Bloodstream infection can rapidly progress to sepsis with each delay to appropriate
therapy. In particular, patients with advanced age, active cancer, hematological disease,
immunocompromised status, or critical illness are prone to severe infection (1). The
main characteristics of infection in these patients are extremely rapid progression
and resistance to conventional broad-spectrum antibiotics. Early identification of
pathogens is imperative for reducing the risk of death (2). However, standard diagnostic
methods rely on culturing, which has poor sensitivity and is not suitable for atypical
infections with fastidious organisms. Even with typical pathogenic microorganisms,
the turnaround time ranges from 48 to 96 hours. Therefore, culturing is always too
slow to guide targeted antimicrobial therapy.
Based on third-generation sequencing technology, we directly incorporated a panel
of microbial tags and developed nanopore-targeted sequencing (NTS). Using real-time
NTS, we successfully identified pathogens under complex host-microorganism settings,
such as whole blood samples. This approach can reduce testing time and clinical turnaround
time to less than 2 and 6 hours, respectively, enabling early targeted therapy.
The detailed protocol and evaluation of NTS have been published elsewhere as a methodological
study (3,4). Briefly, instead of a metagenomic analysis, a panel of 27,668 microbial
tags (including bacteria, fungi, viruses, drug-resistant genes, and atypical pathogens),
were incorporated into the nanopore sequencing platform. Therefore, targeted sequencing,
with full-length read and real-time analytic approaches, was developed. Pathogens
with moderate-to-high abundance were reported within 2 hours after sequencing, and
pathogens with low abundance were reported within 8 hours after sequencing. Thus,
a total clinical turnaround time of 6 to 18 hours can be achieved. Background noise
was filtered out using negative controls. The cut-off value for a positive diagnosis
was 20 reads. The limit of detection was 25 colony-forming units/mL.
This retrospective case series study was registered at the Chinese Clinical Trial
Registry (www.chictr.org.cn, No. ChiCTR2000028904), and was approved by the Ethics
Committee of the Union Hospital affiliated with the Huazhong University of Science
and Technology (IRB approval ID: 2019-S316). Written informed consent was obtained
from all patients to publish this study.
Eleven patients with hematologic disorders who underwent NTS diagnosis between November
and December 2019 were consecutively enrolled and retrospectively reviewed. Ten patients
underwent hematopoietic stem cell transplantation (Table S1). In 6 patients, the procalcitonin
(PCT) or C-reactive protein (CRP) levels were more than 20 ng/mL or 100 mg/L, respectively,
during the first episode of fever. For each infection, routine microbiological culturing
(Appendix 1) was performed at least once, while NTS was performed only once. NTS sampling
was performed at the same time as the culture sampling.
Traditional cultures were able to detect a pathogen in only 2 of the 11 cases. NTS
identified pathogenic microorganisms or conditional pathogenic microorganisms in all
11 cases (100%), with a clinical turnaround time within 6 hours in 9 cases. According
to The Sanford Guide to Antimicrobial Therapy 2018 (5), appropriate antibiotics were
immediately prescribed (Appendix 1). One patient died of severe sepsis (PCT >100 ng/mL)
23 hours after the first episode of hyperpyrexia; and the CRP level in the other 10
cases decreased steadily, and the infection was completely controlled (
Table 1
).
Table 1
Results of nanopore targeted sequencing testing and clinical effect
Case
PCT (ng/mL) & CRP (mg/L)*
Organism culture (clinical turnaround time)†
NTS (clinical turnaround time)
Early targeted antibiotic therapy
Clinical outcome
1
51.8/198
Blood (–), 9 times
First infection: Streptococcus mitis (blood, 18 h, reads 37, coverage 98.7%); Second
infection: Pandoraea sputorum (blood and pharyngeal swab, 6 h, reads 1,875/1,239,
coverage 97.7%/97.7%)
Cefoperazone-sulbactam + daptomycin + caspofungin; followed by Imipenem + sulfamethoxazole
+ caspofungin
Resolution after 41 days
2
1.17/116
Blood (–), 3 times
Pandoraea sputorum (blood, 6 h, reads 2,260, coverage 98.1%)
Imipenem + sulfamethoxazole
Resolution after 10 days
3
1.22/156
Blood (–), 4 times
Enterobacter cancerogenus (blood, 18 h, reads 41, coverage 97.6%)
Meropenem + colistin
Resolution after 10 days
4
0.3/114
Blood (–), twicesputum (–), once
Escherichia coli (blood, 18 h, reads 117, coverage 97.1%)
Cefoperazone-sulbactam
Resolution after 5 days
5
21.05/58.2
Candida tropicalis (blood, 62 h)
Candida tropicalis (blood and anal swab, 6 h, reads 72,295/4,389, coverage 99.2%/99.5%)
Meropenem + caspofungin
PCT increased to more than 100 ng/mL; CRP increased to 231 mg/L; Death from sepsis
within 23 hours
6
0.34/117.3
B (–), 3 times
Candida albicans (blood, 6 h, reads 23,112, coverage 98.7%)
Micafungin
Resolution after 9 days
7
0.85/25
Fecal (–)
Acinetobacter baumannii (fecal and anal swab, 6 h, reads 9,892/32,430, coverage 97.7%/98.9%)
