Role of Hologic® Panther AptimaTM SARS-CoV-2 assay in the detection of SARS-CoV-2: screening or diagnostic technique?

During the multiple waves of COVID-19 suffered all over the world, having a rapid and sensitive diagnostic test has become a priority for microbiology laboratories. The AptimaTM SARS-CoV-2 transcription-mediated amplification (TMA) assay running on the Panther system (Hologic) was presented as a very good option to cover this need. To evaluate this system, 570 respiratory samples were included in the study and were processed both by the Panther (Hologic) system and by qRT-PCR (Thermo Fisher Science, Waltham, USA), current assay for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A high number of false positives (n=76) was obtained with Panther system (Hologic), but the number of false positives decreases as the relative light units (RLU) value increases. These results show that this technique can be a good option for sample screening but checking for positive results should be mandatory, especially those with low RLU values.

[Evaluation of two immunocromatographic tests for the detection of antibodies against SARS-CoV-2]. / Valoración de dos pruebas inmunocromatográficas para la detección de anticuerpos frente a SARS-CoV-2.

OBJECTIVE: Serological tests have been a valuable tool during the SARS-CoV-2 pandemic, supporting molecular methods for detection, and monitoring the immune response, caused by vaccination or by natural infection. Within all these techniques, rapid tests are interesting due to their ease of use, rapid response and low cost. METHODS: Two different immunological techniques were evaluated: Realy Tech and Mikrogen Diagnostik recomLine SARS-CoV-2 IgG. SARS-CoV-2 IgG II Quant antibody test and SARS-CoV-IgG assay, both from Abbott Diagnostics, were used as reference techniques. RESULTS: Mikrogen Diagnostik recomLine SARS-CoV-2 IgG shows the best results (S=0.985; E=0.839). Three techniques offered good positive predictive values, but Realy Tech and Healgen negative predictive values left to be desired. CONCLUSIONS: Mikrogen Diagnostik recomLine SARS-CoV-2 IgG showed good results in the detection of antibodies against SARS-CoV-2 and could be used as an alternative to automated techniques.

In vitro activity of imipenem/relebactam against Gram-negative clinical isolates in two Spanish tertiary hospitals.

OBJECTIVE: The aim of this study was to analyze the activity of the imipenem-relebactam combination (IMI/REL) against a collection of multidrug-resist Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii clinical isolates. METHODS: The study was conducted in two tertiary hospitals in Spain and included 192 clinical isolates of these 3 genera (139 resistant and 53 susceptible to IMI). The MICs for IMI with and without REL (at a fixed concentration of 4 mg/L) were determined by a standard broth microdilution method according to international recommendations. RESULTS: All IMI-susceptible E. coli strains were also susceptible to IMI/REL. Enterobacterales resistant to IMI due to the production of carbapenemases, the MIC50 and MIC90 decreased from 64/256 with IMI to 8/64 mg/L with IMI/REL. This high activity was principally detected among isolates with KPC enzymes. Enterobacterales with class B carbapenemases, P. aeruginosa carrying VIM carbapenemase and A. baumannii strains showed no changes on IMI MIC50 or MIC90 after adding REL. Among P. aeruginosa strains without carbapenemase the MIC for IMI/REL was reduced between 1 to 5 dilutions. CONCLUSIONS: IMI/REL showed high activity against the strains that carry Klebsiella pneumoniae carbapenemase (KPC) and against carbapenem-resistant P. aeruginosa unrelated to the VIM enzyme, mainly AmpC beta lactamase associated with impermeability. Against strains carrying oxacillinase 48 (OXA-48) associated with extended-spectrum beta-lactamase (ESBL), IMI/REL presented activity only slightly better than IMI and had no beneficial effect superior to IMI against A. baumannii.

Comparative study of different SARS-CoV-2 diagnostic techniques.

The rapid spread of SARS-CoV-2 led to the necessity of developing diagnostic tests for rapid virus detection. Many commercial platforms have appeared and have been approved for this purpose. In this study, 95 positive and 5 negative retrospective samples were analyzed by 4 different commercial RT-qPCR kits (TaqMan 2019nCoV Assay, Allplex™SARS-COV-2 Assay, FTD SARS-COV-2 Assay and qCOVID-19). The Hologic Aptima SARS-COV-2 and the Clart-COVID-19 system were also tested. serial dilutions of SARS-COV-2 standard control were included for sensitivity analysis. Among the qPCR tested qCOVID19 and Allplex™SARS-COV-2 Assay were both able to detect all the clinical samples included in the study. All four qPCR evaluated showed high sensitivity for samples with Ct<33. Clart-COVID-19 microarrays detected all samples and controls used in this study whereas Hologic Aptima Panther failed with one of the clinical samples. However, the main problem with this system was the number of invalidated samples despite avoiding the use of medium with guanidine isothiocyanate as recommended by the manufacturer. All the techniques tested were of value for SARS-CoV-2 detection.

