Cytomegalovirus infection after bone marrow transplantation: relation of pneumonia to postgrafting immunosuppressive treatment

Five fatal cases of CMV associated interstitial pneumonia occurred among 20 patients who had received allogenic bone marrow transplantation for acute leukemia or aplastic anaemia. This outcome was analysed in relation to prospectively obtained data on complement fixing (CF) and IgM anti-CMV titres and excretion of CMV in urine or pharynx. Altogether 16 patients had primary or reactivated CMV infection. Five of these patients receiving corticosteroids at the time of infection, at a dose of more than 1 mg/kg/day, failed to mount a significant CF antibody response, excreted large amounts of virus, and died from pneumonia, while the remaining 11 infected patients obtained high CF antibody titres and showed no severe symptoms related to CMV infection. IgM antibodies occurred simultaneously with CF antibodies in most cases of primary and reactivated infection. The results of the study suggest a protective role of the humoral immune response to CMV infection in BMT recipients, and passive immunisation with CMV hyperimmune globulin should be attempted especially in patients given additional immunosuppression with high dose corticosteroids.     

Pattern of anti-cytomegalovirus IgM antibodies determined by immunoblotting

Summary In order to improve the knowledge of the humoral immune response to CMV infection, we developed an immunoblotting technique which allowed a better analysis of the changes in the pattern of anti CMV-polypeptides IgM. We examined 234 sera belonging to 27 renal allograft recipients developing a primary or recurrent CMV infection and 12 non infected recipients. Thus we found that 11 main anti CMV-polypeptides IgM antibodies were present in over 25% of the infected patients. They reacted with proteins whose molecular weights ranged from 32K to 205 K.We showed that anti-p 45–47 IgM antibodies were present in 100% of CMV infected recipients and never in the non-infected population. They appeared very early in the course of the infection (5.43 weeks post-graft for primary infection and 5.00 weeks for recurrent ones) and, therefore, constitute a good marker of active infection. Two other CMV-specific IgM antibodies (anti-p 60–64 and anti-p 100) were found exclusively in the course of primary infections. Anti-p 60–64 IgM was observed at a high frequency (57.1%) and with a mean delay of 6.57 weeks post-graft. Therefore, the anti-p 60–64 IgM detection could be helpful for the diagnosis of primary infection. In almost 100% of both primary and recurrent infections, we observed anti-p 140 and anti-p 38 IgM antibodies. Only about 50% of non-infected patients had low levels of anti-p 140 and anti-p 38 IgM.The follow-up of recurrent infections showed that the anti CMV-polypeptides IgM antibodies appeared earlier than in primary infection. When we compared anti-p 45–47 IgM detection by immunoblotting and anti-CMV IgM detected by ELISA we observed that immunoblotting permitted the diagnosis 2.5 weeks earlier for primary infection, and 1 week earlier for recurrent infection, than ELISA. In addition, the detection of anti-p 45–47 IgM antibodies also occurred earlier than virus excretion.     

Is the measurement of virus-specific antibody in the lungs of transplant recipients with cytomegalovirus pneumonitis of diagnostic or prognostic value?

We have investigated 12 transplant recipients (nine bone marrow transplants, three renal) with 15 episodes of pneumonitis caused by cytomegalovirus (CMV) and ten patients (eight bone marrow transplant recipients, two renal transplant recipients) with 12 episodes of interstitial pneumonitis in whom no CMV was detected, to determine whether levels of CMV-specific IgG in the lung are diagnostic of infection. We have also assessed whether a vigorous specific local antibody response is important for survival. CMV-specific IgG and herpes simplex (HSV)-specific IgG were measured in bronchoalveolar lavage (BAL) fluid and serum using albumin to correct for simple diffusion from serum into the lung. We have found evidence for local production or facilitated transport of CMV-specific IgG in ten patients with CMV pneumonitis but also in six patients with pneumonitis where no CMV was detected. Similar results were found for HSV-specific IgG although only one patient had a demonstrable HSV infection. There was a tendency for those patients producing large amounts of immunoglobulin in the lung to survive compared with those who died, but there were wide variations between patients in each group. The local humoral immune response in transplant patients with pneumonitis was not specific to the infecting agent and is most probably the result of polyclonal B cell activation or facilitated transport of IgG from serum to lung secretions. Measurement of CMV-specific IgG response in the lung should not, therefore, be used for diagnostic or prognostic purposes.     

