Monica Compri

Prevalence and predictors of Lymphogranuloma venereum in a high risk population attending a STD outpatients clinic in Italy

BackgroundWe evaluated LGV prevalence and predictors in a high risk population attending a STI Outpatients Clinic in the North of Italy.MethodsA total of 108 patients (99 MSM and 9 women), with a history of unsafe anal sexual intercourses, were enrolled. Anorectal swabs and urine samples were tested for Chlamydia trachomatis (CT) DNA detection by Versant CT/GC DNA 1.0 Assay (Siemens Healthcare Diagnostics Terrytown, USA). RFLP analysis was used for CT molecular typing.ResultsL2 CT genotype was identified in 13/108 (12%) rectal swabs. All LGV cases were from MSM, declaring high-risk sexual behaviour and complaining anorectal symptoms. Patients first attending the STI Outpatient Clinic received a significant earlier LGV diagnosis than those first seeking care from general practitioners or gastroenterologists (P = 0.0046).LGV prevalence and characteristics found in our population are in agreement with international reports. Statistical analysis showed that LGV positive patients were older (P = 0.0008) and presented more STIs (P = 0.0023) than LGV negative ones, in particular due to syphilis (P < 0.001), HIV (P < 0.001) and HBV (P = 0.001).Multivariate logistic regression analysis revealed that HIV and syphilis infections are strong risk factors for LGV presence (respectively, P = 0.001 and P = 0.010).ConclusionsEven if our results do not provide sufficient evidence to recommend routine screening of anorectal swabs in high-risk population, they strongly suggest to perform CT NAAT tests and genotyping on rectal specimens in presence of ulcerative proctitis in HIV and/or syphilis-positive MSM. In this context, CT DNA detection by Versant CT/GC DNA 1.0 Assay, followed by RFLP analysis for molecular typing demonstrated to be an excellent diagnostic algorithm for LGV identification.

Novel approaches for the taxonomic and metabolic characterization of lactobacilli: Integration of 16S rRNA gene sequencing with MALDI-TOF MS and 1H-NMR.

Lactobacilli represent a wide range of bacterial species with several implications for the human host. They play a crucial role in maintaining the ecological equilibrium of different biological niches and are essential for fermented food production and probiotic formulation. Despite the consensus about the ‘health-promoting’ significance of Lactobacillus genus, its genotypic and phenotypic characterization still poses several difficulties. The aim of this study was to assess the integration of different approaches, genotypic (16S rRNA gene sequencing), proteomic (MALDI-TOF MS) and metabolomic (1H-NMR), for the taxonomic and metabolic characterization of Lactobacillus species. For this purpose we analyzed 40 strains of various origin (intestinal, vaginal, food, probiotics), belonging to different species. The high discriminatory power of MALDI-TOF for species identification was underlined by the excellent agreement with the genotypic analysis. Indeed, MALDI-TOF allowed to correctly identify 39 out of 40 Lactobacillus strains at the species level, with an overall concordance of 97.5%. In the perspective to simplify the MALDI TOF sample preparation, especially for routine practice, we demonstrated the perfect agreement of the colony-picking from agar plates with the protein extraction protocol. 1H-NMR analysis, applied to both culture supernatants and bacterial lysates, identified a panel of metabolites whose variations in concentration were associated with the taxonomy, but also revealed a high intra-species variability that did not allow a species-level identification. Therefore, despite not suitable for mere taxonomic purposes, metabolomics can be useful to correlate particular biological activities with taxonomy and to understand the mechanisms related to the antimicrobial effect shown by some Lactobacillus species.

Evaluation of the Versant CT/GC DNA 1.0 Assay (kPCR) for the Detection of Extra-Genital Chlamydia trachomatis and Neisseria gonorrhoeae Infections.

