Detection of human cytomegalovirus DNA in paraffin

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tion (nPCR). A characteristic 183 base pair (bp) fragment of the HCMV genome could readily be amplified in 4 cases of. HCMVE. In 2 cases of HCMVE, viral.

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J7ournal of Neurology,

Neurosurgery,

211

and Psychiatry 1993;56:211-214

SHORT REPORT

Detection of human cytomegalovirus DNA in paraffin sections of human brain by polymerase chain reaction and the occurrence of false negative results P Gass, M Kiessling, P Schafer, C Mester, H P Schmitt, J E Kuhn

Abstract non-specific nodular encephalitis, that is, mulParaffin-embedded necropsy material tifocal microglial proliferation, occur with from 6 patients with human cytomegalo- increasing frequency in HIV patients. Incluvirus encephalitis (HCMVE) corrobo- sion-bearing cells of Cowdry type A are pathorated by immunocytochemistry and 11 gnomonic but may be missing. HCMV control cases were examined for the pres- infection can be verified by immunocytochemence of human cytomegalovirus (HCMV) istry with specific antibodies against viral DNA by a nested polymerase chain reac- antigens or by nucleic acid hybridization techtion (nPCR). A characteristic 183 base niques even in the absence of HCMV inclusion pair (bp) fragment of the HCMV genome bodies.2 could readily be amplified in 4 cases of Recently, the feasibility of detecting herpes HCMVE. In 2 cases of HCMVE, viral simplex virus type 1 (HSV-1) DNA in paraffin DNA could be demonstrated only sporadi- sections of human necropsy brains by the use cally by PCR, due most likely to inefficient of PCR was reported.4 Compared to in situ DNA extraction or DNA degradation. All hybridisation techniques, PCR is easier to control cases remained negative. The perform and may offer increased sensitivity. nPCR provides a specific method for The purpose of this study was to investigate, detecting HCMV DNA in routinely pro- whether it is possible to detect specific viral cessed biopsy and necropsy material and DNA sequences in archival materials of HCMVE cases. For this purpose, a PCR may be used in archival tissues for the diagnosis of infection. Fixation of samples protocol was applied, which has been successand DNA extraction are, however, crucial fully used to detect HCMV DNA in human steps and require careful control if PCR is peripheral blood and urine samples.5 used for detection of HCMV, to avoid false negative results. Material and methods We examined six cases of HCMVE (table, (3 Neurol Neurosurg Psychiatry 1993;56:21 1-2 14) cases 1-4 from our Institute, cases 5-6 from the Department of Neuropathology, UniverHCMVE occurs in up to 30% of immuno- sity of Oxford, UK). All demonstrated typical compromised patients and occurs occasionally histological features such as inclusion-bearing in immunocompetent hosts as well.' In adults, cells of Cowdry type A, microglial proliferaHCMV infection is increasingly seen as an tion, focal parenchymal necroses and lymphoopportunistic infection complicating the cytic infiltration. Six cases with HSV- 1 acquired immunodeficiency syndrome encephalitis and five cases without neuro(AIDS). Perinatally HCMVE is due mostly to pathological changes were used as controls. intra-uterine transplacental infection. A char- Diagnosis was made by routine histological acteristic histological feature of cerebral examination and corroborated by immunocyHCMV infection is a periventricularly accen- tochemistry with a monoclonal antibody (antituated necrotising encephalitis with haemor- cytomegalovirus, dilution 1:25, DAKO, rhage and calcification, but cases with Hamburg, Germany) in all cases. Necropsy 3

University of Heidelberg, Heidelberg, Germany Institute of

Neuropathology P Gass M Kiessling H P Schmitt Institute of Virology P Schafer Institute of Virology, University of Cologne, Cologne, Germany C Mester J E Kuhn Correspondence to: Dr Gass, Institute of Neuropathology, University of Heidelberg, Im Neuenheimer Feld 220, D-6900 Heidelberg, Germany Received 6 August 1991 and in revised form 13 April 1992. Accepted 29 May 1992

Table Clinical and laboratory data of 6 patients with HCMV encephalitis

Histology

Imniuno-

AgelSex

AIDS

diagnosis

cytochemistry

a

1 2 3

44/Male 47/Male

+ + +

4

2m/Male

50/Male

+

HCMV-pos HCMV-pos HCMV-pos HCMV-pos HCMV-pos HCMV-pos

+ + + +

5 6

-

HCMVE HCMVE HCMVE HCMVE HCMVE HCMVE

Number Case Case Case Case Case Case

49/Male

id/Male

HCM-PCR b

c

f3-globin-PCR nonspecific a

c

inhibition

nd

nd

No No No No No No

6/6

nd

5/5 5/5

>10'

+

102 >104

+

>10' >10' >104

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