Chlamydia trachomatis serovar distribution and other concurrent sexually transmitted infections in heterosexual men with urethritis in Italy.

The distribution of Chlamydia trachomatis serovars among 157 heterosexual male patients with urethritis and the presence of coinfections with other sexually transmitted infections were studied. One hundred seventeen (74.5%) patients, with a mean age of 33.7 years, were Italians, whereas 40 (25.5%) were immigrants coming from eastern European countries, Africa, and South America. All the immigrants and 82 (70.0%) Italian patients reported sex with prostitutes. Out of 157 patients, 73 (46.5%) were found positive for C. trachomatis in urethral secretions and eight different C. trachomatis serovars were identified. The most common serovars were E (n = 18; 24.7%), D (n = 15; 20.5%), G (n = 14;19.2%), and F (n = 12; 16.4%). The sequencing data showed a high degree of conservation of the omp1 gene. Thirty-six (46.7%) out of the 73 C. trachomatis-positive patients were coinfected with another sexually transmitted infection. The most common coinfection was gonorrhoea detected in 22 (30.1%) patients, followed by condyloma in eight (8.2%) patients, syphilis in five (6.8%), and HIV in three (4.1%).

Development of a hamster model of Chlamydophila pneumoniae infection.

The aim of this study was to develop a new experimental model of Chlamydophila pneumoniae infection in the hamster. Intraperitoneal injection of C. pneumoniae purified elementary bodies (EBs) in the hamsters caused a systemic infection, since it was possible to isolate viable chlamydiae from several organs up to 14 days after infection. In particular, spleen infection was detectable up to 7 days post infection in 100% of animals. In contrast, cultures of the organs obtained from intranasally infected animals were far less frequently positive. Systemic infection probably occurred via macrophages, as demonstrated by the presence of intracellular chlamydial inclusions in peritoneal macrophages of peritoneally inoculated animals four days after infection. Furthermore, by infecting LLC-MK2 cells with supernatant preparations obtained from these macrophages, it was possible to observe the development of chlamydial intra-cytoplasmic inclusions after 96 h. Immunization of 18 hamsters with heat-inactivated purified EBs completely protected 16 animals and substantially reduced infection levels in the remaining two. Sera obtained from immunized hamsters prior to challenge reacted mainly against two C. pneumoniae proteins of about 60 kDa, when tested by immunoblot.

Development of transplantable ascites tumours which continuously produce polyclonal antibodies in pristane primed BALB/c mice immunized with bacterial antigens and complete Freund's adjuvant.

Bacterial immunogens (whole cells of Borrelia burgdorferi, elementary bodies of Chlamydia trachomatis and purified proteins of 22 and 24 kDa of Borrelia hermsii) were emulsified with an excess of complete Freund's adjuvant and injected (i.p.) on days 0, 7, 14 and 21, into BALB/c mice treated with pristane on day 6. This procedure induced the development of antibody-producing ascites tumours which could be serially transplanted in pristane-conditioned mice. Ascites tumours continued to yield a consistent amount of specific polyclonal antibody after ten serial transplants. The method described appears to be particularly useful for the production of a large amount of antibody when only small amounts of immunogen are available.

Detection of Chlamydia trachomatis DNA in patients with non-gonococcal urethritis using the polymerase chain reaction.

A practical protocol using the polymerase chain reaction (PCR) was designed for detecting Chlamydia trachomatis in clinical samples. DNA was extracted from material collected on urethral swabs and used as substrate for the PCR. The target was a 600 basepair DNA segment of the multicopy plasmid that is common to all strains of the bacterium. Negative samples were checked for loss of DNA or presence of polymerase inhibitors by a second PCR, targeted to a conserved segment of the human genome. The whole procedure was tested on 216 men with non-gonococcal urethritis (NGU). All patients were independently assessed by tissue culture isolation (60 positive samples) and a commercial immunoenzymatic assay. The PCR protocol, while sufficiently simple for routine application, was reliable and, for the diagnosis of urethritis, at least as good as tissue culture isolation.

A rapid immunoperoxidase assay for the detection of specific IgG antibodies to Chlamydia trachomatis.

A technique, using indirect immunoperoxidase antibody (IPA), was developed for the detection of IgG antibody to Chlamydia trachomatis. The IPA technique employs glass slides with air-dried and acetone-fixed C trachomatis infected cells, which can be stored at -70 degrees C and used for several months. Antibody titres detected by IPA were comparable to those detected by the indirect fluorescent antibody technique.

An immunoperoxidase assay for the detection of specific IgA antibody in Epstein-Barr virus infections.

