Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in genitourinary specimens from men and women by a coamplification PCR assay

A coamplification PCR test for the direct detection of Neisseria gonorrhoeae and Chlamydia trachomatis in urethral and endocervical swabs and urine samples from men and women was compared to standard culture techniques. Processed specimens were amplified in single reaction tubes containing primers for both organisms, and PCR products were detected by a colorimetric microwell plate hybridization assay specific for each pathogen. Of 344 specimens from men, 45 (13.1%) urine specimens were PCR positive for C. trachomatis, 51 (14.8%) urethral swab specimens were PCR positive, and 29 urethral swab specimens (8.4%) were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis were 96.2 and 99.3%, respectively, in urethral swab specimens, compared to 88.2 and 98.6% for urine specimens. Of the 192 specimens from women, 28 (14.6%) urine specimens were PCR positive for C. trachomatis, 32 (16.7%) endocervical specimens were PCR positive, and 19 (9.9%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis for endocervical specimens were both 100% compared to 100 and 99.4%, respectively, for urine specimens from women. In men, 68 (19.8%) urine specimens were PCR positive for N. gonorrhoeae, 73 (21.2%) urethral swabs were PCR positive, and 59 (17.2%) urethral swabs were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 97.3 and 97.0%, respectively, for urethral specimens compared to 94.4 and 98.5% for urine specimens. In women, 18 (9.4%) urine specimens were PCR positive for N. gonorrhoeae, 23 (12.0%) were endocervical swab PCR positive, and 15 (7.8%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 100 and 99.4%, respectively, for endocervical specimens compared to 90.0 and 95.9% for female urine specimens. These results indicate that a multiplex PCR is highly sensitive for detecting both C. trachomatis and N. gonorrhoeae from a single urine or genital swab, providing a more cost-effective way of screening multiple pathogens.     

Healthy puppies and kittens as carriers of Campylobacter spp., with special reference to Campylobacter upsaliensis

Living in a household with a dog or cat has previously been identified as a significant risk factor for acquiring campylobacteriosis, in particular, with reference to Campylobacter upsaliensis infection. In a cross-sectional study carried out in Denmark between August and December 1996, 72 healthy puppies and 42 healthy kittens, aged between 11 and 17 weeks, were sampled for fecal campylobacter shedding by culture of rectal swab specimens on blood-free agar base with cefoperazone at 32 mg/liter and amphotericin at 10 mg/liter and on blood-free agar base with cefoperazone at 8 mg/liter, teicoplanin at 4 mg/liter, and amphotericin at 10 mg/liter. Additionally, with respect to the C. upsaliensis transmission potential of poultry, a chicken cloacal swab sample from each of 100 different broiler flocks was included in the study for comparison. We found 21 (29%) of the puppies positive for Campylobacter spp., with a species distribution of 76% C. jejuni, 5% C. coli, and 19% C. upsaliensis. Of the kittens examined, two (5%) excreted campylobacters; both strains were C. upsaliensis. None of the chicken samples examined were found to be positive for C. upsaliensis. We concluded that young puppies and kittens are potential transmitters of human-pathogenic Campylobacter spp., including C. upsaliensis, while poultry seems negligible in C. upsaliensis epidemiology.     

Long-term follow-up of a randomized trial on adjuvant chemotherapy and hormonal therapy in locally advanced breast cancer

Epidemiological research on respiratory syncytial virus (RSV) infections in children was carried out at the Virology Laboratory, University Teaching Hospital (UTH), in Lusaka, Zambia, from January-December 1996. Specimens including 736 nasal washings and 2424 throat swabs were collected from children with acute respiratory infections (ARI) and tested for RSV by enzyme immunoassay and by virus isolation. RSV was isolated in 62 (4.1%) of 1496 throat swabs collected from March to September and was detected in 99 (16.3%) of 609 nasal washings from March to November. The average RSV isolation rate was 2.6% and the average RSV detection rate was 13.5%. The highest RSV isolation (8.1%) and detection (30.5%) rates were in June 1996. RSV antibody in the 278 serum specimens collected from Zambian children, who were hospitalized in the paediatric ward, UTH, was detected using a standard neutralization test. The antibody positive rate was 60-80% in children > 4 years. It is evident that RSV is one of the main causal agents of ARI in children in Zambia.     

