Uncovering the hidden pathways of a Candida albicans infection cluster through Multilocus Sequence Typing
Imagine a medical mystery where three organ transplant recipients fall ill with the same dangerous fungal infection, and doctors must determine: is this a tragic coincidence or something more concerning? This isn't a television drama but a real-world scenario that confronted a medical team when Candida albicans, a typically harmless fungus that lives on our skin and in our mouths, turned deadly in immunosuppressed patients. The breakthrough technology that cracked this case? Multilocus Sequence Typing (MLST)—a sophisticated genetic detective that examines the DNA of pathogens to track their movement.
MLST provides unprecedented ability to visualize how infections spread between patients and environments.
Genetic evidence helps establish definitive links between infection cases with precision unimaginable decades ago.
MLST represents a revolution in medical mycology, offering scientists an unprecedented ability to trace infection pathways and confirm transmission chains with precision that was unimaginable just decades ago. For organ transplant recipients, who take immunosuppressive drugs to prevent organ rejection, common fungi like Candida can become life-threatening invaders. Understanding how these infections spread is critical to saving lives, and MLST provides the powerful tool needed to visualize these invisible pathways.
Multilocus Sequence Typing is a DNA-based method that examines the genetic blueprint of microorganisms like Candida albicans. The technique focuses on seven housekeeping genes—essential genes that all cells need for basic functions. These genes include AAT1a, ACC1, ADP1, MPIb, SYA1, VPS13, and ZWF1b 7 9 . Unlike more variable genes, housekeeping genes evolve slowly, providing stable markers for comparing different isolates of the same species.
For each of these seven genes, scientists sequence approximately 450-500 base pairs—the fundamental building blocks of DNA 2 5 . When slight variations (called single nucleotide polymorphisms) are found at these locations, they're assigned different "allele numbers." The combination of allele numbers across all seven genes creates a unique genetic profile called a diploid sequence type (DST) for each Candida albicans strain 1 2 .
Unlike methods that rely on interpreting banding patterns, DNA sequence data is unambiguous and reproducible between laboratories 2 .
MLST can distinguish between even closely related strains, a critical capability when investigating potential outbreak clusters 5 .
The investigation began when a 21-year-old woman who died of hypoxic brain injury became an organ donor, providing life-saving organs to multiple recipients 1 . What followed was every transplant team's worst nightmare:
Lost their graft 26 days post-transplant due to Candida arteritis—a dangerous fungal infection of the arteries. Candida albicans was grown from blood cultures and a perinephric blood clot.
Received antifungal prophylaxis after Candida albicans was isolated from the preservation fluid and fortunately developed no complications.
Succumbed to a ruptured pseudoaneurysm of the aortic conduit on post-transplant day 23, with Candida albicans growing from the vascular tissue 1 .
To complicate matters, another liver recipient in the same transplant unit had recently died from similar complications from Candida infection. The pressing question was: were these infections related, originating from a common source?
When traditional microbiology could only confirm that all infections were caused by Candida albicans—a fungus commonly present in the general population—investigators turned to MLST for answers. They applied this sophisticated genetic analysis to isolates from all three recipients linked to the common donor 1 .
The results were revealing: three isolates from recipients linked to donor A were genetically indistinguishable from each other and belonged to the same novel diploid sequence type. Meanwhile, two other isolates were identical but of a different novel DST 1 . These two DSTs differed significantly—at 5 of the 7 genetic loci examined—indicating they were distinct strains.
Scientists first extract DNA from pure cultures of Candida albicans isolates obtained from patient samples 5 .
Using polymerase chain reaction (PCR), they target and make millions of copies of the seven specific housekeeping genes used for Candida albicans MLST 7 .
The amplified genes are then sequenced in both directions to ensure accuracy, using the same primers employed in the initial amplification 5 .
The sequences are compared to reference databases, with each unique sequence assigned an allele number. The combination of alleles defines the diploid sequence type 2 .
Isolates with identical DSTs are considered genetically related, potentially part of the same transmission chain, while those with different DSTs are likely unrelated 1 .
The MLST results provided crucial evidence for understanding the infection cluster:
| Source of Isolate | Diploid Sequence Type (DST) | Genetic Relationship |
|---|---|---|
| Right kidney recipient | Novel DST A | Genetically indistinguishable |
| Liver recipient | Novel DST A | Genetically indistinguishable |
| Other liver recipient | Novel DST B | Different strain (5/7 loci different) |
The discovery that two recipients shared an identical novel DST provided strong evidence of a common source 1 . The fact that this strain had never been previously described in databases indicated it was not a common laboratory contaminant or widespread strain. Instead, it likely originated from the donor or the transplant process itself.
In their investigation, the researchers concluded that contamination from the gastrointestinal tract of a colonized donor was the most probable explanation, despite the absence of a direct isolate from the donor 1 . The preservation fluid—the solution used to store organs between donation and transplantation—represents another potential source, with reported yeast contamination rates in abdominal organ retrieval varying between 0.4% to 4% 1 .
| Reagent/Material | Function in MLST |
|---|---|
| Primers for 7 housekeeping genes | Target specific gene fragments for amplification |
| DNA polymerase | Enzyme that amplifies DNA segments during PCR |
| PCR purification kit | Removes excess primers and nucleotides before sequencing |
| Big Dye Terminators | Fluorescent labels for DNA sequencing reactions |
| ABI PRISM Genetic Analyzer | Capillary electrophoresis system for reading DNA sequences |
| Sabouraud dextrose agar | Growth medium for culturing Candida isolates |
| Zymolyase | Enzyme that breaks down fungal cell walls for DNA extraction |
The utility of MLST extends far beyond this single investigation. Researchers have used it to:
Identify 18 major genetic clades with distinct geographical distributions 9 .
Find that MLST clade 1 strains show higher resistance to certain antifungal drugs 7 .
| Candida Species | Number of Genes in MLST | Notable Characteristics |
|---|---|---|
| C. albicans | 7 | Most common cause of invasive candidiasis |
| C. dubliniensis | 8 | Closely related to C. albicans |
| C. glabrata | 6 | Shows inherent reduced susceptibility to azoles |
| C. tropicalis | 6 | Important cause of candidemia in neutropenic patients |
| C. krusei | 6 | Intrinsically resistant to fluconazole |
The case of the Candida cluster in transplant recipients illustrates how modern molecular techniques like Multilocus Sequence Typing have revolutionized our ability to investigate and understand infectious diseases. What once would have been written off as unfortunate but unrelated infections can now be precisely linked, revealing transmission pathways that inform crucial prevention strategies.
For solid organ transplant recipients—who face a 4.3% risk of developing invasive candidiasis, with a distressing 23.5% mortality rate within 12 weeks—such insights are literally life-saving 4 6 . The decreasing incidence of these infections in recent years 4 suggests that improved surveillance techniques, including molecular methods like MLST, are making a meaningful difference.
As MLST databases grow and sequencing technologies become more accessible, this genetic detective will continue to play an expanding role in safeguarding vulnerable patients, uncovering the hidden movements of pathogens, and turning medical mysteries into solvable cases through the power of DNA.