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ObjectiveTo evaluate the field performance of a multiplex PCR (M-PCR) assay for detection of herpes simplex virus (HSV)-1 and HSV-2, Treponema pallidum (T. pallidum) and Haemophilus ducreyi (H. ducreyi) in genital ulcer disease (GUD) specimens.MethodsGUD M-PCR was performed on 186 remnant specimens, previously collected for HSV testing, by four public health laboratories (PHLs) and the Laboratory Reference and Research Branch (LRRB) at the Centers for Disease Control and Prevention. The results from the PHLs were compared with those of LRRB, which served as the reference testing method, and percentage agreement was calculated.ResultsHSV was detected in 31 of 52 (59.6%), 20 of 40 (50%), 43 of 44 (97.7%) and 19 of 50 (38.0%) specimens from PHL1, PHL2, PHL3 and PHL4, respectively. There were seven discrepant results for HSV, and the overall percent agreement between the PHLs and the LRRB was 94%–100%, with a kappa value of 0.922, which demonstrates high agreement. T. pallidum was identified in 7 of 51 (13.7%) specimens from PHL1 with 94.1% agreement and in 2 of 40 (5.0%) specimens from PHL2 with 100% agreement. The LRRB identified three additional T. pallidum-positive specimens from PHL1. The kappa value (0.849) for T. pallidum testing suggests good agreement. Consistent with the LRRB results, no T. pallidum was detected in specimens from PHL3 and PHL4, and H. ducreyi was not detected at any of the study sites.ConclusionsThe GUD M-PCR assay performed well in four independent PHLs and 12 suspected syphilis cases were identified in this study. The M-PCR assay could provide improved diagnostic options for GUD infections in state and local PHLs.

A guanine mononucleotide repeat in the rpsA (tp0279) gene was evaluated for improved strain discrimination using 72 Treponema pallidum-positive specimens. The tandem repeat combined with the enhanced Centers for Disease Control and Prevention typing system resulted in increased discrimination and should be useful for molecular epidemiologic studies on syphilis especially in outbreaks and among men who have sex with men.

Introduction The WHO yaws eradication strategy consists of one round of total community treatment (TCT) of single-dose azithromycin with coverage of > 90%.The efficacy of the strategy to reduce the levels on infection has been demonstrated previously in isolated island communities in the Pacific region. We aimed to determine the efficacy of a single round of TCT with azithromycin to achieve a decrease in yaws prevalence in communities that are endemic for yaws and surrounded by other yaws-endemic areas. Methods Surveys for yaws seroprevalence and prevalence of skin lesions were conducted among schoolchildren aged 5–15 years before and one year after the TCT intervention in the Abamkrom sub-district of Ghana. We used a cluster design with the schools as the primary sampling unit. Among 20 eligible primary schools in the sub district, 10 were assigned to the baseline survey and 10 to the post-TCT survey. The field teams conducted a physical examination for skin lesions and a dual point-of-care immunoassay for non-treponemal and treponemal antibodies of all children present at the time of the visit. We also undertook surveys with non-probabilistic sampling to collect lesion swabs for etiology and macrolide resistance assessment. Results At baseline 14,548 (89%) of 16,287 population in the sub-district received treatment during TCT. Following one round of TCT, the prevalence of dual seropositivity among all children decreased from 10.9% (103/943) pre-TCT to 2.2% (27/1211) post-TCT (OR 0.19; 95%CI 0.09–0.37). The prevalence of serologically confirmed skin lesions consistent with active yaws was reduced from 5.7% (54/943) pre-TCT to 0.6% (7/1211) post-TCT (OR 0.10; 95% CI 0.25–0.35). No evidence of resistance to macrolides against Treponema pallidum subsp. pertenue was seen. Discussion A single round of high coverage TCT with azithromycin in a yaws affected sub-district adjoining other endemic areas is effective in reducing the prevalence of seropositive children and the prevalence of early skin lesions consistent with yaws one year following the intervention. These results suggest that national yaws eradication programmes may plan the gradual expansion of mass treatment interventions without high short-term risk of reintroduction of infection from contiguous untreated endemic areas., Author summary In this study, we provided a single round of total community treatment (TCT) with azithromycin to the population of a sub-district in Ghana (16,287 people) that is endemic for yaws and surrounded by other yaws-endemic communities to determine whether a sustained decrease in yaws prevalence could be achieved up to one year after the intervention. The efficacy of TCT was assessed by performing a clinical evaluation and serological testing of any yaws-like lesions found as well as serological screening of asymptomatic schoolchildren aged 5–15 years pre-TCT and at one year post-TCT. The results indicate that after a single round of high coverage TCT (89%) with azithromycin, the prevalence of active and latent yaws was significantly reduced. We also found that the use of a dual point-of-care immunoassay to detect non-treponemal and treponemal antibodies among school-going children is a practical alternative to laboratory-based serological testing to evaluate the effectiveness of yaws interventions in resource-poor settings.

