Evaluation of R-Mix FreshCells in shell vials for detection of respiratory viruses.

The performance of a mixture of mink lung and A549 cell lines in shell vials (MSVs) for the detection of respiratory viruses in 159 specimens was evaluated. MSVs, conventional culture, and direct immunofluorescence assay identified 96, 85, and 67% of the influenza A virus-positive specimens, respectively. MSVs provided both a high degree of sensitivity and rapid turnaround times for the detection of influenza A virus.

Use of a single swab in multi-microbe or flex trans transport medium for detection of Chlamydia trachomatis by Roche Amplicor PCR and culture in specimens from two different patient populations.

The Roche Amplicor PCR increased the detection of Chlamydia trachomatis compared with culture in promptly processed clinical specimens from a local clinic (100 and 86.5%, respectively) and in samples with delayed processing transported from distant facilities (100 and 72.7%, respectively). A single swab collected in culture transport medium was used. Two media, Multi-Microbe and Flex Trans, were tested and found to be equally acceptable.

Re-examination of the McCoy cell line for confirmation of its mouse origin: karyotyping, electron microscopy and reverse transcriptase assay for endogenous retrovirus.

BACKGROUND: The McCoy cell line originally derived from human synovial fluid in 1955, has been later found useful for cultivation of Chlamydia trachomatis. This cell line has been subcultured and exchanged between laboratories for many years. In recent years, the McCoy cell line has been widely used in many clinical diagnostic laboratories and has been supplied through commercial companies for the isolation and identification of Chlamydia trachomatis from clinical specimens. OBJECTIVES: Since retrovirus-like particles have been observed in McCoy cells and the species of origin of the currently used cell line has not been adequately documented, further characterization of McCoy cell line obtained from commercial sources was carried out. STUDY DESIGN: This study includes karyotypes analysis using G-banding for the confirmation of species origin of McCoy cells, electron microscopy for examination of virus particles associated with the cells and biochemical assay for reverse transcriptase activity for detection of retrovirus. RESULTS: Our results showed by karyotype analysis that McCoy cells are of mouse origin. Electron microscopic examination revealed the presence of endogenous retrovirus type-A and type-C virions. Biochemical assays of culture supernatant fluids from McCoy cells detected reverse transcriptase activity which required Mg(2+) ions. CONCLUSIONS: The present study has confirmed that McCoy cells currently used by many laboratories are mouse cells, not the original McCoy cells derived from human cells. Laboratory workers should be aware of the presence of endogenous murine retrovirus in this cell line and appropriate precautions should be taken.

An adventitious viral contaminant in commercially supplied A549 cells: identification of infectious bovine rhinotracheitis virus and its impact on diagnosis of infection in clinical specimens.

The isolation and identification of an adventitious viral agent, infectious bovine rhinotracheitis virus, in one lot of A549 cells from a commercial supplier is described in this report. The presence of infectious bovine rhinotracheitis virus in A549 cells was unexpected and has caused problems in the diagnosis of infections in clinical specimens in three laboratories.

Advantages of multiple cell culture systems for detection of mixed-virus infections.

Simultaneous infections by two or more viruses occur frequently, especially in immunosuppressed patients. In order to detect more than one viral agent in a single specimen, multiple cell systems have been employed in our laboratory. Specimens are routinely inoculated into four different cell cultures, namely: MRC-5, a human diploid lung fibroblast cell strain; A549, a human continuous cell line; primary guinea pig embryo (GPE) cell culture, and primary rhesus monkey kidney (RhMK) cell culture. For rapid detection of cytomegalovirus (CMV) antigen, MRC-5 cells grown in shell vials containing coverslips are also inoculated with the same specimens followed by centrifugation. During 1989, nine cases of multiple-virus isolations were obtained in this laboratory. In all nine patients, CMV was detected in MRC-5 cells. Five of the nine cases were co-infected with HSV-1, three were co-infected with adenovirus, and one was co-infected with both HSV-1 and adenovirus. All four adenovirus isolates were obtained in A549 cells. Of the six HSV-1 isolates, one was detected in all three cell cultures, e.g. MRC-5, A549 and GPE; one was detected in both MRC-5 and A549 cells, and four were isolated in a single-cell type only. For nine CMV-positive cases, five were obtained by both conventional and centrifugation cultures, two each were detected by centrifugation or conventional culture only. Thus for a maximum detection of viruses present in a single specimen, it is suggested that multiple-cell-culture systems, together with more than one technique, should be employed.