A technique using indirect immunoperoxidase antibody was developed for the detection of specific serum IgA antibody to Epstein-Barr virus capsid antigen and early antigen. The IgA technique was compared with an immunofluorescence antibody method. Epstein-Barr virus IgA antibody against viral capsid antigen was detected in all nine patients with Epstein-Barr virus associated undifferentiated nasopharyngeal carcinoma, in 13 (72.2%) of 18 patients with infectious mononucleosis, in 21 (28.3%) of 74 patients with acute lymphoblastic leukaemia, and in six (20%) of 30 patients who had recently had kidney transplants. Epstein-Barr virus IgA antibody against viral capsid antigen was also detected in four (10%) of 40 healthy subjects, but it was not found in any of 20 cord blood samples. Epstein-Barr virus IgA antibody to early antigen was detected in six (66.6%) patients with nasopharyngeal carcinoma and in two (2.7%) patients with acute lymphoblastic leukaemia. The immunoperoxidase assay for Epstein-Barr virus specific IgA was simple, reliable, and rapid and correlated well (r = 0.94) with the immunofluorescence antibody technique.

Antigenic specificity of serological response in Chlamydia trachomatis urethritis detected by immunoblotting.

Sera from 19 patients with Chlamydia trachomatis culture positive non-gonococcal urethritis were studied for the presence of antibodies to chlamydial proteins by immunoblotting. Ten C trachomatis negative patients with non-gonococcal urethritis and 10 healthy controls were also studied. Acute phase sera from C trachomatis positive patients with non-gonococcal urethritis reacted only with the major outer membrane protein whereas all the convalescent phase serum samples reacted with the major outer membrane protein and with a 60,000 and a 62,000 molecular weight protein. Some sera also reacted with a 45,000 molecular weight protein. Five of 10 convalescent phase samples from patients with C trachomatis negative non-gonococcal urethritis showed a reaction pattern comparable with that observed in convalescent sera from C trachomatis from C trachomatis positive patients with non-gonococcal urethritis. Sera from healthy seronegative subjects were negative by blotting.

Serum specific IgA antibody to Chlamydia trachomatis in patients with chlamydial infections detected by ELISA and an immunofluorescence test.

Sera obtained from 34 men with Chlamydia trachomatis positive non-gonococcal urethritis, 34 men with C trachomatis negative non-gonococcal urethritis, 42 women with acute salpingitis, 38 healthy women, and 34 healthy men were studied for the presence of specific serum C trachomatis IgA and IgG antibodies. Serological results were correlated with C trachomatis isolation in cell culture. An enzyme linked immunosorbent assay (ELISA) for C trachomatis specific serum IgA was employed using highly purified elementary bodies of C trachomatis serotype L2 grown in LLC-MK2 cells. Results obtained for C trachomatis IgA antibody by the ELISA test were compared with results obtained for the same sera by a single antigen immunofluorescence technique. A good correlation (r = 0.91) was found between two methods. Serum IgG antibody was also determined in the same sera by the immunofluorescence technique. Patients with C trachomatis positive non-gonococcal urethritis had a significantly (p less than 0.0005) higher prevalence (94.1%) of serum IgA antibody by ELISA compared with patients with C trachomatis negative non-gonococcal urethritis (20.5%) or healthy men (5.9%). Similarly, women with acute salpingitis had a significantly (p less than 0.005) higher prevalence of serum IgA antibody (45.2%) compared with healthy controls (5.2%). Comparable results were obtained for C trachomatis serum IgA antibody using the immunofluorescence technique. The prevalence of C trachomatis IgG antibody was significantly higher in patients with C trachomatis positive non-gonococcal urethritis (97.0%) compared with those with C trachomatis negative non-gonococcal urethritis (33.3%) and healthy controls (23.5%). The importance of using specific C trachomatis serum IgA in the identification of chlamydial infection is discussed.

Class specific immunoglobulin response to individual polypeptides of Chlamydia trachomatis, elementary bodies, and reticulate bodies in patients with chlamydial infection.

Sera from 10 women with Chlamydia trachomatis culture positive cervicitis and sera from six men with C trachomatis positive non-gonococcal urethritis were studied for the presence of IgG, IgM, and IgA antibodies to polypeptides of C trachomatis elementary bodies and reticulate bodies using immunoblotting techniques. All the sera with IgG, IgM, or IgA immunoglobulins specific to C trachomatis recognised the major outer membrane protein (MOMP) of elementary bodies. IgG antibodies also detected several other proteins, whereas IgM immunoglobulins recognised only MOMP and proteins of 60 kD, 62 kD, and 66 kD. The IgA reacted with MOMP and the 60 kD and 62 kD proteins in elementary bodies. Class specific antibody response against the proteins of reticulate bodies was similar to that observed for elementary body antigens--with one substantial difference: no reaction was observed in the 60 kD and 62 kD positions. This suggests that 60 kD and 62 kD proteins are deficient in reticulate bodies.