Infections are at their lowest level worldwide, but nongenital cutaneous infections have increased., The global epidemiology of Haemophilus ducreyi infections is poorly documented because of difficulties in confirming microbiological diagnoses. We evaluated published data on the proportion of genital and nongenital skin ulcers caused by H. ducreyi before and after introduction of syndromic management for genital ulcer disease (GUD). Before 2000, the proportion of GUD caused by H. ducreyi ranged from 0.0% to 69.0% (35 studies in 25 countries). After 2000, the proportion ranged from 0.0% to 15.0% (14 studies in 13 countries). In contrast, H. ducreyi has been recently identified as a causative agent of skin ulcers in children in the tropical regions; proportions ranged from 9.0% to 60.0% (6 studies in 4 countries). We conclude that, although there has been a sustained reduction in the proportion of GUD caused by H. ducreyi, this bacterium is increasingly recognized as a major cause of nongenital cutaneous ulcers.

Background Sustained genital tract inflammation caused by sexually transmitted infections (STIs) is known to increase risk of vaginal human immunodeficiency virus (HIV) infections but, to our knowledge, there are no nonhuman primate studies that have evaluated its link to rectal HIV acquisition. Methods Rhesus macaques inoculated with Chlamydia trachomatis (CT) (serovars LGV-L2 and CT-E; n = 7) or saline (n = 7) received up to 20 rectal challenges twice a week of simian/HIV immunodeficiency virus (SHIVSF162p3). SHIV viremia was determined by real-time PCR and Chlamydia infection by APTIMA Combo 2 testing. The rectal cytokine-chemokine levels were evaluated by multiplex bead assays. Results Rectal Chlamydia infection was maintained throughout the study. We did not observe significant differences (P = 1.0) in frequency of SHIV acquisition between the STI and control arms. It took fewer SHIV challenges to infect the STI animals although the difference was not significant (P = 0.59). There were no significant differences in peak plasma viremia between STI and control arms (P = 0.63). The association of plasma viremia with rectal shedding was significantly different by arm (P = 0.038). Conclusions In the first such study in a macaque model, we did not observe an increased risk of SHIV acquisition due to rectal Chlamydia coinfection. This macaque model can be further developed and expanded to better investigate the impact of different rectal STIs on HIV acquisition.

Clinical isolates of Treponema pallidum subspecies pallidum (T. pallidum) would facilitate study of prevalent strains. We describe the first successful rabbit propagation of T. pallidum from cryopreserved ulcer specimens. Fresh ulcer exudates were collected and cryopreserved with consent from syphilis-diagnosed patients (N = 8). Each of eight age-matched adult male rabbits were later inoculated with a thawed specimen, with two rabbits receiving 1.3 ml intratesticularly (IT), and six receiving 0.6 ml intravenously (IV) and IT. Monitoring of serology, blood PCR and orchitis showed that T. pallidum grew in 2/8 rabbits that were inoculated IV and IT with either a penile primary lesion specimen (CDC-SF003) or a perianal secondary lesion specimen (CDC-SF007). Rabbit CDC-SF003 was seroreactive by T. pallidum Particle Agglutination (TP-PA) and Rapid Plasma Reagin (RPR) testing, PCR+, and showed orchitis by week 6. Euthanasia was performed in week 7, with treponemal growth in the testes confirmed and quantified by qPCR and darkfield microscopy (DF). Serial passage of the extract in a second age-matched rabbit also yielded treponemes. Similarly, rabbit CDC-SF007 showed negligible orchitis, but was seroreactive and PCR+ by week 4 and euthanized in week 6 to yield T. pallidum, which was further propagated by second passage. Using the 4-component molecular typing system for syphilis, 3 propagated strains (CDC-SF003, CDC-SF007, CDC-SF008) were typed as 14d9f, 14d9g, and 14d10c, respectively. All 3 isolates including strain CDC-SF011, which was not successfully propagated, had the A2058G mutation associated with azithromycin resistance. Our results show that immediate cryopreservation of syphilitic ulcer exudate can maintain T. pallidum viability for rabbit propagation.

