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Downstream next generation sequencing (NGS) of the syphilis spirocheteTreponema pallidumsubspeciespallidum(T. pallidum) is hindered by low bacterial loads and the overwhelming presence of background metagenomic DNA in clinical specimens. In this study, we investigated selective whole genome amplification (SWGA) utilizing multiple displacement amplification (MDA) in conjunction with custom oligonucleotides with an increased specificity for theT. pallidumgenome, and the capture and removal of CpG-methylated host DNA using the NEBNext®Microbiome DNA Enrichment Kit followed by MDA with the REPLI-g Single Cell Kit as enrichment methods to improve the yields ofT. pallidumDNA in isolates and lesion specimens from syphilis patients. Sequencing was performed using the Illumina MiSeq v2 500 cycle or NovaSeq 6000 SP platform. These two enrichment methods led to 93-98% genome coverage at 5 reads/site in 5 clinical specimens from the United States and rabbit propagated isolates, containing >14T. pallidumgenomic copies/μl of sample for SWGA and >129 genomic copies/μl for CpG methylation capture with MDA. Variant analysis using sequencing data derived from SWGA-enriched specimens, showed that all 5 clinical strains had the A2058G mutation associated with azithromycin resistance. SWGA is a robust method that allows direct whole genome sequencing (WGS) of specimens containing very low numbers ofT. pallidum, which have been challenging until now.ImportanceSyphilis is a sexually transmitted, disseminated acute and chronic infection caused by the bacterial pathogenTreponema pallidumsubspeciespallidum. Primary syphilis typically presents as single or multiple mucocutaneous lesions, and if left untreated, can progress through multiple stages with varied clinical manifestations. Molecular studies often rely on direct amplification of DNA sequences from clinical specimens; however, this can be impacted by inadequate samples due to disease progression or timing of patients seeking clinical care. While genotyping has provided important data on circulating strains over the past two decades, WGS data is needed to better understand strain diversity, perform evolutionary tracing, and monitor antimicrobial resistance markers. The significance of our research is the development of a SWGA DNA enrichment method that expands the range of clinical specimens that can be directly sequenced to include samples with low numbers ofT. pallidum.

Background Ocular syphilis cases continue to be identified in the United States since two clusters were reported in late 2014 into early 2015. Ocular syphilis (OS) is an inflammatory eye condition that can occur at any stage of syphilis with vision loss and blindness reported in some patients. We performed genotyping and whole genome sequencing (WGS) on Treponema pallidum strains from OS cases as part of molecular surveillance activities. Methods A total of 79 specimens from 57 patients with suspected or confirmed OS were received from 14 states between February 2016 and November 2020. Specimens included CSF, whole blood, serum, plasma, vitreous fluid, and a throat swab. T. pallidum DNA was detected with a real-time PCR assay targeting the polA gene. Genotyping was done using the four-component typing scheme (tpr E, G, & J; arp, tp0548, and tp0279). T. pallidum genomic DNA was enriched by selective whole genome amplification (SWGA) using Multiple Displacement Amplification (MDA) with custom oligonucleotides followed by WGS on an Illumina MiSeq v2 500 cycle platform. Results Twenty-three patients (40.4%) were MSM and HIV positive, respectively; 41 (71.9%) identified as White race, 4 (7%) Hispanic, and 3 (5.3%) Black. Twenty-three specimens from 18 (31.6%) patients tested positive for T. pallidum DNA. Thirteen of 23 (56.5%) specimens were CSF, while the remaining 10 included whole blood, serum, vitreous fluid, and a throat swab. Specimens from 3 patients were fully typed, revealing strain types 14b9g, 14d10g, and 14e9f. Six patients had partial genotypes. WGS was successful on 1 CSF and 2 vitreous fluid specimens from 2 cases resulting in 87% – 98% genome coverage with at least 5 reads/site. Phylogenetic analysis showed that the 2 strains belonged to the Street 14 clade. Conclusions Our findings show that multiple strain types are responsible for ocular syphilis in the United States.