Preconditioning with cromakalim improves long-term myocardial preservation for heart transplantation

Polymerase chain reaction (PCR) and ligase chain reaction (LCR) were compared for the diagnosis of Chlamydia trachomatis infections by testing urine specimens from 408 high school female students. After therapy, sequential urine specimens were tested to determine persistence of chlamydial DNA in urine. Baseline PCR of cervical specimens was positive in 53 (13.0%) students, and PCR and LCR of urine specimens were positive in 63 (15.4%) and 60 (14.7%), respectively. After discrepant analysis, 64 (15.7%) patients could be confirmed as truly infected. Follow-up urine specimens from 33 infected patients demonstrated that at 1-3 days after therapy, PCR and LCR were positive for 40% and 73.3%, respectively. Only at 15 days after therapy did all specimens test negative. Urine tests for Chlamydia organisms should not be used as a test of cure within 3 weeks after treatment. Use of urine assays for screening sexually active adolescents has the potential to significantly improve control of chlamydial infections.     

Direct DNA probe assay for Neisseria gonorrhoeae in pharyngeal and rectal specimens

The direct detection of gonococcal DNA in rectal and pharyngeal specimens was evaluated by using a DNA probe-based assay (Gen-Probe, Inc., San Diego, Calif.). Rectal (234) and pharyngeal (608) swab specimens were obtained from 249 men and 372 women attending sexually transmitted disease clinics in Las Vegas and Reno, Nevada. The prevalence of gonococcal infection by culture at the pharyngeal and rectal sites was 2.9% (16 of 548 specimens) in women and 2.7% (8 of 294 specimens) in men. No false-positive reactions were observed among the 234 rectal specimens tested. Two probe-positive, culture-negative specimens were detected among the 361 pharyngeal specimens obtained from women. Both of these samples were confirmed as Neisseria gonorrhoeae by a probe competition assay. The overall correlation of the DNA probe test with pharyngeal and rectal cultures was 99.4% (837 of 842 cultures), with a sensitivity of 87.5% (21 of 24 cultures) and specificity of 99.7% (816 of 818 cultures). The positive and negative predictive values of the DNA assay were 91.3 and 99.8%, respectively. The direct DNA probe assay provides an alternative to culture screening for rectal and/or pharyngeal gonococcal infections.     

Is urine leukocyte esterase test a useful screening method to predict Chlamydia trachomatis infection in women?

We evaluated the use of the leukocyte esterase test (LET) on first-catch urine specimens from women as a screening test to predict infection with Chlamydia trachomatis. For diagnosis, we used Abbott's ligase chain reaction (LCR) on urine specimens and isolation by tissue culture (TC) on cervical brushes. Of 4,053 women attending sexually transmitted disease and family planning clinics, 4.3% (n = 174) were positive by TC and 5.9% (n = 239) were positive by LCR. When LET was compared to TC, the sensitivity, specificity, positive predictive value, and negative predictive value were 54.0, 67.0, 6.8, and 97.0%, respectively. The corresponding performance of LET versus LCR was 53.1, 67.3, 10.1, and 95.8%. Almost half of the laboratory-confirmed chlamydial infections were negative by LET. The low specificity probably reflects multiple causes of pyuria in women and results in a low positive predictive value. LET is neither sensitive nor specific as a predictor of chlamydial infection and cannot be recommended for use as a screening test for C. trachomatis with first-catch urine samples from females from low- or moderate-prevalence populations.     

Detection of Chlamydia trachomatis infections in women by Amplicor PCR: comparison of diagnostic performance with urine and cervical specimens

We used the Roche Amplicor PCR assay to compare urine and cervical swabs as sample material in the detection of Chlamydia trachomatis causing genital infections. The diagnostic performance of Amplicor PCR was compared with that of cell culture and the Gen-Probe PACE 2 assay with cervical specimens. If discrepant from other results, the specimens negative by PCR were diluted and reanalyzed to reveal PCR inhibitors. Of 666 patients, 39 (5.9%) were confirmed to have chlamydial infection. The respective sensitivity and specificity of Amplicor PCR were as follows: urine specimens, 82.0 and 99.7%; cervical specimens, 82.0 and 99.8%. Those for cell culture with cervical specimens were 84.6 and 100%. For the Gen-Probe PACE 2 assay, the sensitivity and specificity with cervical specimens were 79.5 and 100%, respectively. Without the effect of PCR inhibitors, the sensitivity of PCR with urine would have been 97.4%. Provided that the problems currently caused by inhibitors will be solved, the Amplicor PCR assay with urine specimens offers a tempting alternative for the diagnosis of C. trachomatis infection in women.     

