JCM Accepted Manuscript Posted Online 14 February 2018

J. Clin. Microbiol. doi:10.1128/JCM.01782-17

Copyright © 2018 American Society for Microbiology. All Rights Reserved.

1 Retrospective review of T. pallidum PCR and serology results: Are both tests

2 necessary?

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3

4 Anna Brischettoa,# Ian Gassiepa, David Whileyb,c, Robert Nortona

5 Department of Microbiology, Pathology Queensland, Townsville, Australiaa, Department

6 of Microbiology, Pathology Queensland, Brisbane, Australiab, Faculty of Medicine,

7 Centre for Clinical Research, The University of Queensland, Brisbane, Australiac.

9 Running Head: Review of T. pallidum PCR and serology results

10

11 #Address correspondence to Anna Brischetto, anna.brischetto@gmail.com.

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20 Abstract:

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21 There has been a resurgence of syphilis diagnoses in Australia. We investigated

22 whether our Treponema pallidum polymerase chain reaction (PCR) test provides any

23 additional diagnostic information over syphilis serology (chemi-luminescence

24 immunoassay (CMIA), Treponema pallidum particle agglutination (TPPA) and rapid

25 reagin (RPR) flocculation test). A retrospective audit was conducted of all T. pallidum

26 PCR requests that came through our laboratory from January 2010 to June 2017; data

27 collected included age, gender, site of swab, T. pallidum PCR, syphilis serology and

28 HSV 1 and HSV 2 PCR results. A total of 441 T. pallidum PCR tests were performed,

29 with on average three requests for T. pallidum PCR per month in 2011, which increased

30 to 17.2 per month in 2017. There were 323 patients who had both T. pallidum PCR and

31 syphilis serology performed, with 67% of swabs taken from the genitals. T. pallidum

32 PCR was positive in 61/323 (19%) patients, of which 59/61 (97%) also had positive

33 syphilis serology result (sensitivity 68%, specificity 99%, positive predictive value 97%

34 and negative predictive value 89%). Syphilis serology was positive in 91/323 patients

35 (28%) of which 61 (66%) were also T. pallidum PCR positive (sensitivity 97%, specificity

36 88%, positive predictive value 60% and negative predictive value 99%). The Cohen’s

37 Kappa value was 0.74, indicating substantial agreement between the two tests. Our

38 results show most patients with positive T. pallidum PCR results also had positive

39 syphilis serology. Therefore, T. pallidum PCR adds little clinical value over serology for

40 diagnosis of syphilis in certain clinical settings.

41

42
43 Introduction:

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44 Syphilis is a sexually transmitted disease caused by the spirochete Treponema

45 pallidum. It has varied disease manifestations and is classically divided into 3 distinct

46 stages: primary, secondary and tertiary syphilis(1). There has been a resurgence of

47 syphilis in Australia. This has occurred largely in the metropolitan areas, predominantly

48 among the men who have sex with men (MSM), but syphilis is now also increasing in

49 regional Indigenous communities particularly among adolescents and young adults(2-4).

50 The diagnosis of syphilis remains a challenge. Serology has emerged as the mainstay

51 of diagnosing syphilis, and is divided into treponemal and non-treponemal tests. Non-

52 treponemal tests are the Rapid Plasma Reagin (RPR) and the Venereal Diseases

53 Research laboratory test (VDRL). These define disease activity but suffer from poor

54 specificity. Treponemal tests detect antibodies to treponema species, and are used to

55 confirm the presence of a treponemal infection. These include the chemi-luminescence

56 immunoassay (CMIA), Treponema pallidum haemagglutination assay (TPHA) and

57 Treponema pallidum particle agglutination assay (TPPA). Their sensitivity and

58 specificity are not affected by the activity of the infection, and these tests remain

59 positive for life(5, 6).

