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Serology has a pivotal role in the diagnosis of congenital syphilis (CS), but problems arise because of the passive transfer of IgG antibodies across the placenta. The aim of this study was to assess the diagnostic value of a comparative Western blot (WB) method finalized to match the IgG immunological profiles of mothers and their own babies at birth in order to differentiate between passively transmitted maternal antibodies and antibodies synthesized by the infants against Treponema pallidum Thirty infants born to mothers with unknown or inadequate treatment for syphilis were entered in a retrospective study, conducted at St. Orsola-Malpighi Hospital, Bologna, Italy. All of the infants underwent clinical, instrumental, and laboratory examinations, including IgM WB testing. For the retrospective study, an IgG WB assay was performed by blotting T. pallidum antigens onto nitrocellulose sheets and incubating the strips with serum specimens from mother-child pairs. CS was diagnosed in 11 out of the 30 enrolled infants; 9/11 cases received the definitive diagnosis within the first week of life, whereas the remaining two were diagnosed later because of increasing serological test titers. The use of the comparative IgG WB testing performed with serum samples from mother-child pairs allowed a correct CS diagnosis in 10/11 cases. The CS diagnosis was improved by a strategy combining comparative IgG WB results with IgM WB results, leading to a sensitivity of 100%. The comparative IgG WB test is thus a welcome addition to the conventional laboratory methods used for CS diagnosis, allowing identification and adequate treatment of infected infants and avoiding unnecessary therapy of uninfected newborns., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The presence of IgG and IgA antibodies to Simkania negevensis in adult Italian patients with respiratory or gastrointestinal symptoms was investigated by the microimmunofluorescence test. In patients with respiratory infections, IgG (50%) and IgA (13%) seropositivity was consistent with previous data. In patients with gastrointestinal disorders, IgG (68%) and IgA (18%) seroprevalence was significantly higher than in healthy controls. These results, in association with the previously described detection of S. negevensis in water sources, could suggest an oral route of infection other than droplets or close contact, and a possible association of S. negevensis with gastrointestinal infections.

Chlamydiae are obligate, intracellular, gram-negative bacteria that are responsible for important diseases in humans, other mammals, and birds. Studies have shown that chlamydiae could be present in wild ruminants, but the serodiagnostic method most commonly used did not allow identification of chlamydial species. We determined the prevalence of antibodies to Chlamydia pecorum, Chlamydia suis, Chlamydia abortus, and Chlamydia psittaci in 271 red deer (Cervus elaphus) of a central Italian population, by using the microimmunofluorescence test that shows antibody response against genus-specific and species-specific antigens. No sera had detectable antibodies to C. pecorum and C. abortus. Antibodies were detected against C. psittaci (9.6%) and C. suis (3.3%). Antibody response could be related to contact of the red deer with birds and wild boars (Sus scrofa), respectively, and confirm an extended host range of individual Chlamydia species. In view of the potential zoonotic risk related to exposition of C. psittaci, our findings suggest surveillance of wild ruminants as potential reservoirs for chlamydiae.

We used serology to estimate the prevalence of exposure to chlamydiae in Italian populations of wild boars (Sus scrofa). Sera from 173 hunter-killed wild boars harvested during the 2006-2009 hunting seasons in three Italian regions were tested for antibodies to Chlamydia suis, Chlamydophila pecorum, Chlamydophila abortus, and Chlamydophila psittaci by the microimmunofluorescence test. Antibody titers to chlamydiae ≥ 1:32 were detected in 110 of the 173 samples tested (63.6%). Specific reactivity could be assessed only in 44 sera with antibody titers to C. suis that were two- to threefold higher than antibody titers against the other chlamydial species; the other 66 sera had similar reactivity against all the chlamydia species tested. Antibody to C. suis was detected in sera from wild boar populations with rare or no known contact with domestic pigs. These results suggest that the wild boar could be a chlamydia reservoir and may acquire chlamydiae independent of contacts with the domestic pig.

Specific antibodies to plasmid-encoded protein pgp3 are known to be encountered in human Chlamydia (C.) trachomatis infections. In order to verify whether antibodies to this protein could be developed in animals infected with plasmid-carrying chlamydial strains, 454 animal sera were examined using a home-made pgp3 protein ELISA and Western blots (WB) of recombinant pgp3 protein from Chlamydophila (Cp.) psittaci. Likewise, 50 human sera were tested by ELISA and WB of recombinant pgp3 from C. trachomatis. The reactivity against pgp3 protein was compared to the reactivity against chlamydial elementary bodies (EBs) detected by microimmunofluorescence (MIF) test. The presence of pgp3-specific antibodies was demonstrated in most ducks and pigeons with Cp. psittaci infection detected by MIF, as well as in the majority of symptomatic cats and pigs infected with Cp. felis and C. suis, respectively, which reacted at high titres to Cp. felis and C. suis EBs by MIF. Moreover, most of the sera collected from patients with C. trachomatis culture-confirmed infection and seropositive to C. trachomatis by MIF, presented antibodies specific to C. trachomatis pgp3 recombinant protein. Therefore, pgp3 protein could be a useful marker of chlamydial infections in animals, as well as in humans.

