Infrequent detection of cytomegalovirus and Epstein-Barr virus DNA in synovial membrane of patients with rheumatoid arthritis

OBJECTIVE: To study the role of the cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus types 1 and 2 (HSV-1 and 2), varicella zoster virus (VZV), and human herpes virus 6 (HHV-6) in the etiology of rheumatoid arthritis (RA). METHODS: Polymerase chain reaction (PCR) was used to detect DNA of the different herpes viruses in synovial membranes from 31 patients with chronic RA and 14 control patients. Specific antibodies were determined by indirect immunofluorescence and ELISA. RESULTS: Out of 31 patients with RA, CMV DNA was detected in synovial membranes from 2 patients and EBV DNA was detected in synovial membranes from 2 other patients. All samples from the patients with RA were negative for DNA from HSV-1 and 2, VZV, and HHV-6. All samples from the 14 control patients were negative in all PCR assays. No statistically significant differences in IgG antibodies were found for CMV, HSV-1, VZV, and HHV-6 in patients with RA compared to controls. Higher titers of IgG antibodies against EBV viral capsid antigen were found in patients with RA, with a significance of p < 0.05. CONCLUSION: Both CMV and EBV DNA were detected in synovial membranes from 6% of the patients with RA. We cannot exclude the possibility that these viruses were associated with disease development in a minority of patients with RA.     

Diagnosis of Pneumocystis carinii pneumonia in HIV-positive patients. Bronchoalveolar lavage vs. bronchial brushing

OBJECTIVE: To evaluate the contribution of bronchoalveolar lavage (BAL) and bronchial brushing (BB) and the use of different tinctorial stains in the detection of Pneumocystis carinii (PC) in human immunodeficiency virus (HIV)-positive patients. STUDY DESIGN: In a retrospective study, 195 HIV-positive patients suspected of a pulmonary infection underwent bronchoscopy with BAL. In 143 cases subsequent BB was performed. On 135 BAL fluid cytocentrifuge preparations four staining techniques were applied simultaneously: May-Grünwald-Giemsa (MGG), toluidine blue-O (TOL), Papani-colaou (PAP) and Grocott methenamine silver (GRO). RESULTS: PC was recovered in 79 (40.5%) cases. The yields of MGG and TOL were identical (33.3%). PAP and GRO showed lower results, 31.1% and 29.6%, respectively. These differences were not statistically significant. The combination of BAL and BB revealed 64 cases of PC infection. BAL was positive in the vast majority of cases (63, 44.1%). BB was positive in 54 (37.8%). The combination of positive BB with negative BAL was present in one case. However, 10 cases of PC were found with the use of BAL and not detected by BB (P < .01). CONCLUSION: The results of this study indicate that to confirm a PC infection in HIV-positive patients, the use of bronchoalveolar lavage with a single staining technique is appropriate. Bronchial brushing seems to be of limited additional value.     

Comparison of bronchoalveolar lavage and catheter lavage to confirm ventilator-associated lower respiratory tract infection

Lower respiratory tract infection (LRTI) is a well recognised complication of artificial ventilation in intensive care units (ICU). Ideally, specimens for microbiological analysis should be obtained during bronchoscopy, but this is not always possible. Therefore, the microbiological diagnosis of lower respiratory tract infection by broncho-alveolar lavage (BAL) obtained during bronchoscopy was compared with catheter lavage (CL) with a balloon-tipped catheter. Adult patients with clinical evidence of lower respiratory tract infection in an adult ICU were randomly assigned to undergo BAL followed by CL or vice versa. Forty ml of normal saline 0.9% were instilled and then aspirated with a flexible bronchoscope to obtain BAL. A similar volume was instilled and aspirated with a 12-gauge Foley balloon-tipped catheter to obtain a CL sample. The number of inflammatory cells, epithelial cells and organisms seen by microscopy were quantified. Culture results were semi-quantified and classified as negative, positive, equivocal or contaminated. Seventy-nine paired specimens were obtained from 66 patients, including specimens from 10 patients taken on two or more occasions. Only 20% of BAL and 16% of CL had one or more epithelial cells and bacteria were seen in 26 BAL and 21 CL specimens, respectively; 35% of BAL and CL specimens were positive and there was a discrepancy in the culture result in only two cases. Staphylococcus aureus was the pathogen isolated most frequently and polymicrobial lower respiratory infection was diagnosed on 10 occasions (15%). CL fluid is as reliable as BAL in diagnosing lower respiratory tract infection in ICU. This approach does not require bronchoscopic expertise and utilises convenient laboratory techniques.     

