Fourteen heart transplant recipients were monitored for human cytomegalovirus (HCMV) infection based on determination of antigenemia, viremia, and DNAemia (by polymerase chain reaction [PCR]) in peripheral blood polymorphonuclear leukocytes (PMNL). Three patients had symptomatic primary, 10 had recurrent (3 asymptomatic), and 1 (seronegative) had no HCMV infection. Severe clinical symptoms appeared when levels of viremia/antigenemia were greater than 50 infected PMNL/2 x 10(5) cells examined. Of 200 blood samples examined, 93 (46.5%) were positive for viremia/antigenemia and DNAemia, whereas 48 (24.0%) were positive for DNAemia only; 59 (29.5%) were negative in all assays. Follow-up of HCMV infections in heart transplant recipients showed that PCR can detect viral appearance in blood 7-10 days earlier than assays for antigenemia/viremia. On the other hand, viral disappearance from blood, as assessed by PCR, occurred weeks or months later than revealed by other assays. Detection of virus by PCR only was never associated with overt HCMV-related clinical symptoms. Of the 8 symptomatic patients treated with ganiclovir, 2 became PCR-negative at the end of treatment and 1 cleared virus from blood in the following weeks, whereas 5 showed persistent or recurrent infection.

Monitoring of Human Cytomegalovirus Infections and Ganciclovir Treatment in Heart Transplant Recipients by Determination of Viremia, Antigenemia, and DNAemia

Human cytomegalovirus (HCMV) viremia in peripheral blood polymorphonuclear leukocytes (PMNLs) from 187 immunosuppressed patients (79 heart transplant recipients and 108 patients with acquired immunodeficiency syndrome [AIDS]) was investigated. Five mouse monoclonal antibodies (MAbs), one specifically reactive to HCMV immediate-early antigen (IEA) in PMNLs, two specifically reactive to IEA in infected cell cultures, and two specifically reactive to late antigens, were used in immunofluorescence and/or immunoperoxidase test systems for (i) detection of HCMV IEA in human embryonic lung fibroblast (HELF) cell cultures inoculated with PMNL samples, (ii) direct detection of HCMV IEA in PMNL nuclei of cytospin preparations, and (iii) identification of HCMV isolates from PMNL samples in HELF cells. Quantification of viremia was achieved by counting the number of infected PMNLs per 2 x 10(5) cells examined directly on cytospin preparations as well as by counting the number of IEA-positive HELF cells inoculated with 2 x 10(5) PMNLs. A significant correlation was found between the number of infected PMNLs and the number of infected HELF cells. When the number of infected PMNLs per 2 x 10(5) cells was greater than 10, both methods (PMNL IEA and HELF IEA) gave concordant results; when it was greater than 80/2 x 10(5) cells, clinical symptoms were consistently associated with HCMV viremia. Ten patients with heart transplants and three patients with AIDS who had high or increasing levels of HCMV viremia underwent antiviral treatment with ganciclovir. Treatment was discontinued only after disappearance of IEA-positive PMNLs from blood (the last marker of infection to become negative). On the other hand, in the presence of low levels of viremia (less than 10 infected PMNLs per 2 x 10(5) cells), different methods often provided discordant results and overt clinical symptoms were never observed. IEA-negative results with PMNL samples or HELF cells in the presence of positive virus isolation could never be attributed to the inability of IEA MAbs to recognize individual HCMV strains, since all of the relevant viral isolates were recognized by IEA MAbs. In addition, all five MAbs used in the study were capable of identifying all 135 conventional HCMV isolates obtained from the study population. It was concluded that (i) maximal sensitivity in diagnosing HCMV viremia may be achieved by combining different techniques; (ii) direct detection of HCMV IEA in PMNLs, yielding results on the day of blood collection, is a very rapid and sensitive technique, and thus one can rely on it for clinical management of HCMV infections; and (iii) low levels of viremia are clinically irrelevant, whereas high levels are associated with clinical symptoms. Images

Quantification of human cytomegalovirus viremia by using monoclonal antibodies to different viral proteins.

