Treponema & Others

TAXONOMY Back to top

The recognition of spirochetes as human host-associated organisms is believed to date from

nearly 400 years ago, when Van Leeuwenhoek described spiral, nimble “animalcules” in

human oral plaque (61). Determination of taxonomic relationships among spirochetes has

been complicated by their fastidious nature and the refractoriness of many to cultivation.

Numerous phenotypic traits have been examined in attempts to establish taxonomic

hierarchies (113). Paster et al. (119) demonstrated using 16S rRNA sequences that

spirochetes can be grouped into a phylum of five clusters, Treponema, Spirochaeta, Borrelia,

Serpula (now Brachyspira), andLeptospira. The relatedness among members of clusters

varied considerably. Interspecies similarities among borrelia were >97%, suggesting recent

evolutionary divergence. In contrast, the approximate 10% sequence differences among

treponemes pointed toward divergence over a much greater evolutionary time frame.

Many investigators formerly believed that cultivatable treponemes were closely related,

nonpathogenic forms of Treponema pallidum (32, 129, 157). Miao and Fieldsteel (96, 97)

dispelled this idea by demonstrating that T. pallidum DNA shared less than 5% homology

with DNAs of cultivatable treponemes but was indistinguishable from that of a Treponema

pertenue (yaws) strain. Consistent with rRNA data (119), they were unable to detect crosshybridization

between DNAs from many cultivatable treponemes. Their work led to the

reclassification of the agents of venereal syphilis, endemic syphilis, and yaws as T.

pallidum subsp. pallidum, T. pallidum subsp.endemicum, and T. pallidum subsp. pertenue,

respectively, while Treponema carateum, the cause of the skin disease pinta, retained its

status as a distinct species primarily because no isolates were available for study (142).

While Treponema denticola has long been considered the prototype oral treponeme, it

represents only a small fraction of the resident treponemes in patients with gingivitis and

periodontitis (118). In 1997, allSerpulina species were reclassified as Brachyspira (112).

Two Brachyspira species, B. aalborgi and B. pilosicoli, have been identified as causes of

human intestinal spirochetosis (HIS) (14, 98).

DESCRIPTION OF THE AGENTS Back to top

Treponema pallidum Subspecies

The four members of the genus Treponema that cause venereal syphilis, endemic syphilis,

yaws, and pinta are morphologically identical (65) and, despite advances in molecular

differentiation, are distinguished primarily by differences in geographic distribution,

epidemiology, clinical manifestations, and host range in experimental animals (Table 1)

(95, 136). Only T. pallidum subsp. pallidum is transmitted routinely by sexual contact and

vertically from a pregnant woman to her fetus (95, 127). It also is the only subspecies that

regularly breaches the blood-brain barrier (127). The type strain of T.

pallidum subsp. pallidum (Nichols) was isolated in 1912 from the cerebrospinal fluid of an

individual with secondary syphilis (108) and has been propagated since by intratesticular

inoculation of rabbits (87). No strain or subspecies of T. pallidum can be cultivated

continuously in vitro, although limited replication has been achieved by cocultivation with

mammalian cells (29). Rabbits are the animals of choice for studying syphilitic infection and

can be used to recover strains from clinical specimens by rabbit infectivity testing (RIT)

(87).

Host-Associated Spirochetes

Treponemes

Oral treponemes are anaerobic, spiral-shaped organisms ranging from 0.15 to 0.30 μm in

diameter and from 5 to 16 μm in length (80). They can be differentiated based on genotypic

characteristics and biochemical parameters, such as growth requirements, carbohydrate

fermentation, and enzymatic activities (142). T. denticola has been identified in specimens

from healthy patients and in individuals with periodontal disease (104). T. phagedenis, T.

refringens, and T. minutum inhabit the smegma found beneath the prepuce and in other

epithelial folds of the genital region. T. phagedenis and T. refringens are 0.20 to 0.25 μm in

diameter, whereas T. minutum tends to be smaller in diameter (0.15 to 0.20 μm) (80). T.

denticola binds to host cells and extracellular matrix components and also coaggregates with

other bacteria (Porphyromonas gingivalis andFusobacterium nucleatum) in periodontal

pockets (34). Although less invasive than T. pallidum, T. denticolaforms abscesses and

demonstrates a limited degree of hematogenous dissemination in a SCID mouse model

(42).

Brachyspira

B. aalborgi was first isolated in 1982 (66) but remains poorly characterized (150). The type

strain is comma-shaped or helical, 2 to 6 μm long and approximately 0.2 μm in width, with

tapered ends and 4 flagella at each end (66). Isolation requires weeks of incubation under

anaerobic conditions (37). B. aalborgi has not been isolated from animals. B.

pilosicoli colonizes the large intestine of a number of animal species, causes intestinal

spirochetosis in pigs, and is thought to have greater human pathogenic potential (11, 98).

Compared to B. aalborgi, isolates of B. pilosicoli are longer (4 to 12 μm), more coiled, and

have more-pointed ends (153). On blood agar plates, B. pilosicoli isolates display 2

morphologically distinct weakly β-hemolytic colony types (149). Intestinal spirochetes can be

found in all regions of the colon but increase in number from cecum to rectum (75). They

reside in the brush border surrounded by microvilli with their proximal tips embedded in

invaginations of the host cell membrane (Fig. 2). Because of high density and orientation

perpendicular to the mucosal surface, they form a characteristic basophilic fringe often

described as a “false brush border” in histologic samples stained with hematoxylin and eosin

(H&E) (Fig. 2, top) (75). Though generally noninvasive and minimally inflammatory, they

have been observed within colonic epithelial cells, subepithelial cells, and Schwann cells

(117) as well as causing crypt abscesses, ulceration, and necrosis (76). Spirochetemia has

been reported in a small number of critically ill patients with multiple-organ failure (151).

