% vs. 21.5% of Haarlem. Among isolates classified as LAM by MIRU typing, 28.0% did not present the characteristic spoligotype profile with absence of spacers 21 to 24 and 32 to 36 and we designated these conveniently as “LAM-like”, 79.3% of these presenting the LAM-specific SNP fbpC103. The frequency of RDRio and RD174 in the LAM strains, as defined both by spoligotyping and 24 MIRU-VNTR loci, were respectively 11% and 15.4%, demonstrating that RD174 is not always a marker for LAM/RDRio strains. We conclude that, although spoligotyping alone is a tool for classification of strains of the Euro-American lineage, when combined with MIRU-VNTRs, SNPs and RD typing, it leads to a much better understanding of the bacterial population structure and phylogenetic relationships among strains of M. tuberculosis in regions with high incidence of TB.

Tuberculosis (TB) remains one of the most threatening diseases and Nigeria has one of the world’s largest burdens. This study performed high-throughput spoligotyping directly on sputum smears to describe the Mycobacterium tuberculosis strains circulating among new TB patients in Nigeria.MethodsAll State TB control programmes in Nigeria were requested to submit 25–50 smear-positive Ziehl-Neelsen (ZN) stained slides for screening during 2013–2014. DNA was extracted from 929 slides for spoligotyping and drug-resistance analysis using microbead-based flow-cytometry suspension arrays.ResultsSpoligotyping results were obtained for 549 (59.1%) of 929 samples. Lineage 4 Cameroon sublineage (L4.6.2) represented half of the patterns, Mycobacterium africanum (L5 and L6) represented one fifth of the patterns, and all other lineages, including other L4 sublineages, represented one third of the patterns. Sublineage L4.6.2 was mostly identified in the north of the country whereas L5 was mostly observed in the south and L6 was scattered. The spatial distribution of genotypes had genetic geographic gradients. We did not obtain results enabling the detection of drug-resistance mutations.Conclusion/SignificanceWe present the first national snapshot of the M. tuberculosis spoligotypes circulating in Nigeria based on ZN slides. Spoligotyping data can be obtained in a rapid and high-throughput manner with DNA extracted from ZN-stained slides, which may potentially improve our understanding of the genetic epidemiology of TB.

Using Ziehl-Neelsen–positive slides collected from tuberculosis diagnostic centers in Burkina Faso, we showed that 20% of 80 spoligotyping-positive DNA samples had a characteristic Mycobacterium africanum–specific genomic signature. This result suggests that M. africanum is still present in Burkina Faso at almost the same prevalence as 15–20 years ago.

We developed “spoligoriftyping,” a 53-plex assay based on two preexisting methods, the spoligotyping and “rifoligotyping” assays, by combining them into a single assay. Spoligoriftyping allows simultaneous spoligotyping (i.e., clustered regularly interspaced short palindromic repeat [CRISPR]-based genotyping) and characterization of the main rifampin drug resistance mutations on the rpoB hot spot region in a few hours. This test partly uses the dual-priming-oligonucleotide (DPO) principle, which allows simultaneous efficient amplifications of rpoB and the CRISPR locus in the same sample. We tested this method on a set of 114 previously phenotypically and genotypically characterized multidrug-resistant (MDR) Mycobacterium tuberculosis or drug-susceptible M. tuberculosis DNA extracted from clinical isolates obtained from patients from Bulgaria, Nigeria, and Germany. We showed that our method is 100% concordant with rpoB sequencing results and 99.95% (3,911/3,913 spoligotype data points) correlated with classical spoligotyping results. The sensitivity and specificity of our assay were 99 and 100%, respectively, compared to those of phenotypic drug susceptibility testing. Such assays pave the way to the implementation of locally and specifically adapted methods of performing in a single tube both drug resistance mutation detection and genotyping in a few hours.

BackgroundWe aimed to characterize the genetic diversity of drug-resistant Mycobacterium tuberculosis (MTb) clinical isolates and investigate the molecular epidemiology of multidrug-resistant (MDR) tuberculosis from Minas Gerais State, Brazil.MethodsOne hundred and four MTb clinical isolates were assessed by IS6110-RFLP, 24-locus mycobacterial interspersed repetitive units variable-number tandem repeats (MIRU-VNTR), TB-SPRINT (simultaneous spoligotyping and rifampicin-isoniazid drug-resistance mutation analysis) and 3R-SNP-typing (analysis of single-nucleotide polymorphisms in the genes involved in replication, recombination and repair functions).ResultsFifty-seven different IS6110-RFLP patterns were found, among which 50 had unique patterns and 17 were grouped into seven clusters. The discriminatory index (Hunter and Gaston, HGDI) for RFLP was 0.9937. Ninety-nine different MIRU-VNTR patterns were found, 95 of which had unique patterns and nine isolates were grouped into four clusters. The major allelic diversity index in the MIRU-VNTR loci ranged from 0.6568 to 0.7789. The global HGDI for MIRU-VNTR was 0.9991. Thirty-two different spoligotyping profiles were found: 16 unique patterns (n = 16) and 16 clustered profiles (n = 88). The HGDI for spoligotyping was 0.9009. The spoligotyped clinical isolates were phylogenetically classified into Latin-American Mediterranean (66.34 %), T (14.42 %), Haarlem (5.76 %), X (1.92 %), S (1.92 %) and U (unknown profile; 8.65 %). Among the U isolates, 77.8 % were classified further by 3R-SNP-typing as 44.5 % Haarlem and 33.3 % LAM, while the 22.2 % remaining were not classified. Among the 104 clinical isolates, 86 were identified by TB-SPRINT as MDR, 12 were resistant to rifampicin only, one was resistant to isoniazid only, three were susceptible to both drugs, and two were not successfully amplified by PCR. A total of 42, 28 and eight isolates had mutations in rpoB positions 531, 526 and 516, respectively. Correlating the cluster analysis with the patient data did not suggest recent transmission of MDR-TB.ConclusionsAlthough our results do not suggest strong transmission of MDR-TB in Minas Gerais (using a classical 100 % MDR-TB identical isolates cluster definition), use of a smoother cluster definition (>85 % similarity) does not allow us to fully eliminate this possibility; hence, around 20–30 % of the isolates we analyzed might be MDR-TB transmission cases.