Service-Learning, Movies, and Infectious Diseases: Implementation of an Active Educational Program in Microbiology as a Tool for Engagement in Social Justice.

Service-Learning is an educational methodology that allows student learning while addressing community needs. A program in microbiology and infectious diseases was implemented in Universidad Complutense de Madrid, Spain. University lecturers, clinical microbiologists, doctorate students, and undergraduates from several Bachelor Degrees and courses worked in an interdisciplinary team along with social institutions that attend disadvantaged persons. Using commercial movies that deal with infectious diseases, the students learn clinical microbiology, prepare divulgation materials, visit social centers to accompany, and help others to know about illnesses and prevention. The program was developed through two academic years and involved 58 voluntary students, 13 teachers and tutors, and 4 social entities as community partners. Postsurvey evaluation of the program revealed a highly satisfactory achievement of goals: acquiring scientific and personal competencies by university students, including critical analysis and science diffusion, solving problems or collaborative team working, and contributing, together with the tutors, to the social responsibility of the university.

[Usefulness of MALDI-TOF and REP-PCR against PFGE for the epidemiological study of Acinetobacter baumannii]. / Utilidad de MALDI-TOF y REP-PCR frente a PFGE para el estudio epidemiológico de Acinetobacter baumannii.

OBJECTIVE: To evaluate the ability of MALDI-TOF MS and rep-PCR to discriminate Acinetobacter baumannii clones. METHODS: A total of 21 strains of A. baumannii with different epidemiological and phenotipycal characteristics were included in the study. All isolates were analyzed in parallel by MALDI-TOF MS and rep-PCR and the spectra obtained were compared with each other and with the results obtained by pulsed field gel electrophoresis (PFGE). Isolates with a similarity equal to or greater than 87% were considered to be part of the same clonal group. RESULTS: The analysis of the 21 isolates included in the study, resulted in 8 clonal groups using PFGE, 3 groups by MALDI-TOF MS and 7 groups by rep-PCR analysis. The isolates that formed the different groups by the 3 techniques used were totally different, so it can be concluded that there is no equivalence between the results obtained with the three typing methods used. CONCLUSIONS: Despite its simplicity, neither MALDI-TOF MS nor rep-PCR can at this time replace PFGE for the epidemiological study of A. baumannii.

Activity of doripenem against extended-spectrum beta-lactamase-producing Enterobacteriaceae and Pseudomonas aeruginosa isolates.

The in vitro activity of doripenem was evaluated against a recent collection of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae and Pseudomonas aeruginosa isolates (201 ESBL-producing Enterobacteriaceae [153 Escherichia coli and 48 Klebsiella pneumoniae] and 201 P. aeruginosa). Comparator agents included amikacin, tobramycin, ciprofloxacin, cefepime, cefotaxime, ceftazidime piperacillin-tazobactam, imipenem, and meropenem. Both doripenem and meropenem inhibited 100% of the ESBL-producing Enterobacteriaceae at 4 microg/mL. Doripenem is active against ESBL-producing Enterobacteriaceae and P. aeruginosa isolates. Its activity is similar to that of meropenem and slightly better than that of imipenem. The results of this study suggest that doripenem could be an alternative therapeutic agent for infections caused by these organisms.

A combination of tigecycline, colistin, and meropenem against multidrug-resistant Acinetobacter baumannii bacteremia in a renal transplant recipient: pharmacodynamic and microbiological aspects.

Acinetobacter baumannii are emerging as the causal agents of healthcare-associated infections. We describe arenal transplant recipient who developed bacteremia caused by multiresistant A. baumannii, which received a combination of tigecycline, colistin, and meropenem in continuous infusion. The clinical outcome was favorable. In this article we made a molecular study of this multiresistant strain. Our analysis reveals the presence of abla-OXA-72 gene,a class D of oxacillinase belonging to bla-OXA-40-like group,which constitutes the most disseminated familiy of carbapenemases in Spain. Thus, we found different susceptibility patterns of A. baumannii when we used different Mueller-Hinton agars with different manganese concentrations. Lastly, we explain the combination of these three antibiotics administered to increase microbiologic and pharmacodynamic yield.

