Determinants of persistent symptoms after treatment for Lyme borreliosis: a prospective observational cohort study.

BACKGROUND: Patients treated for Lyme borreliosis (LB) frequently report persistent symptoms. Little is known about risk factors and etiology. METHODS: In a prospective observational cohort study with a follow-up of one year, we assessed a range of microbiological, immunological, genetic, clinical, functional, epidemiological, psychosocial and cognitive-behavioral variables as determinants of persistent symptoms after treatment for LB. Between 2015 and 2018 we included 1135 physician-confirmed LB patients at initiation of antibiotic therapy, through clinical LB centers and online self-registration. Two reference cohorts of individuals without LB (n = 4000 and n = 2405) served as a control. Prediction analyses and association studies were used to identify determinants, as collected from online questionnaires (three-monthly) and laboratory tests (twice). FINDINGS: Main predictors of persistent symptoms were baseline poorer physical and social functioning, higher depression and anxiety scores, more negative illness perceptions, comorbidity, as well as fatigue, cognitive impairment, and pain in 295 patients with persistent symptoms. The primary prediction model correctly indicated persistent symptoms in 71.0% of predictions (AUC 0.79). In patients with symptoms at baseline, cognitive-behavioral responses to symptoms predicted symptom persistence. Of various microbiological, immunological and genetic factors, only lower IL-10 concentrations in ex vivo stimulation experiments were associated with persistent symptoms. Clinical LB characteristics did not contribute to the prediction of persistent symptoms. INTERPRETATION: Determinants of persistent symptoms after LB were mainly generic, including baseline functioning, symptoms and cognitive-behavioral responses. A potential role of host immune responses remains to be investigated. FUNDING: Netherlands Organisation for Health Research and Development (ZonMw); the Dutch Ministry of Health, Welfare and Sport (VWS).

Diagnostic parameters of cellular tests for Lyme borreliosis in Europe (VICTORY study): a case-control study.

BACKGROUND: Cellular tests for Lyme borreliosis might be able to overcome major shortcomings of serological testing, such as its low sensitivity in early stages of infection. Therefore, we aimed to assess the sensitivity and specificity of three cellular tests. METHODS: This was a nationwide, prospective, multiple-gate case-control study done in the Netherlands. Patients with physician-confirmed Lyme borreliosis, either early localised or disseminated, were consecutively included as cases at the start of antibiotic treatment. Controls were those without Lyme borreliosis from the general population (healthy controls) and those with potentially cross-reactive conditions (eg, autoimmune disease). We used three cellular tests for Lyme borreliosis (Spirofind Revised, iSpot Lyme, and LTT-MELISA) as index tests, and standard two-tier serological testing (STTT) as a comparator. Clinical data from Lyme borreliosis patients were collected at baseline and at 12 weeks after inclusion, and blood samples were obtained at baseline, 6 weeks, and 12 weeks. Control participants underwent clinical and laboratory assessments at baseline only. FINDINGS: Cases comprised 271 patients with Lyme borreliosis (of whom 245 had early-localised Lyme borreliosis and 26 had disseminated disease) and controls comprised 228 participants without Lyme borreliosis from the general population and 41 participants with potentially cross-reactive conditions. Recruitment occurred between May 14, 2018, and March 16, 2020. The specificity of STTT in healthy controls (216 of 228 samples [94·7%, 95% CI 91·5-97·7]) was higher than that of the cellular tests: Spirofind (140 of 171 [81·9%, 76·1-87·2]), iSpot Lyme (32 of 103 [31·1%, 21·5-40·3]) and LTT-MELISA (100 of 190 [52·6%, 44·9-60·3]). Cellular tests had varying sensitivities: Spirofind (88 of 204 [43·1%, 36·4-50·4]), iSpot Lyme (51 of 94 [54·3%, 44·5-63·7]), and LTT-MELISA (66 of 218 [30·3%, 23·8-36·7]). The Spirofind and iSpot Lyme outperformed STTT for sensitivity, but were similar to the C6-ELISA (C6-ELISA: 135 of 270 [50·0%, 44·5-55·5]; STTT: 76 of 270 [28·1%, 23·0-33·6]). INTERPRETATION: The cellular tests for Lyme borreliosis used in this study have a low specificity compared with serological tests, which leads to a high number of false-positive test results. We conclude that these cellular tests are unfit for clinical use at this stage. FUNDING: Netherlands Organization for Health Research and Development, AMC Foundation (Amsterdam UMC), and Ministry of Health of the Netherlands.

