Visualization of the individual blood microbiome to study the etiology of sarcoidosis.

Introduction: Single microbial pathogens or host-microbiome dysbiosis are the causes of lung diseases with suspected infectious etiology. Metagenome sequencing provides an overview of the microbiome content. Due to the rarity of most granulomatous lung diseases collecting large systematic datasets is challenging. Thus, single-patient data often can only be summarized visually. Objective: To increase the information gain from a single-case metagenome analysis we suggest a quantitative and qualitative approach. Results: The 16S metagenomic results of 7 patients with pulmonary sarcoidosis were compared with those of 22 healthy individuals. From lysed blood, total microbial DNA was extracted and sequenced. Cleaned data reads were identified taxonomically using Kraken 2 software. Individual metagenomic data were visualized with a Sankey diagram, Krona chart, and a heat-map. We identified five genera that were exclusively present or significantly enhanced in patients with sarcoidosis - Veillonella, Prevotella, Cutibacterium, Corynebacterium, and Streptococcus. Conclusions: Our approach can characterize the blood microbiome composition and diversity in rare diseases at an individual level. Investigation of the blood microbiome in patients with granulomatous lung diseases of unknown etiology, such as sarcoidosis could enhance our comprehension of their origin and pathogenesis and potentially uncover novel personalized therapeutics.

Genome sequences of BCG Pasteur ATCC 35734 and its derivative, the vaccine candidate BCGΔBCG1419c.

BACKGROUND: Bacillus Calmette-Guérin (BCG) remains the only vaccine to prevent tuberculosis (TB) during childhood, with relatively low to no efficacy against pulmonary TB in adolescents and adults. BCG consists of close to 15 different substrains, where genetic variations among them might contribute to the variable protective efficacy afforded against pulmonary TB. We have shown that the vaccine candidate, BCGΔBCG1419c, which is based on BCG Pasteur, improved protection against chronic TB in murine models, as well as against pulmonary and extrapulmonary TB in guinea pigs. Here, to confirm deletion of the BCG1419c gene and to detect possible genetic variations occurring as a consequence of the spontaneous mutations that may arise during in vitro culture of mycobacteria, the genomes of BCG Pasteur ATCC 35734 and its isogenic derivative, BCGΔBCG1419c, were sequenced and subjected to a comparative analysis between them and against BCG Pasteur 1173P2. RESULTS: The complete catalog of variants in genes relative to the reference genome BCG Pasteur 1173P2 (GenBank NC008769) showed that the parental strain BCG Pasteur ATCC 35734, from which the mutant BCGΔBCG1419c originated, showed five synonymous mutations, three missense mutations, and five codon insertions, whereas the BCGΔBCG1419c mutant reported the same changes. When BCG Pasteur ATCC 35734 and BCGΔBCG1419c were compared, we confirmed that the latter was devoid of the BCG1419c gene, with only one unanticipated SNP at position 2, 828, 791  which we consider has no role in vaccine properties reported thus far. CONCLUSION: We provide evidence that the mutagenesis performed to remove BCG1419c from BCG Pasteur ATCC 35734 solely deleted this gene, and that compared with the reference strain BCG Pasteur 1173P2, few changes were present confirming that they are BCG Pasteur strains, and that changes in immunogenicity or efficacy observed thus far in BCGΔBCG1419c are most likely derived solely from the elimination of the BCG1419c gene.

Draft Genome Sequences of 77 Endemic Multidrug-Resistant Mycobacterium tuberculosis Strains of SIT41 (TUR) Spoligotype from Bulgaria.

Sequences of multidrug-resistant (MDR) Mycobacterium tuberculosis strains are of particular interest to study the molecular mechanisms of drug resistance evolution. Here, we report the draft genome sequences of 77 endemic multidrug-resistant Mycobacterium tuberculosis strains of SIT41 (TUR) spoligotype from Bulgaria. SIT41 spoligotype is dominant (>40%) among the MDR-TB strains in Bulgaria.

Biodiversity of Mycobacterium tuberculosis in Bulgaria Related to Human Migrations or Ecological Adaptation.