In this study, raising and development of antibody response to Borrelia burgdorferi infection in 66 Italian patients suffering from culture-confirmed Lyme borreliosis erythema migrans (EM) was investigated. Sixty-two of 66 cultures obtained from biopsies were identified as B. afzelii by PCR. A total of 175 serially collected serum samples were tested by using two different sets of commercial assays: Enzygnost Lyme link VlsE/IgG and Enzygnost Borreliosis IgM (DADE Behring, Marburg, Germany) and LIAISON Borrelia IgG and IgM (Diasorin, Saluggia, Italy). Considering only samples obtained at first presentation when EM was clinically evident, 49/66 patients (72.4%) were IgG or IgM positive by Enzygnost, whereas 33/66 (50.0%) patients were IgG or IgM positive by LIAISON. Taking into account the follow-up period, eight patients sero-converted for IgG or IgM by Enzygnost and four by LIAISON. Similar and very good specificity values were obtained by all methods. Testing sera obtained from blood donors (n = 300) and from patients suffering from some of the most common biological conditions possibly resulting in false-positive reactivity in Lyme disease serology (n = 100) showed that Enzygnost Lyme link VlsE/IgG was the more specific (98.3%), followed by LIAISON Borrelia IgG (96.5%), and considering IgM tests, Enzygnost Borreliosis IgM showed to be 95.3%% specific, whereas the LIAISON Borrelia IgM was 92.8% specific. Recombinant VlsE antigens obtained from all three B.burgdorferi genospecies pathogenic to humans (included in Enzygnost Lyme link VlsE/IgG) greatly improved serodiagnosis of Lyme disease.

We tested 731 sera from apparently healthy light horses against Chlamydophila pneumoniae, by a microimmuno-fluorescence (MIF) test. To verify cross-reactions with other species of chlamvdiae, all sera with an antibody titre > or = 32 to C. pneumoniae were tested against both C. psittaci and C. abortus. Antibodies to C. pneumoniae were detected in 194 out of 731 (26.5%) samples tested, with antibody titres ranging from 32 to 1024. No antibody titre > or = 32 was detected in sera to C. abortus. Only few sera with a high antibody titre to C. pneumoniae reacted weakly with C. psittaci at the dilution of 1:32.

The purpose of this study was to evaluate the diagnostic performance of the LIAISON Borrelia Screen (Diasorin, Saluggia, Italy), a new automated immunoassay based on the chemiluminescent technology (chemiluminescence immunoassay). To assess whether a decrease in a negative value in the anti-VlsE immunoglobulin G (IgG) antibody titer was correlated with a positive response to treatment, a group of serially collected serum samples from 67 patients with culture-confirmed erythema migrans was retrospectively studied. All the patients had been treated with antibiotics and were free of disease within 3 to 6 months of follow-up. All the 15 patients who were found to be IgG positive at the time of enrollment and who were bled at least four times during the follow-up became IgG seronegative at 2 to 6 months posttreatment. These results indicate that a decline in the anti-VlsE antibody titer coincides with effective antimicrobial therapy in patients with early localized Lyme disease.

It has long been assumed that parodontal disease can be a cause of false positive results in syphilis serology, but so far there are no definitive data supporting this hypothesis. In this study we tested 250 serum samples obtained from blood donors. All of them were negative when routinely screened for antibodies against Treponema pallidum. Then, all these samples were tested by immunoenzymatic (ELISA) and Western Blot (WB) assays to investigate reactivities against T. denticola. Thirteen samples showed a strong positivity when tested by both methods. When tested by WB against T. pallidum no sample met the positivity criteria. Nevertheless, bands with molecolar weights of about 30-35 KDa (endoflagellar core antigens) were recognized. All the 13 subjects serologically T. denticola positive underwent oral clinical and radiological observation: all showed a very poor parodontal status (CPSS > 103). Eleven crevicular fluid samples out of the total of 13 patients were T. denticola positive by Real Time PCR carried out using a LightCycler system. In this study we demonstrated that the presence of T. denticola in the crevicular fluid samples obtained from patients with a severe periodontal status and/or a positive serology against T. denticola is not a cause of false positive results in syphilis serology.