Retrospective Evaluation of Various Serological Assays and Multiple Parameters for Optimal Diagnosis of Lyme Neuroborreliosis in a Routine Clinical Setting.

Laboratory diagnosis of Lyme neuroborreliosis (LNB) is challenging, and validated diagnostic algorithms are lacking. Therefore, this retrospective cross-sectional study aimed to compare the diagnostic performance of seven commercial antibody assays for LNB diagnosis. Random forest (RF) modeling was conducted to investigate whether the diagnostic performance using the antibody assays could be improved by including several routine cerebrospinal fluid (CSF) parameters (i.e., leukocyte count, total protein, blood-CSF barrier functionality, and intrathecal total antibody synthesis), two-tier serology on serum, the CSF level of the B-cell chemokine (C-X-C motif) ligand 13 (CXCL13), and a Borrelia species PCR on CSF. In total, 156 patients were included who were classified as definite LNB (n = 10), possible LNB (n = 7), or non-LNB patient (n = 139) according to the criteria of the European Federation of Neurological Societies using a consensus strategy for intrathecal Borrelia-specific antibody synthesis. The seven antibody assays showed sensitivities ranging from 47.1% to 100% and specificities ranging from 95.7% to 100%. RF modeling demonstrated that the sensitivities of most antibody assays could be improved by including other parameters to the diagnostic repertoire for diagnosing LNB (range: 94.1% to 100%), although with slightly lower specificities (range: 92.8% to 96.4%). The most important parameters for LNB diagnosis are the detection of intrathecally produced Borrelia-specific antibodies, two-tier serology on serum, CSF-CXCL13, Reibergram classification, and pleocytosis. In conclusion, this study shows that LNB diagnosis is best supported using multiparameter analysis. Furthermore, a collaborative prospective study is proposed to investigate if a standardized diagnostic algorithm can be developed for improved LNB diagnosis. IMPORTANCE The diagnosis of LNB is established by clinical symptoms, pleocytosis, and proof of intrathecal synthesis of Borrelia-specific antibodies. Laboratory diagnosis of LNB is challenging, and validated diagnostic algorithms are lacking. Therefore, this retrospective cross-sectional study aimed to compare the diagnostic performance of seven commercial antibody assays for LNB diagnosis. Multiparameter analysis was conducted to investigate whether the diagnostic performance using the antibody assays could be improved by including several routine (CSF) parameters. The results of this study show that LNB diagnosis is best supported using the detection of intrathecally produced Borrelia-specific antibodies, two-tier serology on serum, CSF-CXCL13, Reibergram classification, and pleocytosis. Furthermore, we propose a collaborative prospective study to investigate the potential role of constructing a diagnostic algorithm using multiparameter analysis for improved LNB diagnosis.

The Performance of Nine Commercial Serological Screening Assays for the Diagnosis of Lyme Borreliosis: a Multicenter Modified Two-Gate Design Study.

