Robert B. Jones

The use of tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes

The determination of the immunoreactivity of protein antigens in complex mixtures has been greatly facilitated by combining their separation via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with electrophoretic transfer to nitrocellulose membrane (NCM), and probing of bound proteins with specific antisera. Methods using various buffers and blocking agents have been published, but no studies have been published which compare these methods with each other or with others of potential merit. We have performed such a comparative study using protein antigens from Chlamydia trachomatis and Neisseria gonorrhoeae. In addition, we describe a method that blocks unoccupied protein binding sites on NCM with the nonionic detergent Tween 20, rather than proteins. This system proved to be equivalent or superior to other methods evaluated in the detection of immunoreactive proteins, and permitted staining of the NCM for protein after immunological probing. Such staining allowed precise identification of immunoreactive proteins. In addition, individual stained proteins could be excised and assessed for bound antibody in an indirect radioimmunoassay.

Multicenter Evaluation of the BDProbeTec ET System for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Urine Specimens, Female Endocervical Swabs, and Male Urethral Swabs

ABSTRACT The performance of the Becton Dickinson BDProbe Tec ET SystemChlamydia trachomatis and Neisseria gonorrhoeaeAmplified DNA Assays (BD Biosciences, Sparks, Md.) was evaluated in a multicenter study. Specimens were collected from 2,109 men and women, with or without symptoms, attending sexually transmitted disease, family planning, and obstetrics and gynecology clinics. Both swab and urine samples were collected, and the results obtained from 4,131 specimens were compared to those from culture and the LCx nucleic acid amplification test (Abbott Industries, Abbott Park, Ill.). PCR and cytospin of the culture transport medium with chlamydia direct fluorescent antibody staining were used to adjudicate chlamydia culture-negative results. Sensitivity and specificity were calculated both with and without use of the amplification control (AC), with little apparent difference in the results. Without the AC result, sensitivity for C. trachomatis and N. gonorrhoeae were 92.8 and 96.6%, respectively, for cervical swabs and 80.5 and 84.9% for urine from women. C. trachomatis and N. gonorrhoeae sensitivities were 92.5 and 98.5%, respectively, for male urethral swabs and 93.1 and 97.9% for urine from men. This amplified DNA system for simultaneous detection of chlamydial and gonococcal infections demonstrated superior sensitivity compared to chlamydia culture and has performance characteristics comparable to those of other commercially available nucleic acid-based assays for these organisms.

Polymorphisms in Chlamydia trachomatis tryptophan synthase genes differentiate between genital and ocular isolates

We previously reported that laboratory reference strains of Chlamydia trachomatis differing in infection organotropism correlated with inactivating mutations in the pathogens tryptophan synthase (trpBA) genes. Here, we have applied functional genomics to extend this work and find that the paradigm established for reference serovars also applies to clinical isolates - specifically, all ocular trachoma isolates tested have inactivating mutations in the synthase, whereas all genital isolates encode a functional enzyme. Moreover, functional enzyme activity was directly correlated to IFN-gamma resistance through an indole rescue mechanism. Hence, a strong selective pressure exists for genital strains to maintain a functional synthase capable of using indole for tryptophan biosynthesis. The fact that ocular serovars (serovar B) isolated from the genital tract were found to possess a functional synthase provided further persuasive evidence of this association. These results argue that there is an important host-parasite relationship between chlamydial genital strains and the human host that determines organotropism of infection and the pathophysiology of disease. We speculate that this relationship involves the production of indole by components of the vaginal microbial flora, allowing chlamydiae to escape IFN-gamma-mediated eradication and thus establish persistent infection.

Correlation between serum antichlamydial antibodies and tubal factor as a cause of infertility

Although salpingitis frequently produces tubal damage and infertility, many women with tubal factor as a cause of their infertility do not have a clinical history of salpingitis. In order to investigate whether or not some such cases might be due to subclinical chlamydial infections, we measured antichlamydial antibodies in the serum of 172 women consecutively undergoing evaluation for infertility. Only 16 (9.3%) had a prior history of salpingitis. Sixty-one (35%) had antichlamydial antibodies (S+), and of these 75% had tubal factor as a sole or contributing cause of their infertility, versus 28% of the seronegative (S-) women (x2 - 34, P less than 0.001). There was no association between chlamydial seropositivity and any infertility factor other than tubal factor in multivariant analyses. Subclinical infections with Chlamydia trachomatitis may be a major cause of tubal infertility in the United States, and chlamydial serologic studies may be useful in identifying the subset of infertile women likely to have tubal factor.