Seroconversion to human herpesvirus 6 following liver transplantation is a marker of cytomegalovirus disease

Human herpesvirus 6 (HHV-6) infection is common after transplantation; HHV-6 is known to interact with other viruses and induce immunosuppression. Whether HHV-6 plays a role in the occurrence of cytomegalovirus (CMV) infection after transplantation was investigated. In a cohort of 247 liver transplant recipients, HHV-6 seroconversion was identified as a significant risk factor for development of symptomatic CMV infection (P < .001), including CMV organ involvement (P < .001), even in the presence of the other significant risk factors: D+/R- CMV serologic status (P < .001) or use of OKT3 after transplantation (P = .002). Subgroup analysis indicated that HHV-6 seroconversion was significantly associated with symptomatic CMV infection in the D+/R+ but not in the D+/R- CMV serologic group (P < .001 and P = .11, respectively). These results indicate that HHV-6 seroconversion is a marker for CMV disease after transplantation and suggest that additional studies using more sensitive diagnostic techniques are warranted to determine the relationship between HHV-6 and CMV infection after transplantation.     

Prospective study of human herpesvirus 6, human herpesvirus 7, and cytomegalovirus infections in human immunodeficiency virus-positive patients

Blood samples from human immunodeficiency virus (HIV)-positive patients were monitored for cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), and HHV-7 by PCR. We detected CMV in 17% of the patients, HHV-6 in 6%, and HHV-7 in 3%. The viral loads of CMV were significantly higher than those of HHV-6 (P = 0.007) or HHV-7 (P = 0.01). Detection of CMV and HHV-6 was associated with low and high CD4 counts, respectively.     

Polymerase chain reaction on cerebrospinal fluid for diagnosis of virus-associated opportunistic diseases of the central nervous system in HIV-infected patients

OBJECTIVE: To assess the diagnostic reliability of polymerase chain reaction (PCR) on cerebrospinal fluid (CSF) for virus-associated opportunistic diseases of the central nervous system (CNS) in HIV-infected patients. DESIGN: CSF samples from 500 patients with HIV infection and CNS symptoms were examined by PCR. In 219 patients the PCR results were compared with CNS histological findings. METHODS: Nested PCR for detection of herpes simplex virus (HSV) type 1 or 2, varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), and JC virus (JCV) DNA. Histopathological examination of CNS tissue obtained at autopsy or on brain biopsy. RESULTS: DNA of one or more viruses was found in CSF in 181 out of 500 patients (36%; HSV-1 2%, HSV-2 1%, VZV 3%, CMV 16%, EBV 12%, HHV-6 2%, and JCV 9%). Among the 219 patients with histological CNS examination, HSV-1 or 2 was detected in CSF in all six patients (100%) with HSV infection of the CNS, CMV in 37 out of 45 (82%) with CMV infection of the CNS, EBV in 35 out of 36 (97%) with primary CNS lymphoma, JCV in 28 out of 39 (72%) with progressive multifocal leukoencephalopathy. Furthermore, HSV-1 was found in one, VZV in four, CMV in three, EBV in three, HHV-6 in seven, and JCV in one patient without histological evidence of the corresponding CNS disease. CONCLUSIONS: CSF PCR has great relevance for diagnosis of virus-related opportunistic CNS diseases in HIV-infected patients as demonstrated by its high sensitivity, specificity, and the frequency of positive findings.     

