When is the best time to order a Western blot and how should it be interpreted?

Despite significant progress in the diagnostics of Lyme borreliosis, including molecular methods, the detection of a specific antibody response remains the mainstay in the laboratory diagnosis of the disease. Current guidelines propose the combination of highly sensitive screening assays, such as ELISAs, with very specific confirmatory tests, such as immunoblots, to guarantee a cost-effective, sensitive and specific diagnostic approach. For a correct interpretation of the serological findings, the investigator must always consider a whole series of clinical and laboratory facts. Here, we summarize current laboratory algorithms in the diagnosis of Lyme borreliosis, with a special emphasis on when to order a Western blot and how to interpret it correctly in the context of additional clinical and laboratory information.

[Lyme borreliosis: research gaps and research approaches. Results from an interdisciplinary expert meeting at the Robert Koch Institute]. / Lyme-Borreliose: Forschungsbedarf und Forschungsansätze. Ergebnisse eines interdisziplinären Expertentreffens am Robert Koch-Institut.

Lyme borreliosis is currently the most frequent tick-transmitted zoonosis in the northern hemisphere. Germany and other European countries are regarded as highly endemic areas; therefore the burden of disease and consequently the costs for the health systems are considered to be high. This report summarises the results of an interdisciplinary workshop on Lyme borreliosis which aimed to identify research deficits and to prioritise areas which need to be addressed. Research needs have been recognised for different areas: diagnosis, epidemiology, immunology, clinics, ecology and health services research. Examples of research areas which have priority are the standardisation of diagnostic tests, the development of markers to detect an active infection, the improvement of the epidemiological database and the analysis of the burden of disease.

Factor H family proteins: on complement, microbes and human diseases.

At present, the human Factor H protein family represents seven multidomain, multifunctional serum proteins. This group includes the complement and immune regulators Factor H, the Factor H-like protein 1 (FHL-1) and five Factor H-related proteins proteins (FHR-1, -2, -3, -4 and -5). Each is exclusively composed of individually folded protein domains, termed short consensus repeats (SCRs) or complement control modules. Structure-function analyses allowed the localization of the complement regulatory domain of Factor H and FHL-1 in the N-terminal region within SCRs 1-4. In addition, multiple binding sites for C3b, heparin and microbial surface proteins were localized in the N-terminus, within the middle region and also in the C-terminus of Factor H and FHL-1. Recent results show a central role for the C-terminus of Factor H, i.e. SCRs 19-20. These particular domains are conserved in all FHRs identified so far, include contact points for C3b, heparin and microbial surface proteins and represent a 'hot-spot' for gene mutations in patients that suffer from the Factor H-associated form of haemolytic uraemic syndrome.

Further characterization of complement regulator-acquiring surface proteins of Borrelia burgdorferi.

The three genospecies Borrelia burgdorferi, Borrelia garinii, and Borrelia afzelii, all causative agents of Lyme disease, differ in their susceptibilities to human complement-mediated lysis. We recently reported that serum resistance of borrelias correlates largely with their ability to bind the human complement regulators FHL-1/reconectin and factor H. To date, two complement regulator-acquiring-proteins (CRASP-1 and CRASP-2) have been identified in serum-resistant B. afzelii isolates (P. Kraiczy, C. Skerka, M. Kirschfink, V. Brade, and P. F. Zipfel, Eur. J. Immunol. 31:1674-1684, 2001). Here, we present a comprehensive study of the CRASPs detectable in both serum-resistant and intermediate serum-sensitive B. afzelii and B. burgdorferi isolates. These CRASPs were designated according to the genospecies either as BaCRASPs, when derived from B. afzelii, or as BbCRASPs, for proteins identified in B. burgdorferi isolates. Each borrelial isolate expresses distinct CRASPs that can be differentiated by their mobility and binding phenotypes. A detailed comparison reveals overlapping and even identical binding profiles for BaCRASP-1 (27.5 kDa), BbCRASP-1 (25.9 kDa), and BbCRASP-2 (23.2 kDa), which bind FHL-1/reconectin strongly and interact weakly with factor H. In contrast, two B. afzelii proteins (BaCRASP-4 [19.2 kDa] and BaCRASP-5 [22.5 kDa]) and three B. burgdorferi proteins (BbCRASP-3 [19.8 kDa], BbCRASP-4 [18.5 kDa], and BbCRASP-5 [17.7 kDa]) bind factor H but not FHL-1/reconectin. Most CRASPs bind both human immune regulators at their C-terminal ends. Temperature-dependent up-regulation of CRASPs (BaCRASP-1, BaCRASP-2, and BaCRASP-5) is detected in low-passage borrelias cultured at 33 or 37 degrees C compared with those cultured at 20 degrees C. The characterization of the individual CRASPs on the molecular level is expected to identify new virulence factors and potential vaccine candidates.

