Detection of herpes simplex virus DNA in genital and dermal specimens by LightCycler PCR after extraction using the IsoQuick, MagNA Pure, and BioRobot 9604 methods.

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 (Orca Research, Inc., Bothell, Wash.) for extraction of herpes simplex virus (HSV) DNA from dermal and genital tract specimens prior to analysis by LightCycler PCR. Of 198 specimens (152 genital, 46 dermal), 92 (46.2%) were positive for HSV DNA by LightCycler PCR after automated extraction of specimens with either the MagNA Pure or BioRobot 9604 instrument. The manual IsoQuick method yielded HSV DNA (total n = 95) from three additional specimens that were negative by the automated method (P = 0.25, sign test). Although the mean numbers of LightCycler PCR cycles required to reach positivity differed statistically significantly among all three of the methods of extraction, the estimated means differed by no more than 1.5 cycles (P < 0.05). Seventy (76%) of the 92 specimens that were LightCycler PCR positive by all three extraction methods were also positive by shell vial cell culture assay. HSV DNA was detected by a lower LightCycler PCR cycle number (26.1 cycles) in specimens culture positive for the virus than in culture-negative samples (33.3 cycles) (P < 0.0001). The manual IsoQuick and automated MagNA Pure and BioRobot 9604 methods provide standardized, reproducible extraction of HSV DNA for LightCycler PCR. The decision to implement a manual versus an automated procedure depends on factors such as costs related to the number of specimens processed rather than on the minimal differences in the technical efficiency of extraction of nucleic acids among these methods.

Diagnosis of varicella-zoster virus infections in the clinical laboratory by LightCycler PCR.

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.

Evaluation of LightCycler PCR for implementation of laboratory diagnosis of herpes simplex virus infections.

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.

Diagnosis of herpes simplex virus infections in the clinical laboratory by LightCycler PCR.

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.

Clinical comparison of the Treponema pallidum CAPTIA syphilis-G enzyme immunoassay with the fluorescent treponemal antibody absorption immunoglobulin G assay for syphilis testing.

Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTIA-positive samples (antibody indices of >/=0.650 and 0.900, the sample was considered positive. Thirteen of 89 (15%) samples had discrepant results. Compared to the FTA-ABS assay, the CAPTIA EIA had a sensitivity and specificity and positive and negative predictive values of 70.7, 97.9, 96.7, and 79.7%, respectively. In another analysis, discrepancies between results were resolved by repeated FTA-ABS testing (technologists were blinded to previous RPR results) and patient chart reviews. Seven CAPTIA-negative samples which were previously interpreted (unblinded) as minimally reactive by the FTA method were subsequently interpreted (blinded) as nonreactive. One other discrepant sample (CAPTIA negative and FTA-ABS positive [at an intensity of 3+], unblinded) was FTA negative with repeated testing (blinded). For the five remaining discrepant samples, chart reviews indicated that one patient (CAPTIA negative and FTA-ABS positive [minimally reactive], blinded) had possible syphilis. These five samples were also evaluated and found to be negative by another treponema-specific test, the Treponema pallidum microhemagglutination assay. Therefore, after repeated testing and chart reviews, 2 of the 89 (2%) samples had discrepant results; the adjusted sensitivity, specificity, and positive and negative predictive values were 96.7, 98.3, 96.7, and 98.3%, respectively. This study demonstrates that the CAPTIA IgG EIA is a reliable method for syphilis testing and that personnel performing tests which require subjective interpretation, like the FTA-ABS test, may be biased by RPR test results.

Detection of Epstein-Barr virus-specific antibodies by an automated enzyme immunoassay. Performance evaluation and cost analysis.