60 Although serological tests are very sensitive at diagnosing secondary syphilis, some

61 studies have estimated the sensitivity of serology for diagnosing primary syphilis at

62 86%(5). This is due to a window period whereby the humoral response to syphilis

63 develops 1-4 weeks after the chancre forms in primary syphilis(7). Polymerase chain

64 reaction (PCR) tests have been developed to help aid in the diagnosis of early, primary

65 syphilis. These tests have proven to be both sensitive and specific in primary syphilis,
66 but less sensitive in secondary syphilis(8, 9). Further studies have also indicated that

67 PCR may in fact be more sensitive than serology in early, primary syphilis and suggest

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68 it is a necessary adjunct to serological testing to ensure no patients in the “window

69 period” are missed(10).

70 Our laboratory introduced an in-house T. pallidum PCR test in 2001 but was only widely

71 adopted in 2010. Serology is usually performed concurrently with the PCR test. The T.

72 pallidum PCR test is performed in the central laboratory in Brisbane, 1400 kilometres

73 away from our facility, increasing turnaround time and costs associated with the

74 transport of specimens. Serology is performed in our local Townsville laboratory. This

75 duplication in testing (i.e. serology plus PCR), in addition to the increased specimen

76 handling and transport, more than doubles the cost of syphilis screening and diagnosis

77 from 25 ($AUS) to 60 ($AUS) on conservative estimates based on the typical costs

78 billed by our facility. For this reason, and because of increasing requests for syphilis

79 PCR, we decided to conduct a study looking at whether the addition of the PCR test

80 resulted in any extra diagnoses of primary syphilis, or whether positive patients would

81 have been captured regardless with positive serological tests.

82

83

84 Results

85 There were 516 requests for T. pallidum PCR between January 2010 and May 2017, on

86 493 unique patients, of which 75 were excluded. Reasons for exclusion were age less

87 than 18 years (51 patients, based on the recommendations of our local Ethics
 88 Committee), the test being cancelled prior to it being performed (11 patients), and the

89 test being performed to investigate congenital syphilis (13 patients). There were on

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90 average three requests for T. pallidum PCR per month in 2011, which increased to 17.2

91 per month in 2017 (see figure 1).

92 Of the 441 patients that had swabs tested for T. pallidum PCR, 323 (73%) also had

93 syphilis serology performed within 14 days. These were the patients included in our

94 analysis.

95 The median patient age was 29 years (IQR, 23-37 years) and 197/323 (61%) were

96 male. The swabs were predominantly taken from the genital region (see table 1). A

97 summary of the T. pallidum PCR, serological and Herpes simplex virus (HSV) 1 and 2

98 PCR results is provided in table 2.

99 T. pallidum PCR was positive in 61/323 (19%) samples. Syphilis serology was positive

100 in 59/61 (97%) of these PCR positive samples. One of the two patients with positive T.

101 pallidum PCR and negative syphilis serology (CMIA) at baseline had a positive CMIA,

102 equivocal TPPA and negative RPR results when performed 2 months later. The other

103 patient had follow up serology (CMIA) performed at 1 month that remained negative.

104 Two months after this their repeat CMIA was positive but TPPA and RPR remained

105 negative. The sensitivity of the T. pallidum PCR test was 68%, the specificity was 99%,

106 the positive predictive value was 97% and the negative predictive value was 89% using

107 syphilis serology as the gold standard (see table 3)

108 There were 91/323 (28%) patients who had positive syphilis serology, of which 61

109 (66%) were also T. pallidum PCR positive (see table 3). The sensitivity of syphilis
110 serology was 97%, the specificity was 88%, the positive predictive value was 60% and

111 negative predictive value was 99% using T. pallidum PCR as the gold standard. The

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112 Cohen’s Kappa value was 0.74, indicating substantial agreement between the two tests.

113 There were 17/30 (57%) patients who were serology positive and PCR negative who

114 had an RPR titer of 16 or greater, compared with 49/61 (80%) of those who were

115 serology positive and PCR positive (see figure 2; p= 0.023).

116 HSV 1 and 2 PCR tests were performed in 260/323 (80%) of patients (see table 2).

117 Overall 5% of patients were positive for HSV-1 and 20% for HSV-2. There was one

118 patient positive for both HSV-1 and 2, and 7 patients who were positive for both HSV-2

119 and T. pallidum PCR.

