Abstract
The yeast Candida albicans is an important opportunistic human pathogen. For C. albicans strain typing or drug susceptibility testing, a single colony recovered from a patient sample is normally used. This is insufficient when multiple strains are present at the site sampled. How often this is the case is unclear. Previous studies, confined to oral, vaginal and vulvar samples, have yielded conflicting results and have assessed too small a number of colonies per sample to reliably detect the presence of multiple strains. We developed a next-generation sequencing (NGS) modification of the highly discriminatory C. albicans MLST (multilocus sequence typing) method, 100+1 NGS-MLST, for detection and typing of multiple strains in clinical samples. In 100+1 NGS-MLST, DNA is extracted from a pool of colonies from a patient sample and also from one of the colonies. MLST amplicons from both DNA preparations are analyzed by high-throughput sequencing. Using base call frequencies, our bespoke DALMATIONS software determines the MLST type of the single colony. If base call frequency differences between pool and single colony indicate the presence of an additional strain, the differences are used to computationally infer the second MLST type without the need for MLST of additional individual colonies. In mixes of previously typed pairs of strains, 100+1 NGS-MLST reliably detected a second strain. Inferred MLST types of second strains were always more similar to their real MLST types than to those of any of 59 other isolates (22 of 31 inferred types were identical to the real type). Using 100+1 NGS-MLST we found that 7/60 human samples, including three superficial candidiasis samples, contained two unrelated strains. In addition, at least one sample contained two highly similar variants of the same strain. The probability of samples containing unrelated strains appears to differ considerably between body sites. Our findings indicate the need for wider surveys to determine if, for some types of samples, routine testing for the presence of multiple strains is warranted. 100+1 NGS-MLST is effective for this purpose.
Highlights
MATERIALS AND METHODSThe yeast Candida albicans is a frequent commensal colonizer of humans, but can cause disease, including lifethreatening candidemia when it reaches the bloodstream in immunocompromised patients (Kim and Sudbery, 2011)
Our aim was to develop a methodology that could detect the presence of multiple strains in a patient sample and determine their multilocus sequence typing (MLST) types
Call frequencies in the thousands of sequence reads per amplicon generated by next-generation sequencing (NGS) (MiSeq; Illumina_Inc, 2017) sequencing of the seven established C. albicans MLST loci [AAT1a, ACC1, ADP1, MPIb, SYA1, VPS13, ZWF1b (Bougnoux et al, 2003)] in DNA extracted from pools of colonies recovered from a patient sample
Summary
The yeast Candida albicans is a frequent commensal colonizer of humans, but can cause disease, including lifethreatening candidemia when it reaches the bloodstream in immunocompromised patients (Kim and Sudbery, 2011). A possible explanation for the differences between the latter and the former studies could be that multiple strains are present in some human samples more frequently than in others. If so it may be misleading to draw wider conclusions from the limited range of sample types analyzed to date. Another limitation of the existing data is that for the vast majority of samples fewer than 10 colonies were typed. Strains present at frequencies of 10–20% would often have been missed
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