RESUMO
BACKGROUND: Many laboratories using R-Mix cell lines inoculate other shell vial cultures to improve the recovery of viruses, and in particular, perform terminal hemadsorption (THad) following 10-14 days of incubation to improve detection of respiratory viruses. We explored the cost-effectiveness and added benefits of THad on conventional shell vial cultures from respiratory samples for laboratories using R-Mix cell lines. OBJECTIVES: To determine if eliminating the practice of THad from conventional shell vial culture when R-Mix cultures are negative, would result in a significant reduction in the number of hemadsorbing respiratory viruses detected. STUDY DESIGN: THad results were retrospectively reviewed for 41,129 respiratory shell vial cultures that were set up concurrently with R-Mix cultures. RESULTS: Greater than 95% of hemadsorbing respiratory viruses were recovered by R-Mix standard protocol within 24h of inoculation, and only 5% were detected by THad at 10-14 days. CONCLUSION: The practice of hemadsorption at days 10-14 for conventional shell vial cultures from respiratory specimens should be discontinued for laboratories using R-Mix due to its low yield, questionable clinical impact of delayed results and additional costs.
Assuntos
Hemadsorção , Infecções Respiratórias/virologia , Viroses/diagnóstico , Vírus/isolamento & purificação , Linhagem Celular , Humanos , Cultura de Vírus/economia , Cultura de Vírus/métodosRESUMO
A quantitative real time PCR assay utilizing an Eclipse minor groove binding hybridization probe was developed to detect and type human herpes 6. A 115 base pair product from the U67 gene was selected for amplification and the assay included a noncompetitive internal control. The probe's melting temperature from the amplified sequence differentiated between HHV6 variants (A and B). In this study, 120 samples (60 spiked and 60 negative) comprising CSF, plasma, and serum were tested at high and medium levels, and near the limit of quantitation. The use of stored standard curves for assay calibration was compared to curves run on each assay, and the stability of liquid frozen versus lyophilized frozen stocks for calibrators and controls was assessed. After 9 months of clinical testing, assay performance was examined to determine the percent positive rate and positive sample reproducibility, as well as to evaluate standard curve stability. We obtained 100% correlation to expected results for positive and negative samples. A stored curve proved easier, more cost effective, and more reliable than running a standard curve on each assay. The use of lyophilized standards contributes substantially to the maintenance of reproducible testing over an extended period of time.
Assuntos
Variação Genética , Herpesvirus Humano 6/classificação , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Infecções por Roseolovirus/diagnóstico , Infecções por Roseolovirus/virologia , Sangue/virologia , Calibragem/normas , Líquido Cefalorraquidiano/virologia , Sondas de DNA , Liofilização , Herpesvirus Humano 6/genética , Herpesvirus Humano 6/isolamento & purificação , Humanos , Padrões de Referência , Sensibilidade e EspecificidadeRESUMO
Toxoplasma gondii is a cyst-forming parasite of clinical relevance in humans primarily because of the neurologic abnormalities it can cause. In some clinical circumstances, it is desirable to detect the pathogen directly. We modified a commercially available Toxoplasma polymerase chain reaction (PCR) probe capture assay by incorporating uracil N-glycosylase (UNG) to prevent carryover amplicon contamination. In addition, UNG inactivation and DNA denaturation were accomplished chemically to simplify the DNA hybridization to the capture probe. The incorporation of UNG effectively eliminated carryover contamination; the probe capture assay showed a log increase in detection sensitivity compared with standard agarose gel electrophoresis. To assess sensitivity and possible inhibition of amplification, different sample types were spiked with Toxoplasma organisms. After DNA extraction and PCR amplification, a sensitivity of 2 tachyzoites for the assay was determined in buffered saline, cerebrospinal fluid (CSF), serum, and amniotic fluid; 20 tachyzoites for whole blood; and 200 tachyzoites for brain tissue. An additional 20 human serum and CSF samples submitted for Toxoplasma serologic testing were run by the PCR method. Of these, only an IgM-positive CSF sample was PCR positive. The Toxoplasma PCR probe capture assay showed good sensitivity and was not substantially inhibited by several different clinically relevant samples.
