Kongressbeiträge

12th Congress of the European Academy of Dermatology and Venereology (EADV)
October 15-18, 2003
Barcelona (Spain)
Stary A
Outpatients´Centre for Infectious Venerodermatological Diseases; Vienna, Austria

The epidemiologic in Europe shows an increase in rates of reportable bacterial infections such as gonorrhoea and syphilis. In addition, in many countries the number of antibiotic-resistant gonorrhoea has increased. Penicillin and quinolone-resistant N. gonorrhoeae (QRNG) strains continue to spread not only in Asian, Carribean, and African countries but also in Europe with a percentage of up to 30%. Elevated MIC´s to azithromycin raise concerns about its recommendation for gonococcal treatment.
In an evaluation performed in the period from 1999 to 2002 an increase of gonococcal infections has been observed in Austria. In three main Centres for Diagnosis of Sexually Transmitted Infections (STI) a 3.7-fold increase from 123 cases in 1999 up to 575 infections in 2002 was recognized in patients screened for gonorrhea and other STIs. During this period, an almost 10 fold increase of QRNG was observed from 3.9% to 33.6%, followed by a 5-fold increase of penicillin resistant strains. In contrast, resistance against cephalosporines and macrolides were only recognized in very few cases.
The increasing number of gonococcal infections in both, men and women, and the upcoming problem of resistance of N. gonorrhoeae to quinolones and other antibiotics underlines the need for the performance of gonococcal culture in order to examine the resistance profile of N. gonorrhoeae before treatment. Furthermore, recommended management guidelines for gonococcal infections in the USA, Europe, and Austria might have to be revised .

12th Congress of the European Academy of Dermatology and Venereology (EADV)
October 15-18, 2003
Barcelona (Spain)
Stary A
Outpatients´Centre for Infectious Venerodermatological Diseases; Vienna, Austria

Methods using an artificial nucleic acid amplification (NAA) are a new advantage in diagnosis of chlamydial and gonococcal infections with a sensitivity of about 90% and a specificity of about 99%.
Commercially available NAA assays for Chlamydia trachomatis and Neisseria gonorrhoeae have long been restricted to the Amplicor PCR, a DNA target amplification assay, and the ligase chain reaction LCR, a DNA probe amplification assay, which has recently been taken off the market. The quality of NAA assays has improved by including an internal control in some of the assays, such as COBAS Amplicor and the strand displacement amplification (SDA) assay. In addition to DNA amplification, RNA amplification is used in the new APTIMA Combo 2 assay, which has a target capture method included in addition to RNA amplification increasing the sensitivity for samples with only small numbers of RNA. These assays provide laboratories with powerful tools with particular impact in the detection of both, chlamydial as well as gonococcal infections. For practical relevance, coamplification of Neisseria gonorrhoeae and C. trachomatis from one sample enables diagnosis of genital infections with these pathogens within few hours.
The high sensitivity of NAA enables the detection of a low number of organisms present in asymptomatic patients and their contact persons, and the detection of nonviable organisms in specimens contaminated with microorganisms such as first void urine and introital vulvovaginal samples. NAA assays offer therefore screening possibilities especially for asymptomatic individuals.

15th Meeting of the International Society for Sexually Transmitted Diseases Research (ISSTDR)
July 27-30, 2003
Ottawa (Canada)
Stary A
Outpatients´Center for Infectious Venerodermatological Diseases; Vienna, Austria