Cytomegalovirus pneumonitis and bone marrow transplantation: identification of a specific high risk group.

AIMS--To study the association between cytomegalovirus (CMV) excretion and interstitial pneumonitis in allogeneic bone marrow transplant (BMT) recipients, with reference to donor and recipient CMV antibody response. METHODS--The incidence of CMV excretion was prospectively studied in 62 allogeneic bone marrow transplantations performed on adults and children. All recipients received CMV seronegative blood products. Prophylaxis with high dose acyclovir and CMV immune globulin was given to high risk patients (donor or recipient, or both, CMV seropositive). RESULTS--CMV excretion was detected in eight of 26 (31%) high risk patients but in only one of 36 low risk patients (donor and recipient both CMV seronegative). Five of the eight (63%) excretors in the high risk category developed CMV, of whom four (80%) belonged to the seropositive recipient/seronegative donor group, and included the three CMV seropositive recipients whose CMV complement fixation antibody titres were 64 or greater before transplantation. CONCLUSIONS--These findings suggest that there is a subgroup of patients at especially high risk of developing CMV.     

Specific IgM of cytomegaloviruses and primary infection in kidney transplant patients

In 20 renal transplant recipients suffering from symptomatic CMV infection we looked if a specific IgM response was indicative of a primary infection. By an ELISA technique we investigated the specific IgG and IgM in a pair of sera taken at the time of the transplantation and later at the time of the CMV isolation. For 7 out of 8 patients exhibiting an IgM response in the late serum, the pretransplant serum did not contain either IgG or IgM specific antibodies. Therefore the specific IgM response was associated in 7 out of 8 patients with a primary CMV infection. Nevertheless in 6 other primary infected patients no specific IgM response was detected. Otherwise rheumatoid factors appeared in each primary infected patient developing a specific IgG response, with or without specific IgM response.     

Immune responses in cardiac transplantation. I. Detection of activated TIa+ cells in the blood during herpes virus infections.

Lymphocyte subpopulations in the blood of cardiac transplant recipients were monitored by single and double marker rosetting tests; using monoclonal antibodies to monomorphic determinants on T cells and 'Ia' antigens. Elevated absolute numbers and percentages of TIa+ cells were found in association with primary cytomegalovirus (CMV), and reactivation of Epstein-Barr virus or Herpes simplex virus. Serial tests showed that in primary CMV TIa+ cells peaked before the maximal IgM and IgG anti-viral titres measured by ELISA. Infection related antiglobulin levels increased in parallel with anti-viral IgM in primary CMV infections. Intravenous methylprednisone and blood transfusions selectively depressed TIa+ cell levels without affecting antibody titres. These results show that patients on maintenance immunosuppression of cyclosporin A and steroids can successfully mount T cell and antibody responses to herpes virus infections.     

Quantitative measurement of cytomegalovirus-specific IgG and IgM antibodies in relation to cytomegalovirus antigenaemia and disease activity in kidney recipients with an active cytomegalovirus infection

