Evaluation of antimicrobial susceptibility testing of Nocardia spp. isolates by broth microdilution with resazurin and spectrophotometry.

BACKGROUND: Nocardia species are ubiquitous in natural environments and can cause nocardiosis. In the present study, the use of Resazurin salt and Spectrophotometry were proposed as alternative methods to reduce subjectivity in the interpretation of susceptibility results to antimicrobials by the broth microdilution method for Nocardia spp. RESULTS: The susceptibility of Nocardia spp. isolates to Amikacin, Ciprofloxacin, Minocycline and Trimethoprim-Sulfamethoxazole was evaluated by Minimum Inhibitory Concentration (MIC) determinations by the broth microdilution method. To verify cellular growth, the colour-changing dye Resazurin was applied, the Optical Densities were measured on a spectrophotometer, and both were compared to Clinical and Laboratory Standards Institute (CLSI) Gold Standard method (visual MIC determination). Percentages of essential and categorical agreements and interpretative categorical errors were calculated within each method (intra-reading) and between them (inter-reading). The Gold Standard visual reading demonstrated 100% of essential and categorical intra-reading agreements for Amikacin, and there was no error when compared with the alternative methods. For Ciprofloxacin, the comparison between the Gold Standard and the Spectrophotometric reading showed 91.5% of essential agreement. In the categorical intra-reading analysis for Minocycline, there were 88.1 and 91.7% in the Gold Standard and in the Spectrophotometric readings, respectively, and 86.4% of concordance between them. High rates of categorical agreement were also observed on the Trimethoprim-Sulfamethoxazole analyses, with 93.7% for the Gold Standard, 84.9% for the Resazurin readings, and 80.5% between them. CONCLUSIONS: The alternative methods with Resazurin and Spectrophotometric readings showed high agreement rates with the Gold Standard.

Modifications of antifungal sensibility testing as suggested by CLSI document M27-A4: proposal for using different culture medium and buffer.

A common strategy in antifungal susceptibility testing is the utilization of the standardized protocol based on the microbroth dilution assay approach as described by the Clinical Laboratory Standards Institute (CLSI) (M27-A4). One major problem for laboratories in resource-limited countries with this protocol arises from the use of expensive culture media like RPMI-1640 and 3-N-morpholinopropanesulfonic acid (MOPS) buffer. One approach of circumventing this problem in cases of economic need is the evaluation of alternative culture media and buffers. The overall goal of this work was to investigate the influence of modifications in the protocol M27-A4 on diagnostic reliability. We performed univariate analyses evaluating (1) 2 different culture media (YNB and modified SAB); (2) three different buffers (sodium bicarbonate, Tris-HCL, and phosphate), as well as the influence of inoculum concentration (102, 103, 104, 105 cells/mL), the influence of incubation time, and the influence of the assessment mode (visual, biological dye, and spectrophotometer). Our results suggested that (1) RPMI-1640 may be substituted by modified SAB and (2) MOPS buffer may be substituted by Tris-HCl buffer for defined analyses. By comparing the CLSI protocol and the alternative protocol proposed in the present study (modified SAB and Tris-HCl buffer) for the assessment of fluconazole susceptibility of eighteen yeasts (clinical isolates), similar results with both methodologies were recorded. We feel that this study should stimulate a discussion on the feasibility and evolution of the M27-A4 protocol in order to include pragmatic alternatives for resource-limited settings.

Adaptación a la realidad de Latinoamérica de la Guía Clínica NASPGHAN/ESPGHAN 2016 sobre Diagnóstico, Prevención y Tratamiento de Infección por Helicobacter pylori en Pediatría / Adaptation to the reality of Latin America of the NASPGHAN/ESPGHAN 2016 Guidelines on the Diagnosis, Prevention and Treatment of Helicobacter pylori Infection in Pediatrics

