Evaluación del cultivo liofilizado de Candida albicans utilizado en esquemas de certificaciones de calidad / Evaluation of the lyophilized culture of Candida albicans used in quality certification schemes

Introducción: el laboratorio de control microbiológico de la UEB Laboratorios Liorad dispone de una colección de cultivos microbianos para la conservación de microorganismos, donde se encuentra depositada la levadura Candida albicans que se emplea en esquemas de certificaciones de calidad establecidos para la evaluación de ensayos como: promoción de crecimiento de los medios de cultivos, validación de técnicas microbiológicas, entre otros. Objetivo: evaluar los resultados de la conservación de esta cepa por el método de liofilización durante un periodo de ocho años. Métodos: para el crecimiento de la cepa se utilizó el medio de cultivo Caldo Saboraud y variantes de sustancias lioprotectoras puras como: (leche descremada al 20 por ciento, glicerol 20 por ciento, sacarosa al 10 por ciento y peptona 5 por ciento) así como la mezcla de lioprotectores (leche 10 por ciento, sacarosa 5 por ciento, glicerol 5 por ciento). Se evaluó viabilidad, pureza y estabilidad genética de esta cepa durante el tiempo objeto de estudio. Resultados: las características propias de la especie estudiada se mantuvieron inalterables con un elevado grado de pureza en todas las variantes estudiadas. En cuanto a la supervivencia, cuando se emplearon las sustancias lioprotectoras puras se evidenció una marcada disminución de la viabilidad. No así al emplear la mezcla de lioprotectores que mantuvo niveles de viabilidad por encima del límite establecido durante todo el tiempo objeto de estudio. Conclusiones: los valores obtenidos en cuanto a la supervivencia de este microorganismo permiten inferir que para la conservación por largos periodos de tiempo la variante donde se empleó mezclas de lioprotectores resultó una buena opción para la conservación de C. albicans(AU)

Introduction: the microbiological control laboratory at the Basic Enterprise Unit Liorad Laboratories stores a collection of microbial cultures for the preservation of microorganisms, including the Candida albicans yeast used in the quality certification schemes established for the evaluation of assays such as fostering of culture medium growth and validation of microbiological techniques, among others. Objective: evaluate the results obtained in the preservation of this strain by the lyophilization method during a period of eight years. Methods: for strain growth, use was made of Saboraud broth culture medium as well as variants of pure lyoprotective substances such as 20 percent skimmed milk, 20 percent glycerol, 10 percent saccharose and 5 percent peptone, and the mixture of lyoprotectors (10 percent milk, 5 percent saccharose, 5 percent glycerol). An evaluation was conducted of the viability, purity and genetic stability of the strain during the study period. Results: characteristics typical of the study species remained unchanged with a high degree of purity in all the variants examined. As to survival, a marked reduction in viability was observed when pure lyoprotective substances were used, but not with the mixture of lyoprotectors, in which case viability levels exceeded the established limit during the entire study period. Conclusions: the survival values obtained for this microorganism indicate that preservation for long periods with mixtures of lyoprotectors was a good option for the preservation of C. albicans(AU)

The management of first episode genital herpes in genitourinary medicine clinics: a national audit in 2006.

A national audit of the management of first episode genital herpes (GH) was undertaken by non-consultant career grade doctors working in genitourinary (GU) medicine clinics in the United Kingdom. In total, 1620 data collection forms were completed (794 men and 826 women). Virus culture is the main detection method (1150, 71%), although polymerase chain reaction (PCR) use is increasing (442, 28%). PCR was significantly associated (P<0.0001) with fewer negative isolates (29/442, 6.6%) compared with virus culture (174/1150, 15%). Herpes simplex virus type 1 was isolated in 552 cases (46%). For 243 cases (15%), there was no evidence of counselling about GH. A total of 1355 (84%) were screened for other sexually transmitted infections. Serological testing for syphilis was undertaken in 72%. GU medicine clinics are managing first episode GH as broadly outlined in the guidelines. Areas identified for improvement are to increase counselling/written information/testing for syphilis towards 100%.

Viral growth assay to evaluate the replicative capacity of HIV-1 isolates.

