g-Yen Chen

New Tests for Syphilis: Rational Design of a PCR Method for Detection of Treponema pallidum in Clinical Specimens Using Unique Regions of the DNA Polymerase I Gene

ABSTRACT A sensitive and specific PCR method to detectTreponema pallidum in clinical specimens was developed. PCR primers were designed based on two unique features of the DNA polymerase I gene (polA). The first distinctive characteristic is that the region codes for a high cysteine content and has low homology with similar regions of DNA polymerase I gene from known microorganisms. The second unique feature is the presence of four insertions in the gene. PCR tests using primers designed on the basis these regions reacted with various pathogenic T. pallidum subspecies but did not react with nonpathogenic treponemal species or other spirochetes. An additional 59 species of bacteria and viruses, including those that cause genital ulcers, tested negative. This PCR method is extremely robust and sensitive. The detection limit is about 10 to 25 organisms when analyzed on gel. However, the analytic sensitivity can be increased by at least 1 log, to a detection limit of a single organism, when the ABI 310 Prism Genetic Analyzer is used to detect fluorescence-labeled amplicons. We further used this test in a clinical setting and compared the results with results from a previously reported multiplex-PCR test (forT. pallidum, Haemophilus ducreyi, and herpes simplex virus). We tested 112 genital ulcer specimens by the polAPCR, obtaining a sensitivity of 95.8% and a specificity of 95.7%. These results suggest that the polA PCR is applicable as a routine clinical diagnostic test for syphilis.

The ferric iron‐binding protein of pathogenic Neisseria spp. functions as a periplasmic transport protein in iron acquisition from human transferrin

The ferric iron‐binding protein (Fbp) expressed by pathogenic Neisseria spp. has been proposed to play a central role in the high‐affinity acquisition of iron from human transferrin. The results of this investigation provide evidence that Fbp participates in this process as a functional analogue of a Gram‐negative periplasmic‐binding protein component, which operates as a part of a general active transport process for the receptor‐mediated, high‐affinity transport of iron from human transferrin. Known properties of Fbp are correlated with those of other well‐characterized periplasmic‐binding proteins, including structural features and the reversible binding of ligand. Predictive of a periplasmic‐binding protein, which functions in the high‐affinity acquisition of iron, is that Fbp is a transient participant in the process of iron acquisition from human transferrin. Evidence for this is demonstrated by results of pulse–chase experiments. Taken together, the data described here and elsewhere suggest that pathogenic Neisseria spp. use a periplasmic‐binding protein‐mediated active transport mechanism for the acquisition of iron from human transferrin.

Molecular Typing of Treponema pallidum in South Africa: Cross-Sectional Studies

ABSTRACT We evaluated a molecular subtyping system for Treponema pallidum for its ability to differentiate between strains obtained from male patients with primary syphilis in South Africa. Of 201 T. pallidum-positive specimens, 161 were typeable, revealing 35 subtypes. The unique subtypes identified in Durban, Cape Town, and Carletonville and the total number of subtypes suggested that the strain population was very diverse and varied geographically.

The molecular diagnosis of lymphogranuloma venereum: evaluation of a real-time multiplex polymerase chain reaction test using rectal and urethral specimens.

Objectives: The objectives of this study were to evaluate the use of a real-time multiplex polymerase chain reaction (M-PCR) assay to differentiate between trachoma and lymphogranuloma venereum (LGV) biovars of Chlamydia trachomatis and to validate its performance with the conventional genotyping method. Study: Swab specimens from 115 patients with anorectal symptoms or syndromes associated with LGV were tested by a real-time M-PCR assay and the results compared with the PCR-based restriction fragment length polymorphism analysis of the major outer membrane protein gene (omp1). Results: A high agreement of 96.5% (111 of 115 specimens) was found between the real-time M-PCR testing and the standard genotyping method for the detection of C. trachomatis DNA (&kgr; value, 0.945, P <0.00001). Both methods identified 53 LGV, 32 non-LGV C. trachomatis, and 26 negative specimens. Conclusions: The real-time M-PCR assay simultaneously detects and differentiates LGV from non-LGV strains using swab specimens. This assay offers a relatively rapid and sensitive alternative for the diagnosis of LGV infection and is a useful tool for screening and for outbreak investigations.

Diagnosis of Gastric Syphilis by Direct Immunofluorescence Staining and Real-Time PCR Testing

ABSTRACT We report on a case of gastric syphilis in a patient with chronic dyspepsia. The diagnosis was established by serology and the demonstration of spirochetes in diffusely inflammed gastric mucosa by staining with a fluorescent monoclonal antibody specific for pathogenic treponemes and by the detection of specific treponemal DNA sequences by a real-time PCR.

A real-time quadriplex PCR assay for the diagnosis of rectal lymphogranuloma venereum and non-lymphogranuloma venereum Chlamydia trachomatis infections.

Objectives: To develop and evaluate a real-time quadriplex PCR for the diagnosis of lymphogranuloma venereum (LGV) and non-LGV chlamydial infections using rectal swab specimens. Methods: The design of the real-time quadriplex PCR assay incorporates an LGV-specific, a non-LGV-specific target sequence, a Chlamydia trachomatis plasmid target, and the human RNase P gene as an internal control. The performance of the quadriplex PCR was compared with a previously reported real-time duplex PCR assay on which LGV diagnosis was based on exclusion. Results: Very good agreement (85 of 89 specimens, 95.5%) was found between the two multiplex PCR assays for the detection of C trachomatis DNA (kappa value 0.93, 95% CI 0.86 to 0.99). Both assays identified 34 LGV, 35 non-LGV C trachomatis and 16 negative specimens. Of two specimens that tested positive for non-LGV by the duplex PCR, one was found to be a mixed infection and the other was positive only for plasmid and RNase P targets by the quadriplex PCR. Two additional specimens that had equivocal results for non-LGV by the duplex PCR also tested positive only for plasmid target and human DNA by the quadriplex PCR. In addition, six specimens that tested negative by the duplex PCR assay were found to be invalid when using the quadriplex PCR. Conclusions: A real-time quadriplex PCR assay has been developed that is capable of detecting LGV, non-LGV, or mixed infections simultaneously in rectal specimens. The assay also contains a supplemental amplification target for the confirmation of C trachomatis infection as well as a human DNA control for monitoring sample adequacy and PCR inhibition.