Lack of clinical utility of bronchoalveolar lavage cultures for cytomegalovirus in HIV infection

This study assessed the presence of cytomegalovirus (CMV) in bronchoalveolar lavage (BAL) in three subpopulations of HIV-infected patients and correlated its presence with clinical status during 3 mo of follow-up. Nineteen asymptomatic volunteers, six patients with CMV retinitis, and 46 patients with acute pulmonary symptoms underwent BAL and were assessed for CMV by cytopathology, conventional shell vial cultures, and antigen detection. Transbronchial biopsies were also obtained when possible and evaluated for histopathologic changes of CMV. All patients were followed for approximately 3 mo. Cytomegalovirus was detected in BAL in nine of 19 (47%) asymptomatic volunteers, in all six patients with CMV retinitis, and in 33 of 46 (72%) patients with pulmonary symptoms. Only one symptomatic patient with a positive CMV BAL culture developed clinically significant CMV pulmonary disease; this patient developed disseminated CMV and died. The only other death occurred in a patient with CMV retinitis who developed staphylococcal bacteremia. None of the asymptomatic volunteers or patients with CMV retinitis developed evidence of CMV pneumonia or any other organ disease with CMV. Cytomegalovirus is frequently detected in BAL from HIV-infected patients regardless of their pulmonary symptoms and its presence does not clinically predict significant pulmonary morbidity or mortality in 3 mo of follow-up.     

Human herpesvirus-6 antibodies in idiopathic facial nerve palsy and sudden deafness

Human herpesvirus-6 (HHV-6) is the causative agent for exanthem subitum. This study investigated the relationship between idiopathic facial nerve palsy (Bell's palsy), sudden deafness and HHV-6 infection. Both Bell's palsy and sudden deafness are syndromes which causes are unknown. Both of them are suspected viral infection as causative agents. Paired sera from 22 patients of Bell's palsy and 39 patients of sudden deafness were examined for reactivity to HHV-6 by the indirect immunofluorescence test. On a case of Bell's palsy and two cases of sudden deafness each of the HHV-6 antibody titers was increased.     

Human herpesvirus 6 reactivation is associated with cytomegalovirus infection and syndromes in kidney transplant recipients at risk for primary cytomegalovirus infection

A potential association between human herpesvirus 6 (HHV-6) and cytomegalovirus (CMV) following kidney transplantation was explored by retrospectively testing serial serum specimens for HHV-6 IgG and IgM antibody. HHV-6 reactivation occurred in 35 (66%) of 53 transplant recipients. Fungal or parasitic opportunistic infections, graft rejection or loss, and mortality were not associated with HHV-6 reactivation. HHV-6 reactivation was associated with primary CMV infection (P=.001) and CMV syndrome (P=.003) and with trends for CMV-related hepatitis (P=.095), CMV-related neutropenia (P=.104), and serious CMV disease (P=.085). After controlling for CMV immune globulin (CMVIG) prophylaxis, the association between HHV-6 reactivation and primary CMV infection and syndrome remained significant (P=.002 and 0.006, respectively). The reduction in CMV syndrome among those receiving CMVIG prophylaxis remained significant (P=.007) after controlling for HHV-6 reactivation. HHV-6 reactivation in kidney transplant recipients at risk for primary CMV infection is associated with CMV infection and CMV-related disease, and these effects are independent of CMVIG prophylaxis.     

Value of CT angiography for postoperative assessment of patients with iliac artery aneurysms who have received endovascular grafts

OBJECTIVE: To determine the pathologic outcome in human immunodeficiency virus (HIV)-seropositive individuals with nonspecific bronchoalveolar lavage (BAL) cytology. STUDY DESIGN: The study group consisted of 126 cytologically negative or nonspecific BAL specimens from HIV-seropositive adults. Concurrent microbial cultures and transbronchial biopsies, as well as subsequent pulmonary cytology, lung biopsy or autopsy results were reviewed. Additionally, the cytologic morphology of specimens from patients found to have a potential bacterial pathogen was reviewed. RESULTS: In the 126 cases with nonspecific BAL cytology, a potential pulmonary pathogen was identified from a concurrent or subsequent pathologic specimen in 27% of cases, while no pathogen was identified in 73% of cases. Bacteria and fungi were the most common pathogens identified. Microbial cultures alone identified the pathogen in 59% of cases, while transbronchial biopsy added information in only 9%. Specimens with marked acute inflammation often yielded bacterial pathogens on microbial culture. CONCLUSION: A potential pulmonary pathogen can be identified in 27% of HIV-seropositive individuals with negative BAL cytology using other diagnostic modalities. Bacterial pathogens are most common and are usually identified by microbial culture. Marked acute inflammation in a BAL specimen is often associated with bacterial pneumonia.     

