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Additional file 1: Figure S1. Design of the algal trap. Figure S2. Morphological characteristics of the unknown fungal isolate. Figure S3. Extraction of genomic DNA (gDNA) from the unknown fungal isolate. Figure S4. Growth of P. sumatraense AQ67100 in different algal-supplemented media. Figure S5. Time-course analysis of GH activities from P. sumatraense AQ67100 cultures as grown in algal-supplemented media. Figure S6. Analysis of GH activities from P. sumatraense AQ67100 cultures upon growth in different cell wall polysaccharide-supplemented media. Figure S7. Evaluation of protease activity in C.v.-filtrate. Figure S8. Protein bands putatively ascribable to the main algal-degrading enzymes. Figure S9. Growth of P. sumatraense AQ67100 in a chitin-supplemented medium.

INTRODUZIONE. I lattobacilli rappresentano un ampio genere di microrganismi anaerobi Gram positivi di forma cocco-bacillare. Essi risiedono in differenti regioni anatomiche umane dove svolgono un ruolo chiave nel mantenere l’omeostasi microbica attraverso effetti diretti e capacità immuno-modulanti. Scopo di questo lavoro è stato quello di utilizzare lo studio dei metaboliti batterici tramite spettroscopia di risonanza magnetica nucleare (1H-RMN), come approccio innovativo per l’identificazione di specie di lattobacilli. MATERIALI E METODI. Un totale di 40 ceppi di lattobacilli di diversa origine sono stati inclusi nello studio. L’identificazione di specie, ottenuta mediante sequenziamento del gene 16s rRNA ha permesso di identificare 7 L. crispatus, 7 L. gasseri, 5 L. acidophilus, 5 L. delbrueckii, 2 L. vaginalis, 2 L. reuteri, 6 L. plantarum, 1 L. pentosus, 2 L. rhamnosus, 2 L. casei/paracasei e 1 L. brevis. L’analisi metabolica in 1H-RMN è stata eseguita con lo spettrometro AVANCE III (Bruker) sia sull’intero set di metaboliti intracellulari, dopo opportuna lisi batterica, sia sul gruppo di metaboliti extracellulari. Partendo dal dendrogramma basato sulle sequenze del gene 16s rRNA, i lattobacilli sono stati suddivisi in 7 gruppi e le differenze nella composizione del metaboloma sono state studiate con opportuni test statistici. RISULTATI. L’analisi in 1H-RMN ha permesso di identificare 30 e 17 molecole nel metaboloma extracellulare e intracellulare, rispettivamente. Tali molecole erano rappresentate prevalentemente da aminoacidi, alcoli, monosaccaridi e acidi organici. I diversi gruppi di lattobacilli hanno mostrato complessivamente lo stesso tipo di metaboliti ma con significative differenze per quanto riguarda la concentrazione di singole molecole. In particolare, 4 molecole nel metaboloma extracellulare (acetoina, acetone, piruvato, glucosio) e 8 in quello intracellulare (AMP, lattato, lisina, NAD+, propionato, succinato, uracile e valina) hanno mostrato una differenza statisticamente significativa. Ad esempio, il gruppo dei L. crispatus ha mostrato il maggior consumo di glucosio (P=1x10-3) mentre la produzione di lattato è apparsa più significativa per le specie L. casei-L. paracasei-L. rhamnosus (P=1x10-3). CONCLUSIONI. L’analisi metabolomica ha permesso di identificare metaboliti marker caratteristici di alcuni gruppi di lattobacilli, che potrebbero chiarire i meccanismi che stanno alla base dell’effetto antimicrobico svolto da alcune specie.

