Epidemiological and clinical insights into the enterovirus D68 upsurge in Europe 2021/22 and the emergence of novel B3-derived lineages, ENPEN multicentre study.

Enterovirus D68 (EV-D68) infections are associated with severe respiratory disease and acute flaccid myelitis (AFM). The European Non-Polio Enterovirus Network (ENPEN) aimed to investigate the epidemiological and genetic characteristics of EV-D68 and its clinical impact during the fall-winter season of 2021/22. From 19 European countries, 58 institutes reported 10,481 (6.8%) EV-positive samples of which 1,004 (9.6%) were identified as EV-D68 (852 respiratory samples). Clinical data was reported for 969 cases. 78.9% of infections were reported in children (0-5 years); 37.9% of cases were hospitalised. Acute respiratory distress was commonly noted (93.1%) followed by fever (49.4%). Neurological problems were observed in 6.4% of cases with six reported with AFM. Phylodynamic/Nextstrain and phylogenetic analyses based on 694 sequences showed the emergence of two novel B3-derived lineages, with no regional clustering. In conclusion, we describe a large-scale EV-D68 European upsurge with severe clinical impact and the emergence of B3-derived lineages.

What Do We Know About the Health Status of Asylum Seekers in the United States?: Identifying Research Gaps Following a Bibliometric Scoping Review of Existing Literature.

Data about US asylum seekers' health are lacking. We assessed the medical literature by performing a bibliometric scoping review of the literature from 1946 to 2020. Of 114 articles identified, 48 (42.1%) were empirical studies and 66 (57.9%) were editorials or commentaries. Empirical studies focused on mental health (60.42%), African asylees (45.83%). Editorials and commentaries focused on detention and medicolegal processes (31.82% and 30.3%). Empirical data on the health of US asylum seekers are limited. Research expansion requires increased commitment, funding, and participatory engagement of asylees. This limited representation in the scientific literature can impact their care and health system preparedness.

Host DNA depletion can increase the sensitivity of Mycobacterium spp. detection through shotgun metagenomics in sputum.

Identification and phenotypic drug-susceptibility testing for mycobacteria are time-consuming and challenging but essential for managing mycobacterial infections. Next-generation sequencing (NGS) technologies can increase diagnostic speed and quality, but standardization is still lacking for many aspects (e.g., unbiased extraction, host depletion, bioinformatic analysis). Targeted PCR approaches directly on sample material are limited by the number of targets that can be included. Unbiased shotgun metagenomics on direct material is hampered by the massive amount of host DNA, which should be removed to improve the microbial detection sensitivity. For this reason, we developed a method for NGS-based diagnosis of mycobacteria directly from patient material. As a model, we used the non-tuberculous mycobacterium (NTM) Mycobacterium abscessus. We first compared the efficiency of three different DNA extraction kits for isolating DNA (quality and concentration). The two most efficient kits were then used in a follow-up study using artificial sputum. Finally, one extraction kit was selected and further evaluated for DNA isolation from a patients' sputum mixture spiked with M. abscessus at three concentrations (final concentrations 108, 107, 106 CFU/ml). The spiked sputum samples were processed with and without saponin treatment (ST) in combination with DNAse treatment prior to bacterial DNA extraction to evaluate the recovery of bacteria and depletion of host DNA by PCR and Illumina sequencing. While Ct values of the qPCR targeting mycobacterial ITS DNA remained rather stable, Ct values in the qPCR targeting the human ß-actin gene increased by five Ct values in ST samples. In subsequent Illumina sequencing, a decrease of 89% of reads mapped to the human genome was observed in ST samples. The percentage of reads mapped to M. abscessus (108 CFU/ml) increased by 89%, and the sequencing depth increased two times when undergoing ST. In conclusion, the sensitivity of M. abscessus detection in artificial sputum was increased using a saponin pre-treatment step. The saponin followed by the DNase I treatment approach could be efficiently applied to detect and characterize mycobacterial infections, including tuberculosis, directly from sputum.