Bulgaria is among the 18 high-priority countries of the WHO European Region with high rates of tuberculosis. The causative agent of tuberculosis is thought to have emerged in Africa 70,000 years ago, or during the Neolithic age, and colonized the world through human migrations. The established main lineages of tuberculosis correlate highly with geography. The goal of our study was to investigate the biodiversity of Mycobacteriumtuberculosis in Bulgaria in association with human migration history during the last 10 centuries. We analyzed spoligotypes and MIRU-VNTR genotyping data of 655 drug-sensitive and 385 multidrug-resistant M. tuberculosis strains collected in Bulgaria from 2008 to 2018. We assigned the genotype of all isolates using SITVITWEB and MIRU-VNTRplus databases and software. We investigated the major well-documented historical events of immigration to Bulgaria that occurred during the last millennium. Genetic profiles demonstrated that, with the exceptions of 3 strains of Mycobacterium bovis and 18 strains of Lineage 2 (W/Beijing spoligotype), only Lineage 4 (Euro-American) was widely diffused in Bulgaria. Analysis of well-documented immigrations of Roma from the Indian subcontinent during the 10th to the 12th centuries, Turkic peoples from Central Asia in the medieval centuries, and more recently Armenians, Russians, and Africans in the 20th century influenced the biodiversity of M. tuberculosis in Bulgaria but only with genotypes of sublineages within the L4. We hypothesize that these sublineages were more virulent, or that ecological adaptation of imported M. tuberculosis genotypes was the main driver contributing to the current genetic biodiversity of M. tuberculosis in Bulgaria. We also hypothesize that some yet unknown local environmental factors may have been decisive in the success of imported genotypes. The ecological factors leading to local genetic biodiversity in M. tuberculosis are multifactorial and have not yet been fully clarified. The coevolution of long-lasting pathogen hosts should be studied, taking into account environmental and ecological changes.

Morphology of blood microbiota in healthy individuals assessed by light and electron microscopy.

Introduction: The blood microbiome is still an enigma. The existence of blood microbiota in clinically healthy individuals was proven during the last 50 years. Indirect evidence from radiometric analysis suggested the existence of living microbial forms in erythrocytes. Recently targeted nucleic acid sequencing demonstrated rich microbial biodiversity in the blood of clinically healthy individuals. The morphology and proliferation cycle of blood microbiota in peripheral blood mononuclear cells (PBMC) isolated from freshly drawn and cultured whole blood are obscure. Methods: To study the life cycle of blood microbiota we focused on light, and electron microscopy analysis. Peripheral blood mononuclear cells isolated from freshly drawn blood and stress-cultured lysed whole blood at 43°C in presence of vitamin K from healthy individuals were studied. Results: Here, we demonstrated that free circulating microbiota in the PMBC fraction possess a well-defined cell wall and proliferate by budding or through a mechanism similar to the extrusion of progeny bodies. By contrast, stress-cultured lysed whole blood microbiota proliferated as cell-wall deficient microbiota by forming electron-dense or electron-transparent bodies. The electron-dense bodies proliferated by fission or produce in chains Gram-negatively stained progeny cells or enlarged and burst to release progeny cells of 180 - 200 nm size. On the other hand, electron-transparent bodies enlarged and emitted progeny cells through the membrane. A novel proliferation mechanism of blood microbiota called by us "a cell within a cell" was observed. It combines proliferation of progeny cells within a progeny cell which is growing within the "mother" cell. Discussion: The rich biodiversity of eukaryotic and prokaryotic microbiota identified in blood by next-generation sequencing technologies and our microscopy results suggest different proliferation mechanisms in whole and cultured blood. Our documented evidence and conclusions provide a more comprehensive view of the existence of normal blood microbiota in healthy individuals.

Culturable and Non-Culturable Blood Microbiota of Healthy Individuals.

