Usefulness of Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry in the Characterization of Leishmania Strains Causing Tegumentary Leishmaniasis in Bolivia versus hsp70 Gene Sequencing.

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is a proteomic technique with proven efficiency in the identification of microorganisms, such as bacteria, fungi, and parasites. The present study aimed to evaluate the usefulness of MALDI-TOF MS for the characterization of Leishmania species circulating in Bolivia using hsp70 gene sequencing as a reference technique. 55 Leishmania strains that were isolated from patients with tegumentary leishmaniasis were analyzed. MALDI-TOF MS identified two species of the L. braziliensis complex (L. braziliensis, n = 26; L. braziliensis outlier, n = 18), one species of the L. guyanensis complex (L. guyanensis, n = 1), one species of the L. lainsoni complex (L. lainsoni, n = 2), and two species of the L. mexicana complex (L. amazonensis, n = 5; and L. garnhami, n = 3). All of the strains were correctly identified at the subgenus, genus, and complex level, but 10 of them (18%) were misidentified as other species within the same complex by the hsp70 gene sequencing, with 7 of these corresponding to possible hybrids. Thus, one L. braziliensis corresponded to L. peruviana, two L. braziliensis corresponded to L. braziliensis/L. peruviana possible hybrids, two L. amazonensis corresponded to L. mexicana, and three L. garnhami and two L. amazonensis corresponded to L. mexicana/L. amazonensis possible hybrids. Accordingly, MALDI-TOF MS could be used as an alternative to molecular techniques for the identification of Leishmania spp., as it is low cost, simple to apply, and able to quickly produce results. In Bolivia, its application would allow for the improvement of the management of patient follow-ups, the updating of the epidemiological data of the Leishmania species, and a contribution to the control of tegumentary leishmaniasis. IMPORTANCE The objective of the study was to evaluate the usefulness of MALDI-TOF MS for the characterization of Leishmania species circulating in Bolivia, in comparison with the sequencing of the hsp70 gene. In our study, all of the isolates could be identified, and no misidentifications were observed at the complex level. Although the equipment implies a high initial investment in our context, MALDI-TOF MS can be used in different areas of microbiology and significantly reduces the cost of testing. Once the parasite culture is obtained, the technique quickly yields information by accessing a free database that is available online. This would allow for the improvement of the management of patients and follow-ups, the updating of the epidemiological data of the species, and a contribution to the control of tegumentary leishmaniasis in Bolivia. Likewise, it can be used to determine a specific treatment to be given, according to the causal species of Leishmania, when there are protocols in this regard in the area.

Autochthonous and imported tegumentary leishmaniasis in Catalonia (Spain): Aetiological evolution in the last four decades and usefulness of different typing approaches based on biochemical, molecular and proteomic markers.

Leishmaniasis is a transmissible disease caused by Leishmania protozoa. Spain is endemic for both visceral and cutaneous leishmaniasis, the autochthonous aetiological agent being Leishmania infantum. Around the world, the L. donovani complex is associated with visceral symptoms, while any species of the Leishmania or Viannia subgenera affecting human can produce tegumentary forms. In a context of growing numbers of imported cases, associated with globalisation, the aim of this study was to analyse the aetiological evolution of human tegumentary leishmaniasis in a region of Spain (Catalonia). Fifty-six Leishmania strains, isolated from 1981 to 2018, were analysed using MLEE, gene sequencing (hsp70, rpoIILS, fh and ITS2) and MALDI-TOF. The utility of these different analytical methods was compared. The results showed an increase in leishmaniasis over the two last decades, particularly imported cases, which represented 39% of all cases studied. Leishmania infantum, L. major, L. tropica, L. braziliensis, L. guyanensis and L. panamensis were identified. The combination of molecular and enzymatic methods allowed the identification of 29 different strain types (A to AC). Strain diversity was higher in L. (Viannia), whilst the different L. major types were relatable with geo-temporal data. Among the autochthonous cases, type C prevailed throughout the studied period (39%). Minor types generally appeared within a short time interval. While all the techniques provided identical identification at the species complex level, MALDI-TOF and rpoIILS or fh sequencing would be the most suitable identification tools for clinical practice, and the tandem hsp70-ITS2 could substitute MLEE in the epidemiological field.

