Comparison of two rapid microscopic methods and culture for detection of Chlamydia trachomatis in ocular and nasopharyngeal specimens from infants.

The data available for the diagnosis of chlamydial infections in infants which compare direct fluorescent-monoclonal-antibody stains (DFAs) with culture are limited to one reagent, MicroTrak (Syva Inc., Palo Alto, Calif.). We therefore performed a comparison of Pathfinder (Kallestad Diagnostics, Chaska, Minn.) and MicroTrak with chlamydia culture. Paired conjunctival and nasopharyngeal specimens for DFAs and cultures were obtained from 56 infants less than 1 month of age with conjunctivitis. The sensitivities for detecting C. trachomatis in conjunctival specimens with MicroTrak and Pathfinder were 93.8 and 88.2%, respectively, and the specificities were 87.5 and 94.9%, respectively. The DFA tests on nasopharyngeal specimens from infants with conjunctivitis did not perform as well. The sensitivities for Pathfinder and MicroTrak were 33 and 50%, respectively. There were a total of six patients with culture-positive chlamydial conjunctivitis whose nasopharyngeal specimens were DFA positive and culture negative; four of the specimens were positive by both DFAs. These six discordant specimens were further evaluated by preparing pellets and smears of the original culture specimens. All six contained typical fluorescing elementary bodies when stained with the Syva DFA reagent.     

Comparison of rapid methods of detection of cytomegalovirus in saliva with virus isolation in tissue culture.

Two rapid methods for the detection of cytomegalovirus (CMV) in saliva from congenitally and perinatally infected children were assessed by comparison with traditional virus isolation in tissue culture (TC). The polymerase chain reaction (PCR) was used to amplify a 300-bp segment of the CMV gB gene which was detected in ethidium bromide-stained agarose gels. A centrifugation-enhanced microtiter culture method with a monoclonal antibody for the detection of early-antigen fluorescent foci (DEAFF) was also used. Saliva specimens were collected with mouth swabs from children who were between the ages of 1 month and 14 years and who had either prenatal or perinatal CMV infection. One hundred sixty samples were tested by PCR and TC; 65 (40.6%) were found positive by TC, and 58 (36.8%) were found positive by PCR. Although four samples were found positive by PCR and negative by TC, saliva from seronegative and seropositive TC-negative adults were never found positive by PCR. One hundred fifty-two samples were tested by DEAFF and TC; 64 (42.1%) were found positive by TC, and 58 (38.2%) were found positive by DEAFF. With TC results as a standard, the sensitivity and specificity of DEAFF were, respectively, 90.6 and 97.7%. Because of the greater ease of collecting saliva than urine from newborns, both of these rapid methods merit evaluation in screening for congenital infection.     

Comparison of standard tube and shell vial cell culture techniques for the detection of cytomegalovirus in clinical specimens.

A monoclonal antibody was used to detect an early antigen of cytomegalovirus (CMV) by fluorescence 16 h after inoculation of MRC-5 monolayers in 1-dram (ca. 3.7-ml) shell vials and low-speed centrifugation. Of 770 specimens (urine, blood, lung tissue, sputum) processed in shell vials, 124 (16%) were positive for the virus at 16 h postinfection. CMV was isolated in standard tube cell cultures (average time, 9 days) from only 88 specimens, but there were no instances (with the exception of 2 blood specimens) in which CMV was recovered from tube cultures but not from shell vials. Additional specimens from 18 patients were positive in the shell vial assay but negative in the conventional tube cell culture assay. Other specimens from 14 of the 18 patients yielded CMV in conventional tube cell cultures. Of the 4 patients from whom CMV was not recovered from other specimens by conventional tube cell culturing, all had evidence of recent CMV infections, as indicated by a fourfold or greater rise in antibody titer. The specificity of the shell vial assay for the detection of CMV is supported by assays of other specimens from the same patients yielding the virus or serological evidence indicating recent infections, the known enhancement of CMV detection after centrifugation of the shell vials, and the distinct and easily recognizable fluorescence confined to the nuclei of CMV-infected cells. Our data indicate that the shell vial cell culture assay for the detection of CMV is as specific as and more sensitive than conventional tube cell culturing for the diagnosis of CMV infections.     