In vitro activities of fluoroquinolones against the spirochete Borrelia burgdorferi.

Little is known to date about the in vitro activity of fluoroquinolones against Borrelia species. Our study aimed at determining the in vitro activities of 15 quinolones against nine isolates of the Borrelia burgdorferi sensu lato complex in addition to one Borrelia valaisiana and one Borrelia bissettii tick isolate. For the determination of MICs, a standardized colorimetric microdilution method was applied. Determination of minimal borreliacidal concentrations providing 100% killing of the final inoculum (MBCs) after 72 h and time-kill experiments were performed by conventional culture in Barbour-Stoenner-Kelly medium in combination with dark-field microscopy. The rank order of potency on a microgram-per-milliliter basis for the substances with in vitro activity against B. burgdorferi was gemifloxacin (MIC at which 90% of the isolates tested are inhibited [MIC(90)], 0.12 microg/ml) > sitafloxacin (MIC(90), 0.5 microg/ml), grepafloxacin (MIC(90), 0.5 microg/ml) > gatifloxacin (MIC(90), 1 microg/ml), sparfloxacin (MIC(90), 1 microg/ml), trovafloxacin (MIC(90), 1 microg/ml) > moxifloxacin (MIC(90), 2 microg/ml), ciprofloxacin (MIC(90), 2 microg/ml) > levofloxacin (MIC(90), 4 microg/ml) > ofloxacin (MIC(90), 8 microg/ml), norfloxacin (MIC(90), 8 microg/ml) > fleroxacin (MIC(90), >16 microg/ml), and pefloxacin (MIC(90), 32 microg/ml) > nalidixic acid (MIC(90), 256 microg/ml). After 72 h of exposure, gemifloxacin was borreliacidal (100% killing) against the isolates investigated at a median MBC of 4 microg/ml. In the other compounds tested, median MBCs were higher (> or =8 microg/ml). Results of electron microscopy and time-kill studies clearly support an in vitro activity of some fluoroquinolones against borreliae. Our study demonstrates for the first time the enhanced in vitro effectiveness of some of the recently introduced 4-quinolones against B. burgdorferi.

Rapid molecular typing of methicillin-resistant Staphylococcus aureus by PCR-RFLP.

OBJECTIVE: To establish a new, rapid, and reliable genotypic fingerprinting technique for methicillin-resistant Staphylococcus aureus (MRSA) typing in routine epidemiological surveillance. DESIGN: The method is based on polymerase chain reaction (PCR) restriction fragment-length polymorphism (RFLP) following HaeII digestion of simultaneously amplified parts of the protein A gene, the coagulase gene, and the hypervariable region adjacent to mecA. A total of 46 MRSA initial isolates were analyzed, including 14 isolates from five countries; the six German epidemic strains; 16 isolates from the Frankfurt metropolitan area, which were known to be heterogeneous by pulsed-field gel electrophoresis (PFGE); and 10 isolates obtained during three epidemics, all of which displayed an identical genotype. RESULTS: Restriction analysis by PCR-RFLP permitted discrimination of 10 of 14 international isolates, all six German epidemic strains, and 15 of 16 national isolates. It also confirmed the homogeneous character of the 10 outbreak isolates. CONCLUSIONS: This new and rapid PCR-RFLP typing method is an attractive tool in routine epidemiological surveillance. Its impressive characteristics are ease of performance and interpretation, while at the same time guaranteeing good discriminatory power, reproducibility, and typeability.