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.

An outer surface lipoprotein of 22 kDa was identified in the avian pathogen Borrelia anserina Ni-NL by using antibody preparations reactive with bacterial surface-exposed proteins. Amino acid sequence analysis of the 22-kDa protein demonstrated 90% identity with VmpA of B. turicatae, suggesting that the protein belongs to the family of 20-kDa outer surface proteins of the genus Borrelia. All of the 60 chicks intramuscularly treated with antibodies specifically reacting with the 22-kDa protein and infected with strain Ni-NL were completely protected from infection, since no spirochetemia was detected, and from death. Control chicks were treated with immune sera raised against apathogenic strain B. anserina Es, which expresses a prominent 20-kDa polypeptide that is also a member of the Vmp family but does not cross-react immunologically with the 22-kDa protein of the Ni-NL strain. These animals, infected with B. anserina Ni-NL, showed a high degree of spirochetemia 10 days after infection, and all died between 14 and 21 days after infection. The results showed that the 22-kDa surface protein of B. anserina Ni-NL is a determinant of the pathogenic potential of the strain and also confirmed that only strain-specific antibodies are protective against B. anserina infection.

Sensitivity and specificity of IgG detection by Western blotting performed with a lysate of Treponema pallidum whole cells were compared with those of the most common assays used in the laboratory diagnosis of syphilis, i.e. fluorescent treponemal antibody absorption test (FTA-ABS) and treponemal haemagglutination assay (TPHA). Thirty-five serum samples obtained from twenty-one patients with a clinical diagnosis of early syphilis, based on the presence of typical chancre or skin or mucous membrane lesions, were studied. In addition, thirty blood samples from donors, ten sera positive for Borrelia burgdorferi and five positive for Leptospira interrogans were tested as controls. The clinical diagnosis was the reference method used to compare the performance of the serological tests. Western blotting performed with a sensitivity and specificity of 100%, whereas the corresponding sensitivity and specificity for FTA-ABS were 88.5% and 98%, respectively. The performance of TPHA showed a sensitivity of 86% and a specificity of 100%.

Antibodies to Chlamydia pneumoniae (IOL-207) were studied by the microimmunofluorescence (MIF) assay and in vitro neutralization in serum samples from 230 healthy adults and 332 patients at risk of C. pneumoniae infection. In the MIF test, the prevalence of immunoglobulin G (IgG) antibody was 53.9% among healthy subjects and 40.7% in the patients. An MIF antibody titre of > or = 1 : 512, consistent with acute infection by C. pneumoniae was documented in 23 out of 332 patients, and in none of the healthy adults. C. pneumoniae complement-dependent neutralizing antibody was detected in 25.8% and 31.2%, respectively of MIF-positive sera from healthy subjects and patients. The neutralizing antibody detection rate was 52.2% among the 23 patients with MIF titres of > or = 1 : 512. Complement-independent neutralization was observed in only 5 sera from healthy subjects and in 3 sera from patients. The complement-dependent neutralizing ability of sera significantly (p < 0.001) correlated with MIF titres.

In order to clarify the supposed involvement of urban pigeons (Columba livia livia) in the epidemiology of Lyme disease, the presence of antibodies against Borrelia burgdorferi and Borrelia anserina in pigeons' sera, collected in 12 areas of northern and central Italy, was evaluated. This evaluation was carried out using a classic immunofluorescence assay (IFA), a surface immunofluorescence assay (SIFA) and a standard Western Blot (WB) assay. A total of 104 out of 3,186 (3.26%) serum samples were positive for both spirochetes when tested by IFA, with titres ranging from 1/40 to 1/1280. All positive specimens showed the same or a higher reactivity against B. anserina than against B. burgdorferi. Of the IFA positive samples, 30 were tested by WB and SIFA to evaluate further the specificity of the antibody response, i.e. to try to clarify against which spirochete the antibodies were raised. The presence of antibodies against the 23 kDa protein exclusive to B. anserina, and against epitopes which are not surface-exposed and which are common to B. anserina and B. burgdorferi, was assessed by WB and SIFA. No serological evidence that B. burgdorferi can infect pigeons was found.

In this report we describe the use of a newly developed immunofluorescence technique performed with living spirochetes to detect serum antibody to B. burgdorferi s.l. in a case of early Lyme borreliosis. The immunofluorescence method used (surface immunofluorescence assay: SIFA) proved useful in the serological evaluation of suspected cases of Lyme disease.

An indirect immunofluorescence assay using a 96 microwell cell culture plate (IFA) was developed for the detection of antibodies to C. pneumoniae. The results obtained by IFA on 230 sera of healthy subjects were compared with those obtained with the microimmunofluorescence test. The correlation coefficient for IgG detection with the two methods was 0.96 showing good agreement.