Background In Russia, the microscopy- and culture-based diagnostics of trichomoniasis is mainly suboptimal. Recent years, domestically produced diagnostic PCR assays have been implemented; however, ...

To determine the aetiology of genital ulcer disease (GUD) and its association with HIV infection in the mining community of Carletonville, South Africa, from two cross sectional surveys of consecutive men presenting with genital lesions during October 1993 to January 1994 and July to November 1998.A multiplex polymerase chain reaction (M-PCR) assay combined with amplicon detection was used to identify DNA specific sequences of Treponema pallidum, herpes simplex virus (HSV), and Haemophilus ducreyi. A real time PCR assay was used to differentiate between HSV-1 and HSV-2.M-PCR detected T pallidum, HSV, and H ducreyi in 10.3%, 17.2%, and 69.4% of 232 GUD patients during 1993-4 and in 12.4%, 36.0%, and 50.5% of 186 GUD patients in 1998. The proportion of patients with more than one agent increased significantly from 7.3% (17/232) in 1993-4 to 16.7% (31/186) in 1998 (p0.01). HSV-2 was detected in a higher proportion of ulcer specimens from HIV infected patients than in specimens from HIV uninfected patients during both time periods (1993-4: 26.2% v 6.7%, p0.001; 1998: 42.1% v 29.6%, p0.09).Based on two cross sectional surveys, 4 years apart, chancroid remained the leading cause of GUD in men who presented at the STD clinic with genital ulcers in the mining community of Carletonville, South Africa. The relative prevalence of primary syphilis has remained low. However, HSV-2 has emerged as a more significant cause of GUD and the proportion of GUD patients infected with more than one agent also increased significantly. HSV-2 DNA was detected in a significantly higher proportion of ulcer specimens from HIV positive patients than from HIV negative patients. No association was found between HIV infection status and the relative prevalence of chancroid or syphilis.

During a survey of yaws prevalence in the Solomon Islands, we collected samples from skin ulcers of 41 children. Using PCR, we identified Haemophilus ducreyi infection in 13 (32%) children. PCR-positive and PCR-negative ulcers were phenotypically indistinguishable. Emergence of H. ducreyi as a cause of nongenital ulcers may affect the World Health Organization's yaws eradication program.

The rate of clearance of Neisseria gonorrhoeae in vaginal specimens harvested from inbred Balb/c and outbred (Institute of Cancer Research [ICR]) white mice, previously primed with saline or beta-estradiol and then challenged with live gonococci, was determined. Survival of gonococci was significantly (P0.05) better in estradiol-primed mice from both inbred and outbred lines at 24 h after challenge. Vaginal clearance of gonococci from ICR mice parenterally primed with a gonococcal PI-B synthetic peptide and then orally immunized with gamma irradiated, protein-III deficient gonococcal cells was significantly (P0.001) faster than in corresponding nonimmunized, estradiol-primed controls. The more rapid vaginal clearance in the orally immunized mice was correlated with an elevated subcutaneous chamber ID50% (100,000 colony forming units) determined for the same groups of mice. Peptide-primed mice orally immunized with irradiated Escherichia coli cells demonstrated a chamber ID50% of100 CFU and were significantly (P0.01) slower to clear gonococci following vaginal challenge. High levels of specific IgA and IgG antibodies to gonococci were detected by an enzyme-linked immunosorbent assay in vaginal wash specimens from orally immunized mice. However, high antibody titers were not always predictive of an increased resistance to vaginal challenge. These models may be helpful in providing more economical and accessible in vivo methods for screening certain gonococcal vaccine candidates before more expensive testing in chimpanzees or humans.

