Case Report of Pediatric Naegleria fowleri Meningoencephalitis

Case Report of Pediatric Naegleria fowleri Meningoencephalitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Case Report of Pediatric Naegleria fowleri Meningoencephalitis Yuanjing Kou, Jiayao Zhang, Dan Wang, Lidan Cui, Qi Sun, Yanqi Lv, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6549687/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Jul, 2025 Read the published version in Infectious Diseases of Poverty → Version 1 posted 5 You are reading this latest preprint version Abstract Background Naegleria fowleri , a pathogenic free-living amoeba, is the causative agent of primary amoebic meningoencephalitis (PAM), a rare but devastating disease. PAM is characterized by its acute onset, rapid progression, and severe clinical manifestations, with mortality rates exceeding 95%. Despite its rarity, the catastrophic outcomes associated with this infection underscore the critical importance of prevention. In this report, we present a rare pediatric fatality caused by PAM in China, highlighting the challenges of diagnosis and treatment. Case presentation We report a pediatric case of PAM that occurred in Lushan County, Pingdingshan City, Henan Province, China. The patient, a 6-year-old child, initially presented with persistent high fever on December 5, 2024, accompanied by headache, vomiting, and altered mental status. After receiving ineffective treatment at a local hospital, the child was transferred to the Eastern District of Henan Children’s Hospital on December 7 for further evaluation and management. Upon admission, cerebrospinal fluid was collected for laboratory analysis, and antimicrobial therapy, including amphotericin B, fluconazole, and rifampicin, was promptly initiated. Despite these interventions, the patient’s condition deteriorated rapidly, and the child succumbed to the infection on December 9. Conclusions The clinical presentation and laboratory findings strongly suggest that the child was infected with Naegleria fowleri , resulting in PAM. Epidemiological investigation suggests that the infection may have been acquired during bathing at a public bathhouse. Given the survival characteristics of the amoeba and the accelerating effects of global warming, which may expand its habitat, this sporadic case serves as a stark reminder of the lethal potential of PAM. With a mortality rate exceeding 95%, early recognition and prompt intervention are crucial. Clinicians should maintain a high index of suspicion and consider PAM in the differential diagnosis of patients presenting with similar symptoms, particularly in regions where exposure to warm freshwater environments is common. Primary amoebic meningoencephalitis Naegleria fowleri Genetic analysis Metagenomic sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Pathogenic amoebae represent a diverse group of organisms widely distributed in natural and artificial environments, including soil, lakes, rivers, hot springs, inadequately chlorinated swimming pools, and water distribution systems [ 1 ] . Among these, Naegleria fowleri ( N. fowleri ), commonly termed the "brain-eating amoeba", is a particularly virulent species [ 2 ] . This free-living amoeba invades the central nervous system (CNS) by entering through the nasal mucosa, migrating along the olfactory nerve through the cribriform plate into the cranial cavity, and ultimately causing primary amoebic meningoencephalitis (PAM) [ 3 ] . PAM is a fulminant disease characterized by acute onset, rapid progression, severe clinical manifestations, and an extremely high mortality rate (> 95%), predominantly affecting children and young adults [ 4 ] . Despite its rarity, the aggressive nature of this infection necessitates urgent clinical attention. Currently, no rapid and reliable diagnostic methods are universally available for amoebic infections, and early symptoms of PAM—including fever, headache, vomiting, and neck stiffness, overlap significantly with those of other CNS infections [ 5 , 6 ] . This clinical mimicry poses substantial diagnostic challenges, often leading to delayed recognition and suboptimal treatment outcomes. In this case report, we present a detailed analysis of the diagnostic utility of multiple detection techniques applied to cerebrospinal fluid (CSF) samples from a suspected pediatric N. fowleri infection. Our