Evaluation of an epitope-blocking enzyme-linked immunosorbent assay for the diagnosis of West Nile virus infections in humans.

An epitope-blocking enzyme-linked immunosorbent assay (b-ELISA) was evaluated for the diagnosis of West Nile virus (WNV) infections in humans. Sera from patients diagnosed with WNV infections from an outbreak in 2003 in Colorado and from patients diagnosed with dengue virus infections from Mexico and Thailand were tested with the b-ELISA. The b-ELISAs were performed using the WNV-specific monoclonal antibody (MAb) 3.1112G and the flavivirus-specific MAb 6B6C-1. Although the WNV-specific b-ELISA was effective in diagnosing WNV infections in humans from Colorado, it was not efficacious for diagnosing WNV infections in serum specimens from Mexico and Thailand. In serum specimens from patients from Colorado, the WNV b-ELISA and the WNV plaque reduction neutralization test showed an overall agreement of 91%. The sensitivity and specificity of the WNV b-ELISA were 89% and 92%, respectively, with a false-positive rate of 5%, based on receiver operating characteristic analysis. In contrast, false-positive rate results in specimens from the countries of Mexico and Thailand, where flaviviruses are endemic, were 79% and 80%, presumably due to the presence of antibodies resulting from previous dengue virus infections in Mexico and/or Japanese encephalitis virus infections or vaccination in Thailand. Thus, in regions where people have experienced previous or multiple flavivirus infections, the use of the b-ELISA for WNV diagnosis is contraindicated.

Testosterone concentrations in women aged 25-50 years: associations with lifestyle, body composition, and ovarian status.

While there is substantial evidence of the importance of endogenous and exogenous estrogen in reproductive health and chronic disease, there is little consideration of androgens in women's health. In the Michigan Bone Health Study (1992-1995), the authors examined the correlates of testosterone concentrations in pre- and perimenopausal women (i.e., age, menopausal status, body composition, and lifestyle behaviors) in a population-based longitudinal study including three annual examinations among 611 women aged 25-50 years identified through a census in a midwestern community. Current smokers had the highest testosterone concentrations with decreasing values in former and nonsmokers (p = 0.0001). Body composition measures (body mass index, body fat (%), weight (kg), lean body mass (kg), and fat mass (kg)) were significantly and positively associated with total testosterone concentrations in a dose-response manner. Hysterectomy with oophorectomy was associated with significantly lower testosterone concentrations. Alcohol consumption, physical activity, and dietary macronutrient intake were not associated with testosterone concentrations. This is one of the first studies to examine correlates of serum testosterone concentrations in anticipation of the growing interest in the role of androgens in women's health. The greater circulating levels of testosterone in obese women and smokers suggest that testosterone concentrations should be considered in the natural history of disease conditions where obesity and smoking are risk factors, including cardiovascular disease.

How adequate is adequate for the collection of endocervical specimens for Chlamydia trachomatis testing?

BACKGROUND: Endocervical specimen adequacy has been assessed by subjective criteria that are based on arbitrarily chosen thresholds for the presence of target cells observed on microscopic slide examinations. GOAL OF THIS STUDY: To assess the relationship of chlamydia test positivity to specimen adequacy with the use of a semi-quantitative cytologic staining method for assessing endocervical specimen collection cellularity. STUDY DESIGN: Endocervical specimens for chlamydia testing (PACE 2, GenProbe, San Diego, CA) and for a slide cytologic examination (n = 3,500) were collected in parallel. A semi-quantitative cytologic examination to determine a specimen adequacy (SA) score was performed for every chlamydia-positive result (n = 163) and approximately every fifth negative result (n = 626). The Chi-square test for linear trends was used to assess the relationship between SA scores and chlamydia positivity. The median SA scores for chlamydia-positive and negative slides were compared. RESULTS: The median SA scores for chlamydia-positive and -negative slides were 27 and 20, respectively (P < 0.001). Chlamydia positivity rates increased with increasing specimen adequacy scores (0-9, 2.7%; 10-19, 15.1%; 20-29, 24.8%; and 30-45, 31.3%; Chi-square for linear trend: P < 0.001). CONCLUSION: These results demonstrate a linear relationship between the numbers of cells observed on an endocervical smear and chlamydia positivity rather than the threshold concept in practice. The semiquantitative cytologic technique offers an objective method for further evaluating specimen adequacy for Chlamydia trachomatis testing.

