JOURNAL OF CLINICAL MICROBIOLOGY, June 2005, p. 2973–2975 Vol. 43, No.

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0095-1137/05/$08.00⫹0 doi:10.1128/JCM.43.6.2973–2975.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Molecular Diagnosis of Lymphogranuloma Venereum in Patients with

Genital Ulcer Disease
Patrick D. J. Sturm, Prashini Moodley, Keshnie Govender, Louise Bohlken, Trusha Vanmali, and
A. Willem Sturm*
Genital Ulcer Disease Research Unit of the University of KwaZuluNatal and the South African Medical Research Council,
Department of Medical Microbiology, University of KwaZuluNatal, Durban, South Africa
Received 9 August 2004/Returned for modification 16 December 2004/Accepted 29 January 2005

The detection of herpes, chancroid, and syphilis in genital ulcers is done by PCR. This is not so for
lymphogranuloma venereum (LGV). We report on the use of a PCR with digestion that differentiates the LGV
biovar from the trachoma biovar. Our findings suggest that the clinical description of LGV in current textbooks
is incomplete.

Current textbooks divide lymphogranuloma venereum LGV. We also describe the clinical presentation of LGV diag-
(LGV) into three stages (7, 31). The primary stage is charac- nosed with this methodology in patients presenting with GUD.

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terized by small, painless herpetiform genital ulcers which are
often not recognized and resolve spontaneously. The second-
ary stage is characterized as lymphadenopathy, mostly without
a genital lesion. Inguinal lymphadenitis with or without a his-
tory of genital lesions is therefore the characteristic presenta-
tion (11, 12, 14). The tertiary or anorectal/elephantiasis stage
results from destroyed inguinal lymphoid tissue. Diagnostic
methods to diagnose LGV are microimmunofluorescence
(MIF), culture, direct immunofluorescence (DIF), and nucleic
acid amplification tests (NAATs). The use of NAATs is the
preferred method to diagnose herpes, chancroid, and syphilis,
but these tests are rarely applied to diagnose LGV. One study
used a PCR targeting the cryptic plasmid without further anal-
ysis (9), and one used sequence analysis of major outer mem-
brane protein gene amplicons (1). MIF and culture are not
widely available and lack sensitivity, while the targets for DIF
and commercially available NAATs are present in all Chla-
mydia trachomatis biovars (4). The lack of sensitive and specific
diagnostic tests is likely responsible for the wide variation in
prevalence of LGV in patients with genital ulcer disease
(GUD) reported from Africa (2, 5, 6). Biovar identification of
C. trachomatis in specimens from genital ulcers is important
because non-LGV ulcers can be contaminated with the tra-
choma biovar from concurrent urethritis or cervicitis (13).
A PCR with restriction digestion of the product to distin-
guish between the C. trachomatis biovars, with the cysteine-rich
outer membrane protein (CrP) gene (GenBank AF 304332) as
the target, has been described previously (15). We were first in
applying this combination of PCR and restriction fragment
length polymorphism in a treatment efficacy study with patients
with GUD (13), resulting in a rise in the prevalence of LGV in
patients with GUD in Durban from 2% to 10% (5, 13). Here
we report on the validity of this NAAT for the diagnosis of
* Corresponding author. Mailing address: Department of Medical
Microbiology, Nelson R. Mandela School of Medicine, Private Bag 7,
Congella 4013, South Africa. Phone: 27-31-2604395. Fax: 27-31-
2604431. E-mail: sturm@kznu.ac.za.
The study cohort was the same as that in the treatment
efficacy study (13) and included 520 consecutive consenting
patients presenting with GUD at the Prince Cyril Zulu Com-
municable Diseases Clinic in Durban between October 2000
and April 2001.
Epidemiological and clinical characteristics of patients with
LGV were compared with those of patients with genital her-
pes, chancroid, syphilis, and/or granuloma inguinale (Table 1).
All infections except granuloma inguinale were diagnosed by
PCR (13). The study was approved by the Ethics Committee of
the Nelson R. Mandela School of Medicine.
Specimen collection and preparation have been described
previously (13). A blood and tissue mini kit (QIAGEN, Frank-
furt, Germany) was used to extract DNA. LGV was diagnosed
by PCR-restriction fragment length polymorphism as previ-
ously described (13, 15). A PCR targeting the 60-kDa cysteine-
rich outer membrane protein was followed by digestion with
AccI. C. trachomatis L2 (ATCC VR-902B), C. trachomatis
biovar trachoma (a laboratory isolate from a cervical speci-
men), and water were included as controls in each run. This
test is further referred to as CrP-PCR-D. Figure 1 shows the
results of one run.
The specimens of 66 (13%) patients tested positive by CrP-
PCR-D, and these specimens were further analyzed. To
confirm the presence of chlamydia, the strand displacement
amplification assay (BDProbeTec ET; Becton Dickinson,
Cockeysville, Md.) was performed on all samples with positive
CrP-PCR-D results. The sensitivity of CrP-PCR-D was mea-
sured by the amplification of 10-fold serial dilutions, in phos-
phate-buffered saline (pH 6.8), of a C. trachomatis culture in
McCoy cells. The number of inclusion bodies per ml in the
undiluted culture was established by immunofluorescence mi-
croscopy (MicroTrak; Trinity Biotech, Wicklow, Ireland). Six
randomly selected PCR products with LGV biovar restriction
patterns and one with a trachoma biovar restriction pattern
were sequenced. PCR products were purified by using the QIA
Quick PCR purification kit (QIAGEN). Sequence reactions
were done by using the BigDye terminator cycle-sequencing

2973
2974 NOTES J. CLIN. MICROBIOL.