EPIDEMIOLOGY AND TRANSMISSION Back to top

Venereal Syphilis

The incidence of syphilis in the United States fell precipitously in the late 1940s following the

introduction of penicillin (107). After a nadir in the 1950s, syphilis rates began to rise with

peaks occurring every 10 years. Grassly et al. (49) contended that the oscillating nature of

syphilis epidemics can be explained by waxing and waning immunity in at-risk populations.

Fenton et al. (39) argued forcefully against this thesis, maintaining that epidemiologic

determinants are the major drivers of syphilis transmission in developed countries. After

steady declines throughout most of the 1990s to historically low levels, and despite the

launching of the National Plan to Eliminate Syphilis in the United States in 1999, rates have

increased annually since 2001 (21). These trends, mirrored throughout Western Europe

(154), reflect the resurgence of risky sexual behaviors among men who have sex with men

(MSM) (56) and also increases in syphilis among women (21). Hispanics and African

Americans, particularly those in the southeastern United States, continue to be

disproportionately affected; a recent serosurvey found the seroprevalence among all adults

aged 18 to 49 years to be 0.71% but 4.3% among non-Hispanic blacks (48, 121). In 1999,

the World Health Organization (WHO) estimated that 12 million persons acquire syphilis each

year (63). The majority of cases occur in underdeveloped countries, particularly sub-Saharan

Africa and South Asia. Eastern Europe and Russia reported dramatic increases in the

incidence of syphilis with the fall of Communism (78). Alarming increases in syphilis rates in

China, attributed to the enormous societal and economic changes in that country during the

past two decades, also have been noted (26).

Transmission of syphilis occurs after contact with primary- and secondary-stage lesions

(Table 1andFig. 3). Estimates of the risk of sexual transmission vary greatly, from 10 to

80%, with the 30% transmission rate reported by Schroeter et al. (138) a commonly quoted

figure. Vertical transmission of syphilis has been recognized for several centuries (139).

Neonates also can become infected from exposure to lesional exudate or infected maternal

blood within the birth canal (139).

Oral Treponemes: Gingivitis, Periodontal Disease, and

Atherosclerosis

The epidemiologic importance of T. denticola and other oral treponemes arises from their

occurrence as components of the polymicrobial consortium that causes gingivitis (62).

Gingivitis is ubiquitous globally in children and adults and is associated with poor oral

hygiene; comprehensive oral hygiene programs are effective in preventing or reducing

gingival inflammation (4). Chronic periodontitis is most common in adults and seniors and is

more common with cigarette smoking, obesity, diabetes, and alcohol consumption (3). The

National Survey of Employed Adults and Seniors and the Third National Health and Nutrition

Examination Survey found periodontal disease in 24% of employed adults and more than

60% of seniors (3, 24). Accumulating evidence links periodontal disease with coronary artery

disease and ischemic stroke (6, 55), although risk varies considerably among studies

(105, 120).

Intestinal Spirochetosis

The frequency of spirochetal colonization of the intestinal tract has declined dramatically in

developed countries during the 20th century but remains high in the developing world

(75). B. aalborgi, lacking animal reservoirs, is probably transmitted via the fecal-oral route

(11). Infection with B. pilosicoli likely occurs by ingestion of water contaminated by feces of

birds, animals, or infected humans (11). In developed countries, HIS prevalence is greatest

in MSM with or without human immunodeficiency virus (HIV) infection, with oral-anal contact

being the presumed mode of transmission (35, 149).

CLINICAL SIGNIFICANCE Back to top

Venereal Syphilis

Figure 3 illustrates the natural history of untreated syphilis, emphasizing the relationship

among the stages of the disease, the presence of infectious treponemes, and reactivity in

serologic tests (see below), while Table 2 describes the criteria required for staging syphilitic

infection. Although the clinical consequences of syphilitic infection may be delayed for

months to years, venereal syphilis typically commences with the appearance of one or more

mucocutaneous lesions days to weeks following inoculation (85). Primary syphilis occurs

when spirochetes replicating at the site of inoculation induce a local inflammatory response,

giving rise to one or more chancres, the defining lesion(s) of primary syphilis. The clinical

consequences of spirochete dissemination, collectively referred to as secondary syphilis,

become manifest 4 to 10 weeks after the chancre. Because chancres often are not visible in

females, women tend to present with secondary disease; a similar trend has been noted in

MSM (160). Although mucocutaneous lesions are the most common presentation, secondary

syphilis can affect any organ (85). Mucocutaneous lesions of secondary syphilis usually

resolve in 3 to 12 weeks, leading to the asymptomatic stage referred to as latency. The Oslo

Study of Untreated Syphilis showed that 25% of patients experience secondary relapses,

mostly within the first year but as late as 5 years (85). All forms of tertiary syphilis have

decreased markedly in incidence in the postantibiotic era; there are no reliable figures on the

relative frequency of late complications.

A statistical association between HIV infection and syphilis became evident in the AIDS

epidemic (72). Although initially thought to reflect similar risk factors, complex epidemiologic

and biologic relationships have been identified (72). Chancres facilitate HIV transmission by

increasing susceptibility or infectiousness (44). A multicenter prospective study sponsored by

the Centers for Disease Control and Prevention found that HIV infection had only a minimal

effect on early syphilis (133).

Early congenital syphilis is analogous to secondary syphilis and can involve almost any fetal

organ, with liver, kidneys, bone, pancreas, spleen, lungs, heart, and brain the most

frequently afflicted (115, 139). Two years of age is used to demarcate early from late

congenital syphilis, which corresponds to tertiary syphilis in the adult. The best known

stigmata of late congenital syphilis are Hutchinson’s teeth, interstitial keratitis, saddle nose

deformity, frontal bossing, and saber shins (139).

