Next-generation sequencing of microbial cell-free DNA for rapid noninvasive diagnosis of infectious diseases in immunocompromised hosts

Introduction

Infections are a leading cause of morbidity and mortality among immunocompromised individuals^1–4. Bacteremia occurs in up to 25% of all patients with neutropenia and fever⁵. Infection is a leading cause of non-relapse mortality among hematopoietic cell transplantation (HCT) recipients⁶. The incidence of bacteremia^7–9 and double-stranded DNA viral reactivation¹⁰ is higher than 40% and 90%, respectively, within the first 100 days post-transplant. The cumulative incidence rates of proven/probable invasive fungal infections during the first year after allogeneic HCT with non-myeloablative conditioning is 19%¹¹. Infection is also a common complication of chimeric antigen receptor-modified T (CAR-T)-cell immunotherapy with 28-day cumulative incidence of 23% after CAR-T-cell infusion¹².

Establishing a microbiological diagnosis of infectious diseases in this vulnerable population is often challenging for a number of reasons. i) Prior exposure to antibiotics and antifungals which confounds the yield of blood cultures; indeed, most patients with neutropenia and fever will have no infectious etiology documented⁵. ii) Low sensitivity of mycobacterial and fungal cultures; some microorganisms, such as fastidious bacteria, mycobacteria and dimorphic fungi require longer incubation periods; and blood cultures in almost half of patients with candidemia are negative^13,14. iii) Tissue biopsies are often precluded due to the risk of bleeding in the setting of thrombocytopenia, coagulopathy in those with liver disease or hemodynamic instability in critically ill patients. A delay in diagnosis in patients with invasive fungal infection results in higher mortality^15,16. Thus, there is an unmet need for novel, rapid, cost-effective, noninvasive diagnostic methods in the field.

Cell-free DNA (cfDNA) technology has been used successfully in noninvasive prenatal testing, organ transplant rejection screening, and oncology liquid biopsies^17–22. In recent years, this technology has been developed for use in infectious disease diagnostics^23,24. Detection of microbial cfDNA by next generation sequencing (NGS) is an accurate and precise way of identifying and quantifying pathogens²⁵. The Karius^® Test relies on sequencing of microbial cfDNA circulating in plasma to identify over 1,000 pathogens, including bacteria, viruses and fungi, from a 5 ml blood sample²⁵. This novel diagnostic tool has been recently validated in a study showing that microbial cfDNA NGS identified 94% of microbes identified by conventional blood culture in patients with sepsis²⁵ and has excellent correlation with quantitative PCR testing in patients with cytomegalovirus (CMV)^23,25.

Recent reports indicate that NGS measuring microbial cfDNA is useful in the diagnosis of cases of Streptococcus pneumoniae-related hemolytic uremic syndrome, Coxiella burnetii endocarditis, invasive Mycobacterium chimaera infection, Nocardia cyriacigeorgica pneumonia, Capnocytophaga canimorsus sepsis, M. tuberculosis complex and M. haemophilum infections, M. bovis aortitis; Candida spp., Aspergillus spp., non-Aspergillus molds invasive infections; Pneumocystis jirovecii pneumonia (PJP), Toxoplasma gondii infection and chorioamnionitis, among others^24,26–33. Among 21 patients with culture-positive infective endocarditis, cfDNA NGS identified the same organism as blood cultures in 20 patients (95% sensitivity) and additionally identified Enterococcus faecalis in one out of the three patients with definitive culture-negative endocarditis³⁴. Of note, in this study the cfDNA NGS test identified pathogens causing endocarditis in patients pre-treated with antibiotics up to 30 days prior to initial sample collection.

Here we evaluated the clinical utility of NGS for detection of microbial cfDNA in plasma in a cohort of ten patients receiving chemotherapy or transplants with episodes of febrile neutropenia, sepsis or documented infection.

