The impact of cytomegalovirus infection on seronegative recipients of seropositive donor kidneys versus seropositive recipients treated with cyclosporine-prednisone immunosuppression.

To assess the impact of cytomegalovirus (CMV) infection in D+R- patients treated with cyclosporine (CsA)-prednisone immunosuppression, we compared the incidence of CMV infection, severity of disease, and the 1, 2, and 3-year actual graft and patient survival rates of CMV-infected D+R- patients with R+ patients from a group of 516 renal allograft recipients at our center. CMV infection occurred more frequently in 27/56 D+R- patients (48%) versus 111/376 R+ patients (29%) (P less than 0.01). The incidence of CMV was also significantly greater in D+R- versus R- patients receiving CAD grafts (59% vs. 32%, P less than 0.01) and first transplants (47% vs. 30%, P less than 0.05). There were no significant differences in CMV disease severity between the aggregate D+R- and R+ patient groups and when subgroups of these patients receiving cadaveric donor (CAD), living-related donor (LRD), first, or retransplant allografts were compared. The actual 1, 2, and 3-year graft survival rates for D+R- patients (68%, 58%, 68%) were not significantly different from rates in R+ patients (83%, 77%, 63%) with CMV infection. When the 1, 2, and 3-year actual graft survival rates in subgroups of D+R- and R+ patients were compared in CAD, LRD, and first and retransplants, there were no significant differences. The actual 1, 2, and 3-year patient survival rates were not significantly different between D+R- (89%, 92%, 100%) and R+ patients (94%, 91%, 86%) with CMV infection, nor were they different when CMV infected D+R- and R+ patients with CAD, LRD, first or retransplant grafts were compared. These data do not support the policy of denying a seropositive kidney to a seronegative recipient, since the severity of CMV disease and the impact of CMV infection is not significantly different comparing D+R- and R+ patients receiving CsA-prednisone immunosuppression.

Reactivity of serologic tests for the detection of antibody specific to cytomegalovirus.

A total of 212 sera were assayed for antibody specific for cytomegalovirus by complement fixation (CF), indirect immunofluorescence (IFA Electro-Nucleonics Laboratory, Inc., Bethesda, MD), ELISA (Cordis Laboratories, Inc., Miami, FL) and the FIAX system (International Diagnostic Technology, Santa Clara, CA). Correlation of CF with IFA, ELISA, and FIAX was 61%, 78%, and 71%, respectively. Quantitative correlation between IFA and FIAX and ELISA was not possible because of the broad range of reaction intensity of the latter two tests in sera with a particular IFA titer.

Detection of non-A, non-B hepatitis antigen by immunocytochemical staining.

Liver tissue obtained from a chimpanzee during the acute phase of an experimental non-A, non-B (NANB) hepatitis virus infection was studied by a sensitive immunocytochemical staining procedure for the presence of NANB viral antigens. Initial investigations were conducted with a model system of hepatitis B virus (HBV) antigens for purposes of comparing two immunocytochemical staining methods. Of these two procedures, an immunoperoxidase procedure, utilizing an avidin-biotinylated enzyme complex, was at least 40-fold more sensitive than a conventional immunoperoxidase technique for the detection of HBV-specific tissue antigens. Utilization of the avidin-biotin-amplified immunoperoxidase staining procedure, in conjunction with four primary convalescent antisera obtained from NANB hepatitis-implicated donors, resulted in the observation of NANB virus-associated antigen in the cytoplasm of hepatocytes from an infected chimpanzee liver. These same human antisera were not reactive with a number of uninfected control cells nor with cells infected with HBV, hepatitis A virus, or cytomegalovirus. Preincubation of one of these convalescent NANB sera, or IgG derived thereof, with an acute-phase serum obtained from a NANB hepatitis virus-infected chimpanzee abolished the antibody reactivity. We conclude from these observations that selected convalescent sera from NANB hepatitis virus-infected patients contain low levels of antibody that specifically react with a cytoplasmic antigen associated with NANB virus-infected hepatocytes.

Case report of spinal epidural abscess caused by Haemophilus paraphrophilus.

Haemophilus paraphrophilus was recovered in pure culture from purulent material collected at surgery from a patient presenting with a spinal epidural abscess and a severe neurological deficit. This is the first report of such an occurrence.

Induction of hepatitis B surface antigen in human hepatoma-derived cell lines.

Cultures of two human hepatoma cell lines were examined for expression of hepatitis B virus surface antigen (HBsAg). The PLC/PRF/5 cells secreted HBsAg continuously into the culture medium, whereas Mahlavu cells did not secrete the antigen. However, cytoplasmic antigen was detected in a low percentage (less than 5%) of the Mahlavu cells. The expression of HBsAg also was assayed in cultures treated wtih dexamethasone (DXM), 5-iodo-2'-deoxyuridine (IdUrd), or both. The results demonstrated that: (i) DXM stimulated secretion of HBsAg by PLC/PRF/5 cells but not by Mahlavu cells; (ii) the percentage of Mahlavu cells expressing cytoplasmic HBsAg was not increased in any cultures if the medium was replaced at 24 t0 48 h intervals but was increased approx. fivefold within 4 days in cultures treated with DXM or IdUrd/DXM if the medium was not changed. However, no increase was noted in the intensity of the immunoperoxidase stain of PLC/PRF/5 cells that expressed cytoplasmic antigen in any DXM cultures; (iii) HBsAg expression was stimulated to a lesser extent in IdUrd/DXM cultures than in DXM cultures and was not enhanced in IdUrd cultures. Thus, DXM enhanced secretion of HBsAg by PLC/PRF/5 cells within 24 h and, after a delay, enhanced expression of cytoplasmic antigen by Mahlavu cells. However, antigen secretion by Mahlavu cells evidently was blocked.