Introduction We evaluated a multiplex PCR assay that can distinguish between syphilis and yaws on children with cutaneous lesions. Also, a rapid syphilis test, Chembio DPP Screen and Confirm Assay was evaluated for serological diagnosis of yaws. Methods Lesions swabs for PCR were obtained from children (5–14 years old) in West Akyem Municipality, Ghana pre- and post-MDA with azithromycin and pre-MDA on Tanna Island, Vanuatu. DPP testing was done on site and blood was collected for serology (RPR and TPPA). Molecular diagnosis and screening for azithromycin resistance markers was done using TaqMan-based real-time multiplex PCR tests. Another duplex PCR test was used to detect H. ducreyi and M. ulcerans. Results Pre-MDA TPPA and RPR dual positivity was 35.8% (63/176) in Vanuatu and 33.6% (109/326) in Ghana and post-MDA was 18.6% (16/43) and 6.5% (3/46), respectively in children with skin lesions. The overall sensitivity and specificity of the DPP treponemal component versus TPPA was 88.2% and 82.7%, and DPP non-treponemal component versus RPR was 84.8% and 94.7%. In children with T. pertenue PCR-positive lesions, dual positive DPP had an overall sensitivity and specificity of 86.3% and 78.6%, and a PPV of 44% and a NPV of 96.7%. 14.9% (27/181) of pre-MDA swab samples from Vanuatu and 17.3% (31/179) in Ghana were PCR-positive for T. pertenue . None of the 49 samples from Ghana were positive for T. pertenue post-MDA. Azithromycin resistance markers were not found in any of the samples. H. ducreyi was detected by PCR in 40.3% (73/181) of samples from Vanuatu, and 27.4% (51/208) from Ghana pre-MDA and 28.6% (14/49) in Ghana post-MDA. Six children were co-infected with T. pertenue and H. ducreyi in Vanuatu and seven in Ghana. M. ulcerans was not detected. Conclusion The DPP test is a useful screening test to exclude yaws in cases with a high index of suspicion on clinical grounds and the real-time PCR is essential for confirmation of a yaws diagnosis. MDA with oral azithromycin is effective for treatment of yaws but has limited impact on H. ducreyi .

Objectives Data regarding the performance characteristics of STI diagnostic tests used in Eastern Europe are very scarce. The objective of this study was to validate the AmpliSens test systems (Central Research Institute for Epidemiology, Moscow, Russia), compared to internationally acknowledged and validated tests, for genetic detection of N gonorrhoeae (NG) , C trachomatis (CT) , M genitalium (MG) and T vaginalis (TV) . Methods In total, 319 females and 127 males were recruited. For evaluation of the TV tests, additional 33 patients with diagnosed trichomoniasis at dermatovenereological dispensaries (VD) were involved. Conventional AmpliSens PCR assay (cPCR), real-time PCR assay (rtPCR), and a real-time nucleic acid sequence-based amplification assay for detection of NG or CT were evaluated. Furthermore, AmpliSens cPCR and rtPCR for detection of MG or TV were tested. As international reference tests an internationally validated real-time N gonorrhoeae porA pseudogene PCR, Cobas Amplicor CT PCR and LightMix 480HT CT PCR, MG rtPCR targeting the MgPa adhesin gene with positive results confirmed by two MG cPCRs targeting the 16S rRNA gene and MgPa gene, and a TV rtPCR targeting a TV specific repeat DNA fragment, respectively, were used. Results The overall prevalence of CT infection was 12.6%. The AmpliSens CT NAATs and the reference methods displayed a high level of concordance (97.9%–99.2%). The prevalence of NG was 2.7% and 16% among the urogenital and extragenital samples, respectively. The AmpliSens NG NAATs and the reference method displayed high level of concordance (99.4–100%). The prevalence of MG was 2.5% among females and 9.6% among males. The highest sensitivity (71.4%–100% in different specimens) was exhibited by the AmpliSens rtPCR. All tests had a 100% clinical specificity. The prevalence of TV was 1.2% among the females, and all additional VD patients tested positive. The sensitivity and the specificity of both Russian TV tests validated was 100%. Conclusion It seems clear that the biomedical industry in Eastern Europe has the potential for producing reliable reagents and tests kits at affordable prices for genetic diagnosis of STIs. This would open new perspectives for the whole region and could also be cost-effective for some other regions experiencing financial constraints. However, more comprehensive evaluations of regionally manufactured tests should be conducted according to internationally accepted guidelines.

