JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1996, p. 49–54 Vol. 34, No.

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0095-1137/96/$04.0010
Copyright q 1996, American Society for Microbiology

Simultaneous PCR Detection of Haemophilus ducreyi, Treponema

pallidum, and Herpes Simplex Virus Types 1
and 2 from Genital Ulcers
KARINA A. ORLE,1* CAROL A. GATES,1 DAVID H. MARTIN,2 BARBARA A. BODY,3 AND JUDITH B. WEISS1
1
Roche Molecular Systems, Alameda, California ; Section of Infectious Diseases, Department of Medicine,
Louisiana State University Medical Center, New Orleans, Louisiana2; and Laboratory
Corporation of America Holdings, Ltd., Burlington, North Carolina3
Received 25 July 1995/Returned for modification 28 September 1995/Accepted 13 October 1995

A multiplex PCR (M-PCR) assay with colorimetric detection was devised for the simultaneous amplification
of DNA targets from Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus (HSV) types 1 and 2.
By using target-specific oligonucleotides in a microwell format, 298 genital ulcer swab specimens collected in
New Orleans during three intervals from 1992 through 1994 were evaluated. The results of the M-PCR assay
were compared with the results of dark-field microscopy and H. ducreyi culture on two different culture media.
HSV culture results were available for 99 specimens collected during the third interval. Confirmatory PCR
assays targeting different gene sequences for each of the three organisms were used to validate the M-PCR
results. Specimens were resolved as positive for the determination of sensitivity if the reference diagnostic test
was positive or if the results of both the M-PCR and the confirmatory PCR were positive. The resolved
sensitivities of M-PCR for HSV, H. ducreyi, and T. pallidum were 100, 98.4, and 91%, respectively. The resolved
sensitivities of HSV culture, H. ducreyi culture, and dark-field microscopy were 71.8, 74.2, and 81%, respec-
tively. These results indicate that the M-PCR assay is more sensitive than standard diagnostic tests for the
detection of HSV, H. ducreyi, and T. pallidum from genital ulcers.

The three major causes of genital ulcer disease (GUD) in
the United States are herpes simplex virus (HSV), Treponema
pallidum (the causative agent of syphilis), and Haemophilus
ducreyi (the causative agent of chancroid). Currently, the di-
agnosis of GUD is based primarily on the clinical presentation
of the ulcer itself, because H. ducreyi cultures are performed in
very few clinical laboratories and dark-field examinations are
not done in many clinical settings (29). However, agent-specific
diagnoses based solely on clinical evaluation are often ob-
is usually made by dark-field microscopic examination of lesion
material or by serology. Currently, serologic tests are useful for
the confirmation of a diagnosis based on clinical presentation
and for follow-up treatment, but sensitivities as low as 30%
have been reported for the early stages of syphilis (21). Direct
examination methods, including dark-field microscopy and im-
munofluorescent-antibody staining, are limited by low degrees
of sensitivity and specificity (15).
The accurate diagnosis of H. ducreyi depends on the ability

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scured by the overlapping patterns of clinical presentation and
the occurrence of multiple and mixed infections (30). Efficient
early treatment of GUD is becoming increasingly important,
because studies report a correlation between GUD and human
immunodeficiency virus transmission (1, 14, 19, 31, 34). Addi-
tionally, since treatment differs for each of these diseases, it is
important to correctly identify the causative agent.
Current methods for detecting HSV include virus isolation
in tissue culture, cytologic examination, immunostaining, and
antigen detection. The ‘‘gold standard’’ is viral culture; this is
followed by confirmation with immunofluorescent-antibody
staining. The sensitivity of HSV detection by culture depends
on the stage and duration of the lesion. Although the sensitiv-
ity of detection approaches 100% for samples obtained from
vesicular lesions, it drops to 30% when samples are obtained
from crusted or healing lesions (7). The presence of HSV in
most specimens will be detected within 48 h of culture; how-
ever, for some specimens up to 14 days may be required.
Recent studies suggest that the current methods used for the
diagnosis of genital HSV in women may miss many cases (6,
18).
T. pallidum cannot be cultured in vitro. Laboratory diagnosis
* Corresponding author. Mailing address: Roche Molecular Sys-
tems, 1145 Atlantic Ave., Alameda, CA 94501. Phone: (510) 814-2886.
Fax: (510) 814-2810. Electronic mail address: karina.orle@roche.com.
to culture the organism; specimens must be cultured on solid
medium within 4 to 6 h after collection from the patient.
