Improved rapid diagnostic tests to detect syphilis and yaws: a systematic review and meta-analysis.

BACKGROUND: Current rapid tests for syphilis and yaws can detect treponemal and non-treponemal antibodies. We aimed to critically appraise the literature for rapid diagnostic tests (RDTs) which can better distinguish an active infection of syphilis or yaws. METHODS: We conducted a systematic review and meta-analysis, searching five databases between January 2010 and October 2021 (with an update in July 2022). A generalised linear mixed model was used to conduct a bivariate meta-analysis for the pooled sensitivity and specificity. Heterogeneity was assessed using the I2 statistic. We used the Quality Assessment of Diagnostic Accuracy Studies (QUADAS) to assess the risk of bias and Grading of Recommendations, Assessment, Development and Evaluations (GRADE) to evaluate the certainty of evidence. RESULTS: We included 17 studies for meta-analyses. For syphilis, the pooled sensitivity and specificity of the treponemal component were 0.93 (95% CI: 0.86 to 0.97) and 0.98 (95% CI: 0.96 to 0.99), respectively. For the non-treponemal component, the pooled sensitivity and specificity were 0.90 (95% CI: 0.82 to 0.95) and 0.97 (95% CI: 0.92 to 0.99), respectively. For yaws, the pooled sensitivity and specificity of the treponemal component were 0.86 (95% CI: 0.66 to 0.95) and 0.97 (95% CI: 0.94 to 0.99), respectively. For the non-treponemal component, the pooled sensitivity and specificity were 0.80 (95% CI: 0.55 to 0.93) and 0.96 (95% CI: 0.92 to 0.98), respectively. CONCLUSIONS: RDTs that can differentiate between active and previously treated infections could optimise management by providing same-day treatment and reducing unnecessary treatment. PROSPERO REGISTRATION NUMBER: CRD42021279587.

Antibody Responses to Two Recombinant Treponemal Antigens (rp17 and TmpA) before and after Azithromycin Treatment for Yaws in Ghana and Papua New Guinea.

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.

Increased Discrimination of Treponema pallidum Strains by Subtyping With a 4-Component System Incorporating a Mononucleotide Tandem Repeat in rpsA.

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.

Molecular strain typing of the yaws pathogen, Treponema pallidum subspecies pertenue.

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.

Community-based mass treatment with azithromycin for the elimination of yaws in Ghana-Results of a pilot study.

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.

Complete Genome Sequences of 11 Haemophilus ducreyi Isolates from Children with Cutaneous Lesions in Vanuatu and Ghana.

Haemophilus ducreyi causes chancroid and has recently been shown to be a significant cause of cutaneous lesions in tropical or subtropical regions where yaws is endemic. Here, we report the draft genome assemblies for 11 cutaneous strains of Haemophilus ducreyi, isolated from children in Vanuatu and Ghana.

Metaanalysis of the Performance of a Combined Treponemal and Nontreponemal Rapid Diagnostic Test for Syphilis and Yaws.

BACKGROUND: The human treponematoses are important causes of disease. Mother-to-child transmission of syphilis remains a major cause of stillbirth and neonatal death. There are also almost 100 000 cases of endemic treponemal disease reported annually, predominantly yaws. Rapid diagnostic tests (RDTs) would improve access to screening for these diseases. Most RDTs cannot distinguish current and previous infection. The Dual Path Platform (DPP) Syphilis Screen & Confirm test includes both a treponemal (T1) and nontreponemal (T2) component and may improve the accuracy of diagnosis. METHODS: We conducted a metaanalysis of published and unpublished evaluations of the DPP-RDT for the diagnosis of syphilis and yaws. We calculated the sensitivity, specificity, and overall agreement of the test compared with reference laboratory tests. RESULTS: Nine evaluations, including 7267 tests, were included. Sensitivity was higher in patients with higher titer rapid plasma reagin (≥1:16) for both the T1 (98.2% vs 90.1%, P < .0001) and the T2 component (98.2% vs 80.6%, P < .0001). Overall agreement between the DPP test and reference serology was 85.2% (84.4%-86.1%). Agreement was highest for high-titer active infection and lowest for past infection. CONCLUSIONS: The RDT has good sensitivity and specificity of the treponemal and nontreponemal components both in cases of suspected syphilis and yaws, although the sensitivity is decreased at lower antibody titers.