[In vitro activities of colistin combinations against Pseudomonas aeruginosa isolated from the intensive care unit]. / Actividad in vitro de terapia combinada con colistina frente a Pseudomonas aeruginosa aisladas en Unidad de Cuidados Intensivos.

Introduction. As the number of multidrug-resistant strains of Pseudomonas aeruginosa has risen in the intensive care unit (ICU) of the San Carlos Clinic Hospital, 12 consecutive isolates from different patients were collected to determine the possibility of an epidemic outbreak caused by the spread of a single strain. We determined the antimicrobial susceptibility to the most common agents used in the treatment of infections caused by this bacteria. The results of susceptibility studies suggest that different strains of P. aeruginosa are responsible for the respiratory tract infections in ICU. Methods. The clonal relationship between the isolates using was determined using BOX and ERIC primers by means of repetitive sequence-based polymerase chain reaction (rep-PCR). The in vitro activity of these strains against colistin, rifampicin, doxicycline and azythromycin was studied to determine in which cases the combination of colistin with any of the other three antibiotics was synergistic. Results. Sensitivity studies point out the presence of several strains of P. aeruginosa as the causal agents of respiratory infections produced by this microorganism in the ICU. Combinations of colistin with doxycicline and colistin with azithromycin were synergistic for some isolates in the synergy studies. Discussion. Clonal studies reveal the presence of five different clones among our isolates. Therefore we can conclude that there was no outbreak of P. aeruginosa in the ICU. Synergistic activity of combinations of colistin plus azithromycin, colistin plus doxicycline and colistin plus rifampicin was less than expected and a high percentage of indifferent results was observed.

Actividad in vitro de terapia combinada con colistina frente a Pseudomonas aeruginosa aisladas en Unidad de Cuidados Intensivos / In vitro activities of colistin combinations against Pseudomonas aeruginosa isolated from the intensive care unit

Introducción. Ante el incremento de aislados de Pseudomonasaeruginosa multirresistentes en la Unidad de CuidadosIntensivos (UCI) del Hospital Clínico San Carlos deMadrid y para determinar la posibilidad de que se tratase deun brote epidémico causado por la diseminación de unaúnica cepa, se recogieron 12 muestras consecutivas de distintospacientes que fueron identificadas y a las que posteriormentese determinó su sensibilidad a los antibióticosutilizados habitualmente para el tratamiento de las infeccionesproducidas por este microorganismo.Métodos. Mediante la amplificación de secuencia basadaen la reacción en cadena de la polimerasa (repetitive sequence-based polymerase chain reaction, rep-PCR) se determinó larelación clonal entre los aislados utilizando los primers BOX yERIC y se estudió la actividad in vitro frente a estas cepas decolistina, rifampicina, doxiciclina y azitromicina para determinaren qué casos la combinación de colistina con alguno de losotros tres antibióticos presentaba actividad sinérgica.Resultados. Los estudios de sensibilidad apuntan a la presenciade varias cepas de P. aeruginosa como responsables delas infecciones respiratorias producidas por este microorganismoen la UCI, hecho que fue corroborado mediante los estudiosclonales realizados. En los estudios de sinergia las asociacionesde colistina con doxiciclina y con azitromicina presentaron actividadsinérgica para alguno de los aislados.Discusión. Los resultados de los estudios clonales revelanla presencia de cinco clones diferentes entre los aisladosseleccionados, por lo que podemos concluir que no se tratade un brote de P. aeruginosa en la UCI. La actividad sinérgicade la asociación de colistina con azitromicina, doxiciclinay rifampicina ha sido menor de la esperada y se observa unelevado porcentaje de resultados indiferentes (AU)

Introduction. As the number of multidrug-resistantstrains of Pseudomonas aeruginosa has risen in the intensivecare unit (ICU) of the San Carlos Clinic Hospital,12 consecutive isolates from different patients were collectedto determine the possibility of an epidemic outbreakcaused by the spread of a single strain. We determinedthe antimicrobial susceptibility to the most commonagents used in the treatment of infections caused by thisbacteria. The results of susceptibility studies suggest thatdifferent strains of P. aeruginosa are responsible for therespiratory tract infections in ICU.Methods. The clonal relationship between the isolatesusing was determined using BOX and ERIC primersby means of repetitive sequence-based polymerase chainreaction (rep-PCR). The in vitro activity of these strainsagainst colistin, rifampicin, doxicycline and azythromycinwas studied to determine in which cases the combinationof colistin with any of the other three antibioticswas synergistic.Results. Sensitivity studies point out the presence ofseveral strains of P. aeruginosa as the causal agents ofrespiratory infections produced by this microorganism inthe ICU. Combinations of colistin with doxycicline andcolistin with azithromycin were synergistic for some isolatesin the synergy studies.Discussion. Clonal studies reveal the presence of fivedifferent clones among our isolates. Therefore we canconclude that there was no outbreak of P. aeruginosa inthe ICU. Synergistic activity of combinations of colistinplus azithromycin, colistin plus doxicycline and colistinplus rifampicin was less than expected and a high percentageof indifferent results was observed (AU)

In vitro activity of tigecycline against Bacteroides species.