Because of its fastidious growth requirements, even on selec-
tive enrichment media, isolation rates have been only 53 to
88% (24). Very few U.S. laboratories are properly equipped
to perform H. ducreyi culture (29), and diagnosis dependent
solely on clinical presentation has a reported accuracy of 33 to
53% (24). Because of these restrictions, it is believed that H.
ducreyi is often underdiagnosed in the United States (29).
PCR assays for each of the three agents have been de-
scribed. T. pallidum DNA has been successfully detected from
genital ulcers, as well as cerebrospinal fluid, amniotic fluid, and
fetal and neonatal sera (2, 11, 13, 25, 37). Fulminant and
asymptomatic shedding of HSV from genital ulcers and cere-
brospinal fluid was detected by PCR with an increased sensi-
tivity compared with that by culture (5, 7, 12, 17, 20). The low
detection limit of PCR offered a further advantage to culture
by identifying HSV in crusted-over lesions, as well as before
and after lesions appeared (7). H. ducreyi DNA has been de-
tected in genital ulcer specimens by PCR, and PCR was more
sensitive than culture (4, 16).
We report here the first multiplex PCR (M-PCR) assay for
the simultaneous detection of these three etiologic agents in a
single genital ulcer swab specimen. The goal of the study was
to evaluate the three-way PCR assay by using specimens col-
lected in two different transport media during periods of a

49
50 ORLE ET AL.
TABLE 1. M-PCR primer and probe sequences
Organism Target Primer or probe
T. pallidum 47-kDa membrane Primer KO3A
immunogen gene (35)
Primer KO4
Probe KO17
HSV Glycoprotein B gene (3) Primer KS30
Primer KS31
Probe KS54
H. ducreyi 16S rRNA gene (9) Primer KO7A
Primer KO8A
Probe KO15
known chancroid outbreak in New Orleans. The M-PCR re-
sults were compared with dark-field microscopic detection of
T. pallidum, Venereal Disease Research Laboratory (VDRL)
and rapid plasma reagin (RPR) tests for T. pallidum serology,
and culture for H. ducreyi and HSV.
(A preliminary report of this work has been presented pre-
viously [26].)
MATERIALS AND METHODS
Specimens. During three intervals from 1992 through 1994, consecutive men
presenting to the New Orleans Sexually Transmitted Disease Clinic with genital
ulcers were enrolled in the study if an adequate specimen for dark-field micros-
copy could be obtained. After first cleaning the ulcer with a sterile swab moist-
ened with saline, specimens were collected in the following order: (i) swab for
HSV culture (last interval only) collected in viral transport medium (tryptose-
phosphate with gelatin), (ii) swab for H. ducreyi culture, (iii) dry swab for M-PCR
which was vigorously agitated in 1 ml of transport medium (see below), ex-
pressed, and discarded, and (iv) a specimen from the base of the lesion collected
for dark-field analysis. Serum was also obtained for syphilis serology.
The first set of 101 specimens was collected during the winter of 1992. The
specimen for M-PCR was collected in 1 ml of phosphate-buffered saline (PBS)
containing chenodeoxycholate (Sigma) at 1 mg/ml. The second set of 97 speci-
mens was collected during the late summer of 1993, and the third set of 100
specimens was collected from May through October 1994. The last two sets were
collected by using Roche AMPLICOR Specimen Transport Medium (STM). All
specimens for M-PCR were frozen and stored at 2708C after collection until
they were thawed and divided into aliquots for use. Unused aliquots were
refrozen and were then thawed when needed.
Comparative diagnostics. For T. pallidum diagnosis, dark-field microscopy, the
serologic RPR test, and the VDRL test were performed. For H. ducreyi isolation,
specimens were inoculated onto two different selective media (16). Specimens
for HSV culture were frozen at 2708C after collection. Prior to culture, peni-
cillin-streptomycin-amphotericin B solution was added, and the specimens were
incubated on ice for 1 h. A specimen (0.25 ml) was inoculated onto two tubes of
low-passage-number MRC-5 cells (BioWhittaker, Inc., Walkersville, Md., and
Bartels Diagnostics, Inc., Bellvue, Wash.), and cultures were examined for cyto-
pathic effects at 24 h and subsequently every 48 h. All cultures showing cytopathic
effects were stained with fluorescent antibody (HSV typing; Bartels Diagnostics,
Inc.) to confirm the presence of HSV type 1 (HSV-1) or HSV-2.
