Selection of primers of optimal sensitivity for the detection of HIV-1 from Africa and Europe by polymerase chain reaction.

In order to facilitate the detection of integrated HIV-1 proviral DNA from African as well as European patients, four new primer pairs for use in the polymerase chain reaction (PCR), localised in the gag, pol, vif and env genes of HIV-1, were constructed. The primer pairs were compared to all accessible HIV-1 sequences from African and European isolates and to some of the earlier published and most commonly used primer pairs. HIV-1 DNA was detected in blood drawn from 13 out of 13 individuals infected in Africa, in three out of three Tanzanian HIV-1 isolates and in three out of three asymptomatic Swedes infected in Europe. The new selection of primer pairs can be used as an alternative to enhance the detection of HIV-1 of different origins.

PIP: In order to facilitate the detection of integrated HIV-1 proviral DNA from African as well as European patients, 4 new primer pairs for use in the polymerase chain reaction (PCR), localized in the gag, pool, vif, and env genes of HIV-1, were constructed. The primer pairs were compared to all accessible HIV-1 sequences from African and European isolates and to some of the earlier published and most commonly used primer pairs. HIV-1 DNA was detected in blood drawn from 13 infected individuals in Africa, in 3 Tanzanian HIV-1 isolates, and in the 3 asymptomatic Swedes infected in Europe. The new selection of primer parts can be used as an alternative to enhance the detection of HIV-1 of different origins.

Improved detection of HIV-2 DNA in clinical samples using a nested primer-based polymerase chain reaction.

A two-step polymerase chain reaction (PCR), with four double (nested) primer pairs, used for the detection of HIV-2 in clinical samples is described. With these four nested primer pairs we could detect HIV-2 DNA in 17 of 17 virus isolates and in blood mononuclear cell samples from 31 of 37 (83.7%) seropositive individuals after ethidium bromide staining of the amplified DNA. The nested primer PCR was also compared with a single primer pair-based PCR followed by hybridization. The sensitivities of the two methods were almost equal, but the nested primer PCR offered obvious technical advantages.

Preparation of sera for typing of adenovirus infections by immunofluorescence.

The conditions for preparation of adenovirus antisera with group, subgroup and type-specific reactivity to be used in the immunofluorescence technique have been evaluated. Group-specific antibodies were prepared by passing an antiserum against virions or hexons of one serotype through an immunosorbent column containing soluble viral components of an adenovirus type belonging to a different subgroup. The group-specific antibodies were recovered from the solid phase by elution at pH 2.8. Reagents specific for subgroup I and II were obtained by passing anti-dodecon sera through an immunosorbent column containing soluble viral components from heterologous subgroups. In this case antibodies not attaching to the absorbent were recovered. Sera against fibers of subgroup III members could be used as subgroupspecific sera without absorption. Type-specific antibodies were prepared by removal of antibodies of other specificities by passing anti-virion or anti-hexon sera through immunosorbent columns containing soluble viral components of an adenovirus type belonging to the same subgroup. Reagents specific for seven adenovirus types representing Rosen's three subgroups were prepared according to the outlined procedures.

Preparation of specific antisera against adenoviruses by affinity bead immunization (ABI).

Certain adenovirus types can be replicated only to low titer in tissue cultures. Other, such as adenovirus strains associated with infantile gastroenteritis, cannot be replicated in vitro. A method which allows preparation of specific antisera has therefore been evaluated. The procedure involves coupling of group-specific antibodies against adenovirus capsid subunits to CNBr-activated Sepharose 4B; reaction of crude virus suspensions with immobilized adenovirus-specific IgG; elimination of contaminating material by extensive washing using a wide pH range; and immunization with adenovirus immunogens immobilized on the beads. Efficient immunization was obtained with immunogen doses of both 50 ng and 50 microgram. The immunization procedure which has been designated affinity bead immunization (ABI) could therefore have a wide applicability in cases where the relevant immunogen constitutes a minor fraction of a crude preparation.

Vaccine protection against HIV-2 infection in cynomolgus monkeys.