Activity of (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC) against guinea pig cytomegalovirus infection in cultured cells and in guinea pigs.

(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, HPMPC, and two HPMPC-related nucleoside analogs, (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine, HPMPA, and (2-phosphonylmethoxyethyl)guanine, PMEG, were evaluated for their antiviral activities against guinea pig cytomegalovirus (GPCMV) infection in guinea pig embryo (GPE) cells and human cytomegalovirus (HCMV) infection in human diploid fibroblast (MRC-5) cells. DHPG, 9-(1,3-dihydroxy-2-propoxymethyl)guanine, was used for comparison. The antiviral activity of HPMPC against GPCMV infection in vivo and its toxicity to Hartley guinea pigs were also evaluated. The 50% antiviral effective doses (ED50) of HPMPC, HPMPA, PMEG and DHPG against GPCMV infection in GPE cells were 0.22, 1.4, 0.07 and 62 microM, respectively; and against HCMV infection in MRC-5 cells, the ED50s were 0.51, 0.72, 0.01 and 17.5 microM, respectively. Their cytotoxic doses (CyD50) in GPE replicating cells were 84, 35, 1.4 and 700 microM, respectively and in MRC-5 cells were approximately 114, 31, 0.86 and 750 microM, respectively. Based on their calculated therapeutic indexes, HPMPC was the most potent and selective of the four compounds tested. In vivo, during acute infection, the spleen indexes of all infected animals that were treated with 1.25 to 5.0 mg/kg/day of HPMPC for 5 days were significantly reduced as compared with sham-treated animals. Virus infectivity titers in blood and various tissues of infected animals treated with HPMPC, 2.5 or 1.25 mg/kg/day were not significantly lower than those of the infected, sham-treated animals; with 5 mg/kg/day, infectivity titers in the blood, spleen, and salivary gland were significantly lower in HPMPC-treated than in sham-treated animals. However, HPMPC was toxic to guinea pigs especially at doses of 5 to 10 mg/kg/day. These data showed that HPMPC was highly active and selective in cultured guinea pig cells and human fibroblast cells against CMV infection but did not effectively inhibit GPCMV infection in guinea pigs at minimum toxic concentrations.

Detection of human cytomegalovirus early and late antigen and DNA production in cell culture and the effects of dimethyl sulfoxide, dexamethasone, and DNA inhibitors on early antigen induction.

Recently a conventional method for the laboratory diagnosis of human cytomegalovirus (HCMV) infection was improved by using centrifugation culture to enhance viral adsorption and by detecting HCMV early antigen and DNA. Comparison of the sensitivity of three rapid methods using commercial diagnostic reagents for the detection of HCMV early antigen (EA), late antigen (LA), and DNA was quantitatively evaluated in centrifugation cultures of human fibroblast cells (MRC-5) infected with HCMV. HCMV-EA was first detected 4 hours after infection, and the number of antigen-positive cells increased rapidly thereafter. Using biotinylated DNA probe, viral DNA was first detected 12 hours postinfection; the number of DNA-positive cells increased slowly. HCMV-LA was first seen 48 hours postinfection, and the number of LA-positive cells also increased thereafter. Thus detection of HCMV-EA was the most rapid and sensitive method for HCMV diagnosis. Several chemical compounds have been used to enhance HCMV replication. The effect of dimethyl sulfoxide (DMSO), dexamethasone (DEX), 5-bromo-2-deoxyuridine (BrdU), 5-fluoro-deoxyuridine (FdU), and cytosine arabinoside (Ara-C) on HCMV-EA induction was evaluated in centrifugation cultures of MRC-5 cells infected with HCMV. Infected cells treated with 1% DMSO alone or with DMSO plus DEX (10(-5) M) have been shown to increase the number of HCMV-EA-positive cells three- to fivefold over the untreated control cultures. The enhancing effects of Ara-C, BrdU, and BrdU plus FdU were demonstrated only occasionally.