Endemic Treponematoses

Clinical features of the endemic treponematoses are summarized in Table 1. Primary yaws

(frambesia) consists of a papule at the site of inoculation that enlarges to a hyper keratotic

papilloma (“mother yaw”) before forming a shallow ulcer that can persist for months to

years. Highly infectious secondary lesions appear weeks to months later, frequently

accompanied by painful periostitis (95). After a several-year period of infectious relapses,

patients enter an asymptomatic late latent period; approximately 10% of untreated patients

progress to tertiary disease, generally consisting of solitary destructive lesions of bone or

mucocutaneous surfaces. Endemic syphilis usually begins as a generalized infection in the

absence of an obvious primary lesion and is characterized by ulcerative lesions of the

oropharyngeal mucosa (mucous patches), angular stomatitis and split papules at the corners

of the mouth, intertrigial condylomata (similar to those of venereal syphilis), painful

periostitis, and rashes (95). Tertiary gummas, most commonly on the skin, in the

nasopharynx, and in bone, may result in severe disfigurement. Pinta is unique in being

limited to skin (95). The initial lesions of primary pinta, small papules, appear after an

incubation period of 1 week to 4 months. Secondary lesions (pintids) usually appear 2 to 6

months later. Tertiary lesions consist of well-defined hyperchromic, hypochromic, achromic,

or dyschromic patches of skin.

Intestinal Spirochetosis

In 1967, Harland and Lee (53) coined the term intestinal spirochetosis to describe a

noninflammatory condition of the large bowel in which spirochetes attached end-on to the

colonic epithelium in a dense, palisade-like arrangement, forming a basophilic “false brush

border” that was easily overlooked on casual inspection (Fig. 2 top). Although they identified

spirochetes in 9 of 100 consecutive biopsy specimens examined, they were unable to relate

these findings to symptomatology. The confusion and controversy over the clinical

significance of HIS have not abated over the years despite extensive phylogenetic and

biochemical characterization of intestinal spirochetes (see above) and advances in

methodologies for their isolation and/or detection (see below).

COLLECTION, TRANSPORT, AND STORAGE OF

SPECIMENS Back to top

Treponema pallidum: Syphilis

Because T. pallidum cannot be cultivated on artificial medium, the diagnosis of syphilis has to

rely on the direct detection of the organism in clinical specimens in conjunction with serologic

tests. Due to the complex nature of syphilitic infection, there is no ideal test for the direct

detection of T. pallidum; test selection is dependent upon clinical presentation and type of

specimen obtained (Table 3). T. pallidum can be detected in lesion exudate by DF

microscopy, direct fluorescent antibody test for T. pallidum (DFA-TP), or PCR, while in some

circumstances, touch preparations or tissue impressions can also be used for DFA-TP.

Cerebral spinal fluid (CSF) is used primarily for VDRL testing, but it also can be examined by

PCR. Tissue biopsies are rarely performed on genital ulcers but are used for diagnosing

nongenital manifestations of secondary and tertiary disease. PCR can be performed on either

fixed or unfixed tissue, although unfixed tissue is recommended. Other specimens, such as

lymph node aspirate and amniotic fluid, although rarely obtained, can be examined by DF

microscopy, DFA-TP, and PCR, while placenta or cord tissue can be examined by silver

staining, immunohistochemistry (IHC), direct fluorescent antibody tissue test for T.

pallidum (DFAT-TP), and PCR.

Serum is the specimen of choice for conventional treponemal and nontreponemal

serodiagnostic tests, but whole blood and plasma also can be used in some assays. When

screening for congenital syphilis, the CDC recommends testing of the mother ’s serum rather

than cord blood. Infant’s serum is the specimen of choice for immunoglobulin M (IgM)-

specific tests, since cord blood specimens can be contaminated by maternal blood. Plasma

can be used for the rapid plasma reagin (RPR) and toluidine red unheated serum test

(TRUST) assays but not for the VDRL test because heat inactivation of plasma enhances

fibrin formation, leading to false-positive results. Also, plasma should be tested within 24

hours to avoid false-positive test results. Whole blood, serum, or plasma can be used for

rapid point-of-care (RPOC) treponemal tests although they are designed for use with whole

blood. Whole blood for PCR testing should be collected in tubes containing EDTA as an

anticoagulant.

The order of sample collection from genital ulcers or moist lesions depends on the tests to be

performed. Samples should be collected first for DF microscopy followed by DFA-TP and PCR.

Ideally, the specimen for both DF microscopy and DFA-TP should be free of red blood cells,

other microorganisms, and tissue debris. A detailed procedure for collecting a specimen for

DF examination has been described by Wheeler et al. (156). Briefly, the site is gently

cleansed and abraded with sterile gauze moistened with physiological saline until serous fluid

appears; the specimen is collected directly onto a clean glass slide and a coverslip is applied.

A specimen for DFA-TP is collected in the same manner but left to air dry for 15 minutes. To

collect a specimen for PCR, a sterile dacron- or cotton-tipped swab should be rolled firmly

along the base of the ulcer or lesion. The swab then should be suspended in a cryotube

containing 1 to 2 ml of nucleic acid transport medium such as Genelock (Sierra Molecular

Corporation, Sonora, CA) or universal transport medium (Copan Diagnostics, Murrieta, CA).

Tissue or fine-needle aspirates (e.g., lymph nodes) for silver staining, IHC, or DFAT-TP

should be fixed in 10% buffered formalin at room temperature immediately and sent to the

laboratory for paraffin embedding and sectioning. To test products of conception for

congenital syphilis, a 3- to 4-cm section of umbilical cord that hasn’t been cleansed with

soap or antimicrobial-containing solution should be obtained distal from the placenta. The

specimen should be taken soon after delivery and fixed in formalin or refrigerated if not

being processed immediately.

