PLoS Negl Trop Dis plos plosntds PLOS Neglected Tropical Diseases 1935-2735 Public Library of Science San Francisco, CA USA 10.1371/journal.pntd.0012451 PNTD-D-24-00106 Research Article Medicine and health sciencesClinical medicineSigns and symptomsFevers Medicine and health sciencesDiagnostic medicine Biology and life sciencesGeneticsGenomicsMetagenomics Biology and life sciencesMolecular biologyMolecular biology techniquesSequencing techniquesRNA sequencing Research and analysis methodsMolecular biology techniquesSequencing techniquesRNA sequencing Biology and life sciencesAnatomyBody fluidsBloodBlood plasma Medicine and health sciencesAnatomyBody fluidsBloodBlood plasma Biology and life sciencesPhysiologyBody fluidsBloodBlood plasma Biology and life sciencesMicrobiologyMedical microbiologyMicrobial pathogensBacterial pathogens Medicine and health sciencesPathology and laboratory medicinePathogensMicrobial pathogensBacterial pathogens Medicine and health sciencesClinical medicineSigns and symptomsPainAbdominal pain Biology and life sciencesMolecular biologyMolecular biology techniquesSequencing techniquesDNA sequencingNext-generation sequencing Research and analysis methodsMolecular biology techniquesSequencing techniquesDNA sequencingNext-generation sequencing Biology and life sciencesComputational biologyGenome analysisTranscriptome analysisNext-generation sequencing Biology and life sciencesGeneticsGenomicsGenome analysisTranscriptome analysisNext-generation sequencing Metagenomic next generation sequencing of plasma RNA for diagnosis of unexplained, acute febrile illness in Uganda Metagenomic next generation sequencing for diagnosis of unexplained, acute febrile illness https://orcid.org/0000-0002-9038-134X Kandathil Abraham J. Conceptualization Data curation Investigation Methodology Writing – original draft Writing – review & editing 1 Blair Paul W. Conceptualization Data curation Investigation Methodology Writing – original draft Writing – review & editing 1 2 Lu Jennifer Data curation Investigation Methodology Software Writing – original draft 3 4 5 Anantharam Raghavendran Data curation Investigation 1 Kobba Kenneth Data curation Investigation 6 Robinson Matthew L. Conceptualization Data curation Investigation Writing – original draft 1 Alharthi Sultanah Investigation 2 Ndawula Edgar C. Investigation 6 Dumler J. Stephen Supervision Writing – original draft 2 5 Kakooza Francis Investigation 6 Lamorde Mohammed Investigation Supervision 6 Thomas David L. Investigation Supervision Writing – original draft 1 Salzberg Steven L. Funding acquisition Methodology Software Supervision Writing – original draft Writing – review & editing 3 4 7 https://orcid.org/0000-0001-8619-5598 Manabe Yukari C. Conceptualization Funding acquisition Methodology Supervision Writing – original draft Writing – review & editing 1 * Division of Infectious Diseases, Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America Department of Pathology, Uniformed Services University, Bethesda, Maryland, United States of America Center for Computational Biology, Johns Hopkins University, Baltimore, Maryland, United States of America Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, United States of America Department of Pathology, Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America Infectious Diseases Institute, Makerere University, Kampala, Uganda Departments of Computer Science and Biostatistics, Johns Hopkins University, Baltimore, Maryland, United States of America Rinaldi Gabriel Editor Aberystwyth University - Penglais Campus: Aberystwyth University, UNITED KINGDOM OF GREAT BRITAIN AND NORTHERN IRELAND

The authors have declared that no competing interests exist.

 * E-mail: ymanabe@jhmi.edu 19 9 2024 9 2024 18 9 e0012451 24 1 2024 11 8 2024 This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Metagenomic next generation metagenomic sequencing (mNGS) has proven to be a useful tool in the diagnosis and identification of novel human pathogens and pathogens not identified on routine clinical microbiologic tests. In this study, we applied mNGS to characterize plasma RNA isolated from 42 study participants with unexplained acute febrile illness (AFI) admitted to tertiary referral hospitals in Mubende and Arua, Uganda. Study participants were selected based on clinical criteria suggestive of viral infection (i.e., thrombocytopenia, leukopenia). The study population had a median age of 28 years (IQR:24 to 38.5) and median platelet count of 114 x103 cells/mm3 (IQR:66,500 to 189,800). An average of 25 million 100 bp reads were generated per sample. We identified strong signals from diverse virus, bacteria, fungi, or parasites in 10 (23.8%) of the study participants. These included well recognized pathogens like Helicobacter pylori, human herpes virus-8, Plasmodium falciparum, Neisseria gonorrhoeae, and Rickettsia conorii. We further confirmed Rickettsia conorii infection, the cause of Mediterranean Spotted Fever (MSF), using PCR assays and Sanger sequencing. mNGS was a useful addition for detection of otherwise undetected pathogens and well-recognized non-pathogens. This is the first report to describe the molecular confirmation of a hospitalized case of MSF in sub-Saharan Africa (SSA). Further studies are needed to determine the utility of mNGS for disease surveillance in similar settings.

 Author summary

Unbiased molecular approaches like metagenomic sequencing have improved our ability to identify not only novel microbes but also known microbes in new settings. We report the results of a metagenomic next generation sequencing approach to identify viral and cell free plasma RNA from a curated panel of 42 unexplained, acute febrile, hospitalized study participants from tertiary referral hospitals in Mubende and Arua, Uganda. In ten study participants, metagenomic sequencing allowed us to identify pathogens including Helicobacter pylori, and Rickettsia conorii that were missed on routine clinical microbiologic testing. Sequence-specific targeted PCR assays and Sanger sequencing confirmed Rickettsia conorii infection in the first hospitalized case of Mediterranean Spotted Fever diagnosed in sub-Saharan Africa. Using appropriate controls, we observed metagenomic sequencing to be unable to consistently detect microbial sequences when plasma circulation levels were below 10,000 copies/mL. These results highlight the need for more studies to determine the utility of metagenomic next generation sequencing approaches for disease diagnosis and surveillance in similar settings.