Methods

Results

Discussion

Here we report our experience using cfDNA NGS in the evaluation of immunocompromised patients—predominantly those with hematological malignancy—with febrile illness or documented invasive infections. The study cohort included a heterogeneous group of clinical scenarios, including deep-seated pyogenic abdominal infection, pulmonary nodules/pneumonia, neutropenic fever, and septic shock. The results of this proof-of-concept study, where most of the patients had an established diagnosis of infection prior to NGS testing, complement recent reports studying the use of cfDNA NGS in immunocompromised hosts. In a recent study of 55 patients with neutropenic fever, cfDNA testing had positive agreement with conventional blood cultures in 9 of 10 patients in whom blood cultures identified a causative organism of sepsis. Using clinical adjudication by three infectious diseases specialists, cfDNA NGS had a sensitivity of 85.4% (41/48) and specificity of 100% (7/7)³⁶. Thus, this test is a promising diagnostic tool in neutropenic fever, a clinical scenario where conventional work up fails to identify an etiological agent in a majority of cases⁵. Another study evaluated 40 patients with prolonged neutropenia and fever (>96h) despite administration of antibiotics for suspected fungal infection (the authors excluded patients who had received antifungal therapy for >3 days); in this study cfDNA NGS identified fungal pathogens including Aspergillus fumigatus, Rhizopus spp., Candida albicans, Candida glabrata and Pneumocystis jirovecii³⁷.

In a recent report by Hong et al.²⁴, in seven out of nine subjects (including seven immunocompromised hosts) with proven deep-seated invasive fungal infection, plasma NGS testing detected the same fungus identified from the biopsy tissue at the genus level. The fungi identified by plasma NGS included Aspergillus spp. and non-Aspergillus molds such as Scedosporium, Rhizopus, and Cunninghamella²⁴. In that report, there was one case where the plasma sample was obtained after at least 15 days of anti-Aspergillus therapy, and NGS testing did not identify the causal organism of invasive fungal infection. Similarly, for the kidney transplant patient reported here with invasive aspergillosis, in whom Aspergillus fumigatus cfDNA levels in plasma were detected below the reporting threshold, several months of antifungal therapy had been administered prior to the time of plasma collection. Thus, prolonged antifungal therapy prior to sample collection (e.g., >7-14 days) might interfere with detection of fungal DNA.

Although NGS has been used for screening of allograft rejection in solid organ transplant recipients^17–19,23, there is limited data with the use of NGS for diagnosis of infections in this population. A recent study demonstrated strong correlation between clinical test results and cfDNA derived from CMV in a cohort of lung transplant recipients²³. In addition, cfDNA revealed undiagnosed cases of infection with microsporidia and pathogenic viruses, including adenovirus and human herpesvirus 6 among lung transplant patients²³.

Recently, Fung et al. reported three patients who received allogeneic HCT transplant in whom NGS cfDNA facilitated the diagnosis of an uncommon presentation of Chlamydia trachomatis and recurrent and metastatic complications of Staphylococcus aureus bacteremia before standard microbiology³⁸.

The fact that in our cohort cfDNA NGS testing identified the cause of febrile illness in two patients with culture-negative sepsis who had a compatible clinical syndrome and responded well to antibiotic therapy supports the notion that NGS testing can be a useful diagnostic tool, particularly when conventional blood cultures are negative. The Karius^® Test pathogen-specific reference ranges have been established using cfDNA levels from healthy donors. Patient #8 had detectable levels of Torque teno virus, which belongs to Anelloviridae family and is considered to lack pathogenic potential; this suggests the possibility that cfDNA NSG might on occasion yield detection of members of the commensal microbiota or viroma. To our surprise, however, even though many of the patients tested had mucosal barrier damage (e.g., mucositis) allowing for bacterial translocation from the gut, the Karius^® Test did not show a non-specific gut flora signal. The test was negative in patients in whom we failed to establish a microbiological diagnosis for their febrile illness, and when positive, typically correlated with conventional laboratory testing. Whether the currently defined cfDNA thresholds are optimal for identifying and quantifying pathogens of clinical relevance in highly vulnerable immunocompromised hosts will require further study. Importantly, the turnaround time for results was consistently within 48 hours, which is quite rapid considering that samples were shipped overnight from our institution located in Florida to the Karius Inc. laboratory in California.

Lack of control group, small number of patients and the heterogeneity of the cohort in terms of underlying diseases and causes of immunosuppression represent major limitations of this report. Larger clinical trials evaluating plasma NGS in patients with cancer and undergoing transplant are ongoing (NCT03226158, NCT03262584, NCT02912117, NCT02804464). Until larger cohort data becomes available, our observations suggest that detection of microbial cfDNA using NGS is valuable for the rapid noninvasive diagnosis of infectious complications following chemotherapy or transplantation.

Conclusion

Our data, along with a number of recent reports, support the clinical utility of the measurement of microbial cfDNA in peripheral blood using NGS for rapid noninvasive diagnosis of infections in immunocompromised hosts. As with other novel laboratory diagnostics used in clinical practice, the results of cfDNA NGS technology need to be interpreted with caution and in conjunction with other laboratory, radiological and clinical findings. Larger studies are needed to validate these findings.

Data availability