|
|
Letter
Real-time Polymerase Chain
Reaction To Diagnose Lymphogranuloma Venereum
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
Suggested
citation for this article
To the Editor: An outbreak of rectal 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 transmitted
diseases but also by gastroenterologists. 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) analysis (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 easily identify LGV strains in 2 hours with equipment
that is available in almost all diagnostic settings.
We used the polymorphic membrane 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 following
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 conditions (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 sample; 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
normally reside in the human perianal and urogenital region and in the
oropharynx. These organisms included gram-positive and gram-negative bacteria
and yeast: Acinetobacter baumannii, Campylobacter jejuni,
Candida albicans, other yeast, Enterobacter agglomerans,
Enterococcus faecalis, Escherichia coli, Streptococcus
spp., Haemophilus influenzae, Klebsiella pneumoniae, Mycoplasma
spp., Neisseria meningitidis, 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.
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. trachomatis–positive
rectal swab (Chlamydia 2SP Collection & Transport Kit [Quelab] by
commercially available PCR (COBAS AMPLICOR, Hoffman-La Roche Ltd). By
using the 2 reference standard 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 infections, 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 preferential
amplification of 1 strain in the omp1 PCR and PCR-based sequencing
methods; in the TaqMan and Rotorgene assays, only LGV strains can be amplified.
Whether this outbreak is partially technically driven must be assessed
in the future by retrospectively 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 present only LGV infections from 2003 to 2005 have
been investigated.
References
- Nieuwenhuis RF, Ossewaarde JM, van der Meijden WI,
Neumann HA. Unusual
presentation of early lymphogranuloma venereum in an HIV-1 infected
patient: effective treatment with 1 g azithromycin. Sex Transm Infect.
2003;79:453–5.
- 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.
- Den Hollander JG, Ossewaarde JM, de Marie S. Anorectal
ulcer in HIV patients, don't forget lymphogranuloma venereum! AIDS.
2004;18:1484–5.
- French P, Ison CA, Macdonald N. Lymphogranuloma
venereum in the United Kingdom. Sex Transm Infect. 2005;81:97–8.
- 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.
- van Weel J. Rare
sexually transmitted disease hits Europe. Lancet Infect Dis. 2004;4:720.
- 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.
- 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.
- 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.
- 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 Chlamydia trachomatis serovars in cervical
scrape specimens. J Clin Microbiol. 2004;42:2935–9.
1These authors contributed equally to this
study.
Suggested citation
for this article:
Morré SA, Spaargaren
J, Fennema JSA, de Vries HJC, Coutinho RA, Peña AS. Real-time polymerase
chain reaction to diagnose lymphogranuloma venereum [letter]. Emerg Infect
Dis [serial on the Internet]. 2005 Sep [date cited]. Available
from http://www.cdc.gov/ncidod/EID/vol11no08/05-0535.htm
|