 European Union RIA2018EF-083 Lamorde Mohammed http://dx.doi.org/10.13039/100000057 National Institute of General Medical Sciences R35-GM130151 Salzberg Steven L. http://dx.doi.org/10.13039/100000026 National Institute on Drug Abuse R01DA048063 Thomas David L. http://dx.doi.org/10.13039/100000026 National Institute on Drug Abuse R21DA053145 https://orcid.org/0000-0002-9038-134X Kandathil Abraham J. http://dx.doi.org/10.13039/100000070 National Institute of Biomedical Imaging and Bioengineering U54EB007958 https://orcid.org/0000-0001-8619-5598 Manabe Yukari C. http://dx.doi.org/10.13039/100000061 Fogarty International Center 5D43TW009771 https://orcid.org/0000-0001-8619-5598 Manabe Yukari C. This study was funded by the CAPA-CT II project (RIA2018EF-083 to ML and YCM) which is part of the EDCTP2 program supported by the European Union. Salary support was provided by the U.S. National Institutes of Health under grants R35-GM130151 for S.L.S., National Institute on Drug Abuse grants R01DA048063 for D.T., R21DA053145 for A.J.K., National Institute for Biomedical Imaging and Bioengineering U54EB007958 and Fogarty International Center 5D43TW009771 for YCM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. PLOS Publication Stage vor-update-to-uncorrected-proof Publication Update 2024-10-08 Data Availability The metagenomic dataset generated and analyzed during the current study have been deposited in Sequence Read Archive with the accession code PRJNA1115716. Introduction

The threat of emerging infections, limitations of target-specific diagnostics, decreasing costs, and improving bioinformatics have contributed to broader interest in unbiased metagenomic next-generation sequencing (mNGS) as a tool to identify the cause of undiagnosed infections [1–4]. This approach has facilitated rapid identification of novel outbreak pathogens which in turn can accelerate development of pharmacological and epidemiologic interventions [5,6]. However, while the agnostic mNGS approach to pathogen identification can be useful to overcome limitations of standard microbiologic diagnostics, questions remain about the optimal approach, patient use case, and net diagnostic gain. Careful sample selection, laboratory protocols, and computational analysis are needed to decrease misinterpretation of results from contamination, transient viremia, or nonpathogenic members of the human virome [7,8]. Additionally, careful selection of samples, knowing the clinical context, and/or performing additional confirmatory tests are important factors for optimizing the value of mNGS.

Some acute febrile illness (AFI) is caused by infections like malaria that can be diagnosed at the point-of-care. However, many cases of AFI remain undiagnosed, either because of limitations in diagnostic testing or the emergence of new pathogens or re-emergence of a known pathogen in an unexpected setting [9].We sought to evaluate the potential for RNA mNGS to identify possible microbial causes of unexplained acute febrile illness (AFI) in East Africa. Plasma metagenomic sequencing was performed on a curated panel of study participants selected from an AFI cohort based in Uganda [10]. We used RNA rather than DNA sequencing to enable the detection of both plasma RNA viremia and highly expressed microbial RNA from emerging and reemerging human infections in Rift Valley of East and Central Africa [11–13]. This approach resulted in the identification and molecular confirmation of a bacterium, Rickettsia conorii, as the cause of locally acquired Mediterranean spotted fever (MSF), causing severe illness, requiring hospitalization in sub-Saharan Africa (SSA).

 Study design and methods Ethics statement

This study was conducted in compliance with the Declaration of Helsinki and Good Clinical Practice Guidelines. The study and informed consent process were reviewed and approved by the Joint Clinical Research Centre (JCRC) Research Ethics Committee (JC1518) and the Uganda National Council for Science and Technology (UNCST), HS 371ES, and Johns Hopkins University School of Medicine Internal Review Board (IRB00176961). All participants signed written informed consents prior to study procedures. All samples were de-identified prior to laboratory testing.

 Study participants

This cross-sectional study sequenced plasma from individuals admitted with acute febrile illness to tertiary referral hospitals in Mubende and Arua, Uganda between 2019 to 2020[10]. In brief, adults (≥18 years of age) in the emergency or outpatient departments to be admitted with a recorded fever (≥ 38.0°C) or clinical history of fever within the past 48 hours and had symptom onset within the past 2 weeks were eligible for enrollment in the previously described parent cohort. After consent, study personnel collected baseline physiologic parameters, plasma and serum for standardized clinical diagnostic tests including HIV testing with consent (Determine HIV1/2: Abbott, OK, USA; Stat-Pak, Chembio Diagnostics Systems, Inc, NY, USA; SD Bioline: Gyeonggi-do, Republic of Korea), malaria rapid test (CareStart malaria Pf HRP2 antigen RDT), and thick malaria smears as previously described [10]. Participants were followed at one-month through in-person or telephone visits for blood collection and for determining survival. The participants in this mNGS study panel were selected based on i) clinical indication of viral infection (i.e., thrombocytopenia, leukopenia) and ii) absence of a validated laboratory diagnosis to explain the clinical presentation. As one of the aims of this study was to identify emerging and remerging human infections, we prioritized sequencing of samples from patients without HIV as these patients often present with febrile illness caused by well-described opportunistic infections [14].

Sample Preparation for next generation sequencing (NGS): i) Nucleic Acid Extraction: Extraction was performed on 200 μL of plasma using ZR-Duet DNA/RNA miniprep kit (Zymo Research, CA, USA) as previously described [15]. Pre-extraction steps done to minimize non-viral sequences included centrifugation of plasma at 1600g for 15 minutes at 4°C followed by filtration of the supernatant using 0.2 μM syringe filters (Whatman, Amersham Place, UK). This extraction protocol allowed for separate elution of the RNA fractions, thereby reducing DNA contamination.