120

121 Discussion:

122 The prevalence of syphilis has been increasing in recent times, which has resulted in

123 the need for our pathology service to provide syphilis diagnostic tests with accuracy and

124 precision that return results in a timely manner(2). The syphilis PCR test was introduced

125 in addition to syphilis serology to increase sensitivity in diagnosing primary syphilis. This

126 was due to the “window period” of the serological tests in early disease(7), with the

127 sensitivity of syphilis serology estimated at 86% in primary syphilis(5). As can be seen

128 from Figure 1 there has been increasing use of the T. pallidum PCR in our laboratory.

129 However, our results indicate that in our setting the syphilis PCR test does not add

130 significant diagnostic value in addition to syphilis serology. Syphilis serology

131 demonstrated excellent negative predictive value (99%) in our study. Only 2/61 patients
132 (3%) with positive syphilis PCR results had negative serology at the time of diagnosis.

133 One of these patients had an equivocal TPPA when serology was re-tested 2 months

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134 later that could be considered as a positive syphilis serology result. It is reasonable to

135 therefore consider that this patient was in the serological window period at the time of

136 initial diagnosis and the syphilis PCR was the true positive result. However, the second

137 patient had a repeat negative CMIA one month after initial testing. Two months after this

138 their repeat CMIA was positive but TPPA and RPR remained negative. This patient’s

139 syphilis serology never became truly positive, and it is possible the original positive T.

140 pallidum PCR was a false positive.

141 Other published studies including a meta-analysis have in contrast shown the added

142 value of PCR in addition to serology, reporting up to 8% of positive T. pallidum PCR

143 results had negative syphilis serology at the time of diagnosis(8, 10). We postulate that

144 our lower rate may be due to patients presenting later in the natural history of their

145 disease than other settings. We provide pathology services to many surrounding

146 communities in which socio-economic disadvantage and difficulty accessing healthcare

147 have been shown to correlate with the prevalence of sexually-transmitted infections(11).

148 Syphilis serology showed poor positive predictive value (60%) in our study, as there

149 were 30 patients who had positive syphilis serology, and negative T. pallidum PCR. This

150 resulted in the calculated sensitivity of the syphilis PCR test using syphilis serology as

151 the gold standard being 67%, which is lower than the 78.4% for ulcers in primary

152 syphilis reported from a recent meta-analysis(10). We postulate that this disagreement

153 between the two tests occurred for the following reasons: To confirm that the positive

154 syphilis serology did indeed represent new infection we could only check for past
155 syphilis results on our local pathology database. Theoretically some of the positive

156 results could be due to past infection if previous positive serology was performed with a

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157 different pathology service. In addition, we believe that as we had very little

158 corresponding clinical data some of these serology positive, PCR negative patients may

159 represent secondary syphilis in which T. pallidum PCR testing, has been shown to have

160 lower sensitivities(8, 9), or late latent infection. The RPR values were significantly lower

161 (see figure 2; p= 0.023) for serology positive, PCR negative patients compared with

162 serology positive, PCR positive patients also indicating less acute infection(5).

163 Our study also enabled us to view how clinicians are using the syphilis PCR test in their

164 diagnostic algorithm. A quarter of patients (26%) who had a syphilis PCR test performed

165 had no syphilis serology requested, including two patients with positive PCR results.

166 Australian and international guidelines recommend performing syphilis serology at

167 baseline so that response to treatment can be monitored

168 (https://www.cdc.gov/std/syphilis/treatment.htm), and no guideline to our knowledge

169 recommends using syphilis PCR alone to diagnose primary syphilis. This suggests we

170 may need to increase education for health professionals regarding this issue.

171 There has been a steady increase in the requests for syphilis PCR coming through our

172 laboratory (see figure 1). At a cost of 35 ($AUS) dollars per test (not including transport

173 and processing costs) this means we now spend on average ~7000 ($AUS) dollars a

174 year on this test, which could increase significantly if the rate of requests continues to

175 increase.