INTRODUZIONE. I lattobacilli rappresentano un vasto genere di batteri che colonizzano diverse sedi dell’organismo umano, dove svolgono un ruolo chiave nel mantenimento dell’omeostasi microbica. Sebbene il loro ruolo come microrganismi ‘health-promoting’ sia ben riconosciuto, la loro identificazione a livello di specie pone ancora numerose difficoltà e si basa sulla combinazione di differenti approcci. Scopo del presente lavoro è stato quello di valutare l’accuratezza della spettrometria di massa MALDI-TOF per l’identificazione di specie di ceppi di lattobacilli, comparando tale metodica con il classico approccio basato sul sequenziamento del gene 16s rRNA. MATERIALI E METODI. Sono stati inclusi nello studio 40 ceppi di lattobacilli, isolati da campioni clinici (n=27), presenti in formulazioni di probiotici (n=8) o di origine alimentare (n=5). L’identificazione di specie è stata ottenuta mediante sequenziamento del gene 16s rRNA e il relativo albero filogenetico è stato costruito mediante software MEGA 6. L’analisi spettrometrica è stata condotta sia direttamente da colonia batterica su terreno solido sia dopo estrazione proteica con acido formico/acetonitrile. I campioni sono stati analizzati con lo strumento Bruker MicroFlex MALDI-TOF MS e l’identificazione è stata ottenuta confrontando lo spettro proteico con il database di riferimento. Inoltre, a partire dallo spettro medio di ogni ceppo, è stato costruito un dendrogramma, tramite il software Biotyper 3.1. RISULTATI. Il sequenziamento del gene 16s rRNA ha portato alle seguenti identificazioni: 7 L. crispatus, 7 L. gasseri, 5 L. acidophilus, 5 L. delbrueckii, 2 L. vaginalis, 2 L. reuteri, 6 L. plantarum, 1 L. pentosus, 2 L. rhamnosus, 2 L. casei/paracasei e 1 L. brevis. L’analisi MALDI-TOF ha evidenziato elevati score identificativi (score medio≥1,9) sia in caso di analisi diretta, sia dopo estrazione proteica. Tale approccio ha consentito di identificare correttamente a livello di specie tutti i ceppi di lattobacilli tranne uno (L. pentosus vs L. plantarum), con una percentuale di concordanza con il sequenziamento del 97,5%. A tale riguardo, la stretta relazione filogenetica fra le due specie spiega questo risultato. In alcuni casi, a differenza del sequenziamento, la spettrometria di massa ha addirittura consentito l’identificazione batterica a livello di sub-specie (es. L. delbrueckii). Infine, il dendrogramma proteomico ha mostrato un’elevata sovrapponibilità con quello costruito mediante analisi genomica. CONCLUSIONI. La spettrometria di massa MALDI-TOF si è dimostrata altamente affidabile nell’identificazione di specie di diversi lattobacilli, rappresentando un’ottima alternativa al sequenziamento genico, grazie alla sua rapidità e semplicità di utilizzo. Inoltre, potrebbe rappresentare un metodo innovativo per studi tassonomici dei lattobacilli.

Background: Despite the existence of highly effective interventions, maternal syphilis still causes perinatal morbidity, even when antenatal health services are strong.Serology has a pivotal role in the diagnosis of congenital syphilis, 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 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. Material/methods: Thirty infants born to mothers with unknown or inadequate treatment for syphilis entered in the retrospective study. The babies were born between January 2007 and January 2014, at St. Orsola-Malpighi Hospital, Bologna, Italy. All 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 mother/child pairs’ serum specimens. Results: Eleven out of the 30 enrolled newborns were diagnosed as highly probable congenital syphilis cases: 9/11 infants received the definitive diagnosis with the first week of life, whereas the remaining 2 were diagnosed only later, because of increasing serological tests titers. In contrast, the use of the comparative WB testing performed with mother/child pairs’ serum specimens allowed a correct diagnosis of congenital syphilis in 10/11 cases. Conclusions: The diagnosis of congenital syphilis was greatly improved by a strategy combining comparative IgG WB with IgM WB results, leading to excellent sensitivity (100% - 95% confidence interval, 85.9% to 99.7%). The comparative IgG WB test is thus a welcome addition to the conventional laboratory methods used for the diagnosis of congenital syphilis, allowing to identify and adequately treat infected infants, avoiding unnecessary therapy and the consequent hospitalization of uninfected newborns.

Objectives Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) infections still represent an increasing problem of public health, worldwide. Although unsafe anal and pharyngeal intercourses are common part of sexual repertoires and different cases of extragenital CT/GC infections are reported, extragenital specimens have not been cleared yet for nucleic acid amplification testing (NAAT). For that reason, many laboratories have performed their own validation studies in order to provide results for the clinical management. In this study, Versant CT/GC DNA 1.0 Assay (Siemens) performances were evaluated by testing rectal and pharyngeal swabs collected from a high STD-risk population. Methods From February 2013 to October 2013, urine samples, pharyngeal and anorectal swabs were collected from patients attending the STD Outpatients Clinic of St. Orsola Hospital, Bologna. The patients were enrolled because of a history of unsafe anal and/or oral intercourses. Swabs and urine specimens were tested by Versant CT/GC 1.0 Assay. GC positive results were confirmed using a “home made’ PCR assay targeting PorA gene. CT positive samples were further genotyped by RFLP analysis. Moreover, microbiological investigations for the main STDs (syphilis, HIV, HCV, HBV) were performed. Finally, Chi2 test and Student’s t test were performed and a P value

