Lumipulse G SARS-CoV-2 Ag assay evaluation using clinical samples from different testing groups.

OBJECTIVES: Compared to RT-PCR, lower performance of antigen detection assays, including the Lumipulse G SARS-CoV-2 Ag assay, may depend on specific testing scenarios. METHODS: We tested 594 nasopharyngeal swab samples from individuals with COVID-19 (RT-PCR cycle threshold [Ct] values ≤ 40) or non-COVID-19 (Ct values >40) diagnoses. RT-PCR positive samples were assigned to diagnostic, screening, or monitoring groups of testing. RESULTS: With a limit of detection of 1.2 × 104 SARS-CoV-2 RNA copies/mL, Lumipulse showed positive percent agreement (PPA) of 79.9% (155/194) and negative percent agreement of 99.3% (397/400), whereas PPAs were 100% for samples with Ct values of <18 or 18-<25 and 92.5% for samples with Ct values of 25-<30. By three groups, Lumipulse showed PPA of 87.0% (60/69), 81.1% (43/53), or 72.2% (52/72), respectively, whereas PPA was 100% for samples with Ct values of <18 or 18-<25, and was 94.4, 80.0, or 100% for samples with Ct values of 25-<30, respectively. Additional testing of RT-PCR positive samples for SARS-CoV-2 subgenomic RNA showed that, by three groups, PPA was 63.8% (44/69), 62.3% (33/53), or 33.3% (24/72), respectively. PPAs dropped to 55.6, 20.0, or 41.7% for samples with Ct values of 25-<30, respectively. All 101 samples with a subgenomic RNA positive result had a Lumipulse assay's antigen positive result, whereas only 54 (58.1%) of remaining 93 samples had a Lumipulse assay's antigen positive result. CONCLUSIONS: Lumipulse assay was highly sensitive in samples with low RT-PCR Ct values, implying repeated testing to reduce consequences of false-negative results.

Detection of 14 human papillomavirus genotypes in cervical samples in women from a central-southern area of Italy showing different Pap test results.

We evaluated the prevalence of human papillomavirus (HPV) infection and correlated the molecular test results with the cytological examination data (PAP test) in 364 women living in central-southern Italy (Molise region), by means of polymerase chain reaction HPV DNA genotyping and of cervical cytology. One hundred and twenty-eight women resulted HPV positive (35.2%), HPV16 being the most frequent genotype. HPV positive women were significantly younger than negative patients (35.9 +/- 8.4 years and 38.2 +/- 9.1, respectively; p = 0.018); women with multiple infections were also significantly younger than those with single infection (31.7 +/- 6.9 and 37.6 +/- 8.3, respectively; p = 0.0002). Moreover, high risk HPV positive patients were significantly younger than low risk HPV positive women (35.1 +/- 7.7 and 40.5 +/- 10.5, respectively; p = 0.008). In the HPV positive group, 14 patients (10.9%) did not show any significant cytological alteration. Conversely, 7 out of 236 HPV negative women (3.0%) showed high grade squamous intraepithelial lesions (HSIL). Furthermore, HPV 16 or 18 were present in more than 70% of women positive for HSIL at cytology. Our data suggest the potential effectiveness of combined cytology and molecular test for further study of clinical cases with apparently laboratory conflicting results.

Recurrent ventriculoperitoneal shunt infection caused by small-colony variants of Staphylococcus aureus.

Phenotypic variants of Staphylococcus aureus may be misidentified by routine microbiological methods, and they may also respond poorly to antibacterial treatment. Using molecular methods, we identified small-colony variants of methicillin-resistant S. aureus (which were misidentified by 3 widely used automated identification systems as methicillin-susceptible coagulase-negative staphylococci) as the cause of recurrent ventriculoperitoneal shunt-related meningitis.

Molecular tools for differentiating probiotic and clinical strains of Saccharomyces cerevisiae.