ii) Metagenomic next generation sequencing (mNGS): The NGS library constructions were performed using Ovation SoLo RNA-Seq (Tecan Genomics, Inc., CA, USA) library preparation kit. The kit was selected based on its ability to generate libraries from low biomass samples like plasma. Additionally, during library preparation extra amplification cycles were incorporated to increase the final concentration of the cDNA libraries. Qualitative assessment of each library was done using a 2100 Bioanalyzer (Agilent Technologies, CA, USA) to visualize fragment size distribution and identify presence of non-specific DNA fragments that could potentially affect read quality. Although the average library size ranged from 300–350 bp, samples that had peaks prior to the library range (likely due to primer dimer or adaptor dimers) were also taken for sequencing as these were smaller in proportion to the library peak. In our experience these do not affect read quality. This was followed by quantitative assessment using the Kappa PCR library kit (Roche, IN, USA). Samples were pooled in batches to yield a final concentration of 4nM for sequencing on a NovaSeq 6000 (Illumina, Inc., CA, USA). Required volumes of each sample library was determined using the pooling calculator made available at https://support.illumina.com/help/pooling-calculator/pooling-calculator.htm. Sequencing of pooled samples was done as technical replicates (two lanes) on a single flow cell using high output mode with a read length of 2X100 bp and 500 million reads/lane.

iii) Controls: Both positive and negative controls were included as extraction and sequencing controls. Positive controls included a) two plasma specimens with known clinical and laboratory diagnosis (Plasmodium falciparum and human immunodeficiency virus [HIV]) and b) AcroMetrix Multi-Analyte (Thermo Fisher Scientific, MA, USA). The multianalyte contained SARS-CoV-2, Flu A/B, and RSV A/B at a concentration of 10,000 copies/mL. Water was used as a negative control. The controls were processed identically to the study samples.

iv) Taxonomic classification of reads: All sequencing reads were aligned to the human genome T2T-CHM13[16] using Bowtie 2[17]. The remaining non-human reads were classified by KrakenUniq [18] against a customized database built from all complete bacterial, archaeal, and viral genomes from NCBI RefSeq, a collection of 256 eukaryotic pathogens from EuPathDB, UniVec laboratory vectors from NCBI (https://www.ncbi.nlm.nih.gov/tools/vecscreen/univec/) and the GRCh38 human genome. The KrakenUniq database includes 46,711 bacterial genomes (5981 species), 13,011 viral genomes (9905 species), and 604 archaeal genomes (295 species), and the full database can be downloaded from https://benlangmead.github.io/aws-indexes/k2

v) Computational analysis: The KrakenUniq reports for all samples were analyzed for potential infectious agents using Pavian [19] to compare read counts between all samples and the sample controls (S1 Table). Using Pavian’s z-score, we isolated all species with significantly higher read counts than most other samples or controls, considering these species as potential microbial infections. Reads for any potential microbial infection were extracted using KrakenTools[20] and realigned using Bowtie 2 to visualize genome coverage of the classified reads. We also looked for indications of false positives by identifying plots that showed localization of reads in only 1–2 regions. Additionally, KrakenUniq algorithm reports a "unique k-mer" count, i.e., reads that match low-complexity regions. e.g. a long (ATATAT…) repeat will have 2 k-mers while a 100bp read should have seventy 31-mers if they are all different. Hence, if KrakenUniq reports 100 reads but only 200 k-mers, that means all of those reads hit the same spot either due low complexity or a contaminant. The presence of the microbial species was considered valid only if the number of uniq k-mers were much greater than the number of reads. We primarily used the calculated z-score to identify potential calls, which were then further validated using both the k-mer counts and the genomic coverage by the reads. Clinical relevance was determined by a committee of three board-certified infectious diseases specialists.

vi) Rickettsia PCR: For confirmation of rickettsial infection, DNA was extracted from serum using QIAamp RNA Mini Kit (Qiagen, Venlo, Netherlands). PCR was performed using previously published methods targeting mRNA of spotted fever group sca0 (OmpA gene), R. conorii-specific sca0, and typhus group 17-kDa outer membrane protein gene, and positives were called only if in duplicate [21,22]. The 127-basepair amplicon from R. conorii-specific sca0 PCR plate was sent for Sanger sequencing and aligned to a reference genome using SnapGene software (GLS Biotech, San Diego, CA, USA).

 Results

The 42 study participants included participants hospitalized in Mubende, Uganda (n = 26) and in Arua, Uganda (n = 16). Participants had a median age of 28 years (IQR:24, 38.5) with 50% identifying as females (Table 1). Based on the study selection criteria, participants had leukopenia and/or thrombocytopenia with a median WBC and platelet count of 5.3 x103 cells/mm3(IQR:3.6 to 9.1) and 114 x103 cells/mm3 (IQR:66.5 to 189.8), respectively. Although we enriched for virions during RNA extraction, the protocol allowed us to simultaneously sequence cell free RNA. Hence, in addition to identification of RNA virome, we were also able to identify infections due to bacteria, fungi, parasites, and DNA viruses.

10.1371/journal.pntd.0012451.t001 Baseline Demographic and Vital Physiological Parameters of the study panel.
Characteristic Total (N = 42) Arua RRH (N = 16) Mubende RRH (N = 26)
Female sex—no. (%) 21 (50.0) 8 (50.0) 13 (50.0)
Age—years, median (IQR) 28.0 (24.0, 38.5) 29.0 (24.0, 37.8) 28.0 (24.0, 39.0)
HIV (%) 2 (4.8) 1 (5.9) 1 (3.8)
Duration of symptoms—days, median (IQR) 4.5 (3.0, 7.0) 4.0 (3.0, 5.3) 5.0 (3.0, 7.0)
Physiological Parameters (IQR)
Heart rate (beats per minute) 110.0 (98.3, 128.8) 98.5 (90.5, 109.0) 116.0 (101.2, 132.0)
Temperature (°C) 38.4 (38.1, 38.8) 38.5 (38.0, 38.6) 38.4 (38.1, 38.8)
Systolic blood pressure (mmHg) 112.0 (100,119.0) 113.5(99.0,119.0) 110.5(101.0,120.0)
Diastolic blood pressure (mmHg) 67.5 (59.3, 75.8) 67.5 (61.3, 75.3) 66.5 (59.3, 77.3)
Respiratory rate (breaths per minute) 22.0 (18.0, 26.8) 21.0 (17.3, 27.3) 22.0 (18.0, 25.8)
Oxygen saturation (%) 98.0 (96.0, 98.0) 98.0 (97.8, 98.3) 97.0 (95.3, 98.0)
Glasgow coma scale 15.0 (15.0, 15.0) 15.0 (15.0, 15.0) 15.0 (15.0, 15.0)
qSOFA score≥ 2—no. (%) 10 (23.8) 4 (25.0) 6 (23.1)
Outcome
Died (%) 10 (23.8) 5 (31.3) 5 (19.2)
Duration of hospitalization—days, median (IQR) 2 (2.0, 4.5) 3 (2.0, 4.8) NA

RNA sequencing of all samples yielded an average of 25 million 100 bp paired reads per sample. 83.5% of all reads were identified as human using Bowtie 2 and KrakenUniq. Removal of human sequences resulted in an average of 1.4 million reads per sample (Table 2).