Preparation of specimens for M-PCR. (i) Chenodeoxycholate specimens. Prior
to PCR analysis, specimens were heated at 1008C for 10 min and were then
diluted 10-fold with Roche AMPLICOR Specimen Diluent containing Tween 20
and 6 mM magnesium chloride. Diluted specimens were incubated for at least 15
min at room temperature before M-PCR was performed.
(ii) AMPLICOR STM specimens. Prior to PCR analysis, specimens were
diluted twofold with the specimen diluent described above and were then incu-
bated for at least 15 min at room temperature.
PCR amplification. The M-PCR mixture contained 50 ml of processed swab
specimen and 50 ml of a mixture that resulted in the following final concentra-
tions of reagents: 10 mM Tris buffer, 3.0 mM MgCl2, 50 mM KCl, 12% glycerol,
200 mM (each) dUTP, dATP, dGTP, and dCTP, 2 U of Taq polymerase (Am-
pliTaq; Perkin-Elmer), 1 U of uracil-N-glycosylase (AmpErase; Roche Molecu-
lar Systems) to prevent amplicon carryover contamination, and 25 pmol each of
the three pairs of biotinylated oligonucleotide primers (Table 1).
Amplification was performed in a thermal cycler (Perkin-Elmer PCR System
9600) on a MicroAmp base (Perkin-Elmer) with the following parameters: 508C
for 2 min to allow uracil-N-glycosylase inactivation of any carryover product;
958C for 5 min to inactivate the uracil-N-glycosylase; and 35 cycles of denatur-
J. CLIN. MICROBIOL.
Sequence (bases)
59-biotinyl-GAAGTTTGTCCCAGTTGCGGTT (537–559)
59-biotinyl-CAGAGCCATCAGCCCTTTTCA (797–776)
59-BSA-CGGGCTCTCCATGCTGCTTACCTTA (700–675)
59-biotinyl-TTCAAGGCCACCATGTACTACAAAGACGT (1022–1050, HSV-1)
59-biotinyl-GCCGTAAAACGGGGACATGTACACAAAGT (1453–1425, HSV-1)
59-BSA-GGTCTCGTGGTCGTCCCGGTGAAA (1237–1214, HSV-1)
59-biotinyl-CAAGTCGAACGGTAGCACGAAG (56–78)
59-biotinyl-TTCTGTGACTAACGTCAATCAATTTTG (495–468)
59-BSA-CCGAAGGTCCCACCCTTTAATCCGA (211–186)
ation (958C), annealing (628C), and extension (728C) for 20 s at each step.
Following temperature cycling, the samples were held at 728C for at least 15 min,
but for no longer than 2 h, prior to detection and analysis by oligonucleotide
capture in a microwell plate assay. All amplifications were performed in dupli-
cate for each patient specimen. Analysis of each set of 40 specimens included
multiple negative controls lacking DNA and positive controls consisting of mix-
tures of DNA from three plasmids containing cloned fragments of the gene
targets.
Detection of PCR products. Detection and identification of amplification prod-
ucts was performed as reported previously for Chlamydia trachomatis (22) by
using three separate microwells containing immobilized oligonucleotide capture
probes and a colorimetric detection system in the AMPLICOR (Roche Molec-
ular Systems) kit format. Oligonucleotide capture probes (Table 1) were bound
to the bottom of separate microwells via a bovine serum albumin (BSA) mole-
cule conjugated to the 59 end of the oligonucleotide (33).
Specimens were scored as positive for a specific pathogen if both duplicate
reactions produced microwell signals with an A450 of $0.25. Specimens with split
positive and negative results (3%) were reamplified in duplicate and were rean-
alyzed until the replicates were concordant.
Confirmatory PCR assays. The T. pallidum, H. ducreyi, and HSV confirmatory
PCR assays were targeted to the T. pallidum basic membrane protein gene (8),
the H. ducreyi groEL gene (27), and the HSV DNA polymerase gene (32),
respectively. Of the 298 specimens in the study, 269 were analyzed by all three
confirmatory assays. Because of limitations in the volume of specimen received,
29 specimens were analyzed by confirmatory assays only when a discrepancy
between the comparative diagnosis and PCR existed or for specimens HSV
positive by M-PCR for which culture results were unavailable.
Analysis of PCR inhibition. (i) First set. All specimens tested by M-PCR that
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were negative for all three gene targets were extracted with phenol-chloroform,
ethanol precipitated (23), and reanalyzed.