Detection of antibodies to specific antigens of Epstein-Barr virus (EBV) has been conventionally performed by an immunofluorescence assay (IFA). The procedure is labor intensive and expensive, and interpretation of results is subjective. We evaluated an automated enzyme immunoassay (EIA) (INC-STAR Corp., Stillwater, MN, USA) using 290 serum specimens submitted for the diagnosis of acute infection with EBV. Antibodies (IgG, IgM) to EBV capsid antigen and IgG class antibodies to the nuclear antigen of the virus were obtained using the LABOTECH Automated Microplate Analyzer (BioChem ImmunoSystems Inc., Allentown, PA, USA) and were compared to the antibody profile results obtained by IFA and Western blot as the "gold standard." For detection of acute infection with EBV (presence of IgM and IgG antibodies to the capsid antigen; absence of antibodies to the nuclear antigen), the EIA had 100% sensitivity (11 of 11) and 99% specificity (275 of 279) compared to IFA and Western blot results. A cost analysis of IFA and EIA procedures, based on an estimated annual volume of 12,000 procedures, indicated that $236,000 direct cost and 1,400 h technologist time could be saved with the automated compared with immunofluorescence procedure. The automated EIA for determination of antibodies provides cost-effective, accurate diagnosis of EBV infections in laboratories processing high numbers of specimens now processed by IFA.

New developments in the diagnosis of viral diseases.

Major technical advances have occurred, especially in the last 5 years, in the laboratory diagnosis of viral infections. Immunologic detection of immediate early antigens in specimens such as bronchoalveolar lavage fluid and blood inoculated into shell vial cell cultures, particularly for herpesvirus (cytomegalovirus, herpes simplex virus, varicella-zoster virus), has provided results 16 to 48 hours after inoculation rather than the several days required for recognition of cytopathic effects in conventional tube cell cultures. Similarly, cytomegalovirus viremia can be detected directly by immunostaining of peripheral blood leukocytes with commercially available reagents the same day the specimen is submitted to the laboratory. Single-test membrane immunoassays have provided rapid (15 minutes) detection of viral antigens (respiratory syncytial virus, rotavirus, influenza virus type A). In the near future, diagnostic virology laboratories will be expected to monitor viral strains for susceptibility to the growing list of antiviral drugs. Amplification of nucleic acid sequences of viruses from cerebrospinal fluid or tissue, which generally does not yield isolates by conventional diagnostic techniques, has added a new dimension to the laboratory diagnosis of viral infection.

Diagnostic virology--then and now.

In the past five years, technologic advances in the shell vial assay and expanding availability of rapid membrane EIA tests have allowed over 90% of the viruses detected in our laboratory to be reported within 24 h postinoculation. PCR technology promises to add a new practical dimension to diagnostic virology especially for the detection of viruses in CSF, tissues, and blood. Extension of these diagnostic capabilities from investigative protocols to general laboratories for routine use will be our biggest challenge and be based on considerations of cost, licensing, and availability of this technology in "kit" formats.

Comparison of shell vials and conventional tubes seeded with rhabdomyosarcoma and MRC-5 cells for the rapid detection of herpes simplex virus.

Shell vials (SV) and conventional tubes (CT) were seeded with rhabdomyosarcoma (RD) and MRC-5 cells and inoculated with clinical specimens, and the systems were evaluated for the rapid diagnosis of herpes simplex virus (HSV) infections by detection of cytopathic effects (CPE) (for CT, for 7 days) and by using fluoresceinated monoclonal antibodies (for SV, 16 h postinoculation). Of 245 genital specimens (16 from males and 229 from females) 56 (23%) seeded with MRC-5 cells (14 type 1 and 42 type 2) and 55 (22%) seeded with RD cells were detected in CT; however, CPE were recognized in only 26 (46%) of the total HSV-positive cultures 1 day postinoculation. Forty-eight (86% sensitivity, MRC-5) and 46 (84% sensitivity, RD) HSV strains were detected immunologically in SV 16 h postinoculation. Early CPE in CT or fluorescent foci in SV were easier to detect in MRC-5 than in RD cell cultures. MRC-5 and RD cells were equally sensitive to infection with HSV. CT cell cultures were more sensitive than SV but less rapid for the detection of HSV infection (P less than 0.01). We recommend using SV for the rapid diagnosis of HSV infections, but in addition, CT must be inoculated with MRC-5 or RD to ensure maximum detection of this virus.

Detection of cytomegalovirus from blood leukocytes separated by sepracell-MN and Ficoll-Paque/Macrodex methods.

