Multisite pooling study using ligase chain reaction in screening for genital Chlamydia trachomatis infections.

BACKGROUND: Ligase chain reaction (LCR), a nucleic acid amplification assay, is a highly specific and sensitive test for detecting Chlamydia trachomatis in cervical and urethral swabs as well as first-void urine specimens. GOAL: To examine the suitability of using the LCR test to detect C trachomatis in pooled cervical specimens. STUDY DESIGN: The performance of LCR in pooled specimens was compared with individual specimen testing at six laboratories using 3,170 cervical swab specimens randomly selected from specimens received for routine testing in the participating laboratories. These samples then were combined consecutively into 634 pools of 5 specimens and 317 pools of 10 specimens. A reduced sample to cutoff ratio of 0.2 or more was used for the pooled specimens. RESULTS: Of the 188 positive specimens (98.9%), 186 were identified when single specimens were analyzed. When pools of 5 or 10 specimens were evaluated, 99.5% and 98.9% of the positive swabs, respectively, were identified correctly. Two positive specimens were detected only through pooling. CONCLUSIONS: Pooling samples for detection of C trachomatis by LCR is sensitive and specific. Depending on the prevalence of infection (positivity), LCR testing may result in cost savings, as compared with individual testing of specimens.

Invasive serotype a Haemophilus influenzae infections with a virulence genotype resembling Haemophilus influenzae type b: emerging pathogen in the vaccine era?

OBJECTIVE: Haemophilus influenzae type b causes severe disease in nonimmune infants and young children; other serotypes are uncommon pathogens and thought to have low virulence. Some have hypothesized that with the virtual elimination of H influenzae type b, other serotypes might acquire virulence traits and emerge as important pathogens of children. We describe the clinical, epidemiologic, and molecular biologic features of 5 cases of severe disease attributable to Haemophilus influenzae type a. METHODS: After observing 4 cases of invasive disease caused by H influenzae type a, we reviewed microbiology records at 3 reference laboratories that perform all serotyping in Utah and surveillance databases. Strains of H influenzae type a and control strains were examined by Southern blotting with the use of the cap probe pUO38 and by pulsed-field gel electrophoresis. The putative virulence mutation, the IS1016-bexA deletion, was detected by polymerase chain reaction amplification and sequencing. RESULTS: During a 10-month period, we observed 5 children with severe invasive disease caused by H influenzae type a. No isolates of H influenzae type a had been submitted to the reference laboratories between 1992 and 1998. The median age of patients was 12 months (range: 6-48 months). Four of 5 had meningitis and bacteremia; 1 had purpura fulminans. Three isolates, representing 1 of 2 pulsed-field gel electrophoresis patterns, contained the IS1016-bexA deletion and were associated with particularly severe disease. CONCLUSIONS: We describe an unusual cluster of severe disease caused by H influenzae type a that resembles the clinical and epidemiologic features of H influenzae type b disease. Our data support the hypothesis that the IS1016-bexA deletion may identify more virulent strains of H influenzae. Haemophilus influenzae, epidemiology, virulence, serotyping, pathogenicity.

The pandemic of Salmonella enteritidis phage type 4 reaches Utah: a complex investigation confirms the need for continuing rigorous control measures.

In 1995, Salmonella Enteritidis (SE) cases in the state of Utah increased fivefold. Isolates were identified as phage type 4 (PT4). Risk factors and sources of infection were investigated in two case-control studies, a traceback of implicated foods, and environmental testing. Forty-three patients with sporadic infections and 86 controls were included in a case-control study of risk factors for infection. A follow-up case-control study of 25 case and 19 control restaurants patronized by case and control patients examined risks associated with restaurant practices. In the first case-control study, restaurant dining was associated with illness (P = 0.002). In the follow-up case-control study, case restaurants were likelier to use > 2000 eggs per week (P < 0.02), to pool eggs (P < 0.05), and to use eggs from cooperative 'A' (P < 0.009). Eggs implicated in separately investigated SE PT4 outbreaks were traced to cooperative 'A', and SE PT4 was cultured from one of the cooperative's five local farms. We conclude that SE PT4 transmitted by infected eggs from a single farm caused a fivefold increase in human infections in Utah.

Rapid detection of Campylobacter jejuni in stool specimens by an enzyme immunoassay and surveillance for Campylobacter upsaliensis in the greater Salt Lake City area.

The Alexon-Trend, Inc. (Ramsey, Minn.), ProSpecT Campylobacter microplate assay was compared with culture on a Campy-CVA plate (Remel, Lenexa, Kans.) and blood-free campylobacter agar with cefoperazone (20 microg/ml), amphotericin B (10 microg/ml), and teicoplanin (4 microg/ml) (CAT medium; Oxoid Limited, Hampshire, England) with 631 patient stool samples. The CAT medium was used to isolate Campylobacter upsaliensis. The enzyme immunoassay (EIA) had a sensitivity and a specificity of 89 and 99%, respectively, and the positive and negative predictive values were 80 and 99%, respectively. Even though we extensively looked for C. upsaliensis in stool samples from patients from the greater Salt Lake City area, we did not isolate this species during the study period. The overall excellent specificity of the EIA allows rapid detection and treatment of positive patients; however, a negative result should be confirmed by culture when clinical suspicion is high.