The aim of this study was to determine if protection against an infectious human immunodeficiency virus type 2 (HIV-2) challenge could be obtained in cynomolgus macaques by active immunization using whole killed virus vaccine. Four monkeys were immunized with killed HIV-2SBL-6669, two of them with five intramuscular (im) injections of viral preparation containing 100 or 300 micrograms protein emulsified in incomplete Freund's adjuvant (IFA) and the two remaining received four im injections of 25-50 micrograms viral protein in iscoms. Each of the four vaccinated cynomolgus monkeys, along with four unvaccinated controls, were challenged intravenously two weeks after the last booster with approximately 100 animal infectious doses (ID50) of live HIV-2SBL-6669. All four immunized monkeys developed antibodies to HIV-2 envelope and core proteins before challenge exposure to HIV-2, but only the two animals vaccinated with virus in IFA developed detectable neutralizing antibodies. The two monkeys immunized with killed virus in IFA have shown no evidence of infection nine months after challenge with live virus. When blood and lymph node cells from these animals were transfused into naive cynomolgus monkeys, the recipients remained free of infection. In contrast, virus was recovered repeatedly in all nonimmunized animals and in the two animals immunized with iscom-associated viral antigens, which had a low content of envelope gp125 antigen. The demonstration of vaccine-induced protection against HIV-2 in a nonhuman primate raises hope for effective immunization against HIV infections in humans as well.

Detection of human papillomavirus in cervical intra-epithelial neoplasia, using in situ hybridization and various polymerase chain reaction techniques.

One hundred and forty-eight randomly chosen neutral-buffered formaldehyde-fixed cervical biopsies in which cervical intra-epithelial neoplasia (CIN) I-III had been diagnosed were tested for HPV (human papilloma virus) DNA by in situ hybridization (ISH) and polymerase chain reaction (PCR). For ISH, we utilized a biotinylated panprobe and type-specific, genomic probe sets. For PCR, we used the general primers GP5/GP6 and their recently described, elongated version GP5+/GP6+, and included the modification of hot-start PCR. Amplified DNA was detected by gel electrophoresis and slot blot hybridization. The positivity rate of ISH was 59% for all biopsies and 69%, 62% and 46% for CIN I, II and III, respectively. The sensitivity of GP5/GP6 was 74% with cold-start PCR and 78% with hot-start PCR. When GP5+/GP6+ was used, the sensitivity increased to 89% with cold-start PCR and to 95% with hot-start PCR. Based on the most sensitive PCR technique, HPV detection was 93%, 95% and 96% in CIN I, II and III, respectively. The number of HPV types decreased with the severity of the lesion, and HPV 16 was the predominant type. Multiple HPVs were rare and almost all HPV-positive cases could be typed. ISH and slot blot hybridization correlated well regarding HPV typing specificity. Our results confirm that distinct HPV types are present in a high proportion of cases of CIN. The sensitivity of ISH is lower than that of PCR. Furthermore, the modified general primers GP5+/GP6+ give a higher yield than GP5/GP6, while hot-start PCR increases sensitivity even further.

Reliable high risk HPV DNA testing by polymerase chain reaction: an intermethod and intramethod comparison.

BACKGROUND: The development of a reproducible, sensitive, and standardised human papillomavirus (HPV) polymerase chain reaction (PCR) test is required to implement HPV testing in cervical cancer screening programmes and for triaging women with mild to moderate dysplasia. AIMS: To determine the intermethod agreement between different GP5+/6+ and MY09/11 PCR based protocols for the detection and typing of high risk (HR) HPV DNA in cervical smears and to assess the intramethod reproducibility of the GP5+/6+ PCR enzyme immunoassay (EIA) for HR-HPV detection. METHODS: For the intermethod comparison, crude aliquots of 20 well characterised cervical smears comprising five HPV negative samples, and six and nine samples containing single and multiple HPV infections, respectively, were coded and sent from reference laboratory (A) to three other laboratories. One of these (laboratory B) used the GP5+/6+ PCR-EIA and was provided with standard protocols. Another laboratory (C) used GP5+/6+ PCR combined with sequence analysis and type specific PCR, whereas two laboratories (D and E) used MY09/11 PCR followed by restriction fragment length polymorphism (RFLP) analysis for the detection and typing of HR-HPV. The intramethod agreement of GP5+/6+ PCR-EIA was analysed in a subsequent study with four other laboratories (F to I) on crude aliquots of 50 well characterised cervical smears, consisting of 32 HR-HPV positive and 18 HPV negative samples. Standardised protocols, primers, and probes were also provided by the reference laboratory for HR-HPV detection. RESULTS: In the intermethod comparison, pairwise agreement of the different laboratories with reference laboratory A for the detection of HR-HPV varied between 75% and 100% (kappa values: 0.5 to 1). Typing data revealed a broader range in pairwise agreement rates between 32% and 100%. The highest agreement was found between laboratories A and B using standardised protocols and validated reagents. In the intramethod evaluation, pairwise comparison of the laboratories F to I with reference laboratory A revealed excellent agreement rates from 92% to 100% (kappa values: 0.88 to 1.0) with an overall sensitivity of 97.5% (195/200) and specificity of 99.5% (199/200). CONCLUSIONS: The detection of HR-HPV as a group is highly reproducible with GP5+/6+ PCR-EIA provided that standardised protocols and validated reagents are used.