The subtype of the Saccharomyces cerevisiae yeast species known as S. cerevisiae Hansen CBS 5926 was formerly believed to be a separate species, Saccharomyces boulardii. It is widely considered non-pathogenic and is used as a probiotic agent for treatment and prevention of diarrhea. The biological properties of Saccharomyces spp. show considerable intraspecies variability and the beneficial properties of probiotic yeasts are considered strain-specific. Septicemia and fungemia caused by S. boulardii have recently been described in both immunocompromised and immunocompetent patients receiving biotherapy with this yeast. It cannot be distinguished from other S. cerevisiae strains by phenotypic criteria, so identification of these infections requires molecular typing. To identify the most effective approach for distinguishing S. boulardii, we typed 35 isolates of S. cerevisiae, of which 27 were from various clinical specimens and 8 were isolates of S. boulardii (6 obtained from probiotic preparations and 2 from clinical specimens) using four different molecular methods, two based on PCR-restriction enzyme analysis or sequencing of rDNA spacer regions, the third based on microsatellite polymorphism analysis of the S. cerevisiae genes YKL139w and YLR177w, and the last based on hybridization analysis with retrotransposon Ty917. Several clinical isolates appeared to be identical to one or more other isolates with the first three methods used, whereas with the Ty917 hybridization method all of the isolates tested appeared to be very heterogeneous. The eight S. boulardii isolates were clearly distinguishable from the clinical S. cerevisiae isolates only with Ty917 hybridization and microsatellite DNA analyses. In the latter method, the eight S. boulardii isolates exhibited an allelic variant at one of loci tested that was not shared with any other strain. Our results suggest that microsatellite polymorphism analysis of the YKL139w and YLR177w genes, as well as the analysis by Ty917 hybridization, are the most useful tools for a correct identification of S. boulardii strains.

Identification of methicillin-resistant isolates of Staphylococcus aureus and coagulase-negative staphylococci responsible for bloodstream infections with the Phoenix system.

We evaluated the reliability of the new Phoenix system (Becton Dickinson Microbiology Systems, Sparks, Md.) in species-level identification and detection of oxacillin (methicillin) resistance among 493 staphylococcal isolates (Staphylococcus aureus, n = 223; coagulase-negative staphylococci, CoNS, n = 270) recovered from patients with bacteremia. Identification results were concordant with those of the ID 32 STAPH system (bioMérieux, Marcy l'Etoile, France) for 100% of S. aureus (223/223) and 97.4% (263/270) of CoNS isolates. For S. aureus isolates, Phoenix oxacillin-susceptibility results fully concurred with those of mecA polymerase chain reaction (PCR) (reference method): 96 mecA-positive isolates identified as resistant, 127 mecA-negative strains as susceptible. Two of the 210 mecA-positive CoNS isolates were misclassified as susceptible by the Phoenix (sensitivity 99%, positive predictive value 97.6%). Five of 60 mecA-negative CoNS isolates were classified as resistant by the Phoenix (specificity 91.7%; negative predictive value 96.5%). The Phoenix system can provide accurate and reliable identification of methicillin-resistant staphylococci responsible for bloodstream infections.

Entamoeba dispar: A Rare Case of Enteritis in a Patient Living in a Nonendemic Area.

Entamoeba dispar, a common noninvasive parasite, is indistinguishable in its cysts and trophozoite forms from Entamoeba histolytica, the cause of invasive amebiasis, by microscopy. To differentiate the two species seems to be a problem for laboratory diagnosis. Recent experimental studies showed that E. dispar can be considered pathogenic too. We present a rare case of enteritis due to E. dispar.

Ru486: del aborto químico a la contracepción de emergencia / Ru486: from chemical abortion to emergency contraception

El uso del mifepriston (RU486), en combinación con análogos de las prostaglandinas, en la inducción del aborto químico requiere una específica reflexión en relación a los principales aspectos farmacológicos y toxicológicos. La actual dialéctica bioética y biopolítica, a menudo ideologizada, impone aún más un tratamiento riguroso basado en evidencias científicas, para aclarar sobre todo los mecanismos de acción y de los eventos adversos. Estos últimos a veces también subvalorados o minimizados. Considerada la iniquidad del aborto voluntario, el artículo se propone también el objetivo de aclarar cómo, a la luz de una reciente bibliografía, el recurso al RU486 representa un significativo riesgo para la salud de las mujeres. Una particular atención está reservada a la aclaración etiopatogenética de las hemorragias y de las sepsis, en las cuales se han evidenciado también distintos decesos. En el artículo están presentes, además, los más actuales desarrollos de la investigación con RU486 sea para el tratamiento experimental de patologías –ginecológicas y no– como para el uso de la molécula de la contracepción hormonal y la contracepción de emergencias.