Prospective study of neonatal genital mycoplasma colonization and infection

Genital mycoplasmas have been implicated in different neonatal diseases as pneumonia, sepsis and meningitis. This prospective study was conducted to specify their role in these diseases. POPULATION AND METHODS--A pharyngeal or tracheal swab specimen for mycoplasmas culture was obtained from 100 infants admitted consecutively to the Neonatal Care Unit (NCU) during the first 24 hours of life. Mycoplasma culture of blood and cerebrospinal fluid was also performed. Pharyngeal and/or tracheal specimens were collected again on days 5, 15 and 28 if the child was still in the NCU. Mycoplasma hominis (Mh) and Ureaplasma urealyticum (Uu) were identified by culture in a modified Hayflick's medium. RESULTS--Three-hundred and ten pharyngeal or tracheal swabs were obtained (100 on day 0, 89 on day 5, 72 on day 15 and 49 on day 28). Twenty-one infants had one or more positive swabs in the first five days of life (20 on day 0 and one on day 5); those forming the "Myco+" group and the others forming the "Myco-" group. Uu was isolated alone from 20 infants, associated with Mh from one. Both groups were similar for gestational age, birth weight, maternal fever during labor, prolonged rupture of the fetal membranes or chorioamnionitis and for the incidence of acute respiratory distress. There was a statistically significant difference for the route of delivery (chi 2 < 0.02). One blood culture (from 92 performed) was positive for Uu and another positive for Uu and Mh. Both children were cured without any specific mycoplasmacidal therapy. Three children had probable Uu infection and were also cured without specific therapy. CONCLUSIONS--A pharyngeal colonization with genital mycoplasmas is common in the first days of life (21%) but our data do not allow us to conclude that they are accountable for newborn infections.     

Use of CHROMagar Candida for genital specimens in the diagnostic laboratory

OBJECTIVE: To evaluate CHROMagar Candida (CA), a new yeast differential medium, for yeast isolation in a clinical laboratory for the routine examination of high vaginal swabs. METHODS: Results of high vaginal swab cultures processed in a standard manner on plates containing equal halves of Sabouraud dextrose agar (SDA) and CA were compared. Non-Candida albicans yeast isolates were further speciated with API 20C AUX or API 32C. To assess the ease of use of CA, laboratory staff lacking in experience of the medium were asked to identify 23 unlabelled yeast cultures on CA by referring to six labelled reference plates. RESULTS: Of the 1784 swab cultures processed, yeasts were isolated from 373 SDA and 368 CA. Of the 78 non-albicans isolates further speciated, CA identified correctly all cultures of C krusei and C tropicalis, and 82% of C glabrata. All the 38 inexperienced laboratory staff achieved 100% accuracy for C albicans and over 90% for C krusei and C tropicalis. CONCLUSIONS: CA is a satisfactory isolation medium for genital specimens, allowing immediate and correct identification of the commonly encountered yeasts and easy recognition of mixed cultures.     

Salmonella enteritidis colonization in turkey poults

An experimental trial on the colonization of two Salmonella enteritidis strains (phage types 4 and 8) originating from meat turkey flocks, were carried out. Three-day old poults were inoculated orally with approximately 10(6) cfu/bird (one group with PT8 and the other with PT4). clinical signs were not observed in any of the groups. The total reisolation rates from cloacal swabs in birds inoculated with PT4 was higher than from birds inoculated with PT8. The inoculated strains could also be detected in contact birds. Examination of internal organs 21 days p.i. revealed higher isolation rates in the group infected with PT4 than with PT8. The results indicate that both phage types of S. enteritidis were able to colonize the intestinal tract and the internal organs of turkey poults. However, the colonization and duration of shedding differed between the two phage types. This may be due to the fact that PT8 strain used was free from the S. e. virulence plasmid 37 MDa.     

