Transmission of drug-resistant Mycobacterium tuberculosis isolates between Finnish- and foreign-born cases, 2014-2021: A molecular epidemiological study.

BACKGROUND: Data on the molecular epidemiology and transmission of drug-resistant Mycobacterium tuberculosis (MTB) in low-incidence settings with immigration from high-incidence settings is limited. METHOD: We included 115 drug-resistant (DR) MTB isolates with whole-genome sequencing data isolated in Finland between 2014 and 2021. Potential transmission clusters were identified using a threshold of 12 single-nucleotide polymorphisms (SNPs). Highly related clusters were identified using a threshold of 5 SNPs. RESULT: Of the 115 DR MTB isolates, 31 (27.0%) isolates were from Finnish-born cases and 84 (73.0%) were from foreign-born cases. The proportion of multidrug-resistant (MDR) MTB isolates (30/84, 35.7%) from foreign-born cases was higher than that of MDR MTB isolates from Finnish-born cases (8/31, 25.8%). Lineage 2 (40/115, 34.8%) and lineage 4 (40/115, 34.8%) were the most prevalent lineages. A total of 25 (21.7%) isolates were classified into eight potential transmission clusters (≤12 SNPs). Furthermore, five highly related clusters (≤5 SNPs) were identified, including three DR MTB isolates from Finnish-born cases and 14 DR isolates from foreign-born cases. CONCLUSION: The risk of DR MTB transmission between Finnish- and foreign-born persons is not negligible. Further research on clustering analysis in drug-susceptible MTB is worth to inform tuberculosis management and control in low-incidence settings with increasing immigration.

Multidrug-resistant tuberculosis in Finland: treatment outcome and the role of whole-genome sequencing.

Treatment of multidrug-resistant tuberculosis (MDR-TB) is a global challenge requiring long treatment with costly drugs. We assessed treatment combinations, outcome and the utility of whole-genome sequencing (WGS) in MDR-TB cases. Clinical, demographic and microbiological data were obtained of all patients with MDR-TB who started treatment in Finland in 2007-2016. Definitions of MDR, pre-extensively drug-resistant (pre-XDR) and XDR tuberculosis were those applicable at the study period. Treatment outcome was defined according to World Health Organization (WHO) guidelines. Mycobacterium tuberculosis isolates were analysed by WGS in addition to routinely performed phenotypic drug susceptibility testing and genotyping. Among the 47 cases, 35 (74%) had a successful treatment outcome. Risk factors for non-successful outcome were Finnish origin and XDR. Almost 90% of our cases had an adverse event for at least one drug. Phenotypic and WGS drug resistance results were fully concordant for isoniazid, fluoroquinolones and amikacin, and >90% concordant for rifampicin, pyrazinamide, kanamycin and capreomycin. >60% of phenotypically ethambutol-susceptible isolates were genotypically resistant. The results of the rifampicin and isoniazid nucleic acid amplification tests (NAATs) performed for the isolates were identical to the WGS results except for three isolates having uncommon resistance mutations not included in the NAATs. WGS did not reveal unexpected clustering. More training is needed for physicians treating MDR-TB, and especially XDR-TB, to improve treatment outcome. Phenotypic drug susceptibility testing was shown to be unreliable for ethambutol. WGS could aid in the selection of optimal treatment regimen in the future.

Transmission of tuberculosis between foreign-born and Finnish-born populations in Finland, 2014-2017.

We describe the epidemiology of tuberculosis (TB) and characterized Mycobacterium tuberculosis (M. tuberculosis) isolates to evaluate transmission between foreign-born and Finnish-born populations. Data on TB cases were obtained from the National Infectious Disease Register and denominator data on legal residents and their country of birth from the Population Information System. M. tuberculosis isolates were genotyped by spoligotyping and Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (MIRU-VNTR). We characterized clusters by age, sex, origin and region of living which included both foreign-born cases and those born in Finland. During 2014-2017, 1015 TB cases were notified; 814 were confirmed by culture. The proportion of foreign-born cases increased from 33.3% to 39.0%. Foreign-born TB cases were younger (median age, 28 vs. 75 years), and had extrapulmonary TB or multidrug-TB more often than Finnish-born cases (P<0.01 for all comparisons). Foreign-born cases were born in 60 different countries; most commonly in Somalia (25.5%). Altogether 795 isolates were genotyped; 31.2% belonged to 80 different clusters (range, 2-13 cases/cluster). Fourteen (17.5%) clusters included isolates from both Finnish-born and foreign-born cases. An epidemiological link between cases was identified by (epidemiological) background information in two clusters. Although the proportion of foreign-born TB cases was considerable, our data suggests that transmission of TB between foreign and Finnish born population is uncommon.

