Analysis of antibiotic resistance and genetic profile of conjunctival bacteria flora before and after cataract surgery.

PURPOSE: To analyze antibiotic resistance and genetic profile of conjunctival bacteria flora before and after cataract surgery with the focus on coagulase-negative staphylococci (CNS) during cataract surgery and discuss the implications of this colonization as a potential risk of acquiring endophthalmitis. METHODS: After approval of the institutional review board and informed consent from patients had been obtained, conjunctival swabs for culture from 59 patients undergoing cataract surgery were taken of the fellow eye at baseline (C0) and from the eye to be operated before (T0) and after (T1) irrigation with povine-iodine 5%, and at the end of surgery (T2). Genes responsible for virulence (mecA, ica and atlE) and antibiotic profile were determined; strain clonality of persistent colonizing Staphylococcus epidermidis strains was established by the Multi-locus sequence typing (MLST). RESULTS: The frequency of CNS was significantly reduced in T1 (13.6%) from 81.4% in T0 and 86.4% in C0. The frequency of mecA, ica and atlE genes was 34.4%, 37.5% and 61.4%, respectively; and methicillin phenotypic resistance was 35.4%. S. epidermidis was the most frequent species isolated in every time point. MLST revealed in 7 patients 100% coincidence of the seven alleles of the S. epidermidis isolated previous to povine-iodine 5% disinfection and at the end of the surgery. CNS isolates from T1 or T2 corresponded to the same species, antibiotic and virulence profile as those isolates from C0 or T0. CONCLUSION: Povidone-iodine 5% prophylaxis before surgery significantly reduced conjunctival contamination; in those that persisted, the source of contamination was mostly the patient's microbiota confirmed by the MLST system.

[Keratitis due to Mycobaterium abscessus. First case report in Paraguay]. / Queratitis por Mycobaterium abscessus. Comunicación del primer caso en Paraguay.

We present a clinical case of keratitis caused by M. abscessus in a 76-year-old female patient, resident in the city of Asunción, without trauma or previous ocular surgery and with a history of herpetic keratouveitis. Because it is a keratitis caused by a rare etiological agent and because of the importance of a correct and timely diagnosis for the establishment of appropriate treatment, the present case is reported, the first of Mycobacteria keratitis in Paraguay.

Queratitis por Mycobaterium abscessus: comunicación del primer caso en Paraguay / Keratitis due to Mycobaterium abscessus: first case report in Paraguay

Resumen Se presenta un caso clínico de queratitis por Mycobacterium abscessus en una mujer de 76 años, residente en la ciudad de Asunción, sin traumatismo ni cirugía ocular previa y con antecedente de una queratouveitis herpética. Por tratarse de una queratitis causada por un agente etiológico poco frecuente y por la importancia de un diagnóstico correcto y oportuno para la instauración del tratamiento adecuado, se comunica el primer caso de queratitis por micobacterias en Paraguay.

Abstract We present a clinical case of keratitis caused by M. abscessus in a 76-year-old female patient, resident in the city of Asunción, without trauma or previous ocular surgery and with a history of herpetic keratouveitis. Because it is a keratitis caused by a rare etiological agent and because of the importance of a correct and timely diagnosis for the establishment of appropriate treatment, the present case is reported, the first of Mycobacteria keratitis in Paraguay.

Especies y sensibilidad a antifúngicos de cepas de Candida spp. aisladas de la cavidad bucal de pacientes con VIH de Paraguay

RESUMEN Candida albicans es la levadura más aislada de la cavidad oral de personas con VIH. Sin embargo, el uso de antifúngicos ha incrementado el aislamiento de especies no C. albicans resistentes o con sensibilidad reducida. El objetivo del estudio fue determinar las especies de Candida y la sensibilidad a los antifúngicos de aislamientos de la cavidad bucal de pacientes con VIH, que acudieron al Instituto de Medicina Tropical entre julio a diciembre de 2019. Las levaduras se sembraron en Agar Cromogénico Candida (CONDA®, España) para la identificación presuntiva. Las colonias verdes se identificaron por PCR dúplex y las de coloración distinta o PCR negativa mediante el sistema comercial VITEK®2. La susceptibilidad antifúngica de C. albicans se determinó por VITEK®2, y las no albicans por el método de difusión de discos. De 278 levaduras, C. albicans se aisló en 70,9 %, seguido de C. tropicalis (8,3 %) y C. krusei (5 %). C. albicans presentó 93 % de sensibilidad y 2 % de sensibilidad dosis dependiente al fluconazol; 87 % sensibles y 3 % con sensibilidad intermedia frente a voriconazol; 97, 98 y 100 % sensibilidad a flucitosina, anfotericina B y equinocandinas, respectivamente. C. parapsilosis y C. tropicalis no registraron resistencias a fluconazol y voriconazol. Todos los aislamientos de C. glabrata fueron sensibles dosis dependientes a fluconazol y todos los aislamientos de C. krusei resultaron sensibles a voriconazol. La portación oral de Candida spp. resistentes a azoles podría llevar a fracasos terapéuticos en pacientes con VIH que desarrollen candidiasis orofaríngea.

