Cross-reactivity of nonspecific treponemal antibody in serologic tests for Lyme disease.

Serum samples obtained from 59 persons who had acute necrotizing ulcerative gingivitis, periodontitis, syphilis, or Lyme disease were tested against Treponema phagedenis biotype Reiter, Treponema denticola, Treponema vincentii, and Treponema scoliodontum by indirect fluorescent-antibody staining methods. Although there were positive reactions for sera representing each of these study groups and for 20 (13%) of 156 samples collected from the general population (premarital screening for syphilis), titration endpoints were relatively low (less than or equal to 1:256). Serum samples from 18 persons who had gingivitis or periodontitis but no history of Lyme borreliosis were tested by enzyme-linked immunosorbent assay for antibodies to Borrelia burgdorferi. Of these, five (28%) had immunoglobulin M antibody and four (22%) contained immunoglobulin G antibodies to this spirochete. Adsorption with either sorbent commercially prepared from T. phagedenis biotype Reiter or with washed, whole cells of T. phagedenis biotype Reiter reduced cross-reactivity in the enzyme-linked immunosorbent assay for Lyme borreliosis.     

Adsorption and biotin-streptavidin amplification in serologic tests for diagnosis of Lyme borreliosis.

Serum samples from persons with Lyme borreliosis, periodontitis, or acute necrotizing ulcerative gingivitis were analyzed by an enzyme-linked immunosorbent assay (ELISA) with and without adsorption and amplification procedures. When biotin and streptavidin reagents were used as an amplification procedure in ELISA without the use of commercially prepared sorbent (Treponema phagedenis biotype Reiter), sensitivity increased. Of the 85 serum samples collected from persons with erythema migrans but no detectable antibodies to Borrelia burgdorferi by standard ELISA, 17 (20%) were reactive after amplification. Adsorption of serum samples with a 1:10 dilution of T. phagedenis biotype Reiter sorbent used in conjunction with amplified ELISA also improved the sensitivity of this method. However, cross-reactivity could not be completely eliminated. An adsorbed-amplified ELISA may be helpful in the diagnosis of Lyme borreliosis in the laboratory, particularly during early weeks of infection, when antibodies to B. burgdorferi can be present at a low concentration.     

Complement fixation test for the diagnosis of Lyme disease.

Sera from 43 patients were tested for complement-fixing antibodies to Borrelia burgdorferi; these patients included 8 with confirmed Lyme disease, 21 who were serologically positive but not likely to have Lyme disease, and 14 who were serologically negative. Seven individuals, all confirmed Lyme disease patients, had complement-fixing antibodies. Complement fixation may be a useful confirmatory test for Lyme disease.     

Surface immunofluorescence assay for diagnosis of Lyme disease.

A surface immunofluorescence assay (SIFA) was analyzed and compared with a conventional indirect immunofluorescence assay (IFA) and whole-cell enzyme-linked immunosorbent assay (ELISA) for detecting immunoglobulin G (IgG) antibodies to Borrelia burgdorferi in sera from patients with Lyme disease. Fifty-five patients with syphilis and 33 patients with rheumatoid arthritis were used as disease controls. The sensitivity of the SIFA was low during the acute phase of Lyme disease (sera from seven of nine patients presenting with erythema chronicum migrans were negative during the first 2 months of illness); later, seroconversion was observed in all patients at various times during convalescence. Sera from five patients with complicated Lyme disease were strongly positive. SIFA was found to be highly specific, since sera from all patients with secondary or latent syphilis and patients with rheumatoid arthritis did not react in the test. Strong cross-reactivity occurred when these sera were tested in conventional IFA and ELISA; sera from 38 (69%) patients with syphilis were positive by IFA and sera from 51 (93%) patients were positive by ELISA, whereas 7 (21%) and 12 (36%) of the serum samples from patients with rheumatoid arthritis were positive by IFA and ELISA, respectively. Immunoblot analysis of SIFA-positive sera showed that the 31- and 34-kDa outer surface proteins (proteins A and B, respectively) of B. burgdorferi were the major reactive antigens involved in the test. The results support a role for SIFA in the investigation of complicated Lyme disease as well as in the differentiation of Lyme disease from other diseases associated with B. burgdorferi cross-reactive antibodies.     

