Secreted small RNAs of Naegleria fowleri are biomarkers for diagnosis of primary amoebic meningoencephalitis

  1. University of Georgia, Department of Infectious Diseases, Athens, United States
  2. University of Georgia, Center for Tropical and Emerging Global Diseases, Athens, United States
  3. AdventHealth Central Florida, Microbiology Department, Orlando, United States
  4. Centers for Disease Control and Prevention, Waterborne Disease Prevention Branch, DFWED/NCEZID, Atlanta, United States
  5. University of Georgia, Department of Cellular Biology, Athens, United States

Peer review process

Revised: This Reviewed Preprint has been revised by the authors in response to the previous round of peer review; the eLife assessment and the public reviews have been updated where necessary by the editors and peer reviewers.

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Editors

  • Reviewing Editor
    Christine Clayton
    Centre for Molecular Biology of Heidelberg University (ZMBH), Heidelberg, Germany
  • Senior Editor
    Joshua Schiffer
    Fred Hutchinson Cancer Research Center, Seattle, United States of America

Reviewer #1 (Public review):

Summary:

Early and accurate diagnosis is critical to treating N. fowleri infections, which often lead to death within 2 weeks of exposure. Current methods are based on sampling cerebrospinal fluid, and are invasive, slow, and sometimes unreliable. Therefore, there is a need for a new diagnostic method. Russell et al. address this need by identifying small RNAs secreted by Naegleria fowleri (Fig. 1) that are detectable by RT-qPCR in multiple biological fluids including blood and urine. SmallRNA-1 and smallRNA-2 were detectable in plasma samples of mice experimentally infected with 6 different N. fowleri strains, and were not detected in uninfected mouse or human samples (Fig. 4). Further, smallRNA-1 is detectable in the urine of experimentally infected mice as early as 24 hours post infection (Fig. 5). The study culminates with testing human samples (obtained from the CDC) from patients with confirmed N. fowleri infections; smallRNA-1 was detectable in cerebrospinal fluid in 6 out of 6 samples (Fig. 6B), and in whole blood from 2 out of 2 samples (Fig. 6C). These results suggest that smallRNA-1 could be a valuable diagnostic marker for N. fowleri infection, detectable in cerebrospinal fluid, blood, or potentially urine.

Strengths:

This study investigates an important problem, and comes to a potential solution with a new diagnostic test for N. fowleri infection that is fast, less invasive than current methods, and seems robust to multiple N. fowleri strains. The work in mice is convincing that smallRNA1 is detectable in blood and urine early in infection. Analysis of patient blood samples shows that whole blood could be tested for smallRNA-1 to diagnose N. fowleri infections. The potential for human blood or urine to be tested for N. fowleri could lead to critical early interventions.

Weaknesses:

There are not many N. fowleri cases, so the authors were limited in the human samples available for testing. It is difficult to know how robust this biomarker is in whole blood, serum, or human urine due to little to no sample material being available for testing. This limitation is examined thoroughly in the discussion section, and additional tests are beyond the scope of this work.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public review):

Summary:

Early and accurate diagnosis is critical to treating N. fowleri infections, which often lead to death within 2 weeks of exposure. Current methods-sampling cerebrospinal fluid are invasive, slow, and sometimes unreliable. Therefore, there is a need for a new diagnostic method. Russell et al. address this need by identifying small RNAs secreted by Naegleria fowleri (Figure 1) that are detectable by RT-qPCR in multiple biological fluids including blood and urine. SmallRNA-1 and smallRNA-2 were detectable in plasma samples of mice experimentally infected with 6 different N. fowleri strains, and were not detected in uninfected mouse or human samples (Figure 4). Further, smallRNA-1 is detectable in the urine of experimentally infected mice as early as 24 hours post-infection (Figure 5). The study culminates with testing human samples (obtained from the CDC) from patients with confirmed N. fowleri infections; smallRNA-1 was detectable in cerebrospinal fluid in 6 out of 6 samples (Figure 6B), and in whole blood from 2 out of 2 samples (Figure 6C). These results suggest that smallRNA-1 could be a valuable diagnostic marker for N. fowleri infection, detectable in cerebrospinal fluid, blood, or potentially urine.

