Evaluation of the CHROMagar Candida Plus medium for presumptive identification of yeasts and MALDI-TOF MS identification.

Rapid and accurate yeasts species identification in clinical laboratories is important for appropriate and timely antifungal treatment. We evaluate the performance of the new medium CHROMagar™ Candida Plus for presumptive identification of yeasts species and MALDI-TOF identification. We identify 303 strains belonging to 60 clinically relevant yeasts species by using the new medium. Presumptive identification was correct at the Candida albicans complex, Candida tropicalis and Pichia kudriavzevii (Candida krusei) species. However, although this medium was able to identify all Candida auris and Candida glabrata strains, other species were misidentified as C. auris or C. glabrata. A total of 215 strains were identified by using MALDI-TOF and evaluated two incubation temperatures (30°C and 37°C) and two incubation times (24 h and 72 h). Most strains (94%; 202/215) were correctly identified at the species (n:190) or complex level (n:12) at both temperatures and incubation times. However, we observed that the time of incubation (24 h vs. 72 h) affects the score values when yeasts are incubated at 37°C, but does not affect score values when yeasts are incubated at 30°C. In conclusion, the new medium has a good performance in the presumptive identification of the C. albicans complex, C. tropicalis and P. kudriavzevii (C. krusei). In addition, this medium is useful for the screening of C. auris and C. glabrata isolates, but identification should be confirmed by other more specific techniques, like MALDI-TOF.

Testing of In Vitro Susceptibility of Neisseria gonorrhoeae to Azithromycin: Comparison of Disk Diffusion and Reference Agar Dilution Methods.

Azithromycin in combination with ceftriaxone is recommended as the first-line treatment for uncomplicated gonorrhea in many countries. Therefore, monitoring of azithromycin susceptibility of Neisseria gonorrhoeae isolates is essential. In 2019, the Clinical and Laboratory Standards Institute (CLSI) listed the MIC breakpoint for a susceptible-only category to azithromycin, but breakpoints for disk diffusion are not yet available. In this study, we evaluated the usefulness of disk diffusion for testing the susceptibility of N. gonorrhoeae isolates to azithromycin. A total of 189 clinical isolates susceptible and nonsusceptible to azithromycin were used. Agar dilution MICs were correlated with inhibition zone diameters of azithromycin disks (15-µg) manufactured by BBL and Oxoid. In addition, an interlaboratory study involving two clinical microbiology laboratories was conducted. There was a strong correlation between disk diffusion and agar dilution for BBL disks (r = -0.74; P < 0.001) and Oxoid disks (r = -0.75; P < 0.001). Using a zone diameter breakpoint of ≥27 mm (susceptible) and ≤26 mm (nonsusceptible) yielded good separation between susceptible and nonsusceptible isolates and the least number of discrepancies. Compared to agar dilution, disk diffusion showed high agreement and kappa values of 95.2% and 0.899 (P < 0.001) for BBL disks and 96.8% and 0.933 (P < 0.001) for Oxoid disks, respectively. Major and very major discrepancies were observed in isolates with azithromycin MICs (1 and 2 µg/ml, respectively) near to the breakpoint. These data illustrate that disk diffusion could be a reliable method in clinical laboratories to test susceptibility to azithromycin in N. gonorrhoeae isolates.

Distribution of PCV13 and PPSV23 Streptococcus pneumoniae serotypes in Argentinean adults with invasive disease, 2013-2017.

Streptococcus pneumoniae is a major cause of severe invasive disease associated with high mortality and morbidity worldwide. To identify the serotypes most commonly associated with infection in adults in Argentina, 791 pneumococcal isolates from 56 hospitals belonging to 16 provinces and Buenos Aires city were serotyped. The isolates were submitted as part of a National Surveillance Program for invasive pneumococcal disease in adults, which started in 2013. Serotypes 3, 8, 12F, 7F and 1 were the most prevalent among adult patients. During the study period there was no significant difference in serotype distribution between the age groups studied (18-64 and ≥65 years old), except for serotype 1, 3 and 23A. Most prevalent serotypes in pneumonia were serotype 7F, 1, 12F, 8, and 3. When the clinical diagnosis was meningitis, serotype 3 and 12F were the most prevalent, whereas when the diagnosis was sepsis/bacteremia the most prevalent was serotype 8. In this work, for the 18-64-year-old group, PPSV23 and PCV13 serotypes accounted for 74.56% and 44.54% respectively of the cases in the studied period. On the other hand, for the ≥65-year-old group, these serotypes represented 72.30% and 41.42% respectively. The aim of this work was to establish the knowledge bases of the serotypes that cause invasive pneumococcal diseases in the adult population in Argentina and to be able to detect changes in their distribution over time in order to explore the potential serotype coverage of the vaccines in current use.