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.

WHO and its partners aim to interrupt yaws transmission in countries of endemicity and to certify others as being yaws-free. Transmission can be assessed using rapid plasma reagin (RPR) tests, reflecting current or recent infection, but RPR is operationally impractical. We evaluated changes in antibody levels against two recombinant treponemal antigens, rp17 (also known as Tp17) and TmpA, after antibiotic treatment given as part of a randomized controlled trial for yaws in Ghana and Papua New Guinea. Paired serum samples from children aged 6 to 15 years with confirmed yaws, collected before and after treatment, were tested for antibodies to rp17 and TmpA using a semiquantitative bead-based immunoassay. Of 344 baseline samples, 342 tested positive for anti-rp17 antibodies and 337 tested positive for anti-TmpA antibodies. Six months after treatment, the median decrease in anti-rp17 signal was 3.2%, whereas the median decrease in anti-TmpA was 53.8%. The magnitude of change in the anti-TmpA response increased with increasing RPR titer fold change. These data demonstrate that responses to TmpA decrease markedly within 6 months of treatment whereas (as expected) those to rp17 do not. Incorporating responses to TmpA as a marker of recent infection within an integrated sero-surveillance platform could provide a way to prioritize areas for yaws mapping.

Summary Background A dose of 30 mg/kg of azithromycin is recommended for treatment of yaws, a disease targeted for global eradication. Treatment with 20 mg/kg of azithromycin is recommended for the elimination of trachoma as a public health problem. In some settings, these diseases are co-endemic. We aimed to determine the efficacy of 20 mg/kg of azithromycin compared with 30 mg/kg azithromycin for the treatment of active and latent yaws. Methods We did a non-inferiority, open-label, randomised controlled trial in children aged 6–15 years who were recruited from schools in Ghana and schools and the community in Papua New Guinea. Participants were enrolled based on the presence of a clinical lesion that was consistent with infectious primary or secondary yaws and a positive rapid diagnostic test for treponemal and non-treponemal antibodies. Participants were randomly assigned (1:1) to receive either standard-dose (30 mg/kg) or low-dose (20 mg/kg) azithromycin by a computer-generated random number sequence. Health-care workers assessing clinical outcomes in the field were not blinded to the patient's treatment, but investigators involved in statistical or laboratory analyses and the participants were blinded to treatment group. We followed up participants at 4 weeks and 6 months. The primary outcome was cure at 6 months, defined as lesion healing at 4 weeks in patients with active yaws and at least a four-fold decrease in rapid plasma reagin titre from baseline to 6 months in patients with active and latent yaws. Active yaws was defined as a skin lesion that was positive for Treponema pallidum ssp pertenue in PCR testing. We used a non-inferiority margin of 10%. This trial was registered with ClinicalTrials.gov, number NCT02344628. Findings Between June 12, 2015, and July 2, 2016, 583 (65·1%) of 895 children screened were enrolled; 292 patients were assigned a low dose of azithromycin and 291 patients were assigned a standard dose of azithromycin. 191 participants had active yaws and 392 had presumed latent yaws. Complete follow-up to 6 months was available for 157 (82·2%) of 191 patients with active yaws. In cases of active yaws, cure was achieved in 61 (80·3%) of 76 patients in the low-dose group and in 68 (84·0%) of 81 patients in the standard-dose group (difference 3·7%; 95% CI −8·4 to 15·7%; this result did not meet the non-inferiority criterion). There were no serious adverse events reported in response to treatment in either group. The most commonly reported adverse event at 4 weeks was gastrointestinal upset, with eight (2·7%) participants in each group reporting this symptom. Interpretation In this study, low-dose azithromycin did not meet the prespecified non-inferiority margin compared with standard-dose azithromycin in achieving clinical and serological cure in PCR-confirmed active yaws. Only a single participant (with presumed latent yaws) had definitive serological failure. This work suggests that 20 mg/kg of azithromycin is probably effective against yaws, but further data are needed. Funding Coalition for Operational Research on Neglected Tropical Diseases.