10.1371/journal.pntd.0012451.t002 KrakenUniq classified non-human read counts per sample.
Sample Classified Non-Human Reads Sample Classified Non-Human Reads
AFI-1 700,577 AFI-29 630,375
AFI-6 1,691,350 AFI-30 3,192,999
AFI-7 862,091 AFI-31 547,573
AFI-8 470,110 AFI-32 12,408,653
AFI-9 928,205 AFI-33 3,561,562
AFI-10 387,198 AFI-34 793,952
AFI-11 643,348 AFI-35 1,630,684
AFI-12 552,810 AFI-36 1,520,607
AFI-13 258,230 AFI-37 3,595,947
AFI-14 45,475 AFI-38 1,422,173
AFI-15 182,264 AFI-39 819,044
AFI-16 3,148 AFI-40 669,243
AFI-17 1,485,407 AFI-41 2,088,918
AFI-18 205,858 AFI-42 1,419,928
AFI-19 943,131 AFI-43 179,161
AFI-20 2,836,251 AFI-44 424,884
AFI-21 490,829 AFI-45 38,618
AFI-22 872,502 AFI-46 186,514
AFI-24 445,745 AFI-47 1,840,597
AFI-25 121,075 AFI-48 2,817,500
AFI-26 529,889 AFI-49 3,060,312
AFI-27 395,909 NTC 993,408
AFI-28 2,900,233 ACROMETRIX 5,944,038

This study sequenced 42 participants, AcroMetrix control, No Template Control (NTC) and 2 with known clinical and laboratory diagnosis (AFI-6 and AFI-12).

Analysis of KrakenUniq read classifications revealed the expected Plasmodium falciparum species signal in the positive control (S2 Table). However, the protocol failed to identify HIV-1 present at a concentration <4 log 10 copies/ml. This is consistent with our previous observation that metagenomic sequencing can give false negative results for viruses at concentrations <4 log 10 copies/mL [15]. Additionally, the KrakenUniq protocol and Pavian analysis identified SARS-CoV-2, Flu A/B, and RSV A/B, all of which were present at a concentration of 4 log 10 copies/mL in the AcroMetrix sample.

Further analysis of the KrakenUniq classifications identified 10 microbes among the 42 study participants with significant Pavian z-scores (Tables 3 and S2). These included organisms that are always considered pathogens when isolated from human specimens (N. gonorrhoeae, H. pylori, P. falciparum, and R. conorii). We also identified microbes that can be normal flora or pathogenic in certain settings (Candida parapsilosis, human herpes virus-8 [HHV-8], Human mastadenovirus C, and Staphylococcus haemolyticus) [23–25]. Pegivirus hominis(also known as pegivirus-C), a commonly observed non-pathogen in blood, was also detected in two participants[26]. Fig 1 displays the read counts for these 10 microbes across all samples.

10.1371/journal.pntd.0012451.g001 KrakenUniq read classifications identified microbes across 42 study samples.

KrakenUniq read classifications for 9 identified microbes across 42 study samples are shown. The identified reads were absent in negative control (NTC), and the AcroMetrix control. Each of these microbes had significantly higher total read counts in 1–2 samples as compared to the remaining samples and controls, as determined by the Pavian z-score.

