Metagenomic sequencing complements routine diagnostics in identifying viral pathogens in lung transplant recipients with unknown etiology of respiratory infection

Abstract

Background: Lung transplant patients are a vulnerable group of immunosuppressed patients that are prone to frequent respiratory infections. We studied 60 episodes of respiratory symptoms in 71 lung transplant patients. Almost half of these episodes were of unknown infectious etiology despite extensive routine diagnostic testing.

Methods: We re-analyzed respiratory samples of all episodes with undetermined etiology in order to detect potential viral pathogens missed/not accounted for in routine diagnostics. Respiratory samples were enriched for viruses by filtration and nuclease digestion, whole nucleic acids extracted and randomly amplified before high throughput metagenomic virus sequencing. Viruses were identified by a bioinformatic pipeline and confirmed and quantified using specific real-time PCR.

Results: In completion of routine diagnostics, we identified and confirmed a viral etiology of infection by our metagenomic approach in four patients (three Rhinovirus A, one Rhinovirus B infection) despite initial negative results in specific multiplex PCR. Notably, the majority of samples were also positive for Torque teno virus (TTV) and Human Herpesvirus 7 (HHV-7). While TTV viral loads increased with immunosuppression in both throat swabs and blood samples, HHV-7 remained at low levels throughout the observation period and was restricted to the respiratory tract.

Conclusion: This study highlights the potential of metagenomic sequencing for virus diagnostics in cases with previously unknown etiology of infection and in complex diagnostic situations such as in immunocompromised hosts.

Fig 1. Overview of visits of lung transplant cohort.

Relative timeline of visits for patients of the indication enrollment (A) and for patients of the baseline enrollment starting at the transplantation (B). Symptoms are color coded as follows: no symptoms in green, airway infection in red, diarrhea in yellow, fever in blue, other symptoms (including dyspnea and decrease in FEV1) in purple, death of the patient in black.

Fig 2. Etiology of respiratory symptoms in lung transplant patients remained undetermined in almost half of analyzed episodes.

A) Number of lung transplant patients enrolled at baseline and by indication (in total 71). Patients with baseline enrollment are grouped into those that never developed symptoms and those that developed at least one symptom (airway infection or dyspnea or decrease in FEV1) during the first 15 months after transplantation. B) Etiology of respiratory symptoms among 60 observed episodes as determined by routine diagnostic testing. Episodes for which all routine diagnostic tests were negative were summarized as unknown etiology.

Fig 3. Virus reads identified were distributed over the whole reference genomes.

Coverage plots for respiratory viruses identified in patients gkc048, nrk011 (09/14), lpt371, rcn630, fyc360. The coverage calculated with samtools depth is shown at every position on the reference genome. The following reference genomes were used for alignment: Human rhinovirus B83 strain ATCC VR-1193 (FJ445161.1) for gkc048, Human rhinovirus A46 (DQ473506.1) for lpt371, Human rhinovirus A21 strain ATCC VR-1131 (FJ445121.1) for rcn630, Human rhinovirus A24 (EF173416.1) for nrk011 (09/14), Human coronavirus HKU1 strain N5P8 (DQ339101.1) for fyc360.

Fig 4. TTV and HHV-7 viral loads did not correlate with symptoms.

TTV virus loads in blood samples (A), TTV viral loads in throat swabs (B) and HHV-7 viral loads in throat swabs (C). HC: healthy controls, B: baseline (time of transplantation) of baseline enrollment patients, S: symptomatic visits of both enrollment scenarios, NS: non-symptomatic visits of both enrollment scenarios. Undetermined values were set to -2 log10 copies/μl for plotting and statistical analysis. Error bars show median and interquartile ranges, p-values are calculated with the Mann-Whitney U test.

Fig 5. TTV virus loads increase under immunosuppressive therapy in the early post transplantation phase.

A) TTV virus loads monitored longitudinally in blood and throat swab samples. Patients with baseline enrollment are grouped into those that never developed symptoms and those that developed at least one symptom (airway infection or dyspnea or decrease in FEV1) during the first 15 months after transplantation. There was no significant difference comparing the two patient groups at time of transplantation, at the visit closest to 5 weeks and at the visit closest to one year after transplantation (Kruskal-Wallis test, data not shown). Undetermined values were set to -2 log10 copies/μl for plotting and statistical analysis. B) Same as in A, but for HHV-7 virus loads.