"Lifeguard lung": endemic granulomatous pneumonitis in an indoor swimming pool.

OBJECTIVES: Two sequential outbreaks of respiratory disease among lifeguards at an indoor swimming pool with water spray features were investigated. METHODS: Questionnaires were administered to recreation center employees following each outbreak. Respondents reporting 2 or more pool-related symptoms were offered clinical evaluation, including bronchoscopy with bronchoalveolar lavage and transbronchial biopsy. Pool air and water were sampled for fungi, bacteria, amoebae, endotoxin, and respirable particulates. RESULTS: Thirty-three lifeguards had noncaseating granulomas on biopsy and/or bronchoalveolar lavage lymphocytosis. Attack rates for the outbreaks were 27% and 65%. Case patients had higher cumulative hours of work and tended to work more hours per week. Analyses indicated increased levels of endotoxin in pool air and water (relative to control pools) and gram-negative bacterial colonization of water sprays. Use of water spray features generated a 5.2-fold increase in the number of respirable particles and up to an 8-fold increase in air endotoxin levels. CONCLUSIONS: Lifeguards in this indoor swimming pool developed granulomatous lung disease associated with endotoxin-containing respirable bioaerosols from water spray features, which ventilation system improvements did not prevent.

An outbreak of salmonellosis among children attending a reptile exhibit at a zoo.

OBJECTIVE: In January 1996, an outbreak of diarrhea caused by Salmonella Enteritidis occurred in children attending a Komodo dragon exhibit at a metropolitan zoo. We sought to determine the extent of the outbreak and mode of transmission. STUDY DESIGN: A case-control study was conducted. Controls were randomly selected from zoo membership lists and matched to patients by age group and date of exhibit visit. RESULTS: Of 65 patients identified, 39 had confirmed and 26 had suspected cases. The median age was 7 years (range, 3 months to 48 years); 55% were enrolled in the case-control study. No patients and two (4%) controls reported touching a dragon; however, 83% of patients but only 52% of controls touched the wooden barrier that surrounded the dragon pen (odds ratio = 4.0, 95% CI 1.2 to 13.9). Washing hands at the zoo after visiting the dragons was highly protective (OR = 0.14, 95% CI 0.03 to 0.7). Cultures from the patients, one dragon, and the exhibit barriers yielded Salmonella Enteritidis, phage type 8. On the basis of an attack rate of 4.3% among exhibit attendees under 13 years old on whom data were collected, we estimate that 315 additional cases of salmonellosis occurred among visitors in this age group. CONCLUSION: This large outbreak demonstrates the importance of environmental contamination in the transmission of Salmonella from reptiles, and the protective value of hand washing. Recommendations regarding reptile exhibits and reptilian pets should emphasize this indirect route.

Use of DNA purification kits for polymerase chain reaction testing of Gen-Probe Chlamydia trachomatis PACE 2 specimens.