INTRODUZIONE Nonostante lo screening per le infezioni rettali e faringee da Chlamydia trachomatis e Neisseria gonorrhoeae sia una componente essenziale del management delle infezioni a trasmissione sessuale, nessun sistema per la determinazione degli acidi nucleici ha ricevuto fino ad oggi la marcatura CE per le analisi di tamponi raccolti da tali siti anatomici. Per questo motivo, negli ultimi anni molti laboratori hanno condotto prove di validazione interna. In questo studio riportiamo la validazione, condotta presso la Microbiologia del Policlinico S. Orsola-Malpighi di Bologna, del test VERSANT® CT/GC DNA 1.0 Assay, Siemens, per la contemporanea determinazione del DNA di clamidia e gonococco. Inoltre è stata effettuata una valutazione dell’idoneità di due diversi sistemi di raccolta per i tamponi: E-SwabTM e eNATTM, Copan. METODI La soglia di sensibilità (limit of detection=LOD) del test VERSANT® su tamponi faringei e rettali è stata determinata analizzando in triplicato un serie di concentrazioni note di DNA di C. trachomatis e N. gonorrhoeae (da 102 a 10-1 copie/ml), appartenenti al programma di controllo qualità QCMD 2013. Per simulare campioni clinici, i diversi DNA sono stati diluiti in un pool di 10 tamponi di origine rettale e altrettanti di origine faringea, raccolti in doppio con E-Swab e con eNAT, da soggetti sani, testati per escludere i più comuni patogeni a trasmissione sessuale, delle vie respiratorie e gastrointestinali e che dichiaravano di non aver avuto rapporti a rischio. Inoltre, come controllo per escludere una eventuale presenza nei liquidi di raccolta di agenti interferenti, sono stati testate concentrazioni note di DNA di clamidia e gonococco, diluite in un pool di 10 sospensioni di liquido di trasporto, rispettivamente per tamponi E-Swab e eNAT. Tutti i campioni sono stati processati per estrazione e successiva amplificazione sul sistema VERSANT®. L’estratto di DNA refluo è stato utilizzato per metodiche di PCR home-made che avevano come bersaglio il gene omp1 e porA, rispettivamente per clamidia e gonococco, al fine di valutare i LOD anche di queste due metodiche. RISULTATI Il LOD per C. trachomatis del test VERSANT®, sia su tamponi clinici simulati che su terreni di trasporto, è risultato pari a 10 copie/ml, mentre quello per N. gonorrhoeae era pari 1 copia/ml. Le metodiche di PCR home-made hanno invece mostrato LOD lievemente più alti: 50 copie/ml per clamidia e 5 copie/ml per gonococco. Non sono state notate differenze significative né riguardo alle sedi di prelievo dei campioni clinici simulati, né per quanto riguarda il tipo di sistema di raccolta (E-Swab o eNAT). CONCLUSIONI Il test VERSANT® CT/GC DNA 1.0 Assay ha mostrato ottimi livelli di sensibilità per la determinazione del DNA di C. trachomatis e N. gonorrhoeae, sia su tamponi faringei che rettali. I LOD estremamente bassi permettono di escludere l’ipotesi di eventuali risultati falsamente negativi dovuti alla presenza di inibitori presenti in sede faringea o rettale. Inoltre, entrambi i sistemi di raccolta (E-Swab o eNAT) sono risultati idonei per essere processati sul sistema VERSANT®.

Objectives Lymphogranuloma venereum (LGV) is a systemic sexually transmitted disease (STD) caused by Chlamydia trachomatis (CT) genotypes L1-L3. Since 2003, a new outbreak of LGV infection, characterized by an anorectal primitive syndrome has been described in Industrialized Countries, mainly in men who have sex with men (MSM). Here, we evaluated LGV prevalence and predictors in a high risk population attending the STD Outpatients Clinic of St. Orsola Hospital of Bologna, in the North of Italy. Methods From January 2012 to April 2013, a total of 108 patients (99 MSM and 9 women), complaining of anorectal symptoms or with a history of unsafe anal intercourses, were enrolled for the study. At clinical examination, the external genitalia, perianal skin and anal mucosa were evaluated for the presence of lesions and genital warts. Anorectal and pharyngeal swabs as well as urine specimens underwent CT and Neisseria gonorrhoeae (GC) DNA detection by Versant CT/GC DNA 1.0 Assay (Siemens Healthcare Diagnostics). omp1 gene semi-nested PCR followed by RFLP analysis was used for CT molecular typing of all the CT positivesamples. Finally, microbiological investigations for the main STDs (HIV, HCV, HBV and syphilis) were performed. Results L2 genotype was identified in 13/108 (12%) rectal swabs, while no urine sample nor pharyngeal swabs were found positive for LGV. All LGV cases were from MSM, declaring high-risk sexual behaviour (several partners in the last six months and occasional condom use). Moreover, all of them complained about various anorectal symptoms. Patients first attending the STD Outpatient Clinic received a significant earlier LGV diagnosis than those first seeking care from general practitioners or gastroenterologists (P=0.0046). All LGV positive patients but one suffered from other STDs; in particular, 9 were HIV-positive. Statistical significant differences between LGV-positive and LGV-negative subjects are shown in table1. 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). Conclusion LGV prevalence and characteristics found in our population are in agreement with international reports. Even if our results do not provide sufficient evidence to recommend routine screening of anorectal swabs in high-risk populations, 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 1.0 Assay, followed by RFLP analysis of omp1 gene amplicons is an excellent diagnostic algorithm for LGV identification.