Resumen: Introducción: Las últimas guías clínicas conjuntas de NASPGHAN y ESPGHAN en relación a la infección por H. pylori publicadas el año 2016, contienen 20 afirmaciones que han sido cuestionadas en la práctica respecto a su aplicabilidad en Latinoamérica (LA); en particular en relación a la preven ción del cáncer gástrico. Métodos: Se realizó un análisis crítico de la literatura, con especial énfasis en datos de LA y se estableció el nivel de evidencia y nivel de recomendación de las afirmaciones mas controversiales de las Guías Conjuntas. Se realizaron 2 rondas de votación de acuerdo a la técnica Delfi de consenso y se utilizó escala de Likert (de 0 a 4) para establecer el "grado de acuerdo" entre un grupo de expertos de SLAGHNP. Resultados: Existen pocos estudios en relación a diagnóstico, efectividad de tratamiento y susceptibilidad a antibióticos de H. pylori en pacientes pediátricos de LA. En base a estos estudios, extrapolaciones de estudios de adultos y la experiencia clínica del panel de expertos participantes, se realizan las siguientes recomendaciones. Recomendamos la toma de biopsias para test rápido de ureasa e histología (y muestras para cultivo o técnicas moleculares, cuando estén disponibles) durante la endoscopia digestiva alta sólo si en caso de confirmar la infección por H. pylori, se indicará tratamiento de erradicación. Recomendamos que centros regionales seleccio nados realicen estudios de sensibilidad/resistencia antimicrobiana para H. pylori y así actúen como centros de referencia para toda LA. En caso de falla de erradicación de H. pylori con tratamiento de primera línea, recomendamos tratamiento empírico con terapia cuádruple con inhibidor de bomba de protones, amoxicilina, metronidazol y bismuto por 14 días. En caso de falla de erradicación con el esquema de segunda línea, se recomienda indicar un tratamiento individualizado considerando la edad del paciente, el esquema indicado previamente y la sensibilidad antibiótica de la cepa, lo que implica realizar una nueva endoscopía con extracción de muestra para cultivo y antibiograma o es tudio molecular de resistencia. En niños sintomáticos referidos a endoscopía que tengan antecedente de familiar de primer o segundo grado con cáncer gástrico, se recomienda considerar la búsqueda de H. pylori mediante técnica directa durante la endoscopia (y erradicarlo cuando es detectado). Con clusiones: La evidencia apoya mayoritariamente los conceptos generales de las Guías NASPGHAN/ ESPGHAN 2016, pero es necesario adaptarlas a la realidad de LA, con énfasis en el desarrollo de centros regionales para el estudio de sensibilidad a antibióticos y mejorar la correcta selección del tratamiento de erradicación. En niños sintomáticos con antecedente familiar de primer o segundo grado de cáncer gástrico, se debe considerar la búsqueda y erradicación de H. pylori.

Abstract: Introduction: The latest joint H. pylori NASPGHAN and ESPGHAN clinical guidelines published in 2016, contain 20 statements that have been questioned in practice regarding their applicability in Latin America (LA); in particular in relation to gastric cancer prevention. Methods: We conduc ted a critical analysis of the literature, with special emphasis on LA data and established the level of evidence and level of recommendation of the most controversial claims in the Joint Guidelines. Two rounds of voting were conducted according to the Delphi consensus technique and a Likert scale (from 0 to 4) was used to establish the "degree of agreement" among a panel of SLAGHNP ex perts. Results: There are few studies regarding diagnosis, treatment effectiveness and susceptibility to antibiotics of H. pylori in pediatric patients of LA. Based on these studies, extrapolations from adult studies, and the clinical experience of the participating expert panel, the following recom mendations are made. We recommend taking biopsies for rapid urease and histology testing (and samples for culture or molecular techniques, when available) during upper endoscopy only if in case of confirmed H. pylori infection, eradication treatment will be indicated. We recommend that selected regional centers conduct antimicrobial sensitivity/resistance studies for H. pylori and thus act as reference centers for all LA. In case of failure to eradicate H. pylori with first-line treatment, we recommend empirical treatment with quadruple therapy with proton pump inhibitor, amoxi cillin, metronidazole, and bismuth for 14 days. In case of eradication failure with the second line scheme, it is recommended to indicate an individualized treatment considering the age of the pa tient, the previously indicated scheme and the antibiotic sensitivity of the strain, which implies performing a new endoscopy with sample extraction for culture and antibiogram or molecular resistance study. In symptomatic children referred to endoscopy who have a history of first or se cond degree family members with gastric cancer, it is recommended to consider the search for H. pylori by direct technique during endoscopy (and eradicate it when detected). Conclusions: The evidence supports most of the general concepts of the NASPGHAN/ESPGHAN 2016 Guidelines, but it is necessary to adapt them to the reality of LA, with emphasis on the development of regional centers for the study of antibiotic sensitivity and to improve the correct selection of the eradication treatment. In symptomatic children with a family history of first or second degree gastric cancer, the search for and eradication of H. pylori should be considered.