Screening for extra-genital Chlamydia trachomatis and Neisseria gonorrhoeae infections is a crucial component for sexually transmitted diseases management, even if at present days no commercial methods have been approved for use on pharyngeal and rectal specimens by the US FDA or have received the conformity CE marking. Here we report the analytical sensitivities of the Versant CT/GC 1.0 assay (Siemens Healthcare Diagnostics, Tarrytown, NY, USA) on rectal and pharyngeal swabs, and an evaluation about the suitability for this assay with two widely used swab collection devices (E-Swab and eNAT, Copan, Brescia, Italy). The limits of detection for rectal and pharyngeal specimens with the Versant assay were 10 copies/ml and 1.0 copies/ml, for C. trachomatis and N. gonorrhoeae, respectively. False positive results due to the presence of non-gonococcal Neisseria species were excluded when clinical rectal and pharyngeal samples containing organisms identified as N. meningitidis, N. sicca, N. flavescens and N. subflava were tested. Due to its sensitivity and specificity, the Versant assay represents a good choice for the diagnosis of chlamydial and/or gonococcal infections not only in genito-urinary samples, but also on rectal and pharyngeal swabs.

Acute Fitz-Hugh-Curtis Syndrome in a Man due to Gonococcal Infection

BACKGROUND Fitz-Hugh-Curtis syndrome is a rare extra-pelvic complication of genital infection involving the perihepatic capsule. Most cases have been described in women in association with pelvic inflammatory disease; in rare cases it has been reported in men. Because the main symptom is acute abdominal pain, and laboratory and imaging findings are frequently nonspecific, the differential diagnosis, considering other gastrointestinal or renal diseases, can be difficult in the early stage of the syndrome, leading to frequent misdiagnosis and mismanagement. CASE REPORT We report a case of Fitz-Hugh-Curtis syndrome in a 26-year-old man who first presented to the emergency department with acute abdominal pain, vomiting, and fever. Diagnosis was possible on the basis of clinical signs of orchiepididymitis, abnormal ultrasound findings, and specialist consultation with the Sexually Transmitted Infection Clinic. An acute gonoccocal infection was revealed, which was complicated by a collection of free perihepatic fluid and a subcapsular hypoechoic focal lesion. Prompt antibiotic therapy was established, with complete resolution of the symptoms within a few days. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Awareness of the clinical presentation, imaging, and laboratory findings during the acute phase of Fitz-Hugh-Curtis syndrome could help emergency physicians to make an early diagnosis and to correctly manage such patients. Improved diagnostic skills could prevent chronic complications that are especially a risk in the case of delayed or minor genitourinary symptoms.

Evaluation of a New Protocol for Retrospective Diagnosis of Congenital Toxoplasmosis by Use of Guthrie Cards

ABSTRACT The aim of this study was to assess the diagnostic value of IgM Western blotting (WB), IgA enzyme immunoassay (EIA), and DNA amplification by real-time PCR on Guthrie cards to retrospectively establish the diagnosis of congenital toxoplasmosis (CT). To this purpose, Guthrie cards were collected from 18 infants born to mothers with primary Toxoplasma gondii infection during pregnancy. Moreover, the analytical sensitivity of T. gondii PCR was assessed by testing mock dried blood specimens set up with several known DNA dilutions. IgM WB was demonstrated to be the most sensitive method. When the results of T. gondii DNA detection and specific IgM recovery were combined, retrospective CT diagnosis by using Guthrie cards was established in 3 out of 6 infected infants (sensitivity, 50%; 95% confidence interval, 26.8% to 73.2%). No positive PCR or serologic results were found in the group of 12 uninfected infants, demonstrating the excellent specificity of the three methods (95% confidence interval, 78.1% to 99.5%). The findings of the present study suggest that, in cases of missed diagnosis of CT at birth, analysis of Guthrie cards for children with compatible clinical findings after the perinatal period, in particular the combination of recovery of specific IgM antibodies and T. gondii DNA amplification, could be helpful. Nevertheless, since suboptimal conditions of storage of dried blood specimens can seriously affect sensitivity, negative results cannot rule out CT diagnosis. In contrast, because of the excellent specificity shown by IgM serologic testing and T. gondii DNA amplification on Guthrie cards, positive results obtained by either of the two methods should be considered diagnostic.