Genetic profiling of Mycobacterium tuberculosis revealed "modern" Beijing strains linked to MDR-TB from Southwestern Colombia.

Beijing strains of Mycobacterium tuberculosis (lineage 2) have been associated with drug-resistance and transmission of tuberculosis worldwide. Most of the Beijing strains identified in the Colombian Pacific coast have exhibited a multidrug resistant (MDR) phenotype. We sought to evaluate the clonality and sublineage of Beijing strains circulating in Southwestern Colombia. Thirty-seven Beijing strains were identified through spoligotyping out of 311 clinical isolates collected in 9 years from 2002-2010. Further analysis by MIRU-VNTR 24 loci was conducted for the Beijing strains. For sublineage classification, deletions of RD105, RD207, and RD131 and point mutations at fbpB, mutT2, and acs were evaluated. Drug-resistance associated mutations to first- and second-line anti-TB drugs were also evaluated. Additionally, two Beijing strains were Illumina-whole genome sequenced (one MDR and one drug-susceptible). Among the 37 Beijing strains characterized, 36 belonged to the SIT190 type from which 28 were MDR, four pre-extensively drug resistant (XDR) TB, and four XDR-TB. The remaining strain was SIT1 and drug susceptible. MIRU-VNTR analysis allowed the identification of three Beijing clusters and two unique strains. Beijing strains were confirmed as "modern" sublineage. The mutations rpoB S531L and katG S315T were the most common among MDR strains. Moreover, the two strains evaluated by whole genome sequencing (WGS) shared most of the genetic features with the sublineage 2.2.1 "modern" Beijing previously characterized from Asian strains. WGS analysis of the MDR strain revealed the presence of eight SNPs previously reported in other MDR "Beijing-like" strains from Colombia. The presence of "modern" Beijing strains in Southwestern Colombia, most of them with MDR phenotype, suggests a different origin of this M. tuberculosis sublineage compared to other Beijing strains found in neighboring South American countries. This work may serve as a genetic baseline to study the evolution and spread of M. tuberculosis Beijing strains in Colombia, which play an important role in the propagation of MDR-TB.

Family Environment in Children With Hearing Aids and Cochlear Implants: Associations With Spoken Language, Psychosocial Functioning, and Cognitive Development.

OBJECTIVES: To examine differences in family environment and associations between family environment and key speech, language, and cognitive outcomes in samples of children with normal hearing and deaf and hard-of-hearing (DHH) children who use hearing aids and cochlear implants. DESIGN: Thirty families of children with normal hearing (n = 10), hearing aids (n = 10), or cochlear implants (n = 10) completed questionnaires evaluating executive function, social skills, and problem behaviors. Children's language and receptive vocabulary were evaluated using standardized measures in the children's homes. In addition, families were administered a standardized in-home questionnaire and observational assessment regarding the home environment. RESULTS: Family environment overall was similar across hearing level and sensory aid, although some differences were found on parental responsivity and physical environment. The level of supportiveness and enrichment within family relationships accounted for much of the relations between family environment and the psychosocial and neurocognitive development of DHH children. In contrast, the availability of objects and experiences to stimulate learning in the home was related to the development of spoken language. CONCLUSIONS: Whereas broad characteristics of the family environments of DHH children may not differ from those of hearing children, variability in family functioning is related to DHH children's at-risk speech, language, and cognitive outcomes. Results support the importance of further research to clarify and explain these relations, which might suggest novel methods and targets of family-based interventions to improve developmental outcomes.

Pyrazinamide resistance-conferring mutations in pncA and the transmission of multidrug resistant TB in Georgia.