The use of mifepristone (RU486), in combination with prostaglandin analogues, in chemical abortion requires a specific reflection on the main aspects in pharmacology and toxicology. The current debate in bioethics and bio-policies, often ideological, imposes a more rigorous treatment based on scientific evidence, especially clarification of the mechanisms of action and severe adverse events. The latter is sometimes underestimated or minimized. Considering the inequity of voluntary abortion, this article aims also to clarify how, in the light of the most recent literature; the use of RU486 represents a significant risk to women’s health. Particular attention is given to etiopathogenetic clarification of bleeding and sepsis, which have also involved several deaths. The article reports the latest developments in research with RU486, whether for experimental treatment of pathologies –gynaecological and others– and for the use of the hormonal contraception molecule and emergency contraception.

ESBL-producing multidrug-resistant Providencia stuartii infections in a university hospital.

OBJECTIVES: To investigate the epidemiological and clinical findings of extended-spectrum beta-lactamase (ESBL)-producing Providencia stuartii infections in a large Italian university hospital. PATIENTS AND METHODS: All consecutive episodes of P. stuartii infection that occurred during 1999-2002 were included in the study. For each patient, we recorded the area of hospitalization and drug susceptibility of the P. stuartii strains. Patients with ESBL-producing P. stuartii infection were considered cases and those with non-ESBL-producing P. stuartii infection were used as controls. RESULTS: One hundred and sixteen (52%) out of 223 P. stuartii strains collected during the study period were found to be ESBL-producing. On the basis of PCR and DNA sequencing experiments, TEM-52 was identified in 87% of isolates and TEM-72 in 13%. All ESBL-producing P. stuartii infections were nosocomially acquired. The prevalence increased from 31% of P. stuartii infections in 1999 to 62% in 2002 (P = 0.04). All 116 strains were classified as ESBL-producing multidrug-resistant P. stuartii, since 88% of the isolates were cross-resistant to ciprofloxacin and amikacin and the other 12% were cross-resistant to ciprofloxacin and gentamicin. At logistic regression analysis, advanced age (P < 0.001), previous hospitalization (P < 0.01), neoplastic disease (P < 0.001) and previous antibiotic therapy (P < 0.001) were independent risk factors for the development of ESBL-producing infections. CONCLUSIONS: This 4 year surveillance of Providencia complaints clearly indicates that infections caused by ESBL-producing multidrug-resistant P. stuartii are an emerging problem.

Identification and characterization of a Cryptococcus neoformans ATP binding cassette (ABC) transporter-encoding gene, CnAFR1, involved in the resistance to fluconazole.

Resistance to fluconazole is a possible event during prolonged suppressive drug therapy for cryptococ-cal meningitis, the most frequently encountered life-threatening manifestation of cryptococcosis. The knowledge of this resistance at the molecular level is important for management of cryptococcosis. In order to identify genes involved in azole resistance in Cryptococcus neoformans, a cDNA subtraction library technique was chosen as a strategy. First, a fluconazole-resistant mutant BPY22.17 was obtained from a susceptible clinical isolate BPY22 by in vitro exposure to the drug. Then, a subtractive hybridization procedure was used to compare gene expression between the obtained strains. We identified a cDNA overexpressed in the fluconazole-resistant strain BPY22.17 that was used as a probe to isolate the entire gene in a C. neoformans genomic library. Sequence analysis of this gene identified an ATP Binding Cassette (ABC) transporter-encoding gene called C. neoformans AntiFungal Resistance 1 (CnAFR1). Disruption of CnAFR1 gene in the resistant isolate (BPY22.17) resulted in an enhanced susceptibility of the knock-out mutant cnafr1 against fluconazole, whereas reintroduction of the gene in cnafr1 resulted in restoration of the resistance phenotype, thus confirming that CnAFR1 is involved in fluconazole resistance of C. neoformans. Our findings therefore reveal that an active drug efflux mechanism can be involved in the development of azole resistance in this important human pathogen.

Characterization of clinical isolates of Enterobacteriaceae from Italy by the BD Phoenix extended-spectrum beta-lactamase detection method.

