A preliminary molecular typing by PCR assays of Clostridium perfringens and Clostridium difficile isolates from dogs

Clostridium perfringens and C. difficile have been associated with acute and chronic large and small bowel diarrhoea, and acute haemorrhagic diarrhoeal syndrome in dogs. The objective of this study was to investigate by toxin gene pro- file and PCR-ribotyping the molecular characteristics of 14 C. perfringens and 10 C. difficile isolates from 95 canine faeces (n = 36, diarrhoeic and n = 59, non-diarrhoeic). Concerning C. perfringens, 13 strains (92.9%) were type A, of which 3 (23.1%) also possessed the beta 2 toxin (CPB2)-encoding gene. One isolate (7.1%) was type D and possessed CPB2 gene. On the whole, 4 of the 14 strains (28.6%) tested cpb2-positive. Six C. difficile isolates (60.0%) demon- strated tcdA+/tcdB+ and cdtA+/cdtB+ genotype and tested positive for, in vitro, toxin production by EIA. Eight distinct ribotypes were observed. In conclusion, the PCR assays may provide useful and reliable tools for C. perfringens and C. difficile molecular typing in routine veterinary diagnostics

Characterization of Clostridioides difficile Strains from an Outbreak Using MALDI-TOF Mass Spectrometry

The epidemiology of Clostridioides difficile infection (CDI) has changed over the last two decades, due to the emergence of C. difficile strains with clinical relevance and responsible for nosocomial outbreaks with severe outcomes. This study reports an outbreak occurred in a Long-term Care Unit from February to March 2022 and tracked by using a Matrix-Assisted Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) typing approach (T-MALDI); subsequently, a characterization of the toxigenic and antimicrobial susceptibility profiles of the C. difficile isolates was performed. A total of 143 faecal samples belonging to 112 patients was evaluated and C. difficile DNA was detected in 51 samples (46 patients). Twenty-nine C. difficile isolates were obtained, and three different clusters were revealed by T-MALDI. The most representative cluster accounted 22 strains and was considered to be epidemic, in agreement with PCR-Ribotyping. Such epidemic strains were susceptible to vancomycin (MIC <= 0.5 mg/mL) and metronidazole (MIC <= 1 mg/mL), but not to moxifloxacin (MIC > 32 mg/mL). Moreover, they produced only the Toxin A and, additionally, the binary toxin. To our knowledge, this is the first reported outbreak referable to a tcdA+/tcdB-/cdt+ genotypic profile. In light of these results, T-MALDI is a valid and rapid approach for discovering and tracking outbreaks

Active surveillance for carbapenemaseproducing Klebsiella pneumoniae and correlation with infection in subjects attending an Italian tertiary-care hospital: a 7-year retrospective study

Objectives The distribution of carbapenemase-producing Klebsiella pneumoniae (CPKP) phenotypes and genotypes in samples collected during 2011–2018 was evaluated. The association between patients with CPKP-positive rectal swab and those with CPKP infection, as well as the overall analysis of CPKP-infected patients, was performed. Setting The study was performed in a tertiary-care hospital located in Northern Italy. Participants Two groups were considered: 22 939 ‘atrisk’ patients submitted to active surveillance for CPKP detection in rectal swabs/stools and 1094 CPKP-infected patients in which CPKP was detected in samples other than rectal swabs. Results CPKP-positive rectal swabs were detected in 5% (1150/22 939). A CPKP infection was revealed in 3.1% (719/22 939) of patients: 582 with CPKP-positive rectal swab (50.6% of the 1150 CPKP-positive rectal swabs) and 137 with CPKP-negative rectal swab. The 49.4% (568/1150) of the patients with CPKP-positive rectal swab were carriers. The overall frequency of CPKP-positive patients (carriers and infected) was almost constant from 2012 to 2016 (excluding the 2015 peak) and then increased in 2017–2018. blaKPC was predominant followed by blaVIM. No difference was observed in the frequency of CPKP-positive rectal swab patients among the different material groups. Among the targeted carbapenemase genes, blaVIM was more significantly detected from urine than from other samples. Conclusions The high prevalence of carriers without evidence of infection, representing a potential reservoir of CPKP, suggests to maintain the guard about this problem, emphasising the importance of active surveillance for timely detection and separation of carriers, activation of contact precautions and antibiotic treatment guidance on suspicion of infection