Comparison of sorbitol MacConkey agar and a two-step method which utilizes enzyme-linked immunosorbent assay toxin testing and a chromogenic agar to detect and isolate enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) and specifically serotype O157:H7 are a significant cause of hemorrhagic gastrointestinal disease and the hemolytic uremic syndrome. Methods currently used in clinical microbiology labs, such as sorbitol-MacConkey (SMAC) agar, reliably detect only O157:H7. We have evaluated a two-step method that has the potential to identify and isolate all EHEC serotypes, including serotype O157:H7. This method utilizes a chromogenic selective-differential medium for the isolation of E. coli together with an enzyme-linked immunosorbent assay (ELISA) that detects the Shiga-like toxins Stx1 and Stx2. Both are commercially available and usable in a wide range of clinical microbiology laboratories. Compared to a Vero cell cytotoxic assay, SMAC had sensitivities of 23.5% for the identification of all EHEC serotypes and of 50.0% for the identification of O157:H7 alone. The two-step method had sensitivities of 76.5 and 100%, respectively. The ELISA alone had a sensitivity of 82.4% in the detection of Stx1 and Stx2. The specificity was 100% in all cases. Overall, 14 EHEC isolates were obtained: 8 (58%) O157:H7, 2 (14%) O26, 2 (14%) O111:NM, 1 (7%) O103:H2, and 1 (7%) O121:H19. All but one were isolated during the months of May to September. The two-step method was found to be considerably more expensive than SMAC for both positive and negative samples.

Persistently negative HIV-1 antibody enzyme immunoassay screening results for patients with HIV-1 infection and AIDS: serologic, clinical, and virologic results. Seronegative AIDS Clinical Study Group.

OBJECTIVE: To describe persons with HIV infection and AIDS but with persistently negative HIV antibody enzyme immunoassay (EIA) results. DESIGN: Surveillance for persons meeting a case definition for HIV-1-seronegative AIDS. SETTING: United States and Canada. PATIENTS: A total of eight patients with seronegative AIDS identified from July 1995 through September 1997. MAIN OUTCOME MEASURES: Clinical history of HIV disease, history of HIV test results, and CD4 cell counts from medical record review; results of testing with a panel of EIA for antibodies to HIV-1, and HIV-1 p24 antigen; and viral subtype. RESULTS: Negative HIV EIA results occurred at CD4 cell counts of 0-230 x 10(6)/l, and at HIV RNA concentrations of 105,000-7,943,000 copies/ml. Using a panel of HIV EIA on sera from three patients, none of the HIV EIA detected infection with HIV-1, and signal-to-cut-off ratios were < or = 0.8 or all test kits evaluated. Sera from five patients showed weak reactivity in some HIV EIA, but were non-reactive in other HIV EIA. All patients were infected with HIV-1 subtype B. CONCLUSIONS: Rarely, results of EIA tests for antibodies to HIV-1 may be persistently negative in some HIV-1 subtype B-infected persons with AIDS. Physicians treating patients with illnesses or CD4 cell counts suggestive of HIV infection, but for whom results of HIV EIA are negative, should consider p24 antigen, nucleic acid amplification, or viral culture testing to document the presence of HIV.

Evaluation of the Abbott LCx ligase chain reaction assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine and genital swab specimens from a sexually transmitted disease clinic population.

The Abbott LCx ligase chain reaction (LCR) assay for the simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae was evaluated by using swab and urine specimens from 562 patients. C. trachomatis results by LCR were compared to those by the Gen-Probe PACE 2 assay, whereas N. gonorrhoeae results by LCR were compared to those by culture. The Gen-Probe and LCR assays were performed according to the manufacturers' instructions. Gram-negative diplococci growing on modified Thayer-Martin medium were confirmed as N. gonorrhoeae by the GonoGen II assay. Supplemental data analysis was performed by major outer membrane protein PCR for C. trachomatis and probes for pilin gene detection for N. gonorrhoeae. A true-positive result for each pathogen was defined as a positive result for all three or two of three assays. Overall agreement among the six assays was 94.8%. C. trachomatis prevalence was 16.2%; N. gonorrhoeae prevalence was 5.5%. The overall sensitivity and specificity, respectively, for each test (after supplemental data analysis) were as follows: for C. trachomatis, Gen-Probe, 65.9 and 100%; LCR on urine, 90.1 and 100%; LCR on swab specimens, 96.7 and 100%; and for N. gonorrhoeae, culture, 80.6 and 100%; LCR on urine, 93.5 and 99.8%; and LCR on swab specimens, 96.8 and 100%. For women, the N. gonorrhoeae culture was very insensitive compared to its performance in men (58.3 versus 94.7%, respectively). For C. trachomatis, the Gen-Probe assay's sensitivity was lower for men than for women (62.3 versus 71.1%, respectively). The sensitivity for C. trachomatis detection by LCR on urethral and cervical swab specimens was 96.2 and 97.4% for men and women, respectively. For men, swab results were slightly better than urine results for both pathogens (sensitivity for C. trachomatis in swab and urine specimens, 96.2 and 92.5%, respectively; sensitivity for N. gonorrhoeae in swab and urine specimens, 100 and 94.7%, respectively), while for women, cervical swabs were superior in sensitivity to urine samples for detecting C. trachomatis (swab, 97.4%; urine, 81.6%) and equivalent for N. gonorrhoeae (swab, 92.3%; urine, 91.6%). The LCx LCR appears to be both sensitive and specific for the detection of C. trachomatis and N. gonorrhoeae when performed on urine or genital swab samples. Swab samples had better sensitivity than urine samples for the detection of both pathogens.