Cost analysis for total hip arthroplasty by measurement of time and material expenditure

BACKGROUND AND OBJECTIVES: An estimated 4 million new cases of chlamydial infection occur each year. This experiment assessed the effects of a vaginally applied gel formulation of 0.25% chlorhexidine gluconate on chlamydial infection and on the vaginal ecosystem. STUDY DESIGN: Twelve monkeys were treated with a single application of 0.25% chlorhexidine gluconate. These animals were assessed for changes in vaginal flora before and at 30 minutes, 1 day, and 2 days postapplication by microbiologic analysis. Cervical and vaginal tissues were assessed by colposcopy at each time point. Five monkeys received a single application of 0.25% chlorhexidine gluconate gel followed (30 minutes) by a cervical inoculation with Chlamydia trachomatis. Four monkeys were inoculated with Chlamydia only. Cervicovaginal tissues were assessed via modified colposcopy, vaginal swabs were collected for assessment of vaginal flora, and cervical swabs were collected for detection of Chlamydia (culture/ligase chain reaction) at baseline and days 1, 2, and 7 postinoculation. RESULTS: Changes in vaginal flora were minimal in all monkeys. Application of 0.25% chlorhexidine gluconate did not affect adversely vaginal colonization by lactobacilli. All chlamydial infection control monkeys were infected, whereas none of the five monkeys pretreated with chlorhexidine gluconate were positive for C. trachomatis by culture or ligase chain reaction. Colposcopic observations remained largely unchanged in all groups. CONCLUSIONS: A 0.25% chlorhexidine gluconate gel was protective against chlamydial infection in all animals tested, had no adverse effect on the vaginal flora, and had minimal effect on cervicovaginal tissues after a single application.     

Does Chlamydia trachomatis MoPn enter a microbiologically-inapparent state during experimental infection of the mouse genital tract?

Microbiologically-inapparent chlamydial infection may contribute towards the immunopathogenesis of these diseases. Although morphologically and physiologically aberrant non-cultivable chlamydiae can be induced reversibly in cell culture, evidence for these forms in infections of animals and humans is indirect. A mouse model of salpingitis caused by the mouse pneumonitis biovar of Chlamydia trachomatis (MoPn) was used to determine the existence of non-cultivable organisms in vivo. Following intravaginal inoculation, mice yielded high chlamydial counts for 7-14 dyas, with a decline in culture-positivity by 21-28 days. A significant elevation of IFN gamma production in infected tissues was measured for 21 days and, from 28-70 days, all mice were culture-negative and developed characteristic hydrosalpinges. MoPn was detected by PCR in vaginal swabs of 80% and 69% respectively of culture-negative animals at 21 and 28 days. In a second study, 100%, 63% and 50% of culture-negative genital tissue homogenates were PCR-positive at 21, 28 and 42 days. Immunosuppression with either cyclophosphamide or hydrocortisone failed to regenerate cultivable chlamydiae. Tissues were disrupted by homogenization and inoculated intranasally to MF1 mice which are extremely susceptible to MoPn, but all culture-negative specimens were non-infectious. The significance of the PCR-positive culture-negative specimens requires further investigation, since these may represent a non-cultivable state in the deeper tissues of the mouse genital tract which may be beyond the reach of reactivating triggers.     

Rapid shell vial culture technique for detection of enteroviruses and adenoviruses in fecal specimens: comparison with conventional virus isolation method