176 HSV-1 and 2 were shown to be important differentials for primary syphilis, with 25% of

177 samples tested positive for HSV-1 or 2. This is consistent with other reported rates and
178 confirms the need to perform HSV-1 and 2 PCR on genital lesions as well as

179 investigating for primary syphilis(8). There were 20% of our patients that did not have

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180 HSV 1 or 2 PCR tests requested.

181 We also noted that 7/61 (11%) T. pallidum PCR-positive patients had HSV-2/ syphilis

182 co-infection. High rates of co-infection have previously been described, particularly in

183 men who have sex with men cohorts, and occur due to shared routes of sexual

184 transmission(12).

185 The major limitation of our study is the lack of clinical information with which we could

186 correlate our laboratory results, including the human immunodeficiency virus (HIV)

187 status, whether the patients had acquired syphilis more than once, or whether any had

188 received prior antibiotic therapy. We assumed for this study that a swab sent of a lesion

189 with a request for syphilis PCR was an investigation for a chancre in primary syphilis.

190 For the purposes of this study we had no means of assessing what these lesions looked

191 like, and how much they resembled a typical syphilitic lesion. We also had no

192 information of how long the lesion or symptoms had been present. This would have

193 allowed us to stage the condition as primary or secondary, and to understand whether

194 patients were indeed presenting late in their illness. A further limitation of this study is

195 that we did not explore whether differences in PCR methodology may have impacted

196 upon the results, for example previous studies have found that nested PCR, or use of

197 the polA gene (rather than the 47kDa target used here) may enhance PCR sensitivity

198 (10).

199 In conclusion, we believe the results of this study indicate that the addition of syphilis

200 PCR to serology in the diagnostic algorithm for primary syphilis may not be necessary in
201 our setting. We had a very low rate of positive syphilis PCR tests that were not also

202 accompanied by positive syphilis serology. As syphilis serology is recommended to be

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203 performed at baseline to allow for monitoring of response to treatment it seems

204 reasonable to reconsider the need to also perform a second diagnostic test, especially

205 in this age of increasing awareness of the need for pathology stewardship(13).

206 However, we acknowledge that other studies have shown different results and advocate

207 for individual health services to assess whether the syphilis PCR is necessary in

208 addition to serological testing.

209

210 Materials and Methods:

211 Study population:

212 The Pathology Queensland laboratory at the Townsville hospital is one of a network of

213 35 public hospital laboratories. In addition to providing laboratory services to the 600

214 bed Townsville hospital the laboratory also provides diagnostic services to hospitals and

215 healthcare facilities across North Queensland, in the north-east of Australia. Some

216 PCR tests, including the T. pallidum PCR are performed by the central Pathology QLD

217 laboratory in Brisbane. Clinical samples in this study came from hospitals and sexual

218 health services in the city of Townsville, as well as the surrounding towns of Ayr, Palm

219 Island, Mornington Island, Hughenden, Mount Isa, Charters Towers and Doomadgee.

220

221 Study design and clinical definitions:

222 A retrospective review of the results of T. pallidum PCR requests from 2010-2017 was

223 performed. We used an extended search of our results database “AUSLAB” with the

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224 request of T. pallidum PCR from 2010-2017 as the primary filter. Patient’s aged less

225 than 18 years of age, and those whose swab was from a placenta were excluded.

226 Results of serology performed within 14 days of the T. pallidum PCR, as well as age,

227 gender, site of swab, and results of HSV 1 and HSV 2 PCR performed on the same

228 swab were also collected.