Next-generation sequencing (NGS) and metagenomics revolutionized our capacity for analysis and identification of the microbial communities in complex samples. The existence of a blood microbiome in healthy individuals has been confirmed by sequencing, but some researchers suspect that this is a cell-free circulating DNA in blood, while others have had isolated a limited number of bacterial and fungal species by culture. It is not clear what part of the blood microbiota could be resuscitated and cultured. Here, we quantitatively measured the culturable part of blood microbiota of healthy individuals by testing a medium supplemented with a high concentration of vitamin K (1 mg/mL) and culturing at 43 °C for 24 h. We applied targeted sequencing of 16S rDNA and internal transcribed spacer (ITS) markers on cultured and non-cultured blood samples from 28 healthy individuals. Dominant bacterial phyla among non-cultured samples were Proteobacteria 92.97%, Firmicutes 2.18%, Actinobacteria 1.74% and Planctomycetes 1.55%, while among cultured samples Proteobacteria were 47.83%, Firmicutes 25.85%, Actinobacteria 16.42%, Bacteroidetes 3.48%, Cyanobacteria 2.74%, and Fusobacteria 1.53%. Fungi phyla Basidiomycota, Ascomycota, and unidentified fungi were 65.08%, 17.72%, and 17.2% respectively among non-cultured samples, while among cultured samples they were 58.08%, 21.72%, and 20.2% respectively. In cultured and non-cultured samples we identified 241 OTUs belonging to 40 bacterial orders comprising 66 families and 105 genera. Fungal biodiversity accounted for 272 OTUs distributed in 61 orders, 105 families, and 133 genera. Bacterial orders that remained non-cultured, compared to blood microbiota isolated from fresh blood collection, were Sphingomonadales, Rhizobiales, and Rhodospirillales. Species of orders Bacillales, Lactobacillales, and Corynebacteriales showed the best cultivability. Fungi orders Tremellales, Polyporales, and Filobasidiales were mostly unculturable. Species of fungi orders Pleosporales, Saccharomycetales, and Helotiales were among the culturable ones. In this study, we quantified the capacity of a specific medium applied for culturing of blood microbiota in healthy individuals. Other culturing conditions and media should be tested for optimization and better characterization of blood microbiota in healthy and diseased individuals.

Complete Genome Sequence, Genome Stability and Phylogeny of the Vaccine Strain Mycobacterium bovis BCG SL222 Sofia.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only live attenuated vaccine available against tuberculosis. The first BCG vaccination was done exactly 100 years ago, in 1921. The BCG vaccine strains used worldwide represent a family of daughter sub-strains with distinct genotypic characteristics. BCG SL222 Sofia is a seed lot sub-strain descending from the Russian BCG-I (seed lot 374a) strain and has been used for vaccine production in Bulgaria since 1972. Here, we report the assembled circular genome sequence of Mycobacterium bovis BCG SL222 Sofia and phylogeny analysis with the most closely related BCG sub-strains. The full circular genome of BCG SL222 Sofia had a length of 4,370,706 bp with an average GC content of 65.60%. After 49 years of in vitro evolution in a freeze-dried condition, we identified four SNP mutations as compared to the reference BCG-I (Russia-368) sequence. BCG vaccination is of central importance for the TB elimination programs in many countries. Since 1991, almost 40 million vaccine doses of the BCG SL222 Sofia have been distributed annually through the United Nations Children's Fund (UNICEF) and the Pan American Health Organization (PAHO) to approximately 120 countries. The availability of the complete reference genome sequence for M. bovis BCG SL222 Sofia, a WHO reference reagent for the Russian BCG-I sub-strain, will facilitate the identity assurance of the genomic stability, will contribute to more consistent manufacturing, and has an important value in standardization and differentiation of sub-strains used in vaccine production. We propose to rename the sub-strain BCG SL222 Sofia to BCG-Sofia for practical and common use.

Complete Genome Sequence of Mycobacterium bovis BCG SL222 Sofia, the First WHO Reference Reagent for the M. bovis BCG Vaccine of the Russian BCG-I Substrain.

Mycobacterium bovis BCG SL222 Sofia is a substrain descending from the Russian BCG-I vaccine strain. Here, we report the complete genome sequence of BCG SL222 Sofia, which will facilitate identity assurance and will contribute to more consistent manufacturing, standardization, and differentiation of substrains used in vaccine production.

Author Correction: Genome-wide analysis of multi- and extensively drug-resistant Mycobacterium tuberculosis.

In the version of this article initially published, the URL listed for TubercuList was incorrect. The correct URL is https://mycobrowser.epfl.ch/. The error has been corrected in the HTML and PDF versions of the article.

Genome-wide analysis of multi- and extensively drug-resistant Mycobacterium tuberculosis.