Detection of the A2058G and A2059G 23S rRNA Gene Point Mutations Associated with Azithromycin Resistance in Treponema pallidum by Use of a TaqMan Real-Time Multiplex PCR Assay

ABSTRACT Macrolide treatment failure in syphilis patients is associated with a single point mutation (either A2058G or A2059G) in both copies of the 23S rRNA gene in Treponema pallidum strains. The conventional method for the detection of both point mutations uses nested PCR combined with restriction enzyme digestions, which is laborious and time-consuming. We initially developed a TaqMan-based real-time duplex PCR assay for detection of the A2058G mutation, and upon discovery of the A2059G mutation, we modified the assay into a triplex format to simultaneously detect both mutations. The point mutations detected by the real-time triplex PCR were confirmed by pyrosequencing. A total of 129 specimens PCR positive for T. pallidum that were obtained from an azithromycin resistance surveillance study conducted in the United States were analyzed. Sixty-six (51.2%) of the 129 samples with the A2058G mutation were identified by both real-time PCR assays. Of the remaining 63 samples that were identified as having a macrolide-susceptible genotype by the duplex PCR assay, 17 (27%) were found to contain the A2059G mutation by the triplex PCR. The proportions of macrolide-susceptible versus -resistant genotypes harboring either the A2058G or the A2059G mutation among the T. pallidum strains were 35.6, 51.2, and 13.2%, respectively. None of the T. pallidum strains examined had both point mutations. The TaqMan-based real-time triplex PCR assay offers an alternative to conventional nested PCR and restriction fragment length polymorphism analyses for the rapid detection of both point mutations associated with macrolide resistance in T. pallidum.

A cross-sectional study of 'yaws' in districts of Ghana which have previously undertaken azithromycin mass drug administration for trachoma control.

Yaws, caused by Treponema pallidum ssp. pertenue, is reportedly endemic in Ghana. Mass distribution of azithromycin is now the cornerstone of the WHO yaws eradication campaign. Mass distribution of azithromycin at a lower target dose was previously undertaken in two regions of Ghana for the control of trachoma. Ongoing reporting of yaws raises the possibility that resistance may have emerged in T. pallidum pertenue, or that alternative infections may be responsible for some of the reported cases. We conducted a cross-sectional survey in thirty communities in two districts of Ghana where MDA for trachoma had previously been conducted. Children aged 5–17 years with ulcerative lesions compatible with yaws were enrolled. Samples for treponemal serology and lesion PCR were collected from all children. 90 children with 98 lesions were enrolled. Syphilis serology was negative in all of them. PCR for T. pallidum ssp pertenue was negative in all children, but Haemophilus ducreyi DNA was detected in 9 lesions. In these communities, previously treated for trachoma, we found no evidence of ongoing transmission of yaws. H. ducreyi was associated with a proportion of skin lesions, but the majority of lesions remain unexplained. Integration of diagnostic testing into both pre and post-MDA surveillance systems is required to better inform yaws control programmes.

A Community-based study of risk factors for Trichomonas vaginalis infection among women and their male partners in Moshi urban district, northern Tanzania.

Objective: The objective of this study was to determine predictors of Trichomonas vaginalis among women and their partners in Moshi, Tanzania. Study Design: Women (N = 1440) and their partners (N = 588) were interviewed and specimens for detection of T. vaginalis and sexually transmitted infections (STIs) were collected. Results: Prevalence of T. vaginalis was 10.7% in women and 6.3% in men. Having a partner with T. vaginalis was the strongest risk factor in women (adjusted odds ratio [OR], 19.44; 95% confidence interval [CI], 7.84–48.25) and men (adjusted OR, 19.01; 95% CI, 6.8–52.40). Risk of T. vaginalis infection was increased in subjects with less education. Other risk factors in women were daily alcohol consumption, being separated, reporting infertility problems, having a partner who had children with other women, and other STIs; and in men, the risk factor was having no income. T. vaginalis was not associated with HIV-1 in women and men. Conclusions: Prevention of T. vaginalis and other STIs among couples is a major priority. Reduction of alcohol consumption in women is an important intervention.

Expression of a functional neisserial fbp gene in Escherichia coli

The ability to acquire iron from a human host is a major determinant in the pathogenesis of Neisseria gonorrhoeae and Neisseria meningitidis. Pathogenic Neisseria spp. do not synthesize siderophores and instead express a receptor‐mediated, high‐affinity iron acquisition system in the iron‐restricted environment of its host. A ferric‐iron‐binding protein (Fbp) of Neisseria spp. is also iron‐regulated and may play a central role in this novel iron‐uptake system. To define the physical properties of Fbp further, we used polymerase chain reaction to synthesize DNA fragments containing the fbp structural gene with and without the sequence encoding the Fbp leader peptide. These fragments were ligated into pUC13 to create in‐frame fusions with the alpha peptide of lacZ. The expression of Fbp was under the control of the lacZ promoter. Both fusion clones produced Fbp in large amounts, facilitating the purification of quantities of Fbp sufficient for elucidating the biochemical, immunologic, and functional properties of this protein.