findings aim to inform clinicians on improving diagnostic accuracy and management strategies for this life-threatening condition. Case presentation 1. Case Summary and History The patient presented with a history of bathing at a local bathhouse 5 days prior to symptom onset, with no reported exposure to rivers, lakes, or environments containing silt or decaying vegetation, as per the mother’s account. On December 5, 2024, the child developed sudden-onset high fever, severe headache, vomiting, and altered mental status. The laboratory findings indicate significant abnormalities in CSF parameters: lactate levels were markedly elevated at 13.51 mmol/L (normal range: 1–2.78 mmol/L), S100 protein showed levels exceeding the reference range at > 39.000 µg/L (normal range: 0–0.105 µg/L), and neuron-specific enolase (NSE) was detected at > 300.000 ng/mL (normal range: 0–16.3 ng/mL). These findings, combined with microbiological analysis confirming Acanthamoeba species infection in the CSF, suggest a severe inflammatory or infectious process affecting the central nervous system. Other clinical findings are included in Supplementary Materials. Initial evaluation and treatment at a local hospital were unsuccessful, prompting transfer to the Eastern District of Henan Children’s Hospital on December 7 for specialized inpatient care. 2. Clinical Course and Outcome Despite escalation of therapy with amphotericin B, fluconazole, and rifampicin, the child’s condition deteriorated rapidly. Progressive neurological decline culminated in deep coma by December 9. The family opted to withdraw treatment, and the patient was discharged against medical advice (AMA). The patient succumbed to fulminant PAM-associated multiorgan failure on the afternoon of that day. 3. Pathogen Genomic Identification On December 10, the Henan Provincial Center for Disease Control and Prevention collected clinical samples from the patient for further analysis. Total nucleic acid was extracted from CSF using the QIAGEN QIAamp DNA Mini Kit with an input volume of 200 µL, followed by quality control checks. Nucleic acid extraction and initial quality assessment were performed by the Jiangsu Institute of Parasitic Diseases, while library preparation and sequencing were conducted by Novogene Co., Ltd. (Beijing). The workflow involved fragmentation of genomic DNA using a Covaris ultrasonicator to generate optimal fragment sizes for Illumina sequencing, followed by end repair, A-tailing, adapter ligation, size selection via AMPure XP beads, PCR amplification, and purification. The finalized libraries were sequenced on the Illumina HiSeq ×10 platform, yielding 10 Gb of paired-end sequencing data. Sequencing reads were analyzed using the Parasite Genome Identification Platform (PGIP), a bioinformatics pipeline developed by the Jiangsu Institute of Parasitic Diseases (accessed via: https://pgip.jipd.com:1443/f/login ), which enabled taxonomic classification and identification of the parasitic pathogen through alignment and matching against the PGIP-curated parasite genome databases. Metagenomic Next-generation Sequencing (mNGS) The metagenomic sequencing results demonstrated high-quality data, with Q20 value of 98.76% and Q30 value of 95.97%. A total of 88,932,436 raw sequencing reads were obtained, of which 2,586,082 clean reads remained after quality control filtering. Taxonomic analysis classified 171,448 reads as parasitic in origin. Subsequent taxonomic analysis identified 50,410 sequencing reads assigned to Naegleria fowleri, accounting for 29.40% relative abundance, providing conclusive evidence of active N. fowleri infection in the CSF sample (Table 1 ). Table 1 Top10 species of reads-based metagenomic analysis Species Mapped reads Relative abundance(%) Naegleria fowleri 50410 29.40% Spirometra erinaceieuropaei 33917 19.78% Toxoplasma gondii 14393 8.39% Wuchereria bancrofti 9785 5.71% Pristionchus exspectatus 7805 4.55% Schmidtea mediterranea 5644 3.29% Onchocerca volvulus 3428 2.00% Dirofilaria immitis 1807 1.05% Parapristionchus giblindavisi 1793 1.05% Pristionchus pacificus 1698 0.99% To further clarify the phylogenetic position of the pathogen, the sequencing data was assembled, and the 18S rRNA gene sequence was extracted for BLAST alignment analysis. Among the alignment results, the longest homologous 18S rRNA gene sequence was selected for phylogenetic