Sharon K. Sahi

Normalization of Serum Rapid Plasma Reagin Titer Predicts Normalization of Cerebrospinal Fluid and Clinical Abnormalities after Treatment of Neurosyphilis

BACKGROUND Success of neurosyphilis treatment is defined by normalization of cerebrospinal fluid (CSF) and clinical abnormalities. The goal of this study was to determine whether normalization of serum rapid plasma reagin (RPR) titer could accurately predict treatment success. METHODS One hundred ten patients who were enrolled in a longitudinal study of CSF abnormalities in syphilis had asymptomatic syphilitic meningitis, symptomatic syphilitic meningitis, or syphilitic eye disease and were treated for neurosyphilis. At 4, 7, and 13 months after treatment, serum RPR titer and CSF and clinical abnormalities were analyzed for normalization. Odds ratios for normalization of each CSF and clinical abnormality when serum RPR titer had normalized and the positive predictive value of normalization of serum RPR titer for normalization of CSF and clinical abnormalities were determined. RESULTS Serum RPR titer had normalized in 63 patients (57%) by 4 months after treatment, in 94 (85%) by 7 months, and in 97 (88%) by 13 months. Except for CSF protein concentration, normalization of serum RPR titer predicted normalization of other CSF and clinical abnormalities in >80% of patients at 4 months, >85% at 7 months, and >90% at 13 months. The odds of normalization of CSF and clinical abnormalities were 28-57-fold higher when serum RPR titer had normalized, compared with when it had not. Normalization of serum RPR titer was consistently less accurate in predicting treatment success in human immunodeficiency virus-infected patients who were not receiving antiretroviral therapy, compared with those who were receiving such therapy. CONCLUSIONS In most instances, normalization of serum RPR titer correctly predicts success of treatment of neurosyphilis, and follow-up lumbar puncture can be avoided.

CXCL13 as a cerebrospinal fluid marker for neurosyphilis in HIV-infected patients with syphilis.

Background: Asymptomatic neurosyphilis is more difficult to diagnose in human immunodeficiency virus (HIV)-infected patients because HIV itself can cause cerebrospinal fluid (CSF) pleocytosis. The proportion of CSF lymphocytes that are B cells is elevated in neurosyphilis, suggesting that the CSF concentration of the B cell chemoattractant, chemokine (C-X-C motif) ligand 13 (CXCL13) concentration may also be elevated. Methods: CSF and blood were collected from 199 HIV-infected patients with syphilis and neurosyphilis. Serum and CSF CXCL13 concentrations were determined. Results: Patients with neurosyphilis had higher CSF and serum CXCL13 concentrations compared to patients with syphilis but not neurosyphilis. The odds of having symptomatic neurosyphilis were increased by 2.23-fold for every log increase in CSF CXCL13 concentration and were independent of CSF white blood cell and plasma HIV RNA concentrations, peripheral blood CD4+ T cell count and use of antiretroviral medications. A cut-off of 10 pg/mL CSF CXCL13 had high sensitivity and a cut-off of 250 pg/mL or evidence of intrathecal synthesis of CXCL13 had high specificity for diagnosis of both symptomatic and asymptomatic neurosyphilis. CSF concentrations of CXCL13 declined after treatment for neurosyphilis. Conclusions: CSF CXCL13 concentration may be particularly useful for diagnosis of neurosyphilis in HIV-infected patients because it is independent of CSF pleocytosis and markers of HIV disease.

Two Mutations associated with Macrolide Resistance in Treponema pallidum: Increasing Prevalence and Correlation with Molecular Strain Type in Seattle, Washington

Background Although azithromycin promised to be a safe and effective single-dose oral treatment of early syphilis, azithromycin treatment failure has been documented and is associated with mutations in the 23S rDNA of corresponding Treponema pallidum strains. The prevalence of strains harboring these mutations varies throughout the United States and the world. We examined T. pallidum strains circulating in Seattle, Washington, from 2001 to 2010 to determine the prevalence of 2 mutations associated with macrolide resistance and to determine whether these mutations were associated with certain T. pallidum strain types. Methods Subjects were enrolled in a separate ongoing study of cerebrospinal fluid abnormalities in patients with syphilis. T. pallidum DNA purified from blood and T. pallidum strains isolated from blood or cerebrospinal fluid were analyzed for two 23S rDNA mutations and for the molecular targets used in an enhanced molecular stain typing system. Results Nine molecular strain types of T. pallidum were identified in Seattle from 2001 to 2010. Both macrolide resistance mutations were identified in Seattle strains, and the prevalence of resistant T. pallidum exceeded 80% in 2005 and increased through 2010. Resistance mutations were associated with discrete molecular strain types of T. pallidum. Conclusions Macrolide-resistant T. pallidum strains are highly prevalent in Seattle, and each mutation is associated with discrete strain types. Macrolides should not be considered for treatment of syphilis in regions where prevalence of the mutations is high. Combining the resistance mutations with molecular strain typing permits a finer analysis of the epidemiology of syphilis in a community.

