Volume: 6 Table of Contents: I. LYMENET: ACP Recommendations Opposed by Laboratory Scientists II. LYMENET: New German Language LD Web Sites Now Available III. EUR J CLIN MICROBIOL INFECT DIS: Analysis of the intrathecal immune response in neuroborreliosis to a sonicate antigen and three recombinant antigens of Borrelia burgdorferi sensu stricto IV. ZENTRALBL BAKTERIOL: Detection of Borrelia burgdorferi in urine specimens from dogs by a nested polymerase chain reaction V. ZENTRALBL BAKTERIOL: Expression of outer surface proteins A and C of Borrelia afzeliiin Ixodes ricinus ticks and in the skin of mice VI. ABOUT THE LYMENET NEWSLETTER Newsletter: *********************************************************************** * The National Lyme Disease Network * * http://www.LymeNet.org/ * * LymeNet Newsletter * *********************************************************************** Volume 6 / Number 11 / 20-NOV-98 INDEX I. LYMENET: ACP Recommendations Opposed by Laboratory Scientists II. LYMENET: New German Language LD Web Sites Now Available III. EUR J CLIN MICROBIOL INFECT DIS: Analysis of the intrathecal immune response in neuroborreliosis to a sonicate antigen and three recombinant antigens of Borrelia burgdorferi sensu stricto IV. ZENTRALBL BAKTERIOL: Detection of Borrelia burgdorferi in urine specimens from dogs by a nested polymerase chain reaction V. ZENTRALBL BAKTERIOL: Expression of outer surface proteins A and C of Borrelia afzeliiin Ixodes ricinus ticks and in the skin of mice VI. ABOUT THE LYMENET NEWSLETTER =====*===== I. LYMENET: ACP Recommendations Opposed by Laboratory Scientists ------------------------------------------------------------------- Editor's note: The following letter was submitted to the Annals of Internal Medicine in response to their position paper on the laboratory diagnosis of Lyme disease. The full text of the original article can be found at: http://38.232.17.254/journals/annals/15dec97/pplyme1.htm -- Letter to the Editor American College of Physicians Independence Mall West 6th St. At Race Philadelphia, PA To Whom It May Concern, We have several concerns with the report entitled Guidelines for Laboratory Evaluation in the Diagnosis of Lyme Disease (Annals Int Med 127, 1106-1123. 1997) * The Centers for Disease Control have developed a set of clinical and diagnostic criteria for surveillance purposes. The authors of these "Guidelines" state, with no substitution, that this criteria is also applicable to the clinical diagnosis of Lyme disease. To our knowledge, no such evidence exists. It would appear that the published "Guidelines" have as a basis, a clinical criteria for Lyme disease diagnosis which has never been tested except for clinical studies published by the authors themselves. There is no external validation to support the claim of equivalence between clinical diagnostic criteria and the CDC surveillance definition. * The authors state that "testing with ELISA is the cornerstone of laboratory diagnosis for Lyme disease". In fact, it is not. The commercially available ELISA assays for Lyme Disease do not meet acceptable criteria according to the group that is responsible for much of the United States Laboratory proficiency testing program in Lyme Disease. The data, from a recent study by Bakken et.al. (J Clin Microbiol 35:537-543,1997), indicated "that" the sensitivity and specificity of the currently used tests for Lyme Disease are not adequate to meet the two-tier test approach being recommended by the CDC/ASTPHLD group". Bakken et. al. (1997) also stated that a screening test must have >95% sensitivity to adequately screen for Lyme disease and that the currently available ELISA tests do not perform at that level. * The authors have not followed the CDC/ASTPHLD recommendation for 2-tiered testing. That is, all indeterminate and reactive ELISAs should be reflexed to Western blot (WB), not just indeterminate ELISAs as the authors of the "Guidelines" suggest. Certainly the authors realize that reactive Lyme ELISA results may be non-specific because of a number of cross reacting antibodies (e.g. antibody to the 41 Kda flagellin protein). The authors have missed some important studies of Western blotting, especially those that may be critical of the recommendations of Dressler et. al. (J. Infect. Dis. 167, 392-400,1993) and the CDC/ASTPHLD. The report by Engstrom et al (J Clin Microbiol 33:419-427, 1995) found, for example, that 20% of their Lyme patients remained seronegative throughout the study and that fewer bands on the IgG WB could be appropriately used for interpretation. They noted that the WB was more specific and more sensitive than the ELISA. Their study also showed that only 19% of patients treated with antibiotics for 20 days still had a positive ELISA antibody response after one year, yet almost 60% of their patients continued to be WB positive at the end of the first