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

Pleural tuberculosis: medical thoracoscopy greatly increases the diagnostic accuracy

Our objective was to evaluate the efficacy of a standardised work-up in the diagnosis of pleural tuberculosis (TB) that included fibreoptic bronchoscopy and medical thoracoscopy. A consecutive series of 52 pleural TB patients observed during the period 2001-2015 was evaluated retrospectively. 20 females, mean (range) age 39.7 (18-74) years, and 32 males, mean (range) age 45.75 (21-83) years, were included (28 non-EU citizens (53.8%)). The diagnosis of TB infections was established by identification (using stains, culture or molecular tests) of Mycobacterium tuberculosis in the pleura, sputum and/or bronchial specimens, or by evidence of caseous granulomas on pleural biopsies. Patients with and without lung lesions were considered separately. The diagnostic yield of the microbiological tests on pleural fluid was 17.3% (nine out of 52 patients). Among the 18 patients with lung lesions, bronchial samples (washing, lavage or biopsy) were positive in 50% of cases (nine patients). Cultures of pleural biopsies were positive in 63% of cases (29 out of 46 patients); pleural histology was relevant in all patients. Without pleural biopsy, a diagnosis would have been reached in 15 out of 52 patients (28.6%) and in four of them only following culture at 30-40 days. An integrated diagnostic work-up that includes all the diagnostic methods of interventional pulmonology is required for a diagnosis of pleural TB. In the majority of patients, a diagnosis can be reached only with pleural biopsy

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

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

Intestinal spirochaetosis associated with hyperplastic and adenomatous colonic polyps: a case report.

Intestinal spirochaetosis is a human colonic infection due to Brachyspira aalborgi and Brachyspira pilosicoli causing various abdominal complaints. Although the presence of healthy epithelial cells was hypothesized to be essential for the adhesion of spirochaetes to the colonic mucosa, their adhesion to hyperplastic and adenomatous colonic polyps has been observed recently. We report a case of a woman with longstanding abdominal symptoms, in whom spirochaetes were found on the colonic mucosa surrounding an adenocarcinoma in the biopsies collected during eight years of follow-up. Spirochaetes were found attached to normal mucosa, to hyperplastic and to adenomatous polyps, but not to the epithelium of the carcinoma. The rectal biopsy collected during the last follow-up colonoscopy was subjected to histopathology and to a specific examination for brachyspires, demonstrating the presence of B. pilosicoli DNA. This report could stimulate microbiological investigations during the follow-up of colonic polyps in order to explain whether the persistence of abdominal symptoms in such patients could be caused by a colonic spirochaetosis susceptible to eradication by a targeted therapy

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