Background Rectal STI coinfection models enhance the understanding of rectal HIV transmission risk factors. Materials and Methods Rhesus macaques (n=9) were exposed to one of three rectal Chlamydia trachomatis (CT) challenges: C. trachomatis L2 (CT-L2); C. trachomatis serovar E (CT-E), followed by CT-L2; or CT-E, treatment/clearance, then CT-L2. Infections were monitored by PCR. Weekly blood and rectal secretion/lavage samples were collected for cytokine analyzes and/or epithelial sloughing, occult, and overt blood determinations. Results Chlamydial infections were successfully established in each animal, with varying degrees of persistence. Mucosal IL-1beta was upregulated in animals consecutively infected with CT-E then CT-L2 (P=.05). Epithelial sloughing was also significantly increased post-infection in this group (P=.0003). Conclusions This study demonstrates successful rectal infection of rhesus macaques with CT-E and CT-L2 and describes measures of assessing rectal inflammation and pathology. Different infection strategies yield varying inflammatory and pathologic outcomes, providing well-described models for future SIV/SHIV susceptibility studies.

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.

Yaws is a neglected tropical disease caused by the bacterium Treponema pallidum subspecies pertenue. The disease primarily affects children under 15 years of age living in low socioeconomic conditions in tropical areas. As a result of a renewed focus on the disease owing to a recent eradication effort initiated by the World Health Organization, we have evaluated a typing method, adapted from and based on the enhanced Centers for Disease Control and Prevention typing method for T. pallidum subsp. pallidum, for possible use in epidemiological studies. Thirty DNA samples from yaws cases in Vanuatu and Ghana, 11 DNA samples extracted from laboratory strains, and 3 published genomic sequences were fully typed by PCR/RFLP analysis of the tpr E, G, and J genes and by determining the number of 60-bp repeats within the arp gene. Subtyping was performed by sequencing a homonucleotide “G” tandem repeat immediately upstream of the rpsA gene and an 84-bp region of tp0548. A total of 22 complete strain types were identified; two strain types in clinical samples from Vanuatu (5q11/ak and 5q12/ak), nine strain types in clinical samples from Ghana (3q12/ah, 4r12/ah, 4q10/j, 4q11/ah, 4q12/ah, 4q12/v, 4q13/ah, 6q10/aj, and 9q10/ai), and twelve strain types in laboratory strains and published genomes (2q11/ae, 3r12/ad, 4q11/ad, 4q12/ad, 4q12/ag, 4q12/v, 5r12/ad, 6r12/x, 6q11/af, 10q9/r, 10q12/r, and 12r12/w). The tpr RFLP patterns and arp repeat sizes were subsequently verified by sequencing analysis of the respective PCR amplicons. This study demonstrates that the typing method for subsp. pallidum can be applied to subsp. pertenue strains and should prove useful for molecular epidemiological studies on yaws.

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.

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.

Rectal STI coinfection models enhance the understanding of rectal HIV transmission risk factors.Rhesus macaques (n=9) were exposed to one of three rectal Chlamydia trachomatis (CT) challenges: C. trachomatis LChlamydial infections were successfully established in each animal, with varying degrees of persistence. Mucosal IL-1beta was upregulated in animals consecutively infected with CT-E then CT-LThis study demonstrates successful rectal infection of rhesus macaques with CT-E and CT-L

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.

We explored the association between violence victimization and increased risk for acquiring sexually transmitted infections (STI) in women by measuring cellular immune barrier properties from the female reproductive tract (FRT). STI-negative participants reporting repeated prior victimization occurrences through the lifetime trauma and victimization history (LTVH) instrument were more likely to exhibit alterations in barrier homeostasis and the composition of critical immune mediators irrespective of demographic parameters or presence of bacterial vaginosis. By combining cellular data with mixed-effect linear modeling, we uncovered differences in local T cells, MHCII+ antigen presenting cells, and epithelial cells indicative of altered trafficking behavior, increased immunosuppressive function, and decreased barrier integrity at sites of STI exposure that correlate with LTVH score. These data evidence a biological link between a history of violence victimization and risk of STI acquisition through immune dysregulation in the female reproductive tract.