(ii) Second and third sets. Twenty copies of plasmid pSYC38 DNA, containing
the HSV primer binding sites, a scrambled HSV amplicon sequence, and an
alternate probe binding site, were incorporated into each M-PCR to flag inhib-
itory specimens. Inhibition was defined as a microwell plate signal with an A450
of ,2.0 for pSYC38 and negative signals for all three gene targets. Samples that
were negative for all three targets and pSYC38 were subsequently phenol ex-
tracted, ethanol precipitated, and then reassayed. No specimens were inhibitory
following phenol extraction. The M-PCR results were scored once the specimens
were free of inhibitors.
Discrepant result analysis. Specimens for which PCR results were discrepant
with the results obtained from the comparison diagnostic tests, H. ducreyi culture,
HSV culture, and dark-field microscopy (after decoding of the clinical diagnostic
results), were reanalyzed by using a fresh aliquot of specimen with the M-PCR
and a confirmatory PCR assay targeting a different gene.
Specimens that were consistently positive for the target DNA by repeat M-
PCR and confirmatory PCR assay but negative by the reference test were re-
solved as being true positives for the target.
Specificity testing. The following strains were used for the inclusivity testing of
M-PCR: (i) T. pallidum Nichols, a gift of Michael Norgard, Southwestern Med-
ical School, Dallas, Tex.; (ii) HSV-1 KOS and HSV-2 G, gifts of Patricia Weber,
Houghten Pharmaceuticals, Inc., San Diego, Calif.; and (iii) H. ducreyi ATCC
27721, ATCC 27722, and ATCC 33940, obtained from the American Type
Culture Collection (ATCC), Rockville, Md. H. ducreyi 174, 175, 176, 178, 179,
181, 182, 186, 187, and 189 representing 10 HindIII ribotypes were obtained from
the Centers for Disease Control and Prevention, Atlanta, Ga.
For exclusivity testing, a panel including commensal and pathogenic microbes
found in the genitourinary tract, organisms that comprise the normal skin flora,
and organisms closely related to the intended target species, was obtained from
ATCC and propagated, with the following exceptions. A spirochete panel was
obtained from the Centers for Disease Control and Prevention. The viral DNA
isolates were obtained from cell culture (Table 2).
VOL. 34, 1996 PCR DETECTION OF H. DUCREYI, T. PALLIDUM, AND HSV 51

TABLE 2. Specificity panel TABLE 3. M-PCR results

Bacterium or virus
Achromobacter xerosis
Acinetobacter calcoaceticus
Acinetobacter lwolfi
Actinomyces israelii
Aerococcus viridans
Aeromonas hydrophila
Agrobacterium radiobacter
Alcaligenes dentrificans
Alcaligenes faecalis
Bacillus subtilis
Bacteroides fragilis
Bifidobacterium adolescentis
Borrelia burgdorferi
Branhamella catarrhalis
Brevibacterium linens
Campylobacter fetus
Campylobacter jejuni
Candida albicans
Candida glabrata
Candida guilliermondii
Candida kruisei
Candida parapsilosis
Candida tropicalis
Chlamydia trachomatis
Chromobacter violaceum
Citrobacter freundii
Clostridium innocuum
Clostridium perfringens
Corynebacterium genitalium
Corynebacterium pseudo-
tuberculosis
Corynebacterium xerosis
Cryptococcus neoformans
Cytomegalovirus
Deinococcus radiopugnans
Derxia gummosa
Edwardsiella tarda
Eikenella corrodens
Enterobacter cloacae
Enterococcus faecium
No. of specimens at collection intervala:
Result Total %b
Haemophilus ducreyi 1 2 3
Haemophilus haemoglobinophilus
Haemophilus haemolyticus HSV 30 33 39 102 34
Haemophilus influenzae T. pallidum 15 36 24 75 25
Haemophilus paracuniculus H. ducreyi 37 19 9 65 22
Haemophilus parahaemolyticus Negative 21 13 29 62 21
Haemophilus parainfluenzae Specimens analyzed 101 97 100 298
Haemophilus segnis
Herpes simplex virus type 1 Coinfections 2 Hd, HSV 2 HSV, Tp; 1 Tp, HSV 7
Herpes simplex virus type 2 2 HSV, Hd
Human papillomavirus type 16 a
Hd, H. ducreyi; Tp, T. pallidum.
Human papillomavirus type 18 b
Percentages add up to more than 100% because of coinfections.
Kingella kingae
Lactobacillus casei
Lactococcus lactis cremoris
Lactococcus lactis lactis when all three targets were present in the same reaction tube.
