Several previous studies have used the CDC method to investigate T. pallidum
subtype prevalence among communities. For example, Sutton and colleagues determined CDC subtypes from T. pallidum
DNA isolated from blood and genital ulcer swabs during an outbreak of syphilis in Phoenix, AZ [2
]. About half of the samples were CDC subtype 14f. Nine other types were identified, including 14d. Subtypes 14d and 14f have been identified in diverse geographic settings, including North and South Carolina [4
], Lisbon [7
], Scotland [8
], South Africa [1
] and China [20
]. In samples examined in our study, CDC subtype 14d was most common, and we did not identify any subtype 14f samples. By enhancing the CDC subtyping method with sequence analysis of a small region of the tp0548
gene, we were able to separate 14 CDC subtypes into 24 different strain types. Importantly, we were able to separate the subtypes with 14 arp
repeats into 8 individual strain types.
We show a change in circulating strain types in Seattle, Washington, between 1999 and 2008, with introduction and disappearance of strain types in the region during that period. Retrospectively, we also saw disappearance of two strains that were evident in Seattle in the 1980s compared to the strains identified between 1999 and 2008. The expansion of the introduced types within our recent, predominantly MSM, cohort is consistent with one or more overlapping sexual networks in the MSM community in Seattle. Moreover, we saw a strong trend toward an association between strain type and HIV status. This finding further suggests that there are separate networks within MSMs in our community and is consistent with serosorting such that patients choose sexual partners based on their HIV status. We identified an unusual type (type 16a/e) in only two individuals in 2004, without subsequent detection in other patients. These two individuals were African American heterosexual partners. In addition, a single African American patient was infected with a type 15e/e strain. The finding of these unique strains in African American patients in the setting of a predominantly white MSM epidemic provides additional evidence for separate sexual networks.
Many of the samples examined in this study have also been tested for 23S rDNA mutations associated with macrolide resistance in T. pallidum
], and we considered including either or both of these mutations in our typing scheme. Although these markers were able to distinguish among strains within some types, we were uncertain about the long term stability of this marker within a strain, particularly in light of studies that have shown the association of the A2058G mutation with prior exposure to macrolides [9
]. Similarly, in a small number of samples, we examined two other molecular targets that proved to be unsuitable for strain typing: sequence analysis of the tp0136
], and determination of the number of 24 bp repeats in the tp0470
gene. Sequence analysis of the tp0136
gene lacked discriminatory ability, and the number of repeats in the tp0470
gene varied within a single bacterial isolate (data not shown).
The association between strain type and neurosyphilis is particularly notable. Although literature from the first part of the 1900s discusses the existence of “neuroinvasive” or “neurotropic” strains of T. pallidum,
this concept has not been rigorously studied in humans. In a rabbit model, we showed that the clinical phenotype of disease differed depending on the inoculating strain [23
]. In that study, rabbits were infected with three different strain types: type 14a/a, 14e/b and 14d/f. Animals infected with one type 14a/a strain and one type 14d/f strain had the greatest degree of neuroinvasion. In the study reported here, we found that patients with neurosyphilis were most commonly infected with type 14d/f, consistent with the rabbit studies. We cannot comment on the neuroinvasive potential of type 14e/b in our human study, as none of the Seattle patients was infected with this strain. T. pallidum
type 14d/f strains may be more neuroinvasive, or may be better able to evade host immune responses in the central nervous system (CNS) than other strain types.
The ability to identify the infecting strain types in patients with syphilis could have important clinical implications. Controversy abounds regarding which patients with syphilis should undergo lumbar puncture to evaluate the possibility of neurosyphilis. Currently, the best indicator of risk is serum RPR titer ≥ 1:32, but that criterion leads to some false positives [17
]. If strain type could be determined in a blood sample, it could help to identify those patients who are at greatest risk for neurosyphilis. These individuals, particularly those with RPR titer ≥ 1:32, could be then targeted for lumbar puncture or empiric neurosyphilis treatment. Our data suggest that future larger investigations of the correlation between strain type and the clinical or laboratory markers that indicate increased risk of neurosyphilis are warranted. In addition, the association between specific strain type and neurosyphilis could lead a new understanding of the molecular mechanisms underlying neuroinvasion.
The enhanced typing method that we describe shows biological and clinical relevance, as well as epidemiological utility. It represents a significant advance in our ability to study the molecular epidemiology of syphilis, and it offers the potential to learn more about the pathogenesis of CNS disease. Future epidemiological studies that combine social network analysis and strain typing data are required to determine the ultimate utility of this new typing method for syphilis investigation and control. Similarly, continued study of risks for neurosyphilis is required to determine the ultimate role of strain typing as part of risk assessment.