 10.1371/journal.pntd.0012451.t003 Metagenomic next generation sequencing diagnosis and clinical presentations.
Study ID (site) Age (yrs) Sex Medical History Diagnosis
Clinical mNGS Relevance
AFI 1 (Arua) 27 F Two days of abdominal pain, fever to 38.1°C, who was HIV-negative, but RPR-positive with a WBC elevated at 14.4 K/mm3 alive at 28 days, discharged after receiving IV ceftriaxone. Sepsis of unknown source and syphilis N. gonorrhoeae +
AFI 10 (Mubende) 20 F Fever of 38.2°C, tachycardic (HR 142 bpm), hypotensive (90/62 mmHg) with generalized systemic symptoms (fatigue, headache, myalgias, arthralgias, nausea, vomiting, rigors) with evidence of urinary tract infection, jaundice, and splenomegaly, HIV-negative, treated with ciprofloxacin and metronidazole discharged after 2 days unable to reach at 28 days. Urinary tract infection Staphylococcus hemolyticus +/-
AFI 13 (Arua) 30 F Fever 38.0°C, HIV+ (CD4 118) on antiretroviral therapy who presented with 7 days of diarrhea, headache, nausea, vomiting for 7 days, anemic (hgb 9 g/dl), low platelets (25 K/mm3), decreased kidney function (creatinine 1.5 mg/dL) and mildly elevated liver enzymes (ALT 65 U/L and AST 101 U/L) who died while hospitalized. Sepsis of unknown source and gastroenteritis Human Herpesvirus-8 +/-
AFI 19 (Arua) 25 M Fever 38.8°C presents with chest pain and dyspnea (anorexia, cough, headache) for 7 days who had been seen previously at a clinic, HR 130 bpm, RR 25 bpm, SpO2 68%, toxic appearing with crackles, urinalysis with pyuria, WBC 9.1 K/mm3, platelets 241 K/mm3, hgb 10.7 g/dL, HIV-negative, treated with cefotaxime, died while hospitalized. Pneumonia and urinary tract infection Candida parapsilosis +/-
AFI 21 (Mubende) 40 F Fever 38.4°C presents with abdominal pain, anorexia, arthralgia, dysuria, fatigue, headache, rigors for 4 days, toxic appearing with stable vital signs, tender abdomen, diagnosed with sepsis and gastritis, WBC 4.6 K/mm3, platelets 157 K/mm3, treated with metronidazole and cefotaxime, alive at follow-up Sepsis and gastritis Helicobacter pylori +
AFI 24 (Mubende) 39 M Farmer with direct contact with goats and chickens the bush) with fever 40.2°C, admitted with abdominal pain, altered mental status, anorexia, diarrhea, fatigue, headache, rash and rigors for 7 days HR 119 bpm, hypotensive 81/47 mmHg, RR 38 bpm, confused, toxic, nuchal rigidity, crackles, tender abdomen, papular rash, WBC 4.7 K/mm3, plt 89 K/mm3, treated with clindamycin, levofloxacin, amikacin and improved after 1 day, clindamycin and kanamycin on day 2. Alive at day 28 Meningitis and pneumonia Rickettsia conorii +
AFI 27 (Arua) 38 M Crop farmer with fever 38.6°C, change mental status, headache. Recurrent seizures for 5 days, 154/101 mmgHg, SpO2 93%, GCS 5, toxic, not opening eyes, nuchal rigidity, malaria smear and RDT negative, WBC 8.2 K/mm3, hgb 10.6 g/dL, platelets 102 K/mm3, AST 306 U/L, ALT 93 U/L, treated with ceftriaxone, metronidazole, patient died while hospitalized. Possible meningitis Plasmodium falciparum +
AFI 28 (Mubende) 23 F Teacher with 5 days of shortness of breath, cough, headache, chest pain, abdominal pain, nausea, vomiting presenting with fever of 38.7°C, heart rate 100 bpm, 210/162 mmHg, SpO2 96% and lung crackles on exam. WBC 5.1 K/mm3, hgb 13.1 g/dL, creatinine 6 mg/dL. Chest X-ray revealed right base opacities. Pneumonia human mastadenovirus C -
AFI 35 (Arua) 19 F Fever 38.0°C with abdominal pain, cough, diarrhea, headache, nausea for 4 days, previously seen at clinic and received ACT, HR 107 bpm, 113/67 mmHg, RR 27 bpm, conjunctival pallor, splenomegaly, hgb 3.8 g/dL, platelet count 124 K/mm3, WBC 6.4 K/mm3, treated with ciprofloxacin, could not reach at 28 days. Gastroenteritis and severe anemia Pegivirus hominis/ pegivirus-C -
AFI 47 (Mubende) 40 M Fever 38.1°C, abdominal pain, anorexia, arthralgias, bleeding, dyspnea, fatigue, headache, myalgias, nausea, sore throat for 5 days, toxic, conjunctival pallor, crackles, tachycardia, hepatosplenomegaly, macular rash, Chest X-ray with infiltrates, hemoglobin 5.1 g/dL, platelet count 550 K/mm3, white blood count 16 K/mm3, treated with ceftriaxone, alive at 28 days. Pneumonia and urinary tract infection Pegivirus hominis/ pegivirus-C -

Note: mNGS: metagenomic next generation sequencing; ALT: alanine transaminase; AST: aspartate transaminase; bpm: beats or breaths per minute; F: female; GCS: Glasgow coma scale; HIV: human immunodeficiency virus; hgb: hemoglobin; HR: heart rate; M:male; RPR: rapid plasma reagin, RR: respiratory rate; WBC: white blood count

 Cases with clinically relevant mNGS results

Among the four cases adjudicated to have had clinically relevant results, the pathogens were not identified on protocolized routine clinical microbiologic testing (Table 2). First, N. gonorrhoeae sequences were found in a 27-year-old woman with abdominal pain and fever. Routine testing revealed a positive anti-treponemal antibody syphilis test. The patient was diagnosed with syphilis and was treated with intramuscular penicillin. This patient received intravenous ceftriaxone (active against N. gonorrhoeae); standard blood cultures were negative. Ceftriaxone was given empirically. Second, H. pylori sequences were detected in a 40-year-old woman with fever and abdominal pain who was clinically diagnosed with gastritis and ulcers. The clinical course was consistent with H. pylori infection, but routine access to H. pylori testing was not available in this setting. Third, P. falciparum sequences were found in a 38-year-old man with fever, altered mental status, seizures who died during admission. Rapid diagnostic and smear testing for malaria were negative.

 Mediterranean spotted fever (R. conorii)

The fourth clinically relevant case is a 39-year-old man with no past medical history who presented 7 days after symptom onset with abdominal pain, altered mental status, a generalized rash, and rigors. He was previously seen in a clinic and prescribed antimalarials (artesunate and quinine tablets) which he took for 6 days without improvement. He was a crop and livestock farmer from Mubende. He reported mosquito bites but no known tick bites. He had goats and chickens around his household. He had a fever of 40.2°C, confusion (Glasgow coma score 14), heart rate 119 bpm, blood pressure of 81/47 mmHg, respiratory rate of 38 breaths per minutes, and oxygen saturation of 87%. On exam he was toxic appearing, had nuchal rigidity, crackles on lung auscultation, tachycardia on heart auscultation, abdominal tenderness, and a papular rash. His white blood count was 4.7 x103 cells/μL, hemoglobin 16.9 g/dL, platelet count 89 x103 cells/μL, AST 92 U/L and ALT 39 U/L. Routine microbiology laboratory results were negative for HIV, malaria, hepatitis A and hepatitis B, and blood cultures were without growth. He was admitted with a diagnosis of meningitis and pneumonia and treated with clindamycin, levofloxacin, and amikacin. He remained febrile at 39.4°C, but heart rate improved to 90 bpm, respiratory rate 22 breaths per minutes, and blood pressure 120/80 mmHg with 97% oxygen saturation. He was discharged by the following day. The participant was alive at 28 days per phone follow-up.