Factors influencing susceptibility testing of antifungal drugs: a critical review of document M27-A4 from the Clinical and Laboratory Standards Institute (CLSI).

Due to the increasing numbers of fungal infections and the emergence of drug-resistant fungi, optimization and standardization of diagnostic methods for the measurement of antifungal susceptibility are ongoing. The M27-A4 document by the US Clinical and Laboratory Standards Institute (CLSI) is presently used for the interpretation of minimum inhibitory concentrations of major opportunistic yeast species as measured by broth microdilution testing in many countries. Although microdilution is considered a benchmark for reproducible and accurate results, increased testing capacity, and limited human bias, the method is often inaccessible to routine clinical laboratories and researchers, especially in low-income countries. Furthermore, several studies suggest that there are still a considerable number of factors that make the estimation of in vitro activity of antifungal agents challenging. This review article summarizes the limitations of the M27-A4 standard which, despite the advances and improvements obtained by the standardization of antimicrobial resistance testing methods by CLSI, still persist.

[Evaluation of sensi-disk elution for colistin susceptibility determination in multidrug resistant gramnegative bacilli]. / Evaluación de la elución de sensidiscos para la determinación de susceptibilidad a colistín en bacilos gramnegativos multi-resistentes.

Using clinical strains of multidrug resistant (MDR) Gram negative bacilli, we compared MICs obtained from both broth microdilution, the reference method, and sensi-disk elution method. We found that, with A. baumannii exception, results were very similar. Sensi-disk elution method could be a good and reliable alternative for colistin resistance determination.

Coagulase-negative staphylococci in outpatient routines: the implications of switching from CLSI to BrCAST/EUCAST guidelines.

Coagulase-negative staphylococci (CoNS) are frequently isolated in clinical specimens and are important reservoirs of resistance genes. In 2019, the Brazilian government set the BrCAST/EUCAST (Brazilian Committee on Antimicrobial Susceptibility Testing) guidelines as the national standard, resulting in changes in the interpretation of CoNS susceptibility tests. From outpatients, disk-diffusion susceptibility of 65 CoNS cultures were evaluated and compared using classification criteria from both CLSI and BrCAST/EUCAST. The isolates were identified using matrix assisted laser desorption ionization-time of flight (MALDI-TOF), and the presence of the mecA gene was determined. The most prevalent species were Staphylococcus saprophyticus (32.3%), S. haemolyticus (18.5%), and S. epidermidis (9.2%). Almost perfect agreement was seen between the guidelines, except concerning oxacillin and gentamicin, and the prevalence of multidrug resistant isolates increased with the use of BrCAST/EUCAST. Of all, 15 (23.1%) isolates, mainly S. epidermidis and S. haemolyticus, were positive for the mecA gene, and only three were detected when using CLSI or BrCAST/EUCAST disk-diffusion screening. This, using either guideline, could reveal the difficulty of determining oxacillin resistance. Using warning zones or molecular methods might well be indicated for CoNS. In conclusion, adoption of the BrCAST/EUCAST guidelines will result in certain artificial changes in epidemiological susceptibility profiles, and clinicians and institutions should be aware of the possible implications.

Evaluación de la elución de sensidiscos para la determinación de susceptibilidad a colistín en bacilos gramnegativos multi-resistentes / Evaluation of sensi-disk elution for colistin susceptibility determination in multidrug resistant gramnegative bacilli

Resumen Utilizando cepas clínicas de bacilos gramnegativos multi-resistentes (MDR), comparamos las CIM obtenidas de la microdilución en caldo, el método de referencia y el método de elución de sensidiscos. Encontramos que, con la excepción de A. baumannii, los resultados fueron muy similares. El método de elución de sensidiscos podría ser una buena alternativa y confiable para la determinación de la resistencia a colistín.