Lesion exudates air dried on slides for DFA-TP staining can be sent to the laboratory at

ambient temperature or on dry ice if slides were frozen after collection (80). Samples

collected in Genelock or universal transport medium for PCR can be shipped at room

temperature or with cool packs overnight. Serum and plasma should be shipped with cool

packs. Transportation of whole blood for serologic testing can be done at ambient

temperature if testing is done on-site; otherwise, samples should be stored at 4°C and

transported overnight with cool packs. Previously frozen plasma or serum must be shipped

on dry ice. CSF for serology can be transported at ambient temperature overnight or on cold

packs if also being used for PCR testing. Tissue samples for silver staining, IHC, and DFAT-TP

can be shipped in formalin at ambient temperature for paraffin embedding. Whole blood for

serology can be stored at 4°C for 48 to 72 h. Serum, plasma, and CSF for serology should be

stored at 4°C if testing will be delayed by more than 4 h and at -20°C or lower if testing will

be done more than 5 days from collection. Slides containing air-dried lesion exudates and

touch preparations for DFA-TP staining can be stored in a slide container at 4 to 29°C for up

to 2 weeks; otherwise, specimens should be fixed with acetone and stored at -20°C until

testing. Samples of unfixed tissue, ulcer exudate, mucosal or skin lesions, CSF, and amniotic

fluid should be stored at −70°C if PCR testing cannot be performed immediately. Formalinfixed

samples should be stored at room temperature prior to embedding and sectioning or

DNA extraction.

T. denticola and Other Oral Treponemes

Specimen collection for detection, isolation, or identification of T. denticola and other

commensal treponemes is not normally performed in the routine clinical management of

gingivitis or periodontitis; however, detailed methods and information for propagating these

organisms for research purposes are available (38).

Brachyspira: Intestinal Spirochetosis

Fecal samples should be collected in sterile containers and transported at ambient

temperature to the laboratory or overnight on ice if culture or DF microscopy will be

performed off-site. Rectal swabs (148) can be collected and transported in Stuart’s medium

(Becton Dickinson, Sparks, MD). Fecal samples and swabs should be processed for culture

within 24 h of collection (16, 148). Fresh colonic or rectal biopsy samples should be used for

culture, while samples for histological examination should be processed as described for T.

pallidum. Biopsy samples placed in physiological saline have been successfully used to

culture B. aalborgi (16). Fecal samples or rectal swabs can be used for isolation of strains

(10) or examination by DF microscopy (135). Cultured spirochetes from solid- or brothbased

media can be observed by phase-contrast microscopy (10). Biopsy specimens

obtained from the colon or rectum can serve as material for culture, PCR, or histological

examination by light microscopy or transmission electron microscopy (TEM) (77, 99, 135).

DIRECT DETECTION Back to top

T. pallidum

DF Microscopy

Touch preparations of primary, secondary, and early congenital syphilis lesions should be

examined by DF microscopy, but other specimens (e.g., lymph node aspirates and amniotic

fluid) may contain enough spirochetes for DF microscopy examination (Table 3). The test

must be performed within 20 min, since it relies on the observation of motile treponemes.

The DF microscopy procedure has been described in detail by Wheeler et al. (156). A stepby-

step instructional video can be obtained without charge from David Cox, Laboratory

Reference and Research Branch, Division of STD Prevention, CDC [ (404) 639-

3446 ; dlc6@cdc.gov]. T. pallidum cannot be distinguished from the other human

pathogenic treponemes using DF microscopy. DF microscopy should not be performed on

oral lesions, since T. pallidum cannot be distinguished easily from commensal oral

spirochetes.

DFA-TP

Touch preparations of lesion exudates (64) or tissue impressions (23) can serve as samples

for DFA-TP, which utilizes either a fluorescein isothiocyanate (FITC)-conjugated polyclonal or

monoclonal antibody for staining. There is no FDA-approved DFA-TP test in the United

States, although labeled polyclonal antibodies can be obtained commercially (Meridian Life

Sciences, Saco, ME; ViroStat, Portland, ME). Results with polyclonal antibodies should be

interpreted with caution, since they are not specific for pathogenic treponemes.

Silver Staining, DFAT-TP, IHC

Silver staining (either Warthin-Starry or Steiner) is used for visualizing treponemes in

paraffin-embedded samples. DFAT-TP is a modification of the DFA-TP test that enables

immunofluorescent labeling of treponemes in tissue samples (68). For DFAT-TP, tissue

sections are deparaffinized and pretreated prior to immunostaining to enhance epitope

accessibility (22). Unabsorbed polyclonal antibodies for IHC are available commercially

(Biocare Medical, Concord, CA; ViroStat, Portland, ME).

PCR

PCR is not used routinely for syphilis testing, and a commercial test is not available.

However, since 1991, numerous studies have been published using PCR for T.

pallidum detection based on several gene targets. Of these, the polA (tp0105) (92)

and tpn47 (tp0574) (116) are most commonly used. Although PCR can be performed on

many sample types, the test is most useful for genital ulcers and exudative lesions, which

can be sampled noninvasively and typically contain large numbers of treponemes.

Genomic DNA for PCR testing is usually extracted from 200 μl of nucleic acid transport

medium containing a genital ulcer swab sample using a commercial kit (e.g., QIAamp DNA

mini kit; Qiagen Inc., Valencia, CA). Laboratories with real-time PCR capability can use the

TaqMan-based multiplex PCR assay developed in the Laboratory Reference and Research

Branch, Division of STD Prevention at the CDC for testing genital ulcer disease (GUD)

samples. This assay simultaneously detects T. pallidum, Haemophilus ducreyi, and herpes

simplex viruses 1 and 2 (HSV-1 and -2), the major causative agents of GUD. Gene targets,

primers, and probes for the assay are shown in Table 4. Laboratories that lack real-time PCR

capability can use the conventional multiplex PCR assay for detection of T. pallidum, H.

ducreyi, and HSV described by Mackay et al. (Table 5) (88). If testing for H. ducreyi will not

be performed, PCR products from the multiplex reaction can be analyzed by agarose gel

electrophoresis. Otherwise, an enzyme-linked amplicon hybridization assay (88) can be used

because the amplicons for H. ducreyi and HSV cannot be distinguished on agarose gels. A

TaqMan-based real-time PCR targeting the polA gene can be used when the primary

diagnostic objective is to determine if a specimen contains just T. pallidum (22). The primers

consist of TP-1 (5′-CAGGATCCGGCATATGTCC-3′) and TP-2 (5′-

AAGTGTGAGCGTCTCATCATTCC-3′) and a probe, TP-3 (5′-

CTGTCATGCACCAGCTTCGACGTCTT- 3′), which is labeled with 6-carboxyfluorescein (FAM) at

the 5′ end and black hole quencher 1 (BHQ1) at the 3′ end. Laboratories unable to perform

real-time PCR can use a conventional assay targeting the polA gene of T. pallidum with

primers F1 (5′-TGCGCGTGTGCGAATGGTGTGGTC-3′) and R1 (5′-

CACAGTGCTCAAAAACGCCTGCACG-3′) using PCR conditions described by Liu et al. (84).

