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.

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.

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.

Diagnostic performances of antigen detection compared to conventional and nucleic acid detection of Entamoeba histolytica in a non-endemic setting.

This study evaluated the immunochromatographic (IC) assay "TECHLAB(®) E. HISTOLYTICA QUIK CHEK™" analysing 36 faecal samples and 7 cultured strains. This assay was compared to the methods performed in our laboratory for the diagnosis of amoebiasis. The IC assay revealed a detection limit of 103 trophozoites/g faeces and no cross-reactivity with other parasites and failed to detect E. histolytica antigen in frozen faeces. In our laboratory located in a non-endemic setting this assay could not replace the methods currently used for the diagnosis of amoebiasis.

Identification of Dermatophyte species after implementation of the in-house MALDI-TOF MS database.

Despite that matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool in the clinical microbiology setting, few studies have till now focused on MALDI-TOF MS-based identification of dermatophytes. In this study, we analyze dermatophytes strains isolated from clinical samples by MALDI-TOF MS to supplement the reference database available in our laboratory. Twenty four dermatophytes (13 reference strains and 11 field isolated strains), identified by both conventional and molecular standard procedures, were analyzed by MALDI-TOF MS, and the spectra obtained were used to supplement the available database, limited to a few species. To verify the robustness of the implemented database, 64 clinical isolates other than those used for the implementation were identified by MALDI-TOF MS. The implementation allowed the identification of the species not included in the original database, reinforced the identification of the species already present and correctly identified those within the Trichophyton mentagrophytes complex previously classified as Trichophyton. tonsurans by MALDI-TOF MS. The dendrogram obtained by analyzing the proteic profiles of the different species of dermatophytes reflected their taxonomy, showing moreover, in some cases, a different clusterization between the spectra already present in the database and those newly added. In this study, MALDI-TOF MS proved to be a useful tool suitable for the identification of dermatophytes for diagnostic purpose.

Accurate identification of the six human Plasmodium spp. causing imported malaria, including Plasmodium ovale wallikeri and Plasmodium knowlesi.

BACKGROUND: Accurate identification of Plasmodium infections in non-endemic countries is of critical importance with regard to the administration of a targeted therapy having a positive impact on patient health and management and allowing the prevention of the risk of re-introduction of endemic malaria in such countries. Malaria is no longer endemic in Italy where it is the most commonly imported disease, with one of the highest rates of imported malaria among European non-endemic countries including France, the UK and Germany, and with a prevalence of 24.3% at the University Hospital of Parma. Molecular methods showed high sensitivity and specificity and changed the epidemiology of imported malaria in several non-endemic countries, highlighted a higher prevalence of Plasmodium ovale, Plasmodium vivax and Plasmodium malariae underestimated by microscopy and, not least, brought to light both the existence of two species of P. ovale (Plasmodium ovale curtisi and Plasmodium ovale wallikeri) and the infection in humans by Plasmodium knowlesi, otherwise not detectable by microscopy. METHODS: In this retrospective study an evaluation of two real-time PCR assays able to identify P. ovale wallikeri, distinguishing it from P. ovale curtisi, and to detect P. knowlesi, respectively, was performed applying them on a subset of 398 blood samples belonging to patients with the clinical suspicion of malaria. RESULTS: These assays revealed an excellent analytical sensitivity and no cross-reactivity versus other Plasmodium spp. infecting humans, suggesting their usefulness for an accurate and complete diagnosis of imported malaria. Among the 128 patients with malaria, eight P. ovale curtisi and four P. ovale wallikeri infections were detected, while no cases of P. knowlesi infection were observed. DISCUSSION AND CONCLUSIONS: Real-time PCR assays specific for P. ovale wallikeri and P. knowlesi were included in the panel currently used in the University Hospital of Parma for the diagnosis of imported malaria, accomplishing the goal of adhering to the recommendations of the World Health Organization to countries that are malaria-free to include the improvement of the early diagnosis of all cases of imported malaria.

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.

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.

An 8-year survey on the occurrence of imported malaria in a nonendemic area by microscopy and molecular assays.

Our study aimed to describe the occurrence of imported malaria in a nonendemic area (Parma, Italy) during the period 2000 to 2007, comparing the data obtained by microscopy and molecular assays targeting plasmodial 18S subunit rRNA gene. The prevalence of imported malaria in Parma was 21.8% by microscopy and 22.7% by polymerase chain reaction (PCR). Plasmodium falciparum accounted for 81.1% of the cases, followed by Plasmodium ovale (8.8%), Plasmodium vivax (3.8%), and Plasmodium malariae (1.9%). Mixed infections accounted for 4.4% of the cases. In this study, PCRs proved to be more sensitive and specific than microscopy and changed the picture of malaria epidemiology in Parma, detecting additional cases of malaria undiagnosed by microscopy and allowing speciation of plasmodia in cases misidentified by microscopy. Generally, imported malaria cases reflect the number of immigrants who visit their native countries, in particular, West Africa, explaining the increased prevalence of P. ovale cases among non-P. falciparum infections in Parma.