Abstract Using clinical strains of multidrug resistant (MDR) Gram negative bacilli, we compared MICs obtained from both broth microdilution, the reference method, and sensi-disk elution method. We found that, with A. baumannii exception, results were very similar. Sensi-disk elution method could be a good and reliable alternative for colistin resistance determination.

[Adaptation to the reality of Latin America of the NASPGHAN/ESPGHAN 2016 Guidelines on the Diagnosis, Prevention and Treatment of Helicobacter pylori Infection in Pediatrics]. / Adaptación a la realidad de Latinoamérica de la Guía Clínica NASPGHAN/ESPGHAN 2016 sobre Diagnóstico, Prevención y Tratamiento de Infección por Helicobacter pylori en Pediatría.

INTRODUCTION: The latest joint H. pylori NASPGHAN and ESPGHAN clinical guidelines published in 2016, contain 20 statements that have been questioned in practice regarding their applicability in Latin America (LA); in particular in relation to gastric cancer prevention. METHODS: We conduc ted a critical analysis of the literature, with special emphasis on LA data and established the level of evidence and level of recommendation of the most controversial claims in the Joint Guidelines. Two rounds of voting were conducted according to the Delphi consensus technique and a Likert scale (from 0 to 4) was used to establish the "degree of agreement" among a panel of SLAGHNP ex perts. RESULTS: There are few studies regarding diagnosis, treatment effectiveness and susceptibility to antibiotics of H. pylori in pediatric patients of LA. Based on these studies, extrapolations from adult studies, and the clinical experience of the participating expert panel, the following recom mendations are made. We recommend taking biopsies for rapid urease and histology testing (and samples for culture or molecular techniques, when available) during upper endoscopy only if in case of confirmed H. pylori infection, eradication treatment will be indicated. We recommend that selected regional centers conduct antimicrobial sensitivity/resistance studies for H. pylori and thus act as reference centers for all LA. In case of failure to eradicate H. pylori with first-line treatment, we recommend empirical treatment with quadruple therapy with proton pump inhibitor, amoxi cillin, metronidazole, and bismuth for 14 days. In case of eradication failure with the second line scheme, it is recommended to indicate an individualized treatment considering the age of the pa tient, the previously indicated scheme and the antibiotic sensitivity of the strain, which implies performing a new endoscopy with sample extraction for culture and antibiogram or molecular resistance study. In symptomatic children referred to endoscopy who have a history of first or se cond degree family members with gastric cancer, it is recommended to consider the search for H. pylori by direct technique during endoscopy (and eradicate it when detected). CONCLUSIONS: The evidence supports most of the general concepts of the NASPGHAN/ESPGHAN 2016 Guidelines, but it is necessary to adapt them to the reality of LA, with emphasis on the development of regional centers for the study of antibiotic sensitivity and to improve the correct selection of the eradication treatment. In symptomatic children with a family history of first or second degree gastric cancer, the search for and eradication of H. pylori should be considered.

A Multicenter Study To Evaluate Ceftaroline Breakpoints: Performance in an Area with High Prevalence of Methicillin-Resistant Staphylococcus aureus Sequence Type 5 Lineage.

Ceftaroline (CPT) is a broad-spectrum agent with potent activity against methicillin-resistant Staphylococcus aureus (MRSA). The sequence type 5 (ST5) Chilean-Cordobés clone, associated with CPT nonsusceptibility, is dominant in Chile, a region with high rates of MRSA infections. Here, we assessed the in vitro activity of CPT against a collection of MRSA isolates collected between 1999 and 2018 from nine hospitals (n = 320) and community settings (n = 41) in Santiago, Chile, and evaluated performance across testing methodologies. We found that our hospital-associated isolates exhibited higher CPT MIC distributions (MIC50 and MIC90 of 2 mg/liter) than the community isolates (MIC50 and MIC90 of 0.5 mg/liter), a finding that was consistent across time and independent of the culture source. High proportions (64%) of isolates were CPT nonsusceptible despite the absence of CPT use in Chile. Across methodologies, the Etest underestimated the MIC relative to the gold standard broth microdilution (BMD) test (MIC50 and MIC90 of 1 and 1.5 mg/liter, respectively). There was low (∼51%) categorical agreement (CA) between Etest and BMD results across CLSI and EUCAST breakpoints. The recent revision of CLSI guidelines abolished "very major error" (VME) from the previous guidelines (81%), which perform similarly to the EUCAST guidelines. The level of concordance between CLSI and EUCAST for BMD testing and Etest was >95%. Disk diffusion performed poorly relative to BMD under CLSI (CA, 55%) and EUCAST (CA, 36%) guidelines. Comparison of EUCAST to CLSI for disk diffusion (with EUCAST used as the reference) showed low agreement (CA, 25%; VME, 70%). In summary, CPT-nonsusceptible MRSA are dominant in clinical settings in Chile. Our results provide data to support the reevaluation of CPT breakpoints and to improve agreement across methodologies and agencies.