Legionella pneumophila Reactions in which 10 copies of a target were analyzed in the
Leprospira interrogans serovar presence of 106-fold excess DNA from the other two organisms
icterohaemorrhagiae resulted in a decreased signal, but the target present at 10
Leptospira interrogans copies was still detected in each case.
Leptospira species Specificity testing. M-PCR assay analysis of unrelated bac-
Micrococcus luteus terial, fungal, and viral DNAs showed no nonspecific amplifi-
Mycoplasma genitalium cation, as indicated by the fact that all signals were below the
Mycoplasma hominis
Mycoplasma pneumoniae
cutoff for the microwell plate assay (A450, ,0.15). In addition,
Neisseria gonorrhoeae gel analysis indicated that all specific DNAs were amplifiable.
Neisseria meningitidis M-PCR detected all H. ducreyi ribotypes assayed.
Peptostreptococcus anaerobicus Analysis of clinical specimens. The following M-PCR results
Propionibacterium acnes were obtained for the 298 genital ulcer lesion specimens ana-
Proteus mirabilis lyzed: 75 were positive for T. pallidum, 65 were positive for H.
Pseudomonas aeruginosa ducreyi, and 102 were positive for HSV (Table 3). Of these,
Staphylococcus aureus four specimens contained both H. ducreyi and HSV DNAs and
Staphylococcus epidermidis three contained both HSV and T. pallidum DNAs. Sixty-two
Streptococcus agalactiae
Streptococcus mitis
specimens were negative for all three targets.
Streptococcus mutans M-PCR versus dark-field microscopy. Dark-field micros-
Streptococcus pneumoniae copy detected T. pallidum in 65 specimens, of which 58 also
Streptococcus pyogenes were positive in the M-PCR assay. The M-PCR assay, as well
Streptococcus sanguis as the confirmatory T. pallidum PCR assay, detected an addi-
Treponema denticola tional 17 specimens that were negative by dark-field analysis

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Enterococcus faecalis Treponema pallidum (Table 4). The M-PCR assay detected T. pallidum DNA in two
Epstein-Barr virus Treponema phagedenis Reiter specimens that were negative by dark-field analysis and the T.
Erysipelothrix rhusiopathiae Treponema refringens Noguchi pallidum confirmatory PCR. These specimens were resolved as
Escherichia coli Trichomonas vaginalis
Flavobacterium meningosepticum Ureaplasma urealyticum
falsely positive for T. pallidum by M-PCR. Of the 283 speci-
Gardnerella vaginalis Varicella-zoster virus mens analyzed by both the M-PCR and the T. pallidum con-
Gemmella haemolysans Vibrio parahaemolyticus firmatory PCR, the confirmatory PCR detected no additional
Haemophilus aegypticus Yersinia enterocolitica positive specimens. The concordance of the M-PCR results
Haemophilus aphrophilus with the confirmatory PCR results was 99.3%. Dark-field mi-
croscopy was not performed on specimens from three patients,
one of which was positive for T. pallidum DNA by M-PCR. A
total of 79 (27%) specimens resolved as positive for the pres-
The organisms for the specificity analysis were subjected to the same sample
preparation protocol as the clinical specimens. For all samples, at least 104
genome equivalents were added to each M-PCR mixture. Bacterial and yeast
samples were determined to be suitable for PCR by amplification with the TABLE 4. Detection of T. pallidum by M-PCR
universal bacterial 16S rRNA gene primers RW01 and DG74 (10) and the fungal versus dark-field microscopya
18S rRNA gene primers NS3 and NS8 (36). Viral DNAs were determined to be
suitable for PCR by amplification with primers specific for each virus. Dark-field No. of specimens
microscopy result M-PCR positive M-PCR negative Total
RESULTS
Positive 58 7 65
Negative 17 213 230
Assay sensitivity. To assess the sensitivity of the M-PCR
Total 75 220 295
assay, purified target DNAs from T. pallidum, H. ducreyi, and
HSV-2 were amplified by M-PCR, either alone or together a
Dark-field microscopy was not performed on three specimens, which were
with the other two DNAs, at input levels ranging from 107 to excluded from analysis. A total of 80 specimens were resolved to be positive. The
sensitivity of M-PCR was 73 of 80 specimens (91%). The sensitivity of dark-field
1022 genome equivalents. The M-PCR assay was found to have microscopy was 65 of 80 specimens (81%). The specificity of M-PCR was 213 of
a sensitivity of 1 to 10 copies when each control DNA was 215 specimens (99%). The specificity of dark-field microscopy was 213 of 213
evaluated independently and a sensitivity of at least 10 copies specimens (100%).