Myiasis of the scalp due to Dermatobia hominis in a traveler returning from Brazil.

This article describes a case of myiasis by Dermatobia hominis diagnosed in a young Italian man returning from a vacation through Brazil. Considering the increasing number of travels to tropical and subtropical areas, clinicians in nonendemic areas must think about the possibility of imported unusual infestations during their daily practice.

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.

Occurrence of human intestinal spirochetosis in comparison with infections by other enteropathogenic agents in an area of the Northern Italy.

The aim of this study was to investigate the occurrence of human intestinal spirochetosis (IS) by a 16S rRNA restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) in a selected group (234) of patients with gastrointestinal complaints and/or potential risk factors for IS in comparison with the occurrence of infections by other enteropathogenic agents. By using 16S rRNA RFLP-PCR, 16 patients (6.8%) with IS were found (11 infected by Brachyspira aalborgi, 3 by Brachyspira pilosicoli, and 2 by both species); moreover, 10 patients with gastroenteric viruses (4.2%), 13 with enteropathogenic bacteria other than intestinal spirochetes (5.5%), and 24 with intestinal parasites (10.2%) were found. This study provides an enhancement of the knowledge about the distribution of IS, suggesting that it may be more frequent than suspected and that clinicians should consider IS when patients present with long-standing diarrhea. Moreover, 16S rRNA RFLP-PCR might be a powerful tool not only for diagnostic purpose but also to investigate the occurrence of IS just on fecal samples, not requiring invasive diagnostic techniques.

Human intestinal spirochaetosis in Parma: a focus on a selected population during 2002-2005.

BACKGROUND AND AIM OF THE WORK: Human intestinal spirochaetosis (HIS) is a large bowel infection characterised by the colonization of the intestinal mucosa by spirochaetes belonging to the genus Brachyspira. The causative agents of HIS are Brachyspira aalborgi and Brachyspirapilosicoli. Symptoms of the infection, even if not specific, are long standing diarrhoea, abdominal pain, meteorism and rectal bleeding and sometimes they can suggest the clinical suspect of inflammatory bowel diseases or rectal carcinoma. Since poor data were available on the prevalence of this infection, the aim of our study was to describe the occurrence of this infection in our area in the period 2002-2005. METHODS: During a period of 4 years we analysed 297 faecal samples from 99 patients selected by potential risk factors and symptomatology suspected for HIS. The diagnosis of HIS was performed by isolation and a molecular assay based on 16S rDNA restriction fragment length polymorphism (RFLP)-polymerase chain reaction (PCR). RESULTS: From 2002 to 2005 we detected 12 cases of intestinal spirochaetosis, 7 caused by Brachyspira aalborgi, 4 by Brachyspirapilosicoli and one by both spirochaetes, which represented the first case of a mixed infection by 2 intestinal spirochaetes in our area. CONCLUSIONS: Despite the fact that HIS seems to be a low prevalence infection in our area, in a strongly selected population we found 12 cases of this infection (12.12%). These results stimulate us to extend the research of intestinal spirochaetosis in the general population, when long standing gastrointestinal disorders and potential risk factors are present.

Prevalence of imported malaria in Parma during 2005-2006.

BACKGROUND AND AIM OF THE WORK: Malaria is a protozoan infection caused by parasites of the genus Plasmodium (P. falciparum, P. ovale, P. vivax, P. malariae) that is transmitted from one human to another by female Anopheles mosquitoes. It can be considered a reemerging imported disease in our area because of increasing of movements from endemic countries, and nowadays it is the most common imported infection in Italy. This study describes the occurrence of imported malaria in our area between January 2005 and May 2006. METHODS: During 17 months we analysed 170 blood samples belonging to 139 patients (95 foreigners and 44 Italians) with the clinical suspect of malaria. Samples were used to prepare orange acridine and Giemsa stained thin blood films for microscopic observation and to perform an immunochromatographic assay for the detection of specific plasmodia antigens. Molecular assays (nested-PCR and Real-time PCR) were also performed in order to confirm the diagnosis. RESULTS: Thirty-six cases of malaria were diagnosed: 35 in foreigners coming from Africa and only one in an Italian who lived in Chad. Thirty-three patients were infected by P. falciparum, 1 by P. ovale, 1 by P. vivax, and a mixed infection by P. falciparum, P. ovale and P. malariae was also found. CONCLUSIONS: Malaria is usually associated with travels within areas where the infection is endemic and our data demonstrated that imported malaria in our area has a prevalence of 25.89%.

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.

Entamoeba histolytica and Entamoeba dispar: comparison of two PCR assays for diagnosis in a non-endemic setting.