Multicenter comparative evaluation of two rapid microscopic methods and culture for detection of Chlamydia trachomatis in patient specimens.

Four hundred and seventy-three men and women at high risk for sexually transmitted disease were tested for the presence of Chlamydia trachomatis in the urethra or the endocervix. Four groups were involved in this multicenter study of two direct fluorescent-antibody microscopy tests, Kallestad Pathfinder and Syva Microtrak, compared with culture techniques. Results from the test sites indicated that there was no significant difference overall in the sensitivity and specificity of the two test kits. However, there was some interlaboratory variation seen in the sensitivity of the microscopy, but little difference in the specificity. Either kit could be an effective screening method for C. trachomatis in high-risk populations.     

Comparison of four methods for rapid detection of Pneumocystis carinii in respiratory specimens.

Four stains for the detection of Pneumocystis carinii in respiratory specimens were compared for sensitivity, specificity, preparation time, and ease of interpretation. One hundred specimens were collected. Of these, 50 were induced sputum specimens and 50 were bronchoalveolar lavage fluid. All specimens were stained with Diff-Quik (DQ) (a modified Giemsa stain), a quick silver stain, and direct and indirect immunofluorescence stains. A positive specimen was defined as any smear positive by two or more of the methods. Fifty-eight percent of specimens were positive. Seventy-four percent of the sputum specimens and 42% of the bronchoalveolar lavages were positive. The sensitivities for detection of P. carinii in sputum were 92% with silver stain, 97% with direct immunofluorescence assay (DFA), 97% with indirect immunofluorescence assay (IFA), and 92% with DQ. The sensitivities for detection in bronchoalveolar lavage were 86% with silver stain, 90% with DFA, 86% with IFA, and 81% with DQ. Preparation times varied from 90 min for the silver stain and IFA to 3 min for DQ. Costs of the tests varied from $1.50 per slide for DQ to $10.00 per slide for the silver stain and DFA. Reading times varied from 10 to 30 min for the silver stain and DQ to less than 5 min for the immunofluorescence assays. We conclude that all of these tests are viable options for the clinical laboratory, and the choice will be influenced by factors such as clinical volume, ability to batch specimens, and expertise of technological support. A reasonable option may be to use the quick and inexpensive DQ as a screening test and to confirm negative smears with a more sensitive assay.     

Comparison of antibodies for rapid detection of cytomegalovirus.

Antibodies were compared for use in the spin-amplified shell vial method for rapid cytomegalovirus detection. Commercial antibodies by Du Pont Co., Whittaker M.A. Bioproducts, Bartels Immunodiagnostic, Virostat, and Serono Diagnostics were compared at incubation times of 16 to 48 h on 22 patient specimens. Only the Du Pont antibody showed 100% sensitivity and specificity and no nonspecific reactions.     

New and sensitive standard cell culture technique for the detection of cytomegalovirus in clinical specimens

Conventional tube cell culture has been recognised as the most sensitive technique available for human cytomegalovirus (HCMV) detection. Low-speed centrifugation of specimen inocula onto cell culture monolayers has been shown to increase the efficiency of infection with the AD 169 strain of HCMV. Therefore a centrifugal force of 900g for 1 hour at 37 degrees C was used to enhance the detection of HCMV cytopathic effect (CPE) in shell vials that contained circular coverslips with a monolayer of human embryonic lung (HEL) fibroblasts. Of 195 specimens, HCMV CPE was detected in 18 specimens (9.02%) on shell vial culture assay, whereas conventional tube cell culture was positive in only 13 specimens (6.6%). The shell vial culture assay was significantly more sensitive (P less than 0.05). Furthermore the development of the cytopathic effect on shell vial culture assay was significantly earlier (P less than 0.01) and more extensive. Urine samples were sonicated and the results obtained with immunofluorescence using human immune serum demonstrated that sonication increased both the intensity of fluorescence and number of fluorescent foci of HCMV-infected cells and also decreased the non-specific fluorescence of the background.     