In this retrospective study, the performance of nine serological screening assays for Lyme borreliosis (LB) diagnostics was evaluated using a study population of LB cases and controls. Sera derived from 74 well-defined LB cases and 122 controls were included. The LB cases were diagnosed with erythema migrans (EM; n = 11), Lyme neuroborreliosis (LNB; n = 35), Lyme arthritis (LA; n = 20), or acrodermatitis chronica atrophicans (ACA; n = 8). Controls comprised 74 age- and gender-matched healthy individuals and 48 patients with other diseases with anticipated high rates of cross-reactivity. The assays under evaluation were selected based on a literature review and expected continued availability with CE marking under the new in vitro diagnostic regulation (European Union) 2017/746. The overall sensitivity (IgG and IgM results combined) among LB cases ranged between 54.5% (6 of 11) and 90.9% (10 of 11) for EM patients and between 97.1% (34 of 35) and 100% for patients with LNB, LA, and ACA. The positivity rate ranged between 8.1% (6 of 74) and 29.7% (22 of 74) among the healthy controls and between 22.9% (11 of 48) and 64.6% (31 of 48) among the cross-reactivity controls. The IgM results were more heterogeneous than the IgG and IgM/IgG results and did not contribute to the overall sensitivity but substantially increased the positivity rates among the controls. In conclusion, all evaluated Borrelia serological screening assays performed comparably with respect to early- and late-disseminated LB. The addition of an IgM assay to the screening of Borrelia-specific IgG antibodies had no added value for the diagnosis of Lyme borreliosis. IMPORTANCE Serology plays an important role in the diagnosis of Lyme borreliosis. Guidelines prescribe a two-tier testing algorithm in which a highly sensitive screening assay is used for screening and reactive sera are retested with an immunoblot to reduce false positivity rates. Recently, two commonly used screening assays were discontinued, including the very well-performing C6 Lyme enzyme-linked immunosorbent assay (ELISA) (Immunetics). This study provides an evaluation of the performance of nine different Borrelia serology screening assays, eight with expected future availably and the C6 Lyme ELISA, using a well-defined study panel of Lyme borreliosis patients, healthy population controls, and cross-reactivity controls. Evaluation data on multiple assays aid diagnostic laboratories in their choice for a reliable Borrelia serology screening assay to improve their diagnostic algorithm for Lyme borreliosis.

The Usefulness of Two CXCL13 Assays on Cerebrospinal Fluid for the Diagnosis of Lyme Neuroborreliosis: a Retrospective Study in a Routine Clinical Setting.

Recent studies have shown elevated levels of the B-cell chemokine (C-X-C motif) ligand 13 (CXCL13) in the cerebrospinal fluid (CSF) of patients with early Lyme neuroborreliosis (LNB). In this retrospective study, we evaluated the diagnostic performance of the Quantikine CXCL13 enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Inc., MN, USA) and the recomBead CXCL13 assay (Mikrogen, Neuried, Germany) for the detection of CXCL13 in CSF. All consecutive patients from whom a CSF and a serum sample had been collected between August 2013 and June 2016 were eligible for inclusion. Patients suspected of LNB were classified as definite, possible, or non-LNB according to the guidelines of the European Federation of Neurological Societies (EFNS). Due to the limited number of LNB patients in the predefined study period, additional LNB patients were included from outside this period. In total, 156 patients (150 consecutive patients and 6 additional LNB patients) were included. Seven (4.5%) were classified as definite, eight (5.1%) as possible, and 141 (90.4%) as non-LNB patients. Receiver operating characteristic (ROC) curve analysis comparing definite-LNB patients with non-LNB patients showed a cutoff value of 85.9 pg/ml for the Quantikine CXCL13 ELISA and 252.2 pg/ml for the recomBead CXCL13 assay. The corresponding sensitivity was 100% (95% confidence interval [CI], 100% to 100%) for both, and the corresponding specificities were 98.6% (95% CI, 96.5% to 100%) for the CXCL13 ELISA and 97.2% (95% CI, 93.6% to 100%) for the recomBead CXCL13 assay. This study showed that CXCL13 in CSF can be of additional value for the diagnosis of LNB.

The Simple One-Step (SOS) Stool Processing Method for Use with the Xpert MTB/RIF Assay for a Child-Friendly Diagnosis of Tuberculosis Closer to the Point of Care.