BACKGROUND AND OBJECTIVES: Confirmation testing using nucleic acid amplification has been shown to improve the sensitivity and specificity of screening tests for Chlamydia trachomatis. However, no critical information on the use of these techniques as an adjunct to Gen-Probe hybridization testing, one of the most common screening methods, has been reported to date. We examined the Roche AMPLICOR PCR C. trachomatis Test (Roche Diagnostic Systems, Branchburg, NJ) as a confirmatory test for the Gen-Probe PACE 2 C. trachomatis Test (San Diego, CA). Further, to mitigate the possible effect of interfering compounds in the Gen-Probe PACE 2 transport medium, we tested various DNA purification techniques. STUDY DESIGN: C. trachomatis elementary bodies were used to spike PACE 2 Transport medium, which was serially diluted, then tested by polymerase chain reaction (PCR). Six parallel dilution series were conducted: (1) saline dilutions tested by the Syva Direct Specimen Test, (2) Roche AMPLICOR transport medium dilutions tested by PCR, and (3-6) dilutions in PACE 2 transport medium purified respectively by GENECLEAN II (BIO101, Vista, CA), Puregene (Gentra Systems, Inc., Research Triangle Park, NC), Microcon 100 (Amicon, Inc., Beverly, MA) DNA isolation kits, and no DNA purification, all tested by PCR. The system giving the best results by in vitro endpoint dilution trials was then used to confirm human specimens previously tested by the Gen-Probe method. RESULTS: PCR detected C. trachomatis at 11 twofold dilutions greater than PACE 2 and equivalent to detection of single elementary body by Syva Direct Specimen Test. DNA purification of spiked PACE 2 transport medium by the Microcon 100 kit produced the most consistent PCR detection endpoints, equivalent to endpoints of spiked AMPLICOR transport medium. Endpoints with no DNA purification step were variable and lower. Of 78 endocervical specimens negative by PACE 2 and Gen-Probe Probe Competition Assay, 12 (15.3%) were positive by Microcon DNA purification/PCR testing. CONCLUSIONS: PCR can be used as confirmation method for Gen-Probe PACE 2 testing, but testing must be performed with a DNA purification procedure.

Performance characteristics of the Gen-Probe Probe Competition Assay used as a supplementary test for the Gen-Probe PACE 2 and 2C assays for detection of Chlamydia trachomatis.

The performance characteristics of the Gen-Probe Probe Competition Assay (PCA) used in conjunction with the Gen-Probe PACE 2 and 2C direct detection assays for Chlamydia trachomatis were examined. Data collected by five public health laboratories by using the Gen-Probe PACE 2 were pooled and analyzed. Of 25,081 endocervical and male urethral specimens tested by the PACE 2 assay, 773 were tested by PCA. Of 334 specimens initially positive by the PACE 2 assay with an initial PACE 2 result of greater than 2,000 relative light units (RLU), 333 (99.7%) were positive by PCA while 242 of 339 (71.4%) specimens with an initial result between the cutoff and 2,000 RLU were positive by PCA, and 35 of 100 (35%) specimens with initial results between 200 RLU and the cutoff were positive by PCA. An additional 10,938 specimens were tested by the PACE 2C assay. Of these, positive PCA results were obtained for 187 of 188 (99.5%) specimens with initial results of greater than 2,000 RLU, 99 of 163 (60.7%) of specimens in the range of cutoff to 2,000 RLU, and 12 of 100 (12%) in the range of 200 RLU to the cutoff. These results indicate that specimens greater than 2,000 RLU do not require a supplemental test and that additional positive results can be obtained by testing specimens with an initial result below the cutoff.

Confirmation of the Syva MicroTrak enzyme immunoassay for chlamydia trachomatis by Syva Direct Fluorescent Antibody Test.