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, ...

Yaws, caused by Treponema pallidum ssp. pertenue, is a neglected tropical disease closely related to venereal syphilis and is targeted for eradication by 2020. Latent yaws represents a diagnostic challenge, and current tools cannot adequately distinguish between individuals with true latent infection and individuals who are serofast following successful treatment. PCR on blood has previously been shown to detect T. pallidum DNA in patients with syphilis, suggesting that this approach may be of value in yaws. We performed real-time PCR for Treponema pallidum ssp. pertenue on blood samples from 140 children with positive T. pallidum Particle Agglutination (TPPA) and Rapid Plasma Reagin (RPR) tests and 7 controls (negative serology), all collected as part of a prospective study of yaws in the Solomon Islands. All samples were also tested by a nested PCR for T. pallidum. 12 patients had clinical evidence of active yaws whilst 128 were considered to have latent yaws. 43 children had high titre rapid plasma reagins (RPRs) of ≥1:32. PCR testing with both assays gave negative results in all cases. It is possible that the failure to detect T. pallidum ssp. pertenue in blood reflects lower loads of organism in latent yaws compared to those in latent infection with T. pallidum ssp. pertenue, and/or a lower propensity for haematogenous dissemination in yaws than in syphilis. As the goal of the yaws control programme is eradication, a tool that can differentiate true latent infection from individuals who are serofast would be of value; however, PCR of blood is not that tool., Author Summary Yaws is a bacterial infection closely related to syphilis. The WHO has launched a worldwide campaign to eradicate yaws by 2020. For each clinically apparent case, many close contacts are infected but do not show clinical signs, which is called latent yaws. Currently, diagnosis for these patients relies on the detection of antibodies using syphilis serology. Reliance on detection of antibodies for diagnosis of latent yaws is problematic as the test may remain positive with a low titre despite successful treatment. This makes the interpretation of surveillance data in a post-mass treatment setting difficult for programme managers. In syphilis, PCR can detect T. pallidum ssp. pallidum in the blood of patients with latent disease. We used similar techniques to try and detect the yaws bacterium in the blood of patients seen as part of a study of yaws in the Solomon Islands. Although many people had positive antibodies for yaws consistent with latent yaws, we were unable to detect the bacterium in the blood. This may reflect the lower virulence of the yaws bacterium compared to syphilis. Currently available molecular techniques are therefore not able to aid programme managers in conducting post-mass drug administration surveillance for latent yaws. The development of alternative diagnostic tests should be considered.

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.

A sensitive and specific PCR method to detect Treponema 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 (for T. pallidum, Haemophilus ducreyi , and herpes simplex virus). We tested 112 genital ulcer specimens by the polA PCR, 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.

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.

Ten novel α-gliadin genes (Gli-ta, Gli-turg1, Gli-turg2, Gli-turg3, Gli-turg4, Gli-turg5, Gli-turg6, Gli-cs1, Gli-cs2, and Gli-cs3) with unique characteristics were isolated from wheat ( Triticum aestivum L.), among which Gli-cs1, Gli-cs2, Gli-cs3, and Gli-turg6 were pseudogenes. Gli-cs3 and nine other sequences were much larger and smaller, respectively, than the typical α-gliadins. This variation was caused by insertion or deletion of the unique domain I and a polyglutamine region, possibly the result of illegitimate recombination. Consequently, Gli-cs3 contained 10 cysteine residues, whereas there were 2 cysteine residues only in the other nine sequences. Gli-ta/Gli-ta-like α-gliadin genes are normally expressed during the development of seeds. SDS–PAGE analysis showed that in-vitro-expressed Gli-ta could form intermolecular disulphide bonds and could be chain extenders. A protein band similar in size to Gli-ta has been observed in seed extracts, and mass spectrometry results confirm that the band contains small molecular mass α-gliadins, which is a characteristic of the novel α-gliadins. Mass spectrometry results also indicated that the two cysteine residues of Gli-ta/Gli-ta-like proteins participated in the formation of intermolecular disulphide bonds in vivo.