ABSTRACT Candida albicans is the most isolated yeast from the oral cavity of people with HIV. However, the use of antifungals has increased the isolation of non-C. albicans resistant species or those with reduced sensitivity. The objective of the study was to determine the Candida species and the sensitivity to antifungals of isolates from the oral cavity of patients with HIV who attended the Institute of Tropical Medicine between July and December 2019. The yeasts were plated in Candida Chromogenic Agar (CONDA®, Spain) for presumptive identification. Green colonies were identified by duplex PCR and those with different staining or negative PCR using the commercial VITEK®2 system. The antifungal susceptibility of C. albicans was determined by VITEK®2, and non albicans by the disk diffusion method. Of 278 yeasts, C. albicans was isolated in 70.9 %, followed by C. tropicalis (8.3 %) and C. krusei (5 %). C. albicans presented 93 % sensitivity and 2 % dose-dependent sensitivity to fluconazole; 87 % sensitive and 3 % intermediate sensitivity to voriconazole; 97, 98, and 100 % sensitive to flucytosine, amphotericin B, and echinocandins, respectively. C. parapsilosis and C. tropicalis did not register resistance to fluconazole and voriconazole. All C. glabrata isolates were dose-dependently sensitive to fluconazole and all C. krusei isolates were sensitive to voriconazole. The oral carriage of Candida spp. resistant to azoles may lead to therapeutic failures in HIV patients who develop oropharyngeal candidiasis.

Sensibilidad in vitro de Escherichia coli a la fosfomicina en urocultivos en un Servicio Asistencial Privado de Asunción / In vitro sensitivity of Escherichia coli to fosfomycin in urine cultures in a private healthcare service in Asunción

La fosfomicina es un antibiótico natural, que actúa sobre la síntesis de la pared celular, con actividad bactericida y de amplio espectro. En este trabajo se evaluó la sensibilidad in vitro de aislados de Escherichia coli (E. coli), incluidos aquellos que producen Beta Lactamasa de Espectro Extendido (BLEE) obtenidos a partir de urocultivos, tomados en diferentes lapsos de colección de datos, en personas de ambos sexos. Fueron incluidos 260 muestras de orina con desarrollo de E. coli provenientes de pacientes que concurrieron al Laboratorio San Roque. El aislamiento e identificación bacteriana se realizó según métodos convencionales y la sensibilidad a los antimicrobianos por el método de difusión de disco. Para la detección de la sensibilidad frente a fosfomicina fueron utilizados discos de 200 µg con el agregado de 50 µg de glucosa 6-fosfato. Se observó frente a los antibióticos evaluados mayor sensibilidad a fosfomicina (98,5%) y nitrofurantoína (97,7%). Ciprofloxacina, trimetoprima y la combinación sulfametoxazol/trimetoprima exhibieron frente a los mismos aislados sensibilidad menor y muy similar entre ellos, con 64,2%, 61,2% y 61,2% respectivamente. En 44 (16,9%) de los aislados de E. coli se detectó la presencia de BLEE y es destacable la alta sensibilidad que mostraron fosfomicina y nitrofurantoína, aún frente a los aislados BLEE positivos, con frecuencias de 90,9% y 93,2%, respectivamente. En resumen, la alta sensibilidad demostrada en el presente estudio por E. coli ante la fosfomicina, abre la posibilidad de considerar a este antibiótico de primera elección en las infecciones urinarias bajas, aún en los casos de gérmenes productores de BLEE, en la población de nuestro país.

Fosfomycin is a natural antibiotic, which acts on the synthesis of the cell wall, with broad spectrum bactericidal activity. In this study, the in vitro sensitivity of Escherichia coli (E. coli) isolates was evaluated, including those that produce Extended Spectrum Beta Lactamase (ESBL) obtained from urine cultures taken at different data collection times, in people of both sexes. Were included 260 urine samples with development of E. coli from patients who attended the San Roque laboratory. Bacterian isolation and identification was carried out according to conventional methods and antimicrobial sensitivity by the disk diffusion method. For detection of sensitivity to fosfomycin, 200 µg discs were used with the addition of 50 µg of glucose 6-phosphate. A greater sensitivity for fosfomycin (98.5%) and nitrofurantoin (97.7%) was observed against the evaluated antibiotics. Ciprofloxacin, trimethoprim and the sulfamethoxazole / trimethoprim combination exhibited in front of the same isolated lower sensitivity and very similar among them, with 64.2%, 61.2% and 61.2% respectively. In 44 (16.9%) of the E. coli isolates the presence of ESBL was detected and the high sensitivity shown by fosfomycin and nitrofurantoin is noteworthy, even compared to the positive ESBL isolates, with frequencies of 90.9% and 93,2%, respectively. In summary, the high sensitivity demonstrated in the present study by E. coli to fosfomycin opens the possibility of considering this first-choice antibiotic in lower urinary infections, even in ESBL-producing germs, in the population of our country.

Diagnóstico de endoftalmitis por método microbiológico convencional y molecular en pacientes de centros oftalmológicos de Paraguay / Diagnosis of endophthalmitis by molecular and conventional microbiological methods in patients from ophthalmologic centers of Paraguay