Two density gradient separation techniques for separation of blood leukocytes were compared for the laboratory diagnosis of cytomegalovirus (CMV) viremia. Of 510 blood specimens processed by both methods, 76 (14.9%) yielded CMV. Of the 76 positive specimens, 66 (87%) and 65 (86%) were processed by the Ficoll-Paque/Macrodex (F-P/M; Macrodex is dextran 70 in normal saline; Pharmacia, Pisataway, N.J.) and Sepracell-MN methods, respectively. Of the 76 CMV-positive blood specimens, 72 (95%) were detected in shell vial cell cultures, whereas only 42 (55%) were detected in conventional tube cell cultures. The time for recognition of specific cytopathic effects due to CMV in tube cell cultures (8.0 versus 7.1 days), the number of fluorescent foci in each positive shell vial culture (19.3 versus 20.1), and the costs of the reagents ($3.50 versus $2.80) were similar and independent of the leukocyte separation method (F-P/M versus Sepracell-MN). Recovery of CMV from heparinized blood (F-P/M method) was similar to that from EDTA-anticoagulated blood (Sepracell-MN method). The Sepracell-MN method is a rapid and sensitive method for detection of CMV from blood specimens and is recommended as a replacement for the more tedious and time-consuming F-P/M procedure.

Effect of treatment of shell vial cell cultures with dimethyl sulfoxide and dexamethasone for detection of cytomegalovirus.

Urine specimens submitted for the diagnosis of cytomegalovirus infection were inoculated into shell vials that had been pretreated with a combination of dimethyl sulfoxide (DMSO) and dexamethasone (DEX). The results were compared with those for inoculated shell vials which had received no drug treatment. Of 664 specimens, 100 (15%) were positive for cytomegalovirus. Of the 100 strains of cytomegalovirus, 88 (88%) were detected in both DMSO-DEX-treated and untreated shell vials. Of the remaining 12 positive specimens, 6 were detected with untreated shell vials exclusively and 6 were detected with DMSO-DEX-treated shell vials alone (not significant by the sign test). The median number of fluorescent foci was not significantly higher in DMSO-DEX-treated shell vials compared with that in untreated cultures (Wilcoxon signed-rank test; P = 0.1). DMSO-DEX-treated monolayers did not enhance the sensitivity detection of cytomegalovirus in shell vial cell cultures.

Evaluation of number of shell vial cell cultures per clinical specimen for rapid diagnosis of cytomegalovirus infection.

Specimens submitted for the diagnosis of cytomegalovirus (CMV) infection were inoculated into three (blood) or two (urine, tissue, bronchoalveolar lavage [BAL]) shell vials seeded with MRC-5 cells for the diagnosis of CMV infection. We evaluated the detection of 993 specimens that were positive for CMV according to the number of shell vial cell cultures inoculated per specimen. For blood cultures, and considering one CMV-positive shell vial as 100%, inoculation of three shell vials versus one increased the detection rate of the virus by 51%. Inoculation of three shell vials compared with two yielded a 20% increase in the detection rate of CMV. For urine, tissue, and BAL specimens, inoculation of two shell vials compared with one resulted in increases of 7, 10, and 5%, respectively. For maximum detection of CMV in shell vial cell cultures, at least three vials should be inoculated with blood specimens, and two vials should be used for urine, tissue, and BAL samples.

Detection of cytomegalovirus infections in specimens other than urine by the shell vial assay and conventional tube cell cultures.

Blood, bronchoscopy-lavage, biopsy (lung, liver, kidney), sputum, and other (cecum, bone) specimens were inoculated into shell 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 less than 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 cell 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 shell 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.

Monoclonal antibody for rapid laboratory detection of cytomegalovirus infections: characterization and diagnostic application.

Monoclonal antibodies to early (2H2.4, molecular weight 72,000 daltons) and late (2F3.0, molecular weight 68,000 daltons) antigens of the AD-169 strain of cytomegalovirus (CMV) were prepared by fusing mouse spleen cells with NS-1 mouse myeloma cells. The 2H2.4 monoclonal antibody produced a dense immunofluorescence with prominent lobular staining within the nucleus of CMV-infected substrate cells, whereas the reaction of 2F3.0 was more diffuse and generally involved the entire nucleus of the cells. Both monoclonal antibodies had little or no neutralizing activity against CMV in plaque-reduction assays. No cross-reactions were observed between these monoclonal antibodies and other members of the herpesvirus group. The 2H2.4 monoclonal antibody to early CMV antigen was used in a shell vial assay with a low-speed centrifugation step for the rapid (within 16 hours after inoculation) diagnosis of CMV infections. Optimal conditions for the test included centrifugation of shell vials at 700 X g for 45 minutes at 36 degrees C. An inoculum volume of 0.2 ml provided a reasonable balance between the optimal sensitivity for detecting specific viral fluorescence and the easy discrimination of the specific immunofluorescence from the background debris. Because of the commercial availability of the monoclonal antibody and the simplicity of the procedures used in the shell vial assay and subsequent fluorescence techniques, this rapid assay can be done in any laboratory that is familiar with cell culture manipulations.