Taxonomic Identification of Different Species of the Genus Aeromonas by Whole-Genome Sequencing and Use of Their Species-Specific ß-Lactamases as Phylogenetic Markers.

Some Aeromonas species, potentially pathogenic for humans, are known to express up to three different classes of chromosomal ß-lactamases, which may become hyperproduced and cause treatment failure. The aim of this study was to assess the utility of these species-specific ß-lactamase genes as phylogenetic markers using whole-genome sequencing data. Core-genome alignments were generated for 36 Aeromonas genomes from seven different species and scanned for antimicrobial resistance genes. Core-genome alignment confirmed the MALDI-TOF identification of most of the isolates and re-identified an A. hydrophila isolate as A. dhakensis. Three (B, C and D) of the four Ambler classes of ß-lactamase genes were found in A. sobria, A. allosacharophila, A. hydrophila and A. dhakensis (blaCphA, blaAmpC and blaOXA). A. veronii only showed class-B- and class-D-like matches (blaCphA and blaOXA), whereas those for A. media, A. rivipollensis and A. caviae were class C and D (blaCMY, blaMOX and blaOXA427). The phylogenetic tree derived from concatenated sequences of ß-lactamase genes successfully clustered each species. Some isolates also had resistance to sulfonamides, quinolones and aminoglycosides. Whole-genome sequencing proved to be a useful method to identify Aeromonas at the species level, which led to the unexpected identification of A. dhakensis and A.rivipollensis and revealed the resistome of each isolate.

Population Structure, Antimicrobial Resistance, and Virulence-Associated Genes in Campylobacter jejuni Isolated From Three Ecological Niches: Gastroenteritis Patients, Broilers, and Wild Birds.

Campylobacter jejuni is the causal agent of the food-borne infection with the highest incidence in Europe. Both poultry and wild birds are a major reservoir. To gain insight into the population structure, virulence potential, and antimicrobial resistance (AMR), a collection of 150 isolates from three different ecological niches (broilers, wild birds, and human patients) was studied. Despite the high genetic diversity found, the population structure defined two distinct clusters, one formed mostly by broiler and human isolates and another one by most wild bird isolates. The ST-21 complex exhibits highest prevalence (in humans and broilers), followed by ST-1275 complex (only in wild birds). The ST-48, -45, and -354 complexes were found in all three niches, but represent only 22 out of 150 studied strains. A higher occurrence of AMR and multidrug resistance was detected among broiler and human isolates. Moreover, significant differences were found in the distribution of certain putative virulence genes. Remarkably, many wild bird strains were negative for either cdtA, cdtB, or cdtC from the canonical strain 81-176, whereas all broiler and human strains were positive. These data suggest that the different variants of the cdt genes might be relevant for the efficient colonization of certain hosts by C. jejuni. Our study contributes to the understanding of the role of the diverse Campylobacter reservoirs in the transmission of campylobacteriosis to humans.

Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays.

BACKGROUND: Polymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately. CONCLUSIONS/SIGNIFICANCE: This is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centers.

Identification of Leishmania by Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry Using a Free Web-Based Application and a Dedicated Mass-Spectral Library.

Human leishmaniases are widespread diseases with different clinical forms caused by about 20 species within the Leishmania genus. Leishmania species identification is relevant for therapeutic management and prognosis, especially for cutaneous and mucocutaneous forms. Several methods are available to identify Leishmania species from culture, but they have not been standardized for the majority of the currently described species, with the exception of multilocus enzyme electrophoresis. Moreover, these techniques are expensive, time-consuming, and not available in all laboratories. Within the last decade, mass spectrometry (MS) has been adapted for the identification of microorganisms, including Leishmania However, no commercial reference mass-spectral database is available. In this study, a reference mass-spectral library (MSL) for Leishmania isolates, accessible through a free Web-based application (mass-spectral identification [MSI]), was constructed and tested. It includes mass-spectral data for 33 different Leishmania species, including species that infect humans, animals, and phlebotomine vectors. Four laboratories on two continents evaluated the performance of MSI using 268 samples, 231 of which were Leishmania strains. All Leishmania strains, but one, were correctly identified at least to the complex level. A risk of species misidentification within the Leishmania donovani, L. guyanensis, and L. braziliensis complexes was observed, as previously reported for other techniques. The tested application was reliable, with identification results being comparable to those obtained with reference methods but with a more favorable cost-efficiency ratio. This free online identification system relies on a scalable database and can be implemented directly in users' computers.