229 Prospective approval for this audit was granted by the Townsville Hospital and Health

230 Service Human Research Ethics Committee (HREC/17/QTHS/87).

231

232 T. pallidum PCR testing

233 Swabs of lesions were sent in either dry sterile containers or in vials containing viral

234 transport medium. If a swab was sent for T. pallidum PCR it was assumed that there

235 was a clinical lesion suggestive of a chancre. PCR testing was performed at the

236 Pathology QLD laboratory in Brisbane. DNA was extracted from swab samples using

237 the Magna pure 96 system (Roche Applied Science, Indianapolis). The PCR targeted

238 the T. pallidum 47 kDa integral membrane lipoprotein gene and was based on a

239 previously described method(14). Amplification and detection were achieved on the

240 Rotorgene Q real-time PCR instrument (Qiagen, Victoria, Australia).

241

242 Syphilis serology testing:

243 Syphilis serology was performed on sera at the Pathology QLD laboratory at Townsville

244 hospital. The initial test was a Treponema pallidum chemi-luminescence immunoassay

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245 (CMIA) performed on the commercially available Architect (Abbott diagnostics, Lake

246 Bluff, Illinois) as per the manufacturer’s instructions. If positive by CMIA, a Treponema

247 pallidum particle agglutination (TPPA) was performed on the Serodia TP-PA test kit

248 (Fujirebio, Tokyo) as per the manufacturer’s instructions to confirm the result, as well as

249 a quantitative rapid reagin (RPR) flocculation test performed on the BD macro-Vue RPR

250 card test kit (Thermo Fisher Scientific, Waltham, MA USA) as per the manufacturer’s

251 instructions. The syphilis serology was considered positive and consistent with a new

252 diagnosis of syphilis if the patient had positive reactive treponemal tests (CMIA and

253 TPPA), with or without a positive RPR. If the patient had previously recorded positive

254 CMIA and TPPA results serology was considered positive and consistent with new

255 infection if the RPR was four-fold higher than previously recorded(5, 7). If the CMIA was

256 positive and the TPPA and RPR were negative, we attached a comment stating that this

257 may be a false positive, and suggested repeat testing in 2-4 weeks.

258

259 Statistical analysis:

260 Descriptive and regression statistics were performed using STATA13 (StataCorp,

261 Texas, USA). The sensitivity, specificity, positive predictive value and negative

262 predictive value of both the T. pallidum PCR and syphilis serology tests were evaluated

263 using the other test as the gold standard.

264 A kappa value was calculated to measure the level of agreement between the two tests,

265 T. pallidum PCR and syphilis serology. Categorical data was tested using the chi-

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266 squared test for dependence with a p-value <0.05 considered significant.

267

268

269 References:

270 1. Lafond RE, Lukehart SA. 2006. Biological basis for syphilis. Clin Microbiol Rev

271 19(1):29-49.

272 2. The Kirby Institute. 2016. HIV, viral hepatitis and sexually transmissible infections

273 in Australia Annual Surveillance report 2016. UNSW, Sydney.

274 3. Stamm LV. 2016. Syphilis: Re-emergence of an old foe. Microb Cell 3(9):363-70.

275 4. Fagan PS, Cannon FM. 2007. Syphilis in remote north Queensland. Commun

276 Dis Intell Q Rep 31(1):125-7.

277 5. Larsen SA, Steiner BM, Rudolph AH. 1995. Laboratory diagnosis and

278 interpretation of tests for syphilis. Clin Microbiol Rev 8(1):1-21.

279 6. Morshed MG, Singh AE. 2015. Recent trends in the serologic diagnosis of

280 syphilis. Clin Vaccine Immunol 22(2):137-47.

281 7. French P. 2007. Syphilis. BMJ 334(7585):143-7.

282 8. Heymans R, van der Helm JJ, de Vries HJ, Fennema HS, Coutinho RA, Bruisten

283 SM. 2010. Clinical value of Treponema pallidum real-time PCR for diagnosis of

284 syphilis. J Clin Microbiol 48(2):497-502.

285 9. Shields M, Guy RJ, Jeoffreys NJ, Finlayson RJ, Donovan B. 2012. A longitudinal

286 evaluation of Treponema pallidum PCR testing in early syphilis. BMC Infect Dis

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287 12:353.

288 10. Gayet-Ageron A, Lautenschlager S, Ninet B, Perneger TV, Combescure C. 2013.