Peripheral blood polymorphonuclear (PMN) cells from 35 immunocompromised patients (22 heart transplant recipients and 13 AIDS patients) and four normal subjects were tested for the presence of human cytomegalovirus (HCMV) immediate early antigen (IEA) (antigenemia) by indirect immunofluorescence (IFA) and IEA-specific monoclonal antibodies (MAb). PMN samples were tested in parallel for HCMV isolation (viremia) by using MAb to viral early antigens (EA) and the IFA technique 24-48 hr after inoculation onto human fibroblast monolayers. HCMV was isolated from 26 of 83 PMN samples examined: of these, 25 were also positive for HCMV IEA (96% sensitivity). Seven additional PMN samples negative for viral isolation resulted IEA-positive (87.7% specificity). Six of the seven discordant samples were taken from four patients during ganciclovir treatment. The transitory dissociation between positive HCMV antigenemia and negative viremia during antiviral treatment was followed, at the end of the therapy, either by virus clearance and disappearance of IEA-positive PMNs (one patient) or by reappearance of viremia (three patients). Among concordant positive samples, a significant correlation was observed between the number of IEA-positive PMN leukocytes and EA-positive nuclei of infected fibroblasts, when the same number of PMNs were used for both tests.

Detection of human cytomegalovirus immediate early antigen in leukocytes as a marker of viremia in immunocompromised patients.

Nine human rotavirus strains from stools of infants with gastroenteritis were serially propagated in MA-104 cell cultures. All strains were identified as subgroup 2 rotaviruses by RNA gel electrophoresis, complement fixation, and enzyme-linked immunosorbent assay. The human rotavirus strains were propagated for 15 to 20 passages and then used for immunization of guinea pigs and rabbits. Animal antisera were also raised against a subgroup 1 human strain purified from stools and against the cell culture-adapted Wa strain, a reference subgroup 2 rotavirus of human origin. Cross-neutralization studies revealed the existence of two distinct serotypes within the cell culture-adapted subgroup 2 human rotaviruses: strains related and unrelated to strain Wa were classified as serotypes 1 and 3, respectively. Results with convalescent-phase sera from infants with primary rotavirus infections confirmed the existence of two serotypes within subgroup 2, and the serotypes responsible for primary subgroup 2 infections could be determined on the basis of the neutralizing reactivity of convalescent sera.

https://iai.asm.org/content/iai/43/2/722.full.pdf

Serotyping of cell culture-adapted subgroup 2 human rotavirus strains by neutralization.

Immunoglobulin G antibody to human cytomegalovirus (CMV)-specific early antigens (EA-Ab) was determined by the immunoperoxidase antibody technique in several cases of congenital, primary, and reactivated CMV infections. Mothers of congenitally infected infants and a group of leukemic children and pregnant women were also studied. In 11 cases of congenital infection, CMV EA-Ab was always associated with CMV excretion whether immunoglobulin M antibody was present or not. Nine mothers of congenitally infected infants had CMV EA-Ab for several months after delivery, but association with CMV elimination was not established when urine and/or saliva were tested for virus isolation. In all nine cases of primary CMV infection, CMV EA-Ab was present, and in five its detection was associated with CMV isolation. In one case, disappearance of EA-Ab occurred when virus excretion ceased. In five cases of reactivated CMV infections, a consistent association between CMV EA-Ab and virus isolation was found. Six of 31 leukemia children had CMV EA-Ab, and virus was isolated from 3 of these. Four of 28 pregnant women showed EA-Ab in their serum, but tests for isolation were not done. These data suggest that CMV EA-Ab is not a marker of a current primary CMV infection, as previously reported, but a marker of an active CMV replication which can take place in primary as well as in congenital and reactivated CMV infections. Images

Immunoglobulin G to virus-specific early antigens in congenital, primary, and reactivated human cytomegalovirus infections.

Sixteen cytomegalovirus (CMV) isolates from both ill and healthy patients were identified by the immunoperoxidase technique (IP). CMV detection was accomplished by direct examination of the primary isolate using either direct (DIP) or indirect immunoperoxidase (IIP) techniques. In thirteen of the isolates, confirmation of identification was achieved by indirect immunofluorescence (IFA) and by demonstration of herpes particles by electron microscopy (EM). Further, in four cases of non-CMV alterations in the tissue culture which might be confused with actual infection, the IP test was negative as were the confirmatory tests. The IIP is preferred over the DIP test since the latter shows a certain amount of background stain of uninfected cells. Tissue culture cells showing focal CMV cytopathic effect contained both nuclear and cytoplasmic inclusions stained by IP technique. Nonspecific staining was associated with cytoplasmic inclusion bodies. The IP technique can detect individual cell CMV infection at 24 hours when EM reveals only unenveloped viral particles. It is sensitive, specific, and allows direct identification of infected cells in the primary isolate in as little as 90 minutes. Furthermore, it can be performed in standard isolation tissue culture tubes, whereas IFA requires the transfer of the infected cells onto slides or the routine use of Leighton tubes.

The immunoperoxidase technique for rapid human cytomegalovirus identification.

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The immunoperoxidase technique for rapid human cytomegalovirus identification.