Towards a New Strategy for Diagnosis of Congenital Trypanosoma cruzi Infection.

The immigration of Latin American women of childbearing age has spread the congenital transmission of Chagas disease to areas of nonendemicity, and the disease is now a worldwide problem. Some European health authorities have implemented screening programs to prevent vertical transmission, but the lack of a uniform protocol calls for the urgent establishment of a new strategy common to all laboratories. Our aims were to (i) analyze the trend of passive IgG antibodies in the newborn by means of five serological tests for the diagnosis and follow-up of congenital Trypanosoma cruzi infection, (ii) assess the utility of these techniques for diagnosing a congenital transmission, and (iii) propose a strategy for a prompt, efficient, and cost-effective diagnosis of T. cruzi infection. In noninfected newborns, a continuous decreasing trend of passive IgG antibodies was observed, but none of the serological assays seroreverted in any the infants before 12 months. From 12 months onwards, serological tests achieved negative results in all the samples analyzed, with the exception of the highly sensitive chemiluminescent microparticle immunoassay (CMIA). In contrast, in congenitally infected infants, the antibody decline was detected only after treatment initiation. In order to improve the diagnosis of congenital T. cruzi infection, we propose a new strategy involving fewer tests that allows significant cost savings. The protocol could start 1 month after birth with a parasitological test and/or a PCR. If negative, a serological test would be carried out at 9 months, which if positive, would be followed by another at around 12 months for confirmation.

Serological Diagnosis of Chronic Chagas Disease: Is It Time for a Change?

Chagas disease has spread to areas that are nonendemic for the disease with human migration. Since no single reference standard test is available, serological diagnosis of chronic Chagas disease requires at least two tests. New-generation techniques have significantly improved the accuracy of Chagas disease diagnosis by the use of a large mixture of recombinant antigens with different detection systems, such as chemiluminescence. The aim of the present study was to assess the overall accuracy of a new-generation kit, the Architect Chagas (cutoff, ≥1 sample relative light units/cutoff value [S/CO]), as a single technique for the diagnosis of chronic Chagas disease. The Architect Chagas showed a sensitivity of 100% (95% confidence interval [CI], 99.5 to 100%) and a specificity of 97.6% (95% CI, 95.2 to 99.9%). Five out of six false-positive serum samples were a consequence of cross-reactivity with Leishmania spp., and all of them achieved results of <5 S/CO. We propose the Architect Chagas as a single technique for screening in blood banks and for routine diagnosis in clinical laboratories. Only gray-zone and positive sera with a result of ≤6 S/CO would need to be confirmed by a second serological assay, thus avoiding false-positive sera and the problem of cross-reactivity with Leishmania species. The application of this proposal would result in important savings in the cost of Chagas disease diagnosis and therefore in the management and control of the disease.

Risk of underdiagnosing amebic dysentery due to false-negative Entamoeba histolytica antigen detection.

Entamoeba histolytica antigen assays on stool are widely used to diagnose amebiasis. We report a case of confirmed amebic colitis with a false-negative antigen detection that became positive after treatment. Our results indicate that these assays may underdiagnose acute amebic infection when used alone and should be used cautiously.

Development of an experimental model of induced bacterial peritonitis in cirrhotic rats with or without ascites.

BACKGROUND: Spontaneous bacterial peritonitis (SBP) is a severe complication of cirrhotic patients associated with a high mortality. AIM: To develop an available experimental model of induced bacterial peritonitis in cirrhosis. MATERIAL AND METHODS: Sprague-Dawley rats with carbon-tetrachloride-induced cirrhosis with (N=22) or without (N=101) ascites were randomized to receive an intraperitoneal administration of different concentrations of Escherichia coli (E. coli) diluted in 1 mL of sterile water in ascitic rats and in different volumes in nonascitic rats. A subgroup of nonascitic animals received ceftriaxone 4 h after E. coli inoculation. Mortality of rats was evaluated 24 h after bacterial inoculation. RESULTS: None of the rats receiving sterile water alone and only one infected with 10(7) cfu of E. coli died. Ascitic rats showed a lower mortality rate than nonascitic rats infected with 10(8) or 10(9) cfu of E. coli (P<0.05). Mortality was higher with 10(9) cfu than with 10(8) cfu of E. coli in ascitic (P NS) and nonascitic (P<0.01) rats. A trend was noted to ward higher mortality in nonascitic rats inoculated with 10(8) cfu with increasing water volumes. A marked peritoneal polymorphonuclear cell response was observed 4 h after E. coli injection in both ascitic and nonascitic rats. Antibiotic therapy significantly reduced the mortality rate of rats infected with 10(8) cfu (P<0.01). CONCLUSIONS: This experimental model of induced bacterial peritonitis in cirrhosis with or without ascites may represent a useful tool for the study of pathogenic events postinfection and for the design of new therapeutic strategies to treat patients with SBP.