Young children cannot easily produce sputum for diagnosis of pulmonary tuberculosis (TB). Alternatively, Mycobacterium tuberculosis complex bacilli can be detected in stool by using the Xpert MTB/RIF (Ultra) assay (Xpert). Published stool processing methods contain somewhat complex procedures and require additional supplies. The aim of this study was to develop a simple one-step (SOS) stool processing method based on gravity sedimentation only, similar to Xpert testing of sputum samples, for the detection of M. tuberculosis in stool samples. We first assessed whether the SOS stool method could provide valid Xpert results without the need for bead-beating, dilution, and filtration steps. We concluded that this was the case, and we then validated the SOS stool method by testing spiked stool samples. By using the SOS stool method, 27 of the 29 spiked samples gave valid Xpert results, and M. tuberculosis was recovered from all 27 samples. The proof of principle of the SOS stool method was demonstrated in routine settings in Addis Ababa, Ethiopia. Nine of 123 children with presumptive TB had M. tuberculosis-positive results for nasogastric aspiration (NGA) samples, and 7 (77.8%) of those children also had M. tuberculosis-positive Xpert results for stool samples. Additionally, M. tuberculosis was detected in the stool samples but not the NGA samples from 2 children. The SOS stool processing method makes use of the standard Xpert assay kit, without the need for additional supplies or equipment. The method can potentially be rolled out to any Xpert site, bringing a bacteriologically confirmed diagnosis of TB in children closer to the point of care.

Consequences of the Edge Effect in a Commercial Enzyme-Linked Immunosorbent Assay for the Diagnosis of Lyme Neuroborreliosis.

The diagnosis of Lyme neuroborreliosis (LNB) is based on neurological symptoms, cerebrospinal fluid (CSF) pleocytosis, and intrathecally produced Borrelia-specific antibodies. In most cases, the presence of intrathecally produced Borrelia-specific antibodies is determined by using an enzyme-linked immunosorbent assay (ELISA). The edge effect is a known phenomenon in ELISAs and can negatively influence the assay reproducibility and repeatability, as well as index calculations of sample pairs which are tested in the same run. For LNB diagnostics, an index calculation is used for which the relative amounts of Borrelia-specific antibodies in CSF and serum are measured to calculate a CSF/serum quotient, which is needed to calculate the Borrelia-specific antibody index (AI). The presence of an edge effect in an ELISA used for LNB diagnostics may thus have implications. In this study, we investigated the intra-assay variation of the commercial Enzygnost Lyme link VlsE/IgG ELISA used for LNB diagnostics and showed the presence of an edge effect. Minor adaptations in the ELISA protocol decreased this effect. The adapted protocol was subsequently used to test 149 CSF-serum pairs of consecutive patients received in a routine diagnostic laboratory. By simulation, we showed that, if the standard protocol would have been used, then the edge effect for this study population could have resulted in 15 (10.1%) false-pathological and two (1.3%) false-normal Borrelia-specific IgG AIs. Thus, the observed edge effect can lead to inaccurate LNB diagnoses. Our study underlines that the edge effect should be investigated when ELISAs are implemented in routine diagnostics, as this phenomenon can occur in any ELISA.

Label-free vibrational imaging of different Aß plaque types in Alzheimer's disease reveals sequential events in plaque development.

The neuropathology of Alzheimer's disease (AD) is characterized by hyperphosphorylated tau neurofibrillary tangles (NFTs) and amyloid-beta (Aß) plaques. Aß plaques are hypothesized to follow a development sequence starting with diffuse plaques, which evolve into more compact plaques and finally mature into the classic cored plaque type. A better molecular understanding of Aß pathology is crucial, as the role of Aß plaques in AD pathogenesis is under debate. Here, we studied the deposition and fibrillation of Aß in different plaque types with label-free infrared and Raman imaging. Fourier-transform infrared (FTIR) and Raman imaging was performed on native snap-frozen brain tissue sections from AD cases and non-demented control cases. Subsequently, the scanned tissue was stained against Aß and annotated for the different plaque types by an AD neuropathology expert. In total, 160 plaques (68 diffuse, 32 compact, and 60 classic cored plaques) were imaged with FTIR and the results of selected plaques were verified with Raman imaging. In diffuse plaques, we detect evidence of short antiparallel ß-sheets, suggesting the presence of Aß oligomers. Aß fibrillation significantly increases alongside the proposed plaque development sequence. In classic cored plaques, we spatially resolve cores containing predominantly large parallel ß-sheets, indicating Aß fibrils. Combining label-free vibrational imaging and immunohistochemistry on brain tissue samples of AD and non-demented cases provides novel insight into the spatial distribution of the Aß conformations in different plaque types. This way, we reconstruct the development process of Aß plaques in human brain tissue, provide insight into Aß fibrillation in the brain, and support the plaque development hypothesis.

