千叶大学基于随机PCR的纳米孔全基因组测序可在血培养阳性前约7小时鉴定真菌血流感染

Bloodstream infections caused by fungi present clinical microbiology laboratories with a difficult combination of urgency and technical complexity. Appropriate antifungal treatment depends upon knowing which organism is responsible for the infection, yet conventional identification requires sufficient microbial growth to occur before the laboratory can move to more definitive testing.

By Tim Sandle 

Researchers at Chiba University in Japan have developed a genomic workflow intended to shorten this diagnostic interval. In a study published in Microbiology Spectrum, the researchers report species-level identification of fungal pathogens from blood culture samples in approximately seven hours, and importantly, before automated blood culture systems indicated that the samples were positive. The research is described by Chiba University.

This development is significant because rapid organism identification represents an important component of effective management of bloodstream infection. Different fungal species can respond differently to antifungal agents, making accurate identification relevant to the selection of appropriate treatment.

Getting ahead of blood culture positivity

Conventional blood culture depends upon microorganisms multiplying until their growth can be detected. According to the Chiba University researchers, culture followed by identification testing can take several days to complete. The new method takes a different approach. Instead of waiting for the culture instrument to signal positivity, the scientists remove a sample while the blood culture is still incubating and seek to recover and characterize microbial DNA directly.

The workflow developed by the group led by Hiroki Takahashi, Professor at the Medical Mycology Research Center, Chiba University, consists of three principal stages.

The first challenge is that blood contains a large quantity of human material. The researchers selectively break down human cells before using benzonase to degrade the released human DNA, while leaving fungal and bacterial DNA unaffected. This increases the relative proportion of microbial DNA available for subsequent analysis. The second stage uses PCR-based whole-genome amplification. This produces multiple copies of DNA fragments from across the genomes contained within the specimen and provides sufficient genetic material for sequencing. 

Finally, the amplified material undergoes nanopore sequencing. Rather than requiring the entire sequencing run to finish before analysis can begin, nanopore technology produces sequence data continuously. The resulting sequences are compared with a purpose-built reference database containing genomic information for fungal and bacterial organisms associated with bloodstream infections. This combination of sample preparation, amplification and real-time sequencing is what enables the researchers to seek an identification before conventional blood culture positivity.

Testing clinical blood cultures

The researchers evaluated the process using 48 clinical blood culture samples covering eight fungal species. Chiba University reports that species-level identification was achieved in approximately seven hours with high accuracy. Organisms identified included Candida albicans, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans and Nakaseomyces glabratus. The last organism is particularly familiar to laboratories under its former name, Candida glabrata, although the Chiba report uses the currently accepted Nakaseomyces nomenclature.

A further interesting feature was the ability to recognize more complicated specimens. The researchers detected mixed infections in some samples, including cultures containing two fungal species and others containing fungal and bacterial organisms. This is potentially important for diagnostic microbiology because a dominant organism can complicate the detection of other microorganisms within a mixed population. That difficulty has not been completely overcome. The researchers specifically identify heavy bacterial growth masking fungal signals in mixed infections as an area requiring further work. They also need to establish the optimum point during blood-culture incubation at which a specimen should be taken for genomic analysis. 

These limitations are important. The work demonstrates the potential of the approach, but it should not yet be interpreted as a replacement for established diagnostic workflows.

Genomics provides another layer of information

Species identification is not the only information potentially available from the sequencing process. Because the method obtains genomic information from the pathogen, the researchers state that it can identify genetic variants in genes associated with antifungal drug resistance. This presents an interesting direction for clinical microbiology. Rapidly determining the likely organism is valuable, but coupling identification with genomic information associated with resistance could make sequencing more useful for antimicrobial stewardship.

Phenotypic susceptibility testing and genomic resistance detection do not, however, answer precisely the same question. Consequently, the practical value will depend upon how reliably particular genetic markers predict clinically meaningful antifungal resistance and how genomic information is incorporated into validated laboratory procedures. The study itself is titled Random PCR-based nanopore whole-genome sequencing enables pre-positivity detection of fungal bloodstream infections and was published online on August 21, 2026, in Microbiology Spectrum.

Why speed matters

The technological advance needs to be considered in the context of treatment decisions. Until the causative microorganism is known, clinicians may need to make treatment decisions with incomplete microbiological information. Chiba University notes that during the conventional diagnostic interval patients may receive broad antifungal treatment, while more targeted therapy has to await further information. 

Faster microbiological identification therefore has the potential to reduce some of this uncertainty. “Our method may enable clinicians to initiate appropriate antifungal treatment earlier, potentially improving outcomes for patients with life-threatening fungal bloodstream infections,” Hiroki Takahashi states.

The word “may” is important. The present study demonstrates analytical and diagnostic potential. It does not, on the evidence reported by the university, demonstrate improved patient outcomes resulting from use of the workflow.

From proof of concept to routine microbiology

For pharmaceutical and clinical microbiologists, perhaps the most interesting aspect of the research is how several existing molecular technologies have been integrated around a particular diagnostic bottleneck. The significant step is not nanopore sequencing in isolation. It is the complete sample-to-identification strategy: removing unwanted host DNA, increasing microbial genomic material through amplification and exploiting real-time sequencing sufficiently early in blood-culture incubation to obtain information before the automated culture signal. 

There are still questions to address before such an approach could progress toward broader routine use. Chiba University says further validation is needed, together with work to optimize the sampling time and improve fungal detection where substantial bacterial growth is present. 

Nevertheless, the study illustrates a broader change taking place within diagnostic microbiology. Culture remains enormously important, but genomic technologies are increasingly creating opportunities to obtain actionable information without waiting for all of the traditional microbiological process to be completed.

For fungal bloodstream infections, where identifying the infectious agent is particularly important to choosing suitable antifungal therapy, moving identification even a day earlier could prove valuable. The Chiba University work suggests that it may be possible to move considerably further upstream, interrogating the microbial genome while the blood culture bottle is still incubating.