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.

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.

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.

Rapid Identification of Escherichia coli Colistin-Resistant Strains by MALDI-TOF Mass Spectrometry.

Colistin resistance is one of the major threats for global public health, requiring reliable and rapid susceptibility testing methods. The aim of this study was the evaluation of a MALDI-TOF mass spectrometry (MS) peak-based assay to distinguish colistin resistant (colR) from susceptible (colS) Escherichia coli strains. To this end, a classifying algorithm model (CAM) was developed, testing three different algorithms: Genetic Algorithm (GA), Supervised Neural Network (SNN) and Quick Classifier (QC). Among them, the SNN- and GA-based CAMs showed the best performances: recognition capability (RC) of 100% each one, and cross validation (CV) of 97.62% and 100%, respectively. Even if both algorithms shared similar RC and CV values, the SNN-based CAM was the best performing one, correctly identifying 67/71 (94.4%) of the E. coli strains collected: in point of fact, it correctly identified the greatest number of colS strains (42/43; 97.7%), despite its lower ability in identifying the colR strains (15/18; 83.3%). In conclusion, although broth microdilution remains the gold standard method for testing colistin susceptibility, the CAM represents a useful tool to rapidly screen colR and colS strains in clinical practice.

Malaria Diagnosis in Non-Endemic Settings: The European Experience in the Last 22 Years.

Accurate, prompt, and reliable tools for the diagnosis of malaria are crucial for tracking the successes or drawbacks of control and elimination efforts, and for future programs aimed at global malaria eradication. Although microscopy remains the gold standard method, the number of imported malaria cases and the risk of reappearance of autochthonous cases stimulated several laboratories located in European countries to evaluate methods and algorithms suited to non-endemic settings, where skilled microscopists are not always available. In this review, an overview of the field evaluation and a comparison of the methods used for the diagnosis of malaria by European laboratories is reported, showing that the development of numerous innovations is continuous. In particular, the combination of rapid diagnostic tests and molecular assays with microscopy represents a reliable system for the early diagnosis of malaria in non-endemic settings.

Rapid Classification of Clostridioides difficile Strains Using MALDI-TOF MS Peak-Based Assay in Comparison with PCR-Ribotyping.

Typing methods are needed for epidemiological tracking of new emerging and hypervirulent strains because of the growing incidence, severity and mortality of Clostridioides difficile infections (CDI). The aim of this study was the evaluation of a typing Matrix-Assisted Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS (T-MALDI)) method for the rapid classification of the circulating C. difficile strains in comparison with polymerase chain reaction (PCR)-ribotyping results. Among 95 C. difficile strains, 10 ribotypes (PR1-PR10) were identified by PCR-ribotyping. In particular, 93.7% of the isolates (89/95) were grouped in five ribotypes (PR1-PR5). For T-MALDI, two classifying algorithm models (CAM) were tested: the first CAM involved all 10 ribotypes whereas the second one only the PR1-PR5 ribotypes. Better performance was obtained using the second CAM: recognition capability of 100%, cross-validation of 96.6% and agreement of 98.4% (60 correctly typed strains, limited to PR1-PR5 classification, out of 61 examined strains) with PCR-ribotyping results. T-MALDI seems to represent an alternative to PCR-ribotyping in terms of reproducibility, set up time and costs, as well as a useful tool in epidemiological investigation for the detection of C. difficile clusters (either among CAM included ribotypes or out-of-CAM ribotypes) involved in outbreaks.

Active surveillance for carbapenemase-producing 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 'at-risk' 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.

Detection of SARS-CoV-2 and Other Infectious Agents in Lower Respiratory Tract Samples Belonging to Patients Admitted to Intensive Care Units of a Tertiary-Care Hospital, Located in an Epidemic Area, during the Italian Lockdown.

The aim of this study was the detection of infectious agents from lower respiratory tract (LRT) samples in order to describe their distribution in patients with severe acute respiratory failure and hospitalized in intensive care units (ICU) in an Italian tertiary-care hospital. LRT samples from 154 patients admitted to ICU from 27 February to 10 May 2020 were prospectively examined for respiratory viruses, including SARS-CoV-2, bacteria and/or fungi. SARS-CoV-2 was revealed in 90 patients (58.4%, 72 males, mean age 65 years). No significant difference was observed between SARS-CoV-2 positives and SARS-CoV-2 negatives with regard to sex, age and bacterial and/or fungal infections. Nonetheless, fungi were more frequently detected among SARS-CoV-2 positives (44/54, 81.4%, p = 0.0053). Candida albicans was the overall most frequently isolated agent, followed by Enterococcus faecalis among SARS-CoV-2 positives and Staphylococcus aureus among SARS-CoV-2 negatives. Overall mortality rate was 40.4%, accounting for 53 deaths: 37 among SARS-CoV-2 positives (mean age 69 years) and 16 among SARS-CoV-2 negatives (mean age 63 years). This study highlights the different patterns of infectious agents between the two patient categories: fungi were prevalently involved among SARS-CoV-2-positive patients and bacteria among the SARS-CoV-2-negative patients. The different therapies and the length of the ICU stay could have influenced these different patterns of infectious agents.