In a longitudinal investigation 103 kidney recipients were studied with respect to the development of cytomegalovirus (CMV) specific antibodies of the IgG and IgM class, in relation to the detection of CMV antigenaemia (immediate early antigen, IEA), in weekly obtained blood samples during the first 3 months after transplantation. In 15 out of 49 (31%) seronegative patients a primary infection occurred, which was characterized by a quick rise in IgM antibody followed by a slower production of IgG antibody, high maximum numbers of IEA+ cells, and a CMV syndrome in 11 patients. In 35 out of 54 (65%) seropositive patients a secondary infection occurred. After a post-operative fall in the IgG antibody, which was also found in patients without an active infection and which was accompanied by a similar drop in serum albumin and IgG, a second dip in IgG antibody was found 6 days before the first IEA+ leucocyte appeared in the blood. This was followed by a significant increase, indicative of an active immune response in consequence of the infection, 18 days later. In 31 of these 35 patients an IgM response was found. This could be ascribed to the presence of rheumatoid factor activity in 20 of them. Eight patients who showed a transient rise in IgG antibody between the two dips could be distinguished from the remaining ones by a lower maximum number of IEA+ cells and less severe disease symptoms. The described results suggest that (i) an adequate humoral immune system may prevent symptomatic CMV disease in secondary infections; and (ii) CMV-specific antibodies may be removed from the circulation by antigens present in infected tissues before CMV antigenaemia becomes detectable.     

Specific IgG and IgA antibodies to herpes simplex virus (HSV)-induced surface antigen in patients with HSV infections and in healthy adults

Herpes simplex virus (HSV)-specific IgG and IgA antibody response in patients with HSV infection and in healthy adults was studied by the immunoperoxidase antibody-membrane antigen (IPAMA) technique. In all HSV infections in which specific IgM antibodies were detected by enzyme-linked immunosorbent assay (ELISA), a significant rise in the titer of HSV IgG and IgA antibodies was found. In contrast, in patients with recurrent herpes labialis in which no specific HSV IgM antibodies were detected by ELISA, HSV IgG and IgA antibodies were not found to fluctuate significantly during the course of infection. A higher geometric mean titer (GMT) for HSV IgG and for IgA antibodies was found in seropositive individuals with a previous history of recurrent HSV than in seropositive individuals without a previous history of recurrent HSV infection. Nineteen of 26 HSV IgG seropositive healthy medical students without a previous history of recurrent HSV infection had HSV IgA antibodies to membrane antigen. The significance of this finding in understanding the mechanism of latency in healthy seropositive individuals without previous history of HSV recurrent infections is discussed.     

Solid-phase radioimmunoassay of human immunoglobulin M and immunoglobulin G antibodies against herpes simplex virus type 1 capsid, envelope, and excreted antigens.

A solid-phase radioimmunoassay developed in our laboratory for detection of human viral immunoglobulin M (IgM) and IgG antibodies was applied to demonstrate human class-specific antibody response against capsid, envelope, and excreted antigens of herpes simplex virus type 1. In primary infections, a clear IgM and IgG antibody response was found predominantly against the envelope components, whereas the IgM and IgG antibodies to the capsid antigen appeared more slowly. Increasing IgG antibody titers to the excreted antigen were also found in primary infections, though appearing more slowly than antibodies to the other subunit antigens. The antibody response against capsid and envelope antigens was not type specific, whereas in primary infections IgG class antibodies against the excreted antigen showed distinct type specificity. In recurrent infections, no significant level of IgM class antibodies was demonstrated, but in the patients with a severe secondary herpes simplex virus infection a definite IgM class antibody response was found against the envelope antigen. In addition, during severe secondary infections the antibody response against the excreted antigen was enhanced. The host IgG antibody response in recurrent infections was directed against the envelope and excreted antigens, whereas the level of the capsid antibodies was relatively stable.     

Comparison of immunoblotting with other serological methods and virus isolation for the early detection of primary cytomegalovirus infection in allograft recipients.

Sequential specimens from nine allograft recipients were examined by using a variety of methods to detect primary cytomegalovirus (CMV) infection as rapidly as possible posttransplantation. Sera were examined for immunoglobulin G (IgG) and IgM antibodies by immunoblotting, enzyme immunoassay, and immunofluorescence and also by complement fixation, latex agglutination, and an immunofluorescence test for antibody to CMV early antigen. Urine and occasionally blood, tissue, and other specimens were centrifuged onto cell cultures to enhance CMV infectivity. Eight of the nine patients showed laboratory evidence of primary CMV infection, and CMV was isolated from seven of the eight: in no case was virus isolated before seroconversion had become evident. However, serological tests differed in their abilities to detect antibody response to CMV infection in different patients; while immunoblotting, latex agglutination, and enzyme immunoassay for IgG antibodies generally detected seroconversion before complement fixation, this was not invariably the case. At present, optimal laboratory detection of CMV infections in these patients can be achieved only by a combination of serological methods and virus isolation.     