Emerging source of infection - Mycobacterium tuberculosis in rescue dogs: a case report.

Rescue dog activity is a heavily increasing form of dog charity. Imported homeless dogs represent a reservoir of zoonotic diseases putting owners, veterinarians and pathologists repeatedly at risk. The clinical signs of tuberculosis in a dog are non-specific and diagnosis is often delayed or dismissed. We present a case of 9 months of possible exposure at home and definite exposure at laparotomy and autopsy to intestinal tuberculosis in a family dog imported from Romania to Finland. Persistent gastrointestinal symptoms started 2 years after the import. Abdominal pain, diarrhoea and vomiting proceeded and led to spontaneous death. Mycobacterium tuberculosis was identified in the liver, lymph nodes and intestine at autopsy. Exposed persons were notified and follow-up was provided, and no further infections were identified within 12 months of follow-up. The heavily increasing import of companion animals presents unexpected public health risks, such as prolonged exposure to tuberculosis, of which the general public is not aware. The dramatic consequences and high costs of tuberculosis could be reduced through accessible information of the risks of imported animals to both the general public and veterinarians, in addition to availability of rapid diagnostics and proper personal protection.

A cluster of multidrug-resistant Mycobacterium tuberculosis among patients arriving in Europe from the Horn of Africa: a molecular epidemiological study.

BACKGROUND: The risk of tuberculosis outbreaks among people fleeing hardship for refuge in Europe is heightened. We describe the cross-border European response to an outbreak of multidrug-resistant tuberculosis among patients from the Horn of Africa and Sudan. METHODS: On April 29 and May 30, 2016, the Swiss and German National Mycobacterial Reference Laboratories independently triggered an outbreak investigation after four patients were diagnosed with multidrug-resistant tuberculosis. In this molecular epidemiological study, we prospectively defined outbreak cases with 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) profiles; phenotypic resistance to isoniazid, rifampicin, ethambutol, pyrazinamide, and capreomycin; and corresponding drug resistance mutations. We whole-genome sequenced all Mycobacterium tuberculosis isolates and clustered them using a threshold of five single nucleotide polymorphisms (SNPs). We collated epidemiological data from host countries from the European Centre for Disease Prevention and Control. FINDINGS: Between Feb 12, 2016, and April 19, 2017, 29 patients were diagnosed with multidrug-resistant tuberculosis in seven European countries. All originated from the Horn of Africa or Sudan, with all isolates two SNPs or fewer apart. 22 (76%) patients reported their travel routes, with clear spatiotemporal overlap between routes. We identified a further 29 MIRU-VNTR-linked cases from the Horn of Africa that predated the outbreak, but all were more than five SNPs from the outbreak. However all 58 isolates shared a capreomycin resistance-associated tlyA mutation. INTERPRETATION: Our data suggest that source cases are linked to an M tuberculosis clone circulating in northern Somalia or Djibouti and that transmission probably occurred en route before arrival in Europe. We hypothesise that the shared mutation of tlyA is a drug resistance mutation and phylogenetic marker, the first of its kind in M tuberculosis sensu stricto. FUNDING: The Swiss Federal Office of Public Health, the University of Zurich, the Wellcome Trust, National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC), the Medical Research Council, BELTA-TBnet, the European Union, the German Center for Infection Research, and Leibniz Science Campus Evolutionary Medicine of the Lung (EvoLUNG).

Improved Differentiation of Streptococcus pneumoniae and Other S. mitis Group Streptococci by MALDI Biotyper Using an Improved MALDI Biotyper Database Content and a Novel Result Interpretation Algorithm.