year. The study by Aguero- Rosenfeld et al (J Clin Microbiol 34:1-9, 1996) reported that 89% of patients, with culture-confirmed erythema migrans (EM), developed specific IgG antibodies by WB, but only 22% of these patients were positive by the interpretive criteria proposed by the CDC/ASTPHLD. They further reported that the duration of the antibody response was related to the duration of the EM. Tilton et. al. (1997) stated that a highly sensitive and specific Western blot is desirable for a 2 tiered test approach or as a primary test. Despite the CDC/ASTPHLD recommendations, many physicians who treat patients for L.D. do not believe that an ELISA is an appropriate screening test and consequently use the Western blot as a primary test. The authors state that patients not be tested for Lyme Disease unless the pretest probability of disease is between 0.20 and 0.80. These recommendations will: a) rule out any laboratory detection of B. burgdorferi antibodies, antigens, and/or DNA in non-endemic areas b) require physicians to screen patients based on epidemiological data which may not be available to them outside their own local area require physicians to know the performance characteristics of a wide variety of Lyme disease tests. The authors in their comment on non-antibody based tests have chosen to overlook a number of publications on the utility of direct detection tests for Lyme disease. A comprehensive review of molecular techniques for diagnosis of Lyme Disease has recently been published by Schmidt (Clin Micro Rev 10, 185-201, 1997). They state "evidence is growing that a positive PCR test can be associated with active disease after adequate therapy, PCR results are usually negative" Manak et. al. (J Spirochet Tick Borne Dis 4., 11-20. 1997) in a well controlled study using the CDC criteria for selection of patients indicated that PCR on serum, plasma, or buffy coat could be effectively used to monitor the efficacy of therapy. Similarly, Harris et. al. (J Spirochet Tick Borne Dis 237, 1995) have validated the Lyme urinary antigen test (LUAT) in more than 700 LD patients and controls. The LUAT has a specificity of >95%. These "Guidelines" only complicate an already complex disease diagnostic process. Richard C. Tilton, Ph.D. Diplomate American Board Medical Microbiology BBI Clinical Laboratories, Inc. 75 North Mountain Rd. New Britain, CT 06053 [email protected] Mary N. Sand, Ph.D. BBI Clinical Laboratories, Inc. 75 North Mountain Rd. New Britain, CT 06053 Nick S Harris, Ph.D., Diplomate American Board Medical Laboratory Immunology IGeneX Inc. Reference Laboratory 797 San Antonio Road Palo Alto, CA =====*===== II. LYMENET: New German Language LD Web Sites Now Available ------------------------------------------------------------- Sender: Marc Gabriel <[email protected]> Two new web sites dedicated to providing German speaking individuals with access to Lyme disease information are now available on the web. 1. http://www.LymeNet.de/ This site mirrors much of the information on the LymeNet web site. German translation has been provided by a German physician. Marianne Steenken is responsible for this site. 2. http://www.ariplex.com/lyme/lyme_top.htm Authored by Aribert Deckers, this site offers a wealth of information, much of it translated from English. A extensive list of links is also provided. Both sites offer a translation of the Burrascano treatment protocol. =====*===== III. EUR J CLIN MICROBIOL INFECT DIS: Analysis of the intrathecal immune response in neuroborreliosis to a sonicate antigen and three recombinant antigens of Borrelia burgdorferi sensu stricto ---------------------------------------------------------------------- AUTHORS: Kaiser R, Rauer S ORGANIZATION: Neurologische Klinik und Poliklinik der Albert-Ludwigs- Universitat Freiburg, Germany. REFERENCE: Eur J Clin Microbiol Infect Dis 1998 Mar;17(3):159-66 ABSTRACT: The intrathecal synthesis of borrelial-specific IgM- and IgG-antibodies was studied in 67 patients with neuroborreliosis and in 14 patients with neurosyphilis (controls). Antibody concentrations in serum and in the cerebrospinal fluid were determined by an enzyme immunoassay (EIA) using, as antigens, a sonicate of Borrelia burgdorferi, the recombinant 14 kDa flagellin fragment, the outer surface protein C (22 kDa), and the high molecular mass protein p83 (83 kDa). In the sonicate EIA, IgG- and/or IgM-antibodies to Borrelia burgdorferi in serum were detected in all patients with neuroborreliosis and in 71% of patients with neurosyphilis. Intrathecal synthesis of borrelial-specific IgG- and/or IgM-antibodies was demonstrated in 82% of patients with neuroborreliosis and in 71% of patients with neurosyphilis. Immunoglobulin G- and/or IgM-antibodies in serum against any of the recombinant antigens were detected in 92% of patients with neuroborreliosis and in none of those with