Epidemiology of encephalitis in children. A prospective multicentre study

We found 175 cases with acute encephalitis in a population of 791,712 children aged 1 month-15 years during a 2-year surveillance period in 1993-1994. The overall incidence was 10.5/100,000 child-years with the highest figure in children < 1 year of age, 18.4/100,000 child-years. The microbial diagnosis was considered proven or suggested in 110 cases (63%); varicella zoster, respiratory and enteroviruses comprised 61% of these, and adeno, Epstein Barr-, herpes simplex and rota viruses comprised 5% each. A clearcut change seems to have occurred in the aetiology of encephalitis. Mumps, measles, and rubella virus associated encephalitides have been almost eliminated. Varicella zoster, respiratory, and enteroviruses have increased in frequency and occur in younger age groups. New causes were identified, especially Chlamydia pneumoniae and HHV-6. Our data should assist in making a specific diagnosis and defining appropriate antimicrobial therapy. CONCLUSIONS: The spectrum of encephalitis in children has changed due to vaccination programs. The incidence, however, appears to be about the same due to increasing frequency of other associated old and new microbes.     

Rapid cytomegalovirus pp65 antigenemia assay by direct erythrocyte lysis and immunofluorescence staining

A rapid cytomegalovirus (CMV) pp65 antigenemia assay with direct erythrocyte lysis (DL) with 0.8% NH4Cl, followed by indirect immunofluorescence staining (IF), was evaluated with 82 blood samples from renal transplant recipients, and the results were compared to those of the conventional antigenemia assay with dextran sedimentation and two-cycle alkaline phosphatase, anti-alkaline phosphatase staining (DS-APAAP). The DL-IF modification gave a higher leukocyte yield compared to DS-APAAP (75.4 versus 54.9%; P < 0.05), with similar leukocyte viability rates of >95%. The DL-IF methodology involved fewer technical steps, and the assay time was shortened from 5 h to less than 3 h. Nineteen of the 82 samples concordantly tested positive for pp65 antigenemia by both assays, and the readings showed a good correlation (r = 0.996; P < 0.01). No discordant results were observed. We conclude that the CMV pp65 antigenemia assay by this novel DL-IF modification is technically simpler, cheaper, and less time-consuming but yields results comparable to those of the conventional DS-APAAP assay. The shortened assay time and increased capacity to handle more samples confer distinct advantages in the rapid diagnosis and prompt treatment of CMV disease in immunosuppressed patients.     

Functionally relevant changes occur in HIV-infected individuals' alveolar macrophages prior to the onset of respiratory disease

OBJECTIVE: We have compared the phenotypic and functional changes found in alveolar macrophages recovered from the lungs of 39 HIV-positive individuals with no respiratory disease with those from 33 HIV-positive individuals with pneumonitis and 31 healthy controls. METHODS: Bronchoalveolar lavage (BAL) cell cytospin preparations were stained using monoclonal antibody immunoperoxidase and double immunofluorescence techniques. Cytokine levels within supernatant BAL were determined using enzyme immunoassay. RESULTS: There were marked differences in alveolar macrophage phenotype between the three groups. In particular, the relative proportion of cells staining RFD1+RFD7- (inducer cells) was reduced in the HIV-positive individuals without respiratory disease. This was correlated with measures of declining systemic immunity. Patients with pneumonitis had the highest levels of measured cytokines [interleukin-1 beta, tumour necrosis factor-alpha and transforming growth factor (TGF)-beta 2], followed by the HIV-positive individuals without respiratory disease. In this latter population a negative correlation was found between active (non acid dissociated) TGF-beta 2 and blood CD4 cell count. CONCLUSIONS: The differences between the three groups suggest that alterations of potential relevance to the pulmonary immune response are occurring in alveolar macrophages prior to the onset of respiratory disease. This study confirms the importance of investigating asymptomatic HIV-positive individuals.     