The replicative capacity of HIV is studied by carrying out replication-competition experiments with the insertion of the gene of interest. These assays cannot capture the complicated patterns of mutations of different genes.A cross sectional study was carried out on 10 HIV-infected nai;ve patients and on 15 patients failing HAART. The CD8-depleted PBMCs, with known proviral DNA and cellular HIV-RNA copy numbers, were cultured. A reference curve was determined using the data obtained from 10 nai;ve patients. The replicative capacity was calculated as the ratio multiplied by 100 of the p24 antigen level of isolates over the p24 antigen level determined on the reference curve.A linear correlation between p24 antigen level and the infectious doses of HIV-DNA alone or plus cellular RNA copy number of PBMCs was found in naive patients (r=0.63, P<0.001 and r=0.67, P<0.001, respectively). Although all patients failing therapy had strains with impaired replicative capacity, a wide range of values (0.1-74.5%) was detected. All strains with a replicative capacity above 10% had non-nucleoside reverse transcriptase inhibitors related mutations.A viral assay to evaluate the HIV replicative capacity is described. The high variability of replicative capacity confirms the need to undertake replicative capacity assay using the whole virus.

Comparison of lateral-flow immunoassay and enzyme immunoassay with viral culture for rapid detection of influenza virus in nasal wash specimens from children.

The performance of two commercially available rapid test kits for influenza virus detection was compared to that of viral culture by using 356 nasal wash specimens collected during the 2001 to 2002 influenza season. Overall, the two rapid tests were easy to perform and showed comparable sensitivities (70.4 and 72.2%) and specificities (97.7 and 98.3%); for both test kit groups, most of the specimens that yielded false-negative results were found to be growing influenza B virus.

Comparison of the Denka-Seiken INFLU A.B-Quick and BD Directigen Flu A+B kits with direct fluorescent-antibody staining and shell vial culture methods for rapid detection of influenza viruses.

The INFLU A.B-Quick and Directigen Flu A+B enzyme immunoassays were compared with direct immunofluorescence and cell culture for detection of influenza A and B viruses in a total of 255 patient specimens. Both assays identified 23 of 42 influenza A viruses (sensitivity, 54.8%; specificity, 100%; positive predictive value [PPV], 100%; negative predictive value [NPV], 91.8%). The INFLU A.B-Quick assay identified 10 of 16 influenza B viruses (sensitivity, 62.5%; specificity, 99.6%; PPV, 90.9%; NPV, 97.5%), and the Directigen Flu A+B assay detected 9 of 16 influenza B viruses (sensitivity, 56.3%; specificity, 99.6%; PPV, 90%; NPV, 97.1%).

The clinical utility of CMV surveillance cultures and antigenemia following bone marrow transplantation.

At our institution, the cytomegalovirus (CMV) prophylaxis protocol for allogeneic bone marrow transplant (BMT) recipients who are CMV-seropositive or receive marrow from a CMV-seropositive donor consists of a surveillance bronchoscopy approximately 35 days posttransplant. Patients with a positive surveillance bronchoscopy for CMV receive pre-emptive ganciclovir. In order to determine the utility of other screening methods for CMV, we prospectively performed weekly CMV antigenemia, and blood, urine and throat cultures from time of engraftment to day 120 post-BMT in 126 consecutive patients. Pre-emptive ganciclovir was given to 11/81 patients (13.6%) because of a positive surveillance bronchoscopy for CMV. Results of CMV blood, urine and throat cultures and the antigenemia assay done prior to or at the time of the surveillance bronchoscopy were analyzed for their ability to predict the bronchoscopy result. The antigenemia test had the highest positive and negative predictive values (72% and 96%, respectively). The ability of these tests to predict CMV disease was evaluated in the 70 patients with a negative surveillance bronchoscopy who did not receive pre-emptive ganciclovir. Of 19 cases of active CMV disease, CMV antigenemia was positive in 15 patients (79%) a mean of 34 days preceding symptoms. Blood cultures were positive in 14/19 patients (74%) a mean of 31 days before onset of disease. CMV antigenemia is useful for predicting the surveillance bronchoscopy result, and also predicts the development of CMV disease in the majority of patients missed by the surveillance bronchoscopy.

