MALDI-TOF MS: A Reliable Tool in the Real Life of the Clinical Microbiology Laboratory.

Matrix-Assisted Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) in the last decade has revealed itself as a valid support in the workflow in the clinical microbiology laboratory for the identification of bacteria and fungi, demonstrating high reliability and effectiveness in this application. Its use has reduced, by 24 h, the time to obtain a microbiological diagnosis compared to conventional biochemical automatic systems. MALDI-TOF MS application to the detection of pathogens directly in clinical samples was proposed but requires a deeper investigation, whereas its application to positive blood cultures for the identification of microorganisms and the detection of antimicrobial resistance are now the most useful applications. Thanks to its rapidity, accuracy, and low price in reagents and consumables, MALDI-TOF MS has also been applied to different fields of clinical microbiology, such as the detection of antibiotic susceptibility/resistance biomarkers, the identification of aminoacidic sequences and the chemical structure of protein terminal groups, and as an emerging method in microbial typing. Some of these applications are waiting for an extensive evaluation before confirming a transfer to the routine. MALDI-TOF MS has not yet been used for the routine identification of parasites; nevertheless, studies have been reported in the last few years on its use in the identification of intestinal protozoa, Plasmodium falciparum, or ectoparasites. Innovative applications of MALDI-TOF MS to viruses' identification were also reported, seeking further studies before adapting this tool to the virus's diagnostic. This mini-review is focused on the MALDI-TOF MS application in the real life of the diagnostic microbiology laboratory.

The Laboratory Diagnosis of Malaria: A Focus on the Diagnostic Assays in Non-Endemic Areas.

Even if malaria is rare in Europe, it is a medical emergency and programs for its control should ensure both an early diagnosis and a prompt treatment within 24-48 h from the onset of the symptoms. The increasing number of imported malaria cases as well as the risk of the reintroduction of autochthonous cases encouraged laboratories in non-endemic countries to adopt diagnostic methods/algorithms. Microscopy remains the gold standard, but with limitations. Rapid diagnostic tests have greatly expanded the ability to diagnose malaria for rapid results due to simplicity and low cost, but they lack sensitivity and specificity. PCR-based assays provide more relevant information but need well-trained technicians. As reported in the World Health Organization Global Technical Strategy for Malaria 2016-2030, the development of point-of-care testing is important for the improvement of diagnosis with beneficial consequences for prompt/accurate treatment and for preventing the spread of the disease. Despite their limitations, diagnostic methods contribute to the decline of malaria mortality. Recently, evidence suggested that artificial intelligence could be utilized for assisting pathologists in malaria diagnosis.

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.

Detection of occult hepatitis B virus infection among subjects with isolated hepatitis B core antibodies: Results from a 3-year survey in an Italian tertiary-care hospital.

BACKGROUND: Hepatitis B virus (HBV) infection causes hepatitis, liver cirrhosis, hepatocellular carcinoma, and death. This study examines the subjects with isolated anti-HBV core antigen antibody (anti-HBcAg), a pattern characterized by the persistent HBV carriage in the absence of HBV surface antigen (HBsAg) and anti-HBsAg antibody. METHODS: Based on medical orders, from 2017 to 2019, serological and molecular assays were performed on serum/plasma samples of 33,048 subjects (71.4% Italians, 28.6% foreigners), who referred to the Virology Unit of the University-Hospital of Parma (Northern Italy) for the laboratory diagnosis of HBV infection. RESULTS: The seroprevalence was 4.6% for HBsAg and 11% for anti-HBcAg. The occurrence of the isolated anti-HBcAg status was 3.1%, with higher frequency in males than in females (66.3% vs. 33.7%, P < 0.0001), in Italians than in foreigners (54.8% vs. 45.2%, P < 0.001), and in outpatients than in inpatients (57.4% vs. 42.6%, P < 0.0001). Foreigners with isolated anti-HBcAg came mostly from Africa (67.9%) and Eastern Europe (26.2%). Among subjects with isolated anti-HBcAg, 14.8% had occult HBV infection, 26.3% hepatitis C virus co-infection, 2% human immunodeficiency virus co-infection, and 3.3% both of these latter co-infections. CONCLUSIONS: The anti-HBcAg assay accurately evaluates the HBV exposure; subjects with isolated anti-HBcAg antibody should be further analysed for HBV DNA. The HBV infection prevalence in Italy is increasing, due to growing migratory flows from endemic areas.

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.

Detection of potential enteric pathogens in children with severe acute gastroenteritis using the filmarray: Results from a three - years hospital-based survey in Northern Italy.

Acute gastroenteritis (AGE) are leading causes of morbidity and mortality in children. Therefore, rapid pathogens identification is needed. The AGE aetiology was investigated from 2018 to 2020 in 2,066 children in Parma (Italy) by FilmArray Gastrointestinal Panel and Enterovirus-targeting RT-PCR. Pathogens were detected in 1,162 (56.2%) stool samples from as many children; 798 (68.7%) were single and 364 (31.3%) mixed infections (68.7% vs 31.3%, P < 0.0001). Children aged 0-5 years showed the highest infection incidence (66.1%). The most frequent pathogens were Enteropathogenic Escherichia coli (EPEC; 19.14%), Clostridioides difficile (10.42%), Norovirus (10.36%), Enterovirus (9.44%), and Campylobacter (9.21%). EPEC, Campylobacter, enteroaggregative E. coli, Norovirus, and Rotavirus showed seasonality. The incidence of pathogens detected decreased between 2018 and 2020 (42.7% vs 20.8%, P < 0.0001), seemingly for the preventive measures imposed by the severe acute respiratory syndrome coronavirus-2 pandemic. A putative aetiology in half the children examined and an estimate of enteric pathogens epidemiology were assessed.