The interplay between local immune response and Epstein-Barr virus-infected tonsillar cells could lead to viral infection control.

Epstein Barr virus (EBV) gains access to the host through tonsillar crypts. Our aim was to characterize microenvironment composition around EBV+ cells in tonsils from pediatric carriers, to disclose its role on viral pathogenesis. LMP1 expression, assessed by immunohistochemistry (IHC), was used to discriminate EBV + and - zones in 41 tonsil biopsies. Three regions were defined: Subepithelial (SE), interfollicular (IF) and germinal center (GC). CD8, GrB, CD68, IL10, Foxp3, PD1, CD56 and CD4 markers were evaluated by IHC; positive cells/100 total cells were counted. CD8+, GrB+, CD68+ and IL10+ cells were prevalent in EBV+ zones at the SE region (p < 0.0001, p = 0.03, p = 0.002 and p = 0.002 respectively, Wilcoxon test). CD4+ and CD68+ cell count were higher in EBV + GC (p = 0.01 and p = 0.0002 respectively, Wilcoxon test). Increment of CD8, GrB and CD68 at the SE region could indicate a specific response that may be due to local homing at viral entry, which could be counterbalanced by IL10, an immunosuppressive cytokine. Additionally, it could be hypothesized that CD4 augment at the GC may be involved in the EBV-induced B-cell growth control at this region, in which macrophages could also participate.

Epstein-Barr virus load in transplant patients: Early detection of post-transplant lymphoproliferative disorders.

High levels of circulating EBV load are used as a marker of post-transplant lymphoproliferative disorders (PTLD). There is no consensus regarding the threshold level indicative of an increase in peripheral EBV DNA. The aim of the study was to clinically validate a developed EBV quantification assay for early PTLD detection. Transversal study: paired peripheral blood mononuclear cells (PBMC), plasma and oropharyngeal lymphoid tissue (OLT) from children undergoing a solid organ transplant with (n=58) and without (n=47) PTLD. Retrospective follow-up: 71 paired PBMC and plasma from recipients with (n=6) and without (n=6) PTLD history. EBV load was determined by real-time PCR. The diagnostic ability to detect all PTLD (categories 1-4), advanced PTLD (categories 2-4) or neoplastic PTLD (categories 3 and 4) was estimated by analyzing the test performance at different cut-off values or with a load variation greater than 0.5log units. The higher diagnostic performance for identifying all, advanced or neoplastic PTLD, was achieved with cut-off values of 1.08; 1.60 and 2.47log EBVgEq/10(5) PBMC or 2.30; 2.60; 4.47loggEq/10(5) OLT cells, respectively. EBV DNA detection in plasma showed high specificity but low (all categories) or high (advanced/neoplastic categories) sensitivity for PTLD identification. Diagnostic performance was greater when: (1) a load variation in PBMC or plasma was identified; (2) combining the measure of EBV load in PBMC and plasma. The best diagnostic ability to identify early PTLD stages was achieved by monitoring EBV load in PBMC and plasma simultaneously; an algorithm was proposed.

Development and standardization of an enzyme-linked inmunosorbent for the detection of orthohantavirus infection in Argentina based on its bacterial-expressed nucleocapside protein.