The rapid plasma reagin test cannot replace the venereal disease research laboratory test for neurosyphilis diagnosis.

Background: The cerebrospinal fluid (CSF) Venereal Disease Research Laboratory (VDRL) test is a mainstay for neurosyphilis diagnosis, but it lacks diagnostic sensitivity and is logistically complicated. The rapid plasma reagin (RPR) test is easier to perform, but its appropriateness for use on CSF is controversial. Methods: RPR reactivity was determined for CSF from 149 individuals with syphilis using 2 methods. The CSF-RPR was performed according to the method for serum. The CSF-RPR-V was performed using the method recommended for the CSF-VDRL. Laboratory-defined neurosyphilis included reactive CSF-fluorescent treponemal antibody absorption test and CSF white blood cells >20/uL. Symptomatic neurosyphilis was defined as vision loss or hearing loss. Results: CSF-VDRL was reactive in 45 (30.2%) patients. Of these, 29 (64.4%) were CSF-RPR reactive and 37 (82.2%) were CSF-RPR-V reactive. There were no instances where the CSF-VDRL was nonreactive but the CSF-RPR or CSF-RPR-V was reactive. Among the 28 samples that were reactive in all 3 tests, CSF-VDRL titers (median [IQR], 1:4 [1:4–1:16]) were significantly higher than CSF-RPR (1:2 [1:1–1:4], P = 0.0002) and CSF-RPR-V titers (1:4 [1:2–1:8], P = 0.01). The CSF RPR and the CSF-RPR-V tests had lower sensitivities than the CSF-VDRL: 56.4% and 59.0% versus 71.8% for laboratory-diagnosed neurosyphilis and 51.5% and 57.6% versus 66.7% for symptomatic neurosyphilis. Conclusions: Compared with the CSF-VDRL, the CSF-RPR has a high false-negative rate, thus not improving upon this known limitation of the CSF-VDRL for neurosyphilis diagnosis. Adapting the RPR procedure to mimic the CSF-VDRL decreased, but did not eliminate, the number of false negatives and did not avoid all the logistical complications of the CSF-VDRL.

Molecular Typing of Treponema pallidum in Ocular Syphilis

Background Syphilis can have many clinical manifestations, including eye involvement, or “ocular syphilis.” In 2015, an increase in reported cases of ocular syphilis and potential case clusters raised concern for an oculotropic strain of Treponema pallidum, the infectious agent of syphilis. Molecular typing was used to examine strains found in cases of ocular syphilis in the United States. Methods In 2015, after a clinical advisory issued by the Centers for Disease Control and Prevention, pretreatment clinical specimens from US patients with ocular syphilis were sent to a research laboratory for molecular analysis of T. pallidum DNA. Molecular typing was conducted on these specimens, and results were compared with samples collected from Seattle patients diagnosed with syphilis, but without ocular symptoms. Results Samples were typed from 18 patients with ocular syphilis and from 45 patients with syphilis, but without ocular symptoms. Clinical data were available for 14 ocular syphilis patients: most were men, human immunodeficiency virus–infected, and had early syphilis. At least 5 distinct strain types of Treponema pallidum were identified in these patients, and 9 types were identified in the Seattle nonocular patients. 14d/g was the most common type in both groups. An unusual strain type was detected in a small cluster of ocular syphilis patients in Seattle. Conclusions Ocular syphilis is a serious sequela of syphilis. In this preliminary study, clear evidence of a predominant oculotropic strain causing ocular syphilis was not detected. Identification of cases and prompt treatment is critical in the management of ocular syphilis.