To characterize the genetic determinants of resistance to antituberculosis drugs, we performed a genome-wide association study (GWAS) of 6,465 Mycobacterium tuberculosis clinical isolates from more than 30 countries. A GWAS approach within a mixed-regression framework was followed by a phylogenetics-based test for independent mutations. In addition to mutations in established and recently described resistance-associated genes, novel mutations were discovered for resistance to cycloserine, ethionamide and para-aminosalicylic acid. The capacity to detect mutations associated with resistance to ethionamide, pyrazinamide, capreomycin, cycloserine and para-aminosalicylic acid was enhanced by inclusion of insertions and deletions. Odds ratios for mutations within candidate genes were found to reflect levels of resistance. New epistatic relationships between candidate drug-resistance-associated genes were identified. Findings also suggest the involvement of efflux pumps (drrA and Rv2688c) in the emergence of resistance. This study will inform the design of new diagnostic tests and expedite the investigation of resistance and compensatory epistatic mechanisms.

Turkish and Japanese Mycobacterium tuberculosis sublineages share a remote common ancestor.

Two geographically distant M. tuberculosis sublineages, Tur from Turkey and T3-Osaka from Japan, exhibit partially identical genotypic signatures (identical 12-loci MIRU-VNTR profiles, distinct spoligotyping patterns). We investigated T3-Osaka and Tur sublineages characteristics and potential genetic relatedness, first using MIRU-VNTR locus analysis on 21 and 25 samples of each sublineage respectively, and second comparing Whole Genome Sequences of 8 new samples to public data from 45 samples uncovering human tuberculosis diversity. We then tried to date their Most Recent Common Ancestor (MRCA) using three calibrations of SNP accumulation rate (long-term=0.03SNP/genome/year, derived from a tuberculosis ancestor of around 70,000years old; intermediate=0.2SNP/genome/year derived from a Peruvian mummy; short-term=0.5SNP/genome/year). To disentangle between these scenarios, we confronted the corresponding divergence times with major human history events and knowledge on human genetic divergence. We identified relatively high intrasublineage diversity for both T3-Osaka and Tur. We definitively proved their monophyly; the corresponding super-sublineage (referred to as "T3-Osa-Tur") shares a common ancestor with T3-Ethiopia and Ural sublineages but is only remotely related to other Euro-American sublineages such as X, LAM, Haarlem and S. The evolutionary scenario based on long-term evolution rate being valid until T3-Osa-Tur MRCA was not supported by Japanese fossil data. The evolutionary scenario relying on short-term evolution rate since T3-Osa-Tur MRCA was contradicted by human history and potential traces of past epidemics. T3-Osaka and Tur sublineages were found likely to have diverged between 800y and 2000years ago, potentially at the time of Mongol Empire. Altogether, this study definitively proves a strong genetic link between Turkish and Japanese tuberculosis. It provides a first hypothesis for calibrating TB Euro-American lineage molecular clock; additional studies are needed to reliably date events corresponding to intermediate depths in tuberculosis phylogeny.

Recombination in pe/ppe genes contributes to genetic variation in Mycobacterium tuberculosis lineages.

BACKGROUND: Approximately 10% of the Mycobacterium tuberculosis genome is made up of two families of genes that are poorly characterized due to their high GC content and highly repetitive nature. The PE and PPE families are typified by their highly conserved N-terminal domains that incorporate proline-glutamate (PE) and proline-proline-glutamate (PPE) signature motifs. They are hypothesised to be important virulence factors involved with host-pathogen interactions, but their high genetic variability and complexity of analysis means they are typically disregarded in genome studies. RESULTS: To elucidate the structure of these genes, 518 genomes from a diverse international collection of clinical isolates were de novo assembled. A further 21 reference M. tuberculosis complex genomes and long read sequence data were used to validate the approach. SNP analysis revealed that variation in the majority of the 168 pe/ppe genes studied was consistent with lineage. Several recombination hotspots were identified, notably pe_pgrs3 and pe_pgrs17. Evidence of positive selection was revealed in 65 pe/ppe genes, including epitopes potentially binding to major histocompatibility complex molecules. CONCLUSIONS: This, the first comprehensive study of the pe and ppe genes, provides important insight into M. tuberculosis diversity and has significant implications for vaccine development.

A validated UPLC-MS/MS method for the surveillance of ten aquatic biotoxins in European brackish and freshwater systems.

