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Background: Decorin adherence is crucial in the pathogenesis of Lyme borreliosis. Decorin-binding proteins (Dbp) A and B are the adhesins that mediate this interaction. DbpA and B of Borrelia garinii, Borrelia afzelii, and Borrelia burgdorferi sensu stricto (ss) differ in their amino acid sequence, but little attention has been paid to the potential difference in their decorin binding.Methods: We expressed recombinant DbpA and DbpB of B. garinii, B. afzelii, and B. burgdorferi ss and studied their binding to decorin. We also generated recombinant Borrelia strains to study the role of DbpA and DbpB in the adhesion of live spirochetes to decorin and decorin-expressing cells. RESULTS. Recombinant DbpA of B. garinii and DbpB of B. garinii and B. burgdorferi ss showed strong binding to decorin, whereas DbpA of B. burgdorferi ss and both DbpA and DbpB of B. afzelii exhibited no or only minor binding activity. DbpA and DbpB of B. garinii and B. burgdorferi ss also supported the adhesion of whole spirochetes to decorin and decorin-expressing cells, whereas DbpA and DbpB of B. afzelii did not exhibit this activity.Conclusions: Dbp A and B of B. garinii and B. burgdorferi ss mediate the interaction between the spirochete and decorin, whereas the same adhesins of B. afzelii show only negligible activity. [ABSTRACT FROM AUTHOR]

Since April 1993 in Finland and March 1994 in Switzerland, polymerase chain reaction (PCR) has been used routinely nationwide for the diagnosis of pertussis. Nasopharyngeal specimens from 3794 patients suspected of having pertussis and 1125 controls were tested. Finnish and Swiss assays found 23% and 36% of clinical specimens positive, respectively. PCR showed a higher incidence of pertussis infection among 1- to 6-year-old children in Switzerland than in Finland (P < .001). This difference may be due to the booster dose of vaccine given at 2 years of age in Finland but not in Switzerland. In Finland, PCR-confirmed asymptomatic cases were more common among children <7 years old than in older children (P < .001), whereas older children tended to have symptomatic infection. The use of PCR markedly improves the diagnosis of pertussis and opens new perspectives for epidemiologic and vaccine efficacy studies. [ABSTRACT FROM AUTHOR]

Background: Bacterial pathogens causing systemic infections disseminate from the initial infection focus to the target organs usually through the blood vasculature. To be able to colonize various organs, bacteria need to adhere to the endothelial cells of the vascular wall, and the adhesion must be strong enough to resist the shear force of the blood flow.Borrelia burgdorferi sensu lato spirochetes, the causative agents of the tick-borne disease Lyme borreliosis, disseminate hematogenously from the tick bite site to the joints, the heart, and the central nervous system of the patient., Methods: We used both wild-type and genetically modified B. burgdorferi s. l. bacteria, recombinant borrelia adhesins, and an array of adhesion assays carried out both under stationary and flow conditions to investigate the molecular mechanisms of borrelial adhesion to human endothelial cells., Results: Borrelia garinii, a member of the B. burgdorferi s. l. complex, adhered to biglycan expressed by human endothelial cells in a flow-tolerant manner. The adhesion was mediated by the decorin-binding protein A (DbpA) and DbpB surface molecules of B. garinii., Conclusions: The proteoglycan biglycan is a receptor molecule for flow-resistant adhesion of the bacterial pathogen B. garinii on human endothelial cells., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Decorin binding proteins A and B (DbpA and B) of Borrelia burgdorferi are of critical importance for the virulence of the spirochete. The objective of the present study was to further clarify the contribution of DbpA and B to development of arthritis and persistence of B. burgdorferi after antibiotic treatment in a murine model of Lyme borreliosis. With that goal, mice were infected with B. burgdorferi strains expressing either DbpA or DbpB, or both DbpA and B, or with a strain lacking the adhesins. Arthritis development was monitored up to 15 weeks after infection, and bacterial persistence was studied after ceftriaxone and immunosuppressive treatments. Mice infected with the B. burgdorferi strain expressing both DbpA and B developed an early and prominent joint swelling. In contrast, while strains that expressed DbpA or B alone, or the strain that was DbpA and B deficient, were able to colonize mouse joints, they caused only negligible joint manifestations. Ceftriaxone treatment at two or six weeks of infection totally abolished joint swelling, and all ceftriaxone treated mice were B. burgdorferi culture negative. Antibiotic treated mice, which were immunosuppressed by anti-TNF-alpha, remained culture negative. Importantly, among ceftriaxone treated mice, B. burgdorferi DNA was detected by PCR uniformly in joint samples of mice infected with DbpA and B expressing bacteria, while this was not observed in mice infected with the DbpA and B deficient strain. In conclusion, these results show that both DbpA and B adhesins are crucial for early and prominent arthritis development in mice. Also, post-treatment borrelial DNA persistence appears to be dependent on the expression of DbpA and B on B. burgdorferi surface. Results of the immunosuppression studies suggest that the persisting material in the joints of antibiotic treated mice is DNA or DNA containing remnants rather than live bacteria.