While genital ulcers are a risk factor in HIV infection, the association of specific agents of genital ulcer disease (GUD) with HIV infection may vary.To determine the etiology of GUD in HIV-infected and HIV-uninfected men attending sexually transmitted disease (STD) clinics in Durban, Johannesburg, and Cape Town, South Africa, and the association of previous and current sexually transmitted infections with HIV infection in men with ulcerative and nonulcerative STDs.A cross-sectional study of 558 men with genital ulcers and 602 men with urethritis.Patients with GUD were more likely to be infected with HIV than patients with urethritis (39.4% versus 21.4%, Por =0.001). Herpes simplex virus 2 (HSV-2) was the most common agent identified in ulcer specimens (35.9%), and was detected in a significantly higher proportion of ulcer specimens from HIV-infected patients than in specimens from HIV-uninfected patients (47.4% versus 28.2%, Por =0.001). Patients infected with HIV-1 were significantly more likely to have HSV-2 infection, as measured by the presence of the antibody to glycoprotein G-2, than patients not infected with HIV (63.1% versus 38.5%, Por =0.001). Patients infected with HIV-1 were also significantly more likely to have initial HSV-2 infection than HIV-uninfected patients with GUD (50.0% versus 31.6%, P = 0.007). Haemophilus ducreyi was detected in 31.7% of ulcer specimens; prevalence did not vary by HIV-infection status. Treponema pallidum DNA was detected significantly less frequently in ulcer specimens from patients infected with HIV than in specimens from patients not infected with HIV (10.2% versus 26%, Por =0.001); no association was found between HIV-infection status and fluorescent treponemal antibody absorption test seroreactivity, even when men with M-PCR-positive syphilis lesions were excluded from the analyses.The authors found that HSV-2 is a more common etiology of GUD than has been suggested by previous studies conducted in South Africa; serologic evidence of HSV-2 infection and current cases of genital herpes are strongly associated with HIV infection among men who present to STD clinics with GUD or urethritis.

Background and objectives Epidemiologic studies on syphilis have been hampered by the fact that strains of Treponema pallidum subspecies pallidum (T. pallidum), the causative agent of this disease, cannot be differentiated by either protein-based or deoxyribonucleic acid-based methods. Syphilis is endemic in many developing countries and is common in some industrialized nations. In addition, the disease has been shown to increase the risk of infection with the human immunodeficiency virus. Goal To develop a molecular subtyping method for T. pallidum. Study design Two genes exhibiting intrastrain variability were identified as potential targets for strain differentiation: the acidic repeat protein (arp) gene, which contains a variable number of 60 base pair repeats, and a member of the treponema pallidum repeat (tpr) gene family. Polymerase chain reaction amplification and restriction endonuclease digestion of polymerase chain reaction products from laboratory strains and clinical specimens were used to develop a molecular subtyping scheme for T. pallidum. Results Determining the number of repeats in the arp gene by polymerase chain reaction resulted in 12 different subtypes among the 63 isolates that were studied. Among those, most (54.2%) had arp genes with 14 repeats. The other 11 subtypes had arp genes with 7 to 21 repeats, each accounting for 2% to 14% of the isolates. Polymerase chain reaction amplification of a member of the tpr gene family from a subset of 46 isolates followed by digestion of the polymerase chain reaction product with MseI resulted in seven restriction fragment length polymorphism patterns designated a to g. Strains with 14 repeats could be grouped into five restriction fragment length polymorphism subtypes. By combining the two systems we observed 16 subtypes among 46 isolates examined. This typing system is stable, reproducible, and easy to perform. In addition, the use of the ABI Genetic Analyzer for the determination of fragment size and banding patterns makes the results unbiased. Conclusion This is the first molecular subtyping system that distinguishes among clinical isolates of T. pallidum.