Immune evasion of Borrelia burgdorferi by acquisition of human complement regulators FHL-1/reconectin and Factor H.

To understand immune evasion mechanisms of Borrelia burgdorferi we compared serum-resistant B. afzelii and serum-sensitive B. garinii isolates for their capacity toacquire human complement regulators. Here we demonstrate that the two borrelial genospecies show different binding of the two important human complement regulators, FHL-1/reconectin and Factor H. All serum-resistant B. afzelii isolates bound FHL-1/reconectin and also Factor H, and all analyzed serum-sensitive B. garinii isolates showed no or a significantly lower binding activity. Using recombinant deletion mutants, the binding domains were localized to the C terminus of FHL-1/reconectin to short consensus repeats 5-7. The borrelial binding proteins were located in the surface of the bacteria as demonstrated by immunofluorescence staining of intact, serum-exposed bacteria and by enrichment of outer membrane proteins. The surface-attached complement regulators maintained complement regulatory activity as demonstrated in a cofactor assay. By ligand blotting two different borrelial binding proteins were identified that were responsible for the surface attachment of FHL-1/reconectin and Factor H. These borrelial complement regulators acquiring surface proteins (CRASP) were further characterized as either CRASP-1, a 27.5-kDa molecule which preferentially binds FHL-1/reconectin and which was present in all serum-resistant borreliae, or CRASP-2, a 20/21-kDa protein which interacts preferentially with Factor H and the expression of which was more restricted, being detected in four of the six isolates analyzed. In summary, we describe a new immune evasion mechanism of B. burgdorferi, as these bacteria acquire human complement regulators to control complement activation on their surface and to prevent formation of toxic activation products.

Mechanism of complement resistance of pathogenic Borrelia burgdorferi isolates.

Borrelia burgdorferi, the causative agent of Lyme disease, differ in their susceptibility to normal human serum and are consequently classified as complement-resistant, complement-sensitive and intermediate complement-sensitive. Most isolates belonging to the genospecies B. afzelii are complement-resistant, while particularly B. garinii isolates were rapidly killed by complement. In general, isolates of the genospecies B. burgdorferi sensu stricto (s.s.) are intermediate complement-sensitive. Independent of the genospecies, all Borreliae were capable to activate the classical and/or the alternative pathway. Deposition of the activation products C3, C6, and TCC is much stronger by B. burgdorferi s.s. and B. garinii isolates than by B. afzelii isolates. The mechanism(s) on how Borreliae evade complement-mediated bacteriolysis has recently been described by showing that complement-resistant B. afzelii isolates but not the complement-sensitive B. garinii isolates absorb human complement regulators FHL-1/reconectin and factor H. Surface-attached FHL-1/reconectin maintains its complement regulatory activity and supports factor I-mediated C3b cleavage to iC3b. In complement-resistant Borreliae, two outer surface proteins, the 27.5 kDa (CRASP-1, complement regulator-acquiring surface protein 1) and the 20/21 kDa (CRASP-2), are responsible for the surface attachment of the two complement regulators. CRASP-1, which is present in complement-resistant Borreliae, binds preferentially FHL-1/reconectin while CRASP-2, which is restrictively expressed, binds preferentially factor H. Thus, complement-resistant Borreliae bind human complement regulators and control complement activation on their surface and prevent the formation of toxic activation products.

In vitro activity of mezlocillin, meropenem, aztreonam, vancomycin, teicoplanin, ribostamycin and fusidic acid against Borrelia burgdorferi.