Reliable distinction of Streptococcus pneumoniae and viridans group streptococci is important because of the different pathogenic properties of these organisms. Differentiation between S. pneumoniae and closely related Sreptococcusmitis species group streptococci has always been challenging, even when using such modern methods as 16S rRNA gene sequencing or matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. In this study, a novel algorithm combined with an enhanced database was evaluated for differentiation between S. pneumoniae and S. mitis species group streptococci. One hundred one clinical S. mitis species group streptococcal strains and 188 clinical S. pneumoniae strains were identified by both the standard MALDI Biotyper database alone and that combined with a novel algorithm. The database update from 4,613 strains to 5,627 strains drastically improved the differentiation of S. pneumoniae and S. mitis species group streptococci: when the new database version containing 5,627 strains was used, only one of the 101 S. mitis species group isolates was misidentified as S. pneumoniae, whereas 66 of them were misidentified as S. pneumoniae when the earlier 4,613-strain MALDI Biotyper database version was used. The updated MALDI Biotyper database combined with the novel algorithm showed even better performance, producing no misidentifications of the S. mitis species group strains as S. pneumoniae All S. pneumoniae strains were correctly identified as S. pneumoniae with both the standard MALDI Biotyper database and the standard MALDI Biotyper database combined with the novel algorithm. This new algorithm thus enables reliable differentiation between pneumococci and other S. mitis species group streptococci with the MALDI Biotyper.

Molecular epidemiology of tuberculosis in Finland, 2008-2011.

In industrialized countries the majority of tuberculosis (TB) cases are linked to immigration. In Finland, most cases are still Finnish born but the number of foreign born cases is steadily increasing. In this 4-year population based study, the TB situation in Finland was characterized by a genotypic analysis of Mycobacterium tuberculosis isolates. A total of 1048 M. tuberculosis isolates (representing 99.4% of all culture positive cases) were analyzed by spoligotyping and MIRU. Spoligotype lineages belonging to the Euro-American family were predominant among the Finnish isolates, particularly T (n=346, 33.0%) and Haarlem (n=237, 22.6%) strains. The lineage signature was unknown for 130 (12.4%) isolates. Out of the 17 multi-drug resistant TB strains, 10 (58.8%) belonged to the Beijing lineage. In total, 23 new SIT designations were given and 51 orphan strains were found, of which 58 patterns were unique to Finland. Phylogeographical TB mapping as compared to neighboring countries showed that the population structure in Finland most closely resembled that observed in Sweden. By combining spoligotyping and MIRU results, 98 clusters comprising 355 isolates (33.9%) were found. Only 10 clusters contained both Finnish and foreign born cases. In conclusion, a large proportion of the M. tuberculosis isolates were from Finnish born elderly patients. Moreover, many previously unidentified spoligotype profiles and isolates belonging to unknown lineages were encountered.

Interactions between Bordetella pertussis and the complement inhibitor factor H.

Bordetella pertussis causes whooping cough in humans, a highly contagious disease of the upper respiratory tract. An increase in cases of whooping cough in adolescents and adults in many countries has been reported, despite high immunization rates in children. To efficiently colonize the host the bacteria have to resist complement, the first defence line of innate immunity. B. pertussis has previously been shown to bind the classical pathway inhibitors C4b-binding protein and C1-inhibitor being thereby able to escape the classical pathway of complement. In this study recent clinical isolates of B. pertussis and B. parapertussis were found to survive alternative pathway attack in fresh non-immune serum better than the reference B. pertussis strain, Tohama I. By using adsorption assays, flow cytometry and a radioligand binding assay we observed that both B. pertussis and B. parapertussis bound the alternative pathway inhibitor factor H (FH) from normal human serum. The surface attached FH maintained its complement regulatory activity and promoted factor I-mediated cleavage of C3b. The main binding region was located to the C-terminal part of FH, into short consensus repeat domains 19-20. In contrast, the avian pathogen B. avium did not bind FH and was sensitive to the alternative pathway of human complement. In conclusion, the human pathogens B. pertussis and B. parapertussis are able to evade the alternative complement pathway by surface acquisition of the host complement regulator FH.

Typing of SHV extended-spectrum beta-lactamases by pyrosequencing in Klebsiella pneumoniae strains with chromosomal SHV beta-lactamase.