BACKGROUND: The ongoing epidemic of multidrug-resistant tuberculosis (MDR-TB) in Georgia highlights the need for more effective control strategies. A new regimen to treat MDR-TB that includes pyrazinamide (PZA) is currently being evaluated and PZA resistance status will largely influence the success of current and future treatment strategies. PZA susceptibility testing was not routinely performed at the National Reference Laboratory (NRL) in Tbilisi between 2010 and September 2015. We here provide a first insight into the prevalence of PZA resistant TB in this region. METHODS: Phenotypic susceptibility to PZA was determined in a convenience collection of well-characterised TB patient isolates collected at the NRL in Tbilisi between 2012 and 2013. In addition, the pncA gene was sequenced and whole genome sequencing was performed on two isolates. RESULTS: Out of 57 isolates tested 33 (57.9%) showed phenotypic drug resistance to PZA and had a single pncA mutation. All of these 33 isolates were MDR-TB strains. pncA mutations were absent in all but one of the 24 PZA susceptible isolate. In total we found 18 polymorphisms in the pncA gene. From the two major MDR-TB clusters represented (94-32 and 100-32), 10 of 15, 67.0% and 13 of 14, 93.0% strains, respectively were PZA resistant. We also identified a member of the potentially highly transmissive clade A strain carrying the characteristic I6L substitution in PncA. Another strain with the same MLVA type as the clade A strain acquired a different mutation in pncA and was genetically more distantly related suggesting that different branches of this particular lineage have been introduced into this region. CONCLUSION: In this high MDR-TB setting more than half of the tested MDR-TB isolates were resistant to PZA. As PZA is part of current and planned MDR-TB treatment regimens this is alarming and deserves the attention of health authorities. Based on our typing and sequence analysis results we conclude that PZA resistance is the result of primary transmission as well as acquisition within the patient and recommend prospective genotyping and PZA resistance testing in high MDR-TB settings. This is of utmost importance in order to preserve bacterial susceptibility to PZA to help protect (new) second line drugs in PZA containing regimens.

Quick and cheap MIRU-VNTR typing of Mycobacterium tuberculosis species complex using duplex PCR.

While minisatellites are usually typed using capillary sequencers or qiaplex systems in developed countries, many low-resource regions cannot afford it. We propose an optimized agarose gel electrophoresis method to genotype Mycobacterium tuberculosis species complex minisatellites in their standardized format (24 MIRU-VNTR). It is based on duplex PCRs combining VNTR loci harboring distinct amplicon sizes whatever the repetition number of each locus. This method performs well both on DNA extracts of good quality and on thermolysates while reducing workload and reagents costs.

Early Expressive Language Skills Predict Long-Term Neurocognitive Outcomes in Cochlear Implant Users: Evidence from the MacArthur-Bates Communicative Development Inventories.

PURPOSE: The objective of the present article was to document the extent to which early expressive language skills (measured using the MacArthur-Bates Communicative Development Inventories [CDI; Fenson et al., 2006]) predict long-term neurocognitive outcomes in a sample of early-implanted prelingually deaf cochlear implant (CI) users. METHOD: The CDI was used to index the early expressive language skills of 32 pediatric CI users after an average of 1.03 years (SD = 0.56, range = 0.39-2.17) of CI experience. Long-term neurocognitive outcomes were assessed after an average of 11.32 (SD = 2.54, range = 7.08-16.52) years of CI experience. Measures of long-term neurocognitive outcomes were derived from gold-standard performance-based and questionnaire-based assessments of language, executive functioning, and academic skills. RESULT: Analyses revealed that early expressive language skills, collected on average 1.03 years post cochlear implantation, predicted long-term language, executive functioning, and academic skills up to 16 years later. CONCLUSION: These findings suggest that early expressive language skills, as indexed by the CDI, are clinically relevant for identifying CI users who may be at high risk for long-term neurocognitive delays and disturbances.

Mechanisms of Phenotypic Rifampicin Tolerance in Mycobacterium tuberculosis Beijing Genotype Strain B0/W148 Revealed by Proteomics.