Challenges and key research questions for yaws eradication.

Yaws is endemic in west Africa, southeast Asia, and the Pacific region. To eradicate yaws by 2020, WHO has launched a campaign of mass treatment with azithromycin. Progress has been made towards achievement of this ambitious goal, including the validation of point-of-care and molecular diagnostic tests and piloting of the strategy in several countries, including Ghana, Vanuatu, and Papua New Guinea. Gaps in knowledge need to be addressed to allow refinement of the eradication strategy. Studies exploring determinants of the spatial distribution of yaws are needed to help with the completion of baseline mapping. The finding that Haemophilus ducreyi causes lesions similar to yaws is particularly important and further work is needed to assess the effect of azithromycin on these lesions. The integration of diagnostic tests into different stages of the eradication campaign needs investigation. Finally, studies must be done to inform the optimum mass-treatment strategy for sustainable interruption of transmission.

Molecular differentiation of Treponema pallidum subspecies in skin ulceration clinically suspected as yaws in Vanuatu using real-time multiplex PCR and serological methods.

We developed a TaqMan-based real-time quadriplex polymerase chain reaction (PCR) to simultaneously detect Treponema pallidum subspecies pallidum, T. pallidum subsp. pertenue, and T. pallidum subsp. endemicum, the causative agents of venereal syphilis, yaws, and bejel, respectively. The PCR assay was applied to samples from skin ulcerations of clinically presumptive yaws cases among children on Tanna Island, Vanuatu. Another real-time triplex PCR was used to screen for the point mutations in the 23S rRNA genes that have previously been associated with azithromycin resistance in T. pallidum subsp. pallidum strains. Seropositivity by the classical syphilis serological tests was 35.5% among children with skin ulcerations clinically suspected with yaws, whereas the presence of T. pallidum subsp. pertenue DNA was only found in lesions from 15.5% of children. No evidence of T. pallidum subsp. pertenue infection, by either PCR or serology was found in ∼59% of cases indicating alternative causes of yaws-like lesions in this endemic area.

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.

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.

Introducing new diagnostics into STI control programmes: the importance of programme science.

Many innovative diagnostic technologies will become commercially available over the next 5-10 years. These tests can potentially transform the diagnosis of sexually transmitted infections but their introduction into control programmes can be hampered by health system constraints, and political, cultural, socioeconomic and behavioural factors. We used the introduction of syphilis rapid tests to illustrate the importance of programme science to address the gap between accruing evidence of acceptable test performance and the complexity of programme design, implementation and evaluation of test deployment to address public health needs and improve patient-important outcomes.

Prevalence and associations of genital ulcer and urethral pathogens in men presenting with genital ulcer syndrome to primary health care clinics in South Africa.

BACKGROUND: This study aimed to determine the prevalence of genital ulcer and urethral pathogens, as well as their association with clinical features, in men with genital ulcer disease (GUD) enrolled in a clinical trial. METHODS: Clinical data were collected by questionnaire. Ulcer swabs were tested for herpes simplex viruses (HSV-1/2), Treponema pallidum, Haemophilus ducreyi, and Chlamydia trachomatis L1-L3. First-pass urine was tested for urethral pathogens, namely Neisseria gonorrhoeae, C. trachomatis, Trichomonas vaginalis, and Mycoplasma genitalium. Pathogens were detected by real-time molecular assays. Blood was tested for HIV, HSV-2, and syphilis-associated antibodies. Pathogens and clinical associations were investigated using the χ test. RESULTS: A total of 615 men with GUD were recruited. Herpes simplex virus (HSV-1, 4.2%; HSV-2, 98.2%) and bacterial pathogens were detected in 451 (73.6%) and 48 (7.8%) of genital ulcers, respectively. Human immunodeficiency virus, HSV-2, and treponemal antibodies were detected in 387 (62.9%), 434 (70.6%), and 141 (23.0%) men, respectively, whereas 54 men (8.8%) were rapid plasmin reagin (RPR) seropositive. A total of 223 urethral infections were diagnosed in 188 men (30.6%), including 69 (11.2%) M. genitalium, 64 (10.4%) T. vaginalis, 60 (9.8%) C. trachomatis, and 30 (4.9%) N. gonorrhoeae infections. Dysuria was reported by 170 men (27.6%), and 69 men (11.5%) had urethral discharge on examination. Urethral pathogens were detected in 102/409 (24.9%) men without these clinical features. CONCLUSIONS: Herpes accounted for most GUD cases and urethral pathogen coinfections were common. Erythromycin, dispensed to treat infrequent chancroid and lymphogranuloma venereum cases, provided additional treatment of some asymptomatic urethral pathogens. Additional antibiotics would be required to treat asymptomatic trichomoniasis and gonorrhea.