INTRODUZIONE Le infezioni faringee e rettali da Chlamydia trachomatis e Neisseria gonorrhoeae sono in aumento, in particolare in gruppi ad alto rischio. Queste localizzazioni possono dare forme asintomatiche che spesso rimangono misconosciute, fungendo così da serbatoio per la trasmissione. Grazie alle loro elevate performance, le tecniche di biologia molecolare rappresentano oggi il metodo di riferimento per la diagnosi di tali infezioni. Nel presente studio riportiamo la prevalenza di infezioni extra-genitali da clamidia e gonococco in una popolazione selezionata afferente all’ambulatorio per le malattie a trasmissione sessuale (MTS) del Policlinico Sant’Orsola-Malpighi di Bologna. METODI Da Gennaio 2013, tutti i pazienti visitati presso l’ambulatorio MTS del Policlinico e che riferiscono rapporti anali e/o orali non protetti, vengono invitati a sottoporsi alla raccolta, mediante tamponi, di secrezioni prelevate da tali sedi anatomiche per la ricerca degli acidi nucleici di C. trachomatis e N. gonorrhoeae, oltre alla raccolta ormai classica di campioni di urina. Le indagini molecolari vengono eseguite tramite multiplex Real-Time PCR (VERSANT® CT/GC DNA 1.0 Assay; Siemens). In caso di positività per gonococco, i risultati sono confermati con una PCR home-made per il gene porA, mentre la tipizzazione molecolare per le clamidie è condotta con analisi RFLP degli amplificati del gene omp1. RISULTATI Da Gennaio 2013 a Marzo 2014 sono stati analizzati campioni provenienti da 146 uomini omosessuali, 29 uomini eterosessuali e 45 donne, per un totale di 220 urine, 182 tamponi faringei e 119 tamponi rettali. Gli omosessuali sono risultati significativamente più sintomatici (P=0.001) e con più alti tassi di infezione da HIV e sifilide (P

Objectives: Nucleic acid amplification testing (NAAT) has become the preferred method to detect Neisseria gonorrhoeae infections, but no commercial tests have been cleared so far for use with rectal or pharyngeal swab samples, despite anal and oral sex practices are common, in particular for MSM (men having sex with men). 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 172 subjects (133 males and 39 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 commercial test Versant CT/GC DNA 1.0 (Siemens). 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. N. gonorrhoeae was isolated in Thayer-Martin medium and identified by API NH assay (bioMérieux). Antimicrobial susceptibility was assessed by Kirby-Bauer Test. Results: Fifty-three patients provided both the rectal and pharyngeal specimens, 96 patients provided only pharyngeal swabs, whereas only rectal specimens were collected from the remaining 23 patients, for a total of 225 specimens. Versant CT/GC DNA 1.0 gave positive results for N. gonorrhoeae in 13 pharyngeal in 4 rectal samples. Interestingly, all the 4 patients having rectal infection by N. gonorrhoeae had also pharyngeal infection. All the Versant CT/GC DNA 1.0 results were confirmed by cppB "home-made" PCR. Prevalence of rectal infection was 5.3% (4 positive out of 76 patients), whereas prevalence of pharyngeal infection was 8.7% (13/149). No woman was found positive. Culture was far less sensitive than NAAT: only 1 pharyngeal sample and 2 rectal specimens were identified. All of them were resistant to quinolones, but they were susceptible to cephalosporins (cefixime and ceftriaxone). Conclusions: Rectal and pharyngeal screening should be an essential part of consultations in STD clinics. N. gonorrhoeae culture demonstrated to be far less sensitive than NAAT. Anyway, the use of one single swab for performing both culture and NAAT could be of some interest for those clinicians who need to provide antimicrobial test-driven therapy.