La endoftalmitis infecciosa es una condición intraocular grave, aunque poco frecuente, que puede llevar a la pérdida de la visión. El diagnóstico etiológico precoz y el tratamiento adecuado llevan a una mejor evolución. Las técnicas microbiológicas convencionales tienen baja sensibilidad en el diagnóstico de la endoftalmitis infecciosa, en cambio la reacción en cadena de la polimerasa (PCR) es un método de mayor sensibilidad que está siendo utilizado ampliamente. El objetivo fue determinar por cultivo y PCR la etiología de la endoftalmitis infecciosa en pacientes que consultaron en centros oftalmológicos de Paraguay. Se estudiaron muestras de humor vítreo obtenidas por punción de 51 pacientes con endoftalmitis. La muestra se dividió en dos alícuotas; una para el estudio microbiológico convencional y la otra para la PCR anidada. Esta última utiliza un par de cebadores basado en secuencias conservadas del gen 16S ribosomal y cebadores específicos para bacterias gram positivas. Se estandarizó la PCR logrando una sensibilidad de detección de 3.10-5 ng/µl para S. aureus, 2,5.10-3 ng/µl para S. pneumoniae y 6.10-7 ng/µl para E. coli. De las 51 muestras, 12 (23,5%) fueron positivas por métodos microbiológicos convencionales y 26 (51,0%) por la PCR anidada. Por cultivo se identificaron predominantemente bacterias grampositivas 8/11 (73%) y un aislado de hongo filamentoso. La PCR anidada presentó muy buena sensibilidad y de gran utilidad en la identificación del origen bacteriano de la endoftalmitis, mejorando la sensibilidad del cultivo en 27,5%. La discriminación bacteriana en grampositivos y gramnegativos por la PCR se correlacionó con el cultivo en el 100% de los casos(AU)

Methicillin resistance and biofilm production of Staphylococcus epidermidis isolates from infectious and normal flora conjunctiva.

PURPOSE: Coagulase-negative staphylococci have been reported to be the most frequent cause of bacterial postoperative endophthalmitis. Biofilm formation is the major virulence factor of Staphylococcus epidermidis and is often associated with methicillin resistance. This study aims at evaluating the presence of biofilm-producing and methicillin resistance genes (mecA) in S. epidermidis. METHODS: S. epidermidis isolated from clinically infected sites (group 1) and from normal human conjunctiva (group 2) were studied. All the isolates were tested for their ability to produce biofilm by the conventional Christensen´s method and the presence of mecA by PCR using the 22-mer oligonucleotides as primers. RESULTS: In total 20 isolates from group 1 and 22 from group 2 were studied. Biofilm and mecA were detected in 15 (75 %) and in 14 (70 %) in group 1 as compared to 8 (36.3 %) and 4 (18.2 %) in group 2 (p = 0.016). Simultaneously, biofilm production and presence of mecA genes were observed in 13/20 (65.0 %) in group 1, and 4/22 (18.2 %) in group 2 (p = 0.002). Multi-resistance was observed in 55 % in group 1 and 9 % in group 2 (p = 0.002); 57 % of the biofilm-producing strains was multi-resistant in contrast to none of the non-producing strains. In all multi-resistant strains, biofilm production was seen. CONCLUSIONS: Biofilm formation capacity was widely distributed, particularly among mecA (+) S. epidermidis strains, which also displayed a high diversity of antibiotic resistance profiles.

Effect of antibiotic prophylaxis on Coagulase-negative Staphylococcus virulence factor profiles in patients undergoing cataract surgery.

PURPOSE: In this prospective study, multiplex polymerase chain reaction (PCR) was used to identify genes encoding virulence factors (ica, atlE and mecA) in Coagulase-negative Staphylococcus (CNS) isolates from the ocular microbiota of patients undergoing cataract surgery and to investigate possible changes in the CNS profile due to antibiotic prophylaxis. METHODS: Between 09/2011 and 08/2013, patients undergoing cataract surgery were recruited at the Department of Ophthalmology, National University of Asuncion, Paraguay. In the eye to be operated on, patients received moxifloxacin 0.5 % eye drops four times at the day before surgery and a last drop 1 hour before surgery (T1). The other eye remained as control (T0). Conjunctival swabs were taken from both eyes 1 hour after the last drop. The presence of genes encoding biofilm formation (ica and atlE) and methicillin resistance (mecA) was detected by a multiplex PCR. RESULTS: Of the 162 patients (162 study eyes, 162 fellow eye as control group), 87 (53.7 %) eyes were positive for CNS at T0 yielding 96 CNS isolates; 70 eyes (43.2 %) were positive at T1 yielding 77 CNS isolates. For this study, 43 CNS isolates (44.8 %) from T0 and 45 (64.3 %) from T1 were used. Of the total isolates, 81.8 % (72/88) had at least one virulence factor gene (37/43 from T0 and 35/45 from T1) (p = 0.314). Simultaneous detection of ica and atlE genes was higher in T0 (58.0 %) than T1 (46.7 %), but the difference was not significant (p = 0.28). CONCLUSION: A high frequency of genes encoding virulence factors was observed in the coagulase-negative Staphylococcus isolates. The use of moxifloxacin did not significantly modify the CNS virulence factor profiles.

Frecuencia de staphylococcus aureus meticilino resistente y del factor de virulencia PVL en pacientes ambulatorios con infección de piel y partes blandas de Asunción, Paraguay / Frequency of methicillin-resistant staphylococcus aureusand PVL virulence factor in ambulatory patients with skinand soft tissue infections of Asuncion, Paraguay