Contribution of a Comparative Western Blot Method to Early Postnatal Diagnosis of Congenital Syphilis

ABSTRACT 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.

P5.086 Diagnosis of Extra-Genital Chlamydia And/Or Gonorrhoea Infections by Versant CT/GC DNA 1.0

Objectives Nucleic acid amplification testing (NAAT) has become the preferred method to detect Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) infections. Anyway, no commercial test has been cleared so far for use with extra-genital swab samples. In this study Versant CT/GC DNA 1.0 (Siemens) performances have been evaluated by testing ocular, rectal or pharyngeal secretions collected by Siemens collection devices. Methods Study group. A prospective study was performed with 7 newborns with conjunctivitis, and 183 subjects attending the STD Outpatients Clinic of St. Orsola Hospital, Bologna. The latter ones were enrolled because having unsafe receptive anal and/or pharyngeal sex intercourses. NAAT methods. All the specimens were tested by Versant CT/GC DNA 1.0. In case of a Versant CT positive result, we collected the corresponding remnant DNA extract and used it as a template for omp1 semi-nested-PCR. RFLP analysis of PCR-positive samples was carried out by using AluI, HinfI and DdeI as restriction enzymes, for genotyping. All the specimens scored GC positive were retested by a “home-made” PCR assay, targeting cppB gene. Results A total of 253 samples were obtained. In particular, we tested 14 conjunctival swabs, 155 pharyngeal swabs and 84 rectal swabs. Versant assay scored as GC positive 13 pharyngeal and 7 rectal samples. All these specimens were confirmed reactive by cppB PCR. Regarding CT infections, Versant assay identified 2 ocular specimens as positive: one was further genotyped as E and the other one as F. Moreover, we found 4 positive pharyngeal specimens (genotypes E, F, J) and 12 rectal samples (genotypes E, H, J, L2). Conclusions Versant CT/GC DNA 1.0 demonstrated to be a very good method to identify extra-genital infections due to chlamydia and/or gonorrhoea. Because of its performances, and the walk-away capability of the system, this assay can be considered an excellent choice for CT/GC diagnosis.

P2.013 Lymphogranuloma Venereum Cases Identified in Patients Attending a STD Outpatients Clinic in Italy

Background Lymphogranuloma venereum (LGV) is a systemic sexually transmitted infection caused by Chlamydia trachomatis (CT)serovars L1-L3. In the recent outbreaks the classic clinical presentation with inguinal syndrome is giving way to anorectal primitive syndrome in men having sex with men (MSM). Here we report about 6 cases of LGV identified during 2012. Methods A prospective study was performed with 78 rectal specimens obtained from MSM attending the STD Outpatients Clinic of S. Orsola Hospital, Bologna. All the patients were enrolled because having unsafe receptive anal sex intercourses. Samples were tested by Versant CT/GC DNA 1.0 (Siemens). Genotyping was performed with RFLP method for ompl gene, using AluI and DdeI as restriction enzymes. Results We found a total of 11 rectal swabs positive for CT. RFLP analysis showed 6 L2 genotypes and 5 non-LGV genotypes (3 were E, and the others H and J). The five non-LGV infected patients showed no symptoms. On the contrary, at the enrollment perianal ulcers, proctitis and painful lymphadenopathy were found in three LGV cases, whereas perianal ulcers and proctitis in the remaining three ones. Before the correct diagnosis the patients had been investigated for several months for a broad range of other conditions, including traumatic warts, and/or gastroenteric syndromes. Three patients had undergone endoscopic procedures and ultrasound scans. All the LGV cases presented at least one more sexually transmitted infection. Treatment with doxycycline (100 mg b.i.d. for 21 days) was successful. At control, case 1 had a positive result for Neisseria gonorrhoeae in his rectal swab, thus demonstrating his high risk sexual behaviour. Conclusion A firm diagnosis and early treatment of LGV can prevent the development of serious sequelae. Since the ulcerative nature of LGV may facilitate transmission and acquisition of other STDs, enhanced surveillance systems and strengthened case ascertainment would be desirable.