tree construction. Phylogenetic analysis revealed that the pathogen in this case showed high consistency with N. fowleri sequences (Fig. 1 ). Targeted PCR Validation Targeted PCR was also carried out for the identification of N. fowleri. Gene primers were synthesized and amplified according to the protocol described in reference [ 7 ] . For N. fowleri , the forward primer Fwl (5'-GTGAAAACCTTTTTTCCATTTACA-3') and reverse primer RV1 (5'-AAATAAAAGATTGACCATTTGAAA-3') were used. For the Naegleria genus, the forward primer Fw2 (5’-GAACCTGCGTAGGGATCATTT-3’) and reverse primer RV2 (5’-TTTCTTTTCCTCCCCTTATTA-3’) were utilized. These primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The PCR reaction mixture consisted of 25 µL of 2× premix, 1.5 µL each of forward and reverse primers (10 µmol/L), 5.0 µL of DNA template, and ddH 2 O to a final volume of 50 µL. The amplification products were identified by 1.5% agarose gel electrophoresis (Fig. 2 ) and then sent to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional sequencing. PCR amplification results showed that the N. fowleri gene bands were approximately 410 bp and 310 bp in size, which matched their theoretical base pair numbers of 408 bp and 310 bp, respectively, confirming the expected fragment sizes. Sequence analysis of the amplification products via BLAST alignment confirmed both sequences as belonging to the Naegleria genus (Fig. 3 ). Morphological Confirmation Definitive morphological confirmation was achieved through microscopic examination of an iodine-stained CSF smear. At 400× magnification, numerous N. fowleri cysts were identified, characterized by round morphology with diameters ranging from 15 to 18 µm and smooth, intact cyst walls. The cysts exhibited a homogeneous eosinophilic cytoplasm following iodine staining, consistent with the pathognomonic features of N. fowleri (Fig. 4 ). Discussion and Conclusions N. fowleri is a thermophilic, free-living amoeba with a life cycle that includes three morphological stages: trophozoites, flagellates, and cysts. Trophozoites are active and proliferate in warm water bodies at temperatures ranging from 25 to 42°C. Flagellates can briefly adapt to low-nutrient environments, while cysts are capable of surviving for several months under extreme conditions [ 8 ] . Recent global warming trends have likely expanded the geographic and temperature ranges conducive to N. fowleri proliferation, thereby amplifying human exposure risks through contaminated water sources [ 9 ] . Human infection with N. fowleri typically occurs through direct contact of the nasal mucosa with contaminated water (e.g., during swimming or diving in warm freshwater). Following entry, trophozoites migrate along olfactory nerve axons, penetrating the cribriform plate to invade the CNS. This rapid invasion culminates in the development of PAM, characterized by severe neuronal destruction [ 10 ] . Pathogenic mechanisms include the secretion of extracellular proteases that degrade host tissues, coupled with contact-dependent cytolytic activity, which synergistically trigger massive inflammatory responses, neuronal apoptosis, and life-threatening cerebral edema [ 11 ] . The median incubation period for N. fowleri infection ranges from 2 to 15 days, with initial symptoms (fever, headache, nausea/vomiting) mimicking bacterial meningitis or viral encephalitis. However, the disease rapidly progresses due to unchecked CNS invasion, often culminating in fatal outcomes within 5–7 days as a result of massive parenchymal destruction, cerebral edema, and brain herniation secondary to intracranial hypertension [ 12 ] . In this case, the child succumbed to the infection just five days after symptom onset, consistent with the typical clinical trajectory of PAM. Since the first reported case of PAM in Australia in 1965, approximately 450 cases have been confirmed globally, with the majority distributed across the United States, Australia, and South Asia [ 13 ] . In China, following the initial case reported in Zhumadian, Henan Province, in 1978, less than 20 cases have been documented in the public literature to date. Among these, Beijing Friendship Hospital reported one case each in 1991 and 1996. Cases from Hainan Province in 2003, Hebei Province in 2006, and Taiwan in 2011 [ 14 ] were also associated with a history of freshwater exposure during the summer