Recurrent genitourinary chlamydial infections in sexually active female adolescents

To determine the recurrence rate of chlamydial infections, we initially screened an urban population of 1308 sexually active female adolescents for chlamydial infection at the urethral and endocervical sites; these young women were followed and had additional examinations for infection. Chlamydial infection was documented by tissue culture in 31.1% (407) of them at some time during the study. After appropriate antibiotic treatment, 68.3% (278/407) returned for test-of-cure cultures within 3 months of their initial infection; of those 278, a total of 254 had sterile cultures. These patients were followed to determine the recurrence rate of chlamydial infections. Of these 254 patients, 177 (69.7%) had one or more follow-up visits; 38.4% (68/177) had a recurrent chlamydial infection. The majority of recurrent infections were documented within 9 months of the initial infection. Recurrent infections with the same serovar were frequent, suggesting reinfection by untreated partners or possible relapse of the initial chlamydial infection. This high rate of recurrent infection suggests that female adolescents should be rescreened frequently for genitourinary chlamydial infections.

Head-to-Head Multicenter Comparison of DNA Probe and Nucleic Acid Amplification Tests for Chlamydia trachomatis Infection in Women Performed with an Improved Reference Standard

ABSTRACT Few evaluations of tests for Chlamydia trachomatis have compared nucleic acid amplification tests (NAATs) with diagnostic tests other than those by culture. In a five-city study of 3,551 women, we compared the results of commercial ligase chain reaction (LCR) and PCR tests performed on cervical swabs and urine with the results of PACE 2 tests performed on cervical swabs, using independent reference standards that included both cervical swabs and urethral swab-urine specimens. Using cervical culture as a standard, the sensitivities of PACE 2, LCR, and PCR tests with cervical specimens were 78.1, 96.9, and 89.9%, respectively, and the specificities were 99.3, 97.5, and 98.2%, respectively. Using either cervical swab or urine LCR-positive tests as the standard decreased sensitivities to 60.8% for PACE 2 and to 75.8 and 74.9% for PCR with cervical swabs and urine, respectively. Specificities increased to 99.7% for PACE 2 and to 99.7 and 99.4% for PCR with cervical swabs and urine, respectively. Sensitivities with a cervical swab-urine PCR standard were 61.9% for PACE 2 and 85.5 and 80.8% for LCR with cervical swabs and urine, respectively. Specificities were 99.6% for PACE 2 and 99.0 and 98.9% for LCR with cervical swabs and urine, respectively. Cervical swab versus urine differences were significant only for PCR specificities (P = 0.034). Overall, LCR sensitivity exceeded that of PCR, and sensitivities obtained with cervical swabs exceeded those obtained with urine specimens by small amounts. These data have substantiated, using a large multicenter sample and a patient standard, that LCR and PCR tests performed on endocervical swabs and urine are superior to PACE 2 tests for screening C. trachomatis infections in women. In our study, NAATs improved the detection of infected women by 17 to 38% compared to PACE 2.

Population-Based Genetic and Evolutionary Analysis of Chlamydia trachomatis Urogenital Strain Variation in the United States