Strengths:

This study investigates an important problem, and comes to a potential solution with a new diagnostic test for N. fowleri infection that is fast, less invasive than current methods, and seems robust to multiple N. fowleri strains. The work in mice is convincing that smallRNA1 is detectable in blood and urine early in infection. Analysis of patient blood samples suggest that whole blood (but not plasma) could be tested for smallRNA-1 to diagnose N. fowleri infections.

Thank you for comments regarding the strengths of this study. We agree that our data for detecting the biomarker in biofluids from mice is convincing. In addition, our spike-in studies with human cerebrospinal fluid, plasma, and urine (Figure 6) suggest these biofluids from humans could be used for diagnosis.

We appreciate the comment regarding plasma and recognize this was not fully explained in the manuscript. We do believe that plasma can be used to assess the biomarker. Firstly, we demonstrated equivalent sensitivity of the method to detect smallRNA-1 in plasma and urine in mice with end-stage PAM (Figure 5). In addition, spike in samples of human plasma, cerebrospinal fluid, and urine demonstrated equivalent sensitivity of detecting the biomarker (Figure 6).

The negative result for human plasma in Figure 6C requires clarification; this sample was convalescent plasma from a survivor. The patient presented to the hospital on August 7, 2016, was treated, made a remarkable recovery, and was released from the hospital later that month. The plasma sample in Figure 6C was collected September 7, 2016, which is a month after treatment was initiated and weeks after the patient was symptom free. Our interpretation of the convalescent plasma result is the patient had cleared the active amoeba infection and that is why we did not detect the biomarker. We have added text in the discussion and in the legend for Figure 6 to clarify the convalescent plasma result.

One additional caveat for consideration is that many of the samples we received from amoebaeinfected humans were stored at room temperatures for undefined periods of time before being moved to <-20°C (see details in Table S9). We can’t rule out possible sample degradation, but this is an unfortunate reality of obtaining human samples from individuals later confirmed to be infected with pathogenic free-living amoebae.

Weaknesses:

(1) There are not many N. fowleri cases, so the authors were limited in the human samples available for testing. It is difficult to know how robust this biomarker is in whole blood (only 2 samples were tested, both had detectable smallRNA-1), serum (1 out of 1 sample tested negative), or human urine (presumably there is no material available for testing). This limitation is openly discussed in the last paragraph of the discussion section.

We agree the extremely limited availability of human samples is a limitation of this study. Given the rarity of these infections in the United States, even prospective studies to systematically collect samples would be very challenging. We hope that by publishing the details of this biomarker detection is that the method can be used by diagnostic reference centers, especially in areas where outbreaks of multiple cases per year have been reported.

(2) There seems to be some noise in the data for uninfected samples (Figures 4B-C, 5B, and 6C), especially for those with serum (2E). While this is often orders of magnitude lower than the positive results, it does raise questions about false positives, especially early in infection when diagnosis would be the most useful. A few additional uninfected human samples may be helpful.

We agree; however, we would like to point out the progression of disease in humans and mice are similar. Typically, patients survive between 10-14 days after presumed exposure and mice have similar survival times following instillation of N. fowleri amoebae into a nare of the mouse. Therefore, detection of this biomarker as early as 72 h in mice is seemingly equivalent to the onset of initial symptoms in humans.

Reviewer #2 (Public review):

Summary:

The authors sought to develop a rapid and non-invasive diagnostic method for primary amoebic meningoencephalitis (PAM), a highly fatal disease caused by Naegleria fowleri. Due to the challenges of early diagnosis, they investigated extracellular vesicles (EVs) from N. fowleri, identifying small RNA biomarkers. They developed an RT-qPCR assay to detect these biomarkers in various biofluids.

Strengths:

(1) This study has a clear methodological approach, which allows for the reproducibility of the experiments.

(2) Early and Non-Invasive Diagnosis - The identification of a small RNA biomarker that can be detected in urine, plasma, and cerebrospinal fluid (CSF) provides a non-invasive diagnostic approach, which is crucial for improving early detection of PAM.