months. With advancements in diagnostic techniques, the number of reported PAM cases in China has increased in recent years. These include one case reported in Zhejiang Province in 2016 [ 15 ] , one case each in Hunan Province [ 16 ] and Fujian Province in 2020, one case each in Jiangsu Province and Hubei Province [ 17 ] in 2021, and one case each in Zhejiang Province [ 18 ] and Henan Province in 2022. However, due to limitations in diagnostic technologies and insufficient clinical awareness, the actual incidence of PAM may be underestimated [ 19 ] . The diagnosis of PAM relies on the identification of cysts via wet mount microscopy of CSF, PCR detection, or histopathological confirmation [ 20 ] . However, early-stage routine CSF analyses—such as elevated white blood cell counts (predominantly polymorphonuclear cells) and reduced glucose levels, lack diagnostic specificity, often leading to misdiagnosis as viral or tuberculous meningitis [ 21 ] . Further complicating diagnosis is the fact that N. fowleri does not grow in standard bacterial culture media, and CSF smear microscopy may fail to detect amoebic cysts due to low pathogen concentration or sampling variability. Given the rarity of cases in China, there is a lack of clinical experience in diagnosing PAM, which often results in delayed targeted treatment. In this study, we initially employed metagenomic sequencing to preliminarily identify the pathogen species, which indicated the presence of N. fowleri infection in the sample. Subsequently, a PCR-based detection method was used to confirm the Naegleria species infecting the patient. Following the methodology described by Pélandakis et al. [ 22 ] , we designed two sets of primers: one specific for N. fowleri and another targeting the genus Naegleria . These primers were designed based on the ITS1-ITS2 regions. Ribosomal ITS sequences have been reported as a valuable tool for detecting inter- and intraspecific differences among various microorganisms, including Cryptosporidium parvum , Naegleria spp., and Vahlkampfia spp. [ 23 – 25 ] . Within the genus Naegleria , interspecies and intraspecies variations are attributed to sequence polymorphisms in the ITS2 and ITS1 regions. The ITS region has also been utilized for the identification of newly discovered Naegleria isolates [ 22 , 26 ] . Based on the sequencing results of the PCR amplification products, it was determined that the patient had PAM caused by the rare pathogen N. fowleri . To further confirm the diagnosis, CSF smears were prepared using iodine staining, and microscopic examination revealed amoebic cysts, ultimately confirming the positive diagnosis. In terms of treatment, the current mainstream approach involves a combination therapy including liposomal amphotericin B, miltefosine, rifampicin, and dexamethasone [ 27 , 28 ] . Despite this multi-drug regimen, the overall survival rate remains below 5%. In this case, although amphotericin B, fluconazole, and rifampicin were added after the CSF examination confirmed the presence of N. fowleri , the prognosis could not be reversed due to brainstem failure, highlighting the extremely narrow window for effective early intervention. Given the 95% mortality rate associated with N. fowleri infections and the potential risks of environmental exposure, it is imperative to implement multi-level preventive and control measures. For instance, the public should avoid activities such as diving or jumping into warm freshwater bodies that are not adequately disinfected, including lakes, hot springs, and slow-flowing rivers. Municipal authorities must strengthen water quality monitoring and chlorine disinfection management, especially during summer when water temperatures exceed 30°C. Clear warning signs should be posted in recreational water areas, and real-time microbial risk data should be made publicly available. Primary healthcare facilities must enhance diagnostic vigilance for N. fowleri -associated PAM, prioritizing patients with recent freshwater exposure who present with the characteristic triad of sudden-onset high fever, severe frontal headache, and meningeal signs [ 21 ] . For such cases, immediate action is critical: rapid diagnostic testing via CSF PCR for N. fowleri DNA and iodine-stained microscopy for cyst/trophozoite identification [ 20 ] must be paired with empiric anti-amoebic therapy initiated