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Human cytomegalovirus (CMV)-induced complications of the central nervous system (CNS) in patients with human immunodeficiency virus (HIV) infection mainly consist of encephalitis (CMV-E) and polyradiculomyelitis. Histologically, patients with CMV-E can present with ventriculoencephalitis, focal parenchymal lesions, or microglial nodules; in addition, isolated cells containing typical CMV inclusions can also be recognized (1). Although diffuse encephalopathy is the clinical correlate of CMV-E, its clinical presentation may not be specific and a definitive diagnosis requires the identification of the virus in the brain or cerebrospinal fluid (CSF). In this regard, PCR of CSF for the detection of CMV DNA is the most reliable method for the diagnosis of CMV-related CNS lesions in vivo in HIV-infected patients, with most of the studies indicating sensitivity and specificity values of 80 and 100%, respectively (4-6, 8-10, 16). The aim of the study described here was to compare the diagnostic reliabilities of two new commercially available assays for the detection of CMV genomes in peripheral blood, the COBAS AMPLICOR HIV Monitor PCR (the COBAS AMPLICOR PCR) and the NucliSens CMV pp67 nucleic acid sequence-based amplification assay (NASBA), with that of an in-house nested PCR test for CSF analysis (4, 5).

(The content of this article was presented at the Neuroscience of HIV Infection 2000, Edinburgh, United Kingdom, 22 to 24 June 2000.)

Patients belonged to a cohort of HIV-infected patients with CNS symptoms admitted to the San Raffaele Hospital, Milan, Italy, between 1992 and 1998. Thirty-one patients with or without CMV encephalitis were selected because of the availability of both postmortem tissues and a CSF sample that had been taken during the 180 days preceding death and that had been stored at 80°C and never previously thawed. Six patients (four with CMV-E and two with other CNS diseases) were included in a previous study (5). Upon admission, the patients underwent a complete neurological and neuroradiological examination and CSF analysis. Additional CSF specimens were drawn as necessary during the subsequent follow-up. All of the patients who died in hospital underwent postmortem examination (5). At autopsy, CMV-E was defined as the presence of inflammatory and/or necrotizing brain lesions, with CMV identified by histology on the basis of typical viral inclusions and confirmed by immunochemistry with a monoclonal mouse anti-CMV antibody (clone CCH2, diluted 1:25; Dakopatts, Glostrup, Denmark).

The nested PCR assay was performed as described previously (4). This test was used to examine the CSF at the time of the diagnostic workup but was repeated for the present study at the same time of the other two assays and using the same stored CSF samples. CSF samples were tested by the COBAS AMPLICOR test with a 200-µl volume and by the manufacturer's recommendation for analysis of plasma (13, 14). For the Organon Teknika NucliSens CMV pp67 NASBA, an initial CSF sample volume of 100 µl was used and the analysis was performed as recommended for analysis of blood (2, 3).

At neuropathological examination, 15 patients had CMV-E and 16 patients had other CNS conditions. All of the patients with CMV-E had ventriculoencephalitis, and 11 also had focal parenchymal lesions. Further details of the postmortem findings are shown in Tables 1 and 2. The analytical sensitivity of the nested PCR and the COBAS AMPLICOR test, evaluated by using 10-fold dilutions of purified full-length CMV Towne DNA, showed a lower detection limit of 100 copies/ml for the nested PCR and 1,000 copies/ml for the COBAS AMPLICOR PCR. By the nested PCR, the COBAS AMPLICOR PCR, and NASBA, the diagnostic sensitivities were 93.3, 86.6, and 93.3%, respectively, and the specificities were 93.7, 93.7, and 87.5%, respectively. In five patients discordant CSF and histopathology results were found by one or more of the assays (Table 1, patients 14 and 15; Table 2, patients 16 to 18). By the nested PCR, the results of the retrospective and prospective analyses were identical, showing interassay reproducibilities of 100%.

By the COBAS AMPLICOR PCR, the median CMV DNA load was 65,600 copies/ml (range, 6,400 to 100,000 copies/ml) for 12 patients with diffuse ventriculoencephalitis and <400 copies/ml (range, <400 to 1,900 copies/ml) for three patients with a focal form (P < 0.01 by the Mann-Whitney test). No significantly different distributions of the CMV DNA loads were observed according to the presence of associated parenchymal lesions, CMV-infected cell score, extracerebral CMV localization, other CNS diseases, or ongoing anti-CMV therapy, although untreated patients tended to have higher viral loads (Table 1).