Brachyspira

Fecal samples, rectal swabs, or colon or rectum biopsy specimens can be cultured using

brain heart infusion agar (10, 77) or Trypticase soy agar medium (77) with 10% bovine

blood, 400 μg/ml of spectinomycin, and 5 μg/ml of polymyxin incubated anaerobically at

37°C. Specimens should be streaked onto agar plates within 1 h of collection (77). Colonies

of B. aalborgi appear light gray and weakly beta-hemolytic with a diameter of 1.2 mm on

brain heart infusion agar medium after 21 days of incubation (10). B. pilosicoli and B.

aalborgi appear as a thin film or as discrete, pinpoint colonies on Trypticase soy agar

medium after 5 to 14 days (10). B. aalborgicultures usually require a longer incubation

period. B. aalborgi has been successfully subcultured on brain heart infusion agar (10) and

propagated in Trypticase soy broth containing 10% fetal calf serum (16). B. pilosicoli is less

fastidious than B. aalborgi and can be subcultured on media used for its isolation.

IDENTIFICATION Back to top

Brachyspira

B. pilosicoli and B. aalborgi strains can be characterized using API-ZYM (bioMerieux, Inc.,

Durham, NC) and using biochemical tests such as indole production and hippurate hydrolysis

(16, 77, 151). A strong hippurate reaction and weak α-galactosidase activity is often used to

identify B. pilosicoli, while B. aalborgi is negative for α-galactosidase activity and gives a

weak hippurate reaction (77).

TYPING SYSTEMS Back to top

T. pallidum

Identification of variable regions within the T. pallidum genome has made possible the

development of a molecular typing system for T. pallidum (123). Typing is based on PCR

amplification and restriction fragment length polymorphism (RFLP) analysis of 3 members of

the tpr gene family (tprE [tp0313], tprG [tp0317], andtprJ [tp0621]) (Fig. 4, top) and

amplification of a variable number of 60-bp tandem repeats within arp (tp0433)(Fig.

4 bottom). To type T. pallidum strains, an approximately 1.8-kb region of tprE, G, and J is

simultaneously amplified using a nested PCR and primer pairs B1, 5′-

ACTGGCTCTGCCACACTTGA-3′, and A2, 5′-CTACCAGGAGAGGGTGACGC-3′, and IP6, 5′-

CAGGTTTTGCCGTTAAGC-3′, and IP7, 5′-AATCAAGGGAGAATACCGTC-3′, followed by

restriction digestion with MseI and RFLP analysis (124). The 60-bp repeat region of arp is

amplified with PCR primers 1A (5′-CAAGTCAGGACGGACTGTCCCTTGC-3′) and 2A (5′-

GGTATCACCTGGGGATGCGCACG-3′); an improved method has recently been developed (73).

Strain typing is performed primarily on specimens from genital ulcers and mucosal lesions,

but other specimens also have been typed (102, 145). The major strain types identified are

14a, 14d, and 14f (124, 145). Only a few specimens from epidemiologically linked cases

have been typed (41).

Nontreponemal Tests

Table 7 presents the salient features of the commercially available nontreponemal tests

along with the corresponding information for the nontreponemal components of three dual

tests, Vira Med ViraBlot (Planegg, Germany), Span SpiroLipin (Surat, India), and ChemBio

DPP Screen and Confirm (Medford, NY), that are not FDA approved. Detailed protocols for

performing the RPR, VDRL, unheated serum reagin (USR), and TRUST assays can be found

in A Manual of Tests for Syphilis published by the American Public Health Association

(http://www.apha.org) (81); an online version can be accessed

at www.cdc.gov/std/syphilis/manual-1998/.

The VDRL test is a quantitative flocculation reaction between a cardiolipin-based antigen and

serum performed on special glass slides (81). The results are read microscopically at ×100;

the highest titer causing flocculation is the endpoint. The VDRL test also is performed on CSF

to identify and manage cases of neurosyphilis. The USR microflocculation test uses a

modified VDRL antigen containing choline chloride, which eliminates the need for heating of

the serum sample (81), and is read microscopically like the VDRL. The USR test is rarely

used, since the RPR test is essentially equivalent but does not require a microscope for

determination of results. The RPR card test, performed on serum or plasma, contains finely

divided charcoal particles as a visualizing agent (81). In the RPR card test, serial dilutions of

serum or plasma (heated or unheated) are prepared on a plastic-coated card after which the

RPR antigen is added. The presence of antibodies causes flocculation, while suspensions

without antibodies remain uniformly gray. The TRUST is a macroflocculation assay, very

similar to the RPR, in which the charcoal is replaced with toluidine red (81). The sensitivity of

the TRUST is similar to that of the RPR (Table 7), while its specificity is slightly higher (80).

The VDRL-CSF is performed identically to the serum VDRL except that the VDRL antigen is

diluted 1:1 with 10% saline (79). RPR testing of CSF is not recommended.