Accurate serology for SARS-CoV-2 and common human coronaviruses using a multiplex approach.

Serology is a crucial part of the public health response to the ongoing SARS-CoV-2 pandemic. Here, we describe the development, validation and clinical evaluation of a protein micro-array as a quantitative multiplex immunoassay that can identify S and N-directed SARS-CoV-2 IgG antibodies with high specificity and sensitivity and distinguish them from all currently circulating human coronaviruses. The method specificity was 100% for SARS-CoV-2 S1 and 96% for N antigen based on extensive syndromic (n=230 cases) and population panel (n=94) testing that also confirmed the high prevalence of seasonal human coronaviruses. To assess its potential role for both SARS-CoV-2 patient diagnostics and population studies, we evaluated a large heterogeneous COVID-19 cohort (n=330) and found an overall sensitivity of 89% (≥ 21 days post onset symptoms (dps)), ranging from 86% to 96% depending on severity of disease. For a subset of these patients longitudinal samples were provided up to 56 dps. Mild cases showed absent or delayed, and lower SARS-CoV-2 antibody responses. Overall, we present the development and extensive clinical validation of a multiplex coronavirus serological assay for syndromic testing, to answer research questions regarding to antibody responses, to support SARS-CoV-2 diagnostics and to evaluate epidemiological developments efficiently and with high-throughput.

Evaluation of Disease Causality of Rare Ixodes ricinus-Borne Infections in Europe.

In Europe, Ixodes ricinus ticks transmit pathogens such as Borrelia burgdorferi sensu lato and tick-borne encephalitis virus (TBEV). In addition, there is evidence for transmission to humans from I. ricinus of Anaplasma phagocytophilum, Babesia divergens, Babesia microti, Babesia venatorum, Borrelia miyamotoi, Neoehrlichia mikurensis, Rickettsia helvetica and Rickettsia monacensis. However, whether infection with these potential tick-borne pathogens results in human disease has not been fully demonstrated for all of these tick-borne microorganisms. To evaluate the available evidence for a causative relation between infection and disease, the current study analyses European case reports published from 2008 to 2018, supplemented with information derived from epidemiological and experimental studies. The evidence for human disease causality in Europe found in this review appeared to be strongest for A. phagocytophilum and B. divergens. Nonetheless, some knowledge gaps still exist. Importantly, comprehensive evidence for pathogenicity is lacking for the remaining tick-borne microorganisms. Such evidence could be gathered best through prospective studies, for example, studies enrolling patients with a fever after a tick bite, the development of specific new serological tools, isolation of these microorganisms from ticks and patients and propagation in vitro, and through experimental studies.

Prevalence and determinants of persistent symptoms after treatment for Lyme borreliosis: study protocol for an observational, prospective cohort study (LymeProspect).

BACKGROUND: After antibiotic treatment of Lyme borreliosis, a subset of patients report persistent symptoms, also referred to as post-treatment Lyme disease syndrome. The reported prevalence of persistent symptoms varies considerably, and its pathophysiology is under debate. The LymeProspect study has been designed to investigate the prevalence, severity, and a wide range of hypotheses on the etiology of persistent symptoms among patients treated for Lyme borreliosis in the Netherlands. METHODS: LymeProspect is a prospective, observational cohort study among adults with proven or probable Lyme borreliosis, either erythema migrans or disseminated manifestations, included at the start of antibiotic treatment. During one year of follow-up, participants are subjected to questionnaires every three months and blood is collected repeatedly during the first three months. The primary outcome is the prevalence of persistent symptoms after treatment, assessed by questionnaires online focusing on fatigue (CIS, subscale fatigue severity), pain (SF-36, subscale pain) and neurocognitive dysfunction (CFQ). Potential microbiological, immunological, genetic, epidemiological and cognitive-behavioral determinants for persistent symptoms are secondary outcome measures. Control cohorts include patients with long-lasting symptoms and unconfirmed Lyme disease, population controls, and subjects having reported a tick bite not followed by Lyme borreliosis. DISCUSSION: This article describes the background and design of the LymeProspect study protocol. This study is characterized by a prospective, explorative and multifaceted design. The results of this study will provide insights into the prevalence and determinants of persistent symptoms after treatment for Lyme borreliosis, and may provide a rationale for preventive and treatment recommendations. TRIAL REGISTRATION: NTR4998 (Netherlands Trial Register). Date of registration: 13 February 2015.