Susceptibility of iron-loaded Borrelia burgdorferi to killing by hydrogen peroxide and human polymorphonuclear leucocytes.

Borrelia burgdorferi grew more slowly in iron-depleted than in iron-sufficient media. The addition of increasing concentrations of iron stimulated borrelial growth and resulted in the intracellular accumulation of this element. Compared with iron-starved borrelia, iron-enriched organisms showed enhanced sensitivity to hydrogen peroxide. Intracellular iron-content did not, however, influence susceptibility to killing by human polymorphonuclear leucocytes [corrected].

Complement-mediated in vitro bactericidal activity of monoclonal antibodies reactive with outer-surface-protein OspB of Borrelia burgdorferi.

Murine monoclonal antibodies (mAbs) were obtained against the outer-surface-protein OspA and OspB and against the 41-kDa flagellar antigen of Borrelia burgdorferi. The specificity of mAb was determined by the Western blotting technique and the surface association of the antigens was inferred by immunofluorescence of living bacteria. In an in vitro assay in the presence of complement, two mAbs reactive with the Ospa were able to kill borreliae, whereas several mAbs reactive with the OspA as well as with the 41-kDa flagellar protein were not.

Partial characterization of an 89-kDa highly immunoreactive protein from Chlamydia psittaci A/22 causing ovine abortion.

An 89-kDa immunogen from Chlamydia psittaci A/22 causing ovine abortion was partially characterized. The 89-kDa protein, localized on the outer membrane complex of chlamydiae, was synthesized relatively early in the developmental cycle. The protein contained cysteine but was not extensively cross-linked by disulfide bonds. Treatment with proteases apparently did not cleave the protein. The infectivity of strain A/22 was partially (60%) reduced by treatment of chlamydial elementary bodies with monoclonal antibody BS/89 specifically reacting with the 89-kDa antigen. Species-specific as well as strain-specific antigenic determinants were present on the 89-kDa protein.

Virological course of herpes zoster in otherwise normal hosts.

The virological course of herpes zoster infection in 42 otherwise normal hosts was studied by virus isolation and antibody titration. Varicella-zoster virus (VZV) was isolated from vesicle fluid from all three patients examined on the first day of the vesicular eruption and from five out of six examined on the second day. The isolation rate fell to one out of six patients on the seventh day of illness and VZV was not isolated from patients at a later stage of the illness. IgG antibodies were detected by IFAMA and ELISA, in sera from all the patients by the end of the first week of illness; IgG antibody titres were highest during the second and the third weeks. IgM antibodies to VZV were detected in sera from six of the 42 patients with herpes zoster after fractionation by ion-exchange chromatography.

Evaluation of recomWell Treponema, a novel recombinant antigen-based enzyme-linked immunosorbent assay for the diagnosis of syphilis.

OBJECTIVE: To evaluate the diagnostic performance of an enzyme immunosorbent assay (recomWell Treponema) for the diagnosis of syphilis. The novel recombinant antigens Tpn47, TpN17 and TpN15 were utilized. METHODS: A total of 782 human serum specimens, belonging to four different categories (blood donors, n = 200; routine laboratory screening for syphilis, n = 400; syphilis patients, n = 122; potential cross-reactors, n = 60), were evaluated to compare the sensitivity and specificity of the recomWell Treponema kit with a standard whole Treponema pallidum cell lysate antigen-based ELISA (Syphilis Screening) and with micro-haemagglutination (MHA-TP). RESULTS: The overall specificity and sensitivity of the recomWell Treponema IgG was 98.9% and 98.3%, respectively. The specificity and sensitivity of Syphilis Screening ELISA was 98.7% and 98.3%, respectively. The agreement between recomWell Treponema and Syphilis Screening was 100%, 97.8%, 95.9% and 95% among the blood donor specimens, screening samples, syphilis specimens and the potential cross-reactors, respectively. Values of concordance varying from 96.7% to 98.3% were found in the different groups of sera between recomWell Treponema and MHA-TP. In addition, recomWell Treponema demonstrated a good diagnostic performance when used to detect the IgM to T. pallidum. No false-positive sera were identified and, in 17/19 samples from primary infection, an IgM immune response was found. CONCLUSIONS: recomWell Treponema was shown to be a highly specific and sensitive method in all stages of syphilis screening and it can be considered as alternative to other ELISA tests based on native antigen preparations.