289 Sensitivity, specificity and likelihood ratios of PCR in the diagnosis of syphilis: a

290 systematic review and meta-analysis. Sex Transm Infect 89(3):251-6.

291 11. Miller GC, McDermott R, McCulloch B, Fairley CK, Muller R. 2003. Predictors of

292 the prevalence of bacterial STI among young disadvantaged Indigenous people

293 in north Queensland, Australia. Sex Transm Infect 79(4):332-5.

294 12. Li D, Yang X, Zhang Z, Wang Z, Qi X, Ruan Y, Zhou Y, Li C, Luo F, Lau JTF.

295 2016. Incidence of Co-Infections of HIV, Herpes Simplex Virus Type 2 and

296 Syphilis in a Large Cohort of Men Who Have Sex with Men in Beijing, China.

297 PLoS One 11(1):e0147422.

298 13. Spelman D.2015. Inappropriate pathology ordering and pathology stewardship.

299 Med J Aust. 202(1):13-5.

300 14. Palmer HM, Higgins SP, Herring AJ, Kingston MA. 2003. Use of PCR in the

301 diagnosis of early syphilis in the United Kingdom. Sex Transm Infect 79(6):479-

302 83.

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307
308 Table 1: Site of swab taken for T. pallidum Polymerase Chain Reaction (PCR)

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Site Number (%)
Genital 216 (67%)
Anal 21 (7%)
Oral 23 (7%)
Other* 9 (3%)
Ulcer/ lesion with no site specified 40 (12%)
No clinical information 14 (4%)
Total 323 (100%)
309 * Swabs taken from thighs, groins, arm, face, and eyes

310

311 Table 2: A summary of all results for the 323 patients tested both by T. pallidum PCR

312 and serology.

Number of Serology T. Herpes Herpes

samples
Chemi- Treponema Rapid Consensus pallidum simplex simplex
∞
luminescence pallidum reagin (Syphilis PCR virus virus

immunoassay particle (RPR) serology (HSV)- (HSV)-

(CMIA) agglutination assed as 1 PCR 2 PCR

(TPPA) positive or

negative

based on

previous

results)

T. pallidum
PCR+
serology+
1 Pos Pos NRα Pos Pos N/Pβ N/P

3 Pos Pos NR Pos Pos Neg Neg

11 Pos Pos Pos Pos Pos N/P N/P

 39 Pos Pos Pos Pos Pos Neg Neg

7 Pos Pos Pos Pos Pos Neg Pos

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T. pallidum
PCR+
serology -
2 Neg N/P N/P Neg Pos Neg Neg

T. pallidum
PCR-
serology +
2 Pos Pos NR Pos Neg N/P N/P

4 Pos Pos NR Pos Neg Neg Neg

7 Pos Pos Pos Pos Neg N/P N/P

17 Pos Pos Pos Pos Neg Neg Neg

T. pallidum
PCR-
Serology -
42 N/AΩ N/A N/A Neg Neg N/P N/P

119 N/A N/A N/A Neg Neg Neg Neg

15 N/A N/A N/A Neg Neg Pos Neg

53 N/A N/A N/A Neg Neg Neg Pos

1 N/A N/A N/A Neg Neg Pos Pos

Total: 323

313 ∞: PCR=Polymerase chain reaction

314 α: NR=Non-Reactive

315 β: N/P=Not Performed

316 Ω: N/A=Not applicable. The syphilis serology was assessed as negative due to a non-

317 reactive CMIA, or serology that was reactive but unchanged from a previous test result

318
319 Table 3: Syphilis serology versus T. pallidum PCR result

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T. pallidum

PCR result

+ - Total

Syphilis Serology + 61 30 91

result - 2 230 232

Total 63 260 323

320

321

322 Figure 1: Average monthly rate of T. pallidum PCR requests by yea

323

324 Figure 2: The Rapid Plasma Reagin (RPR) value for all the patients with positive

325 serological results

326 α: Polymerase chain reaction

327 β: NR= Non-reactive

328

329

330

331
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332

333

334
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