CD1b presents self and Borrelia burgdorferi diacylglycerols to human T cells.

Lyme disease is a common multisystem disease caused by infection with a tick-transmitted spirochete, Borrelia burgdorferi and related Borrelia species. The monoglycosylated diacylglycerol known as B. burgdorferi glycolipid II (BbGL-II) is a major target of antibodies in sera from infected individuals. Here, we show that CD1b presents BbGL-II to human T cells and that the TCR mediates the recognition. However, we did not detect increased frequency of CD1b-BbGL-II binding T cells in the peripheral blood of Lyme disease patients compared to controls. Unexpectedly, mapping the T cell specificity for BbGL-II-like molecules using tetramers and activation assays revealed a concomitant response to CD1b-expressing APCs in absence of BbGL-II. Further, among all major classes of self-lipid tested, BbGL-II responsive TCRs show strong cross-reactivity to diacylglycerol, a self-lipid antigen with structural similarities to BbGL-II. Extending prior work on MHC and CD1b, CD1c, and CD1d proteins, this study provides evidence for cross-reactive CD1b-restricted T cell responses to bacterial and self-antigens, and identifies chemically defined targets for future discovery of self and foreign antigen cross-reactive T cells.

Genomics and Machine Learning for Taxonomy Consensus: The Mycobacterium tuberculosis Complex Paradigm.

Infra-species taxonomy is a prerequisite to compare features such as virulence in different pathogen lineages. Mycobacterium tuberculosis complex taxonomy has rapidly evolved in the last 20 years through intensive clinical isolation, advances in sequencing and in the description of fast-evolving loci (CRISPR and MIRU-VNTR). On-line tools to describe new isolates have been set up based on known diversity either on CRISPRs (also known as spoligotypes) or on MIRU-VNTR profiles. The underlying taxonomies are largely concordant but use different names and offer different depths. The objectives of this study were 1) to explicit the consensus that exists between the alternative taxonomies, and 2) to provide an on-line tool to ease classification of new isolates. Genotyping (24-VNTR, 43-spacers spoligotypes, IS6110-RFLP) was undertaken for 3,454 clinical isolates from the Netherlands (2004-2008). The resulting database was enlarged with African isolates to include most human tuberculosis diversity. Assignations were obtained using TB-Lineage, MIRU-VNTRPlus, SITVITWEB and an algorithm from Borile et al. By identifying the recurrent concordances between the alternative taxonomies, we proposed a consensus including 22 sublineages. Original and consensus assignations of the all isolates from the database were subsequently implemented into an ensemble learning approach based on Machine Learning tool Weka to derive a classification scheme. All assignations were reproduced with very good sensibilities and specificities. When applied to independent datasets, it was able to suggest new sublineages such as pseudo-Beijing. This Lineage Prediction tool, efficient on 15-MIRU, 24-VNTR and spoligotype data is available on the web interface "TBminer." Another section of this website helps summarizing key molecular epidemiological data, easing tuberculosis surveillance. Altogether, we successfully used Machine Learning on a large dataset to set up and make available the first consensual taxonomy for human Mycobacterium tuberculosis complex. Additional developments using SNPs will help stabilizing it.

Epidemiology of isoniazid resistance mutations and their effect on tuberculosis treatment outcomes.

