Two hundred seventy-six Cryptococcus isolates originating from patients with HIV/AIDS were obtained from the Infectious Diseases Laboratory, Los Angeles County Hospital, Los Angeles, California. The isolates were suspended in sterile skim milk and stored at –20°C. The isolates were transferred frozen to the Mycology Laboratory of the Wadsworth Center in Albany, New York, USA, where they were streaked on Niger seed agar plates (3) to check for purity and reconfirmation of their identity; a typical colony was picked for further analysis. The subcultures were placed in long-term storage in sterile 15% glycerol at –70°C. These isolates were further characterized in our laboratory by testing their growth on canavanine-glycine-bromothymol blue (CGB) agar for differentiation of Cryptococcus species (22) and serotyping with Crypto Check Kit (Iatron Laboratories Inc., Tokyo, Japan). Several investigators gave strains to put together a panel of reference isolates that were either currently being used in molecular pathogenesis studies, represented type strains, or were otherwise unique. The details of these 16 reference isolates are listed in Table 1. Six additional A/D hybrid strains, characterized in our earlier study, were also used (18).

A previous report from this laboratory described the use of specific primers for amplification of MFα1 and MFa1 gene fragments, which could be separated as 100-bp and 117-bp fragments on a specialized agarose gel (21). The primer sets V290/V291, which was earlier designed to amplify MFa1 gene from CnVN, did not amplify similar genes from MATa strains of either CnVG or Cg. Multiple alignment of MFa1 indicated that this gene is highly polymorphic among CnVG, CnVN, and Cg (Figure 1). Therefore, 2 new sets of primers were designed to obtain MFa1 amplicons from CnVG and Cg. These primers are listed in Table 2. A multiplex PCR for simultaneous amplification of MFα1 and MFa1 in a 50-μL reaction volume was performed with 5 μL of 10× PCR buffer with 15 mmol/L MgCl₂, 2.5 μL of each of 8 primers (10 μmol/L stock), 3.0 μL dNTP mix (10 μmol/L each), and 2.0 U Taq DNA polymerase (Perkin Elmer, Foster City, CA, USA). The template DNA was 5.0 μL of either a boiled cell suspension or 50 ng genomic DNA. Initial denaturation was conducted at 95°C for 3 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 57.5°C for 1 min, amplification at 72°C for 1 min, and final extension at 72°C for 7 min, in a GeneAmp PCR System 9600 (Perkin Elmer). In preliminary experiments, PCR products (10-μL aliquots) were resolved by electrophoresis on 3.5% MetaPhor agarose (FMC Bio-Products, Rockland, ME, USA) gels in Tris-borate-EDTA (TBE) buffer, and were detected by ethidium bromide staining. The PCR experiments were repeated twice, and identical results were obtained.

The MFα1 sense primer (V190) was labeled with FAM (6-carboxyfluorescein) at the 5´ end, the antisense primer (V191) was labeled with tetrachloro-fluorescein (TET) at the 3´ end, and MFa1 sense primers (V290, V1311, V1313) were labeled with 6-carboxy-2´, 4´, 4´, 5´, 7´, 7´-hexachlorofluorescein (HEX) at the 5´ end. The fluorescent dye–labeled primers were custom ordered (Operon Technologies, Inc., Alameda, CA, USA). FLA and SSCP of the MFα1 and MFa1 PCR amplicons were determined by CE with an ABI PRISM 310 Genetic Analyzer, and the electronic images were analyzed by using GeneScan analysis software (Applied Biosystems Inc., Foster City, CA, USA). The sample preparation for CE consisted of 1 μL MFα1 and MFa1 PCR amplicons, 12 μL highly deionized formamide, and 0.5 μL GeneScan-500 (TAMRA) size standard (Applied Biosystems). The sample mixture was denatured for 5 min at 95°C and was then rapidly cooled on ice before loading on the instrument. For CE-FLA, the samples were analyzed under denaturing conditions (POP-4 polymer [Applied Biosystems] in buffer supplied by manufacturer) at 60°C, and for CE-SSCP, the samples were analyzed under nondenaturing conditions (3% GeneScan polymer in 1× TBE buffer with 10% glycerol) at 30°C. A capillary (47 cm × 50 μm inside diameter) was installed, and POP-4 or 3% polymer was filled according to manufacturer's instructions. The electrophoresis conditions for CE-FLA were 5-s injection time, 15-kV injection voltage, 15-kV electrophoresis voltage, 150-s syringe pump time, 120-s preinjection electrophoresis, and 20-min collection time for each sample, and the run was performed at 60°C. The electrophoresis conditions for CE-SSCP were 5-s injection time, 15-kV injection voltage, 13-kV electrophoresis voltage, 30-s syringe pump time with no preinjection time, and 20-min collection time for each sample, and the run was performed at 30°C. CE-FLA and CE-SSCP standardization experiments were carried out on >4 independent occasions, and unknown sample analyses were repeated at least once.