We conducted a development and standardization of an IgG ELISA assay for serological detection of human orthohantavirus infections using the recombinant antigen rLECH13 produced in bacterial and derived from the LECHV. The evaluation and standardization were carried out by analyzing serum samples from a total of 50 patients with confirmed Hantavirus Pulmonary Syndrome (HPS) diagnosis through the reference technique, 50 negative sera, and 53 patients with other medical conditions. The data from the assay analysis showed a diagnostic sensitivity value of 95% and a diagnostic specificity of 80%. The high sensitivity of this novel assay leads us to conclude that rLECH13 is a feasible option for use in the immunodiagnostic of orthohantavirus infection. Additionally, it is crucial to have an antigen that can be produced under conditions that do not require highly complex laboratories. Furthermore, the new assay is cost-effective, reproducible, and demonstrates excellent performance.

Exploring Streptococcus pneumoniae capsular typing through MALDI-TOF mass spectrometry and machine-learning algorithms in Argentina: Identifying prevalent NON PCV13 serotypes alongside PCV13 serotypes.

Introduction: Laboratory surveillance of Streptococcus pneumoniae serotypes plays a crucial role in effectively implementing vaccines to prevent invasive pneumococcal diseases. The conventional method of serotyping, known as the Quellung reaction, is both time-consuming and expensive. However, the emergence of MALDI-TOF MS technology has revolutionized microbiology laboratories by enabling rapid and cost-effective serotyping based on protein profiles. Objectives: In this study, we aimed to investigate the viability of utilizing MALDI-TOF MS technology as an adjunctive and screening method for capsular typing of Streptococcus pneumoniae. Our approach involved developing classification models based on MALDI-TOF MS to discern between Streptococcus pneumoniae strains originating from PCV13 (13-valent pneumococcal conjugate vaccine) and NON PCV13 isolates. Methods: Firstly, we established a comprehensive spectral database comprising isolates of serotypes present in the PCV13 vaccine, along with the top 10 most prevalent NON PCV13 serotypes based on local epidemiological data. This database served as a foundation for developing unsupervised models utilizing MALDI-TOF MS spectra, which enabled us to identify inherent patterns and relationships within the data. Our analysis involved a dataset comprising 215 new isolates collected from nationwide surveillance in Argentina. Our approach involved developing classification models based on MALDI-TOF MS to discern between Streptococcus pneumoniae strains originating from PCV13 (13-valent pneumococcal conjugate vaccine) and NON PCV13 isolates. Results: Although our findings revealed suboptimal performance in serotype classification, they provide valuable insights into the potential of machine learning algorithms in this context. The sensitivity of the models ranged from 0.41 to 0.46, indicating their ability to detect certain serotypes. The observed specificity consistently remained at 0.60, suggesting a moderate level of accuracy in identifying non-vaccine serotypes. These results highlight the need for further refinement and optimization of the algorithms to enhance their discriminative power and predictive accuracy in serotype identification.By addressing the limitations identified in this study, such as exploring alternative feature selection techniques or optimizing algorithm parameters, we can unlock the full potential of machine learning in robust and reliable serotype classification of S. pneumoniae. Our work not only provides a comprehensive evaluation of multiple machine learning models but also emphasizes the importance of considering their strengths and limitations. Conclusion: Overall, our study contributes to the growing body of research on utilizing MALDI-TOF MS and machine learning algorithms for serotype identification purposes.

First Argentine database for the accurate identification of Brucella to species level by MALDI-TOF MS.

MALDI-TOF mass spectrometry (MS) has proven to be a fast and reliable method for the identification of a large number of taxonomic groups. It offers the advantage of being able to incorporate protein spectra of microorganisms that are absent or poorly represented in commercial databases, such as the genus Brucella. The aim of the study was to build the first database of protein spectra of local biological variants of Brucella in Argentina and of standard strains. First, the identification performance of a panel of 135 strains was evaluated with the Swedish database ¨Folkhälsomyndigheten¨ (containing protein spectra of several international standards of the genus Brucella) imported from the open access site https://spectra.folkhalsomyndigheten.se/spectra/. With this library 100 % of the strains were correctly identified by mass spectrometry to genus level, but not to species level. Due to the limitation found, an in-house database was designed with local Brucella isolates from Argentina and standard strains used in routine bacteriological diagnosis. For its validation, a panel of strains, different from those used to develop the extended local database (n: 177), was used to, simultaneously, challenge both libraries. The samples were processed by triplicate and the results obtained were: 177 strains correctly identified to genus and species level compared to the gold standard method (phenotypic typing), meeting the criteria accepted by the literature and the manufacturer as reliable identification. Only 2 of these isolates had score values lower than 2 (1.862) and were therefore not included in the calculation of results. According to these results, MALDI-TOF MS is a fast and reliable method for the routine identification of the different Brucella species, and even has the advantage of reducing the time of exposure to pathogenic microorganisms for laboratorians. It could be considered a valuable technique to replace, in the near future, the current conventional techniques due to the ease of transferring protein spectra, avoiding the use of reference strains that are difficult to find commercially available and commonly used in phenotypic typing.

