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Molecular Typing of Treponema pallidum in Ocular Syphilis

Oliver, Sara MD, MSPH; Sahi, Sharon K. BS; Tantalo, Lauren C. BS; Godornes, Charmie BS; Neblett Fanfair, Robyn MD, MPH; Markowitz, Lauri E. MD; Lukehart, Sheila A. PhD; Marra, Christina M. MD

doi: 10.1097/OLQ.0000000000000478
The Real World of STD Prevention

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.

Molecular typing identified at least five different Treponema pallidum types in ocular syphilis cases, suggesting that there is not a single type responsible for the increase in reported ocular syphilis.

From the *Centers for Disease Control and Prevention, Atlanta, GA; †Department of Neurology, and ‡Department of Medicine (Infectious Diseases), University of Washington School of Medicine, Seattle, WA

Acknowledgements: The authors would like to thank all patients and clinicians who provided samples for this investigation, as well as state and local health departments who assisted with identification of patients with ocular syphilis. The authors would also like to thank Julia Dombrowski, MD for assistance in obtaining samples from patients with syphilis in the Seattle & King County Public Health STD Clinic, and Tom Peterman, MD with CDC’s Division of STD Prevention for support of this project.

Conflict of interest: None declared.

Sources of support: Portions of this work were supported by National Institute of Health, National Institute of Neurological Disorders and Stroke grant number NS082120.

Correspondence: Sara Oliver, MD, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA 30329. E-mail:

Received for publication February 29, 2016, and accepted April 14, 2016.

Syphilis is a genital ulcer disease caused by the spirochete Treponema pallidum subspecies pallidum and can result in a variety of clinical manifestations. Reported primary and secondary syphilis cases in the United States have been increasing since 2001, with the total number of primary and secondary syphilis cases reported to the Centers for Disease Control and Prevention (CDC) increasing 15.1% from 2013 to 2014.1

Syphilis can have a variety of clinical manifestations, including involvement of the eye. Ocular syphilis, an inflammatory eye disease, can involve almost any eye structure.2 Uveitis is the most common manifestation, and there have been reports of significant vision loss, including blindness.3–5 Ocular syphilis can occur at any stage, with acute ocular inflammation during early (primary, secondary, and early latent) infection and a more indolent “chronic” inflammation during late or tertiary infection.4,6

Historically, limited studies of the expected frequency of ocular syphilis have been performed; results found ocular syphilis in 0%7 to 7.9%8 of patients with secondary syphilis and from 3%9 to 51%10 of patients with neurosyphilis. In a recent study, 7.9% of syphilis patients reported new onset of visual or hearing disturbances; of these, 44% had cerebrospinal fluid (CSF) findings consistent with neurosyphilis.11 However, most studies have not taken a systematic approach for identification of ocular involvement, and ocular syphilis has not been a reportable manifestation to CDC, therefore no “baseline rates” are available.

Molecular typing of T. pallidum can potentially provide additional information on the epidemiology of syphilis. In 1998, a method was published to distinguish various molecular types of T. pallidum using 2 genetic targets (variable number of 60-base pair repeats in the acidic repeat protein [arp] gene and restriction fragment length polymorphism [RFLP] analysis of the T. pallidum repeat [tpr] subfamily II genes)12; a third target involving sequence analysis of the tp0548 gene was added in 2010.13 Since the development of this molecular typing system, substantial diversity has been identified in strains of T. pallidum globally.14 In Seattle, a specific strain type (14d/f) was associated with neurosyphilis, suggesting enhanced neurotropism by certain strains,13 which had been suggested previously in observational studies.15

In early 2015, 2 clusters of ocular syphilis were reported in California and Washington,16 leading CDC to issue a Clinical Advisory in April of that year.17 Since then, many jurisdictions reported an increase in cases of ocular syphilis, raising concern for an “outbreak.”17 Clinical specimens from the ocular syphilis cases in Seattle, WA, were typed, and a strain type (8d/g) not previously identified in Seattle nor widely found in the United States was identified in 2 of the ocular syphilis cases and implicated in a third case. A broader investigation was then initiated to describe molecular types detected in ocular syphilis cases nationally.

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Ocular Syphilis Cases

Prompted by the clinical advisory,17 clinicians or laboratories with preantibiotic clinical samples (whole blood, serum, swabs of lesion exudate, CSF or ocular fluid) from patients with ocular syphilis contacted CDC, which facilitated shipment of the specimens to a research laboratory at the University of Washington. Deidentified demographic information was collected on the patients, as well as information on the patients' syphilis diagnoses and ocular involvement. A case of ocular syphilis was defined as a person with clinical symptoms or signs consistent with ocular disease and syphilis of any stage. Specimens from the convenience sampling of ocular syphilis cases described here were obtained from cases identified in 2015.

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Nonocular Syphilis Cases From Seattle

Blood and oral or genital swab samples were collected anonymously from 88 patients with syphilis in the Seattle & King County Public Health sexually transmitted disease (STD) Clinic in Seattle, WA. None of these individuals had symptoms of ocular syphilis. All samples were collected in 2015.

