Rapid Detection Of Cytomegalovirus Research Articles

Rapid Detection of Cytomegalovirus and Listeria monocytogenes Infection in Symptomatic Newborns by Fluorescence End-Point Detection PCR (FEP PCR)

Background: Congenital CMV infection is the most prevalent congenital infection in the world, and perinatal Listeria infection is an important cause of neonatal sepsis. The identification of these two infections is hard. The PCR technique is a sensitive method, which is not simply available in Iran. Objectives: This study investigated the prevalence of CMV and Listeria in 100 urine samples from under 21-day-old neonates with positive risk factors by PCR electrophoresis and fluorescent end-point detection (FEP) PCR in Tehran referral hospitals. Methods: We used 100 urine samples from neonates admitted to 11 medical centers in Tehran with positive selected clinical symptoms compatible with these infections. Electrophoresis and FEP PCR for CMV and Listeria were performed on all urine samples. For the data analysis, SPSS version 21 software was used, and p values of less than 0.05 were considered significant. Results: We collected 100 urine samples from 100 under 21-day-old neonates, referring to Tehran hospitals. Seven cases were positive for Listeria, and 58 cases were positive for CMV by electrophoresis and FEP PCR. Respiratory distress was the most common clinical finding in both groups. The Mc-Nemar test revealed a statistical agreement between calcification and CMV and meningitis and Listeria (P = 0.18 and 0.07, respectively). There was a positive relationship between the neonates’ age and CMV-positive condition (P = 0.001). This result was not significant among Listeria-positive cases. Conclusions: Screening for CMV and Listeria infections in neonates with clinical features compatible with these intrauterine infections may give important information for their treatment and follow-up.

Management of cytomegalovirus infection and disease after solid-organ transplantation.

Cytomegalovirus (CMV) continues to be a cause of substantial morbidity and death after solid-organ transplantation. There are 3 major consequences of CMV infection: CMV disease, including a wide range of clinical illnesses; superinfection with opportunistic pathogens; and injury to the transplanted organ, possibly enhancing chronic rejection. This article discusses the considerable progress that has been made in elucidating risk factors for CMV disease, in the rapid detection of CMV in clinical specimens, and in the use of antiviral chemotherapy and immunoglobulin to prevent and treat CMV disease after solid-organ transplantation.

Evaluation of CMV-vue antigenemia assay for rapid detection of cytomegalovirus in mixed-leukocyte blood fractions

Evaluation of CMV-vue antigenemia assay for rapid detection of cytomegalovirus in mixed-leukocyte blood fractions

CYTOMEGALOVIRUS INFECTION IN ORGAN TRANSPLANT RECIPIENTS

Cytomegalovirus (CMV) continues to be responsible for a substantial fraction of the morbidity and mortality that follows organ transplantation. The three major consequences of CMV infection are CMV disease (encompassing a range of clinical illness), superinfection with opportunistic pathogens, and injury to the transplanted organ. In the last decade, considerable progress has been made in elucidating risk factors for CMV disease, in rapid detection of CMV in clinical specimens, and in the use of antiviral chemotherapy and immunoglobulin to prevent and treat CMV disease after transplantation. This article discusses the impact of these advances on patient outcome following transplantation and the challenges for the next decade.

Detection of Cytomegalovirus DNA in Serum Correlates with Clinical Cytomegalovirus Retinitis in AIDS

The high sensitivity of nested polymerase chain reaction (PCR) offers the possibility of rapid detection of cytomegalovirus (CMV) DNA in serum. Five consecutive serum samples were examined from 52 human immunodeficiency virus (HIV)-seropositive patients (19 of whom had clinically presumed diagnosis of CMV chorioretinitis). Presence of CMV DNA in serum was shown to precede development of clinical disease. Eleven patients who developed chorioretinitis were positive for CMV DNA in serum samples obtained 3 months before clinical disease, and 3 retinitis patients who initially were negative for CMV DNA became positive with the onset of clinical retinitis. In contrast, 29 of 33 HIV-seropositive subjects without clinical CMV chorioretinitis and matched with respect to age and CD4 T cell numbers were negative for CMV DNA in all 5 serum samples. Thus, the presence of CMV DNA in serum analyzed by PCR is a good predictive marker of CMV retinitis in HIV-seropositive subjects. A positive PCR results supports the clinical diagnosis and may be useful for monitoring response to antiviral treatment.