Detection of enteroviruses and adenoviruses mainly in fecal specimens by rapid culture with inoculation onto cell monolayers in flat-bottom tubes by centrifugation and immunofluorescence staining with genus-specific monoclonal antibodies was compared with that by the conventional virus isolation procedure. For both conventional culture and shell vial culture human lung fibroblast cells and tertiary monkey kidney cells were used. For enterovirus detection, 979 clinical specimens (916 stool specimens, 56 cerebrospinal fluid specimens, and 7 nasopharyngeal swabs) were used. Conventional culture detected 74 enterovirus isolates. A cytopathic effect compatible with the presence of an enterovirus after 3 days of incubation occurred in 25 of the 74 (34%) specimens that eventually became positive. The detection rate for enteroviruses by rapid cell culture after 2 to 3 days of incubation was 42 of 74 (57%). The genus-specific enterovirus monoclonal antibody did not react with strains of echovirus types 22 and 23 or enterovirus type 71. Rapid cell culture for the detection of adenoviruses was performed with 567 clinical specimens (536 stool specimens, 25 cerebrospinal fluid specimens, and 6 miscellaneous specimens), in which 42 adenoviruses were found by conventional culture. Nine of the 42 (21%) adenovirus isolates were detected by conventional culture within 3 days after inoculation, whereas 21 (50%) were found by rapid cell culture within 2 to 3 days. Only two of the nine specimens found to be positive for the enteric adenovirus type 41 by conventional culture as well by a type-specific enzyme-linked immunosorbent assay (ELISA) tested positive by rapid cell culture. In conclusion, the rapid shell vial assay allows the early detection and identification of enteroviruses and adenoviruses in clinical specimens but is markedly less sensitive than the conventional isolation procedure according to the eventual results of the conventional isolation procedure. Conventional cell culture remains a prerequisite for serotyping of enteroviral isolates. On the basis of the results for adenovirus type 41, the rapid detection of adenoviruses was not considered to be useful for the detection of clinically relevant adenoviruses in fecal samples.     

An examination of the occurrence of surgical wound infection following equine orthopaedic surgery (1981-1990)

First-void urine specimens, collected from 309 military recruits, 246 male adolescent gymnasium students and 194 patients consulting venereal disease clinics, were studied for the presence of Chlamydia trachomatis with the use of antigen detection tests--two enzyme immunoassays (EIA) and a direct immunofluorescence test (DIF; Syva MicroTrak). Urethral swabs were collected when discrepancies between the EIA and DIF tests were detected. The patient was regarded as positive when the culture result was positive or when two antigen detection tests corraborated one another. The Syva MicroTrak EIA and DIF tests were more sensitive than the Orion EIA, i.e. 98.5%, 99.2% and 74%, respectively. This was true when testing both low- and high-risk groups, with a prevalence of chlamydial infection ranging from 0.4% to 58.6%. All three tests were highly specific. The positive predictive values for the Syva MicroTrak EIA, the DIF and the Orion EIA were 99.2%, 100% and 100%, respectively and the negative predictive values 99.8%, 99.8% and 94.8%, respectively.     

Comparison of performances of two commercially available tests, a PCR assay and a ligase chain reaction test, in detection of urogenital Chlamydia trachomatis infection

The diagnostic performance of a PCR test (Roche Cobas Amplicor CT/NG Test) and that of a ligase chain reaction (LCR) test (Abbott LCx Chlamydia trachomatis assay) were compared by using endocervical and urethral swab specimen culture as a reference test. First-void urine (FVU) and endocervical and urethral swab specimens were collected from 1,015 unselected patients attending a sexually transmitted disease clinic and a clinic for adolescents in Helsinki, Finland. Chlamydia trachomatis was cultured from samples from the endocervix or urethra. PCR was performed with fresh and frozen urine and the culture transport medium. LCR was performed with fresh and frozen urine and LCx swab transport medium. Diagnostic consistency and diagnostic accuracy were statistically tested. The test results were identical for 984 patients (97%). Discrepant results were observed for 31 patients. Overall, LCR and PCR showed excellent kappa coefficients of consistency for both swab and FVU specimens (0.93 and 0.95, respectively). Sixty-one patients (6%) were culture positive. Testing of FVU by LCR or PCR increased the overall positivity rates to 7.0 and 7.7%, respectively. While PCR of FVU detected the greatest number of C. trachomatis infections (sensitivity, 96.1%), for some PCR-positive FVU specimens the results could not be confirmed (specificity, 99.6%). PCR and LCR were more sensitive than culture (sensitivities, 92 and 93% versus 79% for culture) in the diagnosis of genital C. trachomatis infection. In conclusion, both tests can be recommended for use in the clinical laboratory and for the screening of asymptomatic C. trachomatis infections.     