The in vitro susceptibility profile of Borrelia burgdorferi is not yet well defined for several antibiotics. Our study explored the in vitro susceptibility of B. burgdorferi to mezlocillin, meropenem, aztreonam, vancomycin, teicoplanin, ribostamycin and fusidic acid. Minimal inhibitory concentrations (MICs) and minimal borreliacidal concentrations (MBCs) were measured using a standardised colorimetric microdilution method and conventional subculture experiments. MIC values were lowest for mezlocillin (MIC(90), < or =0.06 mg/l) and meropenem (MIC(90), 0.33 mg/l). Vancomycin (MIC(90), 0.83 mg/l) was less effective in vitro. Borreliae proved to be resistant to aztreonam (MIC(90), >32 mg/l), teicoplanin (MIC(90), 6.6 mg/l), ribostamycin (MIC(90), 32 mg/l), and fusidic acid (MIC(90), >4 mg/l). The mean MBCs resulting in 100% killing of the final inoculum after 72 h of incubation were lowest for mezlocillin (MBC, 0.83 mg/l). This study gathered further data on the in vitro susceptibility patterns of the B. burgdorferi complex. The excellent in vitro effectiveness of acylamino-penicillin derivatives and their suitability for the therapy of Lyme disease is emphasised.

Colorimetric in vitro susceptibility testing of penicillins, cephalosporins, macrolides, streptogramins, tetracyclines, and aminoglycosides against Borrelia burgdorferi isolates.

The spectrum of antibiotic susceptibility of Borrelia burgdorferi has been only partially defined. In the present study the effectiveness of 12 antimicrobials, belonging to six different antibiotic classes have been tested against Borrelia burgdorferi s.s. (N=3), Borrelia garinii (N=3), Borrelia afzelii (N=3), Borrelia valaisiana (N=1), and Borrelia bissettii (N=1) isolates. These isolates were analysed by a new standardised colorimetric minimal inhibitory concentration (MIC) method based upon colour changes that result from actively metabolizing spirochaetes after 72 h of incubation. Piperacillin (MIC90: 0.08 mg/l), ceftriaxone (MIC90: 0. 04 mg/l), cefotaxime (MIC90: 0.15 mg/l), azithromycin (MIC90: 0.015 mg/l), roxithromycin (MIC90: 0.05 mg/l) and quinupristin/dalfopristin (MIC90: 0.12 mg/l) gave the lowest MIC values. Minimal inhibibitory activity of amoxycillin (MIC90: 1.04 mg/l), cefixime (MIC90: 1.33 mg/l), cefoperazone (MIC90: 0.83 mg/l) tetracycline (MIC90: 0.29 mg/l) and minocycline (MIC90: 0.30 mg/l) was slightly lower, whereas borrelia were resistant to amikacin (MIC90: >128 mg/l). Mean minimal borreliacidal concentrations (MBCs) were representatively determined for piperacillin (MBC: 1.8 mg/l), ceftriaxone (MBC: 2.0 mg/l), azithromycin (MBC: 0.82 mg/ml), roxithromycin (MBC: 1.8 mg/l), quinupristin/dalfopristin (MBC: 5.0 mg/l), minocycline (MBC: 5.8 mg/l), and amikacin (MBC: >128 mg/l) by using conventional subculture for three weeks in combination with dark-field microscopy. B. garinii proved to be the most susceptible of the genospecies tested. Our study showed excellent in vitro antimicrobial activity of all classes of antibiotics tested, except the aminoglycosides and hence their suitability for therapy of Lyme disease.

Comparison of two laboratory methods for the determination of serum resistance in Borrelia burgdorferi isolates.