Aqueous exudates from differentiated or undifferentiated basidiospore germlings, as well as differentiated or undifferentiated urediospore germlings, of race 1 of the cowpea rust fungus were tested for their ability to elicit cell necrosis when injected into cowpea leaves. Only exudates from differentiated basidiospore germlings consistently exhibited high clicitation activity with the same cultivar-specificity as the fungus. Intercellular washing fluids from monokaryon-infected, but not from dikaryon-infected, leaves exhibited similar activity and cultivar-specificity to exudates from differentiated basidiospore-derived germlings. The results suggest that the production of the cultivar-specific elicitor of necrosis by the monokaryon is triggered by the formation of infection structures, and that this elicitor is produced in planta. The dikaryon either does not produce this elicitor or elicitor production is restricted to the haustorium.

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 .

BACKGROUND AND PURPOSE: In patients with multiple sclerosis (MS), a few preliminary functional MR (fMR) imaging studies of the visual cortex reveal information about magnitude differences between healthy individuals and patients with MS at only a single luminance level. We therefore investigated whether varying luminance contrast levels can help uncover subtle changes in fMR imaging characteristics of the visual cortex in healthy volunteers and patients with MS. METHODS: Blood oxygenation level–dependent fMR imaging signal changes in the primary visual cortex were examined as a function of luminance contrast at 1.5 T in 10 healthy volunteers and nine patients with MS. Ten axial sections through the calcarine fissure were obtained with an echo-planar T2*-weighted imaging sequence (4000/54/1 [TR/TE/excitation]; field of view, 220 mm; voxel size, 1.72 × 1.72 × 5 mm). The imaging series consisted of an alternating 20-second rest epoch (black screen) with a 20-second activation epoch (flickering checkerboard) repeated six times. Each imaging series used a graded increase of eight luminance contrast levels. A paired t test between rest and activation images was used to analyze significant (P < .001) contiguous voxels in the region of interest (primary visual cortex). RESULTS: A progressive increase in fMR imaging activation across all luminance contrast levels in healthy controls and patients with MS was shown. The patients with MS had a significantly lower magnitude in the number of fMR imaging activated voxels at all luminance contrast levels (P < .001). A statistically significant increase in fMR imaging activation (activation threshold) was seen at the second luminance contrast level in controls and at the seventh level in patients with MS. CONCLUSION: Quantifiable changes in blood oxygenation level-dependent signal and a progressive increase in activated voxels within the primary visual cortex with increasing luminance contrast were demonstrated at 1.5 T in controls. The patients with MS showed a significant decrease in the number of activated voxels and an increase in activation threshold compared with healthy controls.

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.

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.

Neurofibromatosis type I (NF-1) belongs to a family of diseases named phakomatoses, which are characterized by congenital malformations of ectodermal structures. Neurofibromatosis type I affects 1 in 3000 people, and has a diverse clinical presentation as well as an array of imaging findings. In this article the authors review the various neuroimaging findings present in NF-1, including abnormalities of the parenchyma, meninges, vessels, and associated neoplasms.

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.

Multiple-flip-angle, three-dimensional, time-of-flight magnetic resonance (MR) angiography was performed in vitro and in vivo in the carotid artery bifurcation. Composite MR angiographic images were created from multiple data sets. Compared with images obtained with a low (20 degrees) flip angle, composite images obtained with flip angles of 20 degrees and 60 degrees demonstrated improved image quality and statistically significant improvement in signal-to-noise ratio (P < .05).

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.