Development of DNA probes for early diagnosis and epidemiological study of cryptococcosis in AIDS patients

Abstract

We report the isolation of middle-repetitive DNA sequences from Cryptococcus neoformans that are species and variety specific. These probes were used for assessing strain relatedness among cryptococcal isolates from patients with and without AIDS who were from Zaire and the United States. Five distinct hybridization patterns were observed for the 60 isolates examined, regardless of the restriction enzyme used for digestion. The most common pattern among the isolates from the patients without AIDS was also the most common among the isolates from the patients with AIDS who were from the United States and was the only pattern observed for all isolates tested from patients with AIDS who were from Zaire. On the basis of the high specificity and sensitivity of the signals observed by hybridization, we suggest that these sequences provide a means for both biotyping and early diagnosis of C. neoformans.

Involvement of multiple Cryptococcus neoformans strains in a single episode of cryptococcosis and reinfection with novel strains in recurrent infection demonstrated by random amplification of polymorphic DNA and DNA fingerprinting

“…Many studies have shown that clinical and environmental isolates of C. neoformans are highly polymorphic (6,7,16,25,27). Our results reinforce these findings.…”

“…A suitable system for C. neoformans isolate discrimination would greatly facilitate studies of the epidemiology and virulence of this pathogenic yeast. Many systems have been proposed, but they often lack the necessary reproducibility and discriminatory power to be useful in differentiating between individual strains (3,16,24,26). However, recent reports have demonstrated that molecular typing techniques are able to discriminate C. neoformans at the strain level (6,19,25,27).…”

“…Molecular typing methods have been applied successfully to the typing of a number of pathogenic fungi, e.g., Histoplasma capsulatum (11), Aspergillus fumigatus (12), and Candida albicans (9). When similar methods were first applied to the typing of C. neoformans, discrimination at the strain level remained elusive (16,24,26). However, several recent reports have established that genotypic variation in C. neoformans can be identified by molecular techniques.…”

Rapid and easy method to extract and preserve DNA from Cryptococcus neoformans and other pathogenic yeasts

“…The published protocols for DNA isolation from Cr. neofomans include capsule digestion by mixtures of enzymes (Novozyme 234 or Mureinase [4,5,9,101 and Lyticase [8]). These methods are expensive, complicated and time-consuming.…”

Serotyping of Cryptococcus neoformans by dot enzyme assay

“…Alternative means for serotyping new isolates include the use of a combination of selective media and differential staining of yeast cells by indirect immunofluorescence (13). Other investigators have examined genetic variations between serotypes and between isolates within a given serotype by using molecular methods such as karyotyping (23), DNA probes (24), restriction fragment length polymorphism (7,12,14), amplification of polymorphic DNA patterns by PCR (22), and multilocus enzyme electrophoresis (6). Despite the introduction of alternative techniques, no standardized method for serotyping of isolates has been adopted.…”