PCR method for detection of adenovirus in urine of healthy and human immunodeficiency virus-infected individuals.

Adenoviruses (AdV) cause diseases that range from localized, self-limited illnesses to fatal infections in immunocompromised patients. Culture is assumed to be sensitive but requires viable virus and up to 3 weeks for detection, and it can be inhibited by bacterial contamination. A new PCR method amplifying a region of the hexon gene was developed in order to detect AdV in urine more rapidly and with greater sensitivity than obtainable by culture technology. All 18 serotypes tested were detected. Quantitatively, with optimized urine processing, AdV PCR detected 0.2 PFU/ml (serotype 11) and 10 DNA copies/ml (serotype 2). Serially collected urine samples from human immunodeficiency virus (HIV)-infected patients with concurrent cytomegalovirus retinitis were divided into three groups: AdV culture-positive samples, AdV culture-negative or bacterially contaminated samples from patients with a history of AdV culture-positive urines, and AdV culture-negative samples from patients without a history of AdV culture positivity. Urine samples from healthy adults were also tested by culture and PCR to screen for asymptomatic shedding. Amplification was assessed with and without prior DNA purification. AdV was detected by PCR in 90% of culture-positive urines (100% of unclotted samples, e.g., those culture positive after storage for PCR testing), 71% of culture-negative or bacterially contaminated urines from AdV-infected patients, and 28% from AdV culture-negative patients. Healthy volunteers were culture negative for AdV, and 96% were PCR negative. The new AdV PCR method is rapid and sensitive and can detect viral DNA in samples for which culturing is problematic. The role of AdV replication during HIV infection merits further investigation with sensitive tools such as PCR.

Detection and strain differentiation of feline calicivirus in conjunctival swabs by RT-PCR of the hypervariable region of the capsid protein gene.

Reverse transcription-polymerase chain reaction (RT-PCR) was used to direct the amplification of a 670 to 680 base pair segment that included the hypervariable regions of the capsid protein gene of feline calicivirus (FCV). The segment was amplified from 13/13 cultivated FCV strains including 12 field isolates collected over 18 years. The sensitivities of culture and this RT-PCR for the detection of FCV in conjunctival swabs over the course of infection were then compared and correlated with clinical signs in 5 vaccinated and 3 unvaccinated experimentally-infected cats. Conjunctival swabs were taken daily from days 0 to 14 and on days 16, 19, 21 and 24 after challenge. FCV was detected in 19/144 swabs by RT-PCR and 16/144 swabs by culture. Virus detection correlated poorly with clinical signs regardless of the assay used. The Sau3AI restriction profiles of the RT-PCR products amplified from both cultivated strains and clinical samples were compared. All 13 cultivated isolates, including the 12 field isolates, exhibited different profiles, whereas all profiles from the experimentally-infected cats were identical, and matched the profile of the challenge/vaccine strain. This study has established that the RT-PCR assay described is as sensitive as culture for detection of FCV in conjunctival swabs, that a broad range of field isolates can be detected and rapidly differentiated by restriction endonuclease digestion, and that the assay thus meets the requirements for large scale epidemiological studies of FCV infections in cats. However, it has also shown that even the use of RT-PCR on conjunctival swabs alone is insufficient for accurate diagnosis of FCV infection in cats with conjunctivitis.

Novel DNA assay for cytomegalovirus detection: comparison with conventional culture and pp65 antigenemia assay.

We compared conventional cytomegalovirus (CMV) isolation, rapid viral culture, a CMV pp65 antigenemia assay, and a novel CMV DNA hybrid capture system (HCS). A total of 309 blood samples from individuals in different risk groups were assessed by at least two of the methods mentioned above. Leukocytes were recovered either after centrifugation in Leucosep tubes containing 1.080 Ficoll for pp65 assay or after simple differential lysis steps for DNA detection. HCS was based on DNA hybridization with a CMV RNA probe and its capture by antibodies to DNA-RNA hybrids. The CMV pp65 lower matrix protein was detected by fluorescence with c10-c11 monoclonal antibody in formalin-fixed leukocytes. Concordant results were observed for 92.9, 78.3, and 82.7% of the patients when comparing (i) viral culture and the pp65 antigenemia assay, (ii) viral culture and HCS, and (iii) the pp65 antigenemia assay and HCS, respectively. Discordant results were observed between a positive HCS result and negative culture and/or pp65 results. These results were associated with relatively low DNA levels (< 20 pg/10(6) cells) and positive viruria. In conclusion, the pp65 antigenemia assay is a rapid and reliable method of detecting CMV and is preferable to culture, but the Murex HCS appears to be more sensitive for CMV detection.