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.

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.

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.

Host-cell dependent role of phosphorylated keratin 8 during influenza A/NWS/33 virus (H1N1) infection in mammalian cells.

In this study, we investigated the involvement of keratin 8 during human influenza A/NWS/33 virus (H1N1) infection in semi-permissive rhesus monkey-kidney (LLC-MK2) and permissive human type II alveolar epithelial (A549) cells. In A549 cells, keratin 8 showed major expression and phosphorylation levels. Influenza A/NWS/33 virus was able to subvert keratin 8 structural organization at late stages of infection in both cell models, promoting keratin 8 phosphorylation in A549 cells at early phases of infection. Accordingly, partial colocalizations of the viral nucleoprotein with keratin 8 and its phosphorylated form were assessed by confocal microscopy at early stages of infection in A549 cells. The employment of chemical activators of phosphorylation resulted in structural changes as well as increased phosphorylation of keratin 8 in both cell models, favoring the influenza A/NWS/33 virus's replicative efficiency in A549 but not in LLC-MK2 cells. In A549 and human larynx epidermoid carcinoma (HEp-2) cells inoculated with respiratory secretions from pediatric patients positive for, respectively, influenza A virus or respiratory syncytial virus, the keratin 8 phosphorylation level had increased only in the case of influenza A virus infection. The results obtained suggest that in A549 cells the influenza virus is able to induce keratin 8 phosphorylation thereby enhancing its replicative efficiency.

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.

Impact of Human Cytomegalovirus and Human Herpesvirus 6 Infection on the Expression of Factors Associated with Cell Fibrosis and Apoptosis: Clues for Implication in Systemic Sclerosis Development.

Systemic sclerosis (SSc) is a severe autoimmune disorder characterized by vasculopathy and multi-organ fibrosis; its etiology and pathogenesis are still largely unknown. Herpesvirus infections, particularly by human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6), have been suggested among triggers of the disease based on virological and immunological observations. However, the direct impact of HCMV and/or HHV-6 infection on cell fibrosis and apoptosis at the cell microenvironment level has not yet been clarified. Thus, this study aimed to investigate the effects of HCMV and HHV-6 infection on the induction of pro-fibrosis or pro-apoptosis conditions in primary human dermal fibroblasts, one of the relevant SSc target cells. The analysis, performed by microarray in in vitro HCMV- or HHV-6-infected vs. uninfected cells, using specific panels for the detection of the main cellular factors associated with fibrosis or apoptosis, showed that both viruses significantly modified the expression of at least 30 pro-fibrotic and 20 pro-apoptotic factors. Notably, several recognized pro-fibrotic factors were highly induced, and most of them were reported to be involved in vivo in the multifactorial and multistep pathogenic process of SSc, thus suggesting a potential role of both HCMV and HHV-6.

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.

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.

Epidemiology of human respiratory viruses in children with acute respiratory tract infection in a 3-year hospital-based survey in Northern Italy.

Acute respiratory tract infections (ARTIs) are among the leading causes of morbidity and mortality in children. The viral etiology of ARTIs was investigated over 3 years (October 2012-September 2015) in 2575 children in Parma, Italy, using indirect immunofluorescent staining of respiratory samples for viral antigens, cell culture, and molecular assays. Respiratory viruses were detected in 1299 cases (50.44%); 1037 (79.83%) were single infections and 262 (20.17%) mixed infections. The highest infection incidence was in children aged >6 months to ≤3 years (57.36%). Human respiratory syncytial virus (27.12%) and human adenovirus (23.58%) were the most common viruses identified. The virus detection rate decreased significantly between the first and third epidemic season (53.9% vs. 43.05%, P < 0.0001). The simultaneous use of different diagnostic tools allowed us to identify a putative viral etiology in half the children examined and to provide an estimate of the epidemiology and seasonality of respiratory viruses associated with ARTIs.

A Paradigmatic Interplay between Human Cytomegalovirus and Host Immune System: Possible Involvement of Viral Antigen-Driven CD8+ T Cell Responses in Systemic Sclerosis.

Human cytomegalovirus (HCMV) is a highly prevalent opportunistic agent in the world population, which persists as a latent virus after a primary infection. Besides the well-established role of this agent causing severe diseases in immunocompromised individuals, more recently, HCMV has been evoked as a possible factor contributing to the pathogenesis of autoimmune diseases such as systemic sclerosis (SSc). The interplay between HCMV and immune surveillance is supposed to become unbalanced in SSc patients with expanded anti-HCMV immune responses, which are likely involved in the exacerbation of inflammatory processes. In this study, blood samples from a cohort of SSc patients vs. healthy subjects were tested for anti-HCMV immune responses (IgM, IgG antibodies, and T cells to peptide pools spanning the most immunogenic HCMV proteins). Statistically significant increase of HCMV-specific CD8+ T cell responses in SSc patients vs. healthy subjects was observed. Moreover, significantly greater HCMV-specific CD8+ T cell responses were found in SSc patients with a longer disease duration and those with higher modified Rodnan skin scores. Given the known importance of T cells in the development of SSc and that this virus may contribute to chronic inflammatory diseases, these data support a relevant role of HCMV-specific CD8+ T cell responses in SSc pathogenesis.