OBJECTIVES: To ascertain the current susceptibility patterns of members of the Bacteroides fragilis group in our hospital and to assess the in vitro activity of tigecycline against these organisms. METHODS: A total of 400 non-duplicate clinical isolates of the B. fragilis group collected from 2000 to 2002 were studied. Susceptibility testing was performed according to the reference agar dilution method described by the NCCLS. The following antimicrobials were tested: tigecycline, clindamycin, metronidazole, chloramphenicol, cefoxitin, imipenem, amoxicillin-clavulanate and piperacillin-tazobactam. RESULTS: All strains were susceptible to metronidazole and chloramphenicol. For clindamycin and cefoxitin, the overall susceptibility rates were 59.5% and 83%, respectively. Imipenem and piperacillin-tazobactam were the most active beta-lactam agents tested. Tigecycline inhibited 89.8% of the strains at a concentration of 8 mg/L with an MIC range of 16 mg/L. By comparing the MIC50 and MIC90 values of tigecycline among the various species of the group, B. fragilis, Bacteroides thetaiotaomicron and Bacteroides vulgatus were the most susceptible (MIC50/MIC90s of 0.5-1/8 mg/L). CONCLUSIONS: Tigecycline exhibited activity against most isolates of the B. fragilis group tested. These results indicate that tigecycline may be useful in the treatment and prophylaxis of infections involving these organisms.

Resistencia a carbapenemes por metaloenzimas en aislamientos clínicos de Pseudomonas aeruginosa / Carbapenem resistance determined by metalloenzymes in clinical isolates of Pseudomonas aeruginosa

La resistencia a los carbapenemes en aislamientos clínicos de Pseudomonas aeruginosa ha aumentado considerablemente en los últimos años. P. aeruginosa posee diferentes mecanismos de resistencia a estos fármacos. Los más frecuentes son la sobreexpresión de bombas de expulsión activa y la disminución de la expresión de porinas de membrana OprD. No obstante, la aparición de metaloenzimas que pueden hidrolizar carbapenemes está cobrando cada vez más importancia. El objetivo de este estudio fue determinar la presencia de metalobeta- lactamasas en P. aeruginosa aislados de muestras clínicas, y para ello se seleccionaron 133 cepas con una CMI ≥ 4 mg/l para imipenem, meropenem o ambos. Se estudió la sensibilidad a diferentes antimicrobianos, siendo la tobramicina (26,3%) y la colistina (17,3%) los que pre- sentaron un menor porcentaje de resistencias. Para evaluar la presencia de carbapenemasas se utilizaron dos métodos diferentes: la técnica del E-test® imipenem/imipenem + EDTA y la de difusión disco-placa, utilizando también EDTA como inhibidor de las carbapenemasas. Cuatro de las 133 cepas dieron positivo con las dos técnicas, y en ellas se detectaron genes tipo VIM (AU)

Carbapenem resistance in clinical isolates of P. aeruginosa has risen notably in recent years. P. aeruginosa has different mechanisms for carbapenem resistance, such as decreased levels of OprD or overexpression of the MexAB-OprM efflux system. Also, the emergence of metallobeta-lactamases (MBL)-producing bacteria is becoming a severe therapeutic problem. The aim of this study was to determine MBL presence in clinical isolates of P. aeruginosa: 133 with MIC ≥4 mg/l for imipenem and/or meropenem were selected. We studied the sensivity to different antibiotics in these strains. Tobramycin (26.3%) and colistin (17.3%) were the most active antibiotics. To determine the presence of MBL, we used the E-test® with imipenem/imipenem plus EDTA and disk-agar diffusion, also using EDTA as an MBL inhibitor. As a result of the screening test to evaluate the presence of MBL, we obtained four out of 133 strains as probable producers of metalloenzyme. These four strains were positive for the VIM-like gene as determined by a polymerase chain reaction method (AU)

In vitro activities of tigecycline against erythromycin-resistant Streptococcus pyogenes and Streptococcus agalactiae: mechanisms of macrolide and tetracycline resistance.