Usefulness of bronchoalveolar lavage in the renal transplant patient with suspected respiratory infection

In this retrospective study we aimed to assess the diagnostic yield of bronchoalveolar lavage (BAL) in kidney transplant patients who were suspected of having severe respiratory infection or in whom empirical antibiotic treatment had failed. All BAL procedures performed on kidney transplanted patients suspected of having respiratory infections between January 1, 1988 and July 31, 1996 were analyzed. BAL was carried out in the standard way and samples were sent for cytologic and bacteriologic study. Thirty-three patients with a mean age of 48.5 years were enrolled. All had been receiving immunosuppressive treatment and the mean time following transplantation was 320 days. Thirty-one had received antibiotic treatment before BAL. BAL was positive for 21 of the 33 patients (64%). Twenty-two pathogens were identified: 6 Pneumocystis carinii, 4 Cytomegalovirus, 3 Mycobacterium tuberculosis, 2 Aspergillus fumigatus, 2 Herpes simplex type I, 1 Streptococcus pneumoniae, 1 Staphylococcus aureus, 1 Streptococcus mitis, 1 Legionella pneumophila, 1 Legionella longbeachae. BAL was negative for 12 patients, of whom 8 were tentatively diagnosed of bacterial infection, 3 of acute pulmonary edema and one of pulmonary infarction. Based on the results, therapy was changed for 20 patients (61%), 19 (58%) because an unsuspected pathogen was identified and 1 because treatment could be simplified. The diagnostic yield of BAL is high (64%) in kidney transplant patients suspected of respiratory infection and is useful for managing such cases, as evidenced by the fact that a high proportion (19/33) of our patients were infected by pathogens not covered by empirical treatment.     

Cytomegalovirus and human herpesvirus-6 trans-activate the HIV-1 long terminal repeat via multiple response regions in human fetal astrocytes

Cytomegalovirus (CMV) and human herpesvirus-6 (HHV-6) infection stimulated HIV-1 replication and trans-activated the HIV-1 promoter (the long terminal repeat or LTR) to a similar extent in transfected, nonimmortalized, human fetal astrocytes. CMV infection increased basal LTR expression by approximately sevenfold, while HHV-6 infection increased basal LTR expression by fourfold. This enhancing effect required cell-cell contact between CMV-infected or HHV-6-infected and LTR-containing cells. To determine the target regions on the HIV promoter that respond to CMV and HHV-6 trans-activation, several modified LTR-reporter gene constructs were tested. Loss of functional NFkappaB, Sp1, or upstream modulatory sites on the LTR caused significant reduction ofbasal LTR expression in astrocytes. These elements also mediated the trans-activation events during HHV-6 or CMV infection in astrocytes, though to varying degrees. Electrophoretic mobility shift assays (EMSA) indicated that core, enhancer, and upstream modulatory regions of the LTR interacted specifically with nuclear proteins from both uninfected and CMV- or HHV-6-infected human fetal astrocytes. CMV or HHV-6 infection did not appear to induce unique, LTR-specific nuclear binding proteins, but rather enhanced the relative proportion of some of the existing protein complexes, in particular, the complexes formed with the AP-1 binding sites on the HIV-1 LTR (nt - 354 to - 316). Our data suggest that CMV or HHV-6 trans-activation of HIV LTR activity in human fetal astrocytes proceeds via intracellular molecular interactions involving herpesviral gene products, cellular proteins, and multiple sites on the LTR upstream of the TATA box. The pattern of LTR activity in astrocytes suggests that host cell factors modulating HIV expression may differ from those dominant in T-cells or immortalized astroglia, and this could contribute to differences in the astrocyte's ability to support HIV replication.     

Distribution of desmin and fibronectin in chick embryo gonad during testicular cord formation

In order to compare PCR with rapid virus culture for the early detection of CMV in bronchoalveolar lavage (BAL) after bone marrow transplantation, 26 asymptomatic patients were routinely evaluated for the presence of CMV on day 35 using these two techniques. Concurrent blood samples were also analyzed in all cases. CMV was detected synchronously by both culture and PCR in six of 26 (23%) BAL and in five of 26 (19%) blood specimens. Among these positive specimens, three BAL and blood samples were positive in the same patients. Five (19%) BAL and five (19%) blood samples were culture-negative but PCR-positive. No BAL or blood specimens were positive by culture alone. When considering matched BAL-blood samples, five were positive in only one fluid, BAL (n = 3) or blood (n = 2) using culture, while seven were positive in only one fluid, BAL (n = 4) or blood (in = 3) using PCR. Overall, six of 26 (23%) patients had culture-negative but PCR-positive results. Three of these six patients were positive only in BAL and two of them subsequently received antiviral therapy for development of symptoms suggestive of CMV infection. We suggest that asymptomatic patients with negative-culture but PCR-positive results on day 35 in BAL should be subsequently closely monitored for the presence of CMV.     