Introduzione La tendenza all’accorpamento dei laboratori e il conseguente aumento del numero di richieste giornaliere pone sempre più la necessità di adottare sistemi ad elevata automazione. Negli ultimi anni, nell’ambito dello screening in gravidanza per le infezioni del complesso TORCH, sono stati proposti diversi sistemi automatizzati “random access”. In questo studio sono stati confrontati i dati relativi allo screening sierologico per Toxoplasma gondii (TG), Cytomegalovirus (CMV) e virus della Rosolia ottenuti con due diversi analizzatori: IMMULITE® 2000 (Siemens) e BioPlex® 2200 (Bio-Rad). Metodi Sono stati studiati 288 sieri pervenuti al Laboratorio dell’U. O. Microbiologia con richiesta contemporanea di screening IgG e IgM per TG, CMV e virus della Rosolia. Tutti i sieri sono stati analizzati con BioPlex® 2200 e IMMULITE® 2000. I test per il sistema BioPlex® 2200 sfruttano la tecnologia del “Multiplex Flow Immunoassay”, in grado di rilevare contemporanemente la presenza di anticorpi nei confronti di diversi analiti, mentre i kit per IMMULITE® 2000 sono basati sul principio della chemiluminescenza amplificata. Nel caso di risultati discordanti per il dosaggio delle IgM sono stati utilizzati come conferma i test ELFA Vidas (bioMerieux). Risultati La concordanza per IgG tra i due sistemi è stata, rispettivamente, del 98.2% per TG, 95.8% per CMV e 98.6% per Rosolia. Per IgM la concordanza è stata, rispettivamente, pari a 92.7% per TG, 95.8% per CMV e 96.5% per Rosolia. Tutti i sieri con risultati discordanti per IgM sono risultati negativi quando analizzati con i test ELFA. Conclusioni Entrambi i sistemi “random access” hanno dimostrato valide performance analitiche e, grazie all’alta capacità produttiva e alla completa automazione, sia BioPlex® 2200, sia IMMULITE® 2000 risultano adatti nella gestione della routine di laboratorio per lo screening del complesso TORCH. In caso di riscontro di anticorpi di classe IgM, è consigliabile affiancare un adeguato metodo di conferma.

Introduzione Lo screening sierologico per il complesso TORCH riveste un ruolo fondamentale nella prevenzione delle infezioni a trasmissione materno-fetale. L’elevato numero di analisi quotidiane impone ai laboratori l’adozione di nuove metodiche, caratterizzate da tempi rapidi di esecuzione e alto tasso di automazione. Nel presente studio vengono messi a confronto i dati relativi allo screening sierologico per Toxoplasma gondii (TG), Cytomegalovirus (CMV) e virus della Rosolia ottenuti con IMMULITE® 2000 (Siemens), un analizzatore “random access” basato sulla metodica di chemiluminescenza (CMIA), con quelli ottenuti mediante le tradizionali metodiche immunoenzimatiche (EIA) in micropiastra. Metodi Sono stati analizzati 536 sieri pervenuti al laboratorio dell’U.O. di Microbiologia per lo screening sierologico di infezioni da CMV, TG e Rosolia. I sieri sono stati analizzati per la ricerca di IgM e IgG sul sistema BEP III mediante metodica EIA con micropiastre Enzygnost (Siemens) e su IMMULITE® 2000. Come metodi di conferma, sono stati utilizzati i test ELFA Vidas (bioMerieux). Risultati La concordanza dei test IgG IMMULITE® con le rispettive analisi in micropiastra è stata, rispettivamente, del 98.7% per TG, 98.2% per CMV e 99.8% per Rosolia. Infine, per i test IgM la concordanza tra i due sistemi è stata del 97.9% per TG, 99% per CMV e 97.2% per Rosolia. Conclusioni Il sistema IMMULITE® 2000 permette di effettuare fino a 200 test/ora, garantendo al laboratorio un notevole risparmio di tempo rispetto alle analisi in micropiastra. I valori di concordanza ottenuti, unitamente all’alto livello di automazione e alla elevata produttività rendono il sistema IMMULITE® 2000 una buona alternativa per lo screening anticorpale del complesso TORCH. Per quanto riguarda la ricerca di anticorpi di classe IgM, si consiglia di affiancare a tale sistema un adeguato metodo di conferma, per completezza di risposta