Rapid and accurate detection of Shiga toxin-producing Escherichia coli (STEC) serotype O157 : H7 by mass spectrometry directly from the isolate, using 10 potential biomarker peaks and machine learning predictive models.

Introduction. The different pathotypes of Escherichia coli can produce a large number of human diseases. Surveillance is complex since their differentiation is not easy. In particular, the detection of Shiga toxin-producing Escherichia coli (STEC) serotype O157 : H7 consists of stool culture of a diarrhoeal sample on enriched and/or selective media and identification of presumptive colonies and confirmation, which require a certain level of training and are time-consuming and expensive.Hypothesis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a quick and easy way to obtain the protein spectrum of a microorganism, identify the genus and species, and detect potential biomarker peaks of certain characteristics.Aim. To verify the usefulness of MALDI-TOF MS to rapidly identify and differentiate STEC O157 : H7 from other E. coli pathotypes.Methodology. The direct method was employed, and the information obtained using Microflex LT platform-based analysis from 60 clinical isolates (training set) was used to detect differences between the peptide fingerprints of STEC O157 : H7 and other E. coli strains. The protein profiles detected laid the foundations for the development and evaluation of machine learning predictive models in this study.Results. The detection of potential biomarkers in combination with machine learning predictive models in a new set of 142 samples, called 'test set', achieved 99.3 % (141/142) correct classification, allowing us to distinguish between the isolates of STEC O157 : H7 and the other E. coli group. Great similarity was also observed with respect to this last group and the Shigella species when applying the potential biomarkers algorithm, allowing differentiation from STEC O157 : H7Conclusion. Given that STEC O157 : H7 is the main causal agent of haemolytic uremic syndrome, and based on the performance values obtained in the present study (sensitivity=98.5 % and specificity=100.0 %), the implementation of this technique provides a proof of principle for MALDI-TOF MS and machine learning to identify biomarkers to rapidly screen or confirm STEC O157 : H7 versus other diarrhoeagenic E. coli in the future.

[Contribution of the PCR assay to the diagnosis of Mansonella ozzardi in endemic areas of Argentina]. / Aporte de la técnica de PCR en el diagnóstico de Mansonella ozzardi en zonas endémicas de la Argentina.

Mansonella ozzardi is a tissue-dwelling parasitic nematode, the causative agent of mansonelliasis in almost all Latin American countries. It has been described along the Argentine Yungas region. The microscopic diagnosis can yield false-negative test results at low microfilaremia levels. The aim of this study was to optimize the molecular diagnostic technique and compare it with the Knott's method and standard blood smear procedures (thin blood films and thick smears) in 92 blood samples of individuals from an endemic area. The PCR technique followed by the sequencing of the amplified product yielded 100 % sensitivity compared to the Knott's test, which is considered a reference method. Seven more cases of this parasitosis could only be identified with the molecular technique.

Invasive Streptococcus pneumoniae isolates from pediatric population in Argentina for the period 2006-2019. Temporal progression of serotypes distribution and antibiotic resistance.