neurosyphilis. Intrathecal synthesis of IgG- and/or IgM-antibodies to individual recombinant antigens was demonstrated in 67% of patients with neuroborreliosis and in none of those with neurosyphilis. The sensitivity of the recombinant antigens in serum was almost equal to that of the sonicate EIA, whereas the recombinant antigens were clearly less sensitive in the estimation of the intrathecal specific immune response. It was concluded that in suspected cases of neuroborreliosis, the estimation of high specific antibodies in the recombinant EIA will be helpful in confirming the diagnosis. =====*===== IV. ZENTRALBL BAKTERIOL: Detection of Borrelia burgdorferi in urine specimens from dogs by a nested polymerase chain reaction --------------------------------------------------------------------- AUTHORS: Bauerfeind R, Kreis U, Weiss R, Wieler LH, Baljer G ORGANIZATION: Institut fur Hygiene und Infektionskrankheiten der Tiere, Justus-Liebig-Universitat Giessen, Germany. REFERENCE: Zentralbl Bakteriol 1998 May;287(4):347-61 ABSTRACT: A nested PCR (nested flagellin PCR) carrying an internal E. coli DNA control was established and compared with an in-vitro culture method for the detection of Borrelia burgdorferi in urine specimens of dogs. The predicted specific amplicon of the flagellin gene fla was generated from all cultured strains of B. burgdorferi tested (comprising three European genospecies). In contrast, all 13 strains of seven other flagellated bacterial species were negative. The PCR detection limit yielded 20 cells of B. burgdorferi per ml of double-distilled water and approx. 250 bacteria per ml of dog urine. Using the bacterial culture method, urine specimens collected from 216 dogs in Germany were all diagnosed negative for spirochetes by in-vitro culture and dark-field microscopy. In contrast, DNA of B. burgdorferi was detected in 32 specimens (14.8%) by PCR. 31 urine specimens (14.4%) showed inhibitory activity in the PCR assay. However, 94 (44%) were inhibitory in the culture assay. The majority of the PCR-positive dogs exhibited major clinical symptoms which have not been reported in the course of B. burgdorferi infection previously, e.g. cystitis (14/32 dogs) or prostatitis (5/32 dogs). Our results indicate that the analysis of urine specimens by the nested flagellin PCR is a highly valuable procedure for the diagnosis of B. burgdorferi infections in dogs. =====*===== V. ZENTRALBL BAKTERIOL: Expression of outer surface proteins A and C of Borrelia afzeliiin Ixodes ricinus ticks and in the skin of mice --------------------------------------------------------------------- AUTHORS: Leuba-Garcia S, Martinez R, Gern L ORGANIZATION: Institut de Zoologie, University of Neuchatel, Switzerland. REFERENCE: Zentralbl Bakteriol 1998 May;287(4):475-84 ABSTRACT: Several studies have described changes in the expression of proteins, especially of OspA and OspC, of B. burgdorferi sensu stricto during tick feeding. In this study, the expression of OspA and OspC of B. afzelii in unfed and feeding I. ricinus nymphs and in the subsequent adults was followed by means of the immunofluorescence test. Spirochaetes expressing OspA and OspC were observed in 70% and 80%, respectively of the unfed nymphs. In feeding and in fully engorged ticks, spirochaetes expressed OspC, while OspA disappeared 24 hours after the beginning of the blood meal. Spirochaetes expressing OspC in salivary glands were observed in one engorged tick. After molting, in unfed adults spirochaetes again expressed OspA and OspC but did so less frequently (6% and 13%, respectively). The mouse strain (AKR/N or BALB/C) on which ticks had their infectious blood meal influenced OspC expression in the following tick stage. In the skin of AKR/N mice, at the tick feeding site, B. afzelii expressed OspC only, as was shown by immunostaining. =====*===== VI. ABOUT THE LYMENET NEWSLETTER ----------------------------------------------------------------------- For the most current information on LymeNet subscriptions, contributions, and other sources of information on Lyme disease, please refer to the LymeNet Home Page at: http://www.lymenet.org ----------------------------------------------------------------------- To unsubscribe from the LymeNet newsletter, send a message to: [email protected] On the first line of the message, write: unsub lymenet-l ----------------------------------------------------------------------- LymeNet - The Internet Lyme Disease Information Source ----------------------------------------------------------------------- Editor-in-Chief: Marc C. Gabriel <[email protected]> FAX (for contributions ONLY): 908-789-0028 Contributing Editors: Carl Brenner <[email protected]> John Setel O'Donnell <[email protected]> Frank Demarest <[email protected]> Advisors: Carol-Jane Stolow, Director <[email protected]> William S. 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