Comparative susceptibilities of human embryonic fibroblasts and HeLa cells for isolation of human rhinoviruses.

The recovery of human rhinovirus (HRV) from nasal washings and nasal and pharyngeal swabs from volunteers with naturally acquired colds was compared in different cell types. Human embryonic lung fibroblast (HELF) strain WI-38 (sensitivity, 61 to 84%) and HeLa-I, an HRV-susceptible HeLa clone (sensitivity, 86 to 94%), were the most sensitive cell types used. HELF-WI-38 cells showed a cytopathic effect earlier than the other cells used, and the different strains of HRV-susceptible HeLa cells varied in their sensitivities for HRV isolation. HRV was detected in a single cell type in 20 to 35% of the positive samples, suggesting that use of a combination of different HRV-susceptible cell lines is the best approach for the recovery of HRV. Although nasal washings tended to yield more HRV isolates than nasal and pharyngeal swabs, the two sampling methods were not found to be significantly different.

Monitoring of CMV infection: a comparison of PCR from whole blood, plasma-PCR, pp65-antigenemia and virus culture in patients after bone marrow transplantation.

Rapid, specific and sensitive methods are essential for early detection of CMV infection in patients after marrow transplantation. Thus, in a prospective study, PCR from whole blood, plasma-PCR, pp65-antigenemia and virus culture were used and compared in 20 consecutive marrow transplant recipients for early diagnosis of CMV infection and monitoring of antiviral therapy. Moreover, semi-quantification of the viral load in blood samples by PCR from whole blood or plasma and pp65-antigenemia was performed. Fifteen out of 20 patients were found to be CMV positive by PCR from whole blood, plasma-PCR and pp65-antigenemia, whereas only 9/20 developed culture-proven viremia and/or viruria. PCR from whole blood, plasma-PCR and pp65-antigenemia revealed identical results in 96 and discordant results in 13 of 109 blood samples (P < 0.01). The efficacy of antiviral therapy was monitored by semi-quantitative scoring of pp65-antigen-positive leukocytes and/or CMV-DNA levels in blood and plasma samples. Twelve of 13 patients were found to be CMV negative by all methods after 14 days of ganciclovir therapy. A good correlation of the semi-quantitative evaluation of the three assays was demonstrated. Thus, all three highly sensitive assays seem to be suitable for screening patients at risk for CMV infection and monitoring the efficacy of antiviral therapy.

Comparison of a direct antigen enzyme immunoassay, Herpchek, with cell culture for detection of herpes simplex virus from clinical specimens.

Direct enzyme immunoassay (Herpchek) was compared with culture on 21,522 specimens mainly from asymptomatic women for herpes simplex virus detection. Sensitivity and specificity were 73.8 and 97.7%, respectively. The 33% detection rate by enzyme immunoassay in 5 h increased to 43% when the enzyme immunoassay was combined with culture. Herpchek alone was not sensitive enough, but in combination with culture, maximum detection was achieved.

Herpes simplex virus detection from genital lesions: a comparative study using antigen detection (HerpChek) and culture.

The sensitivity of a rapid enzyme immunoassay test (HerpChek Direct Herpes Simplex Virus Antigen Test [DuPont Medical Products, Wilmington, Del.]) for the detection of herpes simplex virus (HSV) antigens in patient specimens was compared with HSV culture. HerpChek positivity for HSV occurred with 179 (65%) of 275 lesion specimens, whereas culture for HSV was positive for 145 (53%) lesions (P = 0.002). HerpChek was twice as sensitive as culture for the detection of HSV in late-stage lesions and was equivalent to culture for the detection of HSV in early lesions. We conclude that HerpChek provides greater sensitivity than culture for HSV detection in late-stage genital lesions.