Staphylococcus aureus (S. aureus) es actualmente el agente etiológico más frecuente en infecciones de piel y tejidos blandos. El S. aureus meticilino resistente (SAMR) aislado en infecciones de pacientes de la comunidad ha ido aumentando, constituyéndose en un problema de salud pública a nivel mundial. En Paraguay existen pocos registros sobre la meticilino resistencia y factores de virulencia a nivel comunitario, por lo que se realizó este estudio observacional descriptivo para determinar la frecuencia de SAMR y del factor de virulencia leucocidina de Panton Valentine (PVL-Panton Valentine leukocidin), así como el perfil de resistencia antimicrobiana acompañante a la meticilino resistencia en S. aureus aisladosde infecciones de piel y partes blandas de pacientes ambulatorios de dos laboratorios de Asunción, Paraguay, entre octubre de 2012 a febrero de 2014. La identificación bacteriana se realizó mediante técnicas microbiológicas convencionales y la susceptibilidad antimicrobiana por la prueba de difusión en disco. Elgen mecA y luk-PV fueron detectados por la técnica de PCR. De los 70 aislados de S. aureus estudiados, el 54,3% (38/70) fue SAMR tanto por método fenotípico como molecular. La frecuenciade PVL fue de 15,7% (11/70), siendo mayor en los SAMR (21%; 8/38) que en los SAMS (9,4%; 3/32). El 2,6% de los SAMR presentó resistencia a ciprofloxacina, no se observó multiresistencia en ningún aislado. Se encontró alta frecuencia de SAMR comparado con reportes previos en Paraguay. Se requiere fortalecer estrategias de vigilancia, prevención y control de la resistencia bacteriana en ambientes hospitalarios y de la comunidad.

Staphylococcus aureus (S. aureus) is currently the most common etiologic agentof skin and soft tissue infections. The isolation of methicillin-resistant S. aureus(MRSA) from infections of patients in the community has increased, becoming a public health problem worldwide. In Paraguay, there are few records aboutmethicillin resistance and virulence factors at community level. Therefore, this descriptive observational study was performed to determine the frequency of MRSA and factor virulence of Panton-Valentine leukocidin (PVL) as well as the antimicrobial resistance profile accompanying methicillin resistance in S. aureusisolated from skin and soft tissue infections in ambulatory patients from two laboratories of Asuncion, Paraguay from October, 2012 to February, 2014. The bacterial identification was performed using conventional microbiological techniques and the antimicrobial susceptibility was determined by disk diffusion. The mecA andluk-PV genes were detected by PCR technique. Out of the 70 S. aureus isolates studied, 54.3% (38/70) was SAMR by phenotypic and molecular methods. PVL frequency was 15.7% (11/70) being higher in MRSA (21%; 8/38) than in the SAMS (9.4%; 3/32), 2.6% of the MRSA was resistant to ciprofloxacin and multidrug resistance was not observed in any isolates. A high frequency of MRSA was found compared with previous reports in Paraguay. It is required to strengthen surveillance, prevention and control of bacterial resistance strategies in hospital and community environments.

Enterobacterias productoras de betalactamasas de espectro extendido aisladas de pacientes ambulatorios y hospitalizados en un laboratorio privado de Asunción / Extended spectrum β lactamases producing Enterobacteriaceae isolated from outpatient and hospitalized patients in a private laboratory in Asunción, Paraguay

Entre los mecanismos de resistencia en Enterobacteriaceae, las ß-lactamasas de espectro extendido (BLEE) juegan un papel de gran importancia. Estas enzimas confieren resistencia a penicilinas, cefalosporinas de primera, segunda y tercera generación y al aztreonam y por su naturaleza plasmídica se diseminan con mucha facilidad. El presente estudio observacional, descriptivo de corte trasverso tuvo como objetivo determinar la frecuencia de enterobacterias productoras de BLEE en pacientes ambulatorios e internados del Laboratorio San Roque de septiembre a noviembre de 2012. La identificación bacteriana fue realizada por métodos convencionales y la determinación de BLEE por dos métodos fenotípicos; sinergismo de doble disco y el de disco combinado. De los 481 aislados, 88,1% era proveniente de pacientes ambulatorios y 11,9% de internados; resultó predominante Escherichia coli, seguido por Klebsiella pneumoniae. Fue confirmada la producción de BLEE por ambos métodos en 47 enterobacterias, 9,8%. Entre estas K. pneumoniae produjo BLEE con mayor frecuencia 30,8%, seguida de E coli, 6,6%. Todas las cepas productoras de BLEE aisladas en este estudio presentaron actividad cefotaximasa. La frecuencia obtenida es menor a lo reportado en países vecinos, lo que podría deberse a que la mayoría de los aislamientos provinieron de pacientes ambulatorios. Coincidente con estudios realizados en Latinoamérica, K. pneumoniae fue la productora de BLEE con mayor frecuencia y la acción de las BLEEs fue preferentemente sobre las cefotaximas. Si bien la cifra de BLEE encontrada no es alta, se debe insistir en tomar medidas para lograr la reducción de la frecuencia, considerando las graves implicancias que tiene la presencia de esta enzima.

Among resistance mechanisms of enterobacteriaceae, the production of ExtendedSpectrum Betalactamases (ESBL) plays an important role. These enzimes are capable ofinactivating, apart from penicillins, first and second generation cephalosporins, oxyiminocephalosporinsand aztreonam, and because of their plasmidic nature, they spread easily.We conducted a cross-sectional descriptive observational study. The objective was todetermine the frequency of ESBL producing enterobacteriaceae in outpatient andhospitalized patients at the San Roque laboratory from September to November 2012. Microbiological ESBL detection was performed by the double-disk synergy and combineddisk tests. Among 481 clinical isolates of Enterobacteriaceae, 88.1% was from outpatientsand 11.9% from hospitalized patients. Escherichia coli was predominant, followed byKlebsiella pneumoniae. ESBL production was confirmed by both methods in 47enterobacteriaceae, 9.8%. Among these, the most frequent producer of ESBL was K.pneumoniae 30.8% followed by E. coli, 6.6%. All ESBL-producing enterobacteriacea in thisstudy presented cefotaximase activity. The frequency obtained is lower than rates reportedin neighboring countries, which could be explained by the fact that most of the isolates werefrom outpatients. Like other studies in Latin America, ESBL- producing K. pneumoniae wasmore frequent and the action of ESBLs was mainly on cefotaximase. While the number ofESBL found is not high, we should insist on taking steps towards reducing the frequency,considering the serious implications of the presence of this enzyme.