Assuntos
DNA Glicosilases , N-Glicosil Hidrolases , Reação em Cadeia da Polimerase/métodos , Toxoplasma/genética , Toxoplasma/isolamento & purificação , Líquido Amniótico/parasitologia , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/líquido cefalorraquidiano , Sangue/parasitologia , Encéfalo/parasitologia , Líquido Cefalorraquidiano/parasitologia , DNA de Protozoário/análise , Sangue Fetal/parasitologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/líquido cefalorraquidiano , Imunoglobulina M/sangue , Imunoglobulina M/líquido cefalorraquidiano , Desnaturação de Ácido Nucleico , Sensibilidade e Especificidade , Uracila-DNA GlicosidaseRESUMO
OBJECTIVE: Enteroviruses are important pathogens in infants, but their true contribution to febrile illness in infants =90 days old is unknown. The purpose of this study was to use the polymerase chain reaction (PCR) for diagnosis of enteroviral (EV) infection in febrile and afebrile infants =90 days of age to improve the understanding of the epidemiology of EV infection in this population. METHODS: Patients included all unimmunized, febrile infants =90 days of age admitted to Primary Children's Medical Center (Salt Lake City, UT) for sepsis evaluation from December 1996 to December 1997. Blood, urine, cerebrospinal fluid, and throat swabs were tested for enteroviruses using a PCR assay (Roche Molecular Systems, Branchburg, NJ). Alternate PCR assays separated polio and nonpolio enteroviruses. Results of bacterial cultures, outcome, and hospital charges were obtained. Blood from afebrile, control infants =90 days old was tested for enteroviruses. RESULTS: A total of 345 febrile infants were enrolled; 89 (25.8%) were positive for enterovirus. The incidence of EV infection ranged from 3.2% in January to 50% in August and October. Five EV-positive, febrile infants (5.6%) had concomitant urinary tract infections, and 1 (1. 1%) had concomitant bacteremia. Infants with confirmed EV infection were significantly less likely to have bacterial infection than those who were EV-negative. All infants infected with an enterovirus recovered. Average length of stay was 3 days, average charges were nearly $4500. Eighty-six afebrile, control infants were enrolled; 6 (6.9%) were positive for enterovirus; 3 had received oral polio vaccine. CONCLUSIONS: Nonpolio EV infections commonly cause fever in infants =90 days of age. Rates of EV positivity are low in afebrile, unimmunized infants. The use of PCR to identify febrile infants with nonpolio EV infections may decrease length of hospital stay, unnecessary antibiotic administration, and charges.
Assuntos
Infecções por Enterovirus/epidemiologia , Reação em Cadeia da Polimerase , Infecções Bacterianas/complicações , Estudos de Casos e Controles , Enterovirus/genética , Enterovirus/isolamento & purificação , Infecções por Enterovirus/complicações , Infecções por Enterovirus/diagnóstico , Infecções por Enterovirus/virologia , Feminino , Febre/virologia , Hospitais Pediátricos , Humanos , Incidência , Lactente , Recém-Nascido , Masculino , Poliovirus/genética , Poliovirus/isolamento & purificação , Sensibilidade e Especificidade , Utah/epidemiologiaRESUMO
The incorporation of a commercially available coprecipitant into the AMPLICOR enterovirus PCR test specimen preparation enhanced the sensitivity and reproducibility of this assay. Fifty-five previously tested archived cerebrospinal fluids (CSF) specimens were tested in a blind study in duplicate with and without Pellet Paint coprecipitant (Novagen, Inc., Madison, Wis.). Of these specimens, 26 had previously been determined to be positive and 29 had previously been determined to be negative. All previously positive CSF specimens were positive when Pellet Paint was used and only 18 were positive without Pellet Paint. No previously negative specimens were positive on repeat testing with or without Pellet Paint. The background signal was not affected by the addition of Pellet Paint. These data support the utility of a coprecipitant in minimizing false-negative results.