Detection of Cytomegalovirus Antibodies in Serum Using the TranSTAT-CMV and CMV Scan Assays

Two hundred serum samples were tested with two assays for rapid detection of cytomegalovirus (CMV) antibodies. The assays were CMV Scan, a two-step latex particle agglutination assay for CMV IgG and IgM antibodies, and TranSTAT-CMV, a newly available six-step solid-phase enzyme immunoassay for CMV IgG antibodies. Comparison of initial results to reference results revealed a sensitivity/specificity of 97.2%/96.8% for CMV Scan and 94.3%/83.0% for TranSTAT-CMV. The same comparison after testing discrepant samples in duplicate increased the sensitivity/specificity to 99.1/100% for CMV Scan and 100%/93.6% for TranSTAT-CMV. Borderline-positive results were observed much more frequently with TranSTAT-CMV than CMV Scan (35 vs. 2), compromising easy interpretation of results. CMV Scan is superior to TranSTAT-CMV on the basis of performance characteristics, simplicity of test performance, and ease of interpretation of results.

Rapid Detection of CMV in Urine and Bronchoalveolar Lavage Specimens

Two hundred eighteen urine specimens and 43 bronchoalveolar lavage (BAL) specimens were cultured for cytomegalovirus (CMV) using a shell vial culture method and stained with a direct fluorescent antibody technique after 2 and 7 days. Results were compared with conventional tissue culture (CTC) incubated for 28 days. CMV was detected in 42 specimens (16.1%) by CTC and in 51 specimens (19.5%) by shell vial culture immunofluorescence (SV-IF). Average time required for detection of CMV by CTC was 11.4 days, whereas SV-IF required a maximum of 7 days. Cost per test was $21.25 for CTC and $14.64 for SV-IF.

Rapid detection of cytomegalovirus in bronchoalveolar lavage specimens from marrow transplant patients: evaluation of a direct fluorescein-conjugated monoclonal antibody reagent

An FITC-conjugated monoclonal antibody reagent containing three CMV-specific monoclonal antibodies was evaluated for the rapid detection of CMV in bronchoalveolar lavage (BAL) cytospin preparations by direct IF (DFA). Eighty-six BAL samples from 72 marrow transplant patients were inoculated into both centrifugation and standard cell culture. CMV was detected in 49/86 (57%) BAL samples. DFA detected 37/46 (80%) samples which were positive in centrifugation culture. While DFA staining lacked the sensitivity (overall sensitivity 38/49, 78%) to replace either standard or centrifugation culture, the total laboratory time needed to complete the DFA was only 1.5 h and its concurrent use with centrifugation culture can provide rapid specific diagnosis of CMV pneumonia.

Increased sensitivity for rapid detection of cytomegalovirus by shell vial centrifugation assay using mink lung cell cultures

A comparative study was made of various human and non-human cell cultures to determine their sensitivity for cytomegalovirus (CMV) as detected by the production of CMV early antigen using the shell vial centrifugation assay. Mink lung cell cultures, frequently used for detection of herpes simplex virus in clinical specimens, were found to be significantly more sensitive to infection by CMV than other cell cultures tested. Using the shell vial centrifugation assay, the mink lung cell cultures were more sensitive than human diploid fibroblasts for the detection of the Davis strain of human CMV and CMV from clinical specimens.

Rapid Detection of Cytomegalovirus by 24-Well Plate Centrifugation with the Use of a Monoclonal Antibody to an Early Nuclear Antigen

Two methods for the detection of cytomegalovirus (CMV) in 457 clinical specimens were compared: (1) centrifugal inoculation of MRC-5 cells seeded on coverslips in 24-well plates and staining with a monoclonal antibody to CMV early nuclear antigen after incubation for both 16-18 hours (EA-1) and four days (EA-4); and (2) conventional tube cell culture. CMV was identified in 50 (11%) specimens from 34 different patients. EA-1 and EA-4 had positive results for CMV in 32 (64%) and 36 (73%) of the specimens, respectively. Positive inclusions were present on only one coverslip in 31% of the cases by EA-1 and in 10% by EA-4. The number of inclusions was not necessarily predictive of tissue culture results. CMV was recovered by conventional tissue culture from 27 specimens (54%) after an average of 17 days (range, 6-26 days). One specimen, positive for CMV by EA-4, yielded herpes simplex virus (HSV), and from 9 of the 407 CMV-negative specimens, another virus was recovered: HSV from 6 specimens and varicella zoster virus, adenovirus, and enterovirus from one specimen each. CMV was detected in significantly more specimens by EA-4 than by tissue culture (P = 0.037). However, there was no significant difference in the detection of CMV between EA-1 and EA-4 or between EA-1 and conventional culture. The authors' data suggest that for maximum recovery of CMV from clinical specimens, both an early antigen assay and conventional tissue culture should be performed. For urine specimens it appears that inoculation of two coverslips followed by staining after overnight incubation is adequate. To optimize the yield of the early antigen assay when testing specimens other than urine, the authors recommend inoculating three coverslips, two of which should be stained after overnight incubation, and, if necessary, the third coverslip could be stained after a more prolonged incubation period.