52 ORLE ET AL. J. CLIN. MICROBIOL.

TABLE 5. Detection of T. pallidum by M-PCR versus serologya
VDRL or RPR No. of specimens
test result M-PCR positive M-PCR negative
Positive 64 24
Negative 11 197
Total 75 221
a
Serology was not performed on two specimens, which were excluded from
the analysis. Concordance 5 (64 1 197)/296 5 88.2%.
ence of T. pallidum. The sensitivity of M-PCR detection of T.
pallidum was 91%, compared with 81% for dark-field micros-
copy. The specificity of M-PCR was 99%, compared with 100%
for dark-field microscopy.
M-PCR and dark-field microscopy versus serology. When
the results of M-PCR detection of T. pallidum DNA in genital
ulcer lesions was compared with serologic detection (positive
by the VDRL or the RPR test) of T. pallidum, the concordance
of the assays was 88% (Table 5). When the RPR and VDRL
test results were analyzed separately, M-PCR detected 11 spec-
imens containing T. pallidum that were negative by the RPR
test and 15 specimens that were negative by the VDRL test.
Results comparing the combined serology with the direct
detection of T. pallidum were also tabulated (Table 6). Of the
58 dark-field microscopy- and PCR-positive specimens, 55
were detected by serology, and of the 15 specimens that were
dark-field microscopy negative and PCR positive, 9 were de-
tected by serology. Serology was positive for an additional 18
patients whose ulcers were negative for T. pallidum by direct
detection.
M-PCR versus H. ducreyi culture. Culture of genital ulcer
lesion swabs for H. ducreyi with two media detected 46 H.
ducreyi-positive specimens, of which 45 were detected by M-
PCR (Table 7). The M-PCR assay was positive for an addi-
tional 17 specimens that were negative by culture. Sixteen of
these were positive by the confirmatory H. ducreyi PCR assay.
One specimen was culture positive but M-PCR negative. The
confirmatory PCR was also negative for this specimen. The
TABLE 7. Detection of H. ducreyi by M-PCR
versus
H. ducreyi culturea
Total
H. ducreyi No. of specimens
88 culture result M-PCR positive M-PCR negative Total
208
296 Positive 45 1 46
Negative 17 226 243
Total 62 227 289
a
Culture was contaminated for nine specimens, which were excluded from the
analysis. A total of 62 specimens were resolved to be positive. The sensitivity of
M-PCR was 61 of 62 specimens (98.4%). The sensitivity of culture was 46 of 62
specimens (74.2%). The specificity of M-PCR was 227 of 228 specimens (99.6%).
The specificity of culture was 227 of 227 specimens (100%).
The concordance between M-PCR and the H. ducreyi confir-
matory PCR was 99.6%. Nine cultures were contaminated, and
data for those cultures were not included in the final calcula-
tions. However, swab specimens corresponding to three of the
contaminated cultures contained H. ducreyi DNA by both the
M-PCR assay and the confirmatory H. ducreyi PCR assay. A
total of 62 specimens (22%) resolved as positive for the pres-
ence of H. ducreyi. The sensitivity of M-PCR detection of
H. ducreyi was 98.4%, compared with 74.2% for culture. The
specificity of the H. ducreyi M-PCR assay was 99.6%, com-
pared with 100% for H. ducreyi culture.
M-PCR versus HSV culture. HSV culture detected 28 pos-
itive specimens, of which 27 were HSV-2 and 1 was HSV-1.
The M-PCR assay detected HSV DNA in all 28 specimens that
were positive for HSV by culture and detected 11 additional
specimens that were negative for HSV by culture (Table 8). Of
the 99 specimens for which culture was performed, 39 (40%)
resolved as positive for the presence of HSV. The sensitivity of
M-PCR was 100%, compared with 71.8% for culture. The
specificities of both M-PCR and culture were 100%.
Detection of HSV in the first and second specimen sets.