The "successful" Russian clone B0/W148 of Mycobacterium tuberculosis Beijing is well-known for its capacity to develop antibiotic resistance. During treatment, resistant mutants can occur that have inheritable resistance to specific antibiotics. Next to mutations, M. tuberculosis has several mechanisms that increase their tolerance to a variety of antibiotics. Insights in the phenotypic mechanisms that contribute to drug tolerance will increase our understanding of how antibiotic resistance develops in M. tuberculosis. In this study, we examined the (phospho)proteome dynamics in M. tuberculosis Beijing strain B0/W148 when exposed to a high dose of rifampicin; one of the most potent first-line antibiotics. A total of 2,534 proteins and 191 phosphorylation sites were identified, and revealed the differential regulation of DosR regulon proteins, which are necessary for the development of a dormant phenotype that is less susceptible to antibiotics. By examining independent phenotypic markers of dormancy, we show that persisters of in vitro rifampicin exposure entered a metabolically hypoactive state, which yields rifampicin and other antibiotics largely ineffective. These new insights in the role of protein regulation and post-translational modifications during the initial phase of rifampicin treatment reveal a shortcoming in the antituberculosis regimen that is administered to 8-9 million individuals annually.

Predominance of modern Mycobacterium tuberculosis strains and active transmission of Beijing sublineage in Jayapura, Indonesia Papua.

Mycobacterium tuberculosis genotype distribution is different between West and Central Indonesia, but there are no data on the most Eastern part, Papua. We aimed to identify the predominant genotypes of M. tuberculosis responsible for tuberculosis in coastal Papua, their transmission, and the association with patient characteristics. A total of 199 M. tuberculosis isolates were collected. Spoligotyping was applied to describe the population structure of M. tuberculosis, lineage identification was performed using a combination of lineage-specific markers, and genotypic clusters were identified using a combination of 24-locus-MIRU-VNTR and spoligotyping. A high degree of genetic diversity was observed among isolates based on their spoligopatterns. Strains from modern lineage 4 made up almost half of strains (46.9%), being more abundant than the ancient lineage 1 (33.7%), and modern lineage 2 (19.4%). Thirty-five percent of strains belonged to genotypic clusters, especially strains in the Beijing genotype. Previous TB treatment and mutations associated with drug resistance were more common in patients infected with strains of the Beijing genotype. Papua shows a different distribution of M. tuberculosis genotypes compared to other parts of Indonesia. Clustering and drug resistance of modern strains recently introduced to Papua may contribute to the high tuberculosis burden in this region.

Genomic characterization of Mycobacterium tuberculosis lineage 7 and a proposed name: 'Aethiops vetus'.

Lineage 7 of the Mycobacterium tuberculosis complex has recently been identified among strains originating from Ethiopia. Using different DNA typing techniques, this study provides additional information on the genetic heterogeneity of five lineage 7 strains collected in the Amhara Region of Ethiopia. It also confirms the phylogenetic positioning of these strains between the ancient lineage 1 and TbD1-deleted, modern lineages 2, 3 and 4 of Mycobacterium tuberculosis. Four newly identified large sequence polymorphisms characteristic of the Amhara Region lineage 7 strains are described. While lineage 7 strains have been previously identified in the Woldiya area, we show that lineage 7 strains circulate in other parts of the Amhara Region and also among foreign-born individuals from Eritrea and Somalia in The Netherlands. For ease of documenting future identification of these strains in other geographical locations and recognizing the place of origin, we propose to assign lineage 7 strains the lineage name 'Aethiops vetus'.

Concept formation skills in long-term cochlear implant users.

This study investigated if a period of auditory sensory deprivation followed by degraded auditory input and related language delays affects visual concept formation skills in long-term prelingually deaf cochlear implant (CI) users. We also examined if concept formation skills are mediated or moderated by other neurocognitive domains (i.e., language, working memory, and executive control). Relative to normally hearing (NH) peers, CI users displayed significantly poorer performance in several specific areas of concept formation, especially when multiple comparisons and relational concepts were components of the task. Differences in concept formation between CI users and NH peers were fully explained by differences in language and inhibition-concentration skills. Language skills were also found to be more strongly related to concept formation in CI users than in NH peers. The present findings suggest that complex relational concepts may be adversely affected by a period of early prelingual deafness followed by access to underspecified and degraded sound patterns and spoken language transmitted by a CI. Investigating a unique clinical population such as early-implanted prelingually deaf children with CIs can provide new insights into foundational brain-behavior relations and developmental processes.