Chlamydia trachomatis is a major cause of ocular and sexually transmitted diseases worldwide. While much of our knowledge about its genetic diversity comes from serotyping or ompA genotyping, no quantitative assessment of genetic diversity within serotypes has been performed. To accomplish this, 507 urogenital samples from a multicenter U.S. study were analyzed by phylogenetic and statistical modeling. No B, Da, or I serotypes were represented. Based on our analyses, all but one previous urogenital B serotype was identified as Ba. This, coupled with the lack of B serotypes in our population, suggests that B has specific tropism for ocular mucosa. We identified a Ba/D recombinant (putative crossover nucleotide 477; P < 0.0001) similar to a B/D mosaic we described previously from an African trachoma patient. Computational analyses of the Ba/D recombinant indicated that upstream changes were less important for tissue tropism than downstream incorporation of the D sequence. Since most serotypes had nonsynonymous/synonymous ratios of <1.0, the major outer membrane protein, encoded by ompA, has many functional constraints and is under purifying selection. Surprisingly, all serotype groups except for J had a unimodal population structure indicating rapid clonal expansion. Of the groups with a unimodal structure, E and Ia and, to a lesser extent, G and K were prevalent, had infrequent incorporation of mutations, and, compared to other groups, had a relatively greater degree of diversifying selection, consistent with a selective sweep of mutations within these groups. Collectively, these data suggest a diverse evolutionary strategy for different serogroups of the organism.

Chlamydia trachomatis in the pharynx and rectum of heterosexual patients at risk for genital infection

Although urogenital infections with Chlamydia trachomatis are well recognized, less is known about infection at other body sites in adults. Pharyngeal specimens obtained from 706 heterosexual men and 686 women, and rectal specimens obtained from 1223 women who were at risk for chlamydia infection were cultured for C. trachomatis. Urogenital specimens were obtained from all patients. Chlamydia trachomatis was isolated from the pharynx in 3.7% of men and 3.2% of women. Recovery of chlamydiae was not associated with the presence of pharyngeal symptoms, but in women, but not men, it was associated with a history of oral-genital sex. The organism was also recovered from the rectum of 5.2% of the women. Rectal isolation did not correlate with a history of rectal symptoms or rectal sex but did correlate with concurrent genital infection. Infection at these sites may be important in the transmission or persistence of C. trachomatis infections.

Efficiency and cost-effectiveness of field follow-up for patients with Chlamydia trachomatis infection in a sexually transmitted diseases clinic.

Unlike contact-tracing procedures for syphilis and gonorrhea, field follow-up to locate and treat patients with Chlamydia trachomatis infections has not been extensively applied in the United States. We implemented two studies to assess the efficiency and cost-effectiveness of using field follow-up for contact of two groups: patients with chlamydial infection detected as part of a screening program and women who were sexual partners of men with nongonococcal urethritis (NGU). Of the 142 patients with chlamydial infection who had not been treated empirically, 112 (79%) returned for treatment when a reminder system was used, as compared with a return rate of 97% (259/266) achieved by field follow-up (P less than 0.0001). Among the 678 men with NGU enrolled in a randomized trial of field follow-up vs. two self-referral methods, field follow-up yielded over three times as many partners returning to the clinic for treatment as did either of the other two methods (P less than 0.001). Analyses using the estimated costs of the intervention strategies and the medical costs associated with an untreated chlamydial infection showed that field follow-up by trained investigators proved to be not only the most efficient method for locating patients with chlamydial infection and/or patients who were at risk for it, but also the most cost-effective in terms of total health-care dollars spent.

Effect of prior sexually transmitted disease on the isolation of Chlamydia trachomatis

In developed nations, Chlamydia trachomatis is the most common sexually transmitted pathogen. To determine whether prior disease affects the probability of subsequent chlamydial infection, we took culture specimens from 2,546 men and 1,998 women attending a sexually transmitted diseases clinic. The men had nongonococcal urethritis and the women were contacts of men who had a positive chlamydial culture or nongonococcal urethritis. Significantly lower isolation rates for those with a history of sexually transmitted diseases were found for both men (29% vs. 38%; P less than 0.0001) and women (27% vs. 36%; P less than 0.0001). In addition, both men and women with previously documented chlamydial infections had a lower isolation rate at the index visit, if the previous infection occurred less than, as opposed to more than, six months earlier (men: 20% vs. 41%; P = 0.0006; women: 14% vs. 35%; P = 0.003). These relationships were found to be independent of age. However, the effect of partial immunity due to prior infection could not be distinguished from that of prior antibiotic therapy, and if such immunity does confer protection against reinfection, that protection appears to be both partial and of relatively short duration.