Coutinho, RA

Fennema, J.S.

Morre, S.A.

Pena, A.S.

Spaargaren, J

Vries, H.J. de

DSpace at VU

LETTERS
Real-time
Polymerase 
Chain Reaction 
To Diagnose
Lymphogranuloma
Venereum
To the Editor: An outbreak of rec-
tal lymphogranuloma venereum
(LGV) has been detected in the
Netherlands among men who have
sex with men (1–4). More cases of
LGV in other European countries
such as Belgium, France, and the
United Kingdom have been reported,
and the first cases have been detected
in the United States as well. This
infection is encountered not only by
clinicians who treat sexually transmit-
ted diseases but also by gastroenterol-
ogists. Both the European
Surveillance of Sexually Transmitted
Infections (http://www.essti.org) and
the Centers for Disease Control and
Prevention (http://www.cdc.gov) are
working on outbreak warning and
response systems to increase the
awareness and the direct management
of the LGV outbreak (5,6).
Different approaches have been
described to diagnose LGV infections
(Figure). The first 3 approaches have
serious disadvantages: cell culture is
rarely available in routine diagnostic
settings, polymerase chain reaction
(PCR)-based restriction fragment
length polymorphism (RFLP) analy-
sis (usually nested PCR approaches
are used) needs post-PCR restriction
enzyme profiling, and sequencing
requires additional analyses of
sequence data to identify the
Chlamydia trachomatis serovar
responsible for infection. In addition,
all 3 techniques are time consuming
(at least 1–4 days to get a result),
laborious, and require specially
trained personnel in a sophisticated
laboratory setting. Therefore, we
developed a real-time PCR approach
(TaqMan and Rotorgene) that can eas-
ily identify LGV strains in 2 hours
with equipment that is available in
almost all diagnostic settings.
We used the polymorphic mem-
brane protein H gene (pmp gene) as a
PCR target because it has a unique gap
in LGV strains of C. trachomatis,
compared to other serovars, which
makes it highly specific. The follow-
ing primers and probes were selected:
LGV-F 5′ CTG TGC CAA CCT CAT
CAT CAA 3′, LGV-R 5′ AGA CCC
TTT CCG AGC ATC ACT 3′, and
LGV MGB-probe 6-FAM-CCT GCT
CCA ACA GT. Real-time PCR condi-
tions (20-µL format) for TaqMan were
as follows: 2× TaqMan Universal
Mastermix (Applied Biosystems,
Foster City, CA, USA), 18 pmol each
primer, 0.2 µmol/L probe, and 2 µL
(LGV L2) DNA or clinical sample; 2
min at 50°C, 10 min at 95°C, and 40
cycles of 15 sec at 95°C and 1 min at
60°C. Conditions for Rotorgene were
as follows: 10× buffer (Hoffman-La
Roche Ltd, Basel, Switzerland), 10
pmol each primer, 0.04 µmol/L probe,
2 µL (LGV L2) DNA or clinical sam-
ple; 2 min at 50°C, 10 min at 95°C,
and 45 cycles of 15 sec at 95°C and 1
min at 60°C. By using a previously
described serial dilution of LGV L2
(7), sensitivity was assessed as 0.01
inclusion-forming units for both real-
time PCR assays.
To determine specificity, we tested
different C. trachomatis serovars and
serovariants A, B, Ba, C, D, Da, D-, E,
F, G, Ga, H, I, Ia, I-, J, Jv, K, L1, L2,
L2b, L3, C. muridarum (MoPn), C.
pneumoniae, C. pecorum, C. psittaci,
and 32 other microorganisms that nor-
mally reside in the human perianal
and urogenital region and in the
oropharynx. These organisms includ-
ed gram-positive and gram-negative
bacteria and yeast: Acinetobacter
baumannii, Campylobacter jejuni,
Candida albicans, other yeast,
Enterobacter agglomerans, Entero-
coccus faecalis, Escherichia coli,
Streptococcus spp., Haemophilus
influenzae, Klebsiella pneumoniae,
Mycoplasma spp., Neisseria meningi-
tidis, Pasteurella spp., Pseudomonas
aeruginosa, Salmonella enteritidis,
Shigella sonnei, Staphylococcus
aureus, and others. Only LGV strains
L1, L2, L2b, and L3 tested positive in
both the TaqMan and Rotorgene
assays, which shows the analytical
specificity of real-time PCR.
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 8, August 2005 1311
Figure. Diagnosis of lymphogranuloma venereum. MIF, microimmunofluorescence; STI,
sexually transmitted infection; PCR, polymerase chain reaction; RFLP, restriction fragment
length polymorphism; PAA, poly acrylamide; BLAST, basic local alignment search tool.
LETTERS
Subsequently, we determined in a
blinded setting the presence of LGV in
a selected group of patients (clinical
spectrum and epidemiology described
elsewhere [8]) according to C. tra-
chomatis–positive rectal swab
(Chlamydia 2SP Collection &
Transport Kit [Quelab] by commer-
cially available PCR (COBAS
AMPLICOR, Hoffman-La Roche
Ltd). By using the 2 reference stan-
dard techniques to type C. trachomatis
serovars (PCR-based RFLP of the
omp1 gene or sequencing the variable
segment 2 [VS-2] of the omp1 gene)
(9,10) with DNA isolated from rectal
swab specimens (standard isopropanol
DNA isolation method), we identified
28 of 125 men as LGV-positive. These
28 samples were also positive in both
the TaqMan and Rotorgene assays. We
also identified 2 additional LGV infec-
tions, which were initially typed and