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.

Neurocognitive risk in children with cochlear implants.

IMPORTANCE: Children who receive a cochlear implant (CI) for early severe to profound sensorineural hearing loss may achieve age-appropriate spoken language skills not possible before implantation. Despite these advances, reduced access to auditory experience may have downstream effects on fundamental neurocognitive processes for some children with CIs. OBJECTIVE: To determine the relative risk (RR) of clinically significant executive functioning deficits in children with CIs compared with children with normal hearing (NH). DESIGN, SETTING, AND PARTICIPANTS: In this prospective, cross-sectional study, 73 children at a hospital-based clinic who received their CIs before 7 years of age and 78 children with NH, with average to above average mean nonverbal IQ scores, were recruited in 2 age groups: preschool age (age range, 3-5 years) and school age (age range, 7-17 years). No children presented with other developmental, cognitive, or neurologic diagnoses. INTERVENTIONS: Parent-reported checklist measures of executive functioning were completed during psychological testing sessions. MAIN OUTCOMES AND MEASURES: Estimates of the RR of clinically significant deficits in executive functioning (≥1 SDs above the mean) for children with CIs compared with children with NH were obtained based on 2 parent-reported child behavior checklists of everyday problems with executive functioning. RESULTS: In most domains of executive functioning, children with CIs were at 2 to 5 times greater risk of clinically significant deficits compared with children with NH. The RRs for preschoolers and school-aged children, respectively, were greatest in the areas of comprehension and conceptual learning (RR [95% CI], 3.56 [1.71-7.43] and 6.25 [2.64-14.77]), factual memory ( 4.88 [1.58-15.07] and 5.47 [2.03-14.77]), attention (3.38 [1.03-11.04] and 3.13 [1.56-6.26]), sequential processing (11.25 [1.55-81.54] and 2.44 [1.24-4.76]), working memory (4.13 [1.30-13.06] and 3.64 [1.61-8.25] for one checklist and 1.77 [0.82-3.83] and 2.78 [1.18-6.51] for another checklist), and novel problem-solving (3.93 [1.50-10.34] and 3.13 [1.46-6.67]). No difference between the CI and NH samples was found for visual-spatial organization (2.63 [0.76-9.03] and 1.04 [0.45-2.40] on one checklist and 2.86 [0.98-8.39] for school-aged children on the other checklist). CONCLUSIONS AND RELEVANCE: A large proportion of children with CIs are at risk for clinically significant deficits across multiple domains of executive functioning, a rate averaging 2 to 5 times that of children with NH for most domains. Screening for risk of executive functioning deficits should be a routine part of the clinical evaluation of all children with deafness and CIs.

Executive functioning skills in preschool-age children with cochlear implants.

PURPOSE: The purpose of this study was to determine whether deficits in executive functioning (EF) in children with cochlear implants (CIs) emerge as early as the preschool years. METHOD: Two groups of children ages 3 to 6 years participated in this cross-sectional study: 24 preschoolers who had CIs prior to 36 months of age and 21 preschoolers with normal hearing (NH). All were tested on normed measures of working memory, inhibition-concentration, and organization-integration. Parents completed a normed rating scale of problem behaviors related to EF. Comparisons of EF skills of children with CIs were made to peers with NH and to published nationally representative norms. RESULTS: Preschoolers with CIs showed significantly poorer performance on inhibition-concentration and working memory compared with peers with NH and with national norms. No group differences were found in visual memory or organization-integration. When data were controlled for language, differences in performance measures of EF remained, whereas differences in parent-reported problems with EF were no longer significant. Hearing history was generally unrelated to EF. CONCLUSIONS: This is the first study to demonstrate that EF deficits found in older children with CIs begin to emerge as early as preschool years. The ability to detect these deficits early has important implications for early intervention and habilitation after cochlear implantation.

Optimization of standard in-house 24-locus variable-number tandem-repeat typing for Mycobacterium tuberculosis and its direct application to clinical material.