Production of extended-spectrum beta-lactamases (ESBLs) is an important mechanism of beta-lactam resistance in Enterobacteriaceae: Identification of ESBLs based on phenotypic tests is the strategy most commonly used in clinical microbiology laboratories. The Phoenix ESBL test (BD Diagnostic Systems, Sparks, Md.) is a recently developed automated system for detection of ESBL-producing gram-negative bacteria. An algorithm based on phenotypic responses to a panel of cephalosporins (ceftazidime plus clavulanic acid, ceftazidime, cefotaxime plus clavulanic acid, cefpodoxime, and ceftriaxone plus clavulanic acid) was used to test 510 clinical isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Providencia stuartii, Morganella morganii, Enterobacter aerogenes, Enterobacter cloacae, Serratia marcescens, Citrobacter freundii, and Citrobacter koseri. Of these isolates, 319 were identified as ESBL producers, and the remaining 191 were identified as non-ESBL producers based on the results of current phenotypic tests. Combined use of isoelectric focusing, PCR, and/or DNA sequencing demonstrated that 288 isolates possessed bla(TEM-1)- and/or bla(SHV-1)-derived genes, and 28 had a bla(CTX-M) gene. Among the 191 non-ESBL-producing isolates, 77 isolates produced an AmpC-type enzyme, 110 isolates possessed TEM-1, TEM-2, or SHV-1 beta-lactamases, and the remaining four isolates (all K. oxytoca strains) hyperproduced K1 chromosomal beta-lactamase. The Phoenix ESBL test system gave positive results for all the 319 ESBL-producing isolates and also for two of the four K1-hyperproducing isolates of K. oxytoca. Compared with the phenotypic tests and molecular analyses, the Phoenix system displayed 100% sensitivity and 98.9% specificity. These findings suggest that the Phoenix ESBL test can be a rapid and reliable method for laboratory detection of ESBL resistance in gram-negative bacteria.

Use of the VITEK 2 system for rapid identification of clinical isolates of Staphylococci from bloodstream infections.

Staphylococci are an increasing cause of bloodstream infections. Rapid reliable identification of these organisms is essential for accurate diagnosis and prompt effective treatment. We evaluated the ability of the VITEK 2 system (bioMérieux, Inc, Hazelwood, Mo.) to identify these organisms rapidly and accurately. A total of 405 clinically relevant nonduplicate staphylococcal isolates (Staphylococcus aureus, n = 130; coagulase-negative staphylococci, n = 275) collected from blood cultures were tested. VITEK 2 results were considered correct when they were identical to those furnished by the comparison method based on the ID 32 STAPH system (bioMérieux, Marcy l'Etoile, France) plus supplementary manual testing. When discrepancies occurred, isolate identity was verified by molecular typing. The VITEK 2 correctly identified 387 (95.6%) isolates at the species level: 379 (including all but one [99.2%] of 130 S. aureus isolates and 249 of 275 [90.5%] coagulase-negative isolates) were identified by the automated reading; for the other eight, supplemental tests suggested by the manufacturer had to be used. Only one strain (0.2%) was misidentified (Staphylococcus hominis as Staphylococcus epidermidis), and four (1%), all S. epidermidis, were not identified. For the remaining 13 strains (including 10 S. hominis), the VITEK 2 system was unable to discriminate among two species, and no supplemental tests were suggested for conclusive identification. Over 90% of results were obtained within 4 h. These results suggest that the VITEK 2 system can provide rapid, accurate, and reliable species-level identification of staphylococci responsible for bloodstream infections, although there is room for improvement in the identification of certain coagulase-negative species, especially S. hominis.

Early detection of negative BACTEC MGIT 960 cultures by PCR-reverse cross-blot hybridization assay.

We evaluated the efficacy of a PCR-reverse cross-blot hybridization assay, a test which permits identification of mycobacteria by means of species-specific probes and a Mycobacterium-specific probe, for early detection of negative BACTEC MGIT 960 mycobacterial cultures. Aliquots of 549 cultures were collected 7 days after the culture media were inoculated with various clinical specimens and tested with the molecular assay. PCR results were compared to those obtained at the end times with the BACTEC MGIT 960 system. Of the 549 specimens analyzed, 484 were found to be negative and 64 were found positive by both methods; one specimen, found to be positive by the BACTEC MGIT 960 system, was identified as negative by the molecular assay. In view of its high negative predictive value (99.8%), the PCR-reverse cross-blot hybridization assay appears to be a valid tool for early detection of negative BACTEC MGIT 960 cultures.