then retested as single-strain infections
with serovars E and D by both PCR-
based RFLP analysis and VS-2
sequencing. This discrepancy is most
likely due to a double infection, which
will, in most cases, result in the prefer-
ential amplification of 1 strain in the
omp1 PCR and PCR-based sequenc-
ing methods; in the TaqMan and
Rotorgene assays, only LGV strains
can be amplified. Whether this out-
break is partially technically driven
must be assessed in the future by retro-
spectively investigating the presence
of these LGV infections in men who
have sex with men and the presence of
the L2b strain in the past, since at pres-
ent only LGV infections from 2003 to
2005 have been investigated.
Servaas A. Morré,*1 Joke
Spaargaren,†1
Johannes S.A. Fennema,† 
Henry J.C. de Vries,†‡ 
Roel A. Coutinho,‡§ 
and A. Salvador Peña*
*VU University Medical Center,
Amsterdam, the Netherlands; †Municipal
Health Service, Amsterdam, the
Netherlands; ‡Academic Medical Center,
Amsterdam, the Netherlands; and
§National Institute for Public Health and the
Environment, Bilthoven, the Netherlands
References
1. Nieuwenhuis RF, Ossewaarde JM, van der
Meijden WI, Neumann HA. Unusual pres-
entation of early lymphogranuloma
venereum in an HIV-1 infected patient:
effective treatment with 1 g azithromycin.
Sex Transm Infect. 2003;79:453–5.
2. Nieuwenhuis RF, Ossewaarde JM, Götz
HM, Dees J, Thio HB, Thomeer MG, et al.
Resurgence of lymphogranuloma venereum
in Western Europe: an outbreak of
Chlamydia trachomatis serovar L2 proctitis
in the Netherlands among men who have
sex with men. Clin Infect Dis.
2004;39:996–1003.
3. Den Hollander JG, Ossewaarde JM, de
Marie S. Anorectal ulcer in HIV patients,
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4. French P, Ison CA, Macdonald N.
Lymphogranuloma venereum in the United
Kingdom. Sex Transm Infect.
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5. Centers for Disease Control and
Prevention. Lymphogranuloma venereum
among men who have sex with men—
Netherlands, 2003–2004. MMWR Morb
Mortal Wkly Rep. 2004;53:985–8.
6. van Weel J. Rare sexually transmitted dis-
ease hits Europe. Lancet Infect Dis.
2004;4:720.
7. Morré SA, Sillekens P, Jacobs MV, van
Aarle P, de Blok S, van Gemen B, et al.
RNA amplification by nucleic acid
sequence–based amplification with an
internal standard enables reliable detection
of Chlamydia trachomatis in cervical
scrapings and urine samples. J Clin
Microbiol. 1996;34:3108–14. 
8. Spaargaren J, Fennema HSA, Morré SA, de
Vries HJC, Coutinho RA. New
Lymphogranuloma venereum Chlamydia
trachomatis variant, Amsterdam. Emerg
Infect Dis. 2005;11:1090–2.
9. Morré SA, Ossewaarde JM, Lan J, van
Doornum GJ, Walboomers JM, MacLaren
DM, et al. Serotyping and genotyping of
genital Chlamydia trachomatis isolates
reveal variants of serovars Ba, G, and J as
confirmed by omp1 nucleotide sequence
analysis. J Clin Microbiol.
1998;36:345–51.
10. Molano M, Meijer CJLM, Morré SA, Pol
R, van den Brule AJ. Combination of PCR
targeting the VD2 of omp1 and reverse line
blot analysis for typing of urogenital
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2004;42:2935–9. 
Address for correspondence: Servaas A. Morré,
VU University Medical Center, Faculty of
Medicine, Laboratory of Immunogenetics, Van
der Boechorststraat 7, 1081 BT, Amsterdam,
the Netherlands; fax: 31-20-44-48418; email:
samorretravel@yahoo.co.uk
SARS Vaccine
Protective in Mice 
To the Editor: Less than a year
after the identification of the severe
acute respiratory syndrome coron-
avirus (SARS-CoV) (1), 3 independ-
ent laboratories reported protection
from SARS-CoV challenge in animal
models using a DNA vaccine or
recombinant forms of the modified
vaccinia Ankara or a parainfluenza
virus, encoding the spike gene (2–4).
Their protective efficacies are encour-
aging because they provide proof that
a SARS-CoV vaccine is feasible.
However, vaccines based on those
technologies are not licensed for
human use, and recommendation and
licensing will likely take many years.
We have developed an inactivated
virus vaccine that induces neutraliz-
ing antibodies and protects against
SARS-CoV challenge.
The vaccine was produced as
described elsewhere (5). Briefly, the
SARS-CoV (strain FRA, GenBank
accession no. AY310120) was grown
in Vero cells, inactivated with β-pro-
piolactone (BPL), and complete inac-
tivation was confirmed by 2 consecu-
tive passages on Vero cells.
Inactivated virus was purified by col-
umn chromatography followed by
sucrose gradient centrifugation. The
fraction containing virus was dialyzed
1312 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 8, August 2005
1These authors contributed equally to this
study.


Real-time polymerase chain reaction to diagnose lymphogranuloma venereum

de Vries, H.J.

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Real-time polymerase chain reaction to diagnose lymphogranuloma venereum

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