We identified, by two-dimensional electrophoretic analysis and microsequencing, a protein of Chlamydia trachomatis elementary bodies which corresponds to the polypeptide (pgp3) encoded by open reading frame 3 (ORF3). Amino acid analysis showed that the first residue (Gly) found in the native protein is the one encoded by the second ORF3 codon, implying a typical bacterial removal of the first Met residue. Relatively large amounts of recombinant pgp3 (r-pgp3) in a stable, water-soluble form were obtained by overexpressing ORF3 in Escherichia coli and purifying the product from periplasmic extracts under nondenaturing conditions. These r-pgp3 preparations allowed specific detection of anti-pgp3 antibodies by enzyme-linked immunosorbent assay. Analysis of a group of 170 sera from healthy blood donors and from patients who were seropositive or -negative for C. trachomatis and Chlamydia pneumoniae showed that an immune response to pgp3 occurs in the majority (ca. 81%) of patients with sexually transmitted diseases who are seropositive for C. trachomatis and generally correlates with the response to cell surface antigens. No reaction between r-pgp3 and C. pneumoniae-positive sera was detected.

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.

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.

IgG, IgA, and IgM antibody titers to Chlamydia trachomatis were determined in sera of 80 infertile women and 100 controls by a single antigen (L-2) immunoperoxidase assay. The infertile women included 50 with unexplained infertility and normal hysterosalpingogram (HSG) and 30 with abnormal HSG. The control sera included 50 from primiparous and 50 from multiparous women. The prevalence of C. trachomatis IgG antibody was significantly higher in infertile women with abnormal HSG as compared with infertile patients with normal HSG and controls (87% v. 20% and 10%, respectively). The geometric mean titer (GMT) of C. trachomatis IgG antibodies of infertile women with abnormal HSG was significantly higher than those of controls (20.7 v. 5.6). A significantly higher prevalence of C. trachomatis IgA antibodies was found in infertile women with both abnormal and normal HSG than in controls (77% and 14% v. 3% respectively). No C. trachomatis IgM antibodies (less than 2) were found in any of the infertile or control groups. The possibility that serum C. trachomatis IgA antibodies may serve as a marker for early recognition of persistent C. trachomatis is discussed.

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.

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.

The Authors report the results of a study on the prevalence of serum antibodies against Chlamydia trachomatis among patients with non-gonococcal urethritis caused by Chlamydia trachomatis and diagnosed on the basis of the isolation of the microorganism in cell cultures, among patients with non-gonococcal urethritis of unknown etiology and in groups of healthy population, in Italy. The search for antibodies was performed both with complement-fixation tests in micro-titer system, using an antigen prepared from chicken embryo yolk sacs infected with the 6BC strain of Chlamydia psittaci, and with indirect immunofluorescence tests. The indirect immunofluorescence tests were carried out using as antigen the whole chlamydial inclusion developed in IUDR pre-treated McCoy cell cultures, grown on coverslips, and infected with the BU/434 strain of the LGV2 immunotype of Chlamydia trachomatis. The immunofluorescence tests proved to be much more sensitive than the complement fixation tests in detecting the presence of anti-chlamydial antibody both in patients with non-gonococcal urethritis and in the healthy population groups. The percentage of subjects possessing anti-chlamydial antibodies among the healthy population suggests a diffuse circulation of Chlamydia trachomatis infection. The possibility of employing the serological data in the diagnosis of the etiology of non-gonococcal urethritis is briefly discussed.

Two enzyme-linked immunosorbent assays (ELISA) using highly purified elementary bodies (EB) of Chlamydia psittaci A/22 strain (ovine) or 6BC strain (psittacine) were set up for the detection of antichlamydial antibodies in sheep. No significant differences were observed between the two ELISAs, whereas these tests proved to be more sensitive than complement fixation test and showed a good correlation (r = 0.75) with immunofluorescence assay. The periodate treatment of chlamydial antigens modified the results of serological responses studied by ELISA, depending on the sera. The average reduction of ELISA values by periodate was 28%, ranging between 5% and 65%. The immunoblot analysis of sheep sera showed high cross reactivity between the polypeptides of A/22 and 6BC strains. However, some differences were observed. The major outer membrane protein (MOMP) of 6BC strain was recognized at different molecular weight position (40,000 kDa) in comparison with the MOMP of A/22 strain (38,000 kDa). In addition, a clear band of 97,000 kDa was detected by all sheep sera tested with A/22 strain. This band was undetectable in the blots performed with 6BC strain.