The results obtained in this study by all three techniques closely correlated with the postmortem diagnoses and showed similar diagnostic sensitivity and specificity values. These values were similar to those obtained for two other patient series in which CSF findings were compared with the histopathological diagnoses (4, 5). Overall, the analytical sensitivities of the assays therefore appeared to be adequate to specifically distinguish patients with CMV-E. Furthermore, the results of the present retrospective evaluation by nested PCR were identical to those prospectively obtained by the same assay at the time of patient evaluation 2 to 8 years earlier, which had allowed a premortem diagnosis for the majority of patients. This indicates the high interassay reproducibility of this technique and the good rate of recovery of amplifiable genomes after longterm storage at 80°C.

The COBAS AMPLICOR PCR is a quantitative assay developed to measure CMV levels in plasma or peripheral blood leukocytes (13, 14). To our knowledge, only Roche's other commercially available PCR assay, the qualitative plasma AMPLICOR CMV test, but not the COBAS AMPLICOR PCR assay, has been described for analysis of CSF (11, 15). In the present study, in which we compared the findings obtained with CSF with the neuropathological findings, we found that the analytical and diagnostic sensitivities of the COBAS AMPLICOR PCR were only slightly lower than those of the nested PCR and NASBA, and the COBAS AMPLICOR test failed to recognize only one patient with focal ventriculoencephalitis. The threshold of 400 copies/ml, calculated as representing the best compromise between sensitivity and specificity in identifying HIV-infected patients at risk for the development of systemic CMV disease (12), therefore also seems to be useful for the identification of patients with CMV-E. Furthermore, by the COBAS AMPLICOR assay we found a significant association between high CSF CMV DNA levels and the presence of diffuse rather than focal periventricular lesions and found that the presence of focal parenchymal lesions did not seem to affect the viral load in CSF significantly. These findings are consistent with the hypothesis that the high CSF CMV DNA burden in patients with ventriculoencephalitis is probably due to the direct release of virus from the adjacent infected ependymal and subependymal tissues. From a practical point of view, the ability to discriminate extended from milder lesions may be important for prognostic considerations.

Whereas the nested PCR and the COBAS AMPLICOR PCR detect viral DNA, the NASBA detects late CMV transcripts. A recent evaluation of the NucliSens CMV pp67 NASBA with CSF from HIV-infected patients with a clinical diagnosis of CMV-associated or other opportunistic CNS diseases showed that the test was highly specific but less sensitive than PCR of DNA for the diagnosis of CMV-related neurological complications (17). The results of the present study, specifically designed to compare the results obtained with CSF with those obtained by histopathological examination, not only confirm the high degree of specificity of the NASBA but also show that its sensitivity is not less than that of the PCR assays with DNA.

Discordance between the findings obtained with CSF and postmortem findings were obtained for five patients. Positive PCR results in the absence of brain lesions caused by CMV can be explained by the possible effects of anti-CMV drugs administered between the time of sampling and death, by the inevitable limitations of neuropathological analysis to sample all the microscopic lesions, or by the possible presence of active CMV replication in the brain or CSF in the absence of established lesions. The detection in CSF of viral DNA originating from the blood might be another explanation for these discordant findings. However, previous reports indicated that CMV DNA is generally recovered in the CNS irrespective of CMV DNA detection in blood or of an extracerebral CMV involvement (4, 5, 10). We constantly used strict laboratory procedures to avoid contamination, and so the presence of exogenous nucleic acids in these CSF-positive samples with discordant results was unlikely. The time that elapsed between CSF sampling and postmortem diagnosis may have resulted in a negative CSF result in the presence of CMV-E. On the other hand, the presence of amplification inhibitors in the clinical specimens was unlikely because both of the commercial assays include amplification controls and no inhibition was ever observed.

In conclusion, the two commercial assays examined in the present study are useful additional tools for the diagnosis of CMV-E in HIV-infected patients. Furthermore, the COBAS AMPLICOR assay enables quantification of DNA in CSF, which not only is important for diagnostic and prognostic factors but might also provide precious information for monitoring of antiviral treatment (7). Although the prevalence of CMV-E in HIV-infected patients has decreased since the introduction of highly active antiretroviral therapy, we believe that monitoring of CMV infection of the CNS is still important because a number of patients cannot tolerate or fail to respond to these regimens. Furthermore, although not evaluated in the present study, these new tests might prove to be useful for the diagnosis of other CMV-associated neurological diseases, such as those observed in transplant recipients, neonates with congenital CMV infection, or immunocompetent patients (1).