The genotypes of Toxoplasma gondii strains isolated from HIV and non-HIV immunocompromised patients with cerebral and extracerebral toxoplasmosis were determined and compared to those of strains isolated from non-immunocompromised patients in order to identify the possible relationships between parasite genotype and morbidity of toxoplasmosis. One hundred and ten strains of T. gondii were obtained, either by cell culture (n = 73), brain biopsy (n = 17) or mouse inoculation (n = 20). Ninety strains isolated from immunocompromised patients (74 HIV+ and 16 non-HIV patients) were compared to 20 strains isolated from immunocompetent patients (17 cases of congenital toxoplasmosis, and three cases of primary acquired infection). Genotyping was performed by PCR/RFLP on locus SAG2, and T. gondii strains were classified as Type I, II or III. Ninety out of 110 strains were successfully genotyped, including 20 strains that had been maintained in mice, 69/73 strains maintained in cell cultures, but only 1/17 strains from formalin-fixed paraffin-embedded brain biopsies. 76.7% of the strains in the study population were of type II, 15.6% were type I and 7.7% were type III. The distribution of strain genotypes in immunocompromised and non-immunocompromised patients was comparable: 14.1% and 21% for type I, 76.1% and 79% for type II and 9.8% and 0% for type III, respectively; no correlation could be established between genotype and clinical presentation, i.e., cerebral or extracerebral toxoplasmosis. These results suggest that the type of infecting parasitic strain does not predominantly influence the pathogenesis of toxoplasmosis in immunocompromised patients and fully supports the need for specific prophylaxis in patients infected by T. gondii, regardless of the strain genotype.

A fatal case of dengue (DEN) infection associated with a spleen rupture and with hepatitis is reported here. Microscopic studies showed numerous areas of spleen rupture with hematomas and revealed necrotic foci in liver samples obtained at autopsy. Although hepatitis was reported in several cases of DEN fever, the mechanism of liver injury remains poorly understood. In this case, immunohistochemistry showed that DEN viral antigens were mostly detected in hepatocytes surrounding the necrotic foci. By in situ detection of DNA fragmentation, apoptotic hepatocytes were found to be colocated with DEN virus-infected hepatocytes. These findings suggest that hepatocytes are the major sites of DEN virus replication in the liver and that DEN virus induces apoptosis of hepatocytes in vivo.

Hepatitis C virus (HCV) detection in the livers of chronically infected patients remains a debatable issue. We used immunohistochemistry, in situ hybridization (iSH) alone or after microwave heating with FITC-labeled probes, RT-PCR with unlabeled primers followed by ISH (RT-PCR-ISH), and in situ RT-PCR with FITC-labeled primers (in situ RT-PCRd) to localize the virus in 38 liver biopsy specimens from 21 chronically infected HCV patients treated with interferon-alpha (IFN-alpha). Biopsies were taken at the beginning and end of IFN-alpha treatment and 1 year later. Results were compared with that of HCV-PCR in serum. RT-PCR-ISH and in situ RT-PCRd showed HCV signal in all liver biopsies even in responders with seronegative HCV PCR. This signal was intranuclear, diffuse, or peripheral, in hepatocytes, bile ductule cells, and lymphocytes. Cytoplasmic signals were occasionally observed. Whereas the percentage of labeled hepatocytes remained constant, the number of labeled lymphoid follicles decreased after INF-alpha therapy. Immunohistochemistry resulted in the same pattern of positivity but it was weaker and inconstant. This study indicates the persistency of HCV latency in IFN-alpha responders 1 year after IFN-alpha treatment cessation, a finding that certainly deserves confirmation.

The prevalence of genital human papillomavirus (HPV) infection was evaluated in 30 consecutive human immunodeficiency virus (HIV) + women by polymerase chain reaction (PCR)-in situ hybridization (ISH) on paraffin-embedded tissue sections and compared with that found with standard ISH. Biopsies were removed from normal or neoplastic areas in the cervix, vagina, and vulva, and ISH was performed with biotinylated or fluorescein isothiocyanate genomic DNA probes. One probe was used for HPV screening and others for HPV typing (types 6, 11, 16, 18, 31, and 33). Sequences were amplified by the "hot-start" PCR method and followed by standard ISH. Among the 30 HIV + women, 90% scored HPV + in one or several locations by PCR-ISH, whereas only 67% were positive by ISH. Oncogenic HPV types were found in 63% by PCR-ISH and in only 43% by ISH. The same HPV types detected by standard ISH were also recognized by PCR-ISH, but with the latter the signal was amplified. Moreover, some HPV types were found with PCR-ISH but not by ISH. We conclude that PCR-ISH is a valuable and sensitive method for specific detection of HPV.

In situ polymerase chain reaction is a recent technique which combines the sensitivity of PCR reaction to intracellular localization of genomic sequences with the same specificity as in situ hybridization. This reaction is based on the in situ annealing and polymerisation of oligonucleotides complementary to nucleotides located at each side of the target DNA sequence to amplify. We describe the Hot Start PCR (DNA) and the Hot Start PCR after reverse transcription step (RNA). It allows to amplify some nucleic sequences to a high level, becoming easier to detect. The vizualisation can be realized by direct in situ PCR, the product obtained being directly identifiable by incorporation of labeled nucleotides or primers, or preferentially by indirect in situ PCR. In this case, the amplification is followed by in situ hybridization with labeled probes. This last procedure is more specific. Numerous controls are essential at each step of the technique for validating results.

In situ polymerase chain reaction is a recent technique which combines the sensitivity of PCR reaction to intracellular localizatin of genomic sequences with the same specificity as in situ hybridization. This reaction is based on the in situ annealing and polymerization of oligonucleotides complementary to nucleotides located at each side of the target DNA sequence to amplify. We describe the Hot Start PCR (DNA) and the Hot Start PCR after reverse transcription step (RNA). It allows to amplify some nucleic sequences to a high level, becoming easier to detect. The visualization can be realized by direct in situ PCR, the product obtained being directly identifiable by incorporation of labeled nucleotides or primers, or preferentially by indirect in situ PCR. In this case, the amplification is followed by in situ hybridization with labeled probes. This last procedure is more specific. Numerous controls are essential at each step of the technique for validating results.