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Laboratory Methods

Frozen samples for determination of strain type included blood in ethylenediaminetetraacetic acid tubes; CSF; sera; ocular fluids; and oral or genital swabs collected in 1 mL 1× lysis buffer (10 mM Tris-HCl, 0.1 M ethylenediaminetetraacetic acid, 0.5% sodium dodecyl sulfate) before freezing. T. pallidum DNA was extracted from blood using a QIAamp DNA Blood Midi kit (Qiagen, Valencia, CA) according to the manufacturer's instructions, precipitated overnight at −20°C with 2.5 volumes of 100% ethanol, 0.1 volumes of 3 M sodium acetate, and 1 μL of 20 mg/mL glycogen and resuspended in a final volume of 50 μL molecular grade water. Treponema pallidum DNA was extracted from samples other than blood using a QIAamp DNA Mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions.

Strain typing was based on analysis of three DNA targets: (1) the number of 60bp repeats in the arp gene; (2) RFLP analysis of the tpr subfamily II genes (tprE [tp0313], tprG [tp0317], tprJ [tp0621]); and (3) sequence analysis of a short region of the tp0548 gene (bp131–215). The first 2 targets were analyzed according to published methods13 except that the tprE, G, and J amplicon was reamplified for 20 cycles in the case of a low yield of DNA and the polymerase chain reaction products were gel extracted using a QIAquick Gel Extraction kit (Qiagen) according to the manufacturer's instructions before RFLP analysis. The tp0548 sequence from blood samples was determined as previously described.13 For samples other than blood, an alternate antisense primer was used (5′ TGC CCT GGT GTC CGC CGT TCT 3′) with the following touchdown cycling conditions: 95°C for 5 minutes, then 10 cycles of 95°C for 1 minutes, 65°C for 30 seconds with a 1°C decrease per cycle, and 72°C for 30 seconds; then followed by 35 cycles of 95°C for 1 minutes, 55°C for 30 seconds, and 72°C for 30 seconds, followed by a final extension for 10 minutes at 72°C. Polymerase chain reaction products were purified using ExoSap-It (Affymetrix, Santa Clara, CA) before sequencing. Assignment of molecular type was accomplished as previously described.13 Because of limiting amounts of DNA in the samples, in some cases, only 1 or 2 molecular typing targets could be defined. When multiple samples from the same patient were tested, the most complete type was assigned to that individual.

Review board at CDC determined this investigation not to be human subjects research, with a primary intent for public health practice or disease control activity. Similarly, samples from syphilis patients in Seattle were collected as a part of disease control activity.

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Ocular Syphilis Cases

The laboratory received samples from 36 subjects with ocular syphilis and attempted molecular typing on 23 CSF samples, 12 whole blood and 5 serum samples, and 7 ocular fluid samples (either vitreous or aqueous fluid). Complete typing results for all 3 molecular targets were available for 8 (22%) samples and an additional 10 (27%) samples had a positive result for at least one of the molecular targets. From those 18 patients, clinical information was available for 14 (78%) who had probable ocular syphilis based on examination by an ophthalmologist. Clinical characteristics, laboratory results, and molecular typing results for these 14 patients are described in Table 1.



Thirteen (93%) of the 14 patients were men, and of those, 8 (62%) reported having sex with men. The median age was 45 years (range, 24–63 years). All patients had been tested for human immunodeficiency virus (HIV) and 8 (57%) were HIV-infected. Ten (71%) had early syphilis (primary, secondary, or early latent); secondary syphilis was the most common stage, seen in 5 (36%). The median rapid plasma reagin titer was 256 (range 64–4096). There was no difference in median rapid plasma reagin titers by HIV status. Specific ocular diagnoses were available for all patients. Eight (62%) had panuveitis, and 5 (38%) had retinitis or chorioretinitis.

At least 5 distinct molecular types of T. pallidum were identified in the ocular syphilis patients (Fig. 1). Three types in the arp gene were identified: types 8, 13, and 14. All samples were tpr RFLP type d. Three tp0548 types were identified: types c, f, and g. Type 8d/g was identified in 2 of the first 3 samples tested; however, that type was not subsequently detected. No specific molecular typing result was associated with any specific ocular diagnosis.

Figure 1

Figure 1

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Seattle Nonocular Types

Complete molecular typing results were available for 30 (34%) of 88 patients, including 2 from blood, 5 from oral swabs, and 23 from genital swabs; partial results were available for an additional 15 (17%) samples including 3 from blood, 7 from oral swabs, and 5 from genital swabs. At least 9 distinct types of T. pallidum were identified among these patients without ocular symptoms. Types from Seattle patients with and without ocular symptoms are displayed in Figure 2.