Electron microscopy for the rapid detection and identification of viruses from clinical specimens.

The advantages of using electron microscopy for rapid diagnosis of virus infection from clinical specimens, for identification of virus isolates with unusual properties, and for monitoring endogenous agents in cell cultures are illustrated by several actual cases that have occurred over the years. The importance of using morphological characteristics of viruses for initial identification is emphasized.

Disseminated adenovirus infection in an immunocompromised host. Pitfalls in diagnosis.

In this report, a bone marrow transplant recipient with rapidly fatal gastroenteritis is presented. The presence of intranuclear inclusions on postmortem light microscopic examination of liver, lung, and small bowel tissue was considered diagnostic of cytomegalovirus infection. However, electron microscopic examination of liver tissue demonstrated adenovirus infection. This was confirmed by isolation of an adenovirus type 2 with unusual laboratory features from liver, lung, colon contents, serum, esophageal swab, and oral ulcerations. Results of a complement fixation test for antibodies to adenovirus performed on postmortem serum samples were negative, and a titer of 1:4 was noted for antibody against cytomegalovirus. This case illustrates the diagnostic pitfalls that may be encountered in establishing a specific viral diagnosis in severely ill patients.

Comparison of fluorescent-antibody-to-membrane-antigen test, indirect immunofluorescence assay, and a commercial enzyme-linked immunosorbent assay for determination of antibody to varicella-zoster virus.

The demand for sensitive and specific assays to determine immune status to varicella can be expected to increase with the anticipated availability of a varicella-zoster virus vaccine for use in nonimmune adults, especially health care personnel, and in immunosuppressed children. Although the fluorescent-antibody-to-membrane-antigen (FAMA) test remains the reference standard to which other tests are compared, simpler alternative assays are needed. In this study, the FAMA was compared with a simple indirect immunofluorescence assay (IFA) and a commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to varicella-zoster virus. One hundred and twelve serum samples were screened by the FAMA test and IFA at a 1:5 dilution, and 100% agreement was found. Of these samples, 101 were available for testing by ELISA, and identical results were obtained with 97 samples (96% agreement). When the samples were screened at a 1:2 dilution, 99 of 101 results agreed. In addition, 31 spinal fluid samples were tested by all three methods. When screening was at a 1:2 dilution, there was 96.8% agreement between the FAMA test and IFA. When the cutoff value established for sera was used for the spinal fluid samples, there was 90.3% agreement between the ELISA and the FAMA test. Thus, both IFA and ELISA can be considered sensitive and specific alternatives to the FAMA test, and in addition, both use commercially available reagents.

Replication of human adenoviruses in guinea pig embryo cells: an ultrastructural study.

Guinea pig embryo (GPE) cells showed different degrees of susceptibility to human adenovirus types as determined by virus infectivity assay and electron microscopic examination. Adenovirus 2 and 5 induced extensive cellular changes and produced high titers of infectious virus in GPE cells as in human cells. Mature progeny virus and protein crystals were observed in both cell types. Adenovirus 7 induced some cellular changes in GPE cells but only a small number of cells yielded progeny virus as determined by electron microscopy. Adenovirus 3, 8 and 31 induced some cellular changes but no progeny virus was found under electron microscopic examination. Characteristic fibers were observed in nuclei of adenovirus 31 infected cells. The ability of human adenovirus 2 and 5 to replicate in GPE cells is an example of an unusual cross-species biological property of certain adenovirus types. This property may be useful as a biological marker for these virus types.

