Clinical utility of capillary polymerase chain reaction for diagnosis of Cytomegalovirus pneumonia.

The purpose of this retrospective study was to assess the diagnostic efficacy of CMV DNA detection by capillary PCR in patients with interstitial pneumonia. Of 882 samples taken from 363 patients, 317 were obtained from sputum, 94 from BAL fluid, 291 from blood and 180 from urine. PCR for CMV was positive in 58 samples (6.6%), with positive detection for 6.9% of sputum, 10.6% of BAL fluid, 4.1% of blood and 7.8% of urine samples. CMV pneumonia was diagnosed retrospectively in 34 (9.4%) of the 363 patients by demonstration of CMV antigen-positive cytomegalic inclusion bodies in lung tissue sections. The positive and negative predictive values were 100% (10/10) and 98.8% (83/84) for the BAL fluid samples and 95.5% (21/22) and 99.7% (294/295) for the sputum samples, respectively. Clinical sensitivity and specificity were 90.9% (10/11) and 100% (83/83) for the BAL fluid samples and 95.5% (21/22) and 99.7% (294/295) for the sputum samples, respectively. However, the blood and urine samples showed poor clinical sensitivity and low positive predictive values. We suggest that the use of capillary PCR for BAL fluid and sputum samples is very useful for diagnosing CMV pneumonia in patients with interstitial pneumonia in whom CMV pneumonia is suspected.

Interleukin-4 is needed for the infection of monocytes by human cytomegalovirus.

OBJECTIVE: We studied the ability of human cytomegalovirus (HCMV) to infect peripheral blood mononuclear cells (PBMC) pretreated with or without Th2-cytokine interleukin-4 (IL-4) in vitro. METHODS: Adherent cells and nonadherent cells were obtained from PBMC. We inoculated these cells with HCMV at concentrations ranging from 0 to 10 ng/ml of IL-4. Immediate-early antigen-1 (IE-1) and glycoprotein H (gH) mRNAs were detected using the reverse-transcription polymerase chain reaction. RESULTS: IE-1 and gH mRNAs could be detected in monocytes pretreated with IL-4. In contrast, no IE-1 mRNA was detected in monocytes pretreated without IL-4. We tested whether higher infectious titers could result in the infection of monocytes whether or not they were pretreated with IL-4. However, no IE-1 mRNA was detected in the monocytes not pretreated with IL-4. To elucidate how HCMV-infected monocytes affect lung tissue, human embryonic lung fibroblasts MRC-5 were cocultured with HCMV-infected monocytes. The cytopathic effects of HCMV were observed microscopically and was confirmed by direct immunoperoxidase staining with a human monoclonal antibody against the HCMV IE-1. CONCLUSION: Our data strongly suggest that the ability of HCMV to infect monocytes may correlate with the presence of IL-4.

In vitro transmission of HCMV between fibroblasts and peripheral blood leukocytes in the presence of IL-4.

We demonstrated transmission of human cytomegalovirus (HCMV) from the human lung fibroblast MRC-5 to peripheral blood leukocytes (PBLs). mRNA of the HCMV immediately-early (IE) antigen was detected in PBLs cultured with IL-2 or IL-2 + IL-4 that made direct contact with HCMV-infected MRC-5, whereas it was not detected in PBLs prevented from making cell-to-cell contact. However, mRNA of HCMV IE was not detected in PBLs cultured with IL-2 and IFN-gamma that made direct contact with HCMV-infected MRC-5. Transmission of the pp65 antigen was increased in culture medium containing IL-4. At a higher viral infection titer, cell-free HCMV infected adherent PBLs cells. The subset, which did not adhere, did not infect cell-free viruses even at a very high multiplicity of infection. Moreover, the adhered subset of PBLs infected with HCMV was able to transmit HCMV to non-infected fibroblasts. Our results suggest that cell-to-cell contact (when PBLs make direct contact with HCMV-infected cells) is important in the mechanism of HCMV transmission and that the adherent cells of PBLs are one of the most important vehicles for HCMV infection. Moreover, we suggest that type 2 cytokines such as IL-4 enhance the transmission of HCMV to PBLs.

Interleukin-4 induces mouse cytomegalovirus interstitial pneumonia in a latent infection model.

To better understand immune mechanisms involved in onset of cytomegalovirus pneumonia, we initially examined the replication of a low virulence strain of mouse cytomegalovirus (MCMV) in nude and BALB/c mice infected by intranasal inoculation. MCMV was detected by plaque assay in the salivary glands of nude mice from days 3 to 16, and in those of BALB/c mice from days 7 to 11. Nude mice became infected with MCMV earlier than BALB/c mice. Moreover, MCMV-DNA was detected in the salivary glands until day 16 after MCMV inoculation in nude and BALB/c mice. However, we did not find evidence of interstitial pneumonia at day 16 in either BALB/c or nude mice. These results suggest that this system represents a latent infection model in BALB/c mice and a persistent infection model in nude mice. We treated latently infected BALB/c mice with methylprednisolone or IL-4 every other day. The mice treated with IL-4 developed interstitial pneumonia, whereas those treated with m-PSL did not. In the present study, we constructed a model of MCMV latent infection that could be used to induce development of interstitial pneumonia. IL-4 appears to be a key cytokine for onset of interstitial pneumonia in mice with latent MCMV infection.

Clinical use of capillary PCR to diagnose Mycoplasma pneumonia.

In the present study, serologic data were compared with data obtained by capillary PCR to establish the efficacy of capillary PCR for the determination of Mycoplasma infection in samples obtained from throat swabs, bronchoalveolar lavage fluids (BALF), and sputum of patients with Mycoplasma pneumonia. We performed PCR analysis for Mycoplasma DNA on a total of 325 samples from 197 patients with community-acquired pneumonia and in whom Mycoplasma pneumonia was suspected. There were 68 PCR-positive specimens. Review of the differences in PCR positivity rates based on the site of specimen collection showed the highest rate of detection (28.6%) from throat swabs. From among the 31 patients with significantly elevated titers of serum Mycoplasma antibodies, the PCR results were positive for 25 patients. Thus, capillary PCR had a sensitivity of 80.6% (25 of 31). Five of the six false-negative results were from throat swab specimens. Moreover, testing (PCR) had been performed only once for these five patients with false-negative results. From among the PCR-positive findings from BALF specimens, there were no false-positive results. BALF specimens were very useful, except for the technical procedures and increased patient burden required to obtain these specimens. We suggest that the use of throat swab specimens in capillary PCR is much more suitable for diagnosing Mycoplasma pneumonia in routine clinical practice; however, careful throat swab specimen collection and an increase in the number of times that the PCR is performed are necessary to reduce the rate of false-negative results.