Objectives: Congenital Toxoplasmosis (CT) in newborns results from primary maternal infection with Toxoplasma gondii (TG). Many infected children are asymptomatic at birth but at high risk of neurological sequelae during early childhood. In case of missed diagnosis at birth, retrospective testing of neonatal Guthrie cards for TG DNA or specific IgM anti-TG detection could help to distinguish congenital from acquired Toxoplasmosis. The aim of the this study was to investigate the sensitivity and specificity of IgM testing by Western Blot (WB) and DNA amplification in dried blood samples (DBS) of infants born from mothers infected by TG during pregnancy. Methods: A retrospective study was performed in 18 infants born from mothers who acquired toxoplasmosis during the second or third trimester of pregnancy. At birth, all mother-child serum pairs were tested by conventional assays (Enzygnost Toxoplasmosis-Siemens; Vidas Toxo-bioMérieux) and by comparative WB (Toxoplasma WB IgG/IgM-LDBio Diagnostics). We collected Guthrie cards of every child (informed consensus was obtained from parents); one DBS was used for TG DNA detection and another one for antibodies elution. Nucleic acids were extracted from DBS with Versant kPCR Sample Preparation system (Siemens) and Toxoplasma Q-PCR Alert Kit (Nanogen) was used for amplification. Specific IgM anti-TG were detected in eluates from DBS by using LDBio Toxoplasma WB IgM. Results: At birth CT was diagnosed in 8 of the 18 newborns, because of IgM/IgA positivity and/or different IgG WB pattern in infant’s serum compared to the corresponding mother’s one. CT was excluded in the remaining 10 children because of their sera were IgM/IgA negative and their IgG titres decreased during the follow-up period; at 1 year of age all these 10 babies were IgG negative. In the present study, we confirmed CT in 4 out of the 8 CT cases. In particular, we were able to amplify TG DNA from one of the cards, while in other 3 cases we found specific IgM anti-TG. Specificity of DBS examination was 100%, since no TG DNA or IgM was found in the group of 10 non-infected babies. Serological test at birth and Guthrie card results of the 8 CT cases are shown in detail in table 1. Conclusions: Although serological evaluation at birth and during the first year remains basic for the laboratory diagnosis ofCT, examination of Guthrie cards could be considered a retrospective method to evaluate infants (>1 year ofage) with clinical signs suggestive of CT.

Objectives. 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 synthesized 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.

Objectives. 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 behavior. 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.

Objectives: Bacterial sexually transmitted diseases (STDs) are a public health problem worldwide, but there are marked differences in National Surveillance Systems across the Europe. For example, in Italy chlamydia data are not reported nor any screening programs are available for STDs; syphilis and gonorrhoea should be reported to our national register, but physicians infrequently do. Therefore their diffusion is highly underestimated and the Italian sentinel system can not collect epidemiological data for a full coverage, completeness, and representativeness of national data. In this study we report on prevalence and characteristics of chlamydia (CT), gonorrhoea (GC) and syphilis infections in patients attending a STD Outpatients Clinic in Italy. Methods: In the period between January and December 2011 we collected data of all the patients attending the STD Outpatients Clinic of S. Orsola Hospital, Bologna and being investigated for syphilis, GC and/or CT infections. A blood sample was tested on ARCHITECT Syphilis TP (Abbott) for serological diagnosis of syphilis; positive results were confirmed by TPHA and RPR tests (Randox) and/or a “home made” Western Blot. A urine specimen was collected for DNA detection of Chlamydia trachomatis and Neisseria gonorrhoeae by VERSANT CT/GC DNA 1.0 Assay (Siemens). Results: Total prevalence of antibodies anti-T. pallidum was 21.6% (406/1887). In 173 cases syphilis was diagnosed for the first time, with a prevalence of 9.2%. A total of 1076 patients were investigated for CT/GC infection, with a prevalence of 11.5% (124/1076) for CT and 4.5% (48/1076) for GC: 8 patients were positive for both pathogens. Characteristics of infected patients are reported in table 1. Conclusions: This study highlights the diffusion of STDs in our Country and the need to have clear available data about the real impact on public health. Infection by CT showed the higher prevalence, as expected, on the basis of data reported from other European countries. Moreover, the high number of asymptomatic women underlines the need to implement CT screening programs. In their absence, young people or non-Italian women, for example, rarely seek for medical care. New outbreaks of bacterial STDs seems to be frequent in specific groups of patients (MSM for GC; immigrate women for syphilis), as already reported in other EU Countries. Correct diagnosis for STDs is important to know their real diffusion, to individuate risk factors and to prevent transmission.

Objectives. 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 practices 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 settings.

Background. 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 E-Nat swabs (Copan). 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.

