Methods For Detection Of Cytomegalovirus Research Articles

Evaluation of rapid and sensitive DNA extraction methods for detection of cytomegalovirus in dried blood spots

BackgroundDried blood spots (DBS), collected universally from newborns in the U.S., could be used as a matrix for the detection of cytomegalovirus (CMV) infection in infants. However, sensitivity to detect CMV in DBS as compared to saliva and urine is variable across studies largely due to the DNA extraction method. Thermal shock, a widely used DNA extraction method, is highly sensitive for the detection of CMV in DBS, however, the processing time required is not practical for high-throughput testing. ObjectiveTo determine if rapid and cost-effective DNA extraction methods amenable to newborn screening (NBS) could achieve the same sensitivity as the thermal shock method. Study designDBS were prepared from CMV positive blood samples from 20 organ transplant recipients. Three DNA extraction methods were compared for relative yield and sensitivity of detection of CMV DNA: thermal shock, KOH Tris buffer, and DNA Extract All. CMV DNA was detected by real-time quantitative polymerase chain reaction (qPCR). ResultsThe KOH Tris and DNA Extract All methods gave higher yields and sensitivity of CMV detection in DBS than thermal shock, which were significantly greater when viral loads were ≤ 10,000 copies/ml blood. Both methods gave faster turnaround times than thermal shock and would be better suited for NBS. ConclusionsThe choice of DNA extraction method greatly influences the ability to detect low levels of CMV DNA in DBS. Moreover, development of highly sensitive yet rapid methods for CMV detection could help facilitate future newborn screening of CMV in DBS.

Corrigendum to ‘Evaluation of diagnostic methods for detection of cytomegalovirus in recipients of allogeneic stem cell transplants’ [JCV 20 (2001) 59–70

Corrigendum to ‘Evaluation of diagnostic methods for detection of cytomegalovirus in recipients of allogeneic stem cell transplants’ [JCV 20 (2001) 59–70

Detection of Cytomegalovirus in Bronchoalveolar Lavage Specimens: Spin Amplification and Staining with a Monoclonal Antibody to the Early Nuclear Antigen for Diagnosis of Cytomegalovirus Pneumonia

To diagnose cytomegalovirus pneumonia in a hetergeneous population of patients, three methods for detection of CMV in bronchoalveolar lavage specimens were compared as follow: (1) spin amplification followed by staining with a monoclonal antibody to the early nuclear antigen (EA-assay); (2) conventional tissue cell culture; and (3) cytology. Cell differentials were performed on most specimens. Cytomegalovirus was detected by one or more method in 55 BAL specimens from 39 patients. Cytomegalovirus (CMV) pneumonia was diagnosed by lung tissue (primarily autopsy) histologic findings and conventional culture results or the presence of CMV in extrapulmonary tissue, fulfillment of specific clinical and radiographic criteria plus failure to recover a pathogen other than CMV from a respiratory specimen. Probable CMV pneumonia was diagnosed if only the latter two criteria were met. The EA-assay was positive in all patients with proven or probable CMV pneumonia and in 92 percent of those without documented pneumonia. Cytologic findings were positive only in patients with CMV pneumonia but were negative in one-third of those patients. As a diagnostic test for CMV pneumonia, the EA-assay, conventional culture, and cytology had positive predictive values of 45, 57, and 100 percent, respectively. Lymphocyte percentages in BAL specimens from patients with CMV pneumonia were significantly decreased compared with those of patients without CMV pneumonia (p less than 0.005). Although the EA-assay should not be used alone as a diagnostic test for CMV pneumonia in our patient population, the combination of alveolar lymphopenia and a positive BAL CMV EA-assay was highly suggestive of disease.

Clinical Comparison of Two Assays for Rapid Detection of Cytomegalovirus Early Nuclear Antigen

Three methods for detection of cytomegalovirus (CMV) in 218 clinical specimens were compared: (1) shell vial assay to detect the early nuclear antigen after incubation for 16 hours and 40 hours (Syva Company); (2) 24-well plate assay to detect the early nuclear antigen after incubation for 16 hours (DuPont); and (3) convention tissue cell culture. CMV was detected in 26 specimens (12%) by one or more of these methods. With the shell vial assay, 12 (46%) and 15 (58%) specimens were positive after incubation for 16 hours and 40 hours, respectively. CMV was detected in 17 specimens (65%) by the 24-well plate assay. There was no significant difference in the detection of CMV between these assays. CMV was identified by conventional tissue culture in 15 of 22 (68%) evaluable cultures after an average of 14.2 days. More specimens were positive by conventional culture than by the 16-hour shell vial assay (P = 0.035). For optimal detection of CMV in clinical specimens, both conventional tissue cell culture and an early antigen assay should be performed. The two early antigen assays evaluated in this study yielded comparable results. However, the 24-well plates are more easily manipulated, and the 24-well plate assay, as performed, was easier to interpret and more cost efficient.