The absence of vaginal human papillomavirus 16 DNA in women who have not experienced sexual intercourse.

OBJECTIVE: To determine whether young women who have not experienced sexual intercourse may harbor genital human papillomavirus (HPV) infection in the vaginal-ectocervical mucosa. METHODS: We included 151 women, 10-25 years of age, attending two adolescent health care units (Stockholm and Uppsala) and one primary health care center (Umeå). The size of the hymenal orifice, use of tampons, and the habit of digital vaginal manipulation were registered. Samples of epithelial cells were collected from the vagina and analyzed for the presence of HPV using polymerase chain reaction. RESULTS: One hundred thirty of 154 samples contained an adequate number of cells. Two samples were HPV 6 DNA-positive. None were HPV 16 DNA-positive. None of the women had external genital warts. In 84%, the hymenal opening was 15 mm or less. Forty-eight percent of the women used tampons during periods. Fifty-four percent had inserted their own finger into the vagina and in 23%, a boyfriend's finger had penetrated the vagina. CONCLUSION: Human papillomavirus is rarely present vaginally in virginal women, even with the use of tampons or digital penetration.

Acetowhitening of the cervix and vulva as a predictor of subclinical human papillomavirus infection: sensitivity and specificity in a population-based study.

OBJECTIVE: To evaluate acetowhite changes of the cervix and vulva as a predictor of human papillomavirus (HPV) infection. METHODS: In this population-based study all women aged 19, 21, 23, and 25 years and registered as living in a primary health care area within the city of Umeå, Sweden were eligible for inclusion. Each participant underwent a gynecologic examination with sampling of epithelial cells for HPV-DNA detection and Papanicolaou smear. Colposcopy was performed 5 minutes after application of 5% acetic acid. A two-step polymerase chain reaction (PCR) technique was employed for HPV-DNA detection. RESULTS: Colposcopy and sampling of epithelial cells could be performed in 535 women. The sensitivity of detection of HPV infection by the acetowhitening of the cervix was 22% (95% confidence interval [CI] 18%, 26%). The specificity of detection of HPV infection by the acetowhitening of the cervix was 90% (95% CI 87%, 93%). The sensitivity of detection of HPV infection by cytology was 13% (95% CI 10%, 16%), and the specificity was 99% (95% CI 98%, 100%). The combination of acetowhitening and cytology did not improve the diagnostic value. CONCLUSION: Acetowhitening of the cervix and vulva has low sensitivity as a predictor of HPV infections as determined by PCR.

Hantavirus antigen detection using human serum immunoglobulin M as the capturing antibody in an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was developed to detect different hantavirus antigens in cell culture; i.e. Puumala (PUU), Hantaan (HTN), and Dobrava (DOB) viruses. The assay was based on binding human serum immunoglobulin M (IgM) antibodies to the solid phase by use of goat anti-IgM antibodies. The captured IgM antibodies were present in the acute phase serum from two patients: one infected in Sweden and the other in Bosnia. Antigens being bound to the solid phase by the human anti-PUU and anti-DOB/HTN IgM antibodies were detected by a broadly reacting polyclonal rabbit anti PUU-recombinant nucleocapsid protein antiserum. The IgM isotype was proven to be at least five times more efficient than IgG when used as the capturing antibody. The sensitivity of the PUU antigen ELISA was approximately 0.5 ng/ml, as measured by titration with a PUU recombinant nucleoprotein antigen. Cell-associated PUU antigen in tissue culture was seen after 48 hr by the PUU-ELISA and after 96 hr by immunofluorescent assay. When tested for capacity to discriminate between PUU, DOB, and HTN viruses, significant differences were found: the Swedish serum detected PUU antigen at high titers, whereas no reactivity was found against DOB and HTN; the Bosnian serum detected both DOB and HTN at high titers but had a low reactivity to PUU. The method was also tested for its usefulness in detecting PUU antigen in bank vole (clethrionomys glareolus) lungs. Of 59 animals captured from the surroundings of patients with nephropathia epidemica, three became positive with a high activity in the PUU-ELISA, but with low reactivity in the DOB/HTN-ELISA. It is concluded that a sensitive ELISA has been developed to detect different hantaviruses in cell culture and lungs of bank voles.

