Rapid detection of Mycoplasma pneumoniae by an assay based on PCR and probe hybridization in a nonradioactive microwell plate format.

A new molecular assay, based on a rapid DNA extraction protocol, PCR, and hybridization to a specific probe in a nonradioactive microwell plate format was used to detect Mycoplasma pneumoniae in bronchoalveolar fluid specimens. The sensitivity of the assay was determined to be 10 to 100 organisms with M. pneumoniae reference strains. Specificity testing with different bacteria capable of producing pneumonia showed no cross-reactivity. In a prospective study, bronchoalveolar lavage fluids obtained from patients with pneumonia were investigated with the PCR assay and compared to culture. Twelve positive samples were detected with the PCR assay. Seven of them were subsequently confirmed by culture. All patients with positive PCR results seroconverted. Application of the PCR assay described may lead to safe and early diagnosis of M. pneumoniae in patients with pneumonia.

Rapid diagnosis of enterovirus infection by a new one-step reverse transcription-PCR assay.

The AMPLICOR Enterovirus Test was evaluated with 103 cerebrospinal fluid (CSF) specimens. Twenty-seven CSF specimens were culture positive. With the AMPLICOR test, enterovirus RNA was detected in 34 specimens. Compared with culture, the AMPLICOR test gave a sensitivity of 96.3% and a specificity of 100%. The sensitivity of culture was 79.4% in comparison with the AMPLICOR test.

Detection of antichlamydial antibodies in tears: a diagnostic aid?

OBJECTIVES: The antibody response in sera and tears of 167 patients with suspected chlamydial conjunctivitis was compared with the antibody response in sera and tears of 45 patients with symptoms of urogenital chlamydial infection to discover whether and which type of antichlamydial antibody detected in tears may be of diagnostic help in chlamydial conjunctivitis. METHODS: Diagnosis was based on chlamydial antigen detection from the conjunctiva and urogenital tract, done by a direct immunofluorescence assay, McCoy cell culture, and polymerase chain reaction. Additionally, antichlamydial immunoglobulin A (IgA) and immunoglobulin G (IgG) were determined in sera and tears of all patients by an immunoperoxidase assay. RESULTS: Two hundred twelve patients were examined--167 with conjunctivitis, 45 with symptoms of urogenital chlamydial infection. Cell culture, direct immunofluorescence assay, and polymerase chain reaction brought identical results. Conjunctival specimens taken from 33 (20%) of the patients with conjunctivitis were Chlamydia antigen positive; specimens taken from 134 (80%) were negative. Antichlamydial antibodies were found in tears of 29 (88%) of the patients with conjunctivitis whose specimens were Chlamydia antigen positive. Fifty-four (40%) of the patients with conjunctivitis whose specimens were Chlamydia antigen negative had antichlamydial antibodies in their tears. Twenty-five patients with urethritis (56%) were Chlamydia antigen positive in urethral swabs; 20 (44%) were negative. Antichlamydial antibodies were found in the tears of eight (32%) of the Chlamydia antigen-positive and two (10%) of the Chlamydia antigen-negative patients with urethritis. In contrast to patients with conjunctivitis, findings for patients with urethritis always were negative for antichlamydial IgG in the tears. CONCLUSION: Antichlamydial antibodies in tears were seen significantly more often in patients with conjunctivitis than in those with urethritis (P < or = 0.05). Antichlamydial IgG was found only in tears of patients with conjunctivitis. Therefore, the authors conclude that the detection of antichlamydial IgG in the tears might be helpful for diagnosis in patients with suspected chlamydial conjunctivitis who have antigen-negative conjunctival swabs.

Evaluation of two nonculture antigen tests and three serotests for detection of anti-chlamydial antibodies in the diagnosis of ocular chlamydial infections.