Leptospira species and serovars identified by MALDI-TOF mass spectrometry after database implementation

Background: Leptospirosis, a spirochaetal zoonotic disease of worldwide distribution, endemic in Europe, has been recognized as an important emerging infectious disease, though yet it is mostly a neglected disease which imparts its greatest burden on impoverished populations from developing countries. Leptospirosis is caused by the infection with any of the more than 230 serovars of pathogenic Leptospira sp. In this study we aimed to implement the MALDI-TOF mass spectrometry (MS) database currently available in our laboratory with Leptospira reference pathogenic (L. interrogans, L. borgpetersenii, L. kirschneri, L. noguchii), intermediate (L. fainei) and saprophytic (L. biflexa) strains of our collection in order to evaluate its possible application to the diagnosis of leptospirosis and to the typing of strains. This was done with the goal of understanding whether this methodology could be used as a tool for the identification of Leptospira strains, not only at species level for diagnostic purposes, but also at serovar level for epidemiological purposes, overcoming the limits of serological and molecular conventional methods. Twenty Leptospira reference strains were analysed by MALDI-TOF MS. Statistical analysis of the protein spectra was performed by ClinProTools software. Results: The spectra obtained by the analysis of the reference strains tested were grouped into 6 main classes corresponding to the species analysed, highlighting species-specific protein profiles. Moreover, the statistical analysis of the spectra identified discriminatory peaks to recognize Leptospira strains also at serovar level extending previously published data. Conclusions: In conclusion, we confirmed that MALDI-TOF MS could be a powerful tool for research and diagnostic in the field of leptospirosis with broad applications ranging from the detection and identification of pathogenic leptospires for diagnostic purposes to the typing of pathogenic and non-pathogenic leptospires for epidemiological purposes in order to enrich our knowledge about the epidemiology of the infection in different areas and generate control strategies