Detección molecular de belactamasas de espectro extendido (BLEE) en enterobacterias aisladas en Asunción / Molecular detection of extended spectrum beta-lactamase (esbl) in enterobacteria isolated in Asunción

Las betalactamasas de espectro extendido (BLEE), son enzimas responsables de la hidrólisis del anillo betalactámico de penicilinas y cefalosporinas, excepto carbapemenes, inhibiendo así su actividad terapéutica. Si bien es posible la detección fenotípica de este mecanismo de resistencia por métodos convencionales, sólo los métodos moleculares permiten la identificación del gen responsable de dicha resistencia. El objetivo de este estudio descriptivo retrospectivo fue identificar los genes blaCTX-M2, blaPER-2, blaSHV y blaTEM, en aislamientos de enterobacterias productoras de BLEE,; de muestras clínicas colectadas entre julio 2007 y abril 2008, provenientes de dos hospitales de referencia de Asunción, Paraguay. La detección molecular de los genes se realizó por reacción en cadena de la polimerasa empleando oligonucleótidos específicos. De los 232 aislados BLEE analizados, el 83% (n=192) portó al menos un gen bla, en el 17% (n=40) restante no fue detectado ninguno de los genes incluido en el estudio. Se observaron las siguientes frecuencias: 49% (94/192) blaCTX-M2, 45% (86/192) blaSHV, 40% (77/192) blaTEM y 7% (13/192) blaPER-2. En el 47% (90/192) se detectó más de un gen, siendo la combinación blaCTX-M2+blaTEM+blaSHV, la más frecuente observada en 32 aislados. El blaCTX-M2 como el gen más frecuente en este estudio; concuerda con lo reportado en nuestro país y en Argentina. Este es el primer reporte de la presencia de blaTEM y blaSHV en Paraguay. Es de gran importancia el estudio de otros genes codificantes de resistencia, considerando la emergencia de otras BLEE en la región como blaCTX-M15 con actividad predominantemente ceftazidimasa.

Extended spectrum beta-lactamases (ESBLs), are enzymes responsible for thehydrolysis of the beta-lactam ring and resistance to both cephalosporins and penicillins,except carbapenems, therefore inhibiting its therapeutics activity. Even though, detectionof the phenotypic resistance mechanism by conventional methods is possible, onlymolecular methods allow identification of the gene responsible for the resistance. Theobjective of this retrospective study was to identify the blaCTX-M2, blaPER-2, blaSHV, blaTEMgenes in ESBL-producing enterobacteriaceae isolates, recovered from clinical samples collected between July 2007 and April 2008, from two reference hospitals in Asunción,Paraguay. Molecular gene detection was performed by polymerase chain reaction usingspecifics oligonucleotides. Out of the tested 232 ESBL-producing isolates, 83% (n=192)carried at least one of the bla genes as follows; 49% (94/192) blaCTX-M2, 45% (86/192)blaSHV, 40% (77/192) blaTEM and 7% (13/192) blaPER-2. In the rest 17% (n=40) none of thegenes included in this study was detected; in 47% (90/192) more than one gene wasdetected, resulting blaCTX-M2 + blaTEM + blaSHV as the most frequent combination in 32isolates. The presence of blaCTX-M2, as the most frequent codifying genes of BLEE is inagreement with previous reports in Paraguay and Argentina. This is the first report of thepresence of blaTEM and blaSHV circulating in Paraguay. It is of much importance the study ofothers codifying resistance genes, taking into account the emergence of other BLEE in theregion, such as blaCTX-M15, predominantly with ceftazidimase activity.

[Demodex spp in chronic blepharitis patients]. / Demodex spp en pacientes con blefaritis crónica.

BACKGROUND: Blepharitis is a very common disease in the ophthalmologic practice generally taking a chronic course with intermittent exacerbations. Several studies have linked the presence of Demodex folliculorum with chronic blepharitis, since the mite has the capacity to perpetuate the follicular inflammatory process. The prevalence of infection by Demodex spp. is variable depending on the population. In Paraguay, information on the frequency of the infestation in patients with chronic blepharitis is not available. AIM: To determine the frequency of Demodex spp, and the ocular microbiota in patients with chronic blepharitis attending the Department of Ophthalmology at the Teaching Hospital of the National University of Asuncion. PATIENTS AND METHODS: Consecutively, 28 patients with chronic blepharitis, who agreed to participate in the study, were included. Eyes lashes from the upper and lower eyelids were extracted for immediate mite search by direct observation under a light microscope. Samples from eyelids were taken with Kimura spatula and then cultured on blood agar and in enrichment media and incubated in 5% CO2 at 35° C for 72 hours. RESULTS: Among participants, females were more frequent (64%), the age ranged from 17 to 87 years (mean: 38.0; SD: ± 13.5 years). The prevalence of Demodex sp was 54%. Bacteria were isolated 92.9% of cases, most frequently coagulase-negative staphylococci (75%). No association was found between socio-demographic or clinical characteristics and the presence of Demodex sp. CONCLUSION: The observed high prevalence of infestation by Demodex spp in patients with chronic blepharitis is consistent with other studies.