Figure 2

Figure 2

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We found at least 5 different T. pallidum types among the ocular syphilis cases from which specimens were collected and able to be typed. This diversity likely represents the breadth of molecular types circulating in local networks, as demonstrated by the typing results from Seattle, and suggests that there is not a single type responsible for an overall increase in reported ocular syphilis. The most common type identified from the Seattle STD clinics was 14d/g. This type was also identified in 4 of the 6 ocular syphilis cases who had clinical information and complete typing results. This is consistent with previous observations that 14d strains are the most commonly identified T. pallidum strains globally.14

Although we did not detect a single predominant strain in patients with ocular syphilis, our findings are not inconsistent with the possibility of oculotropic strains. In the initial Seattle cluster,16 2 sexual partners were diagnosed with ocular syphilis. Molecular typing results were available for one of these partners, who was infected with type 8d/g. It is reasonable to infer that the “untyped partner” was infected with the same 8d/g strain as his partner. Another ocular syphilis case from Seattle was typed as 8d/g, but no epidemiologic link to the two other cases was found. This finding could represent a focal Seattle cluster of 8d/g ocular syphilis caused by an “oculotropic strain” within a background level of ocular syphilis caused by commonly circulating strains. Such a scenario would be similar to observations of possible T. pallidum strain-specific neurovirulence. Specifically, individuals infected with type 14d/f are more likely to have reactive CSF VDRL than individuals infected with other strains, but reactive CSF VDRLs are not exclusively seen in those infected with type 14d/f.13

Since the Clinical Advisory was issued, CDC has been notified of over 200 cases of ocular syphilis from 20 states over the last 2 years. Similar to the epidemiology of syphilis in the United States, most cases of suspected ocular syphilis reported to CDC (over 90%) were in men, especially in men who have sex with men (MSM). Several of the cases have resulted in significant sequelae, including blindness. In some cases, there was a delay in diagnosis and treatment due to the lack of awareness of ocular syphilis. Given the number of cases reported, CDC has worked with both local and national partners to increase awareness of syphilis as a cause of eye inflammation, which can occur in any stage of infection. Syphilis diagnostic testing with treponemal and nontreponemal tests is encouraged in any patient with visual complaints without another known cause among those at risk of syphilis, especially MSM and those who are HIV-infected. Additionally, all patients diagnosed with syphilis should be assessed for any neurologic complaints, including vision or hearing loss. Patients with syphilis and ocular complaints should receive immediate ophthalmologic evaluation.

The 2015 STD Treatment Guidelines state that “Syphilitic uveitis or other ocular manifestations (eg, neuroretinitis and optic neuritis) can be associated with neurosyphilis. A CSF examination should be performed in all instances of ocular syphilis, even in the absence of clinical neurologic findings. Ocular syphilis should be managed in collaboration with an ophthalmologist and according to the treatment and other recommendations for neurosyphilis, even if a CSF examination is normal.”18 Recommended treatment for neurosyphilis and ocular syphilis is 18 to 24 million units intravenous aqueous penicillin G daily, given as continuous infusion or divided into every 4-hour dosing, for 10 to 14 days. In addition, although CSF examination is recommended, this should not delay initiation of treatment.

There are several limitations of this study that are important to note. The specimens from patients with ocular syphilis represent a convenience sample, without any systematic collection. All syphilis molecular typing must be interpreted in the context of the background of local syphilis molecular epidemiology. Although we have provided information on the circulating types in Seattle, Washington in 2015 as an example of the diversity of molecular types seen in 1 city, we do not have current national syphilis molecular epidemiology data for comparison.

Also, as these samples from ocular syphilis patients were not collected with a standardized protocol, some samples were handled in ways that allowed for substantial DNA degradation. This is demonstrated in a lower yield of successful complete molecular typing from the national ocular syphilis samples (22%) compared with the samples collected at the Seattle STD clinic (34%), where samples were frozen immediately after collection. Historically, blood and CSF have lower yield for strain typing compared with samples collected from primary and secondary lesions.14 Additionally, as the molecular methods used to classify T. pallidum strain types are focused on 3 specific genetic loci, they could potentially miss identifying other genetic signatures that may confer an oculotropic phenotype. Further investigations are warranted, including whole genome sequencing of T. pallidum from patients with uncomplicated and ocular syphilis.

Ocular syphilis is a serious complication of syphilis. The increase in reports of ocular syphilis in 2015 in the United States could be due to increased recognition of ocular manifestations in the setting of increased syphilis rates, or a true increase in the percentage of syphilis cases with ocular disease. Additional epidemiological investigations may shed light on other contributors to risk of ocular syphilis, including demographic or clinical factors, or the possibility that increased awareness of ocular complications of syphilis has led to increased case ascertainment. Further investigation is needed to evaluate molecular epidemiology of the syphilis epidemic in general, and specifically of those cases diagnosed with ocular syphilis. However, based on this preliminary study, there does not seem to be a single strain responsible for ocular syphilis nationally. A larger sample size is necessary to support these preliminary conclusions. Prompt identification of cases with potential ocular syphilis, ophthalmologic evaluation, and appropriate treatment is critical in the prevention and management of visual symptoms and sequelae of ocular syphilis.

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