Prospective use of RFLP analysis on amplified Cryptococcus neoformans URA5 gene sequences for rapid identification of varieties and serotypes in clinical samples.

Clinical isolates of Cryptococcus neoformans, whole blood, cerebrospinal fluid, bronchoalveolar lavage fluid from patients with positive cryptococcal antigen latex-agglutination test, and spiked clinical material from healthy individuals, were tested by polymerase chain reaction (PCR) with primers amplifying C. neoformans URA5 gene sequences. To test compatibility of different DNA extraction protocols with the PCR-restriction fragment length polymorphism (RFLP) assay, a commercial DNA extraction kit (XTRAX; Gull Laboratories, UT, USA) was used alongside with the hexadecyltrimethylammonium bromide (CTAB) method on spiked biological fluids. Both methods extracted DNA from spiked clinical samples containing C. neoformans (8 +/- 2 cells ml(-1)) and generated amplification products suitable for restriction enzyme analysis. Alu I digestion differentiated the two varieties of C. neoformans. Three distinct RFLP patterns were obtained upon restriction with MspI corresponding to serotypes A, AD and B, C and D. URA5 PCR followed by RFLP analysis, coupled with a sensitive in-house or commercially available DNA extraction method from clinical samples, could be successfully incorporated into rapid routine diagnostic strategies. It could also provide an expeditious tool for epidemiology-based population genetics studies.

First report of Cryptococcus neoformans var. gattii serotype B from Greece.

A plethora of cases of Cryptococcus neoformans infections have been recorded worldwide in immunocompromised individuals. The number of such cases showed a steady rise before 1981 and increased dramatically thereafter due to the AIDS epidemic. A similar pattern has been seen in Greece but, so far, infections appear to have been caused by C. neoformans var. neoformans. This paper describes for the first time two culture-proven C. n. var. gattii serotype B infections in Greece, one in an AIDS patient and one in a patient with systemic lupus erythematosus.

Molecular epidemiology of vertical human immunodeficiency virus type 1 transmission in Greece: evidence of non-B subtypes.

OBJECTIVES: To investigate the subtype classification of the circulating virus strains among human immunodeficiency virus type 1 (HIV-1)-infected children in Greece. STUDY DESIGN/METHODS: Since the beginning of the acquired immunodeficiency syndrome (AIDS) epidemic in Greece in 1982, 23 children have been reported to be vertically infected with HIV-1. Blood samples were available for 19 of these children, and the C2-C4 env region was successfully amplified by nested polymerase chain reaction (PCR) for 16 subjects. HIV-1 subtype was established by the heteroduplex mobility assay (HMA) in 16 subjects and confirmed by DNA sequencing and phylogenetic analysis in 8 subjects. RESULTS: Most subjects (9; 56%) fell into subtype B. However, a substantial proportion (44%) were classified as subtypes A (3; 19%), C (1; 6%), D (1; 6%), and I (2; 12%). According to epidemiologic information, 5 of 7 children infected with non-B HIV-1 subtypes were born to Greek parents. CONCLUSION: These findings clearly suggest that non-B strains have been introduced into Greece, providing evidence that HIV epidemic in this country will probably change profile over time. In addition, subtype I was identified in 2 HIV-1-infected children, both of whom were born to Greek parents.