High prevalence of hantavirus antibodies in bank voles (Clethrionomys glareolus) captured in the vicinity of households afflicted with nephropathia epidemica.

Puumala virus, the causative agent of nephropathia epidemica (NE), occurs endemically in Europe and is spread mainly by the bank vole (Clethrionomys glareolus). In the vicinity of each of four households afflicted with NE, we studied rodents with regard to population density and prevalence of Puumala virus-specific antibodies. For each case area, a control area was randomly selected 10 km away, without regard to the presence of human settlement. During 6,000 trap nights, 328 rodents were caught, of which 299 were C. glareolus. The mean rodent densities of case and control areas were 6.6 and 3.7 animals per 100 trap nights (P < 0.001). The prevalence of serum antibodies was 15.9% in case areas compared with 5.6% in control areas (P < 0.05). In three of the case areas, where NE had occurred 3-10 weeks before trapping, the rodent density and seroprevalence were much higher than in the fourth area, where NE occurred 38 weeks before trapping. In conclusion, C. glareolus seropositive for Puumala virus occurred more frequently near households afflicted with NE than in control areas 10 km away.

Nonradioisotopic detection and typing of human papillomaviruses by use of polymerase chain reaction and single-strand conformation polymorphism.

The polymerase chain reaction (PCR), used to detect human papillomavirus (HPV), is finding increasing applications in clinical laboratories. The standard method of analysis to detect amplified PCR products is ethidium bromide gel electrophoresis combined with labor intensive blot hybridization. In this study, we describe single-strand conformation polymorphism (SSCP) to detect and genotype simultaneously general primer GP5+/GP6+ amplified HPV DNA using semiautomated electrophoresis on polyacrylamide gels (PAGE) combined with sensitive silver staining. To establish a standard for the band patterns of the various HPV types, we used HPV plasmid DNA, which allowed us to distinguish HPV 6, 11, 16, 18, 31, 33, 35, 45, 51, 52, 56, and 58, covering the most frequently recognized types. All the types tested are separated from each other, demonstrating diverse band patterns, HPV 16 being the most distinct. We also investigated PCR-SSCP for HPV detection and typing of 86 cervical biopsies diagnosed as cervical intraepithelial neoplasia (CIN) I-III and known to be HPV positive by PCR-slot blot hybridization and in situ hybridization. The correlation with SSCP was 91% for in situ hybridization and 98% for PCR-slot blot hybridization. SSCP is reproducible and specific. Its sensitivity is comparable to slot-blot hybridization. The interval to SSCP is approximately 2 h after PCR compared with several days' work when using conventional blot hybridization. We concluded that SSCP may be more advantageous than other PCR-based typing technologies.

A general primer pair for amplification and detection of genital human papillomavirus types.

A general primer pair localized in the E7 and E1 regions was identified and used for the detection of genital human papillomaviruses (HPVs). The genital HPV types 6b, 11, 16, 18, 31 and 33 were amplified and detected by the polymerase chain reaction (PCR) performed at a high stringency annealing temperature (60 degrees C). HPV-2, -3, -7, -13 and -30 were amplified only at lower temperatures. Twelve biopsies from women with invasive cancer in the cervix were analysed with the general primer pair. The amplification product specific for the general primer pair was detected in 11 of the 12 biopsies. The eleven HPV DNA positive specimens were shown to contain HPV-6b, HPV-16 and/or HPV-18 by Southern blot hybridization of the PCR products. The general primers were also used for analysis of 57 cervical scrapes from women with normal cytology, condyloma or CIN. By ethidium bromide staining after agarose gel electrophoresis we could detect 21 positives. Slot-blot analysis of the amplification products from all 57 scrapes confirmed the specificity of the 21 positives and revealed 5 additional positives. Among the 57 scrapes, 15/21 CIN scrapes, 10/21 condyloma scrapes and 1/15 normal scrapes contained HPV DNA. Eight different HPV types were detected. The general primer pair from the E7/E1 region is thus a powerful tool for the detection of HPV in clinical samples. The amplimer obtained offers a possibility for further typing by slot-blot hybridization using HPV-type specific probes.