HSV culture was not performed on the specimens collected
during the first two intervals; however, both the M-PCR assay
and the HSV confirmatory PCR assay detected HSV DNA in

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DNA of the organism cultured from this patient was success- 63 specimens. Of the 279 specimens from all three collection
fully amplified by M-PCR. One specimen was positive for H. intervals analyzed by both assays, the HSV confirmatory assay
ducreyi by the M-PCR assay but was negative for H. ducreyi by did not detect any additional positive specimens, resulting in a
both H. ducreyi confirmatory PCR and culture. This specimen 100% correlation between the two assays for HSV detection.
was resolved as a falsely positive result by H. ducreyi M-PCR. Inhibition and transport media. Two different transport me-
Of the 275 specimens analyzed by both the M-PCR and the H. dia were used over the course of the study. During the first
ducreyi confirmatory PCR, the confirmatory PCR did not de- collection interval, specimens were collected in PBS containing
tect any additional positive specimens undetected by M-PCR. 1 mg of chenodeoxycholate per ml. The PBS-chenodeoxy-
cholate transport medium was found to be optimal for PCR
when it was diluted 10-fold. Specimens that were found to be
TABLE 6. Comparison of T. pallidum serology with negative for all three target DNAs were extracted with phenol
direct detection of T. palliduma and reevaluated. No additional positive results were found
No. of specimens with the
following serology: Assay resultsb
Positive c
Negative TABLE 8. Detection of HSV by M-PCR versus HSV culturea
No. of specimens
55 3 Dk1, M-PCR1, C-PCR1 HSV culture
9 6 Dk2, M-PCR1, C-PCR1 result M-PCR positive M-PCR negative Total
0 1 Dk2, M-PCR1, C-PCR2
6 1 Dk1, M-PCR2, C-PCR2 Positive 28 0 28
13d 184d Dk2, M-PCR2, C-PCR2d Negative 11 60 71
Total 39 60 99
a
Serology was not performed on two specimens.
b
Dk, dark-field microscopy; C-PCR, confirmatory T. pallidum PCR; 1, posi- a
Culture was not performed for one specimen which was excluded from the
tive result; 2, negative result. analysis. A total of 39 specimens were resolved to be positive. The sensitivity of
c
Serology positive was RPR or VDRL test positive. M-PCR was 39 of 39 specimens (100%). The sensitivity of culture was 28 of 39
d
Confirmatory T. pallidum PCR was not performed for 15 dark-field micros- specimens (71.8%). The specificity of M-PCR was 60 of 60 specimens (100%).
copy-negative, M-PCR-negative specimens. The specificity of culture was 60 of 60 specimens (100%).
VOL. 34, 1996 PCR DETECTION OF H. DUCREYI, T. PALLIDUM, AND HSV
after treatment, suggesting that none of the specimens in this
set contained significant amounts of inhibitors to PCR.
During the last two collection intervals, specimens were col-
lected in Roche AMPLICOR STM and were then diluted
twofold in specimen diluent prior to PCR. Twenty copies of
control plasmid pSYC38 DNA were added to each M-PCR
mixture to monitor the inhibition of DNA amplification. For
the second and third specimen collection intervals, a total of 22
(11%) specimens were found to be inhibitory. After extraction
with phenol and ethanol precipitation, none of these speci-
mens remained inhibitory, and four additional positive M-PCR
results were obtained. Control phenol extractions of 44 other
specimens did not result in any additional positive M-PCR
results.
DISCUSSION
The M-PCR assay for the detection of three etiologic agents
of GUD described here provides for the simultaneous detec-
tion of the three agents from a single swab specimen. The
multiplex PCR assay was at least as sensitive as that described
in published reports of individual PCR assays for the three
target organisms (4, 7, 11, 16, 37) and is more efficient and less
prone to error compared with the individual assays.
M-PCR was able to establish the presence of an etiologic
agent in 80% of the collected genital ulcer lesion specimens. In
the present study, 28% (n 5 11) of resolved M-PCR HSV-
positive patient specimens were negative by HSV culture, 26%
(n 5 16) of resolved M-PCR H. ducreyi-positive patient spec-
imens were negative by H. ducreyi culture, and 18% (n 5 14) of
M-PCR T. pallidum-positive patient specimens were negative
by dark-field microscopy.
The individual confirmatory PCR assays were as sensitive
and specific as the simultaneous M-PCR, with a correlation of
results of nearly 100%. Therefore, the confirmatory PCR as-
says were used for resolving specimens discrepant by the stan-
dard diagnostic tests.
The M-PCR and the T. pallidum confirmatory assay did not
detect T. pallidum DNA in 7 specimens that were positive by
dark-field microscopy, while they detected T. pallidum DNA in
15 specimens that were negative by dark-field examination.
The results for the seven specimens that were falsely negative
by PCR may have been due to sampling error or a lower
specificity of dark-field microscopy because of the presence of
commensal spirochetes in the specimen. A monoclonal anti-
body immunostaining test can be used to increase the speci-
ficity of microscopic diagnosis of syphilis (15, 28).