Human cytomegalovirus (HCMV) late-mRNA detection in peripheral blood of AIDS patients: diagnostic value for HCMV disease compared with those of viral culture and HCMV DNA detection.

A reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect a human cytomegalovirus (HCMV) late mRNA in peripheral blood leukocytes (PBL) of 102 human immunodeficiency virus-infected individuals. The clinical value of this new technique for the diagnosis of acute HCMV disease was evaluated in comparison with viral culture and direct amplification of viral DNA (PCR). The sensitivity of the RT-PCR was slightly lower than that of the two other methods, but its specificity was 94%, compared to 55 and 32% for culture and PCR, respectively. Transcription of this late mRNA is linked to viral replication, and its detection in PBL confirms that these cells can support a complete viral cycle. The relationship between complete replicative cycles and HCMV disease makes RT-PCR a useful clinical tool.

Cytomegalovirus (CMV) antigenemia assay is more sensitive than shell vial cultures for rapid detection of CMV in polymorphonuclear blood leukocytes.

We compared the cytomegalovirus (CMV) antigenemia assay with shell vial cultures of polymorphonuclear leukocyte (PMNL)-enriched blood fractions for rapid diagnosis of CMV viremia. PMNL fractions of 280 blood specimens from 171 patients (170 solid-organ transplant recipients and 1 patient undergoing pretransplant evaluation) were inoculated in shell vial and conventional CMV cultures. A commercially available kit (CMV-vue kit; INCSTAR Corp.) was used for the CMV antigenemia assay, in which PMNL preparations were stained with monoclonal antibodies directed against the CMV protein pp65. Mixed-leukocyte blood fractions from the same blood specimens were inoculated in parallel shell vial and conventional cultures. CMV viremia (defined by the isolation of CMV in conventional cultures) was detected in 32 (13%) of 245 PMNL fractions included in the final analysis. Twenty-eight (87.5%) were also positive in the CMV antigenemia assay, whereas 22 (69%) were positive in shell vial cultures. Ten (4%) additional PMNL fractions positive only in the CMV antigenemia assay were from eight patients with active CMV infections (six patients), who had previous or subsequent episodes of CMV viremia (seven patients), or in whom CMV was isolated in cultures of simultaneously obtained mixed-leukocyte fractions (three patients). Overall, the CMV antigenemia assay was significantly more sensitive than shell vial cultures for detection of CMV in the PMNL fraction of blood leukocytes (P < 0.01, McNemar's test), and we recommend it as the method of choice for rapid diagnosis of CMV viremia.

Meningitis bacteriana / Bacterial meningitis

Durante 6 meses se investigo la incidencia de meningitis bacteriana en el Hospital del Nino, comprobandose una incidencia y una mortalidad elevada. El grupo etareo mas afectado fue el de menores de 2 anos, totalizando el 83// de los casos estudiados. La etiologia predominante fue debida a dos germenes: Streptococus pneumoniae y Haemophilus influenzae en proporciones casi similares, La patologia previa mas frecuente y relacionada con el cuadro meningeo fue la infeccion respiratoria aguda. La terapia microbacteriana instalada en todos ellos fue la asociacion penicilina sodica y cloranfenicol a dosis establecidas internacionalmente, cambiando luego de conocer todos los resultados de los cultivos del (LCR) por ampicilina en vez de penicilina sodica en los casos de H influenzae en ningun caso hubo resistencia a las drogas administradas. Las complicaciones y el indice de mortalidad fueron mayores en las infecciones por Streptococcus pneumoniae. Nuestro estudio demuestra que la meningitis por Haemophilus influenzae en el Hospital del Nino representa un porcentaje elevado con cifras parecidas a estudios foraneos y por esto recomendamos comenzar el tratamiento con una combinacion de ampicilina y cloranfenicol, hasta conocer los resultados de los cultivos. Es importante realizar estudios en otros centros para evaluar nuestro estudio, confirmando o negando los resultados presentes.