Antiviral effect of 9-(1,3-dihydroxy-2-propoxymethyl)guanine against cytomegalovirus infection in a guinea pig model.

The antiviral activity of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) against guinea pig cytomegalovirus (GPCMV) was evaluated in guinea pig cell cultures and in Hartley guinea pigs. The 50% effective dose of DHPG against GPCMV replication in cell cultures was 71 microM. Ultrastructural studies revealed that DHPG inhibited the formation of viral cores and the production of nucleocapsids, enveloped virions and dense bodies, but the drug did not prevent the formation of virus induced intranuclear tubular structures. In vivo, guinea pigs inoculated intraperitoneally with GPCMV were treated with DHPG, 25 mg/kg subcutaneously, twice daily. Treatment was initiated 24 h after infection and continued for 7 days. During the acute infection, the average body weights of DHPG-treated, virus infected guinea pigs were approximately 14% lower than the sham-treated counterparts on day 10, 11 and 13 post-virus inoculation. Virus infectivity titers were higher in the lungs of DHPG-treated guinea pigs on day 10 than the sham-treated ones. Although there was no significant difference on histopathologic lesions in the spleen, liver and lungs of the drug-treated and the sham-treated guinea pigs, DHPG treated animals appeared to have fewer virus-induced lesions or inclusions in the kidneys and salivary glands than the sham-treated ones. In addition, virus infectivity titers in the salivary gland of DHPG treated guinea pigs were consistently lower than the sham-treated animals.

Illegal abortion: an attempt to assess its cost to the health services and its incidence in the community.

This article describes a study designed to test a method for assessing the cost to the health services of illegally induced abortion and the feasibility of estimating the incidence of induced abortion by a field interviewing approach. The participating centers included three hospitals in Ankara, Turkey; three hospitals in Ibadan, Nigeria; one hospital in Caracas and one in Valencia, Venezuela; and two hospitals in Kuala Lumpur, Malaysia. Hospitalized abortion cases were classified as induced or spontaneous or as "probably induced," "possibly induced," or "unknown" according to a classification scheme comprising certain medical criteria. The sociodemographic characteristics of induced and spontaneous abortion cases were subjected to discriminant function analysis and the discriminating variables best characterizing the induced versus the spontaneous abortion groups were identified for each center. On the basis of this analysis, the "probably" and "possibly" induced and "unknown" categories were further classified as induced or spontaneous abortion, with stated probabilities. Thus an overall estimate is made of the proportion of all hospitalized abortions that can be considered illegally induced outside the hospital. Selected results on costs of induced and spontaneous abortion are shown. The method further tested the feasibility of obtaining valid survey data on abortion from the communities studied by re-interviewing the women hospitalized for induced and spontaneous abortion six months later in their homes. This exercise showed a degree of under-reporting of abortion that varied widely among centers, even among women who had admitted illegal induction at the time of hospitalization. The feasibility of estimating the incidence of illegal abortion by field studies is discussed in the light of these findings.

Nucleic acid hybridization in the diagnosis of viral infections.