Human respiratory viruses, including SARS-CoV-2, circulating in the winter season 2019-2020 in Parma, Northern Italy.

OBJECTIVES: The aim of this study was to determine the prevalence of respiratory virus infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), during the winter period December 2019 to March 2020, via a tertiary care hospital-based survey in Parma, Northern Italy. METHODS: A total of 906 biological samples from the respiratory tract were analysed by both conventional assays (including culture) and molecular assays targeting nucleic acids of SARS-CoV-2 and other respiratory viruses. RESULTS: Overall, 474 samples (52.3%) were positive for at least one virus, with a total of 583 viruses detected. Single infections were detected in 380 (80.2%) samples and mixed infections were detected in 94 (19.8%). Respiratory syncytial virus (138/583, 23.7%) and rhinovirus (130/583, 22.3%) were the most commonly identified viruses, followed by SARS-CoV-2 (82/583, 14.1%). Respiratory syncytial virus predominated until February, with 129 detections; it then decreased drastically in March to only nine detections. SARS-CoV-2 was absent in the study area until February 26, 2020 and then reached 82 detections in just over a month. SARS-CoV-2 was found in mixed infections in only three cases, all observed in children younger than 1 year old. CONCLUSIONS: This study showed a completely different trend between SARS-CoV-2 and the 'common' respiratory viruses: the common viruses mostly affected children, without any distinction according to sex, while SARS-CoV-2 mostly affected adult males.

SARS-CoV-2 infection diagnosed only by cell culture isolation before the local outbreak in an Italian seven-week-old suckling baby.

SARS-CoV-2 emerged in China in December 2019 and has now been declared a pandemic by the World Health Organization. This paper described the case of a 7-week-old suckling baby from Italy who was SARS-CoV-2-positive only by the cell culture method, with no clinical suspicion of and/or risk factors for SARS-CoV-2 infection. The baby was referred to hospital, with signs and symptoms of upper respiratory tract infection, before the virus had spread to the province. Nasal and pharyngeal swabs and a nasopharyngeal aspirate were used for conventional and molecular diagnostic assays not including the SARS-CoV-2 virus. Bacteria referred to the resident population were revealed in nasal and pharyngeal swabs. No viruses were detected using both immunofluorescence assay and nucleic acid amplification assays in the nasopharyngeal aspirate. The baby was discharged in good condition after 3 days of hospitalisation. Later, a cytopathic effect on the cell monolayers currently used for respiratory viruses was observed and the viral particles were identified as Coronaviridae by transmission electron microscopy. SARS-CoV-2 was identified by RT-PCR performed both on cell culture and on the stored aliquot of the original sample. The virus isolate was named SARS-Cov-2/human/Parma/1/2020. Cell culture still remains the only reference diagnostic method also for emerging viruses, allowing it to reveal cytopathogenic viruses and demonstrate their infectivity.

An unusual cause of Steven-Johnson Syndrome.

SJS is a rare mucocutaneous syndrome characterized by skin and mucous detachment. The main etiological factors are drugs and infections; sometimes the cause remains unknown. In the prodromal phase we observed non-specific symptoms, followed by mucocutaneous manifestation. Due to risk of complications and mortality a multidisciplinary approach is needed.  We present a case of a girl with an atypical presentation of SJS related to Enterovirus.

Rapid microbial identification and phenotypic antimicrobial susceptibility testing directly from positive blood cultures: a new platform compared to routine laboratory methods.

The Accelerate Pheno™ System (APS), a new platform that combines rapid identification (ID) of bacteria and yeasts and phenotypic antimicrobial susceptibility testing (AST) in a single assay, has been evaluated directly from positive blood cultures in comparison to routine laboratory methods. The APS ID results showed an overall sensitivity and specificity of 92.6% and 99.6%, respectively. With regard to AST results, 31 discrepancies (8 single errors and 23 combined errors) were observed, including 13 major errors (3.3%) and 18 minor errors (4.6%) mainly involving Pseudomonas aeruginosa. No very major error was observed. The APS ID results were obtained in 1.5 h and the AST results were available in 7 h, on average 34.1 h before routine laboratory methods. This reduction in AST time-to-result represents one of the main advantages of this technology, reducing the time to provide to the physician the microbiological report.

Contribution of the FilmArray® Gastrointestinal Panel in the laboratory diagnosis of gastroenteritis in a cohort of children: a two-year prospective study.