Comparative antifungal susceptibility analyses of Cryptococcus neoformans VNI and Cryptococcus gattii VGII from the Brazilian Amazon Region by the Etest, Vitek 2, and the Clinical and Laboratory Standards Institute broth microdilution methods.

Early diagnosis, efficient clinical support, and proper antifungal therapy are essential to reduce death and sequels caused by cryptococcosis. The emergence of resistance to the antifungal drugs commonly used for cryptococcosis treatment is an important issue of concern. Thus, the in vitro antifungal susceptibility of clinical strains from northern Brazil, including C. neoformans VNI (n = 62) and C. gattii VGII (n = 37), to amphotericin B (AMB), 5-flucytosine, fluconazole, voriconazole, and itraconazole was evaluated using the Etest and Vitek 2 systems and the standardized broth microdilution (CLSI-BMD) methodology. According to the CLSI-BMD, the most active in vitro azole was voriconazole (C. neoformans VNI modal MIC of 0.06 µg/ml and C. gattii VGII modal MIC of 0.25 µg/ml), and fluconazole was the least active (modal MIC of 4 µg/ml for both fungi). Modal MICs for amphotericin B were 1 µg/ml for both fungi. In general, good essential agreement (EA) values were observed between the methods. However, AMB presented the lowest EA between CLSI-BMD and Etest for C. neoformans VNI and C. gattii VGII (1.6% and 2.56%, respectively, P < .05 for both). Considering the proposed Cryptococcus spp. epidemiological cutoff values, more than 97% of the studied isolates were categorized as wild-type for the azoles. However, the high frequency of C. neoformans VNI isolates in the population described here that displayed non-wild-type susceptibility to AMB is noteworthy. Epidemiological surveillance of the antifungal resistance of cryptococcal strains is relevant due to the potential burden and the high lethality of cryptococcal meningitis in the Amazon region.

MODS-Wayne, a Colorimetric Adaptation of the Microscopic-Observation Drug Susceptibility (MODS) Assay for Detection of Mycobacterium tuberculosis Pyrazinamide Resistance from Sputum Samples.

Although pyrazinamide (PZA) is a key component of first- and second-line tuberculosis treatment regimens, there is no gold standard to determine PZA resistance. Approximately 50% of multidrug-resistant tuberculosis (MDR-TB) and over 90% of extensively drug-resistant tuberculosis (XDR-TB) strains are also PZA resistant. pncA sequencing is the endorsed test to evaluate PZA susceptibility. However, molecular methods have limitations for their wide application. In this study, we standardized and evaluated a new method, MODS-Wayne, to determine PZA resistance. MODS-Wayne is based on the detection of pyrazinoic acid, the hydrolysis product of PZA, directly in the supernatant of sputum cultures by detecting a color change following the addition of 10% ferrous ammonium sulfate. Using a PZA concentration of 800 µg/ml, sensitivity and specificity were evaluated at three different periods of incubation (reading 1, reading 2, and reading 3) using a composite reference standard (MGIT-PZA, pncA sequencing, and the classic Wayne test). MODS-Wayne was able to detect PZA resistance, with a sensitivity and specificity of 92.7% and 99.3%, respectively, at reading 3. MODS-Wayne had an agreement of 93.8% and a kappa index of 0.79 compared to the classic Wayne test, an agreement of 95.3% and kappa index of 0.86 compared to MGIT-PZA, and an agreement of 96.9% and kappa index of 0.90 compared to pncA sequencing. In conclusion, MODS-Wayne is a simple, fast, accurate, and inexpensive approach to detect PZA resistance, making this an attractive assay especially for low-resource countries, where TB is a major public health problem.