Comparison of Western immunoblotting and the C6 Lyme antibody test for laboratory detection of Lyme disease

Three commercial Lyme disease Western immunoblotting (WB) kits and the C6 Borrelia burgdorferi (Lyme) enzyme-linked immunosorbent assay (ELISA) kit were compared using two commercially available performance panels from the Centers for Disease Control and Prevention (CDC) and Boston Biomedica (BBI). Combined, the panels consisted of 52 characterized specimens. Immunoglobulin G (IgG) sensitivity was similar for the three WB products. The BBI and Marblot WBs were more specific for IgG antibodies, while the Virablot was the most sensitive for IgM antibody. The BBI WB was 100% specific for IgM, while Marblot was 97% and Virablot was 77% specific for IgM. The C6 ELISA was found to be 100% sensitive. Four false-positive C6 results were identified in patients that had clinically and microbiologically confirmed Lyme disease but were not detected by the CDC reference methods. No one WB product showed overall superiority. The C6 ELISA shows promise as the first ELISA for Lyme disease that would not require a supplemental test such as a WB.     

Lyme meningoradiculitis: prospective evaluation of biological diagnosis methods

The symptoms of Lyme meningoradiculitis and the value of biological examinations in an endemic area were determined in a prospective study in which data were collected on all patients consecutively hospitalised for Lyme meningoradiculitis at our institution during an 18-month period. Specific antibody titres in the serum and cerebrospinal fluid (CSF) were determined by Vidas enzyme-linked-immunosorbent-assay (IgG + IgM), Dade-Behring enzyme immunoassay (EIA) (IgM; IgG) and Western blot analysis (IgG). We also searched for Borrelia burgdorferi in the CSF by PCR analysis and following culture on a specific medium. A control group was recruited, consisting of 16 consecutive patients who had been referred during the same period with suspected but not confirmed Lyme meningoradiculitis. Eleven patients were included. Borrelia EIA of the serum revealed that 40% of the patients had both elevated specific IgM titres and intrathecal synthesis of specific IgG; 40% of the patients was negative for IgM but had isolated intrathecal synthesis of IgG; 20% of the patients had elevated specific IgM titres without intrathecal synthesis of IgG. PCR analysis and the CSF culture were positive in one case only (B. garinii). The results of this study highlight the importance of systematic serological testing for B. burgdorferi in the CSF in the case of early neuroborreliosis suspicion, even in the absence of IgM serum antibodies, which was the case in 40% of the patients in the present study. Nevertheless, intrathecal anti-B. burgdorferi IgG synthesis, which remains the “gold standard” for the diagnosis of neuroborreliosis, was not detectable in 20% of the patients for whom diagnosis was subsequently confirmed by demonstration of specific serum IgM. Conflict of interest statement: none.     

Two-year evaluation of Borrelia burgdorferi culture and supplemental tests for definitive diagnosis of Lyme disease

Lyme disease is usually diagnosed and treated based on clinical manifestations. However, laboratory testing is useful for patients with confusing presentations and for validation of disease in clinical studies. Although cultivation of Borrelia burgdorferi is definitive, prior investigations have shown that no single test is optimal for Lyme disease diagnosis. We applied high-volume blood culture, skin biopsy culture, PCR, and serodiagnosis to a cohort of patients with suspected Lyme disease acquired in Maryland and southern Pennsylvania. The study was performed to confirm the relative utility of culture and to identify laboratory testing algorithms that will supplement clinical diagnosis. Overall, 30 of 86 patients (35%) were culture positive, whereas an additional 15 of 84 (18%) were seropositive only (51% total sero- and culture positive), and PCR on skin biopsy identified 4 additional patients who were neither culture nor seropositive. Among 49 laboratory test-positive patients, the highest sensitivity (100%) for diagnosis was obtained when culture, skin PCR, and serologic tests were used, although serologic testing with skin PCR was almost as sensitive (92%). Plasma PCR was infrequently positive and provided no additional diagnostic value. Although culture is definitive and has a relatively high sensitivity, the results required a mean of 3.5 weeks to recovery. The combination of acute-phase serology and skin PCR was 75% sensitive, offering a practical and relatively rapid alternative for confirming clinical impression. The full battery of tests could be useful for patients with confusing clinical signs or for providing strong laboratory support for clinical studies of Lyme disease.     