Individuals attending a diagnostic centre for sexually transmitted infections (STI) may ask for chlamydial diagnosis for different reasons such as clinical symptoms in the lower or upper genital tract, contact tracing, and monitoring for an STI infection because of tubal sterility, partner control or an occasional sexual contact. In order to prevent further spreading of the infection it is important to use a sensitive and specific diagnostic method for the diagnosis of C. trachomatis at an early stage.
The use of nucleic acid amplification (NAA) assays for routine diagnosis of chlamydial infections has enhanced the sensitivity of diagnostic procedures and enables to get a better knowledge on the infectious status of STI patients and their partners. It has been shown that, by using NAA assays for diagnosis, transmission of C. trachomatis to the asymptomatic partner and ascension to the upper genital tract occur more often than known until now.
However, a higher degree of technical expertise is required for the performance of NAA assays to minimize the risk of contamination and of false positive results. This requires the access to a confirmation test for positive results. It has also been demonstrated that the presence of amplification inhibitors especially for urine may cause false negative results for NAA assays in a various degree. Strict quality control procedures have therefore to be included. Furthermore, the dependance on technical equipments has to be considered when organizing diagnostic strategies for chlamydia.The higher costs of NAA assays may still avoid a large scale use of NAA assays for chlamydia diagnosis as a routine test in many diagnostic centers in Europe and other parts of the world.

15th Meetin of the International Society for Sexually Transmitted Diseases Research (ISSTDR)
July 27-30, 2003
Ottawa (Canada)
Angelika Stary
Outpatients` Centre for Diagnosis of Infectious Venero-dermatological Diseases, Vienna, Austria

Background: In Austria, gonorrhoea and syphilis are the most common venereal diseases and reportable by law to the Publish Health Office (PHO). In contrast, other bacterial and viral STIs are not legally reported and therefore only data collected in few specialized diagnostic centers are available.
Objectives: To determine the frequency of reported cases of syphilis and gonorrhoea in Austria and to evaluate the resistance pattern of isolated N. gonorrhoeae (NG) strains over the period from 1999 to 2002.
Methods: For reported syphilis cases diagnosis was mainly based on serology and for gonorrhea on culture methods. The nationwide reporting system includes only an anonymous differentiation between infections in men and women but no additional demographic o clinical data are available. For further evaluation, a total of 134,626 patients were screened for the presence of gonorrhoea and other STIs in 3 diagnostic centers in Vienna. An overall of 728 isolated NG strains were tested for resistance to quinolones, cephalosporins, penicillin, macrolids, and tetracycline by the disk diffusion test.
Results: A nationwide 2.3-fold increase of syphilis (from 184 to 420 cases) and of gonorrhoea (from 434 to 985 cases) was observed over a 4 years period. QRNG raised from 3.9% in 1999 to 33.6% in 2002 with a total of 108 strains resistant to all quinolones tested. Penicillin resistance increased 4.8-fold, while resistance to cephalosporins and macrolids was seen only in rare cases.
Conclusion: The increase of venereal diseases and the change of the resistance pattern for NG demonstrate the importance of a legally established surveillance system including demographic data and the proof of the antibiotic resistance for NG.

4th Baltic Congress on Dermato-Venereology
May 22.-25, 2003
Tallinn (Estland)
Angelika Stary
Outpatients` Centre for Diagnosis of Infectious Venero-dermatological Diseases, Vienna, Austria

Chlamydial arthritis as part of Reiter`s syndrome is one of several reasons of reactive arthritis that follows a localized infection of the mucosal surface. In contrast to arthritis during disseminated gonococcal infection, chlamydial arthritis is accompanied by symptoms of nongonococcal urethritis, followed by conjunctivitis, and mucocutaneous manifestations, referred to as sexually acquired reactive arthritis (SARA). Neisseria gonorrhoeae is the most common sexually transmitted pathogen causing SARA as a result from direct synovial or periarticular infection. It differentiates from Reiter`s syndrome by clinical, immunological, and therapeutic characteristics. Only in few cases this syndrome has also been reported after gonococcal infection.
Immunologic studies in Reiter`s syndrome have shown a pathologic role of CD-8 positive cytotoxic cells, stimulated by antigen presentation HLA-B27 class 1 molecules. While the enteric form of Reiter´s syndrome is epidemic, the sexually acquired form is endemic. However, there is not always a clear evidence of sexual intercourse with a new partner and the simultaneous occurrence of nongonococcal urethritis. Genital chlamydial infection has been documented in approximately 50% of men with sexually acquired reactive arthritis.
The symptoms of Reiter´s syndrome may resolve after 2 to 6 months but recovery may last up to one year or even longer in about one third of the patients. Treatment is focused on antibiotics for chlamydial therapy, supported by non-steroidal antiinflammatory agents and immunosuppressive drugs in persistent cases.