Treponemal Tests

The salient features of the commercially available treponemal tests, as well as some that are

still developmental are summarized in Tables 8 to 11. Detailed protocols for the FTA-ABS,

microhemagglutination assay for antibodies to T. pallidum (MHA-TP), and T. pallidum particle

agglutination (TP-PA) tests can be found in A Manual of Tests for Syphilis (81).

FTA-ABS Test

In the FTA-ABS test (Table 8), a serum sample, adsorbed with an extract of T.

phagedenis Reiter (Sorbent) to remove cross-reactive antibodies, is reacted with treponemes

fixed to glass slides; FITC-conjugated anti-human immunoglobulin is used to visualize

antibody-labeled organisms. The FTA-ABS test cannot distinguish between IgG or IgM

antibodies. The serum is subjectively scored based upon the fluorescence intensity.

Standardized controls which produce negative, weak, and strong fluorescence readings must

be included in each assay. The test can be performed with unheated CSF for diagnosis of

neurosyphilis (see below). Because of the subjectivity involved in reading samples and the

need for expensive microscopy equipment, the FTA-ABS is used much less frequently than in

the past.

MHA-TP and TP-PA Tests

The MHA-TP test (Table 8) is a passive hemagglutination assay of formalinized, tanned

erythrocytes sensitized with T. pallidum antigen that can be used to test preabsorbed patient

sera; it has been supplanted by the TP-PA. The TP-PA test (Fujirebio, Inc., Tokyo, Japan) is a

modification of the MHA-TP test that uses gelatin particles sensitized with T.

pallidum antigens to reduce the number of nonspecific interactions (125). With both tests,

agglutination indicates the presence of IgG and/or IgM antitreponemal antibodies.

EIAs

With the exception of the Captia IgG (Trinity Biotech Plc; Bio-Rad Laboratories, Hercules,

CA), all of the EIAs (Table 9) currently being used for syphilis diagnosis employ recombinant

antigens and detect IgG, IgM, or both. The 15-, 17-, 44.5-, and 47-kDa antigens are the

most frequently utilized because they induce strong, persistent antibody responses (52) and

are thought to be expressed only by pathogenic treponemes (110). Although EIAs using

recombinant antigens might be expected to perform better than assays using T.

pallidumlysates (67), this has not been borne out (137). EIAs utilize one of three basic

formats or combinations: (i) direct or “sandwich,” (ii) indirect, and (iii) competitive. The

direct format uses antibody immobilized on the plastic matrix to capture serum antibodies.

Examples are the Captia IgM, TrepCheck IgM (Phoenix Bio-Tech Corporation, Mississauga,

Ontario, Canada), and Murex ICE (Murex Biotech Ltd., Dartford, United Kingdom) which use

recombinant antigen enzyme conjugates to detect serum antibodies captured by the

immobilized antibodies. In contrast, the indirect format uses immobilized antigens to capture

reactive serum antibodies. Reactive antibodies are then detected in one of two ways: either

with anti-antibody enzyme conjugates or with recombinant antigen enzyme conjugates. The

Captia IgG, TrepCheck IgG, and Murex ICE detect reactive antibodies using anti-antibody

conjugates, while the TrepSure (Phoenix Bio-Tech Corporation, Mississauga, Ontario,

Canada), Bioelisa 3.0 (Biokit, Barcelona, Spain), Enzywell (Diesse, Siena, Italy), and Syphilis

EIA II (Newmarket Laboratories Ltd., Newmarket, United Kingdom) use antigen conjugates

for detection. One disadvantage of indirect EIAs using anti-antibody conjugates can be high

background signals which give rise to false-positive results. In the direct and indirect

formats, an increasing signal indicates a more-reactive serum. The competitive format

(Pathozyme Syphilis [Omega Diagnostics, Alloa, United Kingdom], Biomerieux Trepanostika

[Organon, Turnhout, Belgium], and Behring Enzygost [Behring, Marburg, Germany]) uses

immobilized antigen to capture specific IgG and IgM antibodies from patient sera, which then

block binding of an antibody conjugate of identical specificity. With this format, optical

density is inversely related to the amount of antibody bound.

Immunoblot (WB) Tests

Immunoblotting techniques (Table 10) add specificity because they identify specific

treponemal proteins recognized by serum antibodies; they also can separately detect IgG or

IgM antibodies. Three Western blot (WB) tests are available: MarDx (Trinity Biotech,

Wicklow, Ireland), INNO-LIA (Innogenetics NV, Ghent, Belgium), and ViraBlot (ViraMed

Biotech, Planegg, Germany). The MarDx test, which uses whole T. pallidumlysate, is used to

define serum reactivity when screening and confirmatory treponemal tests are discrepant

(51). A positive test requires reactivity with 2 of the 3 major antigens (15, 17, and 47 kDa).

The INNO-LIA and ViraBlot tests are pseudo-WBs that employ four recombinant antigens

(15, 17, 44, and 47 kDa) applied onto a nitrocellulose membrane (33, 51, 89). For the INNOLIA,

a positive test requires reactivity with 3 of the 4 major antigens; if only 1 or 2 lines

react, the result is considered indeterminate. In addition to the same 4 recombinant antigens

as the INNO-LIA, the ViraBlot strip (IgG or IgM) has 5 stripes with increasing quantities of

VDRL antigen, which allows for semiquantitative nontreponemal results. A negative

nontreponemal test has a combined score of 0 (i.e., 0 of 5 stripes reactive), a weak reaction

has a combined score of 1 or 2, a medium reaction has a combined score of 3 to 6, and a

strong reaction has a combined score of 6 to 10. A weak reaction is equivalent to RPR titers

between 1 and 8, a medium reaction is equivalent to RPR titers between 8 to 32, and a

strong reaction is equivalent to RPR titers of >32. A positive treponemal reaction requires

reactivity with 2 or more of the recombinant antigens.

