Performance of the 47-Kilodalton Membrane Protein versus DNA Polymerase I Genes for Detection of Treponema pallidum by PCR in Ulcers

Abstract

Treponema pallidum PCR (Tp-PCR) is a direct diagnostic method for primary and secondary syphilis, but there is no recommendation regarding the best choice of target gene. In this study, we sequentially tested 272 specimens from patients with sexually transmitted ulcers using Tp-PCR targeting the tpp47 and then polA genes. The two methods showed similar accuracies and an almost-perfect agreement.

TEXT

Syphilis has been making a worldwide comeback since the early 2000s (1–3) and was reintroduced in 2006 in Switzerland as being among the notifiable infectious diseases (4, 5). Since 2008 (6, 7), European guidelines have considered Treponema pallidum PCR (Tp-PCR) to be a definitive direct diagnostic tool for primary and secondary syphilis. Similarly, the U.S. Centers for Disease Control and Prevention (CDC) has changed their case definitions and now considers Tp-PCR to be capable of confirming early syphilis when clinical manifestations are present (8). However, no specific gene was recommended as a target for Tp-PCR. The two most widely used genes are tpp47 and polA (9). tpp47 encodes a T. pallidum cytoplasmic membrane protein (10) involved in cell wall synthesis (11) but that is partly specific to T. pallidum subsp. pallidum (12–14). polA encodes DNA polymerase I involved in DNA repair and the replication of most bacteria and shows a number of unique features in T. pallidum subsp. pallidum (15). Other targets have been tested sporadically. However, it remains unclear if one of these targets is preferred for use.

(The results from this study were presented at the 24th European Congress of Clinical Microbiology and Infectious Diseases, 10 to 13 May 2014, Barcelona, Spain, poster no. 1757 [16].)

We conducted a multicenter prospective study between 2011 and 2013 in five European cities, which were Geneva, Lausanne, and Zürich in Switzerland, and Paris and Lyon in France (17). Every patient presenting with a sexually transmitted ulcerative disease suggestive of syphilis was invited to participate. Each patient received the conventional diagnostic tests for syphilis (18), either dark-field microscopy (DFM) or a combination of the following serological assays: enzyme immunoassay (EIA), Venereal Disease Research Laboratory (VDRL) or rapid plasma reagin (RPR) assay, a treponemal microhemagglutination assay (MHA-TP), or a fluorescent treponemal antibody absorption test (FTA-ABS). We distinguished between (i) a confirmed case (positive DFM) and (ii) a probable case (reactive VDRL or RPR result and reactive MHA-TP, FTA-ABS, or EIA result). Finally, we used (iii) an enhanced definition combining clinical information and the results from DFM and serology (17). All patients categorized as having syphilis benefited from standard treatment and were followed at 3, 6, and 12 months after treatment. The treatment response was defined by a 4-fold decline in the VDRL or RPR titer (19). Regarding Tp-PCR, swabs from the ulcers were collected and then analyzed sequentially. First, Tp-PCR targeting the tpp47 gene (tpp47-Tp-PCR) was performed at the laboratory of bacteriology at the Geneva University Hospitals (20, 21). The test was considered positive if two of the three replicates had cycle thresholds (C_T) of <40. Next, all frozen DNA extracts were sent to Lyon (Department of Bacteriology, Hôpital de la Croix-Rousse), where Tp-PCR targeting the polA gene (polA-Tp-PCR) was performed using the primers and probes described elsewhere (22). A single polA-Tp-PCR was performed and was considered positive if the C_T was <40. The limits of detection of the two Tp-PCRs were blindly compared using the same positive control (mixed DNA from rabbit tissues and T. pallidum Nichols strain DNA) at dilution rates from 1:10 to 1:100,000.

During a 2-year period, 273 patients were recruited, and 272 specimens for Tp-PCR were collected (Table 1). Most patients were men presenting with a genital ulceration after a mean of 20 days following homosexual intercourse. Nine patients were diagnosed with human immunodeficiency virus at the initial consultation. Globally, we obtained 77 concordant-positive and 191 concordant-negative Tp-PCR results; two specimens resulted in negative polA-Tp-PCR but positive tpp47-Tp-PCR results, and conversely, two had negative tpp47-Tp-PCR but positive polA-Tp-PCR results. The kappa coefficient was 0.96 (exact 95% confidence interval, 0.93 to 0.99). The two Tp-PCR results had the same indices of diagnostic performance according to the three case definitions (Table 2) (P = 0.99 for all comparisons, McNemar's test). When we considered Tp-PCR to be positive whenever one of the two Tp-PCRs was positive, sensitivity increased, especially in the enhanced definition group.