P3.350 Evaluation of a Comparative Western Blot Method For Early Postnatal Diagnosis of Congenital Syphilis

Background Diagnosis of congenital syphilis (CS) remains difficult. Part of the problem arises because the standard serologic tests are not useful in newborns because IgG transfer across the placenta. Since Western Blot technique allows the recognition of a specific response towards every single protein, it can be useful to compare IgG immunological profiles of mothers and babies at birth, in order to differentiate between passively transmitted maternal antibodies and antibodies synthesised by the infants. Methods Study group. Thirty infants born to syphilis seropositive mothers were enrolled for this study. At birth, routine serological tests were performed (ARCHITECT ® Syphilis TP, Abbott; TPHA and RPR, Randox) on mother/child pairs’ serum specimens. “Home made WB”. Treponema pallidum antigens, separated by SDS-PAGE, were blotted onto nitrocellulose sheets and incubated overnight with mother/child pairs’ serum specimens. Criteria for CS diagnosis were the following: presence of specific bands in the newborn’s IgG WB strip different from those found on the corresponding maternal WB strip and/or recognition on IgM WB strip of at least 2 out the 4 following bands Tp47, TmpA, Tp17 and Tp15, including at least one with low molecular weight. Results Out of the 30 infants born to syphilis seropositive mothers, we found 3 babies with different IgG WB profiles from those of their own mothers. Two out these three newborns had also positive IgM WB result. Routine serological testing results of all the 30 newborns showed similar values to those of their own mothers. Conclusion The use of comparative IgG WB test enabled us to diagnose CS in three cases in which the infection would have not been detected by classical serology techniques. Therefore the routine use of comparative IgG WB assay at birth on newborn-mother pairs could be a welcome addition to the conventional laboratory methods used for the diagnosis of CS.

P5.070 Diagnosis of Pharyngeal and Rectal Neisseria Gonorrhoeae Infections

Background Despite nucleic acid amplification tests (NAAT) are widely used to detect Neisseria gonorrhoeae infections, so far no commercial kit has been cleared for testing rectal or pharyngeal swab samples, even if anal and/or oral sex practises are common. In this study, a comparison between Real Time PCR Versant CT/GC DNA 1.0 (Siemens) and N. gonorrhoeae culture performances has been conducted, testing rectal or pharyngeal secretions collected by E-swabs (Copan). Methods Study group. A prospective study was performed with 171 subjects (130 males and 41 females) attending the STD Outpatients Clinic of St. Orsola Hospital, Bologna. All the patients were enrolled because having unsafe receptive anal and/or pharyngeal sex intercourses. NAAT methods. All the specimens were tested by Versant CT/GC DNA 1.0. As a confirmation, all the specimens scored positive for N. gonorrhoeae were retested, using the same extraction, by a “home-made” PCR assay, targeting cppB gene. N. gonorrhoeae culture. Bacteria were isolated in Thayer-Martin medium and identified by API NH assay (bioMérieux). Antimicrobial susceptibility was assessed by Kirby-Bauer Test. Results A total of 227 samples were obtained. In particular, 56 patients provided both the specimens, 89 patients provided only pharyngeal swabs, whereas only rectal specimens were collected from the remaining 26 patients. Versant CT/GC DNA 1.0 gave positive results for N. gonorrhoeae in 13 pharyngeal in 7 rectal samples, all from MSM. All the Versant reactive results were confirmed by “home-made” PCR. Prevalence of rectal infection was 8.5% (7 positive out of 82 patients), whereas prevalence of pharyngeal infection was 9.0% (13/145). Culture was far less sensitive than NAAT, since only 4 samples were identified. All of them were resistant to quinolones, but susceptible to cephalosporins (cefixime and ceftriaxone). Conclusions Pharyngeal and/or rectal screening for gonorrhoea should be considered essential in consultations for MSM in STD clinics.