Rapid Detection of Cytomegalovirus in Clinical Specimens

Rapid Detection of Cytomegalovirus in Clinical Specimens

Rapid detection of cytomegalovirus by fluorescent monoclonal antibody staining and in situ DNA hybridization in a dram vial cell culture system.

By using dram vial cell culture methods, three commercially available tests for cytomegalovirus (CMV) detection were compared: direct fluorescent monoclonal antibody staining for CMV-specific early and late antigens (direct FA), indirect fluorescent monoclonal antibody staining for a CMV-specific early antigen (indirect FA), and in situ DNA hybridization with a biotinylated CMV-specific DNA probe kit (DNA probe). Of those tests, only the indirect FA provided consistent, reliable virus detection within the initial 24 h postinfection for serial 10-fold dilutions of CMV AD169 (laboratory strain) and for three selected urine samples. However, when used prospectively, the indirect FA failed to detect virus within the initial 10 days postinfection in 15 of 78 consecutive specimens that were eventually positive by cell culture. Although the indirect FA was more sensitive than the direct FA or DNA probe, its utility appeared limited to specimens with high CMV concentrations. On the basis of these data, we recommend that indirect FA be reserved as an adjunct to standard cell culture for selected samples in diagnostic hospital laboratories.

Rapid detection of cytomegalovirus in clinical specimens by immunofluorescent staining of shell vial cultures.

A recently described rapid technique for detection of cytomegalovirus (CMV) was evaluated in clinical specimens utilizing indirect immunofluorescent staining (IFA) of shell vial cultures. A total of 266 clinical specimens received for viral isolation were inoculated to commercially available shell vials seeded with human lung fibroblasts (MRC-5), centrifuged at 700 X g for one hour, and stained after 18 hours incubation with monoclonal antibody to CMV early nuclear protein (Biotech Research Laboratories) and fluorescein conjugated goat antimouse IgG (Cappel Laboratories). All specimens were also inoculated to tubes of human lung fibroblasts and observed for cytopathic effect (CPE) for 28 days. Of 54 specimens positive for CMV, 36 were positive by both IFA and CPE, 3 were positive by CPE only, and 15 were positive by IFA only (P less than 0.01 by the chi-square test). Failure to detect CMV associated CPE in 10 of these 15 samples was probably due to concomitant infection with herpes simplex virus or heavy bacterial or fungal contamination. Nine of the 13 patients with IFA-positive CPE-negative specimens had CMV infection documented by other positive cultures. It was concluded that the shell vial IFA rapid technique for detection of CMV is highly specific, more sensitive than conventional isolation, and well suited for application in a clinical virology laboratory.

Rapid detection of cytomegalovirus using immune scanning electron microscopy

Monoclonal antibodies, specific for human cytomegalovirus, were conjugated to latex microspheres that were already labelled with rabbit anti-mouse immunoglobulin. The beads were incubated with serum or urine from patients, and then collected on a filter surface, which was analyzed in a scanning electron microscope. Size, immunological specificity, and relative quantity of virus particles were determined within 2 h after serum or urine collection by the visualization of virus particles specifically bound to the latex-bead surface. No such binding of virus particles was detected in the various controls. This method was compared with conventional virus isolation by tissue culture. It enables identification of viruses within a few hours in different body fluids. Even without specific antibodies, the filtration method may permit the rapid detection of particles and the determination of their size in various body fluids.

Rapid detection of cytomegalovirus by tissue culture, centrifugation, and immunofluorescence with a monoclonal antibody to an early nuclear antigen

A rapid, sensitive and specific assay for the detection of cytomegalovirus (CMV) was developed utilizing MRC-5 cells in 24-well plates containing round coverslips. Centrifugation expedited the detection of CMV early antigen with monoclonal antibody. Immunofluorescent staining 16 h after inoculation with a stock CMV preparation (AD-169), demonstrated an 11-fold increase in the number of nuclear inclusions when the specimens were centrifuged (18 ± 2.2) as compared to the non-centrifuged specimen (1.6 ± 0.9). However, the number of nuclear inclusions depended on the age of the MRC-5 cells. They were more sensitive to CMV infection between 4 and 11 days after the cells were seeded into plates. Among 159 patient samples cultured for CMV, 23 (14%) were positive by the rapid method (mean of 32 h) and 18 (11%) by routine tissue culture (mean of 12 days). Cytomegalo-virus in urine was detected within 1.3 days, whereas buffy coats (2.3 days) and bronchial washings (2.5 days) took longer. Staining for CMV inclusions at more than one time point was necessary for the optimal detection of CMV by the rapid method. We recommend using this assay system as it is rapid, specific, sensitive and versatile for the detection of CMV in many biological specimens.