TABLE 1.

Patient characteristics

Variablea	Patient data (n = 272)
Center
Paris	140 (51.5)
Lyon	59 (21.7)
Geneva	40 (14.7)
Lausanne	17 (6.2)
Zurich	16 (5.9)
Male gender	251 (92.3)
Age at the time of diagnosis (mean ± SD) (yr)	39.1 ± 12.3
Delay since appearance of ulcer (mean ± SD) (days)	20.4 ± 34.0
Localization of the ulcer
Penile/vaginal	148 (54.4)
Anorectal	97 (35.7)
Oral	27 (9.9)
Route of contamination
Homosexual	184 (71.3)
Heterosexual	70 (27.1)
Unknown	4 (1.6)
Coinfection with HIV
Yes	53 (19.5)
No	172 (63.2)
Unknown	47 (17.3)
Concomitant HIV diagnosis during current episode of ulcerative disease	9 (17.0)
Patients treated with antiretroviral therapy
Yes	36 (67.9)
No	14 (26.4)
Unknown	3 (5.7)
Other comorbidities
Chronic lymphoid leukemia	1 (0.4)
Diabetes	1 (0.4)
Under corticotherapy	1 (0.4)
Diagnosis of syphilis with the reference tests
Dark-field microscopy
Positive	31 (18.3)
Negative	138 (81.7)
Serological assays
Positive	87 (34.2)
Negative	151 (59.5)
Undetermined	16 (6.3)
Enhanced definition
Positive	47 (27.8)
Negative	122 (72.2)
Combination of DFM and serological assays
Positive	93 (34.8)
Negative	174 (65.2)

^aThe data are presented as the no. (%), unless otherwise indicated.

TABLE 2.

Description of the results of the two Tp-PCRs according to the three case definitions, corresponding indices of accuracy and clinical utility for each Tp-PCRs taken separately and combining the results of the two methods

	Tp-PCR result with case definition in (n):
	Dark-field microscopy (169)		Serological assays (254)		Enhanced definition (169)
Tp-PCR assessment	Positive	Negative	Positive	Negative	Positive	Negative
Tp-PCR targeting either tpp47 or polA genea
Positive	29	13	68	10	41	1
Negative	2	125	19	141	6	121
Accuracy and clinical utility of Tp-PCR targeting tpp47 or polA gene (% [95% CI])a
Sensitivity	93.6 (78.6–99.2)		77.0 (66.8–85.4)		87.2 (74.3–95.2)
Specificity	90.6 (84.4–94.9)		93.4 (88.2–96.4)		99.2 (95.5–99.9)
Positive predictive value	69.1 (52.9–82.4)		87.0 (77.4–93.6)		97.6 (87.4–99.9)
Negative predictive value	98.4 (94.4–99.6)		87.6 (81.6–91.8)		95.3 (90.1–97.8)
Positive likelihood ratio	9.9 (5.9–16.8)		11.6 (6.3–21.4)		106.4 (15.1–751.8)
Negative likelihood ratio	0.07 (0.02–0.27)		0.25 (0.17–0.36)		0.13 (0.06–0.27)
Accuracy and clinical utility of Tp-PCR combining the results of the two methods (% [95% CI])b
Sensitivity	93.8 (79.2–99.2)		79.6 (69.6–87.4)		97.7 (87.7–99.9)
Specificity	90.6 (84.4–94.9)		93.4 (88.2–96.8)		95.3 (90.0–98.2)
Positive predictive value	69.8 (53.9–82.8)		87.5 (78.2–93.8)		87.5 (74.8–95.3)
Negative predictive value	98.4 (94.4–99.8)		88.7 (82.7–93.2)		99.2 (95.5–99.9)
Positive likelihood ratio	44.3 (11.0–177.7)		7.7 (5.0–12.0)		106.8 (15.1–754.0)
Negative likelihood ratio	0.31 (0.20–0.48)		0.14 (0.08–0.25)		0.13 (0.06–0.27)

^aWe found the same results with Tp-PCR targeting tpp47 gene and that targeting polA. The presence of 2 discrepancies favoring Tp-PCR targeting the tpp47 gene and 2 discrepancies favoring Tp-PCR targeting the polA gene led to the same results. 95% CI, 95% confidence interval.

^bGlobal results are based on the combination of the results of the two Tp-PCRs; if one of the two Tp-PCRs or the two Tp-PCRs were positive, Tp-PCR was globally considered positive.