Background WHO/UNAIDS recommends National HIV/AIDS and Sexually Transmitted Infection STI) Control Programs to implement integrated HIV, STI and behavioural surveillance to assess the disease burden, and to monitor the temporal trends of HIV/STI prevalence and high-risk sexual behaviours. Despite this recommendation, there is limited STI prevalence data available in many resource poor countries owing to lack of STI laboratory capacity to detect multiple STIs. The CDC STD lab explored the feasibility of a decentralised laboratory strengthening approach by applying more advanced molecular STI diagnostic methods to support the second generation surveillance (SGS) activities in the Central American Region during 2007–2011. Methods CDC STD lab developed a real-time multiplex PCR (MPCR) to detect Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, and Mycoplasma genitalium . In collaboration with regional partners, CDC identified and transferred the diagnostic technology to a regional STI reference laboratory in 2008. The regional reference laboratory provides MPCR testing on specimens collected for surveillance purpose. This standardised SGS study design was implemented at regular intervals in multiple countries in the region to estimate the disease burden and to monitor temporal STI trends. Results During 2007–2010, four national integrated sexual behavioural and biomarker surveys were implemented using CDC9s affiliated regional STI reference center. The quality STI prevalence data was made available to the national HIV/STI programs in Honduras, El Salvador, Nicaragua and Colombia. Similar CDC supported surveillance studies are in-preparation in Guatemala, Belize, Panama, Honduras (second round) and the Dominican Republic. Conclusions Based on the experience from the Central American Region, it is feasible to implement integrated HIV, STI and behavioural surveillance surveys using robust molecular techniques in resource poor settings. In collaboration with regional partners, CDC9s approach to STI laboratory capacity strengthening through establishing a regional reference laboratory should be expanded to other regions.

Background The goal of the study was to evaluate two sequence-based subtyping methods for their ability to further differentiate Treponema pallidum strains characterised by the CDC typing system. Methods 12 clinical specimens with the 14d strain type obtained from patients with GUD in Cape Town in 2000; 14 specimens with the 14d strain type from patients with primary or secondary syphilis in San Francisco collected between 2004 and 2007; and 12 clinical specimens with the 14d strain type collected between 2000 and 2002 from a syphilis outbreak in Vancouver were included in the study. Specimens were previously characterised using the CDC typing method which involves analysis of the sequence variability with tprE, G, and J and, the variable number of 60-bp tandem repeats within the arp gene. Subtyping was performed by PCR amplification of a homonucleotide G" tandem repeat within tp0279 and a variable region within tp0548. PCR amplicons were purified and directly sequenced using the BigDye® Terminator v3.1 cycle sequencing kit and an ABI 3130 sequencer. Results Of the 12 samples from South Africa, sequence analysis of tp0279 revealed three subtypes (9G, 10G, 11G) and combining this data with the CDC typing method produced strain subtypes 14d9, 14d10, and 14d11. Sequence analysis of the variable region within tp0548 resulted in five subtypes designated a, c, f, k, and l among these samples and, combination with strain type using the CDC method produced subtypes 14d/a, 14d/c, 14d/f, 14d/k, 14d/l. All 14 samples from San Francisco had 9G tandem repeats within tp0279 resulting in subtype 14d9. Sequence analysis of the variable region within tp0548 resulted in 3 subtypes designated f, g and j among these 14 samples and incorporation of the CDC typing method produced subtypes 14d/f, 14d/g, and 14d/j. Of the 12 samples from Vancouver, sequence analysis of tp0279 produced two subtypes (8G, 9G) resulting in strain subtypes 14d8 (1/12) and14d9 (11/12). Sequencing of tp0548 also produced two subtypes (e, f) resulting in strain subtypes 14d/e (1/12) and14d/f (11/12). Conclusions The tp0279 subtyping method further differentiated 14d strains into four subtypes while the tp0548 method differentiated 14d strains into eight subtypes. Subtyping of strains from the initial syphilis outbreak in Vancouver suggests clonal spread of T pallidum . Both subtyping methods enhanced the original CDC typing method and appear to be promising tools for molecular epidemiological studies on syphilis.