EMQN best practice guidelines for the laboratory diagnosis of osteogenesis imperfecta

Osteogenesis imperfecta (OI) comprises a group of inherited disorders characterized by bone fragility and increased susceptibility to fractures. Historically, the laboratory confirmation of the diagnosis OI rested on cultured dermal fibroblasts to identify decreased or abnormal production of abnormal type I (pro)collagen molecules, measured by gel electrophoresis. With the discovery of COL1A1 and COL1A2 gene variants as a cause of OI, sequence analysis of these genes was added to the diagnostic process. Nowadays, OI is known to be genetically heterogeneous. About 90% of individuals with OI are heterozygous for causative variants in the COL1A1 and COL1A2 genes. The majority of remaining affected individuals have recessively inherited forms of OI with the causative variants in the more recently discovered genes CRTAP, FKBP10, LEPRE1,PLOD2, PPIB, SERPINF1, SERPINH1 and SP7, or in other yet undiscovered genes. These advances in the molecular genetic diagnosis of OI prompted us to develop new guidelines for molecular testing and reporting of results in which we take into account that testing is also used to 'exclude' OI when there is suspicion of non-accidental injury. Diagnostic flow, methods and reporting scenarios were discussed during an international workshop with 17 clinicians and scientists from 11 countries and converged in these best practice guidelines for the laboratory diagnosis of OI.     

Fabry disease: guidelines for the evaluation and management of multi-organ system involvement.

Fabry disease is an X-linked metabolic storage disorder due to the deficiency of lysosomal alpha-galactosidase A, and the subsequent accumulation of glycosphingolipids, primarily globotriaosylceramide, throughout the body. Males with classical Fabry disease develop early symptoms including pain and hypohidrosis by the second decade of life reflecting disease progression in the peripheral and autonomic nervous systems. An insidious cascade of disease processes ultimately results in severe renal, cardiac, and central nervous system complications in adulthood. The late complications are the main cause of late morbidity, as well as premature mortality. Disease presentation in female heterozygotes may be as severe as in males although women may also remain asymptomatic. The recent introduction of enzyme replacement therapy to address the underlying pathophysiology of Fabry disease has focused attention on the need for comprehensive, multidisciplinary evaluation and management of the multi-organ system involvement. In anticipation of evidence-based recommendations, an international panel of physicians with expertise in Fabry disease has proposed guidelines for the recognition, evaluation, and surveillance of disease-associated morbidities, as well as therapeutic strategies, including enzyme replacement and other adjunctive therapies, to optimize patient outcomes.     

Recombinant chimeric Borrelia proteins for diagnosis of Lyme disease

Current serologic Lyme disease tests use whole borrelia cells as the source of antigen. These assays are difficult to standardize and to optimize for sensitivity and specificity. To help solve these problems, we constructed a library of recombinant chimeric proteins composed of portions of key antigens of Borrelia burgdorferi. These proteins were then used to develop an enzyme-linked immunosorbent assay. We compared our assay with the most sensitive of three whole-cell borrelia assays. We found that the recombinant assay could detect antibodies significantly better from early Lyme disease sera (P<0.05), and had the same sensitivity for late Lyme disease sera, as the most sensitive whole-cell borrelia assay. On potentially cross-reactive sera, the recombinant assay was more specific, but not significantly so, than the best whole-cell borrelia assay. Optimization of the recombinant assay offers the potential for a significant improvement in both sensitivity and specificity.     