This study represents a 2-year picture of the epidemiology of enteric pathogens in children suffering from gastroenteritis using the FilmArray® Gastrointestinal Panel (FA-GP), a multiplex molecular assay that allows to simultaneously detect a large panel of pathogens independently of the etiological suspicion and to evaluate its potential contribution to the diagnosis compared to the conventional methods. A total of 1716 stool samples, collected from children with clinical suspicion of bacterial and/or viral gastroenteritis attending the University Hospital of Parma, was submitted to the FA-GP and, when an adequate aliquot was available, to electron microscopy (n = 1163) for virus detection and to an enterovirus-targeting real-time PCR (n = 1703). Specimens with positive results for Salmonella, Yersinia enterocolitica, Vibrio, diarrheagenic Escherichia coli/Shigella, Campylobacter, Plesiomonas shigelloides and/or parasites by the FA-GP were also submitted to conventional diagnostic methods. The FA-GP gave positive results in 958 (55.8%) cases, 64.8% from inpatients: 647 (67.5%) contained a single agent and 311 (32.5%) multiple agents, for a total of 1374 pathogens. Enteropathogenic E. coli, rotavirus, norovirus, toxigenic Clostridioides difficile, and sapovirus were the most commonly detected pathogens. A total of 812 additional agents (344 of which as single pathogen) was detected by the FA-GP and not included in the clinical suspicion. The overall recovery rate of the conventional methods from stools that resulted positive by the FA-GP was 38.6% for bacteria, 50% and 84.2% for Giardia intestinalis and Cryptosporidium, respectively, and ranged from 3.7% to 64.6% for viruses, if excluding all electron microscopy-negative astroviruses. Enterovirus, an agent not targeted by the FA-GP, was revealed in 9.6% (164/1703) of the examined samples, and in 52 cases it was the only agent detected. The results of this study allowed to extend the range of detectable pathogens independently of the clinical suspicion, to detect co-infections in almost one third of children positive for at least one agent and to show that conventional methods would have missed more than half of the enteric agents detected by the FA-GP.

High prevalence of malaria in a non-endemic setting: comparison of diagnostic tools and patient outcome during a four-year survey (2013-2017).

BACKGROUND: Malaria is no longer endemic in Italy since 1970 when the World Health Organization declared Italy malaria-free, but it is now the most commonly imported disease. The aim of the study was to analyse the trend of imported malaria cases in Parma, Italy, during January 2013-June 2017, reporting also the treatment and the outcome of cases, exploring the comparison of the three diagnostic tests used for malaria diagnosis: microscopy, immunochromatographic assay (ICT) (BinaxNOW®) and Real-time PCR assays detecting Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, and Plasmodium knowlesi. RESULTS: Of the 288 patients with suspected malaria, 87 were positive by microscopy: 73 P. falciparum, 2 P. vivax, 8 P. ovale, 1 P. vivax/P. ovale, 1 P. malariae and 2 Plasmodium sp. All samples were positive by ICT except 6. Plasmodial DNA was revealed in the 87 cases and in 2 additional cases showing P. falciparum-specific bands by ICT, as follows: 75 P. falciparum, 2 P. vivax, 6 P. ovale curtisi, 3 P. ovale wallikeri, 1 P. malariae, and 2 mixed infections. 72 patients were foreigners and 17 Italians travelling for tourism or business. The majority of these patients presented with fever at blood collection and did not have chemoprophylaxis. No fatal cases were observed and the drug mostly used was quinine observing a negative blood smear or a parasitaemia < 0.001% after 48-72 h' therapy. CONCLUSIONS: The study shows an update and a thorough analysis of imported malaria cases in the area of Parma during 4.5 years from the point of view of the total case management, clinical and diagnostic. The prevalence of malaria in such area in the considered period was especially due to immigrants mostly from Africa. Molecular methods were more sensitive and specific than microscopy and ICT, both detecting additional cases of P. falciparum malaria missed by microscopy and correctly identifying the Plasmodium species of medical interest. The data reported in this study may stimulate the clinicians in non-endemic areas to suspect malaria also in cases, where the most typical symptoms are absent, and the parasitologists to confirm the results of microscopy, remaining the reference method, with molecular methods to avoid misdiagnosis.

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.

MALDI-TOF MS as a new tool for the identification of Dientamoeba fragilis.

BACKGROUND: In this study for the first time, a Dientamoeba fragilis protein profile by MALDI-TOF MS was created in order to identify specific markers for the application of this technology in the laboratory diagnosis of dientamoebiasis. In particular, one D. fragilis reference strain was used to create a reference spectrum and 14 clinical isolates to verify the reliability of the obtained results. RESULTS: While 15 peaks were found to be discriminating between the reference strain and the culture medium used, six peaks, observed in all the 14 strains tested, were considered as markers able to identify D. fragilis. CONCLUSIONS: In our hands, MALDI-TOF MS technology was demonstrated as a useful tool to be used in association with or in replacement of the real-time PCR assay for the identification of D. fragilis used in our laboratory on xenic cultures, due to its accuracy, rapidity and low cost.