The 4 sets of primers (MFα1/MFa1) produced reproducible results for control CnVG, CnVN, Cg haploids (Figure 2A), and A/D hybrid strains (Figure 2B). These results validated the robustness of the primers, which had been designed from well within the open reading frames of 2 pheromone genes, to prevent amplification of any nontarget DNA. The latter objective also informed the decision to anchor the 3´ ends of all PCR primers within the characteristic Cys-Val-Ile-Ala (CVIA) motifs; this eliminated any possible amplification of other pheromone genes since this is the only sequence shared among fungal pheromones (18). Even though multiple copies of MFα and MFa genes have been reported in C. gattii and C. neoformans by Southern hybridization and whole genome-sequencing, PCR primers only amplify single amplicons because these genes have identical nucleotide sequences (18,23,24). Although this multiplex method was well suited for identifying mating types and hybrids, further delineation of species and varieties would require restriction digestion with several unique enzymes as we stated previously (21). Therefore, we decided to use CE-FLA and CE-SSCP to further characterize pheromone gene amplicons. These techniques have been successfully used to delineate fragment length as well gene mutations for characterizing various fungal and bacterial isolates (25–27). The SSCP analysis displays migration of the amplified DNA fragment as a function of that fragment's structural conformation. Given that the tertiary structure of a fragment is sensitive to single nucleotide substitutions, this method was shown to be suitable for detecting single nucleotide changes when 100-bp to 300-bp DNA fragments were analyzed (28). Since amplified pheromone fragments yield ≈100- to 120-bp products, they were an ideal substrate for this method of mutant detection.

The 16 reference strains of known Cryptococcus species, varieties, mating types, and hybrids were used to establish a robust CE-FLA protocol with denaturing POP-4 polymer at 60°C. The electrophoretic runs with POP-4 polymer produced a 112-bp DNA fragment for MFa1 and 97-bp fragment for MFα1, which were easily distinguished with the GeneScan software by the characteristic peak sizes (Figure 3). CE-FLA allowed Cryptococcus mating types and hybrids to be identified, but not CnVG, CnVN, and Cg.

CE-SSCP test under nondenaturing conditions with 3% GeneScan polymer at 30°C allowed characteristic peak patterns to be detected in MFα1 and MFa1 genes because of the individual differences within the nucleotide sequences. Distinct patterns obtained for CnVG, CnVN, and Cg by using MFα1 gene are shown in Figure 4. The sense strand (labeled blue) yielded 1 characteristic peak pattern, while the antisense strand (labeled green) yielded 2–3 characteristic peak patterns. We subsequently decided to label only the sense strand to reduce the cost of the PCR primers as well the complexity of the peak patterns observed with antisense strand. For determining an unknown sample, the instrument analyses needed to yield highly reproducible results. Therefore, a sample of each of the Cryptococcus strains was injected on 4 separate occasions into the same capillary, and the precision of the sizing was calculated. The low standard deviations associated with each mean peak value indicated that the assignment of variety or species for an unknown sample, based on pattern sizing information alone, would be highly reliable (Table 3).

Based on our success with MFα1 sense primer in the detection of characteristic peaks for CnVG, CnVN, and Cg, we labeled MFa1 sense strands and analyzed MATa strains. In this case, we had to use individual sets of MFa1 primers because of the substantial polymorphism observed at the 5´ and 3´ end of this gene between Cg and Cn, and within Cn vatieties (Figure 1). Again, the MFa1 peak pattern was unique to each Cn variety and Cg (Table 3). Overall, our results indicated that either MFα1 or MFa1 gene products yielded unique SSCP patterns and could be used for identifying Cn varieties and Cg strains.

We used standardized CE-FLA and CE-SSCP techniques to analyze 276 isolates of Cryptococcus that were obtained from AIDS patients and that were stored at the Infectious Diseases Laboratory, Los Angeles County Hospital, Los Angeles, California. The investigations were fully compliant with Los Angeles County Hospital–University of Southern California (USC) Institutional Review Board guidelines (proposal #924008). CE-FLA showed that all 276 isolates were MATα strains, and no MATa or hybrid strains were found in our samples. CE-SSCP found that among the total 276 clinical isolates, 219 (79.3%) were CnVG, 23 (8.3%) were CnVN, and 34 (12.3%) were Cg. For corroborations, all of these isolates were also tested by growth on CGB agar, and by serotyping with the Crypto Check Kit, both of which yielded results in agreement with those obtained with pheromone typing.