Isoniazid resistance is highly prevalent in Vietnam. We investigated the molecular and epidemiological characteristics and the association with first-line treatment outcomes of the main isoniazid resistance mutations in Mycobacterium tuberculosis in codon 315 of the katG and in the promoter region of the inhA gene. Mycobacterium tuberculosis strains with phenotypic resistance to isoniazid from consecutively diagnosed smear-positive tuberculosis patients in rural Vietnam were subjected to Genotype MTBDRplus testing to identify katG and inhA mutations. Treatment failure and relapse were determined by sputum culture. In total, 227 of 251 isoniazid-resistant strains (90.4%) had detectable mutations: 75.3% in katG codon 315 (katG315) and 28.2% in the inhA promoter region. katG315 mutations were significantly associated with pretreatment resistance to streptomycin, rifampin, and ethambutol but not with the Beijing genotype and predicted both unfavorable treatment outcome (treatment failure or death) and relapse; inhA promoter region mutations were only associated with resistance to streptomycin and relapse. In tuberculosis patients, M. tuberculosis katG315 mutations but not inhA mutations are associated with unfavorable treatment outcome. inhA mutations do, however, increase the risk of relapse, at least with treatment regimens that contain only isoniazid and ethambutol in the continuation phase.

Inferring patient to patient transmission of Mycobacterium tuberculosis from whole genome sequencing data.

BACKGROUND: Mycobacterium tuberculosis is characterised by limited genomic diversity, which makes the application of whole genome sequencing particularly attractive for clinical and epidemiological investigation. However, in order to confidently infer transmission events, an accurate knowledge of the rate of change in the genome over relevant timescales is required. METHODS: We attempted to estimate a molecular clock by sequencing 199 isolates from epidemiologically linked tuberculosis cases, collected in the Netherlands spanning almost 16 years. RESULTS: Multiple analyses support an average mutation rate of ~0.3 SNPs per genome per year. However, all analyses revealed a very high degree of variation around this mean, making the confirmation of links proposed by epidemiology, and inference of novel links, difficult. Despite this, in some cases, the phylogenetic context of other strains provided evidence supporting the confident exclusion of previously inferred epidemiological links. CONCLUSIONS: This in-depth analysis of the molecular clock revealed that it is slow and variable over short time scales, which limits its usefulness in transmission studies. However, the superior resolution of whole genome sequencing can provide the phylogenetic context to allow the confident exclusion of possible transmission events previously inferred via traditional DNA fingerprinting techniques and epidemiological cluster investigation. Despite the slow generation of variation even at the whole genome level we conclude that the investigation of tuberculosis transmission will benefit greatly from routine whole genome sequencing.

Tuberculosis relapse in Vietnam is significantly associated with Mycobacterium tuberculosis Beijing genotype infections.

BACKGROUND: In Vietnam, the Mycobacterium tuberculosis Beijing genotype is associated with multi-drug resistance and is emerging. A possible explanation for this genotype's success is an increased rate of relapse. METHODS: In a prospective cohort study, isolates from patients with smear-positive tuberculosis were subjected to drug susceptibility testing and to spoligotyping and variable number of tandem repeats typing before treatment and after recurrence of tuberculosis. RESULTS: Among 1068 patients who were actively followed up over 18 months for recurrence, 23 relapse cases occurred (1.39 cases/100 person-years). After adjustment for genotype, tuberculosis treatment history, and drug resistance, relapse was significantly associated with the Beijing genotype (adjusted hazard ratio [aHR], 5.48; 95% confidence interval [CI], 2.06-14.55) and isoniazid resistance (aHR, 5.91; 95% CI, 2.16-16.16). CONCLUSIONS: The strongly increased relapse rate in tuberculosis cases caused by Beijing strains probably contributes to the successful spread of this genotype in Vietnam and elsewhere.

Comparative study of IS6110 restriction fragment length polymorphism and variable-number tandem-repeat typing of Mycobacterium tuberculosis isolates in the Netherlands, based on a 5-year nationwide survey.