The baseline enrollment plasma sample was tested using real-time PCR and was positive for the spotted fever group sca0 (ompA) target at a mean cycle threshold of 32.7. Typhus group 17-kDa PCR was negative. Serum samples were positive for R. conorii-specific sca0 primers at a mean Ct of 33.6 in three of four plasma duplicates. The amplicon was Sanger sequenced and had a 100% match with a R. conorii Malish 7 strain reference sequence.

 Discussion

In a hospitalized, acute febrile illness cohort in Uganda, RNA-based mNGS identified clinically relevant pathogens in a subset of participants without a microbiologic diagnosis. This report underscores the potential and limitations of mNGS for diagnostics and pathogen discovery. The findings of this study also include the first report of a molecularly confirmed locally acquired R. conorii infection leading to hospitalization in SSA [27,28].

The use of mNGS allowed identification of pathogens missed by standardized diagnostic tools. The pathogens identified included R. conorii, H. pylori, N. gonorrhoeae, and P. falciparum. There are few descriptions of rickettsial illness among those hospitalized in SSA with cases primarily limited to returning travelers. In the absence of surveillance programs and diagnostics, species causing rickettsial illness among those hospitalized with AFI are also largely unknown. Notably, the MSF participant was initially misdiagnosed with malaria and developed progressive illness including signs and symptoms of meningitis and unstable vital signs. Fortunately, empiric treatment with a fluoroquinolone, to which R. conorii is susceptible, resulted in dramatic recovery and swift discharge. This case report along with recent evidence of MSF circulation in tick pools throughout Uganda provides strong clinical evidence that MSF should be considered in patients with severe non-malarial illness leading to hospitalization in Uganda [29]. Availability and application of NGS for clinical purposes could provide valuable pathogen agnostic approaches to diagnosis after first-line testing is exhausted and to inform regional diagnostic needs. As with the identification of R conorii infection, detection of H. pylori sequences in the plasma of a patient with epigastric pain and ulcers was unexpected and clinically meaningful. This finding has been previously described and likely represents a real pathogen [30]. While clinically ‘expected,’ mNGS was the sole evidence of N. gonorrhoeae and malaria infections despite routine diagnostic testing in two other patients. Molecular detection of P. falciparum with a negative blood smear does not implicate malaria as the primary cause of acute febrile illness. However, identification of P. falciparum could provide context as an infection that could contribute to anemia, splenomegaly, or non-typhoidal infection [31].

Our findings also reflect some of the limitations of mNGS. mNGS is not widely available clinically and computational analysis require specialized bioinformatics tools for precise identification of microbial reads. As with existing blood cultures and even some clinical PCR diagnostics, microbes unlikely to be clinically significant (e.g., coagulase-negative staphylococcus) may be identified and lead to incorrect diagnoses or treatments. Hence, like routine interpretation of laboratory results, careful incorporation of clinical interpretation is needed. We also failed to identify evidence of microbial infection in most persons and failed to detect HIV RNA in one positive control, underscoring the limited sensitivity of the approach in detection of acute infections with low plasma microbial RNAemia (<10,000 copies/mL)[15].

Our study itself also had limitations. We focused on the RNA fraction and may have missed genomic DNA of other microbes like viruses, bacteria, fungi, and protozoa. Nonetheless, our approach allowed us to identify their transcripts [32]. The identification of Staphylococcus haemolyticus and Candida parapsilosis could be due to contamination at the time of collection. The use of negative controls indicate that these were less likely from processing in the laboratory. Contamination at time of collection is a common consideration for blood culture testing which needs to be considered as well for sequencing approaches.

 Conclusion

In this study using curated samples, we observed RNA mNGS to be a clinically useful diagnostic approach in a region with diverse causes of infectious diseases. mNGS identified pathogens that would have otherwise been undetected in a protocolized AFI surveillance study in Uganda. mNGS filled current gaps in diagnostic workflows by identifying pathogens not widely known to be circulating in agrarian countries with diverse causes of infection. In addition, our findings further support setting up prospective surveillance for MSF in SSA to allow for early treatment of R. conorii infections. Early recognition, empiric antibiotics, and/or diagnostics could prevent hospitalization or deaths due to rickettsial illness. In addition to demonstrating the diagnostic utility of mNGS, our study highlights the need to identify the burden of rickettsial infections in SSA. These are essential for development of rapid diagnostics, consideration of empiric treatments, and to inform public health policy.

 Declarations

The contents of this article are the sole responsibility of the authors and do not necessarily reflect the views, assertions, opinions, or policies of the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., the U.S. Department of Defense, the U.S. government, or any other government or agency. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. government. Some of the authors of this work are military service members or employees of the U.S. government. This work was prepared as part of their official duties. Title 17 U.S.C. x105 provides that “Copyright protection under this title is not available for any work of the United States government.” Title 17 U.S.C. x101 defines a U.S. government work as a work prepared by a military service member or employee of the U.S. government as part of that person’s official duties.

 Supporting information Overview of Pavian results.

(XLSX)

 KrakenUniq read classifications and Pavian Z-scores.

(XLSX)

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Epub 20211108. doi: 10.1038/s41598-021-00611-0 34750425; PubMed Central PMCID: PMC8576030. Kumata R, Ito J, Takahashi K, Suzuki T, Sato K. A tissue level atlas of the healthy human virome. BMC Biol. 2020;18(1):55. Epub 20200604. doi: 10.1186/s12915-020-00785-5 32493363; PubMed Central PMCID: PMC7269688. 10.1371/journal.pntd.0012451.r001 Decision Letter 0 Rinaldi Gabriel Academic Editor Wunder Jr Elsio A Section Editor 2024 Rinaldi, Wunder Jr This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 0

24 Apr 2024

Dear Dr. Manabe,

Thank you very much for submitting your manuscript "Metagenomic next generation sequencing of plasma RNA for diagnosis of unexplained, acute febrile illness in Uganda" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

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Gabriel Rinaldi, M.D., Ph.D.

Academic Editor

PLOS Neglected Tropical Diseases

Elsio Wunder Jr

Section Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The study design is clear. This is more of an exploratory study based on the generation of large genomics data sets rather than hypothesis testing. The authors aim to evalute the use of RNA metagenomics in diagnosis for patients with unknown illnesses. It should be possible to state the study objectives more clearly as a hypothesis, if this is required. Statistical approaches based on Kraken were used, a well established metagenomics software.