Variable-number tandem-repeat (VNTR) typing with a panel of 24 loci is the current gold standard in the molecular typing of Mycobacterium tuberculosis complex isolates. However, because of technical problems, a part of the loci often cannot be amplified by multiplex PCRs. Therefore, a considerable number of single-locus PCRs have to be performed for the loci with missing results, which impairs the laboratory work flow. Therefore, the original in-house method described by Supply et al. in 2006 was reevaluated. We modified seven primers and the PCR master mixture and obtained a strongly optimized in-house 24-locus VNTR typing method. The percentage of instantly complete 24-locus VNTR patterns detected in the routine flow of typing activities increased to 84.7% from the 72.3% obtained with the typing conducted with the commercially available Genoscreen MIRU-VNTR typing kit. The analytical sensitivity of the optimized in-house method was assessed by serial dilutions of M. tuberculosis in bronchoalveolar lavage fluid. A 1:10 dilution of the different strains tested was the lowest dilution for the detection of a complete 24-locus VNTR pattern. The optimized in-house 24-locus VNTR typing method will reduce the turnaround time of typing significantly and also the financial burden of these activities.

Preschool speech intelligibility and vocabulary skills predict long-term speech and language outcomes following cochlear implantation in early childhood.

Speech and language measures during grade school predict adolescent speech-language outcomes in children who receive cochlear implants (CIs), but no research has examined whether speech and language functioning at even younger ages is predictive of long-term outcomes in this population. The purpose of this study was to examine whether early preschool measures of speech and language performance predict speech-language functioning in long-term users of CIs. Early measures of speech intelligibility and receptive vocabulary (obtained during preschool ages of 3-6 years) in a sample of 35 prelingually deaf, early-implanted children predicted speech perception, language, and verbal working memory skills up to 18 years later. Age of onset of deafness and age at implantation added additional variance to preschool speech intelligibility in predicting some long-term outcome scores, but the relationship between preschool speech-language skills and later speech-language outcomes was not significantly attenuated by the addition of these hearing history variables. These findings suggest that speech and language development during the preschool years is predictive of long-term speech and language functioning in early-implanted, prelingually deaf children. As a result, measures of speech-language functioning at preschool ages can be used to identify and adjust interventions for very young CI users who may be at long-term risk for suboptimal speech and language outcomes.

Comparison of 14 molecular assays for detection of Mycobacterium tuberculosis complex in bronchoalveolar lavage fluid.

We compared 14 molecular assays for their ability to detect the Mycobacterium tuberculosis complex in bronchoalveolar lavage fluid samples. Three approaches were followed. First, by using DNA from Mycobacterium bovis BCG, we determined the detection limits of the assays using routine molecular methods. Second, in order to determine the analytical sensitivities of the assays, we added one of four M. tuberculosis isolates with various numbers of the insertion sequence IS6110 to N-acetyl-l-cysteine (NALC)-NaOH-treated bronchoalveolar lavage fluid samples in dilutions of 1:10 to 1:10,000,000. Third, intertest variabilities were measured and defined by the standard deviations for the quantitation cycle (Cq) values of three positive test results per dilution per assay. The 14 assays tested had similar analytical sensitivities, except for GeneXpert, which had an analytical sensitivity that was 10- to 100-fold lower than that of the other assays. The MP MTB/NTM test and the in-house TaqMan-10 revealed the best performances for the detection limit and had the highest analytical sensitivities. Most of the tests performed well regarding detection limit and analytical sensitivity for the detection of the M. tuberculosis complex in serial dilutions, and the differences were small. The MP MTB/NTM and the in-house TaqMan-10 assays revealed the best, and GeneXpert the worst, overall performances.

Clustering of tuberculosis cases based on variable-number tandem-repeat typing in relation to the population structure of Mycobacterium tuberculosis in the Netherlands.

The population structure of 3,776 Mycobacterium tuberculosis isolates was determined using variable-number tandem-repeat (VNTR) typing. The degree of clonality was so high that a more relaxed definition of clustering cannot be applied. Among recent immigrants with non-Euro-American isolates, transmission is overestimated if based on identical VNTR patterns.