Direct In Situ Hybridization for Rapid Detection of Cytomegalovirus in Bronchoalveolar Lavage

Rapid detection of cytomegalovirus (CMV) from pulmonary specimens in immunosuppressed persons may provide an origin for pneumonia. In situ DNA hybridization has been effective for detection of CMV in otherwise nondiagnostic histologic material. Studies comparing bronchoalveolar lavage (BAL) with open-lung biopsy have shown the former to be superior in detecting most pulmonary pathogens affecting immunocompromised patients. Fifty consecutive BAL specimens were studied to compare direct in situ DNA hybridization, routine tissue culture, and conventional cytologic examination to assess the efficacy of the hybridization technic to rapidly detect CMV. Using tissue culture as the standard, a sensitivity of 90% (28 of 31) and specificity of 63% (12 of 19) were observed with the CMV probe. Discrepant results between the probe and tissue culture were present in ten cases. There were seven probe-positive, culture-negative cases, three of which had systemic CMV infection, including two patients with inclusions noted by conventional cytologic examination. Three probe-negative, culture-positive cases were found. In the authors' laboratory, the predictive value of a positive CMV probe is 80% (28 of 35). In contrast to the probe, conventional cytologic examination revealed CMV inclusions in only 23% (7 of 31) of the culture-positive cases. An average of 21 days was required for CMV cultures to become positive; probe results were available within 24 hours. The authors conclude direct in situ DNA hybridization is a useful rapid method for the detection of CMV in BAL specimens submitted for cytologic examination.

Rapid detection of cytomegalovirus using immune scanning electron microscopy

Monoclonal antibodies specific for human cytomegalovirus were conjugated to latex microspheres that were already labelled with rabbit anti-mouse immunoglobulin. The beads were incubated with serum or urine from patients, and then collected on a filter surface, which was analyzed in a scanning electron microscope. Size, immunological specificity, and relative quantity of virus particles were determined within 2 h after serum or urine collection by the visualization of virus particles specifically bound to the latex bead surface. No such binding of virus particles were detected in the various controls. This method was compared with conventional virus isolation by tissue culture. It enables identification of viruses within a few hours in different body fluids. Even without specific antibodies, the filtration method may permit the rapid detection of particles and the determination of their size in various body fluids.

An immunofluorescent assay for rapid detection of cytomegalovirus.

Two kinds of immunofluorescent assay, the indirect immunofluorescent assay (IIF) and the improved indirect immunofluorescent assay (improved-IIF), were used to detect the intracellular cytomegalovirus (CMV) antigens. It was found that the improved-IIF could demonstrate CMV antigens not only earlier but also in a smaller amount than the IIF. It is obvious that the improved-IIF is a more sensitive method than the IIF. The possible principles for the enhanced sensitivity of improved-IIF were discussed briefly.

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).

Rapid detection of cytomegalovirus in clinical specimens by using biotinylated DNA probes and analysis of cross-reactivity with herpes simplex virus.

A method for rapid identification of human cytomegalovirus (HCMV) was developed with biotinylated DNA probes. BamHI restriction fragments from HCMV strain AD169 were selected and tested for their ability to detect virus in patient urine samples. All probes detected 30 pg of HCMV AD169 DNA. The BamHI B fragment detected 15 of 29 cell-culture-positive samples (sensitivity, 52%). There were four samples which were probe positive and cell culture negative (specificity, 87%). The D and H fragments used as combined probes detected 17 of 21 cell-culture-positive samples (sensitivity, 81%). There were five probe-positive and cell-culture-negative samples (specificity, 68.8%). The H fragment, when used alone, detected 11 of 14 culture-positive samples, and 5 samples were culture negative and probe positive. Sensitivity (78.6%) and specificity (76.2%) for the H fragment were similar to those for the combined probes, but the color intensity of the positive reactions detected by the H fragment alone was lower. There was unexpected cross-reactivity with herpes simplex type 1 and 2 controls when the combined D and H probes were used. Specific hybridization was demonstrated between subfragments of the HCMV BamHI D fragment and the herpes simplex virus type 1 EcoRI M fragment.