It could be further clarified ablout the sufficient sequencing depth used to perform this study. Data should be given to summarize the numbers of reads per sample (after mapping to the human genome), and to summarize the z-scores for Pavian in a quantitative manner. Are these z-scores sufficient for the study? In particular it would be useful to provide the Pavian z-scores for the 9 microbes mentioned in Fig 1 and indicate how they differ from the rest. Were other pathogenic microbes detected by the study, albeit with less significant z-scores? It would be useful for the authors to provide a full summary of the data, beyond the highlights of interest here, so the reader can get a more general feel for what the method detected, including any background signals.

It would probably be helpful to mention the samples were taken from plasma in the abstract.

Reviewer #2: The Objectives of the study are clearly articulated and appropriate. There are no concerns about the ethical conduct of the study per the approvals and adherence standards provided in the Methods.

The study was appropriate for addressing the stated objective to assess the use-case for mNGS as a epidemiologic & diagnostic tool for AFI in settings with diverse potentially diverse and emerging etiologies.

For the question being asked, the sample size is sufficient, and adequate power is not a necessary consideration for this descriptive epidemiologic/diagnostic evaluation.

The use of positive and negative controls, both clinically derived and commercially sourced, is a notable effort for methodologic rigor. The panel-based adjudication process for establishing clinical relevance of an mNGS detection is acceptable approach.

MAJOR

Lines 65-67: thrombocytopenia & leukopenia thresholds

For the purposes of a reader being able to reconstruct “what was the target patient population?” or “what is the provenance of the participants included in this report?” was there a threshold criterion for thrombocytopenia (e.g., platelets < 100,000?) and was there a threshold criterion for leukopenia (e.g., white blood cell count < 4000?)?

MINOR

The study population is appropriate; description of the study population resides in citation 10. To assist the reader’s experience, I recommend the author’s include a simple sentence/clause BRIEFLY stating the target population/eligibility criteria: adults with a ≥ 38.0 °C temperature or history of fever within 48 h of presentation (taken from the Abstract of citation 10). Such a sentence will help the reader decide “did the authors aim to recruit a generalizable patient population for this study?” without having to go to PubMed to dig up citation 10.

Line 78: extraction protocol to reduce DNA contamination.

For the sake of clarity & methods reproducibility, was there a step after centrifugation at 1600g x 15 minutes before filtration using syringe filtration?

In other words, after centrifugation perhaps a certain fraction (the pellet or some other layer) was pipetted and then this fraction was subjected to the additional filtration?

Lines 88-90: assessment of library quality prior to sequencing

Can the others expand on the criteria used for qualitative assessment of each library by the Bioanalyzer and any criteria used for the quantitative assessment by PCR? For example, was there a minimum quantitative threshold that would have disqualified certain samples from being included on the sequencing run?

Lines 115-116: k-mer count.

Can the authors expand on how the “unique k-mer” count from KrakenUniq reports was used?

At present, it is unclear. A reader could speculate that reads mapping to low complexity regions were considered suspicious for spurious detections rather than true infection…?

Lines 116-117: read counts and genome coverage

Was there a minimum threshold criterion for number of read counts in order to consider a detection bona fide?

In some publications, I have seen >50 reads used as a threshold. While this might be semi-arbitrary, it would still be helpful to readers in the field to know any thresholds or decision criteria you used in making calls.

From Figure 1 one can see that the number of reads that mapped to R conorii was a small number (and in what turned out to be confirmed by PCR and Sanger sequencing). So if there was not a minimum read threshold, it would still be helpful to state this and/or clarify further the interpretive approach that was used.

The same questions apply to genome coverage: was there an operational target that you used?

I realize that with the last 3 comments (library quality assessment, k-mer count, and read count threshold / genome coverage threshold) that there might not be standardized criteria (and perhaps things passed “an eyeball test” (How do I know these were real detections? I know it when I see it.)

So these minor queries are not so much meant to be nit-picky as for the sake of Methods reproducibility that would be of interest to the field and to readers who have interest in making platforms like this clinically relevant and actionable.

HIV Status:

HIV infection status is reported in Table 1 of Results, but there was no accompanying explanation in the Methods how this was established: participant self-report? Rapid antibody testing?

Suggest including a sentence towards the beginning of Methods referring the reader to citation 10 for how the demographic/clinical/physiologic information in Table 1 was collected (assuming that citation 10 covers this data collection).

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The analysis matches the plan. The tables are clear but Figure 1 is of poor quality, and the text is illegible. This needs to be replaced with a higher resolution version.

Reviewer #2: The analysis presented matches the analysis plan, and the results are clearly presented.

Figure 2 image file is fuzzy, but hopefully the resolution of a final version will be better.

MINOR

Lines 173-175: relevance of P falciparum sequence detections

It is arguable whether a detection of P falciparum sequences is indeed clinically relevant in a patient who was smear-negative and malaria RDT negative. The clinical relevance of PCR-based detection of P falciparum is likewise problematic. In other words, this is not a criticism of mNGS, but rather nucleic-acid based detection of P falciparum more generally, as most clinicians would assert that clinically significant P falciparum infection typically includes presence on smear. Was there any evidence that the patient was treated for malaria prior to admission/enrollment?

This detection of P falciparum can still be rendered, in my opinion, as clinically relevant, not so much because malaria would have been the cause of the patient’s illness but because recent or concurrent malaria is associated with non-typhoidal Salmonella (one of the most common causes of meningitis in Africa). I suggest the author’s consider further clarification of this case: whether prior treatment with anti-malarials is known vs unknown and wording that acknowledges it might be problematic to attribute illness to malaria in the context of P falciparum nucleic-acid detection with a negative blood parasite smear.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The conclusions are supported by the data presented, although some additional detail could be given (see the methods section).

Limitations are clearly described in two paragraphs of the discussion.

The data are clearly connected to public health issues and help with the diagnosis of unexplained illness.

Line 239/240: please clarify DNA pathogens and "transcripts of DNA microbes". This is currently non-sensical? Is microbial DNA meant?