Specific antibody deficiency (SAD) to unconjugated pneumococcal vaccine (PPV) is an established primary B cell immunodeficiency. The occurrence and natural history of SAD in children is unclear. We conducted an observational study to identify SAD in children with recurrent respiratory infections. Ninety-nine children, mean age 5·9 (range 2-16) years, with recurrent or severe infections were vaccinated with PPV; serum antibody concentrations for serotypes 4, 6B, 9V, 14, 18C, 19F and 23F were measured before and 2 weeks after vaccination with enzyme immunoassay. The retrospective control group consisted of 89 healthy children matched for age and gender. No children had received previous conjugated pneumococcal vaccine (PCV) or PPV. The structured history of infectious diseases of all participants was collected. Ten of 91 (11%) children (eight excluded due to immunoglobulin G subclass deficiency) with recurrent respiratory infections had SAD. In the control group, three children (3%) responded inadequately to PPV (P = 0·05). Most children with SAD also had many other minor immune defects. After 0·5-5 years (medium 3·8), eight children with SAD were revaccinated with PPV; five responded adequately and three inadequately. Two SAD children were revaccinated with PCV, one developed an adequate and one an inadequate response. Two children with SAD received treatment with intravenous immunoglobulin; the remaining eight children recovered without replacement therapy during the follow-up. SAD is common in young children with recurrent respiratory infections, but it is often transient and resolves itself within a few years without specific treatment., (© 2012 British Society for Immunology.)

Drug resistance and molecular epidemiology of tuberculosis (TB) in the Murmansk region was investigated in a 2-year, population-based surveillance of the civilian population. During 2003 and 2004, isolates from all culture-positive cases were collected (n = 1,226). Prevalence of multi-drug resistance (MDR) was extremely high, as 114 out of 439 new cases (26.0%), and 574 out of 787 previously treated cases (72.9%) were resistant to at least isoniazid (INH) and rifampin (RIF). Spoligotyping of the primary MDR-TB isolates revealed that most isolates grouped to the Beijing SIT1 genotype (n = 91, 79.8%). Isolates of this genotype were further analyzed by IS6110 RFLP. Sequencing of gene targets associated with INH and RIF resistance further showed that the MDR-TB strains are highly homogeneous as 78% of the MDR, SIT1 strains had the same resistance-conferring mutations. The genetic homogeneity of the MDR-TB strains indicates that they are actively transmitted in Murmansk.

We have earlier shown that Borrelia burgdorferi-infected and ceftriaxone-treated mice have viable spirochetes in their body, since immunosuppressive treatment allows B. burgdorferi to be detected by culture. However, the niche of the persisting spirochetes remained unknown. In the present study, we analyzed the tissues of B. burgdorferi-infected and ceftriaxone-treated mice by culture and PCR to reveal the foci of persisting spirochetes. C3H/HeN mice were infected via intradermal needle injection with B. burgdorferi s.s. N40. The mice were treated as follows: (i) short (5 days) and (ii) long (18 days) course of ceftriaxone at 2 weeks of infection and killed after either 10 or 30 weeks, or (iii) the mice received ceftriaxone for 5 days at 18 weeks of infection and were killed 21 weeks after the treatment. All samples of ceftriaxone-treated mice were culture negative, whereas all untreated controls were culture positive. Importantly, B. burgdorferi DNA was detected in the joints of 30-100% of the treated mice. In conclusion, these results combined with earlier results suggest that the joint or a tissue adjacent to the joint is the niche of persisting B. burgdorferi in ceftriaxone-treated mice.

Lyme borreliosis is a tick-borne bacterial infection that in many cases is limited to the skin. However, in some patients the bacterium evades the immune response and disseminates into various organs. Dendritic cells (DCs) are among the first cells to meet invading pathogens in the skin. We have previously shown that CD38, an ectoenzyme involved in the migration of DCs and generally upregulated by microbial stimuli, is not upregulated in Borrelia garinii-stimulated DCs. In this paper, we characterize the cellular events that lead to the absence of CD38 on the DC surface after B. garinii stimulation and investigate the consequences of absent CD38 expression for the migration of DCs in vitro and in vivo. The data show that 1) effective signaling via p38 MAPK (and STAT1 and NF-kappaB) is needed for CD38 expression and 2) TLR2 stimulation, as opposed to TLR4 stimulation, does not induce IFN-beta autocrine loop-dependent expression of CD38 and secretion of IL-12. Further, we show that 3) B. garinii-stimulated DCs do not migrate effectively toward CCL19 and CCL21 and 4) after B. garinii infection of mice, the number of DCs migrating from the infection site to draining lymph nodes is only half that induced by Escherichia coli infection. Our results provide evidence for the first time that different TLR use results in different CD38 expression, which correlates with the migratory potential of DCs.

Dissolution behavior of six bioactive glasses was correlated with the antibacterial effects of the same glasses against sixteen clinically important bacterial species. Powdered glasses (<45 microm) were immersed in simulated body fluid (SBF) for 48 h. The pH in the solution inside the glass powder was measured in situ with a microelectrode. After 2, 4, 27, and 48 h, the pH and concentration of ions after removing the particles and mixing the SBF were measured with a normal glass pH electrode and ICP-OES. The bacteria were cultured in broth with the glass powder for up to 4 days, after which the viability of the bacteria was determined. The antibacterial effect of the glasses increased with increasing pH and concentration of alkali ions and thus with increased dissolution tendency of the glasses, but it also depended on the bacterium type. The changes in the concentrations of Si, Ca, Mg, P, and B ions in SBF did not show statistically significant influence on the antibacterial property. Bioactive glasses showed strong antibacterial effects for a wide selection of aerobic bacteria at a high sample concentration (100 mg/mL). The antibacterial effects increased with glass concentration and a concentration of 50 mg/mL (SA/V 185 cm(-1)) was required to generate the bactericidal effects. Understanding the dissolution mechanisms of bioactive glasses is essential when assessing their antibacterial effects., (Copyright 2009 Wiley Periodicals, Inc.)

Extracellular ATP and adenosine are important regulators of immune responses; however, contribution of purinergic signaling to host defense during persistent microbial infections remains obscure. Lyme borreliosis is a common arthropod-borne infection caused by Borrelia burgdorferi sensu lato. In this study, we investigated whether lymphoid purinergic signaling contributes to the mechanisms by which borreliae species evade the immune system and trigger joint inflammation. Intracutaneous inoculation of Borrelia garinii to C3H/He mice induced symptomatic infection manifested in elevated levels of borrelia-specific IgG Abs, persistent spirochete dissemination into the tissues and joint swelling, as well as approximately 2- to 2.5-fold enlargement of draining lymph nodes with hyperplasia of B cell follicle area and L-selectin shedding from activated T lymphocytes. Purine catabolism was also activated in lymph nodes but not spleen and blood of infected C3H/He mice within the first 4 postinfection weeks, particularly manifested in transient upregulations of adenosine triphosphatase/ectonucleoside triphosphate diphosphohydrolase and ecto-5'-nucleotidase/CD73 on CD4(+)CD8(+) T lymphocytes and adenosine deaminase activity on B220(+) B lymphocytes. Compared with borrelia-susceptible C3H/He strain, lymphocytes from C57BL/6 mice displayed markedly enhanced adenosine-generating capability due to approximately three times higher ratio of ecto-5'-nucleotidase to adenosine deaminase. Borrelia-infected C57BL/6 mice efficiently eradicated the inoculated spirochetes at more chronic stage without any signs of arthritis. Strikingly, deletion of key adenosine-generating enzyme, ecto-5'-nucleotidase/CD73, was accompanied by significantly enhanced joint swelling in borrelia-infected CD73-deficient C57BL/6 mice. Collectively, these data suggest that insufficient basal adenosine level and/or pathogen-induced disordered lymphoid purine homeostasis may serve as important prerequisite for promotion of inflammatory responses and further host's commitment to persistence of bacterial infection and arthritis development.

Background: Bordetella pertussis causes whooping cough or pertussis in humans. It produces several virulence factors, of which the fimbriae are considered adhesins and elicit immune responses in the host. B. pertussis has three distinct serotypes Fim2, Fim3 or Fim2,3. Generally, B. pertussis Fim2 strains predominate in unvaccinated populations, whereas Fim3 strains are often isolated in vaccinated populations. In Finland, pertussis vaccination was introduced in 1952. The whole-cell vaccine contained two strains, 18530 (Fim3) since 1962 and strain 1772 (Fim2,3) added in 1976. After that the vaccine has remained the same until 2005 when the whole-cell vaccine was replaced by the acellular vaccine containing pertussis toxin and filamentous hemagglutinin. Our aims were to study serotypes of Finnish B. pertussis isolates from 1974 to 2006 in a population with > 90% vaccination coverage and fimbrial expression of the isolates during infection. Serotyping was done by agglutination and serotype-specific antibody responses were determined by blocking ELISA., Results: Altogether, 1,109 isolates were serotyped. Before 1976, serotype distributions of Fim2, Fim3 and Fim2,3 were 67%, 19% and 10%, respectively. From 1976 to 1998, 94% of the isolates were Fim2 serotype. Since 1999, the frequency of Fim3 strains started to increase and reached 83% during a nationwide epidemic in 2003. A significant increase in level of serum IgG antibodies against purified fimbriae was observed between paired sera of 37 patients. The patients infected by Fim3 strains had antibodies which blocked the binding of monoclonal antibodies to Fim3 but not to Fim2. Moreover, about one third of the Fim2 strain infected patients developed antibodies capable of blocking of binding of both anti-Fim2 and Fim3 monoclonal antibodies., Conclusion: Despite extensive vaccinations in Finland, B. pertussis Fim2 strains were the most common serotype. Emergence of Fim3 strains started in 1999 and coincided with nationwide epidemics. Results of serotype-specific antibody responses suggest that Fim2 strains could express Fim3 during infection, showing a difference in fimbrial expression between in vivo and in vitro.

Lyme borreliosis (LB) is a tick-borne infection caused by the spirochete Borrelia burgdorferi sensu lato. The disease covers a wide spectrum of clinical manifestations affecting the skin, nervous and musculoskeletal systems, the heart, and the eyes. The diagnosis must be based on clinical suspicion and on symptoms and signs observed during a thorough interview and examination of the patient. Laboratory results either support or oppose the conclusions that are drawn from history and clinical examination. Antibiotic therapy is curative in most patients with LB. Unfortunately, some patients develop chronic symptoms, such as arthritis, that do not respond to antibiotics. In these patients, treatment with non-steroidal anti-inflammatory drugs or corticosteroids is recommended, while the role of immunomodulatory drugs, such as tumour necrosis factor (TNF)-alpha inhibitors, remains open. In this review we focus, after presenting the history and basics of LB, on the pathogenesis, diagnosis, and treatment of LB, as well as on recent advances in selected aspects of the field.

Bioactive glasses (BAGs) of different compositions have been studied for decades for clinical use and they have found many dental and orthopaedic applications. Particulate BAGs have also been shown to have antibacterial properties. This large-scale study shows that two bioactive glass powders (S53P4 and 13-93) and a sol-gel derived material (CaPSiO II) have an antibacterial effect on 17 clinically important anaerobic bacterial species. All the materials tested demonstrated growth inhibition, although the concentration and time needed for the effect varied depending on the BAG. Glass S53P4 had a strong growth-inhibitory effect on all pathogens tested. Glass 13-93 and sol-gel derived material CaPSiO II showed moderate antibacterial properties.

Bioactive glasses (BAGs) have been studied for decades for clinical use, and they have found many dental and orthopedic applications. BAGs have also been shown to have an antibacterial effect e.g., on some oral microorganisms. In this extensive work we show that six powdered BAGs and two sol-gel derived materials have a clear antibacterial effect on 29 clinically important bacterial species. We also incorporated a rapid and accurate flow cytometric (FCM) method to calculate and standardize the numbers of viable bacteria inoculated in the suspensions used in the tests for antibacterial activity. In all materials tested growth inhibition could be demonstrated, although the concentration and time needed for the effect varied depending on the BAG. The most effective glass was S53P4, which had a clear growth-inhibitory effect on all pathogens tested. The sol-gel derived materials CaPSiO and CaPSiO II also showed a strong antibacterial effect. In summary, BAGs were found to clearly inhibit the growth of a wide selection of bacterial species causing e.g., infections on the surfaces of prostheses in the body after implantation.

Outer surface proteins OspA and OspB are among the most prominent Borrelia burgdorferi surface molecules. We constructed OspAB and OspA complementation mutants of B. burgdorferi Osp-less strain B313 and investigated the role of these surface proteins in the interactions of B. burgdorferi, human neutrophils and the complement system. We found that (1) OspB inhibits the phagocytosis and oxidative burst of human neutrophils at low serum concentrations, whereas OspA induces the oxidative burst in neutrophils; (2) OspB may have an inhibiting role in serum sensitivity and complement activation; (3) all studied strains inhibit the chemotaxis of human neutrophils specifically towards fMLP but not towards C5a, regardless of their Osp expression. These results suggest that although OspA and OspB are co-ordinately transcribed, they differ in their effects on human neutrophil functions. Our findings suggest that B. burgdorferi exploits a wide variety of immune evasion mechanisms, besides previously documented complement resistance, to survive in the vertebrate host.

Lyme borreliosis is a disease, which can affect several organs and cause a variety of symptoms. In some patients, the infection may become chronic, even after antibiotic therapy, and cause persisting damage. Dendritic cells (DC) are involved in the initiation of innate and adaptive immune responses. To study interactions between Borrelia garinii (Bg), one of the causative agents of Lyme borreliosis, and human DC, we used a cDNA microarray to compare the Bg-induced DC transcriptional response with the response induced by LPS. The Bg-induced response consisted of a smaller number of genes than the LPS-induced response. The microarray showed that the ectoenzyme CD38, which has an important role in DC chemotaxis and migration to lymph nodes, was strongly up-regulated by LPS but practically not at all by Bg. This finding was confirmed with quantitative RT-PCR and with flow cytometry at the protein level. In addition, RT-PCR showed that CCR7 expression was 11-fold greater in LPS-stimulated than in Bg-stimulated cells. These findings suggest that Bg may affect crucial DC functions by blocking the up-regulation of important molecules in DC migration to lymph nodes, thus affecting further immune responses in Lyme borreliosis infection.

Background: The effect of anti-tumor necrosis factor (TNF)-alpha treatment in Borrelia burgdorferi-infected and ceftriaxone-treated C3H/He mice was evaluated., Methods: Mice were infected with B. garinii A218 or B. burgdorferi sensu stricto N40. At 2 weeks of infection, one group was treated simultaneously with ceftriaxone and anti-TNF-alpha, whereas another received ceftriaxone at 2 weeks and anti-TNF-alpha 4 weeks later. One group received ceftriaxone treatment only. Infected and noninfected control groups were sham treated., Results: At 14 weeks of infection, B. burgdorferi could not be detected by cultivation or by polymerase chain reaction in tissue samples of any mouse treated with ceftriaxone only. However, spirochetes grew from the tissue samples of one-third of the mice treated with anti-TNF-alpha simultaneously or 4 weeks after ceftriaxone. These activated spirochetes showed ceftriaxone sensitivity rates, plasmid profiles, and virulence rates similar to those of bacteria used to infect the mice. All infected control mice and mice given anti-TNF-alpha only were culture positive., Conclusions: This report shows that, after ceftriaxone treatment for 5 days, a portion of B. burgdorferi-infected mice still have live spirochetes in their body, which are activated by anti-TNF-alpha treatment.

Background: Species of the tick-transmitted spirochete group Borrelia burgdorferi sensu lato (B. burgdorferi) cause Lyme borreliosis. Acute borrelial infection of the skin has unusual characteristics with only a mild local inflammatory response suggesting that the interaction between borreliae and the cells of the first-line defence might differ from that of other bacteria. It has been reported that human neutrophils phagocytose motile borreliae through an unconventional mechanism (tube phagocytosis) which is not observed with non-motile borreliae. Therefore, it would be of great interest to visualise the bacteria by a method not affecting motility and viability of borreliae to be able to study their interaction with the cells of the innate immunity. Carboxyfluorescein diacetate, succinimidyl ester (CFSE) labelling has been previously used for studying the adhesion of labelled bacteria to host cells and the uptake of labelled substrates by various cells using flow cytometry., Results: In this study, CFSE was shown to efficiently stain different genospecies of B. burgdorferi without affecting bacterial viability or motility. Use of CFSE staining allowed subsequent quantification of borreliae associated with human neutrophils with flow cytometry and confocal microscopy. As a result, no difference in association between different borrelial genospecies (Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii), or between borreliae and the pyogenic bacterium Streptococcus pyogenes, with neutrophils could be detected. Borrelial virulence, on the other hand, affected association with neutrophils, with significantly higher association of a non-virulent mutant B. burgdorferi sensu stricto strain compared to the parental virulent wild type strain., Conclusion: These results suggest that the flow cytometric assay using CFSE labelled borreliae is a valuable tool in the analysis of the interaction between borreliae and human neutrophils. The results also indicate a clear difference in the association with neutrophils between virulent and non-virulent borrelial strains.

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.

We wanted to study the pathogenesis and the long-term manifestations of Borrelia garinii infection in SJL and C3H/He mice. We report here that B. garinii A218 causes a persisting infection in these mouse strains. Mice infected with intracutaneous inoculation of B. garinii at 4-5 weeks of age developed a disseminated infection and joint swelling within 2 weeks of inoculation and remained infected with joint symptoms until the end of follow-ups of up to 52 weeks. Treatment with ceftriaxone or ampicillin at 18 or 44 weeks of infection did not affect the joint swelling during the follow-ups of 19 and 8 weeks, respectively. However, B. garinii could not be cultured from any of the post mortem tissue samples of the treated mice, whereas the spirochete grew from samples of all untreated infected animals. Borrelia-specific IgG antibodies were detectable after 2 weeks of infection, and in late infection, all mice had high anti-borrelia IgG levels. Antibiotic treatment had no effect on antibody levels. Histology showed only slight changes in the joints of the infected mice with occasional lymphocyte infiltration, synovial proliferation and slight involvement of the Achilles' tendon. No difference was seen in the findings between ceftriaxone-treated and untreated mice. The results suggest that the presence of vegetative spirochetes is no prerequisite for persisting joint symptoms and elevated anti-borrelia IgG levels in these B. garinii-infected mice.

Two commercially available DNA line probe assays, Genotype MTBDR and INNO-LiPA Rif. TB, were evaluated for their abilities to detect resistance to isoniazid (INH) and rifampin (RIF) in 52 Mycobacterium tuberculosis isolates. The test results were compared to those obtained by phenotypic drug susceptibility testing and sequencing. Compared to the results of phenotypic drug susceptibility testing, the Genotype MTBDR test results were concordant for INH for 47 of the 52 (90.4%) isolates, and both the Genotype MTBDR and the INNO-LiPA Rif. TB test results were concordant for RIF for 51 of the 52 (98.1%) isolates. The Genotype MTBDR test results correlated with the sequencing results for 48 of the 52 (92.3%) isolates and the INNO-LiPA Rif. TB results for 50 of the 52 (96.2%) isolates. Both assays are useful for the rapid screening of M. tuberculosis isolates obtained from patients suspected of having multidrug-resistant tuberculosis, but the GenoType MTBDR assay has the advantage of being able to detect resistance to both INH and RIF simultaneously.

Hybridization of bacteria with fluorescent probes targeting 16S rRNA and inspection of hybridized bacteria with fluorescence microscopy (microscopy-FISH, i.e. fluorescence in situ hybridization) have constituted an accessible method for the analysis of mixed bacterial samples such as feces. However, microscopy-FISH is a slow method and prone to errors. Flow cytometry (FCM) enables analysis of bacteria more rapidly, accurately and reliably than microscopy. In this study, a FCM method for the analysis of 16S rRNA-hybridized and DNA-stained fecal bacteria was developed. The results of FCM-FISH were comparable to those of microscopy-FISH, and the coefficients of variation of the FCM analyses were extraordinarily low. In previous FCM-FISH studies, the Eub 338 probe, which is supposed to hybridize all bacteria, has been used to detect all bacteria present in the sample. We found that Eub 338 did not bind to all bacteria, which could be detected by DNA-staining; while SYTOX Orange DNA-stain detected all bacterial species tested and produced high fluorescence intensities enabling clear separation of bacteria from non-bacterial material. Thus, DNA-staining is a method of choice for the detection of all bacteria in FCM-FISH. We conclude that FCM of 16S rRNA-hybridized and DNA-stained bacteria is a rapid and reliable method for the analysis of mixed bacterial samples including feces.

A 33-mer peptide of the alpha-gliadin component of gluten was recently identified as primary initiator of the inflammatory response to gluten in coeliac disease (CD) patients. This proline-glutamine-rich peptide (PG-peptide) is highly homologous to internal sequence of pertactin, an immunogenic protein of Bordetella pertussis. Using enzyme immunoassays, we measured serum antibodies to pertactin and to PG-peptide in 167 Finnish subjects including pertussis vaccine recipients and pertussis patients, CD and non-CD patients and healthy individuals. We found no cross-reactivity between human antibodies to the two different components, suggesting that neither pertussis immunization nor disease contributes to the pathogenesis of CD.

In order to evaluate the proficiency of the GenoType Mycobacteria strip hybridization assay (Hain Lifescience, Nehren, Germany) for the routine identification of mycobacteria, the assay was used to identify 178 clinical isolates during a 6-month prospective study. The GenoType results were compared to the identification results obtained with AccuProbe (GenProbe, San Diego, CA, USA) or 16S rDNA sequencing, and an overall agreement of 89.3% between GenoType and the two reference methods was reached. The GenoType assay is, thus, a rapid and reliable method for the identification of clinically important mycobacteria, and it is well suited for use in a routine laboratory.

The outer-membrane protein pertactin (Prn) of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica is believed to function as an adhesin and is an important immunogen. The emergence of B. pertussis and B. bronchiseptica Prn variants has been reported. The aim of this study was to determine whether similar variation is found in B. parapertussis Prn and to characterize Finnish clinical B. parapertussis isolates that were collected in 1982-2000. Of 76 B. parapertussis isolates studied, seven (9 %) were found to have silent and non-silent nucleotide changes. In addition, one (1 %) had eight PQP repeats instead of nine. Three closely related B. parapertussis XbaI PFGE patterns were found. Genetic variation of B. parapertussis was found to be very limited, suggesting that B. parapertussis is a stable organism that is well-adapted to its own ecological niche.

In this study, the 7H10 agar proportion method was compared with the BACTEC TB-460 and BACTEC MGIT 960 systems (BD Biosciences, USA) for the susceptibility testing of 22 genetically characterized Mycobacterium tuberculosis isolates for isoniazid, rifampin, streptomycin, and ethambutol. The 7H10 agar proportion method agreed with the resistant genotype in 87.3%, BACTEC TB-460 in 92.7%, and the MGIT in 96.4% of the cases, showing the high sensitivity of MGIT in the detection of resistant isolates.

We evaluated PCR for the detection of Bacillus anthracis DNA from simulated clinical specimens relevant for the microbiological diagnosis of anthrax or exposure to B. anthracis spores. In simulated blood specimens, the lowest limit of detection was 400 CFU per mL of blood, which may be sufficient for samples from patients with septic anthrax. Screening nasal swabs by PCR may not be sensitive enough to rule out dangerous exposure to anthrax spores, as a minimum of 2000 spores per sample was required for detectable amplification. As spores survived some standard DNA purification methods, special attention should be paid to laboratory safety when preparing samples possibly containing live spores.

A real-time PCR method was developed and used to detect Aspergillus fumigatus mitochondrial DNA (mtDNA) in bronchoalveolar lavage (BAL) fluids and tissue biopsy specimens. The analytical sensitivity of the assay was one A. fumigatus conidium per reaction, and the assay was linear at least over 4 orders of magnitude above the detection limit. BAL fluids from 66 immunocompromised patients at risk of invasive pulmonary aspergillosis (IPA) and 33 immunocompetent controls and tissue biopsy specimens from 10 immunocompromised patients were analyzed. The results were related to the clinical diagnosis established according to recently published consensus criteria. A. fumigatus mtDNA positivity was encountered in 16 of 81 (20%) BAL fluid specimens from patients at risk and 1 of 33 (3%) specimens from immunocompetent controls. PCRs were positive in six of seven, two of four, and four of five of the patients with proven, probable, and possible IPA, respectively, as well as in four patients at risk but without any other evidence of IPA. With qualitative detection, the diagnostic sensitivity of PCR was 73%, specificity was 93%, and predictive values of positive (PPV) and negative (NPV) results were 73 and 95%, respectively. Using a threshold cycle of <35 as a limit for positive PCR, the specificity and PPV of PCR in the diagnosis of invasive aspergillosis were 100%, but its sensitivity was only 45% and NPV was 92%. PCR was positive in tissue biopsy specimens from all patients with invasive aspergillosis caused by A. fumigatus. Semiquantitative detection of A. fumigatus mtDNA in BAL fluid may be helpful in the diagnosis of IPA. PCR is well suited for the verification of the presence of A. fumigatus in tissue biopsy specimens.

Antigen uptake and the following maturation of dendritic cells (DCs) are pivotal to the initiation of specific antimicrobial immune responses. DCs also play an important role in the recruitment and activation of the cells of the innate immune system. We have examined the interactions of DCs with Borrelia burgdorferi to find explanations for the difficulties the human immune system has in dealing with the bacterium. Phagocytosis of B. burgdorferi by immature DCs and the effect of the bacterium on the maturation and interleukin-8 (IL-8) secretion of DCs were studied. Borreliae were phagocytized and processed into fragments by DCs; narrow tube-like pseudopods and broad pseudopods were used for the engulfment. The immature DC population gained a heterogeneous appearance within 2 h of incubation with the borreliae. A 24 h coculture with borreliae induced maturation and IL-8 secretion in the DCs in a manner comparable with the effect of lipopolysaccharides. All strains studied, including a mutant strain lacking outer surface proteins A and B, were capable of inducing these responses. Thus, our results did not show any clear inadequacy concerning the way DCs are dealing with B. burgdorferi. However, further studies on the subject are required.

Divergence has been found between Bordetella pertussis vaccine strains and circulating strains. Polymorphism in pertactin (Prn) is essentially limited to region 1, which is made up of repeats. Today, the 3 most prevalent Prn variants are Prn1-3. Vaccine strains produce Prn1, whereas Prn2 is the predominant type found in circulating strains. We investigated how variation in region 1 affects the production of human serum antibodies. Individuals infected by Prn2 strains had significantly fewer antibodies to Prn1 did than those infected by Prn3 strains and those immunized with a booster dose of acellular vaccines containing Prn1. Moreover, in contrast to vaccine recipients and subjects infected by Prn3 strains, individuals infected by Prn2 strains had hardly any antibodies specific to the variable region of Prn1. These results indicate that conformational changes have occurred in the variable region of Prn, which may offer a possible explanation for the emergence of Prn2 strains in certain countries.

The molecular epidemiology of tuberculosis (TB) in Finland was studied by spoligotyping 380 Mycobacterium tuberculosis isolates. The isolates were obtained during a 1-year study period from July 2000 to June 2001 and represented 90% of new M. tuberculosis findings by culture in the whole country during the study period. The spoligotyping results were compared to the World Spoligotyping Database of the Institut Pasteur de Guadeloupe, which contains data from >14,000 M. tuberculosis isolates obtained worldwide. A total of 138 different spoligotypes were identified among the 380 M. tuberculosis isolates. Thirty-eight (10%) isolates had unique spoligotypes, while 342 (90%) isolates belonged to 100 shared types. The four most common spoligotypes caused approximately one-third of the Finnish TB cases. Forty-seven of the 138 (34.1%) spoligotypes and 61 (16.1%) of the 380 M. tuberculosis isolates had spoligotypes that had not been previously reported. Only four (1.1%) patients were infected with an isolate belonging to the Beijing genotype. The characterization of Finnish M. tuberculosis isolates by spoligotyping shows that ubiquitous spoligotypes were common, but many spoligotypes specific to Finland were also found. However, Beijing family isolates were rarely encountered, although this spoligotype is predominant in our eastern and southern neighbors.

Altogether, 343 adult and 111 nymphal Ixodes ricinus ticks collected from parks in Turku and suburban and rural islands of the Turku archipelago, Finland, and 100 adult I. ricinus ticks collected from Vormsi Island, Estonia, were included in this study. Using the polymerase chain reaction the ticks were examined for 16S rDNA of the Ehrlichia phagocytophila genogroup and for Borrelia burgdorferi sensu lato recA and flagellin genes. None of the Finnish ticks was found to be infected with E. phagocytophila, whereas 3% of the Estonian ticks were positive for this organism. The rate of Finnish ticks infected with B. burgdorferi sensu lato varied from 0% to 11.6% (mean 5%; 9% for adult and 4% for nymphal ticks). The corresponding rate for Estonian ticks was 15%. Borrelia afzelii was the most common genospecies in both Finnish (2.6%) and Estonian (12%) ticks. B. burgdorferi sensu stricto was detected in 2.0% of the Finnish ticks, but in none of the Estonian ticks. These results suggest that the E. phagocytophila genogroup is very rare in Finnish ticks, although the ticks were collected from an area endemic for Lyme borreliosis. In Estonia, E. phagocytophila is found in ticks and may cause disease.

The fate of borreliae invading a human may depend on the early innate response they induce. The interactions of human complement system and neutrophils with two strains of the Lyme borreliosis spirochete Borrelia burgdorferi were studied. Borrelia burgdorferi sensu stricto B31 (resistant to a 28% concentration of normal human serum (NHS)) and Borrelia garinii Bg A218/98 (sensitive to 7% NHS) were examined. Both strains induced neutrophil oxidative burst in a complement-dependent manner. B31 required the presence of 7% NHS, but Bg A218/98 required the presence of only 0.7% NHS for optimal induction of the burst. At all concentrations of NHS, the proportion of the spirochetes with C3bi on their surfaces and the relative amount of C3bi bound per spirochete were larger with Bg A218/98 than with B31. Bg A218/98 was able to induce an oxidative burst, when provided with serum with blocked classical pathway of complement, whereas B31 required the presence of the classical pathway. We suggest a role for the opsonizing effect of complement in controlling borreliae that are either resistant to direct killing by complement or located in the compartments of the human body at sublethal concentrations of the same.

Two DNA strip assays, INNO-LiPA MYCOBACTERIA and GenoType Mykobakterien, were evaluated for identification of 81 Finnish mycobacterial isolates. The LiPA assay correctly identified 89.4% of the 66 isolates studied, and the GenoType assay identified 95.1% of 81 isolates. The GenoType assay had a wider selection of species and less stringent temperature requirements.

A LightCycler real-time PCR hybridization probe assay was developed for rapid typing of gene variants of the Bordetella pertussis virulence factor pertussis toxin. The assay correctly identified the ptxS1 alleles of all strains tested, comprising 57 Finnish clinical isolates and 2 vaccine strains. The method is simple, reliable, and suitable for large-scale screening of B. pertussis strains.

Background: Chlamydia pneumoniae infection has been associated with atherosclerosis by sero-epidemiological, histopathological and interventional studies, and animal experiments. We hypothesized that if chlamydial infection is causative of atherosclerosis, the occurrence of antibodies against C. pneumoniae should be associated with coronary vasomotor dysfunction - an early sign of atherosclerosis., Aim: To study the association between C. pneumoniae infection and coronary vasomotor function in young men without signs of ischemic heart disease., Methods: Serum IgG and IgA antibody concentrations against C. pneumoniae were determined in 125 clinically healthy subjects undergoing positron emission tomography (PET) studies. Myocardial blood flow was measured at rest and during pharmacologically induced hyperemia using [15O]H2O Coronary flow reserve was calculated as the ratio of hyperemic blood flow to resting blood flow., Results: No association was found between serum C. pneumoniae antibody concentrations and myocardial blood flow parameters. In contrast, more conventional risk factors for coronary artery disease, such as total cholesterol and apolipoprotein B, were inversely associated with hyperemic flow and flow reserve., Conclusions: We found no association between C. pneumoniae antibodies and coronary vasomotor function in subjects without ischemic heart disease. Thus, these results do not support the role of C. pneumoniae infection as an early phase risk factor for coronary artery disease.

Recently, eight allelic variants of the pertactin gene (prn1-8) have been characterized in Bordetella pertussis strains isolated in Europe and the United States. It has been suggested that the divergence of the pertactin types of clinical isolates from those of the B. pertussis vaccine strains is a result of vaccine-driven evolution. Sequencing of the prn, which is relatively time-consuming, has so far been the only method for the differentiation of prn types. We have developed a rapid real-time polymerase chain reaction assay suitable for large-scale screening of the prn type of the circulating strains. This method correctly identified the prn type of all tested 41 clinical isolates and two Finnish vaccine strains. The method is simple and reliable and provides an alternative for sequencing in pertussis research.