Bacterial translocation is downregulated by anti-TNF-alpha monoclonal antibody administration in rats with cirrhosis and ascites.

BACKGROUND/AIMS: TNF-alpha is involved in the development of bacterial translocation in rats with cirrhosis. The aim of the current study was to evaluate the effect of anti-TNF-alpha mAb treatment on the incidence of bacterial translocation and systemic infections in rats with cirrhosis and ascites. METHODS: Thirty rats with cirrhosis and ascites were randomly assigned to receive two intraperitoneal doses of anti-TNF-alpha mAb, distilled water or immunoglobulin on days 0 and 4. On day 10, a laparotomy was performed. RESULTS: One out of 11 animals receiving anti-TNF-alpha mAb treatment, 7 out of 10 of the placebo group (p<0.01), and 5 out of 9 of the IgG group developed bacterial translocation (p<0.05). A significantly reduced number of systemic infections were observed in animals receiving anti TNF-alpha mAb treatment vs animals receiving placebo (p<0.01). TNF-alpha in serum at laparotomy in animals receiving anti-TNF-alpha mAb was higher than that in the rest of groups and was also higher in the overall series of animals showing bacterial translocation. CONCLUSIONS: In the experimental model of CCl(4)-induced rat with cirrhosis and ascitic fluid, anti-TNF-alpha mAb administration decreases the incidence of bacterial translocation, in a TNF-alpha/sTNF-alpha receptor-independent manner, without increasing the risk of systemic infections.

An outbreak of norovirus infection in a long-term-care unit in Spain.

BACKGROUND: Norovirus belongs to the Caliciviridae family and causes outbreaks of infectious enteritis by fecal-oral transmission. In Spain, there have been few outbreaks reported due to this virus. We describe an outbreak on a long-term-care hospital ward. METHODS: Cases were classified as probable, confirmed, and secondary. Stool cultures were performed. Polymerase chain reaction detection of norovirus was also performed. RESULTS: The outbreak occurred from December 7 to 28, 2001, involving 60 cases (32 patients, 19 staff members, 8 patients' relatives, and 1 relative of a staff member). Most (82%) of the cases were female. The most frequently involved ages were 20 to 39 years for staff members and 70 to 89 years for patients. The incubation period of secondary cases in patients' families had a median of 48 hours (range, 1 to 7 days). Clinical symptoms included diarrhea (85%), vomiting (75%), fever (37%), nausea (23%), and abdominal pain (12%). Median duration of the disease was 48 hours (range, 1 to 7 days). All cases resolved and the outbreak halted with additional hygienic measures. Stool cultures were all negative for enteropathogenic bacteria and rotaviruses. In 16 of 23 cases, the norovirus genotype 2 antigen was detected. CONCLUSION: This outbreak of gastroenteritis due to norovirus genotype 2 affected patients, staff members, and their relatives in a long-term-care facility and was controlled in 21 days.

Intestinal mucosal oxidative damage and bacterial translocation in cirrhotic rats.