The mean C_T values among all the positive Tp-PCR results were significantly lower for polA-Tp-PCR than for tpp47-Tp-PCR (28.3 ± 3.7 versus 32.1 ± 5.6, P < 0.001 using Wilcoxon signed-rank test). The limits of detection of the two Tp-PCR results were identical at a threshold 1:100,000. Among the four discordant results, none had been examined by DFM, and all had clearly positive serology results (Table 3). Of note, tpp47-Tp-PCR was considered negative in one patient (one C_T value at 39.0 and two <40), but this patient had a polA-Tp-PCR result that was clearly positive; this suggests a false-negative tpp47-Tp-PCR result. All four patients were considered to have syphilis and were treated for syphilis using 2.4 million units of penicillin G benzathine administered intramuscularly, and all responded to treatment, suggesting that all were true-positive cases.

TABLE 3.

Description of the four discordant results between the two types of Tp-PCR

Results	Data for case no. (center):
	1 (Geneva)	2 (Geneva)	3 (Lyon)	4 (Zurich)
Tp-PCR (CT) targeting:
tpp47 gene	Positive (33.9/33.9/0)	Positive (36.0/35.0/37.0)	Negative	Negative (37.0/0/0)a
polA gene	Negative	Negative	Positive (35.8)	Positive (36.8)
Serological assaysb
VDRL/RPR titer	1:64	1:16	1:32	1:4
MHA-TP titer	1:20,480	1:2,560	1:20,480	1:10,240
FTA-ABS	NAc	1:3,200	NA	Reactive
EIA	NA	NA	45.06	NA
Localization of the ulcer	Anorectal	Oral	Anorectal	Penile
Titer at follow-up after treatment (mo)
3	RPR, 1:8; MHA-TP, 1:10,240	RPR, 1; MHA-TP, 1:1,280	RPR, 1:4; MHA-TP, 1:10,240	RPR, 0; MHA-TP, 1:160
6	RPR, 1; MHA-TP, 1:2,560	RPR, 0; MHA-TP, 1:1,280	RPR, 1:2; MHA-TP, 1:10,240	NA
12	RPR, 0; MHA-TP, 1:1,280	NA	NA	NA

^aThree replicates were performed for Tp-PCR targeting the tpp47 gene. As one replicate among the three was positive, Tp-PCR targeting the tpp47 gene was considered negative.

^bVDRL, Venereal Diseases Research Laboratory; RPR, rapid plasma reagin; MHA-TP, microhemagglutination assay for antibodies to T. pallidum; FTA-ABS, fluorescent treponemal antibody-absorbed test; EIA, enzyme immunoassay.

^cNA, not available.

We demonstrated that the diagnostic performances of the two currently used targets for Tp-PCR were comparable with ulcer specimens, irrespective of the case definition of syphilis. The agreement between the two Tp-PCRs was almost perfect (23). To our knowledge, only one previous study reported a high agreement between tpp47-Tp-PCR and polA-Tp-PCR using a smaller collection of 112 paired specimens from patients with ulcers suggestive of primary syphilis (24). Another study reported similar sensitivities of three types of Tp-PCR (tpp47-Tp-PCR, polA-Tp-PCR, and a combination of the two) in the blood samples from latent syphilis cases, but it did not assess the agreement between the three methods (25). Our results also confirmed the accuracy and clinical value of tpp47-Tp-PCR (26) and polA-Tp-PCR (27) with primary syphilis ulcers. The added value of our study is that we assessed the two Tp-PCR methods with a large number of paired specimens selected by a standardized procedure. Any discrepancies between the two techniques could not be explained by a difference in DNA extraction, since the testing procedures were rigorous and fully standardized. The two Tp-PCR methods had the same analytical limits of detection and thus had comparable sensitivities. However, our study was pragmatic, and the mean number of replication cycles performed was higher for tpp47-Tp-PCR than for polA-Tp-PCR; this might explain why the two discrepancies favored tpp47-Tp-PCR. If two additional replications had been done for polA-Tp-PCR, the chance of a positive result would have been greater.

In conclusion, we confirmed that Tp-PCR is a useful diagnostic tool for ulcers that suggest primary syphilis and that the results do not depend on the target gene, suggesting that either tpp47 or polA can be used in practice. Combining the results of the two Tp-PCRs did not drastically improve the clinical utility of the test. Therefore, the supplemental cost of performing an additional Tp-PCR targeting another gene is not justified.