Type- and subtype-specific detection of influenza viruses in clinical specimens by rapid culture assay.

A rapid culture assay which allows for the simultaneous typing and subtyping of currently circulating influenza A(H1N1), A(H3N2), and B viruses in clinical specimens was developed. Pools of monoclonal antibodies (MAbs) against influenza A and B viruses and MAbs HA1-71 and HA2-76, obtained by immunizing mice with the denatured hemagglutinin subfragments HA1 and HA2 of influenza virus A/Victoria/3/75, were used for immunoperoxidase staining of antigens in infected MDCK cells. MAb HA1-71 reacted exclusively with influenza A viruses of the H3 subtype, while MAb HA2-76 reacted with subtypes H1, H3, H4, H6, H8, H9, H10, H11, and H12, as determined with 78 human, 4 swine, and 10 avian influenza virus reference strains subtyped by the hemagglutination inhibition test. To determine if the technique can be used as a rapid diagnostic test, 263 known influenza virus-positive frozen nasal or throat swabs were inoculated into MDCK cells. After an overnight incubation, the cells were fixed and viral antigens were detected by immunoperoxidase staining. Influenza A viruses of the H1 and H3 subtypes were detected in 31 and 113 specimens, respectively. The subtypes of 10 influenza A virus-positive specimens could not be determined because they contained too little virus. Influenza B viruses were detected in 84 specimens, and 25 specimens were negative. We conclude that this assay is a rapid, convenient, non-labor-intensive, and relatively inexpensive test for detecting, typing, and subtyping influenza viruses in clinical specimens.     

Comparison of culture and the antigenemia assay for detection of cytomegalovirus in blood specimens submitted to a reference laboratory.

We compared the antigenemia assay (AA) with tandem shell vial cultures (SVCs) and tube cultures (TCs) for detection of cytomegalovirus (CMV) in 343 blood specimens. For 249 specimens, the AA was performed in duplicate with two different commercially available monoclonal antibody reagents (Biotest Diagnostic Corporation and Argene Biosoft). Specimens considered true positives were positive in either culture system or both AAs. Only specimens which were negative in both cultures and positive in a single AA were tested retrospectively with a CMV PCR assay. CMV recovery rates were also calculated to determine if increased specimen age resulted in decreased positivity. CMV recovery rates for the AA and the combination of both cultures were 20.0 and 5.0% at 3 to 18 h, 20.2 and 14.0% at 18 to 35 h, 12.5 and 7.8% at 36 to 52 h, and 18.8 and 6.3% at 64 to 75 h, respectively. The sensitivities and specificities of the Biotest AA, the Argene AA, SVC, and TC were 84.4 and 100.0, 100.0 and 99.6, 44.4 and 100.0, and 46.0 and 100.0%, respectively. The AA was significantly more sensitive than either culture method alone and was also more sensitive than the two culture methods used in tandem (the tandem culture sensitivity was 63.5%); the Argene AA identified more positives than the Biotest AA.     

Evaluation of two rapid methods for the detection of herpes simplex virus antigen in patient specimens

An indirect immunofluorescent antibody procedure (IFA) for the detection and typing of herpes simplex virus (HSV) and an enzyme-linked immunosorbent assay (ELISA) procedure were compared with conventional viral culture. Specimens for culture and ELISA were inoculated into serum free viral transport medium (VTM) and, for IFA, onto slides provided in the kit. Tissue cultures (MRC-5 and primary rabbit kidney) were inoculated and examined daily for cytopathogenic effect (CPE). The remaining VTM was frozen at -70 degrees C until tested by the ELISA system. Slides for IFA were stained with HSV common and HSV-2 specific monoclonal antibodies. Of 155 specimens, 47 (30 percent) were unsatisfactory for the IFA test owing to an inadequate number of epithelial cells on the slides. Of 108 adequate specimens, 45 were culture positive; 39 were positive by the IFA test with a sensitivity of 87 percent and a specificity of 90 percent. Of the 39 positives, 29 (75 percent) were correctly classified as type 1 or type 2, six (15 percent) were typed incorrectly, and four (10 percent) were inadequate for typing by the IFA test. All 155 specimens were suitable for testing by the ELISA procedure. Of 55 specimens positive by culture, only 25 (sensitivity 45 percent) were positive by ELISA. However, the specificity was 100 percent. After incubation of two, three, and six days, the tissue cultures detected 71 percent, 89 percent, and 100 percent of the positives, respectively.     