A quantitative adaptation of the Wayne test for pyrazinamide resistance.

BACKGROUND: Pyrazinamide (PZA) is the most important drug against the latent stage of tuberculosis (TB) and is used in both first and second line treatment regimens. The continued increase in multi-drug resistant TB and the prevalence of PZA resistance makes the development of alternative assays for prompt identification of PZA resistance all the more important. METHODS: We standardized and evaluated a quantitative variant of the Wayne assay (QW) for determining PZA resistance in Mycobacterium tuberculosis strains. This assay quantifies M. tuberculosis metabolism of PZA and production of pyrazinoic acid (POA) using visible spectrophotometry. We evaluated this method using PZA concentrations of 400 µg/ml and 800 µg/ml at incubation periods of 3, 5 and 7 days. M. tuberculosis strains from 68 sputum samples were also tested with the standard Wayne assay, Tetrazolium Microplate Assay (TEMA), Bactec 460TB and pncA sequencing. We compared QW and standard Wayne assay against a dichotomous reference classification using concordant Bactec 460TB and pncA sequencing. Secondarily, we determined the quantitative correlation between both QW values and TEMA's minimum inhibitory concentration (MIC) against Bactec 460TB percentage growth. RESULTS: The standard Wayne showed sensitivity of 88% and specificity of 97.5%, giving a Youden Index (YI) of 0.855 against reference tests. The QW showed maximum YI of 0.934 on day 7 at 400 µg/ml PZA with 96% sensitivity and 97.4% specificity. Absorbance OD values for 400 µg/ml PZA were more accurate than 800 µg/ml PZA. Although QW showed high accuracy for PZA susceptibility, it did not correlate quantitatively with Bactec percentage growth. TEMA testing was unreliable and did not correlate with Bactec results. CONCLUSIONS: The proposed QW assay is an inexpensive method capable of providing standardization and automation of colorimetric PZA resistance testing, with better discriminatory than the standard Wayne assay.

Accuracy of a vancomycin brain heart infusion screening plate for the screening of Staphylococcus aureus isolates with increased vancomycin minimum inhibitory concentrations.

Vancomycin susceptibility was determined in 125 S. aureus isolates by disk diffusion, microdilution, Etest and vancomycin brain heart infusion (BHI) plate. A 2.0 mg/L vancomycin BHI was highly sensitive (100% and 91% compared to Etest and microdilution) for detecting a MIC≥2 mg/L, and could be used as a simple and affordable screening test.

Development, optimization and validation of a rapid colorimetric microplate bioassay for neomycin sulfate in pharmaceutical drug products.

Microbiological assays have been used to evaluate antimicrobial activity since the discovery of the first antibiotics. Despite their limitations, microbiological assays are widely employed to determine antibiotic potency of pharmaceutical dosage forms, since they provide a measure of biological activity. The aim of this work is to develop, optimize and validate a rapid colorimetric microplate bioassay for the potency of neomycin in pharmaceutical drug products. Factorial and response surface methodologies were used in the development and optimization of the choice of microorganism, culture medium composition, amount of inoculum, triphenyltetrazolium chloride (TTC) concentration and neomycin concentration. The optimized bioassay method was validated by the assessment of linearity (range 3.0 to 5.0µg/mL, r=0.998 and 0.994 for standard and sample curves, respectively), precision (relative standard deviation (RSD) of 2.8% and 4.0 for repeatability and intermediate precision, respectively), accuracy (mean recovery=100.2%) and robustness. Statistical analysis showed equivalency between agar diffusion microbiological assay and rapid colorimetric microplate bioassay. In addition, microplate bioassay had advantages concerning the sensitivity of response, time of incubation, and amount of culture medium and solutions required.

Reducing communication delays and improving quality of care with a tuberculosis laboratory information system in resource poor environments: a cluster randomized controlled trial.