Over the past few decades, there has been an increased frequency and duration of cyanobacterial Harmful Algal Blooms (HABs) in freshwater systems globally. These can produce secondary metabolites called cyanotoxins, many of which are hepatotoxins, raising concerns about repeated exposure through ingestion of contaminated drinking water or food or through recreational activities such as bathing/swimming. An ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) multi-toxin method has been developed and validated for freshwater cyanotoxins; microcystins-LR, -YR, -RR, -LA, -LY and -LF, nodularin, cylindrospermopsin, anatoxin-a and the marine diatom toxin domoic acid. Separation was achieved in around 9min and dual SPE was incorporated providing detection limits of between 0.3 and 5.6ng/L of original sample. Intra- and inter-day precision analysis showed relative standard deviations (RSD) of 1.2-9.6% and 1.3-12.0% respectively. The method was applied to the analysis of aquatic samples (n=206) from six European countries. The main class detected were the hepatotoxins; microcystin-YR (n=22), cylindrospermopsin (n=25), microcystin-RR (n=17), microcystin-LR (n=12), microcystin-LY (n=1), microcystin-LF (n=1) and nodularin (n=5). For microcystins, the levels detected ranged from 0.001 to 1.51µg/L, with two samples showing combined levels above the guideline set by the WHO of 1µg/L for microcystin-LR. Several samples presented with multiple toxins indicating the potential for synergistic effects and possibly enhanced toxicity. This is the first published pan European survey of freshwater bodies for multiple biotoxins, including two identified for the first time; cylindrospermopsin in Ireland and nodularin in Germany, presenting further incentives for improved monitoring and development of strategies to mitigate human exposure.

Two-Year Monitoring of Water Samples from Dam of Iskar and the Black Sea, Bulgaria, by Molecular Analysis: Focus on Mycobacterium spp.

The coast of the Bulgarian Black Sea is a popular summer holiday destination. The Dam of Iskar is the largest artificial dam in Bulgaria, with a capacity of 675 million m3. It is the main source of tap water for the capital Sofia and for irrigating the surrounding valley. There is a close relationship between the quality of aquatic ecosystems and human health as many infections are waterborne. Rapid molecular methods for the analysis of highly pathogenic bacteria have been developed for monitoring quality. Mycobacterial species can be isolated from waste, surface, recreational, ground and tap waters and human pathogenicity of nontuberculose mycobacteria (NTM) is well recognized. The objective of our study was to perform molecular analysis for key-pathogens, with a focus on mycobacteria, in water samples collected from the Black Sea and the Dam of Iskar. In a two year period, 38 water samples were collected-24 from the Dam of Iskar and 14 from the Black Sea coastal zone. Fifty liter water samples were concentrated by ultrafiltration. Molecular analysis for 15 pathogens, including all species of genus Mycobacterium was performed. Our results showed presence of Vibrio spp. in the Black Sea. Rotavirus A was also identified in four samples from the Dam of Iskar. Toxigenic Escherichia coli was present in both locations, based on markers for stx1 and stx2 genes. No detectable amounts of Cryptosporidium were detected in either location using immunomagnetic separation and fluorescence microscopy. Furthermore, mass spectrometry analyses did not detect key cyanobacterial toxins. On the basis of the results obtained we can conclude that for the period 2012-2014 no Mycobacterium species were present in the water samples. During the study period no cases of waterborne infections were reported.

Beijing lineage of MDR Mycobacterium tuberculosis in Bulgaria, 2007-2011.

To assess the spread of the Mycobacterium tuberculosis Beijing genotype among patients with multidrug-resistant and extensively resistant tuberculosis in Bulgaria, we genotyped 188 (72%) of 261 microbiologically confirmed resistant isolates obtained during 2007-2011. The estimated prevalence of the Beijing genotype among these patients was 3.2%.

Optimizing multiplex SNP-based data analysis for genotyping of Mycobacterium tuberculosis isolates.

BACKGROUND: Multiplex ligation-dependent probe amplification (MLPA) is a powerful tool to identify genomic polymorphisms. We have previously developed a single nucleotide polymorphism (SNP) and large sequence polymorphisms (LSP)-based MLPA assay using a read out on a liquid bead array to screen for 47 genetic markers in the Mycobacterium tuberculosis genome. In our assay we obtain information regarding the Mycobacterium tuberculosis lineage and drug resistance simultaneously. Previously we called the presence or absence of a genotypic marker based on a threshold signal level. Here we present a more elaborate data analysis method to standardize and streamline the interpretation of data generated by MLPA. The new data analysis method also identifies intermediate signals in addition to classification of signals as positive and negative. Intermediate calls can be informative with respect to identifying the simultaneous presence of sensitive and resistant alleles or infection with multiple different Mycobacterium tuberculosis strains. RESULTS: To validate our analysis method 100 DNA isolates of Mycobacterium tuberculosis extracted from cultured patient material collected at the National TB Reference Laboratory of the National Center for Tuberculosis and Lung Diseases in Tbilisi, Republic of Georgia were tested by MLPA. The data generated were interpreted blindly and then compared to results obtained by reference methods. MLPA profiles containing intermediate calls are flagged for expert review whereas the majority of profiles, not containing intermediate calls, were called automatically. No intermediate signals were identified in 74/100 isolates and in the remaining 26 isolates at least one genetic marker produced an intermediate signal. CONCLUSION: Based on excellent agreement with the reference methods we conclude that the new data analysis method performed well. The streamlined data processing and standardized data interpretation allows the comparison of the Mycobacterium tuberculosis MLPA results between different experiments. All together this will facilitate the implementation of the MLPA assay in different settings.