Etimicin + colistin
Resolution after 7 days
8
1.26/148
Pseudomonas aeruginosa (sputum, 50 h); blood (–), 6 times
Escherichia coli (blood and anal swab, 6 h, reads 209, coverage 98.1%); Alcaligenes
faecalis (blood, 6 h, reads 210, coverage 98.7%); Candida tropicalis (blood, 6 h,
reads 4,540, coverage 97.5%)
Imipenem + ciprofloxacin + caspofungin; followed by piperacillin-tazobactam + colistin
+ voriconazole
Resolution after 21 days
9
0.82/37.5
Blood (–), twice
Candida albicans (blood, 6 h, reads 27,682, coverage 97.7%)
Micafungin
Resolution after 11 days
10
0.17/71.2
Blood (–), twice
Candida parapsilosis (blood, 6 h, reads 10,395, coverage 97.2%); Enterobacter cancerogenus
(blood, 18 h, reads 78, coverage 98.9%)
Meropenem + caspofungin + amphotericin B
Resolution after 14 days
11
2.06/21
Blood (–), once
Streptococcus oralis (blood, 6 h, reads 1,868, coverage 98.2%); Pseudomonas gessardii
(blood, 6 h, reads 1,342, coverage 97.9%); Klebsiella pneumonia (blood, 18 h, reads
23, coverage 99.6%)
Gentamicin + linezolid + tegacycline
Resolution after 8 days
The prophylactic antibiotics in this setting of HSCT for each patient are “Moxifloxacin
or Gentamicin” + oral “Fluconazole or Voriconazole”. *, PCT and CRP tests at the first
episode of fever; Normal levels: PCT <0.5 ng/mL, CRP <8 mg/L; †, clinical turnaround
time: from sampling to report. CRP, C-reactive protein; PCT, procalcitonin; NTS, nanopore
targeted sequencing.
As an example, in case 1, a 15-year-old girl diagnosed with acute lymphoblastic leukemia,
underwent haploidentical hematopoietic stem cell transplantation (Figure S1). Laminar-flow
ward admission was used as the reference point for day 1. At first, she received preventive
antimicrobial therapy (gentamicin + fluconazole + ganciclovir). After myeloablative
conditioning, allogeneic transplantation of both the peripheral blood and bone marrow
stem cells from a haploidentical donor (her father) was performed. Upon neutropenia,
PCT and CRP levels rapidly increased to 51.8 ng/mL and 198 mg/L, respectively. Because
of sepsis, broad-spectrum antibiotics were empirically administered (cefoperazone
+ tegacycline + caspofungin). Four days later, the first NTS was performed, which
revealed a bloodstream infection with Streptococcus mitis. The inflammatory markers
decreased gradually after intravenous daptomycin treatment, and she recovered after
granulocyte engraftment. However, recurrent hyperpyrexia and grade 4 oral mucositis
occurred 2 days later. The second NTS revealed highly abundant, multidrug-resistant
Pandoraea sputorum in both blood and pharyngeal swab samples within 6 hours after
sampling (Figure S2). The antimicrobial strategy was immediately replaced with imipenem
combined with sulfamethoxazole. Infection was eventually controlled on day 51. No
microorganisms were detected in additional NTS tests (data not shown). During the
entire course of infection, no pathogens were detected in any of the 9 routine blood
cultures.
Third-generation sequencing technology, including the Oxford Nanopore Technologies
platform, is widely employed in metagenomics studies involving animal, plant, and
microorganism samples (6-9). Nanopore sequencing may overcome many shortcomings faced
by next-generation sequencing as a diagnostic tool. It can generate read-by-read data
with individual read lengths of tens and thousands of nucleotides, by utilizing cost-effective
and real-time long-read sequencing strategy. Nanopore sequencing provides higher resolution,
greater accuracy, and faster turnaround time, than do currently available commercial
techniques. Therefore, its implementation in the clinical setting will markedly improve
real-time point-of-care pathogen diagnosis (10-13). In one study, nanopore metagenomics
was developed to enable the rapid diagnosis of bacterial lower respiratory infections
using respiratory samples. Compared with culturing, the optimized method was 96.6%
sensitive and 41.7% specific for bacteria detection, with a minimum turnaround time
of 6 hours under experimental conditions (14). In contrast, we developed a novel NTS
rather than the metagenomics method, and presented the first clinical report that
real-time NTS can timely identify pathogens in whole blood samples from patients with
severe infection. Targeted microbial tags enable pathogen detection in whole blood
samples, a more complex host-microorganism setting, and further shorten the testing
duration. In this study, NTS detected multiple pathogens including uncommon and atypical
pathogens in each patient within 6 to 18 hours in a clinical context. As a result,
these patients with severe infections benefited from early targeted antimicrobial
therapy. Moreover, our method can be expanded to patients in different clinical settings.
In conclusion, NTS is a promising approach for rapidly and accurately characterizing
pathogens, and guiding antimicrobial treatment. This application should be confirmed
and optimized for future clinical practice.
Supplementary
The article’s supplementary files as
10.21037/atm-21-2923
10.21037/atm-21-2923