within the critical 24-hour window to counteract the disease’s fulminant progression, even prior to confirmatory results [ 27 ] . Concurrently, multidisciplinary consultation involving infectious disease specialists, neurologists, and critical care teams is essential to optimize treatment strategies. Clinicians must also recognize the pathognomonic symptom progression of PAM, such as rapid evolution from meningeal signs to altered mental status within 3–5 days, and integrate public health reporting mechanisms to enable timely environmental risk assessments and case tracking [ 12 ] . These measures are imperative given the disease’s < 5% survival rate and narrow therapeutic window, underscoring the need for heightened diagnostic preparedness and system-wide collaboration in regions with endemic risk [ 22 ] . Abbreviations PAM: Primary amoebic meningoencephalitis N. fowleri : Naegleria fowleri CNS: Central nervous system CSF: Cerebrospinal fluid IVIG: Intravenous immunoglobulin NSE: Neuron-specific enolase PCR: Polymerase chain reaction PGIP: Parasite Genome Identification Platform Declarations Ethics approval and consent to participate This study was approved by the Ethics Review Committee of the Henan Provincial Center for Disease Control and Prevention (Ethics Approval No. 2021-KY-010-02). Consent for publication Informed consent was obtained from the families of the participants for procedures. Availability of data and materials All data generated or analyzed during this study are included in this published article. Competing interests The authors declare that they have no competing interests. Funding Not applicable. Authors' contributions Yan Deng designed the study.Yuanjing Kou and Jiayao Zhang conducted the experimental analysis and drafted the manuscript. Lidan Cui , Qi Sun,Yanqi Lü, and Jun Su recruited patients and collected samples/data. Wang Dan performed lab tests. Ying Liu and Zhiquan He managed data. Yuling Zhao, Hongwei Zhang, and Yaobao Liu edited the manuscript. All authors reviewed/revised the manuscript and approved the final version for submission. Acknowledgements The authors would like to thank all staffs of Eastern District of Zhengzhou Children’s Hospital. References Messenger L A, Bern C. Congenital Chagas disease: current diagnostics, limitations and future perspectives[J]. Curr Opin Infect Dis, 2018, 31(5): 415-421. Grace E, Asbill S, Virga K. Naegleria fowleri: pathogenesis, diagnosis, and treatment options[J]. Antimicrob Agents Chemother, 2015, 59(11): 6677-81. CHEN baojian, XIE hanguo, LIN wanhui. Clinical analysis of the first Naegleria fowleri infection in Fujian Province [J]. Chinese Journal of Zoonoses, 2022, 38(01): 89-92. (In Chinese) Jahangeer M, Mahmood Z, Munir N, et al. Naegleria fowleri: Sources of infection, pathophysiology, diagnosis, and management; a review[J]. Clin Exp Pharmacol Physiol, 2020, 47(2): 199-212. Fong H, Leid Z H, Debnath A. Approaches for Targeting Naegleria fowleri Using Nanoparticles and Artificial Peptides[J]. Pathogens, 2024, 13(8). Güémez A, García E. Primary Amoebic Meningoencephalitis by Naegleria fowleri: Pathogenesis and Treatments[J]. Biomolecules, 2021, 11(9). Panda A, Khalil S, Mirdha B R, et al. Prevalence of Naegleria fowleri in Environmental Samples from Northern Part of India[J]. PLoS One, 2015, 10(10): e0137736. Siddiqui R, Ali I K M, Cope J R, et al. Biology and pathogenesis of Naegleria fowleri[J]. Acta Trop, 2016, 164: 375-394. Stahl L M, Olson J B. Environmental abiotic and biotic factors affecting the distribution and abundance of Naegleria fowleri[J]. FEMS Microbiol Ecol, 2020, 97(1). Cabanes P A, Wallet F, Pringuez E, et al. Assessing the risk of primary amoebic meningoencephalitis from swimming in the presence of environmental Naegleria fowleri[J]. Appl Environ Microbiol, 2001, 67(7): 2927-31. Rojo J U, Rajendran R, Salazar J H. Laboratory Diagnosis of Primary Amoebic Meningoencephalitis[J]. Lab Med, 2023, 54(5): e124-e132. Okuda D T, Hanna H J, Coons S W, et al. Naegleria fowleri hemorrhagic meningoencephalitis: report of two fatalities in children[J]. J Child Neurol, 2004, 19(3): 231-3. Mukhtar F, Wazir M S. NAEGLARIA FOWLERI: THE BRAIN EATING AMOEBA OR AN ENIGMA?