Tuberculosis-spoligo-rifampin-isoniazid typing: an all-in-one assay technique for surveillance and control of multidrug-resistant tuberculosis on Luminex devices.

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.

Combined species identification, genotyping, and drug resistance detection of Mycobacterium tuberculosis cultures by MLPA on a bead-based array.

The population structure of Mycobacterium tuberculosis is typically clonal therefore genotypic lineages can be unequivocally identified by characteristic markers such as mutations or genomic deletions. In addition, drug resistance is mainly mediated by mutations. These issues make multiplexed detection of selected mutations potentially a very powerful tool to characterise Mycobacterium tuberculosis. We used Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for dispersed mutations, which can be successfully applied to Mycobacterium tuberculosis as was previously shown. Here we selected 47 discriminative and informative markers and designed MLPA probes accordingly to allow analysis with a liquid bead array and robust reader (Luminex MAGPIX technology). To validate the bead-based MLPA, we screened a panel of 88 selected strains, previously characterised by other methods with the developed multiplex assay using automated positive and negative calling. In total 3059 characteristics were screened and 3034 (99.2%) were consistent with previous molecular characterizations, of which 2056 (67.2%) were directly supported by other molecular methods, and 978 (32.0%) were consistent with but not directly supported by previous molecular characterizations. Results directly conflicting or inconsistent with previous methods, were obtained for 25 (0.8%) of the characteristics tested. Here we report the validation of the bead-based MLPA and demonstrate its potential to simultaneously identify a range of drug resistance markers, discriminate the species within the Mycobacterium tuberculosis complex, determine the genetic lineage and detect and identify the clinically most relevant non-tuberculous mycobacterial species. The detection of multiple genetic markers in clinically derived Mycobacterium tuberculosis strains with a multiplex assay could reduce the number of TB-dedicated screening methods needed for full characterization. Additionally, as a proportion of the markers screened are specific to certain Mycobacterium tuberculosis lineages each profile can be checked for internal consistency. Strain characterization can allow selection of appropriate treatment and thereby improve treatment outcome and patient management.

"Spoligoriftyping," a dual-priming-oligonucleotide-based direct-hybridization assay for tuberculosis control with a multianalyte microbead-based hybridization system.

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.

Dynamics of the laboratory results in patients with pulmonary tuberculosis.

The process of infection and disease development is difficult to follow-up before tuberculosis (TB) confirmation. The laboratory analysis mirrors the infection with the possible subsequent breakdown to clinical TB. To better define the dynamics of the laboratory results in suspected and already confirmed patients with pulmonary TB, we studied the analysis of 1467 pathologic samples collected during the hospitalization of 841 patients. The samples were analyzed by 3 laboratory methods--direct microscopy, culture, and polymerase chain reaction (PCR). It was found that compared to cultures, the PCR method is more sensitive. For few cases, we demonstrate that the PCR result is positive about 2 weeks before the first positive culture. During the treatment follow-up, the PCR result remains positive for a long time, up to 4 to 5 months after the last positive culture. For better definition of the period during which microscopy and culture results remain positive, we studied the laboratory results of 100 casually selected patients with pulmonary TB positive on culture. The median periods during which these patients were found to be microscopy and culture positive were 10 and 25 days, respectively. Second to the dynamics of the laboratory results, we demonstrate that TB development is very rapid, whereas the period of recovery is long. The PCR results have to be reproducibly negative to accept that the process of active therapy is completed and the patient can remain under surveillance. On the basis of the laboratory data obtained, we propose empiric models for the dynamics of the laboratory results for patients with pulmonary tuberculosis.