Utility of terminal hemadsorption for detection of hemadsorbing respiratory viruses from conventional shell vial cultures for laboratories using R-Mix cultures.

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.

Use of lyophilized standards for the calibration of a newly developed real time PCR assay for human herpes type six (HHV6) variants A and B.

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.

Evaluation of a PCR probe capture assay for the detection of Toxoplasma gondii. Incorporation of uracil N-glycosylase for contamination control.

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.

A polymerase chain reaction-based epidemiologic investigation of the incidence of nonpolio enteroviral infections in febrile and afebrile infants 90 days and younger.

OBJECTIVE: Enteroviruses are important pathogens in infants, but their true contribution to febrile illness in infants

Enhancement of the AMPLICOR enterovirus PCR test with a coprecipitant.

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.