Molecular methods in the diagnosis of Clostridium difficile-associated disease

Clostridium difficile è un bacillo anaerobio, Gram-positivo, sporigeno, responsabile di una malattia diarroica e/o di coliti di varia gravità fino alla colite pseudomembranosa nonché uno dei maggiori responsabili di epidemie intraospedaliere. I principali fattori di virulenza di C. difficile sono rappresentati da due tossine principali: l’ enterotossina A (TcdA), la citotossina B (TcdB), codificate dai geni tcdA e tcdB localizzati nel Locus di patogenicità di C. difficile (PaLoc), e da una tossina addizionale, la tossina binaria (CDT), sintetizzata a partire da due diversi geni, cdtA e cdtB, localizzati esternamente al PaLoc. Alcuni ceppi di C. difficile producono tossine A e B varianti e presentano delezioni e/o mutazioni anche in altre regioni del PaLoc. Dal momento che queste mutazioni sono più frequenti in presenza dei geni codificanti per la tossina binaria, la presenza dei geni cdtA and cdtB è pertanto considerata un buon indicatore per la rivelazione di ceppi varianti di C. difficile. Ad oggi sono stati identificati 27 tipi di varianti, denominate da I a XXVII In questo studio sono stati analizzati 438 ceppi di C. difficile, isolati nel corso di setti anni (2000-2006) da 334 pazienti. Per caratterizzare la tossinogenicità dei ceppi, sono state applicate una duplex PCR per la ricerca dei geni tcdA e tcdB ed una PCR in grado di rilevare i geni cdtA e cdtB. Inoltre, è stata eseguita mediante PCR-ribotyping la tipizzazione dei ceppi isolati, allo scopo di tracciare un quadro epidemiologico sulla circolazione dei ribotipi di C. difficile nell’azienda ospedaliero-universitaria di Parma. Infine, è stato approfondito lo studio molecolare dei ceppi di C. difficile risultati varianti (cdtA/B+) con l’analisi del PaLoc mediante toxinotyping e successiva analisi del gene regolatore negativo tcdC, nonché con la verifica della loro sensibilità in vitro ad alcuni chemioantibiotici, fluorochinoloni in particolare come “marker” di patogenicità. Dei 438 ceppi complessivamente esaminati, 390 sono risultati tossinogenici e di questi 125 (32,1%) hanno presentato i geni codificanti per la tossina binaria, che caratterizza i ceppi varianti. Questi ultimi rappresentano il 44% dei ceppi tossinogenici isolati negli anni 2001-2003 e solo il 13% dei ceppi isolati negli anni 2004-2006, un dato che appare in controtendenza rispetto a quelli osservati in altri Paesi. Analizzando i 438 ceppi mediante PCR-ribotyping sono stati trovati 76 differenti ribotipi, arbitrariamente denominati. In particolare, nell’ambito dei 125 ceppi ctdA+ ctdB+ sono stati trovati 8 differenti ribotipi. Il ribotipo più diffuso tra i ceppi varianti è il ribotipo 1, che caratterizza il 94,4% di questi ceppi (118 su 125 totali); di questi ultimi 103 ceppi (82,4%) appartengono al sottotipo 1a risultato responsabile dell’epidemia, che ha coinvolto 15 pazienti ricoverati nei Reparti geriatrici (dic. 2002 - apr. 2003). I nostri ceppi ribotipo 1a sono stati riclassificati mediante pulsed-field gel electrophoresis (PFGE) e rinominati internazionalmente mediante PCR-ribotyping come NAP7/ribotipo 078. Quarantatrè dei 125 ceppi potenzialmente CDT+ sono stati selezionati, in base al ribotipo ed all’appartenenza allo stesso paziente, per le successive fasi di analisi del PaLoc e della sensibilità in vitro ai chemioantibiotici. Di questi l’ 86% ha mostrato di appartenere al tossinotipo V, che caratterizza anche la totalità dei ceppi ribotipo 1, mentre quattro sono stati tipizzati come tossinotipo XXIV ed hanno mostrato ribotipi differenti: 5 (2 ceppi), 11 e 16. Relativamente all’analisi mediante PCR-ribotyping dei 313 ceppi non varianti è stata rilevata la presenza di 4 ribotipi predominanti: il ribotipo 13 (45 ceppi, 14,4%), il ribotipo 17 (85 ceppi, 27,3%), il ribotipo 18 (32 ceppi, 10,3%) e il ribotipo 33 (28 ceppi, 9%). Tra questi, un ceppo appartenente al ribotipo 17 è risultato responsabile di una epidemia, che ha coinvolto 42 pazienti ricoverati nei Reparti pneumologici (apr.-sett. 2006). L’analisi del gene regolatore negativo tcdC ha messo in evidenza che i ceppi ribotipo 1 tossinotipo V posseggono un allele, già descritto in letteratura, denominato A1 che, oltre ad una delezione di 39 pb, presenta una mutazione non senso con la conseguente produzione di una proteina tronca non funzionale. Il quadro molecolare emerso suggerisce che questi ceppi sono da considerarsi iperproduttori delle tossine principali A e B. Questi ceppi ribotipo 1a tossinotipo V si sono dimostrati anche altamente resistenti ai fluorochinoloni, come riportato in letteratura. Una variabilità maggiore, al contrario, si è osservata con i ceppi che presentavano ribotipo diverso da 1. Sono state trovate, infatti, 5 nuove sequenze alleliche e tre proteiche non ancora descritte in letteratura. Inoltre, da segnalare è il ceppo ribotipo 2 tossinotipo XXIV, che ha presentato mutazioni insolite per questo tossinotipo, normalmente dotato di un PaLoc identico a quello del ceppo VPI 10463. Interessante è stato anche ritrovare in un ceppo ribotipo 2 un nuovo tossinotipo, non ancora descritto in letteratura. Il nostro studio sottolinea inequivocabilmente l’importanza dell’analisi molecolare, quando condotta per monitorare attentamente la circolazione dei ceppi di C. difficile in ambiente ospedaliero e particolarmente di