Evaluation of a modified meropenem hydrolysis assay on a large cohort of KPC and VIM carbapenemase-producing Enterobacteriaceae.

Carbapenem-resistant Enterobacteriaceae (CRE) have spread globally and represent a serious and growing threat to public health. The introduction of rapid and sensitive methods for the detection of carbapenemase-producing bacteria is of increasing importance. The carbapenemase production can be detected using non-molecular methods (such as the modified Hodge test, the synergy test, the Carba NP test and the antibiotic hydrolysis assays) and DNA-based methods. In this study, we propose a modified version of a previously described meropenem hydrolysis assay (MHA) by MALDI-TOF MS for the phenotypic detection in 2h of carbapenemase-producing Enterobacteriaceae. The MHA was successfully applied to detect carbapenemase activity in 981 well-characterized Enterobacteriaceae strains producing KPC or VIM carbapenemases, and in 146 carbapenem fully susceptible strains. This assay, applied also to NDM and OXA-48-producing strains and to CRE with resistance mechanisms other than carbapenemase production, has proved to be able to distinguish between carbapenemase-producing and -nonproducing Enterobacteriaceae. As already stated and as observed in our hands, MHA by MALDI-TOF MS analysis is independent from the type of carbapenemases involved, it is faster and easier to perform/interpret than culture-based methods. On the other hand, it cannot detect other carbapenem resistance mechanisms, such as porin alterations and efflux mechanisms.

Identification of different respiratory viruses, after a cell culture step, by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS).

In this study matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), a reliable identification method for the diagnosis of bacterial and fungal infections, is presented as an innovative tool to investigate the protein profile of cell cultures infected by the most common viruses causing respiratory tract infections in humans. MALDI-TOF MS was applied to the identification of influenza A and B viruses, adenovirus C species, parainfluenza virus types 1, 2 and 3, respiratory syncytial virus, echovirus, cytomegalovirus and metapneumovirus. In this study MALDI-TOF MS was proposed as a model to be applied to the identification of cultivable respiratory viruses using cell culture as a viral proteins enrichment method to the proteome profiling of virus infected and uninfected cell cultures. The reference virus strains and 58 viruses identified from respiratory samples of subjects with respiratory diseases positive for one of the above mentioned viral agents by cell culture were used for the in vitro infection of suitable cell cultures. The isolated viral particles, concentrated by ultracentrifugation, were used for subsequent protein extraction and their spectra profiles were generated by MALDI-TOF MS analysis. The newly created library allowed us to discriminate between uninfected and respiratory virus infected cell cultures.

MALDI-TOF mass spectrometry as a potential tool for Trichomonas vaginalis identification.

BACKGROUND: Trichomonas vaginalis is a flagellated protozoan causing trichomoniasis, a sexually transmitted human infection, with around 276.4 million new cases estimated by World Health Organization. Culture is the gold standard method for the diagnosis of T. vaginalis infection. Recently, immunochromatographic assays as well as PCR assays for the detection of T. vaginalis antigen or DNA, respectively, have been also available. Although the well-known genome sequence of T. vaginalis has made possible the application of proteomic studies, few data are available about the overall proteomic expression profiling of T. vaginalis. The aim of this study was to investigate the potential application of MALDI-TOF MS as a new tool for the identification of T. vaginalis. METHODS: Twenty-one isolates were analysed by MALDI-TOF MS after the creation of a Main Spectrum Profile (MSP) from a T. vaginalis reference strain (G3) and its subsequent supplementation in the Bruker Daltonics database, not including any profile of protozoa. This was achieved after the development of a new identification method created by modifying the range setting (6-10 kDa) for the MALDI-TOF MS analysis in order to exclude the overlapping of peaks derived from the culture media used in this study. RESULTS: Two MSP reference spectra were created in 2 different range: 3-15 kDa (standard range setting) and 6-10 kDa (new range setting). Both MSP spectra were deposited in the MALDI BioTyper database for further identification of additional T. vaginalis strains. All the 21 strains analysed in this study were correctly identified by using the new identification method. CONCLUSIONS: In this study it was demonstrated that changes in the MALDI-TOF MS standard parameters usually used to identify bacteria and fungi allowed the identification of the protozoan T. vaginalis. This study shows the usefulness of MALDI-TOF MS in the reliable identification of microorganism grown on complex liquid media such as the protozoan T. vaginalis, on the basis of the proteic profile and not on the basis of single markers, by using a "new range setting" different from that developed for bacteria and fungi.