TABLE 1. Demographics and clinical presentation of male and female patients with genital ulcer disease with and without LGVa
Value for:

Patient parameter Males Females

LGV (n ⫽ 34) Non-LGV (n ⫽ 345) P value LGV (n ⫽ 19) Non-LGV (n ⫽ 110) P value

Demographics and history

Median age (yrs [range]) 25 (18–39) 26 (16–64) 0.5 25 (18–47) 25 (16–46) 0.9
Median duration of sexual 10 (2–21) 9 (0–46) 0.9 8 (1–28) 7 (0–27) 0.7
exposure (yrs [range])
Median total no. of partners in 12 (3–50) 10 (1–100) 0.2 4 (1–20) 3 (1–30) 0.4
lifetime (range)
Last sex contact, median (days 13 (3–42) 7 (1–395) 0.8 7 (3–60) 9 (1–2,190) 0.3
[range])
Previous discharge (%) 59 66 0.5 63 60 0.99
Previous ulcer (%) 44 41 0.83 11 68 0.03

HIV positive (%) 79 70 0.38 95 85 0.47

Symptoms
Median duration (days [range]) 12 (2–90) 7 (1–365) 0.05 7 (1–60) 7 (1–90) 0.4
Inguinal pain (%) 24 14 0.23 5 13 0.69

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Swelling in the groin (%) 6 4 0.64 0 1 1
Concurrent discharge (%) 24 24 0.89 79 74 0.24

Examination
Palpable inguinal nodes (%) 90 62 0.003 44 43 0.81
Painful ulcer on examination 94 94 1 84 92 0.37
(%)
Single ulcer (%) 91 83 0.36 94 77 0.19
Site ulcer (%)
Male
Shaft 0 8 0.14
Glans 19 28 0.39
Prepuce inside 44 44 0.89
Prepuce outside 3 15 0.1
Coronal sulcus 31 22 0.34
Phrenulum 6 1 0.06
Scrotum 0 3 1
Perineum 6 1 0.04
Groin 0 0.003 1
Pubis 0 0.003 1

Female
Labia majora 6 34 0.04
Labia minora 41 48 0.82
Perineum 41 19 0.06
Vaginal wall 12 8 0.54
Clitoris 6 1 0.27
Thigh 0 2 1
Groin 0 3 1
a
Ten patients with mixed LGV infections (three males and seven females) and two patients with unconfirmed LGV infections were excluded. Significant P values
are shown in bold.

ready reaction kit (Applied Biosystems, Foster City, Calif.).
Products were analyzed on an ABI 3100 analyzer.
Human immunodeficiency virus (HIV) infection was diag-
nosed by the Determine HIV1/2 test (Abbott Laboratories,
Chicago, Ill.). Positive results were confirmed by the Capillus
HIV1/2 (Trinity Biotech) test.
Groups were compared by use of Wilcoxon nonparametric
tests for numeric data and the chi-square test or Fisher’s exact
test for categorical data.
The CrP-PCR-D was positive with McCoy cell dilutions con-
taining as few as 5 to 10 inclusion bodies. This is in keeping
with what has been reported previously (15). All 66 specimens
positive by CrP-PCR-D also tested positive with the strand
displacement amplification assay. Identified sequences matched
those of the CrP gene of C. trachomatis in GenBank and
demonstrated the expected numbers and locations of restric-
tion sites for AccI in both biovars (15).
For two specimens (both from male patients), the signal
obtained with the CrP-PCR-D was repeatedly faint and the
restriction pattern was considered unreliable. Of the remaining
64, 63 were positive for LGV and 1 positive for the trachoma
biovar. This suggests that the detection of chlamydiae in gen-
ital ulcer specimens by culture, DIF, or non-biovar-specific
NAATs has an acceptable specificity for LGV.
VOL. 43, 2005
FIG. 1. Gel electrophoresis of PCR products of specimens investi-
gated for the presence of Chlamydia species by CrP-PCR-D. Lane 1,
molecular weight marker; lane 10, undigested 552-bp product; lanes 2
and 9, digested products (280 and 272 bp) of C. trachomatis, LGV
genotype (seen as one line); lanes 4 through 6, digested products (280,
195, and 77 bp) of C. trachomatis, urogenital genotype.
The prevalence of LGV in women was higher than that of
LGV in men (19% versus 10%; P ⫽ 0.006). Characteristics of
patients diagnosed with LGV only were compared with those
of patients with no LGV after stratification for gender (Table
1). Ulcers were, subjectively, equally as painful in LGV and
non-LGV cases on specimen collection. Men, but not women,
with LGV had more frequently palpable inguinal lymph nodes
than men without LGV. These lymph nodes had no particular
features, e.g., groove sign.
Patients included in this study all had primary LGV. Early
publications acknowledge the difficulty in differentiating LGV
lesions from chancroid lesions (3, 7, 8, 10), while current text-
books compare LGV lesions with genital herpes lesions (11,
12). This change in the description of the lesions over time may
have resulted from the application of MIF as the standard
diagnostic test. Being an antibody detection test, MIF is likely
to produce positive results only with cases of advanced disease.
NOTES 2975
LGV was not associated with HIV. The clinical presenta-
tions of LGV for the 45 HIV-infected patients were similar to
those for the 8 non-HIV-infected patients with LGV, as was
the case for patients with non-LGV ulcers (data not shown).
Although changes in clinical presentation of LGV due to HIV
infection cannot be excluded, the similarity between our find-
ings and the early descriptions (3, 7, 8, 10) suggests that this is
not the sole explanation.
In conclusion, the presence of C. trachomatis biovar LGV in
painful ulcers of appreciable size suggests that the description
of the clinical features of LGV in current textbooks is incom-
plete.
This work was supported by the South African Medical Research
Council via funding of the Genital Ulcer Disease Research Unit (di-
rector, A. Willem Sturm).
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