Small volume bronchoalveolar lavage used in diagnosing Pneumocystis carinii pneumonia in HIV-infected patients

To determine the volume of bronchoalveolar lavage (BAL) fluid necessary to diagnose Pneumocystis carinii pneumonia (PCP) in immunocompromised patients, specimens from 25 patients were evaluated. Twenty-one patients were HIV infected. BAL was performed using three to four 60-mL aliquots of room temperature, sterile, saline solution. Each syringe of BAL effluent was numbered and its volume was measured. Immunofluorescent stains were performed on about 8-mL aliquots of the initial, final, and aggregate BAL specimens, and a modified Giemsa stain was also performed on a 0.4-mL aliquot of the aggregate specimen. Of 25 patients, Pneumocystis carinii organisms were identified in 9 with HIV infection, in whom all BAL specimens were positive with both immunofluorescence and Giemsa stains. In 16 patients, BAL specimens were negative for P carinii on both immunofluorescent and modified Giemsa testing. Both staining methods were 100% specific (95% confidence interval [CI], 83 to 100%) and 100% sensitive (95% CI, 72 to 100%). The volume of BAL effluent in the initial specimens positive for P carinii ranged from 15 to 25 mL. We conclude that in this small group of patients, PCP was accurately diagnosed from a single 60-mL BAL specimen stained with immunofluorescence methods.     

Human herpesvirus-6 (HHV-6)-associated necrotizing encephalitis in Griscelli's syndrome

We report a male caucasian German pediatric patient of no Arab or Mediterranean ancestry with virus associated CNS lesions in Griscelli's syndrome (GS; McKusick No. 214450). The boy presented with recurrent infections, and meningitis with subsequent progressive signs of increased intracranial pressure leading to death at 32 weeks of age. At autopsy, various sites of the CNS revealed necroses in gray and white matter. CNS histology revealed numerous and massive predominantly perivascular CD8 positive lymphohistiocytic infiltrates. These findings were associated strictly with the presence of human herpesvirus-6 (HHV-6) genome or the HHV-6 specific late antigen H-AR 3, found in neurons, oligodendrocytes, and astrocytes. The search for HHV-6 replication dependent antigen, HHV-7 DNA, CMV, adenovirus, Coxsackie B1, B2, and B4-antigens, and mycobacteria was not successful. Detection of viruses was attempted using immunohistochemistry, in situ hybridization or nested polymerase chain reaction, respectively. Lymphocyte typing was carried out immunohistochemically. In GS, virus induced CNS damage does not seem to require necessarily active virus replication. It may also appear as a consequence of an immune reaction triggered by antigen expression.     

In vitro activation of human herpesviruses 6 and 7 from latency

Human herpesviruses 6 and 7 (HHV-6 and HHV-7) are prevalent lymphotropic viruses that infect more than 80% of children at infancy or during early childhood. Infection ranges from asymptomatic to severe disease. HHV-6B causes exanthem subitum. The virus can be recovered from peripheral blood mononuclear cells during the acute phase of exanthem subitum, but the host remains latently infected throughout life. In immunocompromised patients undergoing kidney, liver, or bone marrow transplantation latent HHV-6B is reactivated, at times causing severe or fatal disease. Here, we describe the establishment of an in vitro system for reactivation of HHV-6B and HHV-7 from latency. HHV-7 is reactivated from latently infected peripheral blood mononuclear cells by T-cell activation. HHV-6B could not be reactivated under similar conditions; however, the latent HHV-6B could be recovered after the cells were infected with HHV-7. Once reactivated, the HHV-6B genomes became prominent and the HHV-7 disappeared. We conclude that HHV-7 can provide a transacting function(s) mediating HHV-6 reactivating from latency. Understanding the activation process is critical for the development of treatments to control the activation of latent viruses so as to avoid these sometimes life threatening infections in transplant recipients.