In order to switch from IS6110 and polymorphic GC-rich repetitive sequence (PGRS) restriction fragment length polymorphism (RFLP) to 24-locus variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates in the national tuberculosis control program in The Netherlands, a detailed evaluation on discriminatory power and agreement with findings in a cluster investigation was performed on 3,975 tuberculosis cases during the period of 2004 to 2008. The level of discrimination of the two typing methods did not differ substantially: RFLP typing yielded 2,733 distinct patterns compared to 2,607 in VNTR typing. The global concordance, defined as isolates labeled unique or identically distributed in clusters by both methods, amounted to 78.5% (n = 3,123). Of the remaining 855 cases, 12% (n = 479) of the cases were clustered only by VNTR, 7.7% (n = 305) only by RFLP typing, and 1.8% (n = 71) revealed different cluster compositions in the two approaches. A cluster investigation was performed for 87% (n = 1,462) of the cases clustered by RFLP. For the 740 cases with confirmed or presumed epidemiological links, 92% were concordant with VNTR typing. In contrast, only 64% of the 722 cases without an epidemiological link but clustered by RFLP typing were also clustered by VNTR typing. We conclude that VNTR typing has a discriminatory power equal to IS6110 RFLP typing but is in better agreement with findings in a cluster investigation performed on an RFLP-clustering-based cluster investigation. Both aspects make VNTR typing a suitable method for tuberculosis surveillance systems.

Clustering of Beijing genotype Mycobacterium tuberculosis isolates from the Mekong delta in Vietnam on the basis of variable number of tandem repeat versus restriction fragment length polymorphism typing.

BACKGROUND: In comparison to restriction fragment length polymorphism (RFLP) typing, variable number of tandem repeat (VNTR) typing is easier to perform, faster and yields results in a simple, numerical format. Therefore, this technique has gained recognition as the new international gold standard in typing of Mycobacterium tuberculosis. However, some reports indicated that VNTR typing may be less suitable for Beijing genotype isolates. We therefore compared the performance of internationally standardized RFLP and 24 loci VNTR typing to discriminate among 100 Beijing genotype isolates from the Southern Vietnam. METHODS: Hundred Beijing genotype strains defined by spoligotyping were randomly selected and typed by RFLP and VNTR typing. The discriminatory power of VNTR and RFLP typing was compared using the Bionumerics software. RESULTS: Among 95 Beijing strains available for analysis, 14 clusters were identified comprising 34 strains and 61 unique profiles in 24 loci VNTR typing ((Hunter Gaston Discrimination Index (HGDI = 0.994)). 13 clusters containing 31 strains and 64 unique patterns in RFLP typing (HGDI = 0.994) were found. Nine RFLP clusters were subdivided by VNTR typing and 12 VNTR clusters were split by RFLP. Five isolates (5%) revealing double alleles or no signal in two or more loci in VNTR typing could not be analyzed. CONCLUSIONS: Overall, 24 loci VNTR typing and RFLP typing had similar high-level of discrimination among 95 Beijing strains from Southern Vietnam. However, loci VNTR 154, VNTR 2461 and VNTR 3171 had hardly added any value to the level of discrimination.

Drug-resistant tuberculosis in the WHO European Region: an analysis of surveillance data.

To review the latest information about levels of anti-tuberculosis (TB) drug resistance in the European Region of the World Health Organization (WHO) and time-trends in multidrug-resistant TB (resistance to isoniazid and rifampicin; MDR-TB) over the past fifteen years. We analysed data on drug resistance among new and previously treated TB cases reported from 1997 to 2012. Data are collected in surveys of representative samples of TB patients or from surveillance systems based on diagnostic drug susceptibility testing. A total of 15.7% (95% confidence limits (CI): 9.5-21.9) of new and 45.3% (95%CI: 39.2-51.5) of previously treated TB cases are estimated to have MDR-TB in the Region. Extensively drug-resistant TB (MDR-TB and resistance to fluoroquinolones and second-line injectables; XDR-TB) had been reported by 38 of the 53 countries of the region (72%). The proportion of MDR-TB cases with XDR-TB is 11.4% (95%CI: 8.6-14.2). Between 1997 and 2012, population rates of MDR-TB declined in Estonia, Latvia and Germany and increased in the United Kingdom, Sweden and Tomsk Oblasts of the Russian Federation. Surveillance of drug resistance has been strengthened in the WHO European Region, which has the highest proportions of MDR-TB and XDR-TB ever reported globally. More complete data are needed particularly from the Russian Federation.