BACKGROUND: Lost, delayed or incorrect laboratory results are associated with delays in initiating treatment. Delays in treatment for Multi-Drug Resistant Tuberculosis (MDR-TB) can worsen patient outcomes and increase transmission. The objective of this study was to evaluate the impact of a laboratory information system in reducing delays and the time for MDR-TB patients to culture convert (stop transmitting). SETTING: 78 primary Health Centers (HCs) in Lima, Peru. Participants lived within the catchment area of participating HCs and had at least one MDR-TB risk factor. The study design was a cluster randomized controlled trial with baseline data. The intervention was the e-Chasqui web-based laboratory information system. Main outcome measures were: times to communicate a result; to start or change a patient's treatment; and for that patient to culture convert. RESULTS: 1671 patients were enrolled. Intervention HCs took significantly less time to receive drug susceptibility test (DST) (median 11 vs. 17 days, Hazard Ratio 0.67 [0.62-0.72]) and culture (5 vs. 8 days, 0.68 [0.65-0.72]) results. The time to treatment was not significantly different, but patients in intervention HCs took 16 days (20%) less time to culture convert (p = 0.047). CONCLUSIONS: The eChasqui system reduced the time to communicate results between laboratories and HCs and time to culture conversion. It is now used in over 259 HCs covering 4.1 million people. This is the first randomized controlled trial of a laboratory information system in a developing country for any disease and the only study worldwide to show clinical impact of such a system. TRIAL REGISTRATION: ClinicalTrials.gov NCT01201941.

Interlaboratory variability of Caspofungin MICs for Candida spp. Using CLSI and EUCAST methods: should the clinical laboratory be testing this agent?

Although Clinical and Laboratory Standards Institute (CLSI) clinical breakpoints (CBPs) are available for interpreting echinocandin MICs for Candida spp., epidemiologic cutoff values (ECVs) based on collective MIC data from multiple laboratories have not been defined. While collating CLSI caspofungin MICs for 145 to 11,550 Candida isolates from 17 laboratories (Brazil, Canada, Europe, Mexico, Peru, and the United States), we observed an extraordinary amount of modal variability (wide ranges) among laboratories as well as truncated and bimodal MIC distributions. The species-specific modes across different laboratories ranged from 0.016 to 0.5 µg/ml for C. albicans and C. tropicalis, 0.031 to 0.5 µg/ml for C. glabrata, and 0.063 to 1 µg/ml for C. krusei. Variability was also similar among MIC distributions for C. dubliniensis and C. lusitaniae. The exceptions were C. parapsilosis and C. guilliermondii MIC distributions, where most modes were within one 2-fold dilution of each other. These findings were consistent with available data from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (403 to 2,556 MICs) for C. albicans, C. glabrata, C. krusei, and C. tropicalis. Although many factors (caspofungin powder source, stock solution solvent, powder storage time length and temperature, and MIC determination testing parameters) were examined as a potential cause of such unprecedented variability, a single specific cause was not identified. Therefore, it seems highly likely that the use of the CLSI species-specific caspofungin CBPs could lead to reporting an excessive number of wild-type (WT) isolates (e.g., C. glabrata and C. krusei) as either non-WT or resistant isolates. Until this problem is resolved, routine testing or reporting of CLSI caspofungin MICs for Candida is not recommended; micafungin or anidulafungin data could be used instead.

Anti-Aspergillus activity of green coffee 5-O-caffeoyl quinic acid and its alkyl esters.

The antifungal activities of 5-O-caffeoyl quinic acid (5-CQA) and of methyl, butyl, octyl, and dodecyl esters or 5-CQA, were tested on five toxigenic moulds from the Aspergillus genus (Aspergillus flavus, Aspergillus nomius, Aspergillus ochraceus, Aspergillus parasiticus, Aspergillus westerdijkiae). These mycotoxin producers' moulds may contaminate many types of food crops throughout the food chain posing serious health hazard to animals and humans. The use of chemical methods to decrease mycotoxin producer moulds contamination on food crops in the field, during storage, and/or during processing, has been proved to be efficient. In this work, the antifungal effect of 5-CQA and a homologous series of 5-CQA esters (methyl, butyl, octyl, dodecyl), was investigated using the microdilution method and the minimum inhibitory concentrations (MIC50 and MIC80). All molecules presented antifungal activity, and two esters showed a MIC for all fungi: octyl (MIC50 ≤ 0.5-0.75 mg/mL, MIC80 = 1.0-1.5 mg/mL) and dodecyl (MIC50 = 0.75-1.25 mg/mL) chlorogenates. Dodecyl chlorogenate showed a MIC80 (1.5 mg/mL) only for A. parasiticus. The maximum percent of growth inhibition on aspergillii was observed with octyl (78.4-92.7%) and dodecyl (54.5-83.7%) chlorogenates, being octyl chlorogenate the most potent antifungal agent. It was thus concluded that lipophilization improved the antifungal properties of 5-CQA, which increased with the ester alkyl chain length, exhibiting a cut-off effect at 8 carbons. As far as we know, it is the first report demonstrating that lipophilization may improve the antifungal activity of 5-CQA on five toxigenic moulds from the Aspergillus genus. Lipophilization would be a novel way to synthesize a new kind of antifungal agents with a good therapeutic value or a potential use as preservative in food or cosmetics.