Demodex spp en pacientes con blefaritis crónica / Demodex spp in chronic blepharitis patients

Background: Blepharitis is a very common disease in the ophthalmologic practice generally taking a chronic course with intermittent exacerbations. Several studies have linked the presence of Demodex folliculorum with chronic blepharitis, since the mite has the capacity to perpetuate the follicular inflammatory process. The prevalence of infection by Demodex spp. is variable depending on the population. In Paraguay, information on the frequency of the infestation in patients with chronic blepharitis is not available. Aim : To determine the frequency of Demodex spp, and the ocular microbiota in patients with chronic blepharitis attending the Department of Ophthalmology at the Teaching Hospital of the National University of Asuncion. Patients and Methods: Consecutively, 28 patients with chronic blepharitis, who agreed to participate in the study, were included. Eyes lashes from the upper and lower eyelids were extracted for immediate mite search by direct observation under a light microscope. Samples from eyelids were taken with Kimura spatula and then cultured on blood agar and in enrichment media and incubated in 5% CO2 at 35° C for 72 hours. Results: Among participants, females were more frequent (64%), the age ranged from 17 to 87 years (mean: 38.0; SD: ±13.5 years). The prevalence of Demodex sp was 54%. Bacteria were isolated 92.9% of cases, most frequently coagulase-negative staphylococci (75%). No association was found between socio-demographic or clinical characteristics and the presence of Demodex sp. Conclusion: The observed high prevalence of infestation by Demodex spp in patients with chronic blepharitis is consistent with other studies.

Introducción: La blefaritis es una enfermedad muy común en la práctica oftalmológica, generalmente de curso crónico con exacerbaciones intermitentes. Varios estudios han relacionado la presencia del Demodex folliculorum con la blefaritis crónica, por su capacidad potencial de perpetuar el proceso inflamatorio a nivel folicular. La frecuencia de D. folliculorum varía de acuerdo a la población. Objetivo: Determinar la presencia de Demodex spp y, caracterizar la microbiota ocular en pacientes con blefaritis crónica y dar a conocer estos resultados. Pacientes y Métodos: Las muestras fueron obtenidas de pacientes que consultaron en la Cátedra de Oftalmología del Hospital de Clínicas de la Universidad Nacional de Asunción (UNA). Fueron incluidos en forma consecutiva 28 pacientes con blefaritis crónica que accedieron a participar en forma voluntaria en el estudio. Se extrajeron las pestañas del párpado superior e inferior de los pacientes para la búsqueda inmediata del ácaro por observación directa bajo el microscopio óptico. Para el cultivo de microorganismos se tomaron muestras del párpado con espátula de Kimura, las que fueron cultivadas en agar sangre y en medios de enriquecimiento e incubadas en CO2 al 5% a 35°C durante 72 h. Resultados: El sexo femenino fue más frecuente (64%), el rango de edad estuvo entre 17 y 87 años con una media de 37,9 ± 13,5. Se observó la presencia de Demodex sp en 54% y aislamiento de bacterias en 92,9%; Staphylococcus coagulasa negativa fue el más frecuente (75%). No se encontraron asociaciones entre las características socio-demográficas o clínicas y la presencia de Demodex sp. Conclusión: La alta infestación por Demodex spp observada en los pacientes con blefaritis crónica coincide con otros estudios.

Queratitis por Lasiodiplodia theobromae: comunicación de un caso y revisión de la literatura / Keratitis by Lasiodiplodia theobromae: a case report and literature review

We report a case of mycotic keratitis caused by Lasiodiplodia theobromae in a 60-year-old man with a history of ocular trauma with vegetable matter. Ophthalmological assessment with slit-lamp and microbiological evaluation of the corneal ulcer by conventional microbiological techniques were performed. Mycology study of the corneal scraping showed the presence of fungal filaments and the isolate was identified as Lasiodiplodia theobromae. Patient was treated with natamycin 5% and fluconazole 0.2% for 37 days. The infection was controlled but the corneal scars required a cornea transplant. This is the first case of keratitis by Lasiodiplodia theobromae in Paraguay. Difficulties in the management of these cases, which often requires surgical procedures, are discussed.

Se presenta un caso clínico de queratitis causada por Lasiodiplodia theobromae en un agricultor de 60 años de edad, con antecedentes de un trauma ocular con un vegetal. Se realizó un examen oftalmológico con lámpara de hendidura y estudio microbiológico de la úlcera corneal por técnicas microbiológicas convencionales. El examen micológico del raspado corneal reveló la presencia de hifas septadas y el cultivo fue identificado como Lasiodiplodia theobromae. El paciente fue tratado con natamicina al 5% y fluconazol al 0,2% durante 37 días. La infección fue controlada, sin embargo, el paciente quedó con cicatrices corneales y con necesidad de trasplante. Es el primer caso de queratitis por Lasiodiplodia theobromae en Paraguay. Se discute la dificultad de manejo de estos casos que a menudo requieren procedimientos quirúrgicos y trasplante de córnea.