Detection of Entamoeba histolytica, the causative agent of amoebiasis, is an important goal of the clinical parasitology laboratory. The identification of Entamoeba dispar as a morphologically identical but non-pathogenic species has highlighted the need for non-microscopic detection methods able to differentiate between the two organisms. In this study we evaluated the utility of conventional PCR and real-time PCR as methods for identification and differentiation of E. histolytica and E. dispar. The second aim of this study was to determine the relative proportions of infections caused by E. histolytica and the non-pathogenic E. dispar, allowing a picture of the epidemiological situation in a non-endemic setting to be obtained. One hundred and sixty-six clinical samples (faecal and liver abscess samples and one intestinal biopsy) belonging to 108 patients were analysed. More patients with E. dispar infection (8.3%) than patients with E. histolytica infection (5.6%) were found by both PCR assays. It is concluded that routine diagnosis of invasive amoebiasis performed by a combination of microscopy, culture and serology should be complemented with a PCR assay such as real-time PCR that offers a practical and clinically acceptable alternative for rapid and accurate diagnosis of amoebic infection in patients presenting with symptoms indicative of this disease.

Prevalence of intestinal parasites in the area of Parma during the year 2005.

BACKGROUND AND AIM OF THE WORK: Intestinal parasitosis represent a relevant clinical problem, especially in developing countries, where they are responsible for morbidity and mortality in adults and children and many epidemiological data are available for these areas. The actual situation of intestinal parasitosis in Europe is not yet well investigated since they are usually not notified. We describe the occurrence of intestinal parasitosis in our laboratory from January to December 2005. METHODS: We considered all patients (1117) whose stool samples were sent to our laboratory with the suspect of intestinal parasitosis during the year 2005. Each specimen was subjected to macroscopic and microscopic examination to demonstrate the presence of worm eggs, larvae, protozoan trophozoites or cysts and to an immunochromatographic assay to detect Giardia intestinalis and Cryptosporidium spp. specific antigens. Cultures for protozoa and helminths were carried out and a PCR specific for Entamoeba histolytica/Entamoeba dispar was also performed. RESULTS: Our results indicated that 148 patients (13.24%) were affected by intestinal parasitosis. Among the 951 Italians, 96 (10%) were infected, while out of a total of 166 foreigners 52 had intestinal parasitosis (31%). Moreover, we found that 113 infections were caused by only one parasite while 35 were mixed infections. CONCLUSIONS: Intestinal parasitosis represent a remarkable cause of gastrointestinal disease and our study demonstrates that these infections are quite common in our area, affecting both Italians and non European citizens from developing countries.

Comparison between two real-time PCR assays and a nested-PCR for the detection of Toxoplasma gondii.

BACKGROUND AND AIM OF THE WORK: In recent years, the diagnosis of toxoplasmosis has been improved by Real-time PCR assays. In this study we compared the performances of two Real-time PCRs (FRET and TaqMan protocols) already described in the literature, and one nested-PCR, currently used in our laboratory for the molecular diagnosis of toxoplasmosis. METHODS: We evaluated the sensitivity and the specificity of a FRET- and a TaqMan-based Real-time PCRs targeting a 529 bp repeat region and the 18S RNA gene, respectively, and a nested-PCR, targeting the B1-gene of Toxoplasma gondii. We also tested, through nested-PCR, 46 biological samples obtained during a period of 29 months from pregnant women or immunocompromised patients with suspected T. gondii infection. RESULTS: The analytical sensitivity of nested and TaqMan PCRs was approximately 10(3) tachyzoites/ml. FRET assay showed a sensitivity of 102 tachyzoites/ml. Three out of 46 biological samples were nested-PCR-positive and these results were also confirmed by both Real-time PCRs. CONCLUSIONS: Nested- and real-time PCRs evaluated in this study resulted very sensitive and specific; in particular FRET PCR resulted more sensitive than the other assays, probably because of the greater copy number of the target sequence. Real-time PCR assays are easy-to-use, producing results faster than conventional PCR systems and reducing contamination risks.

MALDI-TOF mass spectrometry for the detection and differentiation of Entamoeba histolytica and Entamoeba dispar.

Detection of Entamoeba histolytica and its differentiation from Entamoeba dispar is an important goal of the clinical parasitology laboratory. The aim of this study was the identification and differentiation of E. histolytica and E. dispar by MALDI-TOF MS, in order to evaluate the application of this technique in routine diagnostic practice. MALDI-TOF MS was applied to 3 amebic reference strains and to 14 strains isolated from feces that had been differentiated by molecular methods in our laboratory. Protein extracts from cultures of these strains (axenic cultures for the 3 reference strains and monoxenic cultures for the 14 field isolates) were analyzed by MALDI-TOF MS and the spectra obtained were analyzed by statistical software. Five peaks discriminating between E. histolytica and E. dispar reference strains were found by protein profile analysis: 2 peaks (8,246 and 8,303 Da) specific for E. histolytica and 3 (4,714; 5,541; 8,207 Da) for E. dispar. All clinical isolates except one showed the discriminating peaks expected for the appropriate species. For 2 fecal samples from which 2 strains (1 E. histolytica and 1 E. dispar) out of the 14 included in this study were isolated, the same discriminating peaks found in the corresponding isolated amebic strains were detected after only 12h (E. histolytica) and 24h (E. dispar) of incubation of the fecal samples in Robinson's medium without serum. Our study shows that MALDI-TOF MS can be used to discriminate between E. histolytica and E. dispar using in vitro xenic cultures and it also could have potential for the detection of these species in clinical samples.