Detection of viral and chlamydial antigens in open-lung biopsy specimens.

The recovery of viruses and Chlamydia trachomatis from cell cultures and the detection of their antigens in impression smears prepared from open-lung biopsy (OLB) specimens from immunocompromised adults were compared. Touch impression smears were prepared on three slides, each containing eight wells. OLB tissue was homogenized (Stomacher) and inoculated into MRC-5, primary monkey kidney, and McCoy cell cultures. The direct and indirect immunofluorescence (IF) tests were used to detect antigens to the following organisms: herpes simplex virus, adenovirus, parainfluenza types 1 and 3, respiratory syncytial virus, cytomegalovirus (CMV), Chlamydia trachomatis, influenza types A and B, and varicella-zoster virus. Of 105 OLB specimens, 21 viral isolates (20%) were recovered in cell culture; 20 were CMV and one was an influenza virus type A (H3N2). Both culture and IF results were positive with 12 specimens, but in nine instances a virus was isolated and IF was negative or eight times culture results did not yield the organism but IF test results were positive. Chlamydia trachomatis was never isolated or detected by IF. The authors recommend that for optimal detection of CMV from OLB specimens a new rapid centrifugation-enhanced cell culture system be used in preference, or in conjunction with a preliminary IF screen of impression smears for CMV detection.

Detection and serotyping of herpes simplex virus in MRC-5 cells by use of centrifugation and monoclonal antibodies 16 h postinoculation.

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 cell cultures and shell 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 shell vials containing MRC-5 cells permitted detection of this virus in all positive specimens with serotype determination within 16 h postinoculation.

Effects of trimethoprim-sulfamethoxazole and incubation atmosphere on isolation of group A streptococci.

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.

Pseudospirochetes, a cause of erroneous diagnoses of leptospirosis.

Motile filaments of varying lengths and thicknesses were observed by darkfield microscopy in a blood culture of a specimen from a patient who had fever of unknown origin. Similar structures were also seen in cultures inoculated with donor blood from healthy controls. Since the movement and configuration of the structures were not characteristic for spirochetes, the morphologic features were further examined by electron microscopy. The filaments observed were identical in appearance to pseudospirochetes described more than 50 years ago. The authors' observations indicate that they are derived from erythrocytes. Because of possible confusion of these pseudospirochetes with living organisms, the diagnosis of leptospirosis by darkfield microscopy should be confirmed by cultural or serologic tests.

Sputum counterimmunoelectrophoresis in the diagnosis of pneumococcal pneumonia.

Fifty-six patients with pneumonia were grouped according to degree of clinical certainty that the etiologic agent was Streptococcus pneumoniae. Of 14 patients with definite or probable pneumococcal pneumonia, 12 had pneumococcal antigens detected in sputum by counterimmunoelectrophoresis (CIE), 13 had a positive sputum culture, and 12 had a Gram-stained smear of sputum suggestive of the diagnosis. Of 9 patients with definite nonpneumococcal pneumonia, none had pneumococcal antigens detected by CIE, but one had pneumococci isolated from sputum culture, and one had a Gram stain of sputum suggestive of pneumococci. Of 34 control patients without pneumonia, five had a positive CIE, 11 had a positive culture, and 15 had a positive Gram stain. When used to differentiate pneumococcal from other types of pneumonia, CIE of sputum appears to be a sensitive and specific test. Among patients without pneumonia, however, CIE lacks specificity. Additionally, sputum Gram stain may correlate as well as CIE with pneumococcal pneumonia, but further substantiation of this observation is necessary.