Reduced Treponema pallidum–Specific Opsonic Antibody Activity in HIV-Infected Patients With Syphilis

BACKGROUND Human immunodeficiency virus (HIV)-infected individuals may have poorer serological responses to syphilis treatment and may be more likely to experience neurosyphilis. Treponema pallidum is cleared from sites of infection by opsonization, ingestion, and killing by macrophages. METHODS Serum samples from 235 individuals with syphilis were tested for T. pallidum-specific opsonic activity. Blood T. pallidum concentrations were determined by real-time polymerase chain reaction amplification of the tp0574 gene, and T. pallidum was detected in cerebrospinal fluid (CSF) by reverse-transcriptase polymerase chain reaction of 16S ribosomal RNA. RESULTS Opsonic activity was higher with higher serum rapid plasma reagin titers (P < .001), and in those treated for uncomplicated syphilis before serum collection (P < .001). Opsonic activity was lower in HIV-infected than in HIV-uninfected individuals even after the above factors were taken into account (P = .006). In participants in whom blood T. pallidum was detectable, those with the highest opsonic activity had lower blood T. pallidum concentrations. In multivariable analyses, there was not a significant relationship between opsonic activity and detection of T. pallidum in CSF or CSF-VDRL reactivity. CONCLUSIONS Serum T. pallidum-specific opsonic activity is significantly lower in HIV-infected individuals. Impaired T. pallidum-specific immune responses could contribute to differences in the course of disease or treatment response.

Toll-like receptor polymorphisms are associated with increased neurosyphilis risk.

Background Single-nucleotide polymorphisms (SNPs) in toll-like receptors (TLR) 1, 2, and 6 impair cell signaling in response to spirochetal lipoproteins. We investigated whether common SNPs in TLR1, TLR2, or TLR6 were associated with laboratory- or clinically-defined neurosyphilis. Methods Polymorphisms in the genes for TLR1 (a T→G mutation at position 1805), TLR2 (a G→A mutation at position 2258), and TLR6 (a C→T mutation at position 745) were sought in 456 white patients with syphilis. Laboratory-defined neurosyphilis included a reactive cerebrospinal fluid (CSF)–Venereal Disease Research Laboratory (VDRL) test. Clinically-defined neurosyphilis included new vision or hearing loss. Controls had CSF white blood cells of 5/&mgr;L or less, nonreactive CSF–Venereal Disease Research Laboratory, and no vision or hearing loss. Results Overall, 26.2% of patients had laboratory-defined and 36.2% had clinically-defined neurosyphilis. Compared with controls, patients with any of the 3 SNPs were more likely to have laboratory-defined neurosyphilis. Those with TLR2 or TLR6 SNPs were more likely to have clinically-defined neurosyphilis. These associations were independent of serum rapid plasma reagin titer. Conclusions A common TLR1 polymorphism is associated with an increased risk of laboratory-defined neurosyphilis, and common TLR2 and TLR6 polymorphisms are associated with an increased risk of both laboratory- and clinically-defined neurosyphilis. These data suggest that host factors impact the natural history of syphilis.

Macrolide Resistance in Treponema pallidum Correlates With 23S rDNA Mutations in Recently Isolated Clinical Strains

Background High rates of 23S rDNA mutations implicated in macrolide resistance have been identified in Treponema pallidum samples from syphilis patients in many countries. Nonetheless, some clinicians have been reluctant to abandon azithromycin as a treatment for syphilis, citing the lack of a causal association between these mutations and clinical evidence of drug resistance. Although azithromycin resistance has been demonstrated in vivo for the historical Street 14 strain, no recent T. pallidum isolates have been tested. We used the well-established rabbit model of syphilis to determine the in vivo efficacy of azithromycin against 23S rDNA mutant strains collected in 2004 to 2005 from patients with syphilis in Seattle, Wash. Methods Groups of 9 rabbits were each infected with a strain containing 23S rDNA mutation A2058G (strains UW074B, UW189B, UW391B) or A2059G (strains UW228B, UW254B, and UW330B), or with 1 wild type strain (Chicago, Bal 3, and Mexico A). After documentation of infection, 3 animals per strain were treated with azithromycin, 3 were treated with benzathine penicillin G, and 3 served as untreated control groups. Treatment efficacy was documented by darkfield microscopic evidence of T. pallidum, serological response, and rabbit infectivity test. Results Azithromycin uniformly failed to cure rabbits infected with strains harboring either 23S rDNA mutation, although benzathine penicillin G was effective. Infections caused by wild type strains were successfully treated by either azithromycin or benzathine penicillin G. Conclusions A macrolide resistant phenotype was demonstrated for all strains harboring a 23S rDNA mutation, demonstrating that either A2058G or A2059G mutation confers in vivo drug resistance.

Point of Care Treponemal Tests for Neurosyphilis Diagnosis

Background The laboratory diagnosis of neurosyphilis rests upon identifying cerebrospinal fluid (CSF) abnormalities, including CSF–Venereal Disease Research Laboratory (VDRL) reactivity. The CSF-VDRL may not be available in the parts of the world where neurosyphilis is most common. Treponemal immunochromatographic strip tests (ICSTs) have been developed as point-of-care tests on blood for syphilis diagnosis in resource-limited settings. Methods We optimized 3 commercial ICSTs for performance on CSF and tested CSF samples from 217 patients with syphilis. The Syphicheck-WB test (Qualpro Diagnostics, Goa, India; “Syphicheck”) was chosen for further study based on agreement with CSF-VDRL test results. We determined CSF-Syphicheck titers for 152 samples. We modified the CSF-Syphicheck for point-of-care testing in a US sexually transmitted diseases clinic and compared results on 102 paired centrifuged and uncentrifuged CSF samples obtained in the laboratory to the results obtained at point of care; results of samples diluted 1:4 were compared in a subset. Results The diagnostic sensitivity of a reactive CSF-Syphicheck (62%–64%) and the diagnostic specificity of a CSF-Syphicheck titer at or above 1:4 (79%–81%) were equivalent to the CSF-VDRL (54%–69% sensitivity, 73%–75% specificity) for laboratory and clinical neurosyphilis diagnoses. The CSF-Syphicheck normalized after neurosyphilis therapy similarly to the CSF-VDRL. The modified CSF-Syphicheck performed well at the point of care, albeit with better performance on cell-free compared with uncentrifuged CSF. Conclusions Cerebrospinal fluid treponemal ICSTs hold promise for point-of-care neurosyphilis diagnosis in regions where the CSF-VDRL is not available. Further study should address the performance of CSF ICSTs in resource-limited settings.

Cerebrospinal Fluid Treponema pallidum Particle Agglutination Assay for Neurosyphilis Diagnosis

ABSTRACT Limited data suggest that the cerebrospinal fluid Treponema pallidum particle agglutination assay (CSF-TPPA) is sensitive and a CSF Treponema pallidum hemagglutination assay (CSF-TPHA) titer of ≥1:640 is specific for neurosyphilis diagnosis. CSF-TPPA reactivity and titer were determined for a convenience sample of 191 CSF samples from individuals enrolled in a study of CSF abnormalities in syphilis (training data set). The sensitivity of a reactive test and the specificity for reactivity at serial higher CSF dilutions were determined. Subsequently, CSF-TPPA reactivity at a 1:640 dilution was determined for all available samples from study participants enrolled after the last training sample was collected (validation data set, n = 380). Neurosyphilis was defined as (i) a reactive CSF Venereal Disease Research Laboratory test (CSF-VDRL), (ii) detection of T. pallidum in CSF by reverse transcriptase PCR, or (iii) new vision loss or hearing loss. In the training data set, the diagnostic sensitivities of a reactive CSF fluorescent treponemal antibody absorption test (CSF-FTA-ABS) and a reactive CSF-TPPA did not differ significantly (67 to 98% versus 76 to 95%). The specificity of a CSF-TPPA titer of ≥1:640 was significantly higher than that of lower dilutions and was not significantly different from that of CSF-VDRL. In the validation data set, the diagnostic specificity of a CSF-TPPA titer of ≥1:640 was high and did not differ significantly from that of CSF-VDRL (93 to 94% versus 90 to 91%). Ten CSF samples with a nonreactive CSF-VDRL had a CSF-TPPA titer of ≥1:640. If a CSF-TPPA titer of ≥1:640 was used in addition to a reactive CSF-VDRL, the number of neurosyphilis diagnoses would have increased from 47 to 57 (21.3%). A CSF-TPPA titer cutoff of ≥1:640 may be useful in identifying patients with neurosyphilis when CSF-VDRL is nonreactive.