Frequency and specificity of varicella zoster virus IgM response

A direct ELISA was developed for determination of IgM antibody to varicella zoster virus (VZV). With this sensitive method VZV IgM antibodies were detected in all patients with a varicella and in 84% with a herpes zoster infection. All but one of 28 renal allograft recipients had previously had varicella. A primary infection was seen in the last patient, and reactivated infections in 11 of the others. The VZV IgM response seems to be specific since patients with a herpes simplex virus (HSV) infection and a heterotypic VZV IgG titer rise did not have detectable VZV IgM. An indirect enzyme-linked immunosorbent assay (ELISA) for detection of IgG antibodies has been used for serodiagnosis of VZV infections and to determine the immune status. After injection of zoster immune globulin, it was possible to measure passively transferred antibodies.     

Detection of virus-specific IgA antibodies in serum of kidney transplant patients with recurrent cytomegalovirus infection by enzymeimmuno and radioimmunoassay techniques.

The feasibility of using human cytomegalovirus (CMV)-specific IgA antibody determinations as a signal for early detection of recurrent CMV infections in eight renal transplant recipients was analyzed. Solid phase radioimmunoassay (RIA), enzyme-liked immunosorbent assay (ELISA) and immunoperoxidase assay (IPA) techniques were used for IgA antibody determinations. In parallel, IgG antibodies to CMV were studied by immunoperoxidase assay. A significant rise of CMV-specific IgG antibody titre was observed in all of these patients between 5 and 53 weeks post-transplantation. CMV-specific IgA antibody production was detected close to the time a rise in CMV IgG antibody was observed in seven out of eight patients studied by RIA and ELISA, and in six out of eight patients studied by IPA. In two patients specific CMV IgA antibodies were detected by all three methods before a significant rise of CMV IgG antibody titre was demonstrated. In these patients CMV IgA was detected by RIA earlier than by ELISA and IPA. The potential application of CMV-specific IgA antibody determination for early detection of recurrent CMV infection in renal transplant patients is discussed.     

Increased IgM and IgM immune complex-like material in the circulation of renal transplant recipients with primary cytomegalovirus infections.

Twelve of 60 consecutive adult recipients of cadaver kidney transplants had increased polyethyleneglycol (PEG) precipitable IgM immune complex-like material in their circulation in the first 4 months after transplantation. All 10 recipients with primary CMV and two of four with secondary CMV infections had significant elevations in PEG precipitable IgM that coincided with rises in their CMV antibody titres. Ultracentrifuge analysis demonstrated two peaks of PEG precipitable material with sedimentation rates of about 20S and 40S. Total IgM immunoglobulin levels also were increased in transplant recipients with CMV infections, but this was less specific and occurred in patients without CMV infections. The Clq binding assay, which is more sensitive for IgG than IgM containing complexes, was positive in only three of 10 patients with primary CMV and none of four with secondary CMV. Granular deposits of IgM, but not IgG, were detected in the glomeruli of six of seven transplants biopsied during CMV infection. The PEG-IgM assay was not influenced by rejection or prednisone therapy. Thus, transplant patients, who develop primary CMV infections, produce elevated levels of circulating IgM and IgM immune complex-like material. These findings may help to differentiate CMV infection from transplant rejection as well as to increase our understanding of the special pathogenic properties of CMV in transplant recipients.     