BACKGROUND AND OBJECTIVES: The Syva Micro Trak enzyme immunoassay (EIA) is used widely for screening women infected with Chlamydia trachomatis. Confirmatory tests used in conjunction with EIA screening have shown that false-positive results are common. GOALS: To evaluate the specificity of the Syva MicroTrak EIA by confirmation of positive specimens with the Syva Direct Fluorescent Specimen Test. STUDY DESIGN: Of 6,039 endocervical specimens collected from women attending Colorado family planning clinics, 328 positive EIA results (5.4%) were obtained by Syva MicroTrak EIA. A random subset of 136 positive specimens was tested by Syva Direct Specimen Test. Twenty of 136 specimens (14.7%) negative by Syva Direct Specimen testing were also tested by Syva blocking antibody tests (9 of 20 positive, 45%) and Roche Amplicor polymerase chain reaction (PCR; 6 of 20 positive, 30%). Of 20 specimens positive by Syva MicroTrak EIA and negative by Syva Direct Specimen Test, 11 (55%) were also negative by blocking antibody and PCR, including three specimens with initial EIA sample-to-cutoff ratios greater than 2. CONCLUSIONS: Confirmatory testing of Syva MicroTrak EIA positive specimens with Syva Direct Specimen Test showed that 14.7% were false positive. Coupling the Syva Direct Specimen test with either blocking antibody or PCR reduces the rate of false-positive results to 8%.

Recovery of uncommon bacteria from blood: association with neoplastic disease.

Table 6 is a summary of the organisms discussed with a listing of the environmental source, the endogenous source, the predisposing factors including neoplasms, and the postulated mechanisms by which the organism can gain access to the circulation. The evidence considered indicates that the entrance of one of these microorganisms into the bloodstream of a human being depends on the presence of multiplicity of predisposing factors. In the majority of cases of bacteremia due to one of these unusual organisms, two or more predisposing factors are present. Certain predisposing factors, such as cancer chemotherapy or intravenous catheterization, often provide a barrier break, while others, such as liver disease, may render the host immune system less capable of clearing organisms from the circulation. For organisms such as Campy-lobacter, Listeria, and Salmonella spp., attributes that allow the invasion of a healthy host are present and seem to be enhanced by the simultaneous presence of a predisposing condition, such as liver disease, in the host. Although somewhat fragmentary, a number of individual case reports describe bacteremia due to one of these organisms occurring weeks to years after surgery and after other therapeutic measures had effected a supposed cure of a cancer. It may be speculated that cancer patients, even after a cure, are still susceptible to bloodstream invasion by one of the aforementioned organisms by virtue of the presence of one or more predisposing metabolic, physiologic, or immunologic factors, even though these factors may be cryptic. The predominance of hematologic malignancies among cases of bacteremia due to these unusual organisms is also apparent. Although, as pointed out by Keusch (169), the reduction in the performance of immune function in hematologic malignancies compared with solid tumors is likely to be responsible, other associations of certain organisms with specific neoplasms warrant further examination. The frequency of bloodstream infections of Salmonella typhimurium and Capno-cytophaga canimorsus in Hodgkin's disease patients seems likely due to a particular mechanism which infection by these species is favored. The specific nature of these mechanisms remains to be determined. The recovery of any unusual bacterium from blood should warrant a careful consideration of the possibility of underlying disease, especially cancer. Microbiologists should advise clinicians of the unusual nature of the identified organism and provide the counsel that certain neoplastic processes, often accompanied by neutropenia, render the human host susceptible to invasion by almost any bacterium. The recovery of such organisms as C. septicum or S. bovis should prompt the clinician to aggressively seek to identify an occult neoplasm if one has not yet been diagnosed.

Incidence of Neisseria gonorrhoeae isolates negative by Syva direct fluorescent-antibody test but positive by Gen-Probe accuprobe test in a sexually transmitted disease clinic population.