Multiplex PCR-based assay for detection of Bordetella pertussis in nasopharyngeal swab specimens

A multiplex PCR-based assay was developed for the detection of Bordetella pertussis in nasopharyngeal swab specimens. The assay simultaneously amplified two separate DNA targets (153 and 203 bp) within a B. pertussis repetitive element and a 438-bp target within the beta-actin gene of human DNA (PCR amplification control). PCR products were detected by a sensitive and specific liquid hybridization gel retardation assay. A total of 496 paired nasopharyngeal swab specimens were tested by both the PCR-based assay and culture. Although 30 (6%) of the specimens inhibited the amplification of the beta-actin target, in all 29 specimens studied, the inhibition disappeared on repeat testing or was easily overcome with a 1:8 dilution or less of specimen digest. Of the 495 specimen pairs yielding a final evaluable result by the PCR-based assay, 19.0% were positive by the PCR-based assay, whereas 13.9% were positive by culture (P < 0.0001). After resolving the PCR-positive, culture-negative results by testing an additional aliquot from these specimens by the multiplex PCR-based assay, the PCR-based assay had a sensitivity and specificity of 98.9 and 99.7%, respectively, compared with values of 73.4 and 100%, respectively, for culture. In comparison with patients with culture-confirmed pertussis, those with PCR-positive, culture-negative results were older and more likely to have had prolonged cough, immunization with pertussis vaccine, or treatment with erythromycin. This multiplex PCR-based assay is substantially more sensitive than culture and identifies specimens that contain inhibitors of PCR.     

Resistance to tetracycline of E. coli of chicken intestines after prophylactic treatment of animal feed with bioptivet GB

This study describes the influence of bioptivet GB on minimum inhibitory concentrations (MIC) for oxytetracycline (OTC) of the intestinal E. coli flora of young broiler chickens after oral administration at a dosage equivalent to a prophylactic course of treatment. From day 6 until day 15 one group of 50 birds received a diet containing 124 ppm OTC, another group of 75 birds served as non medicated control. Investigated E. coli had been isolated from cloacal swabs and from caecal contents. MIC of 1581 E. coli strains were determined by agar dilution test procedures. MIC of OTC for the investigated strains were either > or = 128 micrograms/ml (resistant) or < or = 4 micrograms/ml (susceptible). Even from undosed birds resistant strains were isolated frequently, especially from samples of caecal contents. Administration of bioptivet GB resulted in a statistically significant increase in the average MIC. Statistically higher average MIC were recorded among isolates from cloacal swabs only during application of the drug. For strains from caecal contents this could be demonstrated until the end of the experiment.     

Herpesviruses in tortoises: investigations into virus isolation and the treatment of viral stomatitis in Testudo hermanni and T. graeca

Various studies were done during a spontaneous outbreak of stomatitis-rhinitis-complex (mouth rot) in a collection of Mediterranean land tortoises (21 Testudo hermanni, Hermann's tortoises, and three Testudo graeca, spur-thighed tortoises) in southern Germany. These studies were intended to help diagnose the causative agent, establish a possible diagnostic method in vivo and provide information on the efficacy of aciclovir and ganciclovir against chelonian herpesviruses. Thirteen T. hermanni and no T. graeca died within a period of 6 weeks following the introduction of one apparently healthy T. graeca. Two of the dead Testudo hermanni were submitted for post-mortem examination. In addition, blood samples from 11 of the 12 tortoises still surviving at the beginning of this study were cultured for virus content and for the presence of neutralizing antibodies to chelonian herpesviruses and swabs from conjunctiva, pharynx and cloaca were cultured for the presence of viruses. Herpesviruses were isolated from tissues of the two dead Testudo hermanni (tongue, intestine, trachea, lung, spleen, heart and brain). Peripheral leukocytes from one of 11 blood samples were positive for herpesvirus isolation, indicating viremia in at least one animal. Nine of 11 pharyngeal swabs but none of the conjunctival and cloacal swabs yielded herpesviruses. Circulating neutralizing antibodies were present in two of two tested T. graeca, but absent in all of the nine samples from T. hermanni. Aciclovir and ganciclovir were effective when tested in vitro against one of the herpesvirus isolates.