quelli ipervirulenti, come dimostrano i ceppi Nap7/Ribotipo078 tossinotipo V, comparsi nel nostro ospedale nel 2002-2003, responsabili di una grave epidemia nosocomiale.Clostridium difficile is a Gram-positive spore-forming anaerobic bacterium, often involved in severe C. difficile-associated disease (CDAD) and outbreaks in hospital settings. The main virulence factors of C. difficile are represented by two large toxins: toxin A (TcdA, enterotoxin), toxin B (TcdB, cytotoxin), encoded by tcdA and tcdB genes, located in the pathogenicity Locus of C. difficile (PaLoc), and an additional toxin, the binary toxin (CDT), codified by two different genes, cdtA and cdtB, located outside the PaLoc. Some strains produce variant toxin A and B and present deletions and/or mutations also in other regions of the PaLoc. Since these mutations are more frequent in presence of the genes encoding for the binary toxin, the presence of cdtA and cdtB genes is therefore considered a good marker to detect variant C. difficile strains. To date, 27 variant types have been identified, named from I to XXVII. In this study, 438 C. difficile strains have been investigated, isolated from 334 patients over a seven year period (2000-2006). A duplex PCR for the detection of tcdA and tcdB genes and a PCR to detect the cdtA and cdtB genes have been applied in the attempt to characterize their toxinogenicity. Furthermore, all the C. difficile isolates have been typed by PCR-ribotyping in order to draw an epidemiological feature of the different ribotypes circulating in the environment of the university hospital in Parma. In addition, a thorough molecular study of the C. difficile variant strains (cdtA/B+) has been done by toxinotyping and subsequent analysis of the negative regulator gene tcdC. Finally, the in vitro susceptibility to some antibiotics has been tested, in particular to fluoroquinolones as a marker of pathogenicity. Out of the 438 C. difficile isolates examined in total, 390 were toxigenic and, among them, 125 (32.1%) harboured the genes encoding for the binary toxin, which characterizes variant strains. These strains account for the 44% of the toxigenic strains isolated in the years 2001-2003 and only for the 13% of the strains isolated in the years 2004-2006, data which appear in contrast with those observed in other countries. Moreover, 76 different ribotypes, arbitrarily named, have been found among these 438 C. difficile strains by PCR-ribotyping. All the 125 variant strains ctdA+ ctdB+ belonged only to 8 different ribotypes and the ribotype 1 accounted for the 94.4% of the total strains. In particular, 103 strains (82.4%) have been classified to belong to the subtype 1a, which was responsible of a severe outbreak, involving 15 patients hospitalised in geriatric wards (Dec. 2002 - Apr. 2003). Of interest, the strains ribotype 1a have been reclassified by pulsed-field gel electrophoresis (PFGE) and renamed internationally by PCR-ribotyping as NAP7/ribotype 078. Forty-three of the 125 potentially CDT+ strains have been selected, on the basis of the ribotype and their belonging to the same patient, for the subsequent phases of the PaLoc analysis and in vitro susceptibility to antibiotics. The 86% of these variant strains belonged to toxinotype V, which also included all the strains ribotype 1. Four of them were typed as toxinotype XXIV, but belonged to three different ribotypes: 5 (2 strains), 11 and 16. With regard to the 313 non variant strains analysed by PCR-ribotyping 4 different ribotypes have been predominantly detected: ribotype 13 (45 strains, 14.4%), ribotype 17 (85 strains, 27.3%), ribotype 18 (32 strains, 10.3%) and ribotype 33 (28 strains, 9%). Worthy of note, a C. difficile strain belonging to ribotype 17 has been proven to be responsible for an outbreak, which involved 42 patients hospitalised in pneumology wards (April-Sept. 2006). The analysis of the negative regulator gene tcdC has shown that the strains ribotype 1a toxinotype V had an allele, named A1, previously described in the literature. This allele, in addition to a 39-bp deletion, exhibited a non sence mutation with the consequent production of a truncated non-functional protein. This molecular framework suggests that these strains have to be considered hyper producers of the main toxins A and B. As expected, these ribotype 1a toxinotype V strains turned out in our hands to be highly resistant to fluoroquinolones. On the contrary, a greater variability was observed with the strains that showed ribotypes different from 1. In fact, 5 new allelic sequences and three proteins have been found, that had not described yet in the literature. Interestingly, the strain ribotype 16 toxinotype XXIV presented mutations that were unusual for this toxinotype, which normally has the same PaLoc of the reference strain VPI 10463. Moreover, worthy of note has been to find among the strains belonging to the ribotype 2 a new toxinotype, not yet described in the literature. Our study clearly highlights the importance of the molecular investigations in the attempt to monitor carefully the circulation of C. difficile strains in a hospital environment and particularly, of those hyper virulent strains, such as the Nap7/Ribotipo078 toxinotype V ones, which appeared in our hospital during the 2002-2003 period and were proven to be responsible for a severe nosocomial outbreak