To determine the accuracy of the Syva (Palo Alto, Calif.) direct fluorescent-antibody (DFA) test in comparison with the Gen-Probe (San Diego, Calif.) Accuprobe culture confirmation test, we tested 395 isolates of Neisseria gonorrhoeae from cultures obtained from patients attending a sexually transmitted disease clinic from 1 July 1991 through 30 June 1992. All isolates were tested for DFA reactivity with a polyclonal reagent (Difco Laboratories, Detroit, Mich.) and a monoclonal reagent (Syva, Inc., direct specimen test) and for specific molecular probe reactivity by the Gen-Probe Accuprobe culture confirmation test for N. gonorrhoeae. The 395 isolates gave positive results for the Gen-Probe culture confirmation test and the Difco polyclonal direct specimen test. However, 18 (4.6%) of the isolates were negative for N. gonorrhoeae by the Syva DFA test. With the exception of six beta-lactamase-positive isolates, all isolates that were negative by Syva DFA were sensitive to penicillin, tetracycline, spectinomycin, and ceftriaxone by disk-diffusion susceptibility testing. Auxotyping and serotyping studies indicated that strains negative by Syva DFA consisted of several variants. The frequency of N. gonorrhoeae isolates showing negative results by Syva DFA in this patient population ranged from 0 to 11.5%/month. Laboratories using only the Syva DFA test for confirmation of N. gonorrhoeae may incur a significant risk of misidentification.

Use of polymerase chain reaction in an epidemiologic investigation of Pontiac fever.

In June 1992, 13 (38%) of 34 resort guests experienced illness that met a symptom-based case definition of Pontiac fever. Each ill guest reported using an indoor hot tub compared with 6 (29%) of 21 nonill guests (P < .001). Water samples from the indoor hot tub were culture-negative for legionellae using standard techniques, coculture with amebae, and intraperitoneal inoculation of guinea pigs. However, polymerase chain reaction (PCR) testing of the water samples indicated the presence of Legionella pneumophila. Direct fluorescent antibody testing identified the organism as serogroup 6. Seroconversion to L. pneumophila serogroup 6 occurred in 7 (64%) of 11 ill guests and none of 5 nonill guests (P = .03). These results suggest that in certain circumstances, culture of environmental samples should be supplemented with additional tests such as PCR. These results are also consistent with the concept that Pontiac fever can be caused by nonviable legionellae.

Confirmatory testing of Chlamydia trachomatis Syva enzyme immunoassay gray zone specimens by Syva direct fluorescent antibody test.

A series of gray zone specimens with sample to cutoff ratio of 0.7 to 0.99 encountered in routine use of the Syva Microtrak (Syva, Inc.) Chlamydia trachomatis enzyme immunoassay (EIA) test for urogenital specimens were subjected to repeat testing in duplicate and high-speed centrifugation with direct fluorescent antibody testing of the centrifugate. Immunofluorescent C. trachomatis elementary bodies were observed in high-speed centrifugates in more than 40% of two series of gray zone specimens examined indicating that a C. trachomatis gray zone result would require confirmatory testing.

Cost-benefit analysis of selective screening criteria for Chlamydia trachomatis infection in women attending Colorado family planning clinics.

Women attending family planning clinics in Colorado during 1988 were screened for Chlamydia trachomatis infection by enzyme immunoassay (EIA, Chlamydiazyme, Abbott Laboratories; Abbott Park, IL). Cervical specimens from 11,793 women attending 22 family planning clinics were analyzed. Patient history and physical exams were used to assess risk factors for infection. A total of 913 individuals (7.7%) had positive culture results for C. trachomatis. Multivariate analysis showed that infection was significantly related to endocervical bleeding, cervical mucopurulent discharge, a new sexual partner in the last 3 months or multiple previous sexual partners (greater than 3) in the last year, pregnancy, the use of oral contraceptives, and age. Increased odd ratios were observed for the combination of endocervical bleeding and mucopurulent discharge and sexual history that included partners over the previous year as well as the most recent 3 months. A combination of these criteria was used to selectively screen women attending Colorado family planning clinics on an ongoing basis. A cost-benefit analysis employing a model reported previously showed a significant financial benefit associated with universal screening over either selective screening or no screening for C. trachomatis in this population.

Evaluation of enzyme-linked immunoassay systems for detection of human immunodeficiency virus type 1 antibody from filter paper disks impregnated with whole blood.