A major antigenic domain for the human humoral response to Puumala virus nucleocapsid protein is located at the amino-terminus.

Nephropathia epidemica (NE), the major form of hemorrhagic fever with renal syndrome in Europe, is caused by the hantavirus serotype Puumala (PUU). The PUU virus nucleocapsid protein (N) has been shown to be highly immunogenic both in laboratory animals and in man. We aimed to locate domains important in humoral immune reactivity and to use this information to develop a specific and sensitive enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of NE. Escherichia coli poly-histidine fusion protein expression vectors containing over-lapping gene segments encoding the PUU virus N (PUU rN) were constructed. The resulting gene products were examined by immunoblots and ELISA with polyclonal and monoclonal antibodies. The dominating antigenic region of PUU rN was located between amino acids (aa) 7 and 94. A recombinant fusion protein containing aa 7-137 of PUU virus N (PUU rN delta 5) was used for the detection of specific IgG and IgM responses in NE. ELISA based on PUU rN delta 5 was found to have equal sensitivity and specificity as compared to the full length recombinant PUU rN by ELISA, for both acute serological diagnosis of NE and for seroepidemiological screening purposes. Furthermore, this protein is easier to handle than full length PUU rN due to its higher solubility in aqueous solutions.

Nested PCR assays with novel primers yield greater sensitivity to Tanzanian HIV-1 samples than a commercial PCR detection kit.

To investigate the efficacy of the SK431/SK145 primer pair and two nested primer assays in amplifying African HIV-1 samples, a total of 35 Tanzanian PBMC samples were examined. These were assayed by two HIV-1 specific nested in-house PCR assays and a commercial HIV-1 PCR kit (GeneAmp) using SK431/SK145 as the primer pair. One of the nested PCR assays has been evaluated previously (old assay), whereas the modified assay was constructed from the HIV-1 sequence alignment released in August 1993. The modified nested primer assay showed increased sensitivity in the gag and env regions compared to the old nested primer assay. However, both the old and the modified nested primer assays displayed higher sensitivity for the detection of Tanzanian HIV-1 proviruses than the GeneAmp assay. When two regions were used (gag and env) as targets for the amplification, the modified nested primer assay detected 97.1% (34/35) of the proteinase K lysed samples, compared to 68.6% (24/35) using the SK431/SK145 primer pair (P < 0.01**). The results indicate that the SK431/SK145 primer pair may be less suitable when HIV-1 samples from Africa are analysed. The results also show that continuous modification of primer sequences can improve and maintain high sensitivity for the detection of highly divergent HIV-1 strains.

Development of humoral cross-reactivity to the nucleocapsid protein of heterologous hantaviruses in nephropathia epidemica.

A hantavirus infection is followed by a prominent antibody response to the viral nucleocapsid protein. Antibodies from patients infected with one hantavirus cross-react to varying degrees with the nucleocapsid protein of other viruses of the genus. We studied the cross-reactivity in serially obtained blood samples from 17 patients with nephropathia epidemica, a European form of hemorrhagic fever with renal syndrome caused by Puumala virus. Recombinant truncated nucleocapsid protein (aa 1-117) of Puumala virus and four other hantaviruses, Hantaan, Seoul, Dobrava and Sin Nombre viruses, were used as antigens in an indirect ELISA. In most patients, an IgG response to the Puumala virus derived recombinant protein was detected within 2-8 days of onset of disease, remained high for 2-5 months, and declined gradually within 2-3 years. All patients had IgG antibodies cross-reacting with the nucleocapsid protein of Sin Nombre virus. The ratio of the ELISA values obtained with Sin Nombre vs. Puumala virus protein as antigen increased with time after onset of disease. To a lesser extent, cross-reacting IgG antibodies also occurred to Hantaan, Seoul, and Dobrava virus antigens. In the acute phase of the disease, two patients showed IgG antibodies to one or more of these viruses whereas 2-5 months later, 11 of 16 patients had IgG antibodies to all three viruses. IgM and IgA responses to the nucleocapsid protein of Puumala virus were transitory and cross-reactivities were weak. In conclusion, after onset of nephropathia epidemica the IgG response to the Puumala virus nucleocapsid protein was associated with a gradually increasing cross-reactivity to the nucleocapsid protein of heterologous hantavirus. Our findings have implications for the interpretation of serological data, both in the diagnostics of nephropathia epidemica and in seroprevalence studies.