Influence of different media, incubation times, and temperatures for determining the MICs of seven antifungal agents against Paracoccidioides brasiliensis by microdilution.

MIC assays with Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis, had been conducted with variable protocols, employing both macrodilution and microdilution tests and including differences in inoculum preparation, media used, incubation periods, and temperatures. Twenty-one clinical and environmental isolates of Paracoccidioides were tested using amphotericin B, itraconazole, ketoconazole, fluconazole, sulfamethoxazole, sulfamethoxazole-trimethoprim, and terbinafine, according to the National Committee for Clinical Laboratory Standards (National Committee for Clinical Laboratory Standards, document M27-A2, 2002), with modifications such as three medium formulations (RPMI 1640 medium, McVeigh and Morton [MVM] medium, and modified Mueller-Hinton [MMH] medium), two incubation temperatures (room temperature [25 to 28 °C] and 37 °C), and three incubation periods (7, 10, and 15 days). The antifungal activities were also classified as fungicidal or fungistatic. The best results were obtained after 15 days of incubation, which was chosen as the standard incubation time. The MICs for most individual isolates grown for the same length of time at the same temperature varied with the different media used (P < 0.05). Of the isolates, 81% showed transition from the yeast to the mycelial form in RPMI 1640 medium at 37 °C, independent of the presence of antifungals. MMH medium appears to be a suitable medium for susceptibility testing of antifungal drugs with P. brasiliensis, except for sulfamethoxazole and the combination of sulfamethoxazole-trimethoprim, for which the MVM medium yielded better results. The incubation temperature influenced the MICs, with, in general, higher MICs at 25 °C (mycelial form) than at 37 °C (P < 0.05). Based on our results, we tentatively propose a microdilution assay protocol for susceptibility testing of antifungal drugs against Paracoccidioides.

[Resistance to extended-spectrum cephalosporins in non-inducible AmpC enterobacteria: evaluation of the new MIC breakpoints]. / Resistencia a cefalosporinas de espectro extendido en enterobacterias sin AmpC inducible: Evaluación de los nuevos puntos de corte.

The aims of this study were to evaluate the in vitro activity of extended-spectrum cephalosporins (ESC) in non-inducible AmpC enterobacteria throµgh phenotypic and genotypic characterization of the mechanisms of resistance (ESBL, plasmid-mediated AmpC and KPC) and to evaluate the interpretation criteria proposed by the existing recommendations and the new breakpoints established by the CLSI and the EUCAST. Susceptibility tests and PCR multiplex for b/aSHV and b/aCTX-M and amplification using specific primers was performed. One hundred sixty nine resistant isolates: K/ebsie//a pneumoniae (95), Escherichia co/i (55), and Proteus mirabi/is (19) were recovered. ESC resistance was 56.2 %, 32.6%, and 11.2 %, respectively. ESBL was detected in 152 (90 %) isolates, plasmid-mediated AmpC in 12 (7 %) and KPC in 5 (3 %). The CLSI 2009 recommendations and the breakpoints sµggested by the CLSI 2010 and the EUCAST for ceftriaxone were efficacious to detect ESBL, whereas the different breakpoints for ceftazidime presented discrepancies. The CLSI 2010 breakpoints only detected 55 % of the ESBL-producing isolates due to the endemic presence of CTX-M ESBLs in our country. Regarding the plasmid-mediated AmpC producers, the recommendations of the CLSI 2010 and the EUCAST 2010 proved to be more efficient than the old ones.