Streptococcus pneumoniae is a major cause of severe invasive disease associated with high mortality and morbidity worldwide. A total of 2908 pneumococcal isolates were analyzed between 2006 and 2019. Gold standard pneumococcal serotyping (the Neufeld-Quellung reaction) was performed to identify the serotypes associated with infection in children < 5 years in Argentina and agar dilution method was carried out to determine their profiles to 14 antimicrobial agents. In 2012, the 13-valent pneumococcal conjugate vaccine (PCV13) was included in the National Immunization Program. In this work we have analyzed the local epidemiology of invasive pneumococcal diseases before and after the introduction of this vaccine in order to understand the epidemiological relevance and impact of PCV13. During the periods compared in the present study there was a significant increase in the proportion of non-PCV13 serotypes, serogroup 24 (246.7%) and 12F (85.7%), and a significant decrease in PCV13 serotypes, including serotypes 14 (91.2%), 5 (95.6%) and 1 (84.6%) among others. Another observation was that serotypes 3 (7.4%) and 19A (4.9%) still remain among the most frequent serotypes despite being part of the PCV13 formulation. Regarding antimicrobial resistance, in the present study we observed an increase in erythromycin resistance during the period of study mainly associated to serotype 14 in the pre-PCV13 period and to serogroup 24 in the post-PCV13 period, which also was the major NVT serotype associated with antimicrobial resistance and MDR. Serotypes 14, 24A/B/F and 19A were in the first three places among isolates resistant to all the antibiotics tested. Our data highlight the importance of continuous surveillance to assess the impact of pneumococcal vaccines and the use of antibiotics in the dynamic of pneumococcal serotypes.

New enzyme-linked immunoassay for the detection of specific antibodies against multiple Leptospira serogroups in bovine sera.

Leptospirosis is a zoonotic disease with worldwide endemicity in Argentina, it is a significant public health problem in low-income populations. Bovine leptospirosis is a serious economic problem for cattle production, causing abortions, reduced milk yield, mortality in calves and decreased daily weight gain. We developed an enzyme-linked immunosorbent assay (ELISA) with sonicated Leptospira interrogans serogroup Icterohaemorrhagiae serovar Copenhageni M 20. We evaluated its performance for the detection of specific antibodies against multiple Leptospira serogroups in bovine. The microscopic agglutination test (MAT) was used as the gold standard. The performance of this ELISA was evaluated with a panel of sera (118 MAT confirmed positive and 97 MAT negative). The overall sensitivity was close to 85.6 % and the specificity was 83.5 %, according to the MAT reference method. Analytical specificity of the IgG-ELISA was evaluated using 50 bovine serum samples from animals showing serum antibodies against other pathogens that cause abortion in bovine, such as Brucella sp., Neospora sp. and Bovine Viral Diarrhea (BVD), and no cross-reaction was observed. This IgG-ELISA can be an alternative to the MAT for diagnosis of leptospiral infection in bovine.

Evaluation of RT-qPCR and Loop-Mediated Isothermal Amplification (LAMP) Assays for the Detection of SARS-CoV-2 in Argentina.

Our aim was to evaluate the analytical and clinical performance of the SARS-CoV-2 molecular detection kits used in Argentina. Nine real-time reverse-transcription polymerase chain reaction (RT-qPCR) and three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays were evaluated using the World Health Organization (WHO) recommended test as reference method. A secondary standard calibrated for the E, N and RdRp genes against the Pan American Health Organization-World Health Organization-International Standard was used to calculate the limit of detection (LoD). A panel of artificial clinical samples, 32 positive and 30 negative for SARS-CoV-2, were analyzed to estimate the kappa concordance (κ) and the diagnostic performance. Differences among the LoD values for the target genes amplified by each kit were >1 log copies/reaction. The κ for the RT-qPCR kits was greater than 0.9, whereas that for the RT-LAMP assays ranged from 0.75 to 0.93. The clinical performance of RT-qPCR kits showed 100% specificity and high sensitivity, although with variations according to the gene analyzed. The E and N genes provided greater clinical sensitivity, whereas the RdRp gene increased the clinical specificity. The RT-LAMP assays revealed a variable diagnostic performance. The information provided can be useful to choose the most appropriate diagnostic test and may contribute to the establishment of a consensus in the diagnosis of SARS-CoV-2 in Argentina and the region.

Prospective multicenter evaluation of real time PCR Kit prototype for early diagnosis of congenital Chagas disease.