BACKGROUND: Bacterial translocation plays an important role in the pathogenesis of spontaneous bacterial peritonitis mainly due to intestinal bacterial overgrowth. Alterations in the functional integrity of the intestinal barrier caused by an increased production of free radical metabolites as a consequence of portal hypertension could also facilitate bacterial translocation in cirrhotic rats. OBJECTIVE: The aim of the study was to determine intestinal mucosal lipid peroxidation and neutrophil infiltration and their relationship with portal hypertension and bacterial translocation in cirrhotic rats. DESIGN: Eighteen male Sprague-Dawley rats with cirrhosis induced by carbon tetrachloride, administered by gavage, and eight control rats were included in the study. METHODS: Samples of jejunum, ileum and caecum were obtained by laparotomy for the determination of malondialdehyde and myeloperoxidase as indexes of lipid peroxidation and neutrophil infiltration, respectively. Samples of ascitic and pleural fluids, mesenteric lymph nodes and ileal stools were obtained for the culture of microoganisms. RESULTS: The concentration of malondialdehyde was significantly higher in ileal and caecal, but not in jejunal mucosa, in cirrhotic rats, mainly in those with ascites (P< 0.01), as compared to control rats (P< 0.01), and in cirrhotic rats with bacterial translocation compared to those without bacterial translocation (P< 0.01). No differences between groups were observed in the concentrations of myeloperoxidase in jejunum, ileum or caecum. A direct correlation between ileal malondialdehyde and portal pressure was observed (P< 0.01). CONCLUSIONS: Cirrhotic rats, particularly those with ascites and bacterial translocation, show increased malondialdehyde levels in ileal and caecal mucosa. These results suggest that mucosal oxidative damage in ileum and caecum could favour bacterial translocation in cirrhotic rats.

Effect of Lactobacillus johnsonii La1 and antioxidants on intestinal flora and bacterial translocation in rats with experimental cirrhosis.

BACKGROUND/AIMS: Probiotics and antioxidants could be alternatives to antibiotics in the prevention of bacterial infections in cirrhosis. The aim of the present study was to determine the effect of Lactobacillus johnsonii La1 and antioxidants on intestinal flora, endotoxemia, and bacterial translocation in cirrhotic rats. METHODS: Twenty-nine Sprague-Dawley rats with cirrhosis induced by CCl(4) and ascites received Lactobacillus johnsonii La1 10(9)cfu/day in vehicle (antioxidants: vitamin C+glutamate) (n=10), vehicle alone (n=11), or water (n=8) by gavage. Another eight non-cirrhotic rats formed the control group. After 10 days of treatment, a laparotomy was performed to determine microbiological study of ileal and cecal feces, bacterial translocation, endotoxemia, and intestinal malondialdehyde (MDA) levels as index of intestinal oxidative damage. RESULTS: Intestinal enterobacteria and enterococci, bacterial translocation (0/11 and 0/10 vs. 5/8, P<0.01), and ileal MDA levels (P<0.01) were lower in cirrhotic rats treated with antioxidants alone or in combination with Lactobacillus johnsonii La1 compared to cirrhotic rats receiving water. Only rats treated with antioxidants and Lactobacillus johnsonii La1 showed a decrease in endotoxemia with respect to cirrhotic rats receiving water (P<0.05). CONCLUSIONS: Antioxidants alone or in combination with Lactobacillus johnsonii La1 can be useful in preventing bacterial translocation in cirrhosis.

Comparison of macrorestriction analysis of genomic DNA by pulsed-field gel electrophoresis and ribotyping with conventional methods for differentiation of Escherichia coli 0124 isolates.

OBJECTIVE: To evaluate the usefulness of different phenotypic and genotypic markers for epidemiological typing of enteroinvasive Escherichia coli 0124 (EIEC 0124). METHODS: Seven sporadic EIEC 0124 isolates and 22 isolates from two different outbreaks were characterized. Chromosomal deoxyribonucleic acid (DNA) macrorestriction analysis with XbaI resolved by pulsed-field gel electrophoresis (PFGE) and ribotyping with each of the three restriction endonucleases BglII EcoRI, and ClaI were compared with biotyping, antimicrobial susceptibility patterns and plasmid profiles. RESULTS: Biotypes and antimicrobial resistance profiles of the outbreak-associated strains showed considerable variation, thereby limiting the usefulness of such phenotypic markers. Only 57% of the sporadic isolates harbored plasmids. Three different ribotypes based on 5 to 7 bands were recorded among sporadic isolates whereas all outbreak-associated strains showed the same ribotype. BglII appeared to give the best discrimination whereas EcoRI and ClaI provided no additional information. Sporadic EIEC 0124 isolates showed a marked diversity of macrorestriction patterns (similarity coefficient 58 to 93%) and five different patterns were detected. In contrast, the outbreak isolates were closely related (similarity coefficient 90 to 100%). Genomic DNA macrorestriction analysis correlated well with ribotyping, but PFGE was more discriminating. CONCLUSIONS: PFGE is a useful method for epidemiological comparison and differentiation of EIEC 0124 isolates.