The molecular diagnosis of sexually transmitted genital ulcer disease.

Highly sensitive and specific nucleic acid amplification tests (NAATs) have emerged as the gold standard diagnostic tests for many infectious diseases. Real-time PCR has further refined the technology of nucleic acid amplification with detection in a closed system and enabled multiplexing to simultaneously detect multiple pathogens. It is a versatile, fast, and high-throughput system for pathogen detection that has reduced the risk of PCR contamination, eliminated post-PCR manipulations, and improved the cost-effectiveness of testing. In addition, real-time PCR can be applied to self-collected noninvasive specimens. Here, we describe an in-house developed TaqMan-based real-time multiplex PCR (M-PCR) assay for the diagnosis of sexually transmitted genital ulcer disease (GUD) and discuss briefly on issues associated with validation of assay performance.

Cost-effectiveness of a dual non-treponemal/treponemal syphilis point-of-care test to prevent adverse pregnancy outcomes in sub-Saharan Africa.

BACKGROUND: A dual nontreponemal/treponemal point-of-care test (Dual-POC) that simultaneously detects both nontreponemal and treponemal antibodies has been developed and evaluated. In this study, we compare the health and economic outcomes of the new test with existing syphilis tests/testing algorithms in a high prevalence setting. METHODS: We used a cohort decision analysis model to examine 4 testing/screening algorithms; the Dual-POC test, the laboratory-based rapid plasma reagin and Treponema pallidum haemagglutination assay (RPR+TPHA) algorithm, an onsite RPR testing, and point-of-care treponemal immunochromatographic strip (ICS) testing. Outcomes included miscarriage, stillbirth, congenital syphilis, low birth weight, and neonatal death. Disability-adjusted life-years were estimated for all health outcomes. The analytic horizon was the life expectancy for the mother and child. RESULTS: For a cohort of 1000 pregnant women in a historically high syphilis prevalence population (10% infected and 15% previously infected), the model predicted a total of 39 adverse pregnancy outcomes if no serologic screening were performed; 13 for the laboratory-based RPR+TPHA; 11 for the on-site RPR strategy; 5 for the Dual-POC strategy; and 2 for the ICS strategy. On the basis of assumption that the cost of ICS and the Dual-POC tests were the same, the ICS strategy was the most cost saving (saved $30,000) followed by the Dual-POC strategy (saved $27,000). CONCLUSIONS: The dual-POC test may help save cost in resource-poor settings where disease prevalence (and loss to follow-up) is high, while substantially reducing overtreatment.

Laboratory-confirmed case of yaws in a 10-year-old boy from the Republic of the Congo.

We report a case of yaws in a patient with puritic cutaneous eruption who was initially suspected of infection with monkeypox. The diagnosis was established by real-time PCR and sequencing of specific treponemal DNA sequences. This is the first report describing the use of DNA sequencing to identify Treponema pallidum subsp. pertenue-specific sequences in a patient with active yaws.

Evaluation of three serological tests manufactured in Belarus for the diagnosis of syphilis.

The performance of three serological tests manufactured in Belarus for the diagnosis of syphilis, i.e. a microprecipitation reaction (MPR) and two enzyme-linked immunosorbent assays (ELISAs) were compared with internationally recognized assays, namely the rapid plasma reagin test and the Treponema pallidum passive particle agglutination assay (TPPA). Sera from 392 consecutive patients attending Brest (Belarus) regional dermatovenereological dispensaries were tested. The sensitivity of the MPR test was low (77.3%) compared with the rapid plasma reagin test, while the specificity was high (100%). In contrast, both Belarusian ELISAs performed well when compared with the TPPA (sensitivities of 99.2% and 100%, specificities of 98.7% and 99.0%, respectively). There is a clear need to improve the sensitivity of the existing Belarusian MPR test or to use a more sensitive screening test in order to improve diagnosis of the disease in Belarus.