Effects of enhancing agents on detection of cytomegalovirus in clinical specimens.

Dimethyl sulfoxide, dexamethasone, and calcium were tested in combination for their enhancing effects on cytomegalovirus detection in shell vial cultures on 1,579 clinical specimens obtained primarily from adult solid-organ transplant recipients. Fluorescent-focus counts were elevated for the cytomegalovirus-positive urine specimens (P < 0.01) and throat washings (P < 0.05) but not for the tissue biopsy or blood samples. Epidermal growth factor also increased focus counts but provided no additional benefit when used in combination with the other agents. The triple-combination treatment did not increase the number of positive specimens identified.     

Comparison of two leukocyte extraction methods for cytomegalovirus antigenemia assay.

We carried out a prospective, parallel, and blind study on 113 blood samples from immunocompromised patients in order to compare two leukocyte extraction methods (6% dextran sedimentation and Polymorphprep separation) for cytomegalovirus (CMV) antigenemia assay. CMV was detected in 38 samples by antigenemia assay (34 by dextran sedimentation and 35 by Polymorphprep separation). No differences either in the number of positive specimens (P = 1) or in the mean CMV-positive cell counts (P = 0.41) were observed between the two leukocyte extraction methods. In conclusion, the two methods performed equally well for this assay.     

Comparison of two rapid streptococcal antigen detection assays with culture for diagnosis of streptococcal pharyngitis.

In this study, 801 pharyngeal specimens were cultured for group A streptococci and tested with the Biostar Strep A Optical Immunoassay (Strep A OIA). The respective sensitivities and specificities were as follows: culture, 97.1 and 100%; Strep A OIA, 91.5 and 94.8%. Of the 801 specimens, 597 were also tested with the Abbott TestPack Strep A Assay (TP-ST). For those specimens tested by all three methods, the respective sensitivities and specificities were as follows: culture, 98.1 and 100%; Strep A OIA, 92.3 and 95.4%; and TP-ST, 79.4 and 100%. The Strep A OIA is significantly more sensitive than TP-ST and compares favorably with culture.     

DNA probe technology for rapid detection of Haemophilus influenzae in clinical specimens.

In a previous study, we reported that a 5-kilobase Haemophilus influenzae DNA fragment involved in penicillin-binding protein expression could be used as a probe for specific detection of H. influenzae strains (F. Malouin and L. E. Bryan, Mol. Cell. Probes 1:221-232, 1987). Here, we report the ability of this probe to detect H. influenzae in clinical specimens. In a bacterial dot experiment, there was strong hybridization of the 32P-labeled probe to nonencapsulated and serotype a through f H. influenzae strains. The detection of H. influenzae in body fluids was then evaluated by using pooled human serum, urine, cerebrospinal fluid, and sputum as dilution media for H. influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, and Escherichia coli cells. At 65 degrees C, the probe hybridized to H. influenzae and H. aegyptius (greater than or equal to 10(5) cells) in all fluids. There was no hybridization with the E. coli negative control, and H. parainfluenzae hybridized when greater than or equal to 10(7) cells were used. Experiments performed at 73 and 80 degrees C permitted elimination of H. parainfluenzae hybridization. The detection of H. influenzae in 232 sputa from patients with respiratory tract infections was very specific (96 to 97%) and sensitive (74 to 100%) when the total time of the procedure was sufficient (6 to 24 h) and when the experiments were performed at 80 degrees C. In addition, the probe detected three of three and four of four H. influenzae-infected cerebrospinal fluids and blood cultures, respectively, and did not react with pneumococcus- or streptococcus-infected cerebrospinal fluids. Finally, by using a small-scale procedure, the probe rapidly detected H. influenzae in cerebrospinal fluid and sputum specimens (4 and 8 h, respectively). These results imply prompt diagnosis of H. influenzae infections caused by nonencapsulated and serotype a through f strains.     