BACKGROUND: Diagnosis of chlamydial conjuctivitis is difficult in chronic diseases because chlamydial elementary bodies are mostly undetectable in conjunctival scrapings by cell culture. We therefore compared two nonculture antigen tests and three different serotests for anti-chlamydial antibodies with McCoy cell culture, the "gold standard" of chlamydial diagnosis. Conjunctival scrapings and serum samples of 93 patients attending the outpatient eye clinic in Graz because of chronic follicular conjunctivitis were tested. METHODS: A total of 558 conjunctival scrapings and 93 serum samples were investigated. Chlamydial antigen detection was done by McCoy cell culture, polymerase chain reaction (PCR; Amplicor, Roche), and direct immunofluorescence assay (DFA; Microtrak, Syva). Antichlamydial IgA and IgG antibodies in the sera were detected by an immunoperoxidase assay (IPAzyme, Savyon) and two different enzyme-linked immunosorbent assays (SeroELISA, Savyon and rELISA, medac). RESULTS: Cell culture and PCR yielded identical results. The positivity rate for chlamydial conjunctivitis was 8.6% (8 of 93 patients). PCR proved most sensitive and most specific. IPAzyme was 75% sensitive for IgA and 100% for IgG; SeroELISA and rELISA were less sensitive. IPAzyme was 81% specific for IgA and 47.3% for IgG. SeroELISA and rELISA were less specific for IgA, but more specific for IgG. Post-test likelihood of disease was greatest in IPAzyme. CONCLUSIONS: PCR proved to be a good alternative to cell culture; DFA is useful for quick diagnosis. Genus-specific serotests cannot compete with chlamydial antigen detection. They differ in sensitivity and specificity because of the antigen type they present. They are still of only supportive value in cases where chlamydial antigen detection is not possible. Recently introduced species-specific antibody tests should be of greater value.

Detection of herpes simplex virus DNA from cerebrospinal fluid by PCR and a rapid, nonradioactive hybridization technique.

A molecular assay for the detection of herpes simplex virus (HSV), including a novel, nonradioactive hybridization technique, was evaluated with a total of 123 cerebrospinal fluid specimens. After DNA extraction, specific HSV DNA sequences were amplified with digoxigenin-labeled primers derived from the DNA polymerase gene-coding region from HSV. Amplified products were detected by the Enzymun-Test DNA detection assay (Boehringer, Mannheim, Federal Republic of Germany), which uses biotinylated probes. Amplification with nonlabeled primers and then Southern blotting and nonradioactive detection of hybrids by the digoxigenin technique was the reference system. The sensitivities of the molecular assays were determined with 10-fold dilutions of plasmid pS4 with the SalI restriction fragment of the DNA polymerase gene obtained from the HSV type 1 strain Angelotti. The Enzymun assay was able to detect all of the 16 positive samples, giving 100% agreement with the Southern blot hybridization results. Optical density values were widely separated for the positive and negative groups of specimens. Ten copies of plasmid pS4 per microliter could be distinctly detected by the Enzymun assay. The cutoff was determined for the hybridization assay, and an equivocal zone was defined. The whole molecular assay including the Enzymun-Test DNA detection proved to be sensitive and easy to use. It may contribute to the rapid and safe detection of HSV DNA in cerebrospinal fluid.

Rapid detection of Chlamydia trachomatis in conjunctival, pharyngeal, and urethral specimens with a new polymerase chain reaction assay.

GOAL OF THIS STUDY: The Amplicor polymerase chain reaction (PCR) assay for the detection of Chlamydia trachomatis (Roche Molecular Systems, Branchburg, NJ) was evaluated on conjunctival, pharyngeal, and urethral swabs. STUDY DESIGN: A total of 515 conjunctival, pharyngeal, and urethral swabs. The reference system was culture with McCoy cells in shell vials with fluorescent immunostaining. One swab was used for both cell culture and the molecular assay. Initial storage took place in 2-SP medium. After transfer to Amplicor specimen transport medium the molecular assay was done using the Amplicor Chlamydia trachomatis amplification and detection kits. RESULTS: The total positive rate was 6.6%. Specificity of culture was 100%. The evaluated molecular assay gave a specificity of 99.8%. Sensitivities of PCR and culture were 100% and 85.3%, respectively. CONCLUSIONS: Because of the high sensitivity, specificity, and ease of use, the molecular assay was found to be a good alternative to culture for detection of C. trachomatis in conjunctival, pharyngeal, and urethral specimens.