Gonococcal resistance: are cephalosporins next?

The development of resistance to multiple antibiotics has limited treatment options for gonorrhea in many countries. Currently, the Centers for Disease Control and Prevention only recommend cephalosporin antibiotics for treatment of uncomplicated gonorrhea. Although the cephalosporins remain effective, the demonstrated ability of Neisseria gonorrhoeae to develop resistance has raised concerns about the possibility of multidrug-resistant N. gonorrhoeae strains, which include cephalosporin resistance. This article provides a review of global trends in cephalosporin susceptibility among gonococcal isolates, recent findings that deepen our understanding of genetic mechanisms of resistance, and the public health and clinical implications of the potential emergence of cephalosporin-resistant gonorrhea.

Serologic testing for syphilis in the United States: a cost-effectiveness analysis of two screening algorithms.

BACKGROUND: The introduction of automated treponemal enzyme immunoassays and chemiluminescence assays (EIA/CA) tests has led some laboratories in the United States to use new syphilis screening algorithms that start with a treponemal test. We compared the economic and health outcomes of this new algorithm with the standard algorithm from the perspective of the United States health system. METHODS: We used a cohort decision analysis to estimate the expected costs and effects (including follow-ups and overtreatment) of the 2 algorithms from a health-care system perspective. In the standard algorithm, rapid plasma reagin (RPR) is followed (if reactive) by EIA/CA (Nontreponemal-First). In the new algorithm, EIA/CA is followed (if reactive) by RPR. If the RPR is negative, Treponema pallidum passive particle agglutination assay (TP-PA) test is used (Treponemal-First). RESULTS: For a cohort of 200,000 individuals (1000 current infections and 10,000 previous infections), the net costs were $1.6 m (Treponemal-First) and $1.4 m (Nontreponemal-First). The Treponemal-First option treated 118 more cases (986 vs. 868) but resulted in a substantially higher number of follow-ups (11,450 vs. 3756) and overtreatment (964 vs. 38). Treating the additional 118 cases might prevent 1 case of tertiary syphilis. The estimated cost-effectiveness ratios were $1671 (Treponemal-First) and $1621 (Nontreponemal-First) per case treated. The overtreatment was a function of the specificity of the EIA/CA and the lack of independence of EIA/CA and TP-PA. CONCLUSION: The Treponemal-First option costs slightly more and results in more unnecessary treatment.

The tale of two serologic tests to screen for syphilis--treponemal and nontreponemal: does the order matter?

BACKGROUND: Standard syphilis screening involves an initial screening with a nontreponemal test and confirmation of positives with a treponemal test. However, some laboratories have reversed the order. There is no detailed quantitative and qualitative evaluation for the order of testing. In this study, we analyzed the health and economic outcomes of the order of testing for the 2 serologic tests used in syphilis screening under pure screening settings. METHODS: We used a cohort decision analysis to examine the health and economic outcomes of the screening algorithms for low and high prevalence settings. The 2-step algorithms were nontreponemal followed by treponemal (Nontrep-First) and treponemal followed by nontreponemal (Trep-First). We included the 1-step algorithms (treponemal only [Trep-Only] and an on-site nontreponemal only [Nontrep-Only]) for comparison. We estimated overtreatment rates and the number of confirmatory tests required for each algorithm. RESULTS: For a cohort of 10,000 individuals, our results indicated that the overtreatment rates were substantially higher (more than 3 times) for the 1-step algorithms, although they treated a higher number of cases (over 15%). The 2-step algorithms detected and treated the same number of individuals. Among the 2-step algorithms, the Nontrep-First was more cost-effective in the low prevalence setting ($1400 vs. $1500 per adverse outcome prevented) and more cost-saving ($102,000 vs. $84,000) in the high prevalence setting. CONCLUSIONS: The difference in cost was largely due to the substantially higher number of confirmatory tests required for the Trep-First algorithm, although the number of cases detected and treated was the same.