Double HPV infection in African cervical cancer detected by polymerase chain reaction.

Biopsy samples from 13 Kenyan patients with squamous cell carcinoma of the cervix were analysed for the presence of type specific HPV DNA by polymerase chain reaction (PCR). HPV 16 was confirmed in 11 (85%) and HPV 18 in 9 (69%) samples. HPV 6 DNA was detectable in only 3 (23%) samples and no HPV 33 was found. Infection with either HpV16 or 18 was seen in 12 (92%) and infection with both in 8 (62%) cases. The prevalence of double infection found is higher than in previous reports. The significance of this and possible effects of parity on cervical neoplastic changes are discussed.

HPV 18 and cervical adenocarcinomas.

Using Southern blot or in situ hybridization, several studies have recently reported an association between HPV 18 and adenocarcinoma of the uterine cervix. In this study PCR was used to analyse the presence of HPV 16 and 18 in paraffin embedded biopsies of cervical adenocarcinoma in Northern Sweden. HPV DNA was confirmed in 11 (42%) of 26 cases. Seven (27%) were positive for HPV 18 and 4 (15%) for HPV 16. Nine of 13 premenopausal cases were HPV positive compared to only 2 of 13 postmenopausal cases (p less than 0.015). There was also a tendency towards previous negative cytology in HPV 18 positive premenopausal patients. The significance of these findings in relation to epidemiology of cervical adenocarcinoma is discussed.

Aetiology and epidemiology of acute gastro-enteritis in Swedish children.

In a prospective 1-year study, 144 children attending or admitted to hospital and 272 children outside hospital with acute gastro-enteritis and 200 controls were investigated by a broad panel of diagnostic methods for enteropathogenic agents in the faeces and for related antibody responses. Enteropathogens were identified in 77% of the inpatients, 63% of the outpatients and 8% of the controls. Rotavirus and Yersinia enterocolitica were detected significantly more often among inpatients. Altogether, viral, bacterial and parasitic agents were found in 58%, 14% and 1% of diarrhoeal patients, respectively. The isolation of more than one pathogenic agent was uncommon (6.5%). Rotavirus (45%) and enteric adenoviruses 40 and 41 (7.9%) predominated among the viruses, while Campylobacter jejuni (4.8%) was most common among the bacteria. Clostridium difficile and/or its cytotoxin, which were found in 14% of the children with gastroenteritis and in 15% of the controls, were significantly associated with antibiotic therapy but not with gastro-intestinal illness. Diarrhoeal infections of unknown aetiology exhibited a seasonal peak in the autumn. The duration of excretion of enteropathogens was investigated. Rotavirus particles were detectable by solid-phase immune electron microscopy for 14-25 days after the diarrhoea had ceased. Transmission of rotavirus and bacterial pathogens within families was studied also.

The associations between risk behaviour and reported history of sexually transmitted diseases, among young women: a population-based study.

The aim of this study was to determine the associations between risk behaviour and women's reported sexually transmitted diseases (STDs). All the women aged 19, 21, 23 and 25, residing in a specified housing area, were invited to answer a questionnaire regarding their sexual behaviour, smoking and alcohol consumption and previous history of STD. Of the 611 women participating, one out of 4 women had a history of at least one STD. In an univariate analysis, self-reported STD was found to be related to age, having more than 4 lifetime sexual partners, having practised intercourse at first date, inconsistent use of condoms, alcohol consumption of more than 3 bottles of wine per month and smoking. These factors were, however, not independent of each other and when subjected to a multivariate logistic regression analysis 2 factors, i.e. the lifetime number of sexual partners (more than 4 partners vs one; OR 7.94, (3.41-18.50)) and coitus on first date (practised more than once vs never, OR 2.99 (1.55-5.78)) emerged as independently associated with a previous STD.