BACKGROUND: Current algorithm for Congenital Chagas Disease (cCD) diagnosis is unsatisfactory due to low sensitivity of the parasitological methods. Moreover, loss to follow-up precludes final serodiagnosis after nine months of life in many cases. A duplex TaqMan qPCR kit for Trypanosoma cruzi DNA amplification was prospectively evaluated in umbilical cord (UCB) and peripheral venous blood (PVB) of infants born to CD mothers at endemic and non-endemic sites of Argentina. METHODS: We enrolled and followed-up 370 infants; qPCR was compared to gold-standard cCD diagnosis following studies of diagnostic accuracy guidelines. FINDINGS: Fourteen infants (3·78%) had cCD. The qPCR sensitivity and specificity were higher in PVB (72·73%, 99·15% respectively) than in UCB (66·67%, 96·3%). Positive and negative predictive values were 80 and 98·73% and 50 and 98·11% for PVB and UCB, respectively. The Areas under the Curve (AUC) of ROC analysis for qPCR and micromethod (MM) were 0·81 and 0·67 in UCB and 0·86 and 0·68 in PVB, respectively. Parasitic loads ranged from 37·5 to 23,709 parasite equivalents/mL. Discrete typing Unit Tc V was identified in five cCD patients and in six other cCD cases no distinction among Tc II, Tc V or Tc VI was achieved. INTERPRETATION: This first prospective field study demonstrated that qPCR was more sensitive than MM for early cCD detection and more accurate in PVB than in UCB. Its use, as an auxiliary diagnostic tool to MM will provide more accurate records on cCD incidence. FUNDING: FITS SALUD 001-CHAGAS (FONARSEC, MINCyT, Argentina) to the Public-Private Consortium (INGEBI-CONICET, INP-ANLIS MALBRAN and Wiener Laboratories); ERANET-LAC-HD 328 to AGS and PICT 2015-0074 (FONCYT, MinCyT) to AGS and FA.

Toxicological study of bee venom (Apis mellifera mellifera) from different regions of the province of Buenos Aires, Argentina.

Samples of Apis mellifera mellifera venom from different hives in two regions of the Buenos Aires province and its pool were analyzed for their lethal potency, myotoxic, defibrinogenating, hemolytic and inflammatory-edematizing activity and for the histological alterations they produce in the heart, lungs, kidneys, skeletal muscle and liver of mice. In vitro studies focused on the venom's hemolytic activity in different systems and species (horse, man, sheep and rabbit), the cytotoxicity in cellular lines, and on the proteolytic and coagulant activity in plasma and fibrinogen. Hemolytic activity, either observed in vitro or in vivo, showed similar toxicity levels for all samples. Erythrocytes of different species varied in their sensitivity to the venom pool, equines being the most sensitive and sheep the most resistant to direct hemolytic action. Local and systemic myotoxicity was evidenced by either the elevation of serum creatine kinase and/or histopathological lesions, observed in different muscles. All samples caused significant pathological alterations; pulmonary, cardiac, renal and skeletal muscle lesions were substantive and can be related to the pathophysiological mechanisms of envenomation. The venoms from different apiaries and regions of the Buenos Aires province showed very similar toxicological characteristics. These results suggest that severity of envenomation in case of a swarming could therefore be more related to the number of bees than to the differential toxicity of the venom from different regions of the province. This is the first study on the toxicity and toxicological characteristics of Apis mellifera venom in Argentina.

Utility of platforms Viteks MS and Microflex LT for the identification of complex clinical isolates that require molecular methods for their taxonomic classification.

Mass spectrometry has revolutionized the clinical microbiology field in America's and Europe's industrialized countries, for being a fast, reliable and inexpensive technique. Our study is based on the comparison of the performance of two commercial platforms, Microflex LT (Bruker Daltonics, Bremen, Germany) and Vitek MS (bioMérieux, Marcy l´Etoile, France) for the identification of unusual and hard-to-diagnose microorganisms in a Reference Laboratory in Argentina. During a four-month period (February-May 2018) the diagnostic efficiency and the concordance between both systems were assessed, and the results were compared with the polyphasic taxonomic identification of all isolates. The study included 265 isolates: 77 Gram-Negative Bacilli, 33 Gram-Positive Cocci, 40 Anaerobes, 35 Actinomycetales, 19 Fastidious Microorganisms and 61 Gram-Positive Bacilli. All procedures were practiced according to the manufacturer's recommendations in each case by duplicate, and strictly in parallel. Other relevant factors, such as the utility of the recommended extraction protocols, reagent stability and connectivity were also evaluated. Both systems correctly identified the majority of the isolates to species and complex level (82%, 217/265). Vitex MS achieved a higher number of correct species-level identifications between the gram-positive microorganisms; however, it presented greater difficulty in the identification of non-fermenting bacilli and a higher number of incorrect identifications when the profile of the microorganism was not represented in the commercial database. Both platforms showed an excellent performance on the identification of anaerobic bacteria and fastidious species. Both systems enabled the fast and reliable identification of most of the tested isolates and were shown to be very practical for the user.