In a study of other lesion specimens, we have analyzed swabs
collected first and fourth in the collection order. We found that
some of the specimens of the first swab that tested positive by
M-PCR (chiefly T. pallidum-positive specimens) were negative
when the fourth swab was tested (unpublished data). In the
present study, during collection intervals one and two, the
M-PCR swab specimen was collected second; it was third dur-
ing the last collection interval. Thus, sampling error could have
accounted for at least some of the false-negative T. pallidum
M-PCR results.
The combined T. pallidum serology tests detected an addi-
tional 18 specimens not detected by M-PCR or dark-field mi-
croscopy. This is not unexpected because many sexually trans-
mitted disease clinic patients will have had prior or untreated
syphilis. There is also the possibility that some of the serology-
positive, dark-field microscopy- and PCR-negative specimens
could be due to serologic false-positive results because trepo-
nemal serologic tests were not used to confirm the serologic
results. The two nontreponemal serologic tests used for the
53
present study are known to give false-positive results in some
patient populations, and without some other evidence for the
diagnosis of syphilis, a nonreactive treponemal test does not
define a current T. pallidum infection (21). The RPR and
VDRL tests are considered to be generally equivalent in sen-
sitivity. In the present study, of the specimens from 69 patients
with confirmed T. pallidum infections on which both VDRL
and RPR tests were performed, the VDRL test detected 53
positive specimens and the RPR test detected 57 positive spec-
imens.
M-PCR and the T. pallidum confirmatory assay detected
nine specimens that the combined T. pallidum serologic tests
did not detect. This is also not unexpected since nontrepone-
mal tests, such as the VDRL and RPR tests, have a sensitivity
of 75% in patients with early syphilis (21).
Both the M-PCR assay and the H. ducreyi confirmatory PCR
assay did not detect H. ducreyi DNA in one specimen that was
positive by culture for H. ducreyi. However, the H. ducreyi
strain isolated from this patient was successfully amplified by
both the H. ducreyi M-PCR and H. ducreyi confirmatory PCR
assays. The negative M-PCR result with the swab specimen
collected for M-PCR was most likely due to a lack of target
DNA in this particular swab specimen. In the present study,
the sensitivity of H. ducreyi culture was greater or equivalent to
that in a previously published report (24).
Detection of HSV by M-PCR assay was clearly more sensi-
tive than HSV culture. PCR not only detected all 28 culture-
positive specimens but also detected 11 specimens that were
negative by HSV culture. The sensitivity of the HSV culture
may have been compromised because of specimen freezing. It
is interesting that seven specimens contained HSV DNA in
addition to T. pallidum or H. ducreyi DNA. We do not know
whether these represent genital ulcers resulting from two etio-
logic agents, a latent HSV infection reactivated by the pres-
ence of another GUD agent, or the detection of asymptomatic
HSV shedding.
The presence of substances in the analyzed specimens in-
hibitory to PCR was not unexpected, given the nature of the
lesions and frequent attempts by patients at self-treatment with
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salves and ointments. The difference in the frequency of oc-
currence of specimens containing substantial amounts of in-
hibitors between the two transport media was probably due to
the dilution factor used prior to PCR. The PBS-chenodeoxy-
cholate transport medium was diluted 10-fold to avoid inhibi-
tion of the M-PCR because of the substances in the medium
itself, and no specimens were found to be inhibitory. However,
STM required only a twofold dilution with the same sample
diluent to abrogate PCR inhibition because of the medium,
and 11% of the specimens were inhibitory to the PCR. A
10-fold dilution of STM specimens decreased the level of in-
hibitors to a background level in most of these specimens (data
not shown). Other commercial collection and transport media
as well as improved sample preparation protocols are being
investigated for use with the M-PCR assay.
Because of the speed, superior sensitivity, and ability of the
M-PCR to give three diagnoses from one assay, M-PCR could
be used not only for the definitive diagnosis of these sexually
transmitted diseases but also as an epidemiologic tool in prev-
alence studies and for the determination of the etiologic base
of sexually transmitted disease in various locales.
ACKNOWLEDGMENTS
We thank the following people: Shari Kurita for providing graphics,
Sheng-Yung Chang for constructing plasmid pSYC38, Catherine Cam-
marata for coordinating the clinical results, and Stephen A. Morse for
facilitating the study.
54 ORLE ET AL.
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