A growth inhibition assay (GIA) and an immunofluorescence test detecting deposited complement components C6 and C9 were compared for their ability to classify Borrelia isolates with respect to their resistance to non-immune human serum (NHS). In both assays a total of 34 Borrelia isolates of all three human pathogenic genospecies were tested. Interestingly, 95% of the serum-sensitive or intermediate serum-sensitive isolates belonged to the genospecies B. burgdorferi s. s. and B. garinii, whereas most B. afzelii isolates (83%) proved serum-resistant. Consequently, a strong correlation between the assignment of the isolates to the different genospecies and their degree of serum sensitivity was seen. These findings were supported strongly by the quantitative analysis of the deposited complement components and the location of the terminal complement complex on the bacterial surface as detected by means of immunoelectron microscopy. The GIA displayed an obvious lack of sensitivity to slow growing isolates, whereas the IFA allowed classification of all Borrelia isolates. Discrimination between serum-sensitive and serum-resistant isolates in the IFA was the most specific provided that the detection of C6 and C9 was incorporated into the final classification of isolates. Accordingly, both assays, turned out to be effective and reliable tools for the investigation of borrelial serum sensitivity. The IFA, however, is regarded as superior to the GIA owing to the obvious ease of performance and its rapid capability for the classification of even very slow growing isolates.

New colorimetric microdilution method for in vitro susceptibility testing of Borrelia burgdorferi against antimicrobial substances.

A newly developed colorimetric microdilution method was used to analyze the activity of 12 antimicrobial agents against nine Borrelia burgdorferi isolates, including all three genospecies pathogenic for humans. In addition, in vitro antimicrobial resistance patterns of Borrelia valaisiana and Borrelia bissettii tick isolates were investigated. The applied test system is based upon color changes that occur in the presence of phenol red and result from the accumulation of nonvolatile acid produced by actively metabolizing spirochetes. After 72 h of incubation, minimal inhibitory concentrations (MICs) were determined from the decrease of absorbance by software-assisted calculation of growth curves. MIC values were lowest for azlocillin (MIC, < or = 0.125 microg/ml), ceftriaxone (MIC range, < or = 0.015-0.06 microg/ ml), and azithromycin (MIC range, < or = 0.015-0.06 microg/ml). Whereas tobramycin (MIC range, 8-64 microg/ml) exhibited little activity, spectinomycin (MIC range, 0.25-2 microg/ml) showed in vitro antimicrobial activity against Borrelia burgdorferi. The MICs of penicillin G for Borrelia afzelii isolates were ten times higher than those for Borrelia burgdorferi, Borrelia valaisiana, and Borrelia bissettii isolates (P<0.05) and 100 times higher than those for isolates belonging to the genospecies Borrelia garinii (P < 0.05). Further significant differences with respect to the MIC values of the other antimicrobial agents tested were not noted. The colorimetric microdilution method offered the advantages of reliability, reproducibility, and convenience and could handle large numbers of isolates and antibiotics.

Growth inhibitory and bactericidal efficacy of sera from Lyme borreliosis patients on B. burgdorferi strains.

Two B. afzelii strains EB1 and FEM1, classified in normal human sera (NHS) as serum-resistant, and an intermediate serum-sensitive B. burgdorferi s.s. strain 297, were tested in regard of their serum sensitivity in immune sera (IS) of patients at all stages of Lyme borreliosis by a growth inhibition assay (GIA). Fifty-four per cent (13/24) of the tested IS were GIA positive, while the sera of patients in stage III disease inhibited the growth more frequently than did the patients with sera of stage II or stage I disease. Growth inhibition was predominantly directed against strain FEM1 (12/24), less against strain EB1 (4/24) and strain 297 (2/24). A growth inhibiting effect on two strains was only detectable for two IS and merely one stage III serum inhibited all three strains. Positive results in the GIA required fresh serum and resulted in the killing of the borreliae. The detection of the deposited complement components C3 and C9 on the surfaces of the inhibited strains by means of immunofluorescence assays confirmed the role of complement. In Westernblot analyses of strain FEM1, it was striking that GIA-positive IS reacted 3- to 5-fold more often with proteins of molecular masses of 48.9-, 38.6-, 27.5-, 25-, 23.1- (OspC), 21.7-, and 16-kDa, than did GIA-negative IS. Furthermore, two proteins of approximately 20- and 31.2-kDa reacted exclusively with GIA-positive IS. Antibodies reacting with these proteins could play a role in the growth inhibition of NHS-resistant borrelial strains, OspC.