The classical Bordetella species and MALDI-TOF technology: a brief experience.

PURPOSE: The aim of this work was to evaluate and optimize the identification of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica (usually known as the classical Bordetella species) using Bruker Biotyper matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS). METHODOLOGY: A set of 106 previously characterized clinical isolates was used. The results were interpreted according to the manufacturer's recommendations and, in addition, a new score value cutoff was used for species identification. Further, the 10 % rule (previously adopted by other authors) and the new 5 % breaking point (proposed in this work) were evaluated in order to optimize identification rates.Results/Key findings. Our results suggest that it is possible to distinguish different species of the classical Bordetella species by following a simple algorithm without additional testing being required. CONCLUSION: MALDI-TOF might be a reliable tool for the identification of this group of bacteria when a combination of cutoff scores is used. This procedure allows us to increase the identification rates for the classical Bordetella species significantly; however, more studies will be required to determine the applicability of the method to other difficult-to-distinguish organisms.

Comparison of disk diffusion and agar dilution methods for gentamicin susceptibility testing of Neisseria gonorrhoeae.

Gentamicin is a promising antibiotic for the treatment of multidrug-resistant gonorrhea. The aim of this study was to analyze the suitability and reliably of disk diffusion to monitor the susceptibility to gentamicin. We studied 237 Neisseria gonorrhoeae isolates obtained in 2013 and 2015. Reference MICs were correlated with inhibition zone diameters (in millimeters) of gentamicin 10 µg disks manufactured by BBL and Oxoid. The Pearson correlation between disk diffusion and agar dilution was r = -.68 (P < 0.001) for BBL disk and r = -.71 (P < 0.001) for Oxoid disk. No very major or major discrepancies were detected. However, a high percentage of minor discrepancies was observed (44.7%, BBL disk) and (21.9%, Oxoid disk). By adjusting the susceptible breakpoint to S ≥ 17 mm, the minor discrepancies rate was reduced to 19.4% (BBL disk) and 10.1% (Oxoid disk). The disk diffusion may be a screening method in clinical laboratories to detect the gentamicin susceptibility of N. gonorrhoeae.

Comparative Study and Analytical Verification of PCR Methods for the Diagnosis of Congenital Chagas Disease.

Congenital infection is currently the first cause of new cases of Chagas disease in Argentina and nonendemic areas worldwide. Its diagnosis is of utmost importance to guarantee curative treatment. To improve such diagnosis, a transfer process of PCR tests to the national laboratory network has been initiated. We performed a comparative study of four PCR assays [two end-point PCR and two duplex real-time quantitative PCR (qPCR) procedures] to detect Trypanosoma cruzi DNA in blood samples. Because satellite DNA and kinetoplastid DNA qPCR methods showed the best performance and the use of two different molecular targets for confirmatory purposes has been recommended, these methods were selected to perform the transfer process and, in consequence, subjected to an analytical verification protocol based on international guidelines. The anticipated reportable range was verified between 0.25 and 105 parasite equivalents per milliliter of blood (par. eq./mL) for both qPCR methods, and the limit of detection was estimated to be 0.87 (95% CI, 0.62-1.24) and 0.43 (95% CI, 0.32-0.59) par. eq./mL for satellite DNA and kinetoplastid DNA qPCR methods, respectively. In addition, both qPCR methods showed trueness and verified precision in the highest and the lowest concentrations tested. This work provides critical knowledge of the technology transfer process planned to cover laboratories of the regional network with known installed facilities.