Monoclonal antibodies (Syva Co., Palo Alto, Calif.) were used for the detection and serotyping of herpes simplex virus (HSV) isolates by immunofluorescence 16 h after inoculation of MRC-5 monolayers in 3.7-ml shell vials and after low-speed centrifugation. A total of 119 specimens were inoculated into conventional tube celi cultures and sheil vials. Of 98 specimens inoculated on the same day of receipt in the laboratory (fresh specimens), all 23 (23.5%) HSV-positive specimens were identified by serotype in 16 h in shell vials by immunofluorescence, whereas only 8 of 23 HSV-positive specimens (34.8%) produced cytopathic effects in conventional tube cell cultures in this time period. Similarly, of 21 original specimen extracts previously determined to be culture positive for HSV (stored specimens), all were detected and serotyped by the immunofluorescence test with monoclonal antibodies 16 h postinoculation compared with the recognition of only 8 of these isolates (38.1 %) by cytopathic effect that soon. This technique of centrifugal inoculation of HSV in sheli vials containing MRC-5 cells permitted detection of this virus in ail positive specimens with serotype determination within 16 h postinoculation.

No abstract

The LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) is an automated instrument which can monitor the development of amplified target nucleic acid by fluorescence resonance energy transfer after each amplification cycle. The instrument provides rapid (30 to 40 min) PCR results by precise air-controlled temperature cycling; most importantly, the amplification and detection of an amplified product occur in a closed system, which virtually eliminates the likelihood of carryover contamination. In three evaluations that involved 877 dermal and genital and a few ocular specimens and yielded 285 herpes simplex virus (HSV)-and 44 varicella-zoster virus (VZV)-positive results, LightCycler PCR produced a greater sensitivity for the detection of HSV (22%) and VZV (91%) than did the shell vial cell culture assay for the laboratory diagnosis of these viral infections (5, 6, 7). Based on these performance characteristics, together with comparable cost analysis for LightCycler PCR and shell vial cell culture, we implemented the molecular amplification procedure in May 2000 for the routine diagnosis of HSV dermal and genital and VZV dermal infections.Preliminary to LightCycler PCR, we extracted nucleic acids from dermal and genital specimens by the manual IsoQuick (Orca Research, Inc., Bothell, Wash.) procedure. In the present study, we evaluated two automated systems, MagNA Pure (Roche Molecular Biochemicals, Indianapolis, Ind.) and BioRobot 9604 (Qiagen, Inc., Chatsworth, Calif.) as effective replacements for the manual IsoQuick method with the goal of implementing a cost-efficient, standardized system for the processing of clinical specimens. MATERIALS AND METHODSSpecimens and shell vial assay. Genital (n ϭ 152) and dermal (n ϭ 46) swab specimens from patients suspected of having HSV infections were extracted into 2-ml volumes of serum-free medium; the specimen extracts were then divided into four equal aliquots. Each of two shell vial MRC-5 cell cultures received 200 l of inoculum from one aliquot. The vials were centrifuged, incubated overnight at 36°C, and stained by the indirect immunofluorescence test as previously described (10). Nucleic acids were extracted from the remaining aliquots by three different extraction techniques and processed for amplification of HSV DNA by LightCycler PCR. Nucleic acid extracts obtained by each method were stored at 4°C for a maximum of 2 weeks before PCR amplification.IsoQuick nucleic acid extraction. Nucleic acids were extracted manually from a 200-l volume of serum-free extract of genital or dermal swab specimens by the IsoQuick procedure (Orca Research, Inc.), which utilizes guanidine thiocyanate and a noncorrosive extraction reagent, in accordance with the manufacturer's instructions (4, 7).MagNA Pure nucleic acid extraction. A second aliquot (200 l) was extracted by the MagNA Pure LC automated extractor (Roche Molecular Biochemicals) by using the DNA isolation extraction kit produced by the same manufacturer.Qiagen BioRobot 9604 nucleic acid extraction. Another 200-l ...

Herpes simplex virus (HSV) causes several clinical manifestations in both normal and immunocompromised hosts; this agent is the most frequently detected virus in diagnostic laboratories. Recovery of the virus in cell culture is considered the “gold standard” for detection of this virus from sources other than cerebrospinal fluid. LightCycler is a newly developed, commercially available system designed to rapidly perform PCR, with real-time detection of PCR products by a fluorescence resonance energy transfer assay. We compared the detection of HSV for 200 specimens (number of genital specimens, 160; number of dermal specimens, 38; number of ocular specimens, 2) by shell vial cell cultures (MRC-5) and by LightCycler PCR. Of a total of 88 (44%) HSV strains detected, 69 (78%) were detected by both shell vial cell cultures and LightCycler PCR (DNA polymerase target). A total of 19 (22%) specimens were detected exclusively by LightCycler PCR. No specimens were positive by the shell vial assay only. All 19 discrepant samples had HSV DNA detected by an independent PCR directed to the thymidine kinase gene of the virus. The melting curve analysis feature of the LightCycler instrument identified identical genotype results for HSV type 1 (HSV-1) and HSV-2 from 84 of 88 (96%) positive samples. Specimens can be extracted, target HSV DNA can be amplified, and HSV PCR products can be identified by genotype within 2 h after receipt of specimen into the laboratory. The increased level of accurate identification (all 88 positive samples) compared with that of shell vial cell culture (69 of 88 samples identified as positive) and the agreement of LightCycler PCR results with all shell vial positive results indicate the potential for routine implementation of this technology for laboratory diagnosis of HSV infections.