Detection of cytomegalovirus DNA in short term cultures

Detection of human cytomegalovirus (CMV) by in situ DNA hybridisation six days after incubation of human diploid fibroblasts (ISDH-6) was evaluated pro-spectively in 205 urine samples, obtained from 57 kidney transplant and 17 bone marrow transplant recipients. The results were compared to those of conventional virus isolation (CVI) and the detection of CMV early antigens after one day of cultivation (EA-1). Of 42 samples positive for CMV by at least one of these methods, 40 (95%) were detected with ISDH-6. Thirty-five (83%) and 34 (81%) positive samples were found with CVI and EA-1, respectively. These data indicate that ISDH-6 is a sensitive method for detection of CMV. It can be used as a rapid and sensitive alternative to CVI in combination with EA-1

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.

Detection of cytomegalovirus by 24-well plate centrifugation assay using a monoclonal antibody to an early nuclear antigen and by conventional cell culture

During a 12-month period, two methods for detection of cytomegalovirus (CMV) in 1624 clinical specimens were compared: (1) centrifugal inoculation of MRC-5 cells on coverslips in 24-well plates and staining with a monoclonal antibody to CMV early nuclear antigen after incubation for 40 h (EA assay), and (2) conventional tube cell culture. CMV was identified in 183 (11.3%) specimens from 113 different patients. The EA assay was positive for CMV in 144 183 specimens (79%), and CMV was detected by recognition of specific cytopathic effect (CPE) in conventional cell culture in 143 183 (78%). Both methods yielded CMV in 56% of the specimens ( 104 183 ). CMV was detected by EA assay alone in 22% ( 40 183 ) and only by CPE in 21% ( 39 183 ) of the positive specimens. When all specimen types were considered, there was no significant difference in the detection of CMV between the two methods. However, bronchoalveolar lavage (BAL) fluids yielded CMV more frequently by EA assay than by CPE (58 compared to 48 of 574, p = 0.0178), and CMV was detected in blood specimens more often by CPE than by EA assay (20 compared to one of 149, p < 0.0001). In addition to CMV, other viruses were recovered by conventional tube cell culture, including herpes simplex virus (HSV) type 1 from 17 BAL fluids (two of which were positive for CMV by EA assay) and one liver biopsy and adenovirus serotype 4 from four separate urine specimens and three gastrointestinal tract biopsies from one patient. Our data strongly suggest that all specimens submitted for isolation of CMV should be processed for both EA assay and conventional cell culture to provide optimal recovery of CMV. This would also aid in the detection of other viruses, primarily HSV, that may be present in the specimen and play a role in the patient's disease.

Monoclonal antibodies for rapid detection of viral antigens in shell vials

Diagnosis of Cytomegalovirus Infections and Development of Monocional Antibodies Cytomegalovirus (CMV) is a common infection that can cause serious disease in infants who have acquired the virus in utero, in patients immunosuppressed for organ transplantation, and in other individuals who receive chemotherapy or high levels of corticosteroids for treatment of neoplastic and other medical processes. Only 4 years ago, the standard method for detecting CMV infection in the laboratory was the inoculation of diploid fibroblast cell cultures in tubes with subsequent identification of typical cytopathic effects that required an average time period of 7 8 days for recognition (3). Immunologic methods for rapid detection of CMV in clinical specimens waned for lack of high quality homotypic antisera that could produce sensitive and specific results and, importantly, be commercially available. Subsequently, two monoclonal antibodies were prepared against an early and a late antigen of the ADI69 strain of CMV by Shuster et al. at the Mayo Clinic. These monoclonal antibodies provided the key to the development of a rapid test for detection of the virus (10). Since 1973, we have used shell vials to diagnose Chlamydia trachomatis infections. This bacterium is .only 3 4 times larger than the CMV virion; nevertheless, centrifugation of the specimen inoculum at low speed (700 x g) has been shown to enhance the rate and ultimate infectivity of this organism and viruses in susceptible cell cultures. Because we wanted to be able to detect CMV as rapidly as possible after inoculation of cell cultures in the laboratory, we used the monoclonal antibody prepared to an early antigen (72,000 d) to assay for the viral components synthesized after 16 hr incubation by the indirect immunofluorescence test (monoclonal antibody available from DuPont Specialty Diagnostics, Wilmington, DE).

Recent developments in laboratory methods for the rapid detection of cytomegalovirus

Recent developments in laboratory methods for the rapid detection of cytomegalovirus