Detection of Cryptococcus neoformans DNA in Tissue Samples by Nested and Real-Time PCR Assays

Two PCR protocols targeting the 18S rRNA gene of Cryptococcus neoformans were established, compared, and evaluated in murine cryptococcal meningitis. One protocol was designed as a nested PCR to be performed in conventional block thermal cyclers. The other protocol was designed as a quantitative single-round PCR adapted to LightCycler technology. One hundred brain homogenates and dilutions originating from 20 ICR mice treated with different azoles were examined. A fungal burden of 3 × 10¹ to 2.9 × 10⁴ CFU per mg of brain tissue was determined by quantitative culture. Specific PCR products were amplified by the conventional and the LightCycler methods in 86 and 87 samples, respectively, with products identified by DNA sequencing and real-time fluorescence detection. An analytical sensitivity of 1 CFU of C. neoformans per mg of brain tissue and less than 10 CFU per volume used for extraction was observed for both PCR protocols, while homogenates of 70 organs from mice infected with other fungi were PCR negative. Specificity testing was performed with genomic DNA from 31 hymenomycetous fungal species and from the ustilaginomycetous yeast Malassezia furfur, which are phylogenetically related to C. neoformans. Twenty-four strains, including species of human skin flora like M. furfur and Trichosporon spp., were PCR negative. Amplification was observed with Cryptococcus amylolentus, Filobasidiella depauperata, Cryptococcus laurentii, and five species unrelated to clinical specimens. LightCycler PCR products from F. depauperata and Trichosporon faecale could be clearly discriminated by melting curve analysis. The sensitive and specific nested PCR assay as well as the rapid and quantitative LightCycler PCR assay might be useful for the diagnosis and monitoring of human cryptococcal infections.     

A randomized trial of the discontinuation of primary and secondary prophylaxis against Pneumocystis carinii pneumonia after highly active antiretroviral therapy in patients with HIV infection. Grupo de Estudio del SIDA 04/98

BACKGROUND: Prophylaxis against Pneumocystis carinii pneumonia is indicated in patients with human immunodeficiency virus (HIV) infection who have less than 200 CD4 cells per cubic millimeter and in those with a history of P. carinii pneumonia. However, it is not clear whether prophylaxis can be safely discontinued after CD4 cell counts increase in response to highly active antiretroviral therapy. METHODS: We conducted a randomized trial of the discontinuation of primary or secondary prophylaxis against P. carinii pneumonia in HIV-infected patients with a sustained response to antiviral therapy, defined by a CD4 cell count of 200 or more per cubic millimeter and plasma HIV type 1 (HIV-1) RNA level of less than 5000 copies per milliliter for at least three months. Prophylactic treatment was restarted if the CD4 cell count declined to less than 200 per cubic millimeter. RESULTS: The 474 patients receiving primary prophylaxis had a median CD4 cell count at entry of 342 per cubic millimeter, and 38 percent had detectable HIV-1 RNA. After a median follow-up period of 20 months (758 person-years), there had been no episodes of P. carinii pneumonia in the 240 patients who discontinued prophylaxis (95 percent confidence interval, 0 to 0.85 episode per 100 person-years). For the 113 patients receiving secondary prophylaxis, the median CD4 cell count at entry was 355 per cubic millimeter, and 24 percent had detectable HIV-1 RNA. After a median follow-up period of 12 months (123 person-years), there had been no episodes of P. carinii pneumonia in the 60 patients who discontinued prophylaxis (95 percent confidence interval, 0 to 4.5 episodes per 100 person-years). CONCLUSIONS: In HIV-infected patients receiving highly active antiretroviral therapy, primary and secondary prophylaxis against P. carinii pneumonia can be safely discontinued after the CD4 cell count has increased to 200 or more per cubic millimeter for more than three months.     

Candida arthritis in an immunocompetent patient without predisposing factors

Candida arthritis is infrequent in patients who are not intravenous drug users. We describe a patient who was not an intravenous drug user and without other predisposing factors who developed knee arthritis caused byCandida albicans. We believe that this is the first report of such a case.     

Detection and identification of putative bacterial endosymbionts and endogenous viruses in tick cell lines

As well as being vectors of many viral, bacterial, and protozoan pathogens of medical and veterinary importance, ticks harbour a variety of microorganisms which are not known to be pathogenic for vertebrate hosts. Continuous cell lines established from ixodid and argasid ticks could be infected with such endosymbiotic bacteria and endogenous viruses, but to date very few cell lines have been examined for their presence. DNA and RNA extracted from over 50 tick cell lines deposited in the Roslin Wellcome Trust Tick Cell Biobank (http://tickcells.roslin.ac.uk) were screened for presence of bacteria and RNA viruses, respectively. Sequencing of PCR products amplified using pan-16S rRNA primers revealed the presence of DNA sequences from bacterial endosymbionts in several cell lines derived from Amblyomma and Dermacentor spp. ticks. Identification to species level was attempted using Rickettsia- and Francisella-specific primers. Pan-Nairovirus primers amplified PCR products of uncertain specificity in cell lines derived from Rhipicephalus, Hyalomma, Ixodes, Carios, and Ornithodoros spp. ticks. Further characterisation attempted with primers specific for Crimean-Congo haemorrhagic fever virus segments confirmed the absence of this arbovirus in the cells. A set of pan-Flavivirus primers did not detect endogenous viruses in any of the cell lines. Transmission electron microscopy revealed the presence of endogenous reovirus-like viruses in many of the cell lines; only 4 of these lines gave positive results with primers specific for the tick Orbivirus St Croix River virus, indicating that there may be additional, as yet undescribed ‘tick-only’ viruses inhabiting tick cell lines.