Reviewer #2: The conclusions are supported by the data presented. Limitations of mNGS are described. Public health relevance of R conorii detection and the strengths and limitations of mNGS as a surveillance modality are discussed.

Conclusion about MSF in SSA:

The phrasing in the Abstract is technically correct “first report to describe the molecular confirmation of a hospitalized case of MSF in SSA.”

However, in the Conclusion, the rendering is slightly more problematic: “first report of patient admitted with molecularly confirmed locally acquired R conorii infection in SSA.”

AJTMH 2005;73:1086 describes an MSF case acquired in Kenya where the patient returned to Japan and was admitted to hospital in Japan. The Parola et al 2013 global review of rickettsioses (Clin Microbiol Rev 2013;26:657) cites the case of a South African man who acquired MSF in South Africa and then presented with a hemorrhagic fever-like while traveling in Brazil. So I think both of these would be cases of MSF acquired in SSA that required hospital admission.

I recommend the phrasing in the Discussion mirror the phrasing in the Abstract.

Regardless of these semantics, I do think this genetically confirmed detection of MSF in Uganda is significant, as fatal spotted fever rickettsiosis (no speciation) has been described in Kenya and the detection presented in this study is, to my knowledge, only the second genetically confirmed detection of MSF in East Africa, where many assume that the less virulent R africae / African Tick Bite Fever is the main spotted fever rickettsiosis.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: This paper is acceptable with minor revisions.

Reviewer #2: See aforementioned comment about the blurriness of the Figure.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The authors have applied RNA metagenomics to 42 patients from Uganda wtih unexplained acute febrile illness. The data were analysed using Kraken, a well established computational method designed for this kind of study. The data identify pathogenic organisms that had been missed by other approaches, thus demonstrating the utility of the methodology for diagnosis. Some further detail and summary statistics could be given about the analysis, but otherwise this appears to be a fairly routine application of metagenomics to a proof of concept study. It clearly demonstrates the utility of metagenomics for treating patients with unknown illnesses, and has significance for public health issues and medical diagnosis.

The data sets are not yet available, although the authors promise they will be made available on publication. It is useful for reviewers to check that the correct details and URLs are provided in the manuscript before publication.

Reviewer #2: Overall, I think this work adds value to the field 1) showing the strengths (unbiased nature) and limitations (diminished sensitivity and overall low detection frequency) of mNGS as a surveillance tool. 2) The detection of R conorii is a public health relevance as this is a neglected disease and severe disease is more common with R conorii than with R africae.

As stated in my comments above, I think the manuscript would benefit from some additional linkage to the parent study (e.g., providing the inclusion criteria), expanding on some methodologic details (namely for mNGS interpretive methods but also providing details of the selection criteria for thrombocytopenia and leukopenia). 3) I have suggested some nuance around the novelty of the R conorii detection in Uganda.

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Reviewer #1: No

Reviewer #2: No

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References

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 10.1371/journal.pntd.0012451.r002 Author response to Decision Letter 0 Submission Version 1

4 Jun 2024

Submitted filename: Reviewer_PloS_Edited_5232024.docx

 10.1371/journal.pntd.0012451.r003 Decision Letter 1 Rinaldi Gabriel Academic Editor Wunder Jr Elsio A Section Editor 2024 Rinaldi, Wunder Jr This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 1

17 Jul 2024

Dear Dr. Manabe,

Thank you very much for submitting your manuscript "Metagenomic next generation sequencing of plasma RNA for diagnosis of unexplained, acute febrile illness in Uganda" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Gabriel Rinaldi, M.D., Ph.D.

Academic Editor

PLOS Neglected Tropical Diseases

Elsio Wunder Jr

Section Editor

PLOS Neglected Tropical Diseases

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The authors have mainly addressed my previous comments well and the paper is very nearly suitable for publcation. However, I do have a couple of very minor issues.

The authors have left the phrase "DNA microbes" in the paper. I find this phrase confusing. It is clear from their comment that they mean DNA viruses - which is perfectly sensible. But do they intend to include other microbes? It surely makes no sense to talk about DNA bacteria for instance. Would it be clearer to change "DNA microbes" to "DNA viruses"? The paper they now cite from Kumata et al only appears to refer to DNA viruses, I could not find "DNA microbes" in that paper. However, I will leave this issue to the authors discretion.

The authors state that the data have been uploaded under project ID PRJNA1115716, but this ID gives no hits on the SRA or NCBI. Perhaps it is incorrect or not yet released? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, and so I assume the paper should not be published until this has been sorted out.

Reviewer #2: The authors have responded adequately to my concerns and suggestions. Of note, the line numbering in their responses did not align with the revised manuscript version I received, so it did make the process of reviewing their responses more onerous than one would have hoped. Overall, though, their additions and edits have improved the manuscript and increased its informativeness.

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Reviewer #1: No

Reviewer #2: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

 10.1371/journal.pntd.0012451.r004 Author response to Decision Letter 1 Submission Version 2

6 Aug 2024

Submitted filename: Reviewer_PloS_Edited_07232024.docx

 10.1371/journal.pntd.0012451.r005 Decision Letter 2 Rinaldi Gabriel Academic Editor Wunder Jr Elsio A Section Editor 2024 Rinaldi, Wunder Jr This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 2

11 Aug 2024

Dear Dr. Manabe,

We are pleased to inform you that your manuscript 'Metagenomic next generation sequencing of plasma RNA for diagnosis of unexplained, acute febrile illness in Uganda' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Gabriel Rinaldi, M.D., Ph.D.

Academic Editor

PLOS Neglected Tropical Diseases

Elsio Wunder Jr

Section Editor

PLOS Neglected Tropical Diseases

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 10.1371/journal.pntd.0012451.r006 Acceptance letter Rinaldi Gabriel Academic Editor Wunder Jr Elsio A Section Editor 2024 Rinaldi, Wunder Jr This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

13 Sep 2024

Dear Dr. Manabe,

We are delighted to inform you that your manuscript, "Metagenomic next generation sequencing of plasma RNA for diagnosis of unexplained, acute febrile illness in Uganda," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases