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Diagnosis of early Lyme disease by polymerase chain reaction amplification and culture of skin biopsies from erythema migrans lesions.

ABSTRACT: Current laboratory diagnosis of Lyme disease relies on tests for the detection of antibodies to Borrelia burgdorferi, the etiologic agent of the disease. These tests are often unreliable because of a lack of sensitivity and specificity and test-to-test variability. The purpose of this study was to evaluate the sensitivity and specificity of polymerase chain reaction (PCR) amplification for detection of B. burgdorferi in skin biopsy specimens. Forty-six 2-mm skin biopsy samples were obtained from 44 patients with a clinical diagnosis of erythema migrans, 9 of whom were receiving antibiotic therapy at the time of biopsy. Specimens were ground in BSK medium with separate aliquots taken for culture and PCR. Of the specimens from the untreated group, 57% (21 of 37) were positive by culture and 22% (8 of 37) were culture negative; 22% (8 of 37) of the cultures were uninformative because of contamination. By comparison, 22 (59%) of 37 specimens were positive by PCR amplification. Of 21 culture-positive samples, 13 (62%) were also positive by PCR analysis. Thus, the sensitivity of the PCR was 59 to 62%, based on either a clinical or cultural diagnosis of untreated Lyme disease. None of the nine specimens from antibiotic-treated patients grew in culture, whereas two of the nine were positive by PCR analysis. Given the complexity and time required for culture, PCR is a promising technique for the diagnosis of early Lyme disease.

SUBMITTER: Schwartz I

PROVIDER: S-EPMC270592 | biostudies-other | 1992 Dec

REPOSITORIES: biostudies-other

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Diagnosis of early Lyme disease by polymerase chain reaction amplification and culture of skin biopsies from erythema migrans lesions.

Schwartz I I Wormser G P GP Schwartz J J JJ Cooper D D Weissensee P P Gazumyan A A Zimmermann E E Goldberg N S NS Bittker S S Campbell G L GL Pavia C S CS

Journal of clinical microbiology 19921201 12

Current laboratory diagnosis of Lyme disease relies on tests for the detection of antibodies to Borrelia burgdorferi, the etiologic agent of the disease. These tests are often unreliable because of a lack of sensitivity and specificity and test-to-test variability. The purpose of this study was to evaluate the sensitivity and specificity of polymerase chain reaction (PCR) amplification for detection of B. burgdorferi in skin biopsy specimens. Forty-six 2-mm skin biopsy samples were obtained from ...[more]

PMID: 1452688

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Project description:BackgroundInvasive meningococcal disease (IMD) is a highly lethal disease. Diagnosis is commonly performed by culture or Realtime-PCR (qPCR).AimsOur aim was to evaluate, retrospectively, whether culture positivity correlates with higher bacterial load and fatal outcome. Our secondary aim was to compare culture and qPCR sensitivity.MethodsThe National Register for Molecular Surveillance was used as data source. Cycle threshold (CT), known to be inversely correlated with bacterial load, was used to compare bacterial load in different samples.ResultsThree-hundred-thirteen patients were found positive for Neisseria meningitidis by qPCR, or culture, or both; 41 died (case fatality rate 13.1%); 128/143 (89.5%) blood samples and 138/144 (95.8%) CSF were positive by qPCR, 37/143 (25.9%) blood samples and 45/144 (31.2%) CSF were also positive in culture. qPCR was 3.5 times (blood) or 3.1 times (CSF) more sensitive than culture in achieving a laboratory diagnosis of IMD (OR 24.4; 95% CI 12.2-49.8; p < .10-4; Cohen's κ 0.08 for blood and OR 49.0; 95% CI 19.1-133.4; p<10-4; Cohen's κ 0.02; for CSF). Positivity of culture did not correlate with higher bacterial loads in blood (mean CT 27.7±5.71, and CT 28.1±6.03, p = 0.739 respectively in culture positive or negative samples) or in CSF (mean CT 23.1±4.9 and 24.7±5.4 respectively in positive or negative CSF samples, p = 0.11).CT values in blood from patients who died were significantly lower than in patients who survived (respectively mean 18.0, range 14-23 and mean 29.6, range 16-39; p<10-17). No deaths occurred in patients with CT in blood over 23. Positive blood cultures were found in 10/25 (40%) patients who died and in 32/163 (19.6%) patients who survived, p = 0.036, OR 2.73; 95% CL 1.025-7.215), however 60% of deaths would have remained undiagnosed with the use of culture only.ConclusionsIn conclusion our study demonstrated that qPCR is significantly (at least 3 times) more sensitive than culture in the laboratory confirmation of IMD. The study also demonstrated that culture negativity is not associated with lower bacterial loads and with less severe cases. On the other side, in patients with sepsis, qPCR can predict fatal outcome since higher bacterial load, evaluated by qPCR, appears strictly associated with most severe cases and fatal outcome. The study also showed that molecular techniques such as qPCR can provide a valuable addition to the proportion of diagnosed and serotyped cases of IMD.

Project description:BACKGROUND:Laron syndrome (LS) is an autosomal recessive hereditary condition affecting only 1/1000000 births. The cause is associated with mutations in the growth hormone (GH) receptor (GHR), leading to GH insensitivity. LS patients typically present with severe growth retardation, obesity, and abnormal sexual maturation. Currently, LS diagnosis is performed post-delivery. Therefore, we assessed the efficiency of Pre-implantation Genetic Testing (PGT) coupled with monoplex-polymerase chain reaction (PCR) technology for detecting this monogenic disease in embryos from a couple confirmed as LS heterozygous carriers. CASE SUMMARY:The couple LS-carriers were confirmed by the presence of a first child born with LS. The couple underwent a standard in vitro fertilization (IVF) protocol. DNA was collected from trophectoderm cells from day 5 embryos. Whole genome amplification (WGA) was performed using a Sureplex DNA Amplification System and analyzed by PCR, targeting the deletion of the exons 5 and 6 in the GHR gene as well as PGT by Next-generation Sequencing (Illumina). Eleven embryos were collected and analyzed. 27.3% were the wild type for GHR, 45.5% were heterozygotes, and 18.2% homozygous mutants. One embryo yielded no results. Three 2-embryos transfers were performed; 2 normal homozygous and four heterozygous carriers were selected for transfer. The first two transfers were unsuccessful, whereas the final transfer with two heterozygous embryos resulted in clinical pregnancy. The genomic composition of the fetus was verified, applying the same techniques using amniocytes, extracted after 21 wk of the ongoing pregnancy. The fetus was confirmed as GHR deletion in exon 5-6, carrier. A non-affected baby was born. CONCLUSION:Here, we present a case demonstrating that using WGA as a template in addition to PCR targeting specific gene regions, exons 5 and 6 on the GHR gene, could identify LS carrier embryos. This provides evidence that WGA and PCR serve as an excellent tool to detect this specific monogenic disease in IVF embryos, thus allowing selection of candidate embryos for transfer successfully when a specific inherited genetic mutation/disease is suspected.

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Diagnosis of early Lyme disease by polymerase chain reaction amplification and culture of skin biopsies from erythema migrans lesions.

Publications

Diagnosis of early Lyme disease by polymerase chain reaction amplification and culture of skin biopsies from erythema migrans lesions.

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