IgM antibody detection of ppUL80a and ppUL32 by immunoblotting: An early parameter for recurrent cytomegalovirus infection in renal transplant recipients

The value of IgM detection for the early diagnosis of an active cytomegalovirus (CMV) infection in renal transplant recipients was evaluated prospectively. Sequential serum samples obtained from 22 allograft recipients with active CMV infection were tested for the presence of CMV-specific immunoglobulin M antibodies (IgM) by an enzyme-linked immunosorbent assay (ELISA) and a microparticle enzyme immunoassay (MEIA) and were compared with the Western-immunoblotting technique (IB). The time course of CMV IgM antibody detection was evaluated in relation to the shell vial assay (SVA), CMV disease, and immunosuppressive regimen. By IB, IgM antibodies against the capsid protein ppUL80a and the basic matrix phosphoprotein ppUL32 were detected in all 22 recipients with active CMV infection. Using the MEIA and the ELISA, the presence of CMV IgM antibodies was detected in 17 (77%) and ten (46%) of these 22 recipients, respectively. The SVA was the earliest parameter for detection of primary CMV infection in seven of nine (78%) recipients, in contrast to two of 13 (15%) patients with recurrent CMV infection (P < .05). The detection of IgM antibodies by IB was the earliest parameter for detection of recurrent CMV infection in seven out of 13 (54%) recipients in contrast to one out of nine (11%) patients with primary CMV infection (P < .05). During a primary CMV infection, the development of an abundant IgM antibody response was associated with recovery from CMV disease and the end of the viremic phase. © 1996 Wiley-Liss, Inc.     

Antibodies to cytomegalovirus in renal allograft recipients: correlation with isolation of virus.

A cohort of 47 renal transplant recipients was studied prospectively for up to one year after transplantation. Cytomegalovirus (CMV) was isolated from 21 of the patients. The first time the virus was isolated seven patients were IgM positive, nine showed a significant rise in IgG titres, and 12 had a four-fold or greater rise in complement fixation titre. There was no significant difference in the time at which virus was first detected following transplantation between patients with primary CMV infection and those with reinfection or recurrent infection. In general, patients with primary infection shed virus consistently over long periods. Those with reinfection or recurrent infection shed virus intermittently or not at all. There were considerable differences between individual patients in the timing and pattern of the immune response. Taken overall, a four-fold rise detected by the complement fixation test correlated best with the onset of CMV shedding in primary infection. There was more variation in the pattern of antibody response in cases of reinfection or recurrent infection, with no single serological test correlating better than the others. It is concluded that serology is of limited value in the detection of active CMV infection after renal transplantation.     

Postsplenectomy cytomegaloviral mononucleosis: marked lymphocytosis, TCRgamma gene rearrangements, and impaired IgM response

People who have undergone splenectomy mount a poor IgM response to bacterial polysaccharide vaccines. Whether this defect is true during natural bacterial and viral infections is unknown. We present 2 cases of postsplenectomy cytomegalovirus (CMV)-induced mononucleosis with impaired IgM but normal to augmented IgG response. The cases presented initial diagnostic challenges owing to a prolonged course of infection, marked lymphocytosis (peak lymphocyte count, 27,900/microL [27.9 10(9)/L]), clonal T-cell proliferation with T-cell receptor g gene rearrangements, and remote history of splenectomy. However, the acute nature of the infections, serial determinations of the anti-CMV IgM and IgG, exclusion of other causes, and detection of CMV in the blood established the diagnosis and revealed the deranged antibody response. The infections resolved without specific treatment. These cases suggest that the spleen might be a primary site for specific anti-CMV IgM response.     

Specific IgM class antibody production following infection with cytomegalovirus

Specific IgM class antibody production was studied in different groups of patients with characterized cytomegalovirus (CMV) infections using a radioimmunoassay (RIA). In pregnant women, IgM antibodies were detected only following primary infection and generally persisted less than 4 months. The demonstration of CMV-specific IgM during pregnancy is therefore diagnostic of recent primary CMV infection. In patients with symptomatic CMV infections, the appearance of IgM antibody was shown to be closely related to the onset of symptoms and coincided with production of complement fixing (CF) antibody. IgM antibodies were at maximum levels 3-4 weeks after presentation but generally declined to low or undetectable levels by 3-4 months. The significance of the results of testing for CMV-specific IgM in relation to clinical and other serological findings in these patients is discussed. IgM antibody production was also demonstrated in renal transplant patients with primary infections and in 6 of 21 recipients with secondary infections. In both groups the antibodies became detectable 3-6 weeks after transplantation but the titres were much higher following primary infection. IgM antibodies persisted throughout follow-up periods of up to 2 years after transplantation in some cases.     