Preliminary molecular analysis of Clostridium difficile isolates from healthy horses in northern Italy

Clostridium difficile, associated with a wide spectrum of diseases in humans, as well as in several animal species, is an important cause of colitis in adult horses and foals. The aim of this study was to investigate by toxin gene profile and PCR-ribotyping the molecular characteristics of 14 C. difficile strains isolated from 42 faeces of healthy horses. Both toxin genes, tcdA and tcdB, were present in only 1 isolate (7.1%). Six isolates (42.9%) demonstrated tcdA-. /tcdB+ genotype, and seven isolates (50.0%) were tcdA-/. tcdB-. All strains were binary toxin genes negative (cdtA-/. cdtB-). The PCR-positive strains, except for the tcdA+/. tcdB+ isolate, tested negative for, in vitro, A and/or B toxins production by EIA. Eleven distinct ribotypes were observed.In conclusion, C. difficile can be present in the normal intestinal flora of healthy adult horses, in addition to foals. These animals could therefore play an important role as potential reservoirs of toxigenic strains

Respiratory Tract Infections and Laboratory Diagnostic Methods: A Review with A Focus on Syndromic Panel-Based Assays

Respiratory tract infections (RTIs) are the focus of developments in public health, given their widespread distribution and the high morbidity and mortality rates reported worldwide. The clinical spectrum ranges from asymptomatic or mild infection to severe or fatal disease. Rapidity is required in diagnostics to provide adequate and prompt management of patients. The current algorithm for the laboratory diagnosis of RTIs relies on multiple approaches including gold-standard conventional methods, among which the traditional culture is the most used, and innovative ones such as molecular methods, mostly used to detect viruses and atypical bacteria. The implementation of molecular methods with syndromic panels has the potential to be a powerful decision-making tool for patient management despite requiring appropriate use of the test in different patient populations. Their use radically reduces time-to-results and increases the detection of clinically relevant pathogens compared to conventional methods. Moreover, if implemented wisely and interpreted cautiously, syndromic panels can improve antimicrobial use and patient outcomes, and optimize laboratory workflow. In this review, a narrative overview of the main etiological, clinical, and epidemiological features of RTI is reported, focusing on the laboratory diagnosis and the potentialities of syndromic panels