Recombinant DNA technology, including molecular cloning and nucleic acid hybridization, is now being applied to problems in clinical virology. Although viral isolation in cell culture remains the most sensitive and specific diagnostic test for many viruses, for some viruses, isolation in cell culture is lengthy or difficult or has not yet been achieved. Utilization of hybridization techniques has already resulted in important new information concerning the pathogenesis of a number of viruses, such as Epstein-Barr virus, hepatitis B virus, and human papillomavirus. In addition, time to diagnosis for viruses such as cytomegalovirus, Epstein-Barr virus, and varicella-zoster virus can be significantly shortened to 36 to 48 hours, a great improvement over standard isolation with obvious importance for patient management. Hybridization techniques have also been applied to screening of antiviral agents. Although results of studies to date have been encouraging, significant problems remain to be solved before these techniques can be applied in a routine diagnostic laboratory. First, more sensitive assays must be developed. One approach is the generation of probes with higher specific activities. Synthesis of single-stranded probes using recombinant M13 bacteriophage as a template results in probes of higher specific activities that also cannot re-anneal to themselves because they are not complementary. Thus, more probe is available to anneal to sample DNA. Synthesis of cRNA probes that form more stable hybrids with DNA is another approach that is receiving attention. A second problem is reagent safety and stability. The most sensitive and commonly used label in the studies reviewed in this article has been 32P. With its half-life of 2 weeks, potential hazards to personnel, and disposal problems, it is probably not suitable for clinical laboratories. A major step in the development of nonradioactive, stable probes has been synthesis of biotinylated nucleotide analogues that can be efficiently incorporated into DNA or RNA. Biotinylated probes are stable for 1 to 2 years at -20 degrees C, and their use obviates the need for autoradiography, thus shortening reaction times. In addition, very high concentrations of probes can be used without the background problems encountered with radiolabels. To date, biotinylated probes have been significantly less sensitive than those labeled with 32P, but continued efforts to improve sensitivity have yielded promising results.(ABSTRACT TRUNCATED AT 400 WORDS)

The placenta as a site of cytomegalovirus infection in guinea pigs.

The development of cytomegalovirus (CMV) infection in the placenta was studied in Hartley guinea pigs inoculated at midgestation, and its role in determining the outcome of fetal CMV infection was assessed. A hematogenous spread of CMV from the mother to the placenta occurred early during the course of the infection. However, the virus remained present in placental tissues long after CMV had been cleared from maternal blood (i.e., 3 and 4 weeks postinoculation). At that time, the virus was able to replicate in placental tissues in the presence of specific maternal antibodies. Viral nucleocapsids were seen within nuclei of trophoblastic cells, and virions were present surrounding infected cells. In addition, typical CMV-induced histopathological lesions bearing CMV antigens were consistently localized at the transitional zone between the capillarized labyrinth and the noncapillarized interlobium. Whenever CMV infection of the fetus occurred, virus was isolated from the associated placenta. Among placental-fetal units with CMV-infected placentas, only 27% of the fetuses were found to be infected. In addition, there was a delay in the establishment of the infection in the fetus in relation to the placenta, although frequencies of virus isolation in placental and fetal tissues peaked at 3 weeks after CMV inoculation. These results suggest that during primary CMV infection of pregnant guinea pigs, the placenta not only serves as a reservoir for CMV but also acts to limit transmission of the virus to the fetus.

Antiviral activity of CP-20,961 against herpes simplex viruses in vitro.

The antiherpesvirus activity of CP-20,961 [N,N-dioctadecyl-N',N'-bis (2-hydroxyethyl) propanediamine, or Avridine] was investigated in cultured guinea pig embryo (GPE) cells. Plaque formation of herpes simplex virus type 1 (HSV1) and type 2 (HSV2) was inhibited, but vesicular stomatitis virus replication was not inhibited, in GPE cells treated with CP-20,961 before infection. The ID50 concentration of CP-20,961 for HSV was about 50 micrograms/ml for 3-4 days of pretreatment. After virus adsorption and penetration, the same concentration of CP-20,961 had no effect on HSV plaque formation. The compound showed no detergent-like properties nor did it elicit any detectable interferon activity. Thus, the anti-HSV activity of CP-20,961 appeared to be associated with blocking the adsorption or penetration of the virus or both.

Distinctive characteristics of crude interferon from virus-infected guinea-pig embryo fibroblasts.

Crude interferon preparations from primary guinea-pig embryo cells infected with vesicular stomatitis virus strain T1026R1 were shown to be more sensitive to heat (37 degrees C), pH 2.0, and SDS than crude mouse interferon. Since the proportion of antiviral activity lost after each treatment was nearly the same, the existence of a single fraction of antiviral activity sensitive to all three treatments was suggested. Support for this possibility was given by the finding that subjecting this guinea-pig interferon to any one of the treatments rendered it insensitive to the effects of the other two.