In Klebsiella pneumoniae, the cooccurrence of chromosomal and plasmid-mediated beta-lactamases can hinder their accurate molecular detection. We developed a fast and reliable method that allows the typing of isolates carrying more than one SHV gene. The method is based on pyrosequencing the DNA sequence corresponding to amino acid positions 35, 238, and 240.

Immunity to pertussis 5 years after booster immunization during adolescence.

BACKGROUND: We conducted a 5-year follow-up study on the persistence of pertussis-specific antibody and cell-mediated immunity after booster immunization of adolescents aged 11-13 years with a tricomponent acellular pertussis vaccine (Boostrix; trials diphtheria-tetanus-acellular pertussis [Tdap]-004/030). METHODS: Cellular and humoral immunity to pertussis toxin (PT), filamentous hemagglutinin, and pertactin were measured in adolescents (age, 16 years) 5 years after booster immunization. Similar investigations were performed for control adolescents who had received only diphtheria and tetanus booster vaccination. RESULTS: Five years after pertussis booster vaccination, the geometric mean concentrations of immunoglobulin G (IgG) elicited by each of the 3 pertussis vaccine antigens decreased from 1-month and 3-year postvaccination levels, but with the exception of PT IgG, were still higher than the prevaccination levels. PT IgG levels were undetectable in 28% of the subjects, but 44% of those subjects still tested positive for cell-mediated immunity to PT. Filamentous hemagglutinin IgG and pertactin IgG levels were significantly higher in Tdap-boosted adolescents than in the control subjects. Antibody concentrations at 1 month after vaccination strongly predicted antibody persistence. Cell-mediated immunity levels to PT, filamentous hemagglutinin, and pertactin persisted above the prebooster levels measured 5 years earlier. CONCLUSIONS: The results of the present study of adolescents indicate that the interval between acellular pertussis booster immunizations might be extended beyond 5 years.

Identification of alpha-hemolytic streptococci by pyrosequencing the 16S rRNA gene and by use of VITEK 2.

Alpha-hemolytic streptococci are very difficult to identify by phenotypic methods. In this study, a pyrosequencing method for the identification of streptococcal species based on two variable regions of the 16S rRNA gene is described. Almost all studied streptococcal species (n = 51) represented by their type strains could be differentiated except for some closely related species of the Streptococcus bovis or S. salivarius group. The pyrosequencing results of alpha-hemolytic streptococci isolated from blood (n = 99) or from the normal pharyngeal microbiota (n = 25) were compared to the results obtained by the VITEK 2 with GP card (bioMérieux, Marcy l'Etoile, France). As expected, the results of the two methods did not completely agree, but 93 (75.0%) of the isolates assigned to the same streptococcal group by both methods and 57 (46.0%) reached consistent results at the species level. However, 10 strains remained unidentified by VITEK 2, and 4 isolates could not be assigned to any streptococcal group by pyrosequencing. Identification of members of the S. mitis and S. sanguinis groups proved difficult for both methods. Furthermore, the pyrosequencing analysis revealed great sequence variation, since only 43 (32.3%) of the 133 isolates analyzed by pyrosequencing had sequences identical to a type strain. The variation was greatest in the pharyngeal isolates, slightly lower in the blood culture isolates, and nonexistent in invasive pneumococcal isolates (n = 17) that all had the S. pneumoniae type strain sequence. The resolution of the results obtained by the two methods is impeded by the lack of a proper gold standard.

Bordetella holmesii DNA is not detected in nasopharyngeal swabs from Finnish and Dutch patients with suspected pertussis.