20th World Congress of Dermatology
July 1-5, 2002
Paris (France)
Angelika Stary
Outpatients` Centre for Diagnosis of Infectious Venero-dermatological Diseases, Vienna, Austria

Methods using the artificial amplification of nucleic acid or signals after hybridization are a new advantage in diagnosis of STDs. Commercially available nucleic acid amplification (NAA) assays for Chlamydia trachomatis provide laboratories with powerful tools with particular impact in the detection of genital chlamydial infections and have shown a performance pattern with a higher sensitivity when compared to other techniques and a high specificity comparable with culture. The high sensitivity of NAA enables the detection of a low number of organisms present in asymptomatic patients and their contact persons without signs of inflammation. Furthermore, the detection of nonviable organisms offers the opportunity to detect specific DNA or RNA in specimens contaminated with microorganisms such as first void urine and introital vulvovaginal specimen types with similar sensitivities when compared to cervical and urethral specimens in both, men and women. NAA assays offer therefore screening possibilities especially in asymptomatic individuals by testing non-invasive samples. Coamplification of Neisseria gonorrhoeae and C. trachomatis enables diagnosis of genital infections with these pathogens within few hours.
A positive result obtained by NAA is indicative for therapeutic intervention in infected individuals. Treatment should be initiated as soon as possible by single-dose regimens with azithromycin as the drug of first choice or with doxycycline for up to seven days. Prenatal screening of pregnant women can prevent chlamydial infections among neonates and is especially recommended in women <25 years of age or who have new or multiple sex partners. For pregnant women, erythromycin base is recommended for one week. For treatment of infections in infants the same treatment recommendation with erythromycin base is given. Clinical treatment failure associated with in vitro chlamydial resistance has been reported, but further studies are needed to assess the relationship between resistance to antimicrobials and clinical treatment failure or persistence.

Tenth International Symposium on Human Chlamydial Infections
June 16-21, 2002
Antalya (Turkey)

A. Stary, A. Bilina, M. Kerschbaumer, A. Leitner and M. Mück

Outpatients´Center for Diagnosis of Infectious Venerodermatological Diseases, Vienna, Austria

Introduction
Diagnostic procedures making use of amplification specific nucleic acid sequences have provided laboratories with powerful tools with particular impact in the detection of bacterial genital infections such as Chlamydia trachomatis and Neisseria gonorrhoeae. Compared with cell culture methodologies nucleic acid amplification (NAA) assays such as PCR, LCR, and TMA have been highly sensitive and specific for the detection of genital chlamydial infections in symptomatic and asymptomatic men and women, detecting up to one third more infected individuals (1). NAA technologies have already replaced other diagnostic methods for routine diagnosis and screening purposes of C. trachomatis using noninvasive specimen types such as first void urine (FVU) or vulvovaginal swabs (2,3). In contrast to chlamydial diagnosis, conventional diagnosis of N. gonorrhoeae by culture methods is specific and sensitive and NAA assays provide accurate gonococcal diagnosis in case of improper storage or transport of genital specimens and allows the use of noninvasive specimens.
The Gen-Probe APTIMA Combo 2 assay is a new generation of NAA technology and combines the technologies of target capture, transcription mediated amplification (TMA) and Dual Kinetic Assay.
The aim of this study was to compare the performance of the APTIMA Combo 2 assay to LCR for both, chlamydial and gonococcal infections in men and women using FVU, vulval, and penile swabs, and compare the results with those from cervical and urethral specimens.