(3) High Sensitivity and Rapid Detection - The RT-qPCR assay developed in the study is highly sensitive, detecting the biomarker in 100% of CSF samples from human PAM cases and in mouse urine as early as 24 hours post-infection. Additionally, the test can be completed in ~3 hours, making it feasible for clinical use.

(4) Potential for Disease Monitoring - Since the biomarker is detectable throughout the course of infection, it could be used not only for early diagnosis but also for tracking disease progression and monitoring treatment efficacy.

(5) Strong Experimental Validation - The study demonstrates biomarker detection across multiple sample types (CSF, urine, whole blood, plasma) in both animal models and human cases, providing robust evidence for its clinical relevance.

(6) Addresses a Critical Unmet Need - With a >97% case fatality rate, PAM urgently requires improved diagnostics. This study provides one of the first viable liquid biopsy-based diagnostic approaches, potentially transforming how PAM is detected and managed.

Thank you for summarizing the strengths of the study.

Weaknesses:

(1) Limited Human Sample Size - While the biomarker was detected in 100% of CSF samples from human PAM cases, the number of human samples analyzed (n=6 for CSF) is relatively small. A larger cohort is needed to validate its diagnostic reliability across diverse populations.

As noted in response to Reviewer #1 above, we agree this is a limitation of the study; however, we were fortunate to obtain even 15 µL samples of cerebrospinal fluid, plasma, serum, or whole blood from as many patients as we did. There is an urgent need for more systematic collection and storage of samples for rare diseases like primary amoebic meningoencephalitis so that advancements in diagnostics and biomarker discovery can be conducted. It is our sincere hope that by publishing our detailed methods and experimental results in this manuscript, that additional hospitals and research centers can replicate our studies and help advance this or other techniques for early diagnosis of PAM.

(2) Lack of Pre-Symptomatic or Early-Stage Human Data - Although the biomarker was detected in mouse urine as early as 24 hours post-infection, there is no data on whether it can be reliably detected before symptoms appear in humans, which is crucial for early diagnosis and treatment initiation.

It is difficult to envision a method to obtain these biofluids from infected humans prior to onset of symptoms. More likely the best we can hope for is that physicians include primary amoebic meningoencephalitis in their assessment of patients that present with prodromal symptoms of meningitis.

(3) Plasma Detection Challenges - While the biomarker was detected in whole blood, it was not detected in human plasma, which could limit the ease of clinical implementation since plasma-based diagnostics are more common. Further investigation is needed to understand why it is absent in plasma and whether alternative blood-based approaches (e.g., whole blood assays) could be optimized.

See response to Reviewer #1 above.

Reviewer #1 (Recommendations for the authors):

(1) What is the evidence that these small RNAs are secreted specifically in EVs? I believe that they are, and ultimately it doesn't impact the conclusions, but I think the evidence here could be either stronger or presented in a more obvious way.

Our data demonstrates that smallRNA-1 is present in N. fowleri-derived EVs (Figures 2 and Supplemental Figure 7) and in the intact amoebae (Figure 3B). Initial sequencing data to identify these smallRNA biomarkers came from PEG-precipitated EVs (Figure S1), by using methods we previously published (22). The PEG-precipitated EVs were extracted specifically for spike in studies. Finally, the smallRNAs in EVs were confirmed after extraction of EVs from 7 N. fowleri strains (Figure 2). We do not have evidence that they are secreted outside of EVs.

(2) The figure legends would be more useful with some additional information. For example: why are there two points for Nf69 in Fig 2B? In Figure 3A-B, please add more detail as to what the graphs are showing (are they histograms binned by a number of amoebae? This does not seem obvious to me).

We agree the Figure legends should be edited for clarity and to add additional information. Both Figure legends have been updated.

In Figure 2B, each point represents the mean of three technical replicates of EV preps for each N. fowleri strain.

In Figure 3 the points indicate the Copy#/µL of a well from a 96-well plate. The histograms show the mean of these observations for each condition.