Bordetella holmesii is a Gram-negative bacterium first identified in 1995. It can cause pertussis-like symptoms in humans. B. holmesii contains insertion sequences IS481 and IS1001, two frequently used targets in the PCR diagnosis of Bordetella pertussis and Bordetella parapertussis infections. To investigate the prevalence of B. holmesii in Finnish and Dutch patients with pertussis-like symptoms and whether B. holmesii has caused any false-positive results in diagnostic PCRs, B. holmesii-specific real-time PCRs were developed. The Finnish methods were conventional IS481 PCR and B. holmesii-specific real-time PCR (LightCycler, Roche) targeting the B. holmesii recA gene. The Dutch methods were IS481 and IS1001 PCRs with conventional or real-time formats and B. holmesii-specific real-time PCR targeting the homologue of IS1001. Of 11,319 nasopharyngeal swabs, 2804 were collected from Finnish patients from 2000 to 2003, and 8515 from Dutch patients from 1992 to 2003. B. holmesii DNA was not found in the samples analysed. The results suggest that B. holmesii is not among the causative agents of pertussis-like symptoms in Finnish and Dutch patients and thus does not in practice confound IS481 and IS1001 PCRs.

Detection and quantification of macrolide resistance mutations at positions 2058 and 2059 of the 23S rRNA gene by pyrosequencing.

A pyrosequencing method for detection and quantification of macrolide resistance mutations at positions 2058 and 2059 (Escherichia coli numbering) of the 23S rRNA gene is described. The method was developed and tested for Streptococcus pneumoniae, Streptococcus pyogenes, Mycobacterium avium, Campylobacter jejuni, and Haemophilus influenzae.

Pertussis-specific cell-mediated and humoral immunity in adolescents 3 years after booster immunization with acellular pertussis vaccine.

We evaluated pertussis-specific cell-mediated immunity (CMI) and humoral immunity in adolescents 3 years after they received an acellular pertussis booster immunization. Two hundred sixty-four adolescents were examined for immunoglobulin G antibodies, and 49 were examined for CMI against Bordetella pertussis antigens 40 months after receiving the booster. A control group of similarly aged adolescents who had received diphtheria and tetanus vaccination 3 years earlier was included for comparison. Pertussis-specific CMI persisted at greater than prebooster immunization levels. Although they had decreased by the 3-year follow-up, antibody levels remained significantly higher than prebooster immunization levels. Antibodies against pertussis antigens and CMI against filamentous hemagglutinin and pertactin were significantly higher in vaccinated adolescents than in control subjects. The acellular pertussis booster immunization provides long-term CMI and humoral immunity lasting for >or=3 years. The significantly higher immunity observed in the diphtheria, tetanus, and acellular pertussis vaccine recipients, compared with that in control subjects, indicates that these responses are more likely to have resulted from the booster immunization than from the boosting effects of natural B. pertussis infection.

Antimicrobial susceptibility patterns and macrolide resistance genes of viridans group streptococci from normal flora.

OBJECTIVES: Our aim was to study the antimicrobial susceptibilities and macrolide resistance mechanisms of viridans group streptococci isolated from the normal flora. METHODS: In vitro susceptibilities of 16 antimicrobials were studied for 161 viridans streptococci (on average 5.8 isolates per person) from the normal flora of 28 elderly persons. Resistance mechanisms of erythromycin-resistant isolates were studied by the double disc test and PCR. RESULTS: In all, 16.8% of the isolates were non-susceptible (MIC > or =0.25 mg/L) to penicillin, but none showed high-level resistance (MIC > or =4 mg/L). Resistance to erythromycin, tetracycline, quinupristin/dalfopristin, levofloxacin and moxifloxacin was found in 22.4, 27.3, 13.0, 1.9 and 1.9% of the isolates, respectively. Combined resistance to erythromycin and tetracycline was found in 13.0% of the isolates. Erythromycin-resistant isolates were isolated from 57% of the study persons. Of the erythromycin-resistant isolates 80.6% were of the M phenotype and 19.4% were of the macrolide-lincosamide-streptogramin B (MLSB) phenotype (one isolate with constitutive and six with inducible expression). Isolates with the M phenotype were the least susceptible to telithromycin, a new ketolide. The mef(A) gene was found in the isolates with the M phenotype and the erm(B) gene in the isolates with the MLSB phenotype. CONCLUSIONS: The distribution of phenotypes among the viridans streptococci resembles that found in Streptococcus pyogenes, with predominance of the M phenotype. However, the coding gene for the MLSB phenotype, erm(B), is the same in viridans streptococci as in Streptococcus pneumoniae. Viridans group streptococci carrying different resistance traits provide a pool of resistant bacteria that may transfer resistance determinants to more pathogenic organisms.