Chronic Lyme borreliosis in the laboratory mouse.

C3H/HeJ mice were inoculated intraperitoneally with 10(7) uncloned Borrelia burgdorferi at 4 weeks of age and examined on days 30, 90, 180, and 360. Spirochetes were isolated from multiple tissues at all intervals. Joint and heart disease were present in all mice at 30 days and resolved after 90 days. At 180 and 360 days, some mice had mild recurrent joint and heart disease, and most had peripheral segmental periarteritis. The protein electrophoretic migration of 360-day isolates differed from the original inoculum. The experiment was repeated with C3H/HeN and BALB/cByJ mice inoculated intradermally with 10(4) cloned B. burgdorferi. Characterization of infection and disease at 180 and 360 days were similar to those of the first experiment, but spirochetal proteins of isolates from both intervals displayed no protein variation in electrophoretic mobilities. Spirochetes isolated at 360 days were fully pathogenic in naive mice. Sera from infected mice showed an initial immunoglobulin M response, followed by a sustained immunoglobulin G response, involving IgG1, IgG2a, IgG2b and IgG3, with expanding reactivity against multiple antigens over time. These results indicate that immunocompetent mice sustain persistent infections and develop early acute joint and heart lesions that resolve and then recur intermittently.     

Lyme disease: a review.

In the last decade, Lyme borreliosis has emerged as a complex new infection whose distribution is worldwide. The multisystem disorder, which primarily affects the skin, joints, heart and nervous system at different stages, is caused by the tick-borne spirochaete Borrelia burgdorferi. After the first weeks of infection almost all patients have a positive antibody response to the spirochaete and serological determinations are currently the most practical laboratory aid in diagnosis. Treatment with appropriate antibiotics is usually curative.     

Critical evaluation of urine-based PCR assay for diagnosis of Lyme borreliosis

Many approaches were made in recent years to establish urine PCR as a diagnostic tool for Lyme borreliosis, but results are contradictory. In the present study, a standardized protocol spiking urine from healthy donors with a defined amount of whole Borrelia or Borrelia DNA was established. The development of a nested real-time PCR targeting ospA enabled a highly sensitive and quantitative analysis of these samples. We show the following. (i) Storage of spiked urine samples for up to 6 months at--20 degrees C had no negative effect on spike recovery. (ii) Centrifugation of 10 ml of urine at 40,000 x g for 30 min resulted in a concentration of both spikes, i.e., whole Borrelia and DNA. (iii) The inhibition of DNA spike recovery in 48% (11 of 23 samples) of urine samples tested could be attributed to nuclease activity. This was abrogated by alkalizing the urine or by working with the samples on ice. Despite optimized conditions, analysis of urine samples of 12 patients with erythema migrans, the clinical stage considered to be associated with the highest bacterial load, revealed a positive result in only one sample. All 12 samples were negative by an alternative PCR targeting flagellin. The results of our study support doubts that urine is a suitable material for diagnosis of Lyme borreliosis.     

Comparison of whole-cell antibodies and an antigenic flagellar epitope of Borrelia burgdorferi in serologic tests for diagnosis of Lyme borreliosis.

A recombinant protein (p41-G) of an antigenic region of flagellin was used in a standard and amplified enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Borrelia burgdorferi, the causative agent of Lyme borreliosis. Comparable sensitivities (88 to 94%) were noted when sera from 17 persons who had erythema migrans and antibodies to whole-cell B. burgdorferi were tested against the p41-G antigen. In tests of a second study group of 36 persons who had erythema migrans but no detectable antibodies to whole-cell B. burgdorferi, 3 (8%) were positive when the p41-G antigen was used. Assay specificity likewise increased when the p41-G fragment was included in an ELISA with human sera containing treponemal antibodies. Recombinant flagellar proteins of B. burgdorferi, such as the p41-G antigen, can be used in an ELISA and may help confirm Lyme borreliosis during early stages of infection and improve specificity.