Comparison of methods available for assay of chloramphenicol in clinical specimens.

Eight methods for the assay of chloramphenicol in clinical samples were compared with our own modification of a plate diffusion technique using Sarcina lutea and yeast extract agar. Six of the eight methods were less sensitive than originally reported, and five of them were considered unsuitable for use in clinical microbiology practice. The remaining three methods together with the S. lutea/yeast extract modification were used to assay chloramphenicol in 20 samples of serum. Twenty samples of cerebrospinal fluid were also assayed by the S. lutea/yeast extract method. Our results indicate that only the Bacillus subtilis (sensitivity 6x0 mg/l) and the S. lutea (sensitivity 2x5 mg/l) diffusion methods are suitable for use with clinical samples in routine practice. The problems of chloramphenicol toxicity, appropriate dosage regimens, and the need for assay of the drugs are considered.     

Comparison of quantitative cytomegalovirus antigenemia assay with culture methods and correlation with clinical disease.

Blood samples, obtained predominantly from human immunodeficiency virus-infected patients and solid-organ and bone marrow transplant recipients, were submitted to the clinical laboratory for detection of cytomegalovirus (CMV) and were processed by three methods: conventional culture, centrifugation culture, and CMV antigenemia assay with monoclonal antibodies (Clonab CMV; Biotest Diagnostic Corporation, Denville, N.J.) to CMV antigens. Of 496 blood samples tested, 107 were positive by one or more methods: 56 were positive by conventional culture, 27 were positive by centrifugation culture, and 97 were positive for CMV antigen (Ag) by the antigenemia assay. Forty-seven samples were positive by the CMV antigenemia assay only; in these samples, a mean of 12 Ag-positive cells was detected per 200,000 polymorphonuclear leukocytes examined. In contrast, samples positive by the CMV antigenemia assay and both culture methods had a mean of 193 Ag-positive cells, and samples positive by the CMV antigenemia assay and conventional culture alone had a mean of 157 Ag-positive cells. In the antigenemia assay, paraformaldehyde fixation resulted in superior cell morphology when compared with acetone fixation. Use of immunofluorescence staining reduced sample processing time and the complexity of reagent preparation in comparison with immunoperoxidase staining. Differences in the sensitivities between the immunofluorescence and immunoperoxidase staining techniques for detection of antigenemia were minor, with discrepant samples showing only one or two Ag-positive cells. Clinical disease was generally associated with high-level antigenemia, but exceptions were noted. The CMV antigenemia test is a rapid, quantitative assay that greatly facilitated the rapid diagnosis of CMV infection. However, quantitation of antigenemia is labor-intensive, requires processing of samples soon after collection, and does not always correlate with clinical disease in the individual patient.     

Comparison of SHARP signal system and Southern blot hybridization analysis for detection of cytomegalovirus in clinical specimens by PCR.

Thirty cytomegalovirus cell culture-positive samples were tested by the SHARP Signal System. Twenty-seven specimens (100% agreement) were identified by both methods. The SHARP Signal System is rapid (4 h), easy to perform, and potentially adaptable to automation.     

Comparison of monoclonal antibodies for rapid detection of cytomegalovirus in spin-amplified plate cultures.

Cytomegalovirus (CMV) was detected in 56 of 275 specimens (20%); 50 of 56 (89%) were detected by conventional culture, and 37 (66%) were detected by rapid assay at 72 h with a commercial monoclonal antibody and a pooled monoclonal antibody. Although the two antibodies were equally sensitive at 72 h, the pooled antibody gave a brighter, more easily detected signal. Other viruses were isolated from 9 specimens (3.3%) by conventional culture. Use of rapid assays alone fails to detect slow-growing CMV and non-CMV viral pathogens.