Portación de Staphylococcus aureus multiresistentes a antimicrobianos en cavidad bucal de niños que concurren para un tratamiento en una clínica odontológica, Paraguay / Carriage of Multidrug-resistant Staphylococcus aureus in the Oral Cavities of Children Presenting for Treatment at a Dental Clinic in Paraguay

Introducción: Staphylococcus aureus es un microorganismo que posee características particulares de virulencia y resistencia a los antibióticos para uso clínico cuya diseminación es de gran importancia en salud pública. Objetivo: Identificar S. aureus sensibles y resistentes a antimicrobianos, colonizantes en cavidad bucal de niños que concurrieron para un tratamiento a una Clínica Odontológica en Asunción. Materiales y Métodos: En este estudio descriptivo, observacional realizado de agosto a septiembre de 2013, se incluyeron niños de 2 a 15 años de edad. Hisopados de cavidad bucal fueron colectados y cultivados en agar-manitol-sal. Los aislados de S. aureus fueron caracterizados respecto a la susceptibilidad a 11 antibióticos y por métodos moleculares se buscó la presencia del gen mecA y del factor de virulencia pvl. Se empleó un cuestionario sobre datos socio-demográficos y factores de riesgo asociados a infección con S. aureus. Resultados: Se incluyeron 112 niños. Se aisló S. aureus en 37 (33%) hisopados y 35 (94,6%) aislados presentaban resistencia a al menos un antibiótico. Resultaron resistentes a: Penicilina (89%), Cloranfenicol (16,2%), Oxacilina (10,8%), Eritromicina (8%), Clindamicina (8%), Gentamicina (5,4%) y un aislado con resistencia intermedia a Ciprofloxacina (2,7%). Cuatro aislados de S. aureus presentaban resistencia a múltiples drogas, los mismos portaban el gen mecA. No se detectó portación del factor de virulencia PVL. Conclusión: La elevada portación en cavidad oral de S. aureus multiresistentes en niños sin factores de riesgo como hospitalización previa o consumo de antibióticos, implica un alto riesgo para posibles infecciones endógenas, además del potencial de transferencia de determinantes de resistencia entre gérmenes de flora bucal normal .

Introduction: Staphylococcus aureus isa bacterium with special characteristicsof virulence and resistance to theantibiotics used in clinical practice, andwhose spread presents a significantpublic health challenge...

Perfil de susceptibilidad a anti fúngicos de aislados de Candida spp por el método de microdilución. Nuevos puntos de cortes para fluconazol / Anti-fungal susceptibility profile of Candida spp isolates by the microdilution method. New cutoff points for fluconazole

Las infecciones por levaduras tienen gran importancia debido a su relativa alta frecuencia e incremento de la resistencia a los antifúngicos, especialmente a los derivados azólicos y, en particular, al fluconazol. El objetivo del estudio fue determinar la concentración inhibitoria mínima (CIM) del fluconazol y anfotericina B de aislados de Candida de pacientes ambulatorios y hospitalizados en Paraguay de mayo del 2010 a agosto del 2011, aplicando criterios nuevos y antiguos para el fluconazolYeast infections are very important because of their relatively high frequency and increased resistance to antifungal agents, especially to azole derivatives, and in particular, to fluconazole. The objective of this study was to determine the minimum inhibitory concentration (MIC) of fluconazole and amphotericin B in Candida isolates in clinically significant samples from hospitalized patients in Paraguay from May 2010 to August 2011 using the old and new criteria for fluconazole. The MICs of the antifungals were determined by the microdilution method (M27 A-2) according to CLSI recommendations. Fifty isolates were evaluated, being C. albicans the predominant species (40%) followed by C. parapsilosis and C. glabrata in equal proportion (20%), C. tropicalis (16%) and C. krusei (4%). Using the old cutoff, five isolates were resistant to . La CIM de los anti-fúngicos se determinó por el método de microdilución según la CLSI. Fueron incluidos 50 aislados de Candida, resultando predominante C. albicans (40%) seguido de C. parapsilosis y C. glabrata en igual proporción (20%), C. tropicales (16%) y C. krusei (4%). Con el antiguo criterio, cinco aislados resultaron resistentes al fluconazol (10%), dos fueron C. krusei con resistencia natural, dos C. tropicalis y uno C. albicans. Tres aislados tuvieron sensibilidad dosis dependiente (SDD), uno de cada especie de C. tropicales, C. parasilopsis y C. glabrata. Con el nuevo criterio, la resistencia aumentó a siete aislados (14%), dos...

Yeast infections areveryimportantbecause of their relatively high frequency andincreased resistance to antifungal agents, especiallytoazole derivatives, and inparticular, to fluconazole. Theobjective of thisstudy was to determine the minimuminhibitory concentration (MIC) of fluconazole and amphotericin B inCandidaisolatesinclinically significant samples from hospitalized patients in Paraguay from May 2010 toAugust 2011 using the old and new criteria for fluconazole. The MICs of the antifungalswere determined by the microdilution method (M27 A-2) according to CLSIrecommendations. Fifty isolates were evaluated,beingC. albicansthe predominantspecies (40%) followed byC. parapsilosisandC. glabratain equal proportion (20%),C.tropicalis(16%) andC. krusei(4%).Usingthe old cutoff,five isolates were resistant to fluconazole (10%), two naturally resistant isolates (C. krusei), twoC. tropicalisand oneC. albicans.ThreeC. glabrataand one isolates of each species ofC. tropicales, C.parasilopsisyC. glabratashowed sensitivity dose dependent (SDD). With the new cutoff,the number of resistant isolates increased to seven (14%), of which two were SDD basedon the old criteria. NineC. glabrataand twoC. albicans,previouslyclassified as sensitive,changedto SDD. All isolates were susceptible to amphotericin B. With the newcutoffpoints,important variations were observed in the isolates susceptibility profiles,particularly inC. glabrata,supportingtheneedof species identification,permanentsurveillance ofthe isolatesresistancetoanti-fungalsand correlationof the sensitivityresultswith theclinicalresponseto treatment.