RPOC and Other Rapid Tests

Dual rapid tests that can use finger stick blood for nontreponemal and treponemal tests are

RPOC tests (Table 10) and are distinguished from rapid tests that require serum and provide

only treponemal test results. Rapid tests come in 2 formats: lateral flow and flowthrough

cassette. The ChemBio Screen and Confirm (ChemBio Diagnostic Systems, Inc., Medford,

NY) is a lateral-flow RPOC test that requires only 5 μl of whole blood or serum and can be

read visually or with a digital card reader for quantitative results. The Span Spirolipin (Span

Diagnostics Ltd., Surat, India) is a flowthrough RPOC test in which control, nontreponemal,

and 17-kDa antigens are located, respectively, at the 12, 4, and 8 o’clock positions. Both

RPOCs have undergone testing at the CDC and currently are being evaluated under field

conditions (18a, 18b).

Light-Based Bead-Capture Technology

The final group of serologic tests which use bead-capture technology consists of two

platforms: Luminex and chemiluminescence. Performed in 96-well microtiter plates, this

format enables high throughput. The Luminex assay differs from EIAs in two ways: (i) the

capture antibody is attached to a suspension of polystyrene beads instead of the wells, and

(ii) the polystyrene beads are dyed with fluorophores of differing intensities that confer on

each bead a unique fingerprint, enabling multiplex antibody detection. After the sandwich

immunoassay, the bead suspension is analyzed using a dual laser flow cytometry detection

system. Currently, there are three Luminex-based assays, all treponemal tests: the Abbott

Architect (Abbott Laboratories, Abbott Park, IL), the Bio-Rad BioPlex (Bio-Rad Laboratories,

Hercules, CA), and the Zeus Athena (Zeus Scientific, Branchburg, NJ). The first two are FDAapproved

and currently in use; only the Abbott Architect has been evaluated in a published

independent study (90, 159). The DiaSorin Liaison (DiaSorin S.p.A., Vercelli, Italy) is a

chemiluminescence-based assay which captures reactive patient antibodies on magnetic

beads. Unbound antibodies are removed by wash cycles, and an isoluminol-antigen

conjugate is used to bind to reactive antibodies. Positive sera are detected by the addition of

chemical reagents which produce a flash-chemiluminescent signal (91).

ANTIMICROBIAL SUSCEPTIBILITIES Back to top

T. pallidum

Routine antimicrobial susceptibility testing of T. pallidum is not practical due to the lack of a

suitable in vitro cultivation system. T. pallidum strains are highly susceptible to penicillin G,

which has been used successfully for the treatment of syphilis over the past 65 years (158).

Ceftriaxone is highly active against T. pallidum in vitro and is unquestionably effective in the

treatment of early syphilis when a sufficient number of doses is given (158). Because

efficacy data for ceftriaxone are limited, its use as an alternative therapy for syphilis is

recommended only when better-established regimens are contraindicated (158). Tetracycline

has long been the second-line drug recommended for treatment of syphilis in patients

allergic to penicillin; doxycycline, which has a much longer half-life, is equivalent to

tetracycline (47, 158). Erythromycin treatment failures were recognized many years ago

and, in one case from which an isolate was obtained, was shown to be due to infection with

an erythromycin-resistant strain (144). Enthusiasm for azithromycin, which can be

administered orally in a single 2-g dose, has been tempered by the discovery of

geographically widespread macrolide-resistant strains of T. pallidum associated with an

A2058G mutation in both copies of the bacterium’s 23S rRNA genes (86). In vitro studies

indicate that quinolone compounds have low antimicrobial activity against T. pallidum (111).

Brachyspira

The in vitro antimicrobial susceptibilities of B. pilosicoli isolates from a number of geographic

locations have been tested using an agar dilution method (10). Isolates were found to be

susceptible to amoxicillin-clavulanic acid, ceftriaxone, chloramphenicol, meropenem,

tetracycline, and metronidazole. Metronidazole remains the drug of choice for

treating B. pilosicoli infections; resistance has not been reported (8).

EVALUATION, INTERPRETATION, AND REPORTING OF

RESULTS Back to top

Direct Detection of T. pallidum

DF Microscopy and DFA-TP

Identification of a single motile T. pallidum by DF microscopy is sufficient for diagnosis. The

predictive value of DF microscopy has been difficult to discern (54), with one study (156)

claiming a 97% positivity rate in patients with clinically diagnosed primary syphilis (80% for

patients positive on their first clinic visit). The development of PCR has provided a much

needed standard for assessing the sensitivity and specificity of DF microscopy. In two

independent studies in which conventional multiplex PCR was used to evaluate the etiology

of genital ulcers (116, 130), the sensitivity and specificity of DF microscopy ranged from 39

to 81% and 82 to 100%, respectively. DF microscopy has fallen into disfavor because it

requires a specialized microscope and highly trained laboratory personnel. However, the

continued need for DF microscopy proficiency is underscored by studies demonstrating that

substantial percentages of DF microscopy-positive primary syphilis patients lack detectable

antibodies (Fig. 3) (80). Although DFA-TP is at least as sensitive as DF microscopy and more

specific (80), it has never gained wide acceptance and is unlikely to see increased usage in

the PCR era.

Visualization of T. pallidum in Tissues

Silver impregnation is the traditional method for T. pallidum detection in formalin-fixed

tissues (155) and should be performed when routine histopathologic findings suggest

syphilis. When performed by a credible laboratory and accompanied by reactive serologic

tests, visualization of spirochetes by silver staining can be considered definitive evidence for

syphilis (Table 2). Of note, rare cases of seronegative secondary syphilis in HIV-infected

patients are described in which silver staining was instrumental in establishing a diagnosis

(60,74). Silver staining has three principal drawbacks. First, it is prone to staining artifacts.

Second, its sensitivity is limited but not well determined. The Dieterle stain is said to be

more sensitive than the Warthin-Starry stain (9). Third, it is not T. pallidum specific

(e.g., Borrelia burgdorferi might be mistaken for T. pallidum). Recently, impressive results

have been obtained using commercially available polyclonal anti-T. pallidum antibodies and

avidin-biotin immunoperoxidase staining in paraffin-embedded skin biopsy specimens from

secondary syphilis patients (13, 126).

