We prospectively compared the performance of culture, direct fluorescent-antibody testing (DFA), and an in-house-developed PCR test targeting the repeated insertion sequence IS481 for the detection of Bordetella pertussis in nasopharyngeal swab specimens. We tested 319 consecutive paired specimens on which all three tests were performed. A total of 59 specimens were positive by one or more tests. Of these, 5 were positive by all three tests, 2 were positive by culture and PCR, 16 were positive by PCR and DFA, 28 were positive by PCR only, and 8 were positive by DFA only. Any specimen positive by culture was considered to be a true positive, as were specimens positive by both PCR and DFA. Specimens positive only by PCR or DFA were considered discrepant, and their status was resolved by review of patient histories. Patients with symptoms meeting the Centers for Disease Control and Prevention clinical case definition for pertussis and who had a specimen positive by PCR or DFA were considered to have true B. pertussis infections. Of the 28 patients positive by PCR only, 20 met the clinical case definition for pertussis, while 3 of the 8 patients positive by DFA only met the clinical case definition. After resolution of the status of discrepant specimens, the sensitivity, specificity, positive predictive value, and negative predictive value were 15.2, 100, 100, and 87.5%, respectively, for culture; 93.5, 97.1, 84.3, and 98.9%, respectively, for PCR; and 52.2, 98.2, 82.8, and 92.4%, respectively, for DFA. The actual positive predictive value of PCR was probably greater, as several PCR-positive patients who did not meet the clinical case definition had symptoms consistent with typical or atypical pertussis. PCR is a sensitive and specific method for the detection of B. pertussis.
West Nile virus (WNV) can cause severe, potentially fatal neurological illnesses, which include encephalitis, meningitis, Guillain-Barré syndrome, and anterior myelitis. Because of the short viremic phase, WNV infection is most commonly diagnosed by detection of immunoglobulin M antibody to WNV in serum or cerebrospinal fluid (CSF). We describe a patient with T cell lymphoma who had undergone a T cell-depleted bone marrow transplantation and developed fatal WNV infection. The results of serological tests of blood samples and of CSF tests were negative. Diagnosis was made postmortem by a positive result of reverse-transcriptase polymerase chain reaction (ABI 7700; TaqMan) for WNV in stored CSF and serum samples.
No standardized PCR method is available for the laboratory diagnosis of the pertussis syndrome. Consensus recommendations for the use of PCR in the diagnosis of Bordetella pertussisinfections have been proposed, and the aim of this study was to develop a method that fulfills all of these criteria. A rapid-cycle shared-primer PCR method with a microwell format and probe hybridization detection step (POR) was developed using novel oligonucleotides targeted to the outer membrane porin gene (Bordetella spp.). In specimens positive forBordetella spp., B. pertussis was differentiated from Bordetella parapertussis andBordetella bronchiseptica by hybridization with organism-specific oligonucleotide probes. An internal control was developed using overlap extension PCR and mouse β-actin DNA. The analytical specificity was 100%. The analytical sensitivity was comparable to that of nested IS481 and IS1001PCR (∼1 organism per reaction). The clinical sensitivity and specificity were ascertained using 705 specimens (from 705 patients). The results were compared to those of a nested-PCR method targeting the insertion sequences IS481 and IS1001. Fifty-one specimens were positive for B. pertussis by POR and IS481 PCR. Two specimens which fulfilled a clinical definition of pertussis were positive by POR and negative by IS481 PCR. A total of 652 specimens were negative by both methods. B. parapertussis was not detected in any specimens. PCR inhibition was detected in 21 out of 705 specimens (2.98%). Thus, a rapid (4 h, including specimen preparation) PCR method which fulfills all of the consensus recommendations was developed and validated for the detection of B. pertussis.
An Escherichia coli O157:H7 subtyping method based on PCR amplification of variable DNA sequences between the repetitive element IS3 was developed. Template DNA was prepared by boiling cells in Chelex. Two separate IS3 PCR amplifications were performed for each isolate: one with a single primer (primer IS3A) and one with two primers (primers IS3A and IS3B). The IS3 PCR subtyping method was applied to 35 epidemiologically related and unrelated E. coli O157:H7 isolates that had been previously characterized by pulsed-field gel electrophoresis (PFGE). PFGE identified 25 different subtypes (difference of one or more bands). PCR with single primer IS3A and primer pair IS3A-IS3B identified 6 and 14 different subtypes, respectively. By combining the results of the two PCR amplifications, 15 different IS3 PCR subtypes were identified. While not as sensitive as PFGE, IS3 PCR subtyping grouped all outbreak-related isolates. IS3 PCR banding patterns were reproducible between amplifications and between subcultures. IS3 PCR could serve as a simple, rapid screening method for the identification of unrelated E. coli O157:H7 isolates.
In Iowa, public concern regarding Lyme disease has increased markedly over the last decade. In response to these concerns, a statewide surveillance program was initiated in 1990 based on ticks received by the Department of Entomology at Iowa State University. Ticks were received from health care professionals, state government agencies, and the general public. A total of 5,343 ticks from all 99 Iowa counties were identified during the 12 years of this study. Dermacentor variabilis was the most numerous species, followed by Amblyomma americanum, and Ixodes scapularis. Dermacentor variabilis were distributed statewide, but A. americanum came primarily from southern Iowa counties. Prior to 1996, most I. scapularis came from counties along the Mississippi River. In the last 5 years, I. scapularis have been received from several counties in central and western Iowa and there is molecular evidence of infection with Borrelia burgdorferi in a substantial percentage of ticks. All I. scapularis were tested for the presence of B. burgdorferi. During the 12 years of this study, the presence of B. burgdorferi in I. scapularis varied from a low of zero percent in 1991-1995 to 18% in 1996. On average, fewer than 10% of all ticks examined per year were I. scapularis. In the 2000 tick season, the number of I. scapularis per year increased to 22% of submissions. This species further increased to 36.6% of ticks received in 2002.
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