Absence of detectable antibody in a patient infected with human immunodeficiency virus.

Infection with human immunodeficiency virus (HIV) is routinely and easily diagnosed with use of enzyme immunoassay (EIA) test kits. We describe an unusual patient who developed AIDS despite testing negative for antibodies to HIV 35 times over a 4-year period. HIV infection was confirmed by the results of p24-antigen assays and polymerase chain reaction amplification of proviral DNA. Sequence analysis of the virus demonstrated that it was closely related to a strain obtained from the patient's sexual partner. The explanation for this patient's persistently negative EIA results is unclear. However, this case does suggest that physicians who treat patients with AIDS-defining conditions but for whom standard HIV antibody testing is negative should consider the possibility that HIV infection is present and may be identified by additional testing procedures.

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.

Comparison of membrane filters for recovery of legionellae from water samples.

The procedure currently used for isolating legionellae from environmental samples recommend filtration through a 0.2-microns-pore-size polycarbonate filter. In this study we evaluated the performance of 23 other filters composed of various materials and having various pore sizes. We prefer the 0.2-micron-pore-size Gelman Supor filter because of its high level of recovery, faster filtration rate, and ease of handling.

Hemolytic-uremic syndrome during an outbreak of Escherichia coli O157:H7 infections in institutions for mentally retarded persons: clinical and epidemiologic observations.

PURPOSE: To describe an outbreak of Escherichia coli O175:H7 infection resulting in a high rate of progression to hemolytic-uremic syndrome, and to attempt to identify predictors of and risk factors for progression. DESIGN: Case-control study among employees and comparison of daily clinical features in two groups: infected residents with subsequent development of HUS and those who had no complications. SETTING: Two institutions for retarded persons in Utah. PATIENTS: Twenty residents with E. coli O157:H7 infection (13 culture confirmed, 2 probable, and 5 possible); HUS developed in 8, and 4 died. Thirty-one infected employees (3 with culture-confirmed, 6 with probable, and 22 with possible infection). MEASUREMENTS AND MAIN RESULTS: In a case-control study among employees, infection was independently associated with eating ground beef from a single lot prepared at several barbecues and with close contact with a resident who had diarrhea. Five of eight residents in whom HUS developed had received trimethoprim-sulfamethoxazole, compared with none of seven who had no subsequent complications (p = 0.026); this finding may reflect antimicrobial treatment of patients with more severe illness. Compared with infected residents without complications, persons with HUS were younger (median age 13 vs 27 years, p = 0.043) and, by the third day of illness, had higher leukocyte counts (median 23.7 X 10(9)/L vs 9.1 X 10(9)/L, p = 0.018) and temperature (median 38.5 degrees C vs 37.0 degrees C, p = 0.016). Leukocytosis peaked on day 4, more than 24 hours before signs of HUS appeared. CONCLUSIONS: Food-borne outbreaks of E. coli O157:H7 in institutions may have devastating effects. Leukocytosis and fever may precede and predict HUS in patients with E. coli O157:H7 infection.

Survey on testing criteria and reporting methods for human immunodeficiency virus serologic tests in Veterans Administration Medical Centers.

Guidelines for the indications for use, requirements for consent, and mechanisms for reporting of serologic tests for human immunodeficiency virus (HIV) infection are not standardized. In trying to establish such guidelines for our hospital, we surveyed all Veterans Administration Medical Centers regarding their current approach to testing both patients and employees. Infection control practitioners from 67 hospitals representing 37 states responded. Patients are likely to be tested for diverse reasons, unlikely to be counseled about the test or be required to consent to it, and test results are given no special precautions. Although 66% of the respondents do not use any extra precautions concerning patient confidentiality, 80% utilize more stringent criteria for testing and result-reporting with employees than patients. Thus, while the majority of hospitals maintain that current modes of confidentiality are acceptable for patients, practice suggests that these modes are considered inadequate for employees.