Five commercial enzyme-linked immunoassay systems for the detection of human immunodeficiency virus type 1 antibody from filter paper disks impregnated with whole blood were evaluated for technical and operational performance. All five systems performed adequately in the technical challenges posed, with specificities in excess of 99% for 1,020 specimens. In a serial dilution sensitivity challenge, all of the kits were able to detect specific antibody within one dilution of a Western blot (immunoblot) standard, except for a Du Pont Co. kit, which detected antibody within two dilutions of the standard. The Du Pont assay showed the least variation in control values between test runs and between lots. All of the systems produced acceptable results, but their operational parameters differed significantly.

Comparative evaluation of commercial fluorescent treponemal antibody-absorbed test kits.

We describe a comparative evaluation of commercial fluorescent treponemal antibody-absorbed test kits, using 150 selected patient sera. The ability of the test kits to detect reactive sera varied from 82.5 to 95%; that for nonreactive sera varied from 80.9 to 96.4%. Reproducibility of reactive and nonreactive results, measured by between-assay and within-assay studies, averaged 42%. The results showed substantial variation in performance characteristics among the kits, with important clinical implications for the diagnosis of syphilis. We recommend the development of an immunological standard for use in the manufacture of fluorescent treponemal antibody-absorbed test kits, with the goal of obtaining uniform performance characteristics among commercial test kits.

Physician utilization of a gonococcal antibody screening test.

A gonococcal antibody test was introduced for use by the physician-clients of a private reference laboratory accompanied by distribution of literature regarding recommended use and interpretation of the test. The pattern of use of the tests was analyzed, and a telephone survey of clients was conducted to determine the manner of physician utilization of the test with regard to 69 patients with reactive test results. Despite recommendations to the contrary, the group surveyed used the test for males (22%) and symptomatic individuals (61%). Culture for Neisseria gonorrhoeae was not performed for 54% of patients with reactive tests for antibody to N. gonorrhoeae. Most physicians interviewed did not heed recommendations for the use of the test or correctly interpret test results.

Bacteremia due to Bifidobacterium, Eubacterium or Lactobacillus; twenty-one cases and review of the literature.

Twenty-one cases of bacteremia due to Bifidobacterium, Eubacterium and Lactobacillus are described. Transient bacteremic episodes with these organisms may follow trauma to the mouth, intestine, or vagina. The majority of the patients were female and most had an underlying condition that may have predisposed to bacteremia. Ten of the patients died despite antibiotic treatment.

Effect of dilution on recovery of bacteria from blood.

The multiplication rate of bacteria in undiluted blood containing sodium polyanethol sulfonate was compared with growth rates obtained in dilutions of blood ranging from 1:2 to 1:8. Although all organisms tested grew in the undiluted blood, increased growth rates were seen in the 1:2 dilution. Further dilution resulted in growth rates equivalent to that obtained with the 1:2 dilution. In view of these results, we question the present recommendations that blood be diluted 1:10 or 1:20.

Critical analysis of hypertonic medium and agitation in detection of bacteremia.

Over 18,000 clinical specimens collected in Vacutainer tubes with sodium polyanethol sulfonate were inoculated into modified Columbia broth (MCB) with and without 10% sucrose. The effects of venting and shaking on recovery were studied. The volume of the blood had a definite effect on the recovery rate. When inoculum size was held constant, recovery of aerobic and facultative organisms was maximal in vented and shaken bottles; the presence of sucrose had no demonstrable effect, recovery of anaerobes was maximal using an unvented bottle incubated under stationary conditions; a significantly greater recovery of facultatives and a marginally greater recovery of anaerobes was obtained with the hypertonic formulation. We conclude that a hypertonic formulation of MCB offers no advantage in the recovery of anaerobes but is of value in the recovery of facultatives and anaerobes. It is recommended that blood cultures be routinely inoculated into isotonic MCB and then vented and shaken for at least 4 hours, and hypertonic MCB incubated without venting or shaking.