(3) In Figure 2E, the FBS seems like it has near detectable levels of smallRNA-1 compared to Ac and Bm (albeit N. fowleri has 4 orders of magnitude higher levels than the FBS). Because cows are likely exposed to N. fowleri and have documented infections (e.g. doi: 10.1016/j.rvsc.2012.01.002), is it possible this signal is real?

Thank you for making this interesting observation. We agree that cows are likely to have significant exposure to N. fowleri, yet documented infections are rare. In this case we do not believe the near detectable levels of smallRNA-1 in FBS was due to an infected donor animal. This noise was likely due to extracting RNA from concentrated FBS rather than FBS diluted in cell culture media. In addition, as shown in Supplemental Figure 4, the qPCR product from EVs extracted from FBS were not the same as that from the N. fowleri-derived EVs. Please note we used a PEG extraction reagent that separates lipid particles, so this is additional evidence the smallRNAs are present in EVs.

(4) In Figure 6A, why was the sample size greater for water and unspiked urine? Similarly, why is the number of infected mice so variable in Figure 4B?

In Figure 6A we assayed de-identified biofluids provided by Advent Hospital in Orlando, Florida. The plasma and serum samples were pooled from multiple individuals; whereas, individual urine samples (n=8) were provided for this experiment. We have updated the legend for Figure 6A to include these details.

For Figure 4B we used plasma collected at the end-stage of disease following infections with five different strains of N. fowleri. The sample sizes varied for two reasons. First, Nf69 was the strain used most by our lab and we had plasma from several in vivo experiments. The lower sample sizes for the other strains came from an experiment with 8 mice per group. Some of these strains were less virulent and did not succumb to disease with the number of amoebae inoculated in this experiment. Thus, plasma was only collected from animals that were euthanized due to severe N.

fowleri infections. In follow up studies (e.g., Figure 5B), plasma was collected every 24 hr for analysis.

Very minor points:

(1) The number of acronyms (FLA, PAM, EVs, CNS, CSF, LOD) could be reduced to make this paper more reader-friendly.

Acronyms that were used infrequently in the manuscript (FLA, CNS, LOD, mNGS, UC) have been edited to spell out the complete names. We kept the acronyms EVs and CSF because they are each used more than twenty times in the manuscript.

(2) The decimal point in the Cq values is formatted strangely.

The decimal points have been edited to normal format in both the manuscript and supplementary material.

(3) Figure 3C is not intuitive. I do not understand the logic for the placement of the different samples (was row A only amoebae, B only Veros, C blank, D a mix, and F more Veros?).

Thank you for this comment; we agree the microtiter plate schematic (Fig 3C) was misleading. We have revised Figure 3C to make the point that we tested amoebae alone, Vero cells alone, and we combined supernatants from Vero cells (alone) plus amoebae (alone) to confirm that 1) smallRNA-1 was only detected in amoeba-conditioned media, and 2) that Vero-conditioned media does not affect detection of smallRNA-1.

Reviewer #2 (Recommendations for the authors):

Minor corrections:

The abbreviation 'Nf' for Naegleria fowleri is not appropriate in a scientific publication. According to taxonomic conventions, the correct way to abbreviate a scientific name is as follows:

The first mention should be written in full: Naegleria fowleri.

In subsequent mentions, the genus name should be abbreviated to its initial in uppercase, followed by a period, while the species name remains in lowercase: N. fowleri.

The same rule applies to Balamuthia mandrillaris and Acanthamoeba species, which should be abbreviated as B. mandrillaris and Acanthamoeba spp. after their first mention.

We agree and each of the scientific names have been updated to the proper format. Please note Nf69 is the accepted nomenclature for this N. fowleri strain, so no changes were made when referring to this specific strain.

Temperatures should be expressed in international units (°C). Please update the temperatures reported in Fahrenheit (°F) in the 'Materials and Methods' section, specifically in the 'Animal Studies' subsection.

These changes were made in the revised manuscript.

  1. Howard Hughes Medical Institute
  2. Wellcome Trust
  3. Max-Planck-Gesellschaft
  4. Knut and Alice Wallenberg Foundation