Blood, bronchoscopy-lavage, biopsy (lung, liver, kidney), sputum, and other (cecum, bone) specimens were inoculated into sheli vials and conventional cell tube cultures seeded with MRC-5 cells over a 23-month period. Of 1,472 specimens, 182 (12.4%) yielded cytomegalovirus (CMV)-positive results from 81 patients. Significantly more CMV-positive specimens were detected in shell vials (n = 154; 84.6%) than in conventional tube cell cultures (n = 126; 69.2%) (P < 0.01). We found that 98 (53.8%) of the total 182 and 41 (42.7%) of the 96 blood specimens positive for CMV were detected by both the shell vial assay and conventional tube celi cultures. However, 56 (30.7%) of the total 182 and 31 (32.3%) of the 96 blood specimens positive for CMV were obtained exclusively in shell vials after detection with monoclonal antibody. Alternatively, 28 (15.4%) of the total 182 and 24 (25%) of the 96 blood specimens positive for the virus were isolated only in conventional tube cell cultures. Thus, although the sheil vial assay was more sensitive and rapid than the conventional tube cell culture method, both systems must be used, especially for blood specimens, for the laboratory diagnosis of CMV infections.

The effects of selective media and incubation atmosphere on the isolation of group A beta-hemolytic streptococci were evaluated. A higher percentage of group A streptococci was isolated on sheep blood agar incubated in air than in CO2 or anaerobic atmospheric conditions. Fewer non-group A beta-hemolytic streptococci were isolated on sheep blood agar incubated in air than in CO2 or anaerobically. Group A streptococcal isolation was not significantly affected by different incubation atmospheres on sheep blood agar containing trimethoprim-sulfamethoxazole, but detection time was longer than on sheep blood agar alone. No significant difference was found between isolation of group A streptococci on sheep blood agar incubated in air and that on sheep blood agar containing trimethoprim-sulfamethoxazole and incubated in 5 to 10% CO2; however, more group A streptococci were isolated on sheep blood agar in air within 24 h. Sheep blood agar incubated at 35 degrees C in air is, therefore, recommended for the isolation of group A streptococci from throat swabs.

Varicella-zoster virus (VZV) causes vesicular dermal lesions which are clinically evident as varicella (primary infection) or zoster (reactivated) diseases. The LightCycler system (Roche Molecular Biochemicals) is a newly developed commercially available system designed to rapidly perform PCR with real-time detection of PCR products using a fluorescence resonance energy transfer. We compared the detection of VZV from dermal specimens by shell vial cell culture (MRC-5) and by LightCycler PCR. Of 253 specimens, VZV was detected in 23 (9.1%) by shell vial cell cultures and 44 (17.4%) by LightCycler PCR directed to a nucleic acid target sequence in gene 28. Twenty-one of 44 (47.7%) specimens were exclusively positive by LightCycler PCR; the shell vial cell culture assay was never positive when DNA amplification was negative (specificity, 100%). VZV DNA was detected in 39 of 44 (88.6%) specimens positive during cycles 10 through 30 of the LightCycler PCR. These VZV DNA-positive specimens (cycles 10 to 30) and 5 of 11 other PCR positive specimens (cycles 31 to 36) were confirmed by another LightCycler PCR directed to another (gene 29) target of the viral genome. For routine laboratory practice, all specimens yielding amplified DNA to the VZV gene 28 target can be considered positive results. The increased sensitivity (91%) of the LightCycler PCR for detection of VZV, rapid turnaround time for reporting results, virtual elimination of amplicon carryover contamination, and equivalent costs compared to shell vial cell culture for detection of VZV indicate the need for implementation of this technology for routine laboratory diagnosis of this viral infection.

Five hundred specimens (288 genital, 192 dermal, and 20 ocular) were extracted by technologists, and the DNA was assayed by LightCycler PCR (DNA polymerase and thymidine kinase [TK] gene targets) and by conventional tube and shell vial cell culture. One hundred fifty-eight confirmed (by cell culture and TK target PCR) positive and LightCycler-positive specimens were detected during the first 30 PCR cycles. LightCycler PCR-positive results for cycles 31 to 45 (39 of 67 [58.2%]) required confirmation by another PCR target (TK). LightCycler PCR is more sensitive (n = 197; 23.1%) than cell cultures (n = 150) for the routine laboratory detection of herpes simplex virus infections.