Methods
In this ongoing study, genital and urine specimens were collected from a selected high-risk group of men and women attending an outpatients´center for STDs in Vienna. In addition to FVU, cervical and vulval swabs in women and urethral and penile specimens in men were collected in random order for the APTIMA Combo 2 assay (GenProbe, Incorporated, San Diego, Calif.) and LCR (Abbott Park, Ill.) and for gonococcal culture. The LCR assay was processed according to the manufacturer´s recommendation after storage at 4°C and tested within 1 to 4 days. Swabs and 2mL of the FVU were transported in APTIMA Combo 2 transport tubes according to manufacturer recommendations and were processed after storage at 2°C to 30°C within 30 days of collection. In the first step, specific target nucleic acids were separated and potentially inhibiting substances removed by the target capture procedure. Amplification occurred by the replication of the rRNA from C. trachomatis and the rRNA from N. gonorrhoeae via DNA intermediates during different incubation periods at temperatures of 62°C and 42°C. Final diagnosis of chlamydial and gonococcal positive specimens was assessed by the dual kinetic assay in the hybridization, selection, and detection step in the luminometer.
In case of discrepant results, both NAA assays were repeated and urine sediment was tested by DFA. Final calculation of the test performances was based on the number of infected individuals using an expanded gold standard, defined by a positive result in at least one sampling site by both amplifying methods or by either the APTIMA Combo 2 or LCR as confirmed by DFA. Further discrepant analysis of Combo 2 false positives will be performed by TMA amplification of an alternative target sequence of chlamydial rRNA at the end of the study to determine if these are truly false positive. For gonococcal diagnosis culture was included in the evaluation.

Results
Identification of men infected with C. trachomatis and N. gonorrhoeae.
Of the 182 men evaluated in the present study, 55 (30.2%) were found to be infected with C. trachomatis and 40 (22%) with N. gonorrhoeae based on the final calculation of the expanded gold standard. Fourteen gonococcal positive men (35%) were coinfected with chlamydia. Of the 55 chlamydia infected men, 50 (90.9%) were positive with all three sample types when tested by APTIMA Combo 2 assay and 53 (96.4%) when evaluating the urethral, penile, or urine samples separately. One additional sample was repeatedly equivocal in one infected individual, while in 2 noninfected men the equivocal outcome tested negative when repeated. Of the 10 men with a false positive outcome either of the urethral or penile swabs or of urine in the first run of the APTIMA Combo 2 assay, the RLU values were low positive and negative when retested again. Of the 55 infected men tested by LCR, 44 (80%) were positive with all three sample types, and with 52 (94.5%), 50 (90.9%), and 49 (89.1%) urethral, penile, and urine specimens, respectively, when evaluating each one separately. Most of the false positive samples in the LCR had a low extinction rate and were negative when retested for a second time. Of the 40 men with gonorrhea, the APTIMA Combo 2 assay detected all infected men with FVU, and 39 (97.5%) in urethral and penile swabs, while the detection rate for LCR was 95%, 92.5%, and 90% for urethral, penile and urine specimens, respectively. The performance characteristics were high for both NAA assays (Table 1).

Identification of women infected with C. trachomatis and N. gonorrhoeae.
Of the 166 women included in the evaluation, 32 (19.3%) and 11 (6.6%) were infected with C. trachomatis and N. gonorrhoeae, respectively, with a coinfection with C. trachomatis in 45.5% of gonococcal positive women. Of the 32 chlamydia infected women, 81.3% and 78.1% were positive with all specimens by the APTIMA Combo 2 test and LCR, respectively. By testing endocervical swabs only, 27 (84.4%) were positive by both NAA assays. The APTIMA Combo 2 assay detected 32 (100%) and 30 (93.8%) of the infected women with vulval swabs and FVU, respectively, with corresponding data for the LCR of 96.9% and 84.4%, respectively. Of women with a singular false positive outcome in the first run of the APTIMA Combo 2 assay, the RLU values were low positive or equivocal and negative when retested again. Few of the patients had received antibiotic treatment within the last 3 weeks, which has to be taken into account in the final calculation.
For gonococcal diagnosis, all 11 women with gonorrhea were detected in all specimens by the APTIMA Combo 2 and in all but one endocervical swab by LCR. The preliminary data of sensitivities and specificities of both NAA assays for C. trachomatis and N. gonorrhoeae are listed in table 1.