The diagnosis of erythema migrans (EM) is not always easy, and reports of culture- or PCR-confirmed diagnosis as well as reports of EM with simultaneous disseminated disease are few. Characteristics and incidence of EM in addition to frequency of early dissemination of B. burgdorferi were studied in the archipelago of South-Western Finland prospectively using questionnaires, skin biopsies and blood samples. Clinical EM was recognized in 82 patients (incidence 148/100,000 inhabitants/year). Of skin biopsy samples, 35.5% were positive by PCR (the majority B. garinii), and 21.5% by cultivation (all B. garinii). Of blood samples, 3.8% were positive by PCR, and 7.7% by cultivation. Of the patients, 30.9% were seropositive at the first visit, and 52.9% 3 weeks later. Of the patients with laboratory confirmed diagnosis, the EM lesion was ring-like in 31.8% and homogeneous in 65.9%. Dissemination of B. burgdorferi, based on culture or PCR positivity of blood samples, was detected in 11.0% of the patients. The frequency of generalized symptoms was nearly the same in patients with as in those without dissemination (22.2% vs 27.4%). Only 21.4% of the patients with culture-positive EM recalled a previous tick bite at the site of the EM lesion. We conclude that EM lesions are more often homogeneous than ring-like. B. burgdorferi may disseminate early without generalized symptoms.

Background: Commercial nucleic acid amplification tests, designed for the detection of Mycobacterium tuberculosis DNA/RNA in respiratory samples, are often applied also in nonrespiratory specimens in order to verify the diagnosis of extrapulmonary tuberculosis. AIM. To evaluate the value of the Abbott LCx Mycobacterium tuberculosis assay for the diagnosis of pulmonary and extrapulmonary tuberculosis based on routine clinical laboratory results., Methods: The assay was used to analyse 350 respiratory and 826 nonrespiratory specimens from 961 patients, of whom 3.6% had culture-proven tuberculosis. The results obtained by the LCx assay were compared with the records on mycobacterial isolates of the national reference laboratory and, in the case of positive findings, with clinical data., Results: In comparison with culture, the sensitivity, specificity and positive/negative predictive value of the assay on respiratory specimens were 87.5%, 99.7%, 93.3% and 99.4%, respectively. With nonrespiratory specimens, the overall sensitivity, specificity and positive/negative predictive value of the LCx assay were 73.3%, 98.0%, 40.7% and 99.5%, respectively. When clinical and histological data were also included, the positive predictive value of LCx with nonrespiratory specimens was 45.8%., Conclusion: Critical interpretation of the nucleic acid amplification results obtained from nonrespiratory specimens is necessary in both laboratory and clinical settings.

Despite widespread awareness of the most classical clinical presentation with central clearing of erythema migrans, a pathognomonic sign of infection with Borrelia burgdorferi, diagnosis of other forms of erythema migrans remains more difficult. We describe a case of a patient with secondary lesions of erythema migrans that within three months formed a complicated pattern and affected at last nearly the entire lower limb of the patient. In addition, the erythema appeared to be posture-dependent in the way that the lesion was with central clearing in the supine and with homogeneous appearance in the upright position. The borrelial infection was confirmed by PCR sequencing that detected DNA of B. afzelii in the skin biopsy specimen. The lesions disappeared during antibiotic therapy. This case shows how posture can be important in the examination of patients with a suspected erythema migrans.

To differentiate the Borrelia burgdorferi sensu lato genospecies, LightCycler real-time PCR was used for the fluorescence (SYBR Green I) melting curve analysis of borrelial recA gene PCR products. The specific melting temperature analyzed is a function of the GC/AT ratio, length, and nucleotide sequence of the amplified product. A total of 32 DNA samples were tested. Of them three were isolated from B. burgdorferi reference strains and 16 were isolated from B. burgdorferi strains cultured from Ixodes ricinus ticks; 13 were directly isolated from nine human biopsy specimens and four I. ricinus tick midguts. The melting temperature of B. garinii was 2 degrees C lower than that of B. burgdorferi sensu stricto and B. afzelii. Melting curve analysis offers a rapid alternative for identification and detection of B. burgdorferi sensu lato genospecies.

We evaluated the usefulness of the ligase chain reaction (LCR) (Abbott LCx Mycobacterium tuberculosis assay) during the initial diagnosis of tuberculosis. LCx was carried out in parallel with conventional methods for the analysis of clinical samples. Out of 86 patients who were examined clinically, 53 were suspected of having pulmonary tuberculosis, eight had residual X-ray scars from previous tuberculosis and 25 served as asymptomatic controls. Ten bronchoscopy samples and 237 sputum samples were analysed by direct microscopy, culture and LCx. All 11 smear-positive and two of three smear-negative tuberculosis patients had at least one LCx-positive specimen. All samples that were both LCx- and smear-positive were culture-positive for M. tuberculosis. The smear-positive samples from the five patients with atypical mycobacteriosis were LCx-negative. There were three false-positive results: one in a smear-negative sample from a patient with M. malmoense infection and two from two pneumonia patients. All samples from controls and patients with previous tuberculosis were LCx-negative. The sensitivity, specificity and the positive and negative predictive values of LCx in patient analysis were 92.9%, 95.8%, 81.3% and 98.6%, respectively. LCx assay of M. tuberculosis is useful in rapid confirmation of tuberculosis or atypical mycobacteriosis from a smear-positive sample and may aid in diagnosing smear-negative tuberculosis.