A rare case of SARS-CoV-2 and influenza A virus super-infection

We report the first Italian case of SARS-CoV-2 and influenza A virus super-infection. Laboratory diagnosis revealed the presence of both agents' RNA specific sequences by molecular methods and infectious influenza A virus by cell culture methods

Prevalence of Intestinal Parasitoses in a Non-Endemic Setting during a 10-Year Period (2011–2020): A Focus on Dientamoeba fragilis

Dientamoeba fragilis is a cosmopolitan and neglected protozoan. Although little is known concerning its pathogenicity and its true prevalence worldwide, its role as enteric pathogen is emerging, as the occurrence of dientamoebiasis has increased also in industrialised countries. This study investigated the occurrence and prevalence of intestinal parasites, focusing on D. fragilis in a 10-year period (2011–2020) in a single tertiary-care hospital located in Northern Italy. A statistical evaluation of the correlation between dientamoebiasis and specific signs other than gastrointestinal-related ones was performed. The laboratory diagnosis was performed on 16,275 cases of suspected intestinal parasitoses. Intestinal parasites were detected in 3254 cases, 606 of which were associated to D. fragilis, which represented 18.6% (606/3254) of all the intestinal parasitoses with a 3.7% (606/16,275) prevalence and an increasing trend during the last five years (2011–2015: 2.8% vs. 2016–2020: 4.8%). D. fragilis was commonly detected in foreigners, especially those from developing countries, as well as in children; prevalence was equal in males and females. With regard to the clinical aspect, the only putative sign statistically related to dientamoebiasis was anal pruritus. Despite the controversial epidemiological knowledges on dientamoebiasis, the prevalence of D. fragilis found in this study highlights the need to consider this parasite in any differential diagnosis of gastrointestinal disease

MALDI-TOF mass spectrometry as innovative tool applied to parasites identification

increasingly utilized as a rapid technique to identify microorganisms by their molecular fingerprint and/or by biomarker detection and it represents a first-line method for the accurate routinely identification of bacteria and fungi; its application in parasitology is on the contrary very limited. In this study MALDI-TOF MS was used to identify Trichomonas vaginalis, Dientamoeba fragilis and to differentiate Entamoeba histolytica and E. dispar. Introduction Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is Materials and Methods In this study, an aliquot of one cultured reference strain for each parasite were submitted to formic acid/acetonitril protein extraction and to MALDI-TOF MS analysis. The spectrum obtained for T. vaginalis was suplemented in the Bruker Daltonics database (Bruker Daltonics, Germany) and a new identification method was created by modifying the range setting for the MALDI-TOF MS analysis in order to exclude the overlapping of peaks derived from the culture media used in this study. The spectra obtained for E. histolytica, E. dispar and D. fragilis were analysed and subsequently imported into the ClinProTools software version 2.2. (Bruker Daltonics) to perform a statistical analysis in order to check the presence of specific peaks for each parasite. To verify the reliability of the system 21 T. vaginalis, 6 E. histolytica, 8 E. dispar and 13 D. fragilis clinical isolates, respectively, were used. Results After implementation and modification of the paramenters' setting, the protein spectra of T. vaginalis clinical isolates were correctly identified. Five discriminating peaks between E. histolytica (2 peaks) and E. dispar (3 peaks) and 6 discriminating peaks for D. fragilis were found, respectively. When the spectra belonging to the clinical isolates were analysed, all the identifications matched those obtained by a specific Real-time PCR, except for one E. histolytica strain. Dicussion and Conclusions Although the massive number of entries in the available commercial database, the absence of reference spectra of parasites does not allow their identification. Our study demonstrated that MALDI-TOF MS can be applied to the identification of parasites by using two different approaches: i) the comparison of the obtained spectra with a database that can be suitably implemented also modifying the parameters setting, ii) the detection of specific protein biomarkers. For the unique discordant result regarding a E. histolytica strain isolated from a patient with dysentery also positive for E. histolytica antibodies, the presence of amino acid/posttranslational differences as compared to the reference strain could be hypothesized