Discussion
The preliminary data of this ongoing study shows that both amplification methods gave high performance patterns in this selected group of men and women. There was little variation in chlamydial detection rates by the APTIMA Combo 2 assay for invasive and noninvasive genital swabs and urine. Most of the truly infected individuals were chlamydia positive in all specimens, with a higher number in men (90.9%) than in women (81.3%) and a lower number when using the LCR assay (men 80%, women 78.1%). For gonococcal diagnosis, the numbers were even higher (women 100%, men 97.5%) for RNA amplification, and again slightly lower for LCR (women 90.9%, men 87.5%). Testing urine by RNA amplification gave a high sensitivity in both, men and women. This is in contrast to a previous study using the first generation NAA TMA assay (AMP CT) on female urine with a detection rate of 76% for chlamydia infected women compared with 96% when using the LCR (4). In men, the APTIMA Combo 2 assay showed a high sensitivity and specificity not only with urethral and urine specimens, but also with penile swabs, indicating its ability to detect even a low number of specific nucleic acids for both, C. trachomatis and N. gonorrhoeae. In women, the detection rate for both NAA assays was higher with vulval than with endocervical specimens, which can be explained by additional detection of urethral infections. This outcome is similar to previous observations (4,5). The quantitative evaluation of both NAA assays shows that results with a low or equivocal RLU in the APTIMA Combo 2 assay or a low extincition rate in the LCR should be repeated in order to distinguish between truly infected individuals and a false positive result.
In summary, the preliminary data of the present study demonstrate high performance characteristics for the APTIMA Combo 2 assay for both, C. trachomatis and N. gonorrhoeae for all specimen types included in the study with a high overall agreement with LCR. Vulval and penile specimen types can be recommended for chlamydial and gonococcal coamplification by the APTIMA Combo 2 assay as alternative noninvasive sample types and may be even more suitable than FVU.
Table 1
Sensitivities and specificities of APTIMA Combo 2 assay and LCR for the detection of C. trachomatis and N. gonorrhoeae in urogenital specimens
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C. trachomatis N. gonorrhoeae
Specimen Sensitivity Specificity Sensitivity Specificity APTIMA LCR APTIMA LCR APTIMA LCR APTIMA LCR
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Urethra 96.4 94.5 94.5 96.9 97.5 95.0 93.0 100
Glans 96.4 90.9 96.1 100 97.5 92.5 97.9 100
Endocervix 84.4 84.4 91.0 98.5 100 90.9 96.1 100
Vulva 100 96.9 90.3 100 100 100 99.4 100
FVU men 96.4 89.1 98.4 97.6 100 90.0 96.5 99.3
FVU women 93.8 84.4 98.6 100 100 100 98.1 98.7
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References
1 Schachter J, et al. Ligase chain reaction to detect Chlamydia trachomatis infection of the cervix. J Clin Microbiol 1994; 32:2540-2543.
2 Lee HH, et al. Diagnosis of Chlamydia trachomatis genitourinary infection in women by ligase chain reaction assay of urine. Lancet 1995;345:213-216.
3 Stary A, et al. Vulval swabs as alternative specimens for ligase chain reaction detection of genital chlamydial infections in women. J Clin Microbiol 1997; 35:836-838.
4 Stary A, et al. Performance of transcription-mediated amplification and ligase chain reaction assays for detection of chlamydial infection in urogenital samples obtained by invasive and noninvasive methods. J Clin Microbiol 1998; 36:2666-2670.
5 Hook EW III, et al. Diagnosis of genitourinary Chlamydia trachomatis infections by using the ligase chain reaction on patient-obtained vaginal swabs. J Clin Microbiol 1997; 35:2133-2135.