Introduzione. Il linfogranuloma venereo (LGV) è una malattia a trasmissione sessuale (MTS), sostenuta dai sierotipi L1, L2, L3 di Chlamydia trachomatis (CT). L’infezione sta riemergendo nei Paesi industrializzati in uomini omosessuali (MSM) e tende a manifestarsi con sintomi ano-rettali. Nel presente studio riportiamo i dati epidemiologici dell’infezione da LGV in una popolazione ad alto rischio afferente all’ambulatorio MTS del Policlinico Sant’Orsola-Malpighi, Bologna. Metodi. Da gennaio 2012, a tutti i pazienti con sintomi ano-rettali o che riportavano rapporti anali non protetti, è stato offerto uno screening per LGV. Un campione di urina e un tampone rettale sono stati analizzati con il kit Versant CT/GC DNA 1.0 (Siemens) e in caso di positività per CT, la corrispondente genotipizzazione è stata eseguita con metodica RFLP per il gene omp1. Risultati. Sono stati arruolati 108 pazienti, di cui 99 MSM e 9 donne. Dei 23 tamponi rettali positivi per CT, 13 (12%) sono stati identificati come L2 grazie alla tipizzazione molecolare. Nessun campione di urina ha mostrato positività per genotipi L. Tutti i casi di proctite da LGV erano sintomatici e sono stati identificati in MSM con multipli partner sessuali. In 12 di loro è stata confermata la presenza di altre MTS. L’analisi statistica ha rivelato una differenza significativa fra i pazienti LGV-positivi e LGV-negativi per quanto riguarda la presenza di sintomi (100% vs. 20%), l’età media (43 vs. 34 anni) e la coinfezione da HIV (69,2% vs. 10,5%), sifilide (69,2% vs. 16,8%) e HBV (30,8% vs. 3%). All’analisi multivariata, HIV e sifilide sono risultati fattori di rischio per infezione da LGV. Conclusioni. Le caratteristiche dell’infezione da LGV nella popolazione studiata sono in linea con la letteratura internazionale. I nostri dati suggeriscono di eseguire uno screening per infezione da LGV in tutti gli MSM positivi a HIV o sifilide, in presenza di sintomi ano-rettali.

AIMS Congenital Toxoplasmosis (CT) in newborns results from primary maternal infection with T. gondii (TG). Most infected children show no symptoms at birth but are at great risk of sequelae during the first year of life or in early childhood [1]. Polymerase chain reaction (PCR) analysis of dried blood samples on the Guthrie card has been proposed as a sensitive method to screen for congenital CMV infection, but there are no data about the use for TG screening. The aim of present study was to assess the utility of PCR analysis of dried blood samples for the retrospective diagnosis of CT. METHODS A retrospective study was performed with 18 infants born between January 2010 and June 2012. Transmitters mothers seroconverted in the second trimester of pregnancy (mean 23.5 ± 7.9 weeks). At birth, serological tests (Enzygnost Toxoplasmosis IgG, IgM, IgA-Siemens Healthcare Diagnostics; Vidas Toxo IgMbioMerieux) as well as IgM-IgG WB (LDBio Toxoplasma WB IgG/IgM-LDBio Diagnostics) were performed in all mother-child pairs.Nucleic acids were extracted from Guthrie cards with VERSANT kPCR Sample Preparation system (Siemens) and Toxoplasma Q-PCR Alert Kit (Nanogen) was used for the amplification of TG target region AF 146527. RESULTS In 7/18 (38.9%) infants, CT was diagnosed by IgM-WB positivity at birth. The remaining 11 were considered non-infected (61.1%) and became IgG negative within 12 months of life. Infected infants received one-year therapy (pyrimethamine/sulfadiazine) and were followed according to our protocol. Four of these had a pathological neuroimaging (4/4 calcifications, 2/4 ventriculomegaly). None had hearing loss. TG DNA was detected in only one of the Guthrie cards of the infected newborns, while all the others were negative. CONCLUSIONS Although serological methods remain basic in the diagnosis of CT, TG DNA detection in Guthrie cards could be considered a retrospective method to evaluate infants (> 1 year of age) with clinical signs suggestive of CT. More studies with a larger number of infected cases are needed to assess the sensitivity of this method.

Introduzione. I nati da madri con infezione da Chlamydia trachomatis (CT) sono a rischio di infezioni oculari e polmonari. Nel presente studio riportiamo i casi di due neonati, entrambi nati da madri con infezioni asintomatiche da CT, che nella seconda settimana di vita hanno sviluppato una congiuntivite follicolare. Metodi. I tamponi congiuntivali dei neonati e i campioni di urina delle madri sono stati analizzati in Real-Time PCR con il kit commerciale Versant CT/GC DNA 1.0 (Siemens). E’ stata successivamente eseguita la genotipizzazione, con metodica RFLP per il gene omp1. Risultati. Entrambi i neonati, normali per età gestazionale, sono nati con parto spontaneo e sono stati dimessi in terza giornata in buone condizioni cliniche. Il successivo ricorso alle cure dell’U.O. Neonatologia è avvenuto in un caso per presenza di ittero neonatale e controlli per sospetta infezione congenita da Toxoplasma gondii, e nell’altro per presenza di difficoltà alimentari, scarso accrescimento ponderale ed edema palpebrale. La consulenza oculistica richiesta ha permesso in entrambi i casi di diagnosticare una congiuntivite follicolare. I tamponi congiuntivali effettuati sono risultati positivi per CT e la tipizzazione ha evidenziato rispettivamente il genotipo F ed E. In entrambi i neonati è stata instaurata una terapia con Claritromicina, 10 mg/kg/die in due somministrazioni, proseguita per 2 settimane; in un caso si è aggiunta una terapia topica con Ofloxacina collirio 0.3%, 1 goccia per occhio, 4 volte/die. La visita oculistica di controllo ha dimostrato per entrambi completa risoluzione della congiuntivite. Le madri sono state inviate presso l’ambulatorio MTS per ulteriori accertamenti e terapia. Conclusioni. L’infezione da CT nelle donne decorre spesso in maniera asintomatica: da qui deriva la necessità di effettuare in gravidanza uno screening per CT, almeno nelle donne più giovani o con fattori di rischio (numero elevato di partner, nuovo partner) per prevenire infezioni, anche gravi, nei neonati.