Modified spin-amplified adsorption procedure with conventional tissue culture tubes for rapid detection and increased recovery of herpes simplex virus from clinical specimens.

Conventional culture tubes were used in a modification of the spin-amplified adsorption procedure for recovery of herpes simplex virus (HSV) from clinical specimens. The sensitivity of isolation of HSV from 864 specimens adsorbed by the spin-amplified method was 100% (127 of 127), compared with 88.2% (112 of 127) for stationary-phase-adsorbed specimens. Cytopathic effect developed more rapidly in 32.1% (36 of 112) of isolates adsorbed by spin amplification than in those adsorbed by stationary means. In a separate quantitative study, cultures of HSV type 1 adsorbed by spin amplification yielded higher antigen levels and greater cytopathic effect than stationary-phase-adsorbed cultures. Cells grown in conventional tissue culture tubes may be used in a spin-amplified adsorption for rapid detection and increased sensitivity of HSV isolation.

Enhanced recovery of cytomegalovirus in conventional tube cultures with a spin-amplified adsorption.

Low-speed centrifugation-mediated adsorption was evaluated as an enhancement of infectivity of clinical and laboratory strains of cytomegalovirus (CMV) occurring with cells grown in conventional culture tubes. The time required for reporting of primary isolates of CMV from urine specimens adsorbed onto monolayers of WI-38 cells in culture tubes was calculated. Of 668 specimens adsorbed by the stationary phase (SP) method, 98 were positive by cytopathic effect (CPE) that required an average of 16.8 days for recovery in culture. However, the appearance of CPE required a shorter average time of 11.9 days for 70 CMV strains isolated from 283 specimens adsorbed in tube cultures by the spin-amplified (SA) method. In another phase of clinical CMV recovery, urine specimens were adsorbed by the SA method onto cell cultures grown in both shell vials and test tubes. Of 594 specimens inoculated, a total of 74 were positive by either CPE in test tubes or immunostaining-localized early antigen in shell vials. Approximately one-third of these CMV isolates were recovered only by CPE from specimens adsorbed by the SA method in test-tube cultures. In a related study to further evaluate differences between adsorption methods, the AD-169 laboratory strain of CMV was adsorbed by SP and SA methods onto MRC-5 cells grown in both culture vessels. Early antigen detection by immunomicroscopy was found in the infected cells at least 2 to 4 days prior to the appearance of CPE, regardless of adsorption procedure. In both vessels, the replication of AD-169 virus in cultures adsorbed by the SA method consistently exceeded that of virus adsorbed by the SP procedure. CPE occurred 24 to 48 h earlier and progressed two to four times more extensively; early antigen was expressed two- to fourfold greater within 24 to 48 h postinfection; and foci of infected cells containing late antigen were two to four times greater in number at 1, 2, and 5 days postinfection. Overall, the replication and enhancement of infectivity of laboratory and clinical strains of CMV as determined by CPE and early and late antigen expression occurred most efficiently with specimens adsorbed by the SA method onto cultures grown in conventional tubes or shell vials.

Posttransfusion hepatitis type A.

Hepatitis A virus (HAV) transmission through blood is a rare but potential cause of posttransfusion hepatitis. We can now document such a case supported by laboratory evidence of HAV in the donor blood. A 10-year-old girl manifested icteric hepatitis A 31 days after receiving a single unit of packed RBCs from a donor who subsequently experienced hepatitis A and died in hepatic failure. Hepatitis A virus antigen was detected in the donor's hepatocytes and in plasma obtained from the original donor unit. The density in cesium chloride of the HAV antigenic activity from the liver and plasma ranged from 1.33 to 1.37 g/cu cm, which is similar to that reported for infectious HAV particles. The implicated donor plasma had normal aminotransferase levels and was negative for antibody to HAV. Inoculation of this plasma into a chimpanzee resulted in the development of hepatitis A 23 days later based on the appearance of fecal HAV antigen, hepatitis, and IgM anti-HAV seroconversion. These data clearly document the presence of HAV in the donor sample that produced posttransfusion hepatitis A.

Evaluation of new transport medium for detection of herpes simplex virus by culture and direct enzyme-linked immunosorbent assay.

The transport medium Multi-Microbe Media (M4) was evaluated prospectively by culture and direct enzyme-linked immunosorbent assay (ELISA) for detection of herpes simplex virus from 473 specimens. In addition, 377 specimens in Bartels Viral Transport Medium were evaluated. By using culture as a "gold standard," the ELISA sensitivity was approximately 85%, while the specificities exceeded 96% for both media.