PCR Detection of T. pallidum

PCR enhances detection of T. pallidum in genital ulcer exudates. An additional advantage of

PCR for diagnosis of GUD is that multiplex analysis for other causes of GUD, most

importantly HSV-1 and HSV-2 (see above), is possible. CSF has also been examined

extensively by PCR. In a multicenter study, PCR in conjunction with RIT confirmed the longheld

view that neuroinvasion by T. pallidum occurs at high frequency in early syphilis

patients without neurologic symptoms (133). Unfortunately, very little is known about the

utility of CSF PCR for diagnosing symptomatic neurosyphilis. A study from South Africa found

that 56% of 50 patients with suspected neurosyphilis had positive CSF PCRs (102), but these

findings need corroboration. Cumulative evidence suggests that PCR is also useful for

detection of treponemes in fresh and processed tissues. Comparative analysis of paraffinembedded

secondary syphilis skin biopsy specimens revealed that PCR is equivalent to IHC

staining and is easier to perform (9). Studies using conventional and real-time PCR indicate

that analysis of blood has diagnostic utility, although results have varied (46, 92, 145).

Nevertheless, PCR assays of blood should be used sparingly in suspected cases of acquired

syphilis pending a consensus on the specific clinical scenarios in which it complements

conventional diagnostic methods.

Serologic Tests

Serologic tests supplement the direct detection methods for diagnosis of primary and open

lesions of secondary syphilis, essential for confirming a diagnosis in suspected tertiary

disease, and are the only means for diagnosing latent infection when patients are

asymptomatic (Table 2 and Fig. 3). Many problems in syphilis management stem from the

fact that antibody responses are poor surrogate markers for syphilitic infection. Moreover,

available assays measure two distinctly different kinds of antibody reactivities with different

kinetics during untreated and treated infection (Fig. 3). None of the currently available

serologic tests can distinguish venereal syphilis from the endemic treponematoses (Table 1).

Nontreponemal Tests

Nontreponemal antibody tests are reported as the highest dilution giving a fully reactive

result. A fourfold change in titer is required for a clinically significant difference between two

nontreponemal test results using the same assay. Titers for the same serum can differ by

two- to fourfold when tested using microscopic versus macroscopic nontreponemal tests,

underscoring the importance of using the same method for serial serologic tests, preferably

in the same laboratory. Sera with extremely high nontreponemal test titers can give weak,

atypical, or even negative “rough” reactions at low dilutions when antibody excess prevents

agglutination. This prozone phenomenon occurs in 1 to 2% of patients with secondary

syphilis (71). Most laboratories circumvent this problem by routinely determining the titers of

all samples to at least 16 dilutions.

Traditional algorithms utilize nontreponemal tests as the primary screening tests for

suspected syphilis. Nontreponemal tests must be interpreted according to the suspected

stage of syphilis as well as the population being tested. Reactive results should be confirmed

using a treponemal test, since the proportion of false-positive tests increases with decreasing

prevalence of syphilis. Approximately 30% of those with early primary syphilis have

nonreactive nontreponemal test results on the initial visit (Fig. 3 and Table 7), underscoring

the importance of direct detection methods for genital ulcers and the use of treponemal tests

for diagnosis. In secondary syphilis, nearly all patients have nontreponemal test endpoint

titers of >8. Approximately one-third of patients with tertiary disease have nonreactive

nontreponemal tests (85).

Conditions other than treponemal infection can elicit antilipoidal antibodies that cause BFP

reactions, defined as reactivity in a nontreponemal test with a negative treponemal test

result. Review articles cite a number of conditions and diseases that cause BFPs (106); in our

opinion, many of these lack strong scientific evidence. It is important to emphasize that BFPs

are not necessarily indicative of any disease state. A study of 19,067 Jamaicans revealed

that 0.59% of the general population were BFP by the VDRL test (143), and in some

populations, the frequency may be as high as 1% (106). BFP reactions can be classified into

two groups. Acute BFP reactions, which last less than 6 months, are associated with transient

diseases or conditions such as malaria (70), brucellosis (18), mononucleosis (90, 131), viral

hepatitis (58), lymphogranuloma venereum (141), viral pneumonias (5), tuberculosis (131),

and chancroid (141). More recently, smallpox vaccination was confirmed to cause an

increased frequency of BFP reactions (103). Causes of chronic BFP reactions, which last more

than 6 months, include autoimmune diseases, particularly systemic lupus erythematosus

(28), HIV infection (7, 58), intravenous drug use (58), and leprosy (45). Aging (132) and

pregnancy (90, 132) also have been cited as causes of BFP reactions, but it is likely that

these studies did not identify the true causes of the false-positive tests. For example, in a

Swedish study of 5,170 pregnancies, 9 pregnant women with BFPs and 13 matched negative

controls were examined for autoimmune antibodies to ascertain the nature of their reactions

(57). Eight of the 9 BFPs were positive for reasons other than pregnancy, including

antinuclear antibodies, lupus anticoagulant antibodies, anti-DNA and antimitochondrial

antibodies, and several other factors. In the matched control group, the rate of these

conditions was not significantly different. Another study (106) reported that the rate of true

BFPs was about 14 per 10,000 pregnancies. These findings indicate that the frequency of

pregnancy as a contributing factor of BFP reactions may be far less than previously believed.

A drop in nontreponemal test titer is the only means for monitoring therapeutic response

once disease manifestations resolve. It was once believed that nontreponemal tests revert to

nonreactive in the majority of treated patients with primary and secondary syphilis (40).

However, a substantial percentage of patients do not serorevert during the monitoring period

(12). In a longitudinal study of syphilis patients following therapy, Romanowski and

coworkers (134) confirmed that the serologic response to therapy is slow and often

incomplete. This study, as well as anecdotal experience, brought about a substantial