Animal and human antibodies reactive with the outer surface protein A and B of Borrelia burgdorferi are borreliacidal, in vitro, in the presence of complement.

Polyspecific antibodies present in ascitic fluids of mice (pMIAFs) immunized with whole Borrelia burgdorferi cells exerted borreliacidal activity in vitro when tested with complement and homologous antigen but not with heterologous B. hermsii. Similarly, monospecific mouse antibodies obtained by immunizing mice with purified preparations of outer surface protein A and B of B. burgdorferi were borreliacidal. On the contrary, mouse monospecific antibodies raised against the 41-kDa flagellar protein of B. burgdorferi did not kill borreliae in the presence of complement. A complement-mediated, in vitro, borreliacidal activity was observed in human sera from patients with Lyme disease when antibodies against OspA and/or OspB were detectable in sera by the Western blotting technique. The in vitro borreliacidal activity of human sera was evident after 14 h incubation with live B. burgdorferi spirochaetes and complement, whereas antibodies present in mouse immune ascitic fluids killed borreliae after 1 h incubation.

A simple immunoperoxidase method for detecting enteric adenovirus and rotavirus in cell culture.

A technique which includes the use of indirect immunoperoxidase antibody (IPA) has been developed for detecting enteric adenovirus and rotavirus antigens in cell cultures and has been compared with immunofluorescence antibody assay (IFA). The IPA technique was as sensitive as the IFA. The number of positive cells detected by both techniques in tissue cultures was the same; false positive results were not observed. The applicability of IPA in clinical virology is discussed.

Evaluation of antibodies to Epstein-Barr virus in Italian patients with nasopharyngeal carcinoma.

An association of Epstein-Barr virus (EBV) with nasopharyngeal carcinoma (NPC) has been established serologically in Italian patients. The finding of IgA antibodies to EBV-capsid antigen (VCA) and to early antigen (EA) showed a high degree of correlation for patients with NPC, thereby confirming previous reports. In addition, when considered together, IgA anti-VCA and IgG anti-EA antibody titres appeared to distinguish NPC-patients from those in control populations. Poorly differentiated and undifferentiated carcinomas with lymphoid cellular infiltrations showed the highest frequency of association with positive EBV serological tests.

Evaluation of a new latex agglutination test for detecting human rotavirus in faeces.

Four methods for detecting rotaviruses (latex agglutination, electron microscopy, immunofluorescence and ELISA) have been compared on 57 faecal samples from children with acute diarrhoea. Complete agreement among the four techniques was found in 38 samples. One sample was positive by ELISA and latex agglutination but negative by the other two. For all the other samples there was agreement among three of the techniques only. In a blocking ELISA test, samples positive by ELISA only, turned out to be falsely positive. Assuming true positive or negative for those samples for which at least three techniques were in agreement, electron microscopy, ELISA and latex agglutination were more sensitive (96 per cent) than immunofluorescence (84 per cent). Electron microscopy was the most specific (96.4 per cent), followed by immunofluorescence (92.9 per cent), ELISA (89.4 per cent) and latex agglutination (85.9 per cent).

In-vitro activity of ofloxacin against Chlamydia trachomatis.

The in-vitro activity of ofloxacin was evaluated against recently isolated Chlamydia trachomatis strains from patients suffering from non-gonococcal urethritis. The minimal inhibitory concentration (MIC) proved to be 1 mg/l against 8 of the 10 strains assayed (the MICs for the other two strains were 0.5 and 2 mg/l). The data obtained confirm that ofloxacin is active against Chlamydia trachomatis at concentrations achievable with the routine dosage regimen. The drug may thus be regarded as potentially useful for the treatment of non-gonococcal urethritis due to Chlamydia.

In vitro activity of azithromycin against Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis in comparison with erythromycin, roxithromycin and minocycline.

The in vitro activity of azithromycin against 40 strains of Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis was investigated in comparison with erythromycin, roxithromycin and minocycline. All C. trachomatis strains were inhibited by azithromycin at a concentration < or = 0.5 microgram/ml. The initial minimum inhibitory concentration (MIC) of the drug for U. urealyticum was 4 microgram/ml, whereas some resistance against the drug was shown by M. hominis. Erythromycin and roxithromycin presented almost comparable activities, whereas minocycline was slightly more active than macrolides against C. trachomatis (MIC < or = 0.25) and more active against M. hominis (initial MIC < or = 1 micrograms/ml). Only 97% of U. urealyticum strains were susceptible to 8 micrograms/ml of minocycline.