[Keratitis by Lasiodiplodia theobromae: a case report and literature review]. / Queratitis por Lasiodiplodia theobromae: comunicación de un caso y revisión de la literatura.

We report a case of mycotic keratitis caused by Lasiodiplodia theobromae in a 60-year-old man with a history of ocular trauma with vegetable matter. Ophthalmological assessment with slit-lamp and microbiological evaluation of the corneal ulcer by conventional microbiological techniques were performed. Mycology study of the corneal scraping showed the presence of fungal filaments and the isolate was identified as Lasiodiplodia theobromae. Patient was treated with natamycin 5% and fluconazole 0.2% for 37 days. The infection was controlled but the corneal scars required a cornea transplant. This is the first case of keratitis by Lasiodiplodia theobromae in Paraguay. Difficulties in the management of these cases, which often requires surgical procedures, are discussed.

[Clinically significant coagulase-negative staphylococci: most frequent species and virulence factors]. / Staphylococcus coagulasa-negativa clínicamente significativos: Especies más frecuentes y factores de virulencia.

BACKGROUND: Coagulase-negative staphylococci have emerged as responsible for a large number of infections. However, it is often difficult to assess its pathogenic role or to discard it as a contaminant. AIM: The goal of this study was to identify clinically significant coagulase-negative staphylococci to the species level and their virulence factors. Isolates came from patients consulting at the San Roque Laboratory from 2009 to 2011. MATERIAL AND METHODS: Species identification was performed by De Paulis et al simplified method. Production of biofilm, hemolysins, lipases, lecithinases and DNase were determined by conventional methods; methicillin-resistance by diffusion method and mecA and Panton-Valentine genes, by multiplex PCR. RESULTS: Out of 64 isolates, 40.6% were S. epidermidis; 20.3%, S. haemolyticus, and 15.6%, S. lugdunensis. Biofilm production was detected in 73.1% of S. epidermidis, 53.8% of S. haemolyticus and 40% of S. lugdunensis. mecA gene was identified in 69.2% of S. epidermidis, 92.3% of S. haemolyticus and none of S. lugdunensis. 83% of mecA (+) S. epidermidis isolates were biofilm producers as compared to 50% of the mecA (-). CONCLUSION: The frequency of S. lugdunensis, the most virulent coagulase-negative staphylococci species, was relatively high. The main virulence factor in S. epidermidis was biofilm production, being higher in those resistant to methicillin.

Staphylococcus coagulasa-negativa clínicamente significativos: Especies más frecuentes y factores de virulencia / Clinically significant coagulase-negative staphylococci: Most frequent species and virulence factors

Coagulase-negative staphylococci have emerged as responsible for a large number of infections. However, it is often difficult to assess its pathogenic role or to discard it as a contaminant. Aim: The goal of this study was to identify clinically significant coagulase-negative staphylococci to the species level and their virulence factors. Isolates came from patients consulting at the San Roque Laboratory from 2009 to 2011. Material and Methods: Species identification was performed by De Paulis et al simplified method. Production of biofilm, hemolysins, lipases, lecithinases and DNase were determined by conventional methods; methicillin-resistance by diffusion method and mecA and Panton-Valentine genes, by multiplex PCR. Results: Out of 64 isolates, 40.6% were S. epidermidis; 20.3%, S. haemolyticus, and 15.6%, S. lugdunensis. Biofilm production was detected in 73.1% of S. epidermidis, 53.8% of S. haemolyticus and 40% of S. lugdunensis. mecA gene was identified in 69.2% of S. epidermidis, 92.3% of S. haemolyticus and none of S. lugdunensis. 83% of mecA (+) S. epidermidis isolates were biofilm producers as compared to 50% of the mecA (-). Conclusion: The frequency of S. lugdunensis, the most virulent coagulase-negative staphylococci species, was relatively high. The main virulence factor in S. epidermidis was biofilm production, being higher in those resistant to methicillin.

Staphylococcus coagulasa-negativa ha emergido como responsable de un gran número de infecciones. No obstante, con frecuencia es difícil asegurar su rol patógeno o descartarlo como contaminante. Objetivo: Estudiar a nivel de especies Staphylococcus coagulasa-negativa clínicamente significativos y sus factores de virulencia, de aislados provenientes de pacientes del Laboratorio San Roque de Asunción, Paraguay entre los años 2009 y 2011. Material y Métodos: Para la identificación de especies fue utilizado el método simplificado de De Paulis y cols. La producción de biopelícula, hemolisinas, lipasas, lecitinasas, AD-Nasa, fue determinada por métodos convencionales; la resistencia a meticilina por difusión y los genes mecA y Panton-Valentine por RPC múltiple. Resultados: De 64 aislados, 40,6% correspondió a S. epidermidis, 20,3% S. haemolyticus y 15,6% S. lugdunensis. La producción de biopelícula fue detectada en S. epidermidis en 73,1%, S. haemolyticus 53,8% y S. lugdunensis 40%. El gen mecA fue identificado en 69,2% de S. epidermidis, 92,3% de S. haemolyticus y en ninguno de S. lugdunensis. El 83% de S. epidermidis mecA (+) fue productor de biopelícula en comparación a 50% de los mecA (-). Conclusión: La frecuencia de S. lugdunensis, una de las especies más virulentas de Staphylococcus coagulasa-negativa fue relativamente alta; y el principal factor de virulencia en S. epidermidis fue la producción de biopelícula, siendo mayor en los resistentes a meticilina.