Background To develop a TaqMan-based real-time allelic discrimination assay for the simultaneous detection of two point mutations (A2058G and A2059G) in the 23S rRNA gene of T pallidum that have been associated with azithromycin treatment failures. Methods Initially, two TaqMan-based real-time duplex PCR assays were used to detect the A2058G and A2059G point mutations within the 23S rRNA gene of T pallidum. Genotyping results from these assays were then compared to a previously described real-time PCR assay using fluorescence resonance energy transfer (FRET) probes and melting curve analysis that is specific for the detection of the A2058G point mutation. Subsequently, a real-time triplex PCR was developed to distinguish the A2058G from the A2059G point mutation in a single assay and the results were confirmed by pyrosequencing. Results Sixteen of 67 (23.9%) T pallidum-positive specimens collected from patients with genital ulcer disease in the US were found to have the A2059G point mutation. These strains were previously characterised as having azithromycin-susceptible genotypes (no point mutations in the 23S rRNA gene). The A2059G mutation was confirmed by a real-time duplex PCR assay containing the TaqMan probe specific for the mutation and by pyrosequencing. None of the T pallidum strains examined to date had both point mutations. The real-time triplex PCR assay was able to differentiate azithromycin-susceptible genotypes from resistant genotypes containing either the A2058G or A2059G point mutation in a single assay. The limit of detection of the assay was 1–10 copies using 23S rRNA gene fragments that were cloned into a plasmid. Conclusions The previously reported real-time PCR detection platforms were designed to detect only the A2058G point mutation and were unable to differentiate T pallidum strains with susceptible genotypes from resistant genotypes with the A2059G mutation. The new TaqMan-based real-time allelic discrimination assay offers an alternative to the previously described PCR/RFLP method to rapidly detect both point mutations associated with azithromycin resistance in T pallidum. The prevalence of T. pallidum strains that harbour point mutations in the 23S rRNA gene associated with resistance to azithromycin might be much higher than previously estimated.

Background To evaluate the potential use of the Whatman Indicating FTA Elute Micro Card for collection, transport, and storage of rectal swab specimens for subsequent detection of STD pathogens in MSM using a TaqMan-based real-time multiplex PCR. Methods Two rectal swabs were obtained by the physician from each participant in a study to determine carriage of STD pathogens among MSM. One of the swabs was placed into a tube containing 1 ml of Genelock transport medium and while the other swab was firmly pressed onto an FTA Card with three side to side motions held at approximately 60° angle each time. Genomic DNA was eluted from three discs (3 mm diameter) punched out of each card after storage at room temperature for up to 6 months and tested by a real-time multiplex PCR assay which simultaneously detects lymphogranuloma venereum (LGV), non- LGV, Chlamydia trachomatis (CT), and Neisseria gonorrhoeae (GC), and human DNA control. For comparison, an aliquot of sample from genelock tube was extracted using the Qiagen DNA mini kit and tested with same real-time multiplex PCR assay. Results Using purified DNA obtained from the Genelock specimens, the real-time multiplex PCR assay detected nine GC and 17 non-LGV CT; while 7 GC and 15 non-LGV CT were detected in DNA samples eluted from the FTA Cards and used directly for PCR. There were three GC and non-LGV co-infections and no LGV was detected using both specimen collection methods. A substantial number of specimens were found to be PCR inhibitory either collected in Genelock (18.3%) or on FTA Cards (15.4%). Conclusions The FTA Elute Micro Card allows stable storage and convenient transport of rectal swab specimens at room temperature. DNA can be eluted from the card with simple processes instead of numerous purification procedures for downstream real-time PCR amplification and aetiology detection. This preliminary evaluation shows the potential use of FTA Card for rectal specimen collection and PCR testing, and may also provide a cost-saving alternative to expensive international shipping of specimens on dry ice from remote study sites.