Serologic evidence for tick-borne pathogens other than Borrelia burgdorferi (TOBB) in Lyme borreliosis patients from midwestern Germany.

The seroprevalence of antibodies against the human granulocytic ehrlichiosis agent (HGE) and Babesia microti was retrospectively determined in 76 Lyme borreliosis patients and in 44 asymptomatic individuals with a positive borreliosis serology, in comparison to 100 healthy blood donors from the Rhein-Main area. Additionally, seroreactivity for tick-borne encephalitis virus (TBEV) was investigated. For antibody detection, commercially available immunofluorescence assays (MRL Diagnostics, USA) and a TBEV-ELISA (Immuno, Germany) were used. In the control group, the positivity rate for anti-Borrelia burgdorferi (IgG/IgM) and anti-Babesia microti-antibodies in the population of the Rhein-Main area (Midwestern Germany) may be estimated at 15% and 8%, respectively. Examination for both HGE and TBEV demonstrated seroreactivity (IgG) in 1% of tested individuals. Specific anti-HGE IgG and/or IgM antibodies were more often discovered in cases of early Borrelia infection (stage I: 13.6%, stage II: 18.4%) than in patients with stage III disease (0%) or in seropositive but asymptomatic patients (6.8%). Investigation for TBEV revealed seroreactivity for IgG in 13% of these cases. No TBEV-IgM was found. Interestingly, the prevalence of anti-HGE and anti-TBEV antibodies among Lyme borreliosis patients and seropositive patients without active Lyme disease symptoms was significantly higher than that in the control group of healthy blood donors (p < 0.05). Likewise, antibody titers reflecting a recent infection with Babesia microti could be demonstrated more often in patients with Lyme borreliosis stage I or II (p < 0.05). Analysis of 50 samples from patients with florid or recent syphilis infection revealed no crossreactivity between Babesia microti, HGE and Treponema pallidum. Our findings suggest that concomitant or serial infection due to TOBB may be common in tick exposed patients from the Rhein-Main area and in European countries in general. Hence, in addition to TBEV, human babesiosis and HGE should always be considered by European physicians in the differential diagnosis of acute febrile illness following a tick bite.

The molecular basis for the natural resistance of the cytochrome bc1 complex from strobilurin-producing basidiomycetes to center Qp inhibitors.

Mitochondria from the strobilurin A producing basidiomycetes Strobilurus tenacellus and Mycena galopoda exhibit natural resistance to (E)-beta-methoxyacrylate inhibitors of the ubiquinol oxidation center(center Qp) of the cytochrome bc1 complex. Isolated cytochrome bc1 complex from S. tenacellus was found to be highly similar to that of Saccharomyces cerevisiae with respect to subunit composition, as well as spectral characteristics and midpoint potentials of the heme centers. To understand the molecular basis of natural resistance, we determined the exon/intron organization and deduced the sequences of cytochromes b from S. tenacellus, M. galopoda and a third basidiomycete, Mycena viridimarginata, which produces no strobilurin A. Comparative sequence analysis of two regions of cytochrome b known to contribute to the formation of center Qp suggested that the generally lower sensitivity of all three basidiomycetes was due to the replacement of a small amino acid residue in position 127 by isoleucine. For M. galopoda replacement of Gly143 by alanine and Gly153 by serine, for S. tenacellus replacement of a small residue in position 254 by glutamine and Asn261 by aspartate was found to be the likely causes for resistance to (E)-beta-methoxyacrylates. The latter exchange is also found in Schizosaccharomyces pombe, which we found also to be naturally resistant to (E)-beta-methoxyacrylates.