The activity of tigecycline was tested against erythromycin-resistant streptococci (107 Streptococcus pyogenes and 98 Streptococcus agalactiae strains). The presence of erythromycin and tetracycline resistance genes was determined by PCR. Among S. pyogenes strains the most prevalent gene was mef(A) (91.6%). The erm(B) gene was the most prevalent (65.3%) among S. agalactiae strains. Tigecycline proved to be very active against all the isolates tested (MIC at which 90% of the isolates tested were inhibited, 0.06 micro g/ml), including those resistant to tetracycline.

[Carbapenem resistance determined by metalloenzymes in clinical isolates of Pseudomonas aeruginosa]. / Resistencia a carbapenemes por metaloenzimas en aislamientos clínicos de Pseudomonas aeruginosa.

Carbapenem resistance in clinical isolates of P. aeruginosa has risen notably in recent years. P. aeruginosa has different mechanisms for carbapenem resistance, such as decreased levels of OprD or overexpression of the MexAB-OprM efflux system. Also, the emergence of metallo-beta-lactamases (MBL)-producing bacteria is becoming a severe therapeutic problem. The aim of this study was to determine MBL presence in clinical isolates of P. aeruginosa: 133 with MIC > or =4 mg/l for imipenem and/or meropenem were selected. We studied the sensitivity to different antibiotics in these strains. Tobramycin (26.3%) and colistin (17.3%) were the most active antibiotics. To determine the presence of MBL, we used the E-test with imipenem/imipenem plus EDTA and disk-agar diffusion, also using EDTA as an MBL inhibitor. As a result of the screening test to evaluate the presence of MBL, we obtained four out of 133 strains as probable producers of metalloenzyme. These four strains were positive for the VIM-like gene as determined by a polymerase chain reaction method.

Outbreak of TEM-24-producing Enterobacter aerogenes in a Spanish hospital.

Organisms encoding multidrug resistance genes are becoming increasingly prevalent. During a 2-month period (December, 2000, to January, 2001), 83 consecutive isolates of Enterobacter spp. were collected in our microbiology department. Antibiotic susceptibility was determined using the Vitek II automatic system. We selected strains with decreased susceptibility to extended-spectrum cephalosporins. The double-disk potentiation test was positive in 10 of these strains, indicating the presence of extended-spectrum beta-lactamases (ESBLs). Polymerase chain reaction (PCR), isoelectric focusing (IEF), and sequencing identified TEM 24 beta-lactamase in the 10 selected E. aerogenes. Random amplification of polymorphic DNA (RAPD-PCR) revealed the same clonal origin for all the strains tested and strongly suggest an outbreak of multidrug-resistant E. aerogenes. To follow up the trends in ESBLs-producing Enterobacter infections in the hospital over time, we repeated the study 1 year later (December, 2001, to February, 2002). Only three ESBLs-producing Enterobacter were found. All of them corresponded to the previously characterized clone.

Erythromycin and clindamycin resistance and telithromycin susceptibility in Streptococcus agalactiae.

The rates of resistance to erythromycin and clindamycin among Streptococcus agalactiae strains isolated in our hospital increased from 4.2 and 0.8% in 1993 to 17.4 and 12.1%, respectively, in 2001. Erythromycin resistance was mainly due to the presence of an Erm(B) methylase, while the M phenotype was detected in 3.8% of the strains. Telithromycin was very active against erythromycin-resistant strains, irrespective of their mechanisms of macrolide resistance.

Comparative activity of linezolid and other new agents against methicillin-resistant Staphylococcus aureus and teicoplanin-intermediate coagulase-negative staphylococci.

The activity of linezolid was determined against 225 recently isolated methicillin-resistant Staphylococcus aureus (MRSA) and 20 methicillin-resistant coagulase-negative staphylococci (CoNS) with decreased levels of susceptibility to teicoplanin. Linezolid activity was compared with other new agents (quinupristin-dalfopristin, trovafloxacin, moxifloxacin, levofloxacin and telithromycin) and six other antimicrobials (erythromycin, clindamycin, gentamicin, vancomycin, teicoplanin and rifampicin). The in vitro activity of linezolid was similar to that of vancomycin. Linezolid inhibited all MRSA strains at between 0.1 and 2 mg/L and all CoNS strains tested at between 0.2 and 0.5 mg/L. These results suggest that linezolid would be useful for the treatment of infections involving these organisms.