Introduzione La diagnosi molecolare di infezioni respiratorie a partire da campioni fortemente mucosi, quali aspirati faringo-nasali o lavaggi bronco-alveolari (BAL), può essere problematica per difficoltà operative, sia nella fase di estrazione, che in quella di amplicazione degli acidi nucleici. In questo studio proponiamo l’uso della soluzione SL (Sputum Liquefying Solution, Copan), per la processazione di materiali respiratori sui quali eseguire successivamente indagini molecolari per la ricerca di Chlamydia pneumoniae e Mycoplasma pneumoniae. Metodi All’arrivo in laboratorio, i materiali respiratori (aspirati faringo-nasali o BAL) sono stati trattati con SL Solution, un mucolitico pronto all’uso che permette l’emulsione del muco rendendo il campione più omogeneo e idoneo per le successive manipolazioni. Da tali materiali è stata eseguita l’estrazione del DNA su sistema automatizzato Nuclisens® easyMag (bioMérieux). In seguito sono stati amplificati i geni bersaglio di C. pneumoniae e M. pneumoniae con metodica di Real-time PCR su strumento Applied Biosystems ABI PRISMTM, con i rispettivi kit CHLAMYDOPHILA pn. Q – PCR Alert kit e MYCOPLASMA pn. Q-PCR Alert Kit (Nanogen Advanced Diagnostics). Risultati Prove interne, condotte testando in parallelo campioni con e senza pre-trattamento con SL Solution, hanno evidenziato l’assoluta validità nell’utilizzo di tale mucolitico. Per questo motivo proponiamo il seguente semplice protocollo di processazione dei materiali respiratori su cui eseguire metodiche NAAT: 1. All’arrivo in laboratorio, i campioni vengono diluiti 1:1 con SL solution in un volume finale di 1 ml. 2. Le provette vengono agitate su vortex per circa 30 secondi. 3. Le provette vengono lasciate riposare a temperatura ambiente per almeno 15 minuti. 4. I materiali vengono congelati a -20°C fino al momento dell’indagine molecolare. 5. Prima della dispensazione per estrazione le provette vengono nuovamente agitate. Conclusioni La soluzione SL si è dimostrata idonea per il trattamento dei materiali mucosi su cui eseguire l’indagine molecolare di infezione respiratoria da batteri atipici.

An Italian HIV-positive man having sex with men (MSM) attended the STIs Outpatients Clinic of Sant'Orsola Hospital in Bologna complaining of anal pain and constipation. According to patient's sexual history and repertoires, NAAT testing for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) was performed. Pharyngeal and anal swabs resulted positive for CT DNA detection and the following molecular genotyping identified a L2 serovar, coming to the final diagnosis of pharyngeal and rectal lymphogranuloma venereum (LGV) infection. After an antibiotic therapy with doxycycline 100 mg twice a day for 3 weeks, the patient completely recovered and the test of cure was negative for LGV infection.

Chlamydia trachomatis (CT) is the causative agent of the most common bacterial sexually transmitted infection (STI) worldwide. CT molecular typing plays a fundamental role in finding associations between serovars and epidemiological features and can be crucial for therapeutic appropriateness. The aim of this study was to assess CT infection prevalence and serovar distribution in a high density urban area in the North of Italy, by comparing different groups of subjects divided on the basis of care providers they referred to. From January 2011 to May 2014, data about all the urogenital and extra-genital samples (anorectal and/or pharyngeal swabs) submitted to the Microbiology of St. Orsola Hospital in Bologna for CT detection were collected. The specimens were obtained from three different groups of patients: subjects attending the STI Outpatients Clinic of the Hospital, patients attending gynecological clinics or people referring to general practitioners. All the samples were processed by a real-time PCR (Versant CT/GC DNA 1.0 Assay; Siemens) and in case of positivity, molecular genotyping based on RFLP analysis was performed. A P < 0.05 was considered statistically significant. Overall CT infection prevalence was 8.1% with significant differences between sub-groups (P

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.