A serological investigation of human herpesvirus 6 infections in liver transplant recipients and the detection of cross-reacting antibodies to cytomegalovirus.

Sera from 50 orthotopic liver transplant recipients were examined for antibodies to human herpesvirus 6 (HHV-6) and cytomegalovirus (CMV), and the findings correlated with the clinical condition of the patients. Both primary and secondary HHV-6 infections were detected serologically following liver transplantation. Interpretation of serological assays is complicated by CMV and HHV-6 antibody cross reactions which were common. Sera from 5 patients became HHV-6 antibody negative following absorption with CMV infected cells. Thirty patients were initially seronegative for HHV-6 antibodies, 12 remained so following transplantation, 5 developed cross reacting antibodies, and 13 seroconverted. The seroconversions occurred at 4 to 8 weeks post-transplant in the same time period as CMV antibody rises. HHV-6 IgM was detected in only 4 of the 13. Of the 7 patients who had serological evidence of active HHV-6 infections but no evidence of CMV infection, 4 (56%) had fever, 1 (14%) hepatitis, 1 (14%) lung dysfunction, and 3 (42%) neurological disorders. In the 12 patients who remained HHV-6 antibody negative, there were fewer fevers and neurological disorders.     

Evidence for transfer of cellular and humoral immunity to cytomegalovirus from donor to recipient in allogeneic bone marrow transplantation.

In order to study the importance of the immune status of the donor in the development of immunity after allogeneic bone marrow transplantation (BMT), we monitored 23 cytomegalovirus (CMV) antibody-positive BMT recipients for humoral and cellular immunity to CMV, of whom 12 had a CMV antibody-positive and 11 a CMV antibody-negative marrow donor. Lymphocyte proliferation to CMV recovered significantly earlier after BMT in recipients of marrow from a CMV+ donor (10.4 weeks after BMT) compared with the recipients of marrow from CMV- donors (16.7 weeks after BMT, P less than 0.05). This seemed to be specific, as lymphocyte proliferation to phytohaemagglutinin and Candida were not different between the to groups. IgM responses after active infection were seen in both groups, but initial IgG rises without IgM were seen only in recipients of marrow from CMV+ donors (P less than 0.05). Lymphocyte proliferative or humoral immune responses to CMV were not detected in any of the patients in a control group consisting of nine CMV- recipients. These results indicate that T cell memory to CMV is transferred with donor marrow from CMV+ donors, leading in most patients to direct IgG anti-CMV responses and to quicker recovery of cellular immunity to CMV.     

Immunohistological detection of human herpes virus 6 in formalin-fixed,paraffin-embedded lung tissues

Human herpes virus type 6 (HHV-6) infection is widespread in healthy individuals. The only definite disease association is with exanthem subitum in infants though the virus has been linked with a variety of other diseases including interstitial pneumonitis in bone marrow allograft recipients. In order to investigate the role of HHV-6 in the latter disease we have developed an optimised staining method for the demonstration of specific antigen in routinely processed post-mortem tissues. Formalin-fixed, paraffin-embedded lung tissue from 8 immunocompromised patients who died from interstitial pneumonitis was subjected to immunoperoxidase staining with monoclonal antibodies against HHV-6, cytomegalovirus (CMV), and adenovirus, using a modified avidin-biotin complex (ABC) method. Staining for HHV-6 was obtained in 6 of the 8 patients studied and was present in pneumocytes and macrophages. CMV and adenovirus antigens were identified in 4 and 6 patients, respectively. Whilst the lung tissue of 6 patients contained more than one virus, there was no evidence of cross-reactivity between the monoclonal antibodies. We demonstrated that accurate localisation of HHV-6 using monoclonal antibodies is possible in post-mortem lung tissue and conclude that either HHV-6 alone or in combination with other viruses may play a role in the development of interstitial pneumonitis following bone marrow transplantation or chemotherapy.