Relationship between cytomegalovirus DNA load in epithelial lining fluid and plasma of lung transplant recipients and analysis of coinfection with Epstein-Barr virus and human herpesvirus 6 in the lung compartment.

Cytomegalovirus (CMV) is a significant cause of morbidity and mortality in lung transplant recipients (LTRs). The aim of the present study was to elucidate the relationship between the CMV DNA load in the lung compartment and that in plasma. For CMV load determination, the level of CMV DNA in plasma and bronchoalveolar lavage (BAL) samples was measured in a total of 97 paired BAL and plasma samples obtained from 25 LTRs. The original virus concentration in the epithelial lining fluid (ELF) was calculated from the BAL samples by correcting for dilution using the urea dilution method. In addition, the load of Epstein-Barr virus (EBV) and that of human herpesvirus 6 (HHV-6) DNA also were determined in BAL samples, recalculated for their concentrations in the ELF, and compared with the CMV DNA load. CMV DNA was found more frequently and at significantly higher levels in the lung compartment than in plasma (P<0.001, Wilcoxon test), and the CMV load in the ELF was associated with symptomatic CMV disease. EBV and HHV-6 were detected in 43.6% and 21.7% of the ELF samples, respectively. A statistically significant association was found between the CMV and EBV DNA loads in the ELF (P<0.001; Spearman's rho=0.651). Thus, in LTRs, determination of the CMV DNA load in the lung compartment may be advantageous compared to monitoring only viremia. The significant relationship between EBV and CMV DNA loads in the ELF of LTRs and its clinical impact require further investigation.

Detection of Mycoplasma pneumoniae in serum specimens from patients with mycoplasma pneumonia by PCR.

There are few data on detection of Mycoplasma pneumoniae from blood, serum or plasma, and systematic studies on this diagnostic approach in community-acquired pneumonia (CAP) are scarce. Compared to testing respiratory specimens, this approach has the advantages that it is less dependent on proper specimen collection, serum is easily stored and handled, and the pathogen is detected in a primary sterile site, where colonization can be ruled out. In this study, acute-phase serum specimens from 29 patients of Vienna University Hospital (treated between 11/1994 and 6/2004; female: 14, male: 15; median age: 31 years, range: 15-66 years) with CAP and serologically verified M. pneumoniae infection, who had not received anti-mycoplasma therapy prior to serum collection, were tested for M. pneumoniae by conventional PCR and real-time PCR. Conventional PCR yielded negative results for all specimens, but real-time PCR detected M. pneumoniae in 15/29 patient sera (52%). These findings indicate that M. pneumoniae is present in the bloodstream of a substantial proportion of patients with mycoplasma pneumonia. Despite the possible adherence of M. pneumoniae to human erythrocytes, the pathogen can be detected from serum, if a method with enhanced sensitivity is applied. However, the negative predictive value of PCR from serum with regard to etiological diagnosis is low. With regard to the potential clinical benefit of blood-based PCR diagnosis of mycoplasma pneumonia the diagnostic accuracy of this approach using either serum or whole-blood specimens should be addressed by large-scale studies.

Low proportion of recent human immunodeficiency virus (HIV) infections among newly diagnosed cases of HIV infection as shown by the presence of HIV-specific antibodies of low avidity.

The time between human immunodeficiency virus (HIV) infection and diagnosis is mostly unknown. Two hundred five newly diagnosed patients were investigated for the duration of their HIV infection by avidity testing. Recent HIV infection was identified in 27.3% of the cases. Early diagnosis was achieved significantly less frequently in heterosexually infected persons than in other patients.

Serum Epstein-Barr virus DNA load in primary Epstein-Barr virus infection.

Specific viral laboratory diagnosis of primary Epstein-Barr Virus (EBV) infection is usually based on antibody-detection assays. During acute, lytic phase of infection, viral DNA can also be detected in serum. In the present study, the diagnostic utility of EBV DNA detection and quantitation in serum in primary EBV infection was investigated. The level of EBV DNA in the serum of 98 immunocompetent patients aged 1-47 years with symptomatic, antibody-confirmed EBV primary infection was assessed using a quantitative real-time PCR assay. The association between viral load and time after onset of disease, age and clinical and laboratory data was investigated. Quantitative PCR detected EBV DNA in 93 of 98 samples (94.9%), and the measured viral loads ranged from 3.8 x 10(1) to 6.6 x 10(4) copies/ml. EBV DNA detection exhibited a sensitivity of 94.9% and a specificity of 97.4% for primary EBV infection. EBV DNA was always detectable until day 12 after onset of symptoms, whereas no further positive PCR results were found after a period of 22 days after onset of disease. Detection of EBV DNA also showed a clearer association with the clinical manifestation of disease than the presence of EBV specific VCA IgG antibodies of low avidity. EBV DNA load was found to correlate inversely with the time after onset of disease (P < 0.001), and higher viral load levels were detected in younger (P = 0.009) and in hospitalized patients (P = 0.038). The results indicate that real-time PCR is a reliable tool for diagnosis of primary EBV infection early in the course of disease. In addition, EBV DNA detection may serve as a useful diagnostic supplement in serologically indeterminate EBV infections.

Association of cytomegalovirus DNA concentration in epithelial lining fluid and symptomatic cytomegalovirus infection in lung transplant recipients.

BACKGROUND: The authors have investigated the effectiveness of virus detection from bronchoalveolar lavage (BAL) samples for the identification of symptomatic cytomegalovirus (CMV) infection in lung transplant recipients. METHODS: Thus, 275 BAL samples taken from 105 lung transplant recipients during follow-up were analyzed by quantitative polymerase chain reaction (PCR) and virus isolation. RESULTS: Quantitative PCR detected virus in all 24 BAL samples taken at onset of symptomatic disease, and virus culture in only 7 samples (29.2%). Twenty-three of 251 BAL samples (9.2%) were positive by PCR, although they were obtained in an asymptomatic phase. Quantitation of CMV DNA from BAL allowed no discrimination between symptomatic and asymptomatic infection in individual cases. However, when the urea dilution method was used to recalculate the CMV DNA concentration for the epithelial lining fluid (ELF) diluted in the BAL, a CMV DNA level of more than 10 copies/mL ELF was clearly associated with symptomatic disease. CONCLUSION: The CMV DNA level in ELF may be a marker for symptomatic CMV infection.

Detection of hepatitis C virus (HCV) RNA in normal cervical smears of HCV-seropositive patients.

The presence of hepatitis C virus (HCV) in normal cervical smears (CS) obtained from 22 HCV-seropositive and 50 HCV-seronegative patients was assessed by reverse-transcriptase-polymerase chain reaction (RT-PCR). The presence of HCV in serum was established by use of enzyme-linked immunosorbent assay, Western blot test, and RT-PCR. HCV was detected in 36.4% (n=8) of CS cells recovered from 22 HCV-seropositive patients, but not in CS samples obtained from 50 HCV-seronegative patients. Furthermore, cells from the CS of 2 seropositive/smear-positive patients and 1 seropositive/smear-negative patient were isolated; HCV RNA was detectable in the cervical lymphocytes of the 2 smear-positive patients, but not in epithelial cells or granulocytes. HCV RNA is detectable in the CS of some HCV-seropositive women. The clinical importance of these data requires further study.