[J]. J Ayub Med Coll Abbottabad, 2015, 27(3): 735-6. Tung M C, Hsu B M, Tao C W, et al. Identification and significance of Naegleria fowleri isolated from the hot spring which related to the first primary amebic meningoencephalitis (PAM) patient in Taiwan[J]. Int J Parasitol, 2013, 43(9): 691-6. Zhang L L, Wu M, Hu B C, et al. Identification and molecular typing of Naegleria fowleri from a patient with primary amebic meningoencephalitis in China[J]. Int J Infect Dis, 2018, 72: 28-33. Zhou W, Ouyang Y, Zhang D, et al. Case Report and Literature Review: Bacterial Meningoencephalitis or Not? Naegleria fowleri Related Primary Amoebic Meningoencephalitis in China[J]. Front Pediatr, 2022, 10: 785735. Tian Keqing, Wang Yi, He Liangcai, et al. An investigation of one death case probably caused by Naegleria fowleri infection in Hubei, 2021 [J]. Disease Surveillance, 2023, 38(01): 124-127. (In Chinese) CHEN Rui, YUAN Qiong-hui, XIA Wan-bao. Laboratory diagnosis of a rare case of primary amebic meningoencephalitis [J]. Chinese Journal of Parasitology and Parasitic Diseases, 2022, 40(05): 616-621. (In Chinese) Huang S, Liang X, Han Y, et al. A pediatric case of primary amoebic meningoencephalitis due to Naegleria fowleri diagnosed by next-generation sequencing of cerebrospinal fluid and blood samples[J]. BMC Infect Dis, 2021, 21(1): 1251. Kang H, Seong G S, Sohn H J, et al. Effective PCR-based detection of Naegleria fowleri from cultured sample and PAM-developed mouse[J]. Eur J Protistol, 2015, 51(5): 401-8. Hall A D, Kumar J E, Golba C E, et al. Primary amebic meningoencephalitis: a review of Naegleria fowleri and analysis of successfully treated cases[J]. Parasitol Res, 2024, 123(1): 84. Pélandakis M, Serre S, Pernin P. Analysis of the 5.8S rRNA gene and the internal transcribed spacers in Naegleria spp. and in N. fowleri[J]. J Eukaryot Microbiol, 2000, 47(2): 116-21. Aurongzeb M, Fatima S Z, Hussain S I, et al. Detection and identification of Naegleria species along with Naegleria fowleri in the tap water samples[J]. BMC Med Genomics, 2025, 18(1): 6. Tiewcharoen S, Junnu V, Sassa-Deepaeng T, et al. Analysis of the 5.8S rRNA and internal transcribed spacers regions of the variant Naegleria fowleri Thai strain[J]. Parasitol Res, 2007, 101(1): 139-43. Gao Z, Jousset A, Kowalchuk G A, et al. Five Groups in the Genus Allovahlkampfia and the Description of the New Species Vahlkampfia bulbosis n.sp[J]. Protist, 2022, 173(3): 125870. Sheehan K B, Fagg J A, Ferris M J, et al. PCR detection and analysis of the free-living amoeba Naegleria in hot springs in Yellowstone and Grand Teton National Parks[J]. Appl Environ Microbiol, 2003, 69(10): 5914-8. Jhulki S, Bhowmik B, Pal A. Enlightening the promising role of nanoparticle-based treatments against Naegleria fowleri-induced primary amoebic meningoencephalitis: A brain-eating disease[J]. Microb Pathog, 2025, 199: 107234. Vargas-Zepeda J, Gómez-Alcalá A V, Vásquez-Morales J A, et al. Successful treatment of Naegleria fowleri meningoencephalitis by using intravenous amphotericin B, fluconazole and rifampicin[J]. Arch Med Res, 2005, 36(1): 83-6. Supplementary Files Graphicalabstract.jpg Graphical abstract :Neighbor-Joining Tree inferred from partial 18S rRNA sequences (1843bp) from mNGS analysis of N. fowleri SupplementaryMaterials.docx Cite Share Download PDF Status: Published Journal Publication published 17 Jul, 2025 Read the published version in Infectious Diseases of Poverty → Version 1 posted Reviewers agreed at journal 11 May, 2025 Reviewers invited by journal 09 May, 2025 Editor assigned by journal 08 May, 2025 First submitted to journal 07 May, 2025 Editorial decision: Minor revision 06 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6549687","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":454184859,"identity":"3c1e557e-a79b-4ede-8ea5-3f8ef08f6f1e","order_by":0,"name":"Yuanjing Kou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYBACCSjNzMbAwPggoaKGNC3MBg/OHCNeCwiwST5sYSasRbK9x0yap+YOO590+7WKxAY2Bv727gS8WqR5zgC1HHvGzCZzpuxG4g4ZBokzZzfg1SInkQPUwnaYmU0iJ+1G4hk2BgOJXGK0/INoKUhsYyasRRqkhbcNpCX9GANRWiR7jhVbzu0D28IskXDmGA9Bv0gcb9544823w8nyM9IffvxRUSPH396LXwsQsIDiJpmBgccAxOMhpBwEmD8ACTsGBvYHxKgeBaNgFIyCEQgAShtCBhn9e0IAAAAASUVORK5CYII=","orcid":"","institution":"Henan Province CDC: Henan Province Center for Disease Control and Prevention","correspondingAuthor":true,"prefix":"","firstName":"Yuanjing","middleName":"","lastName":"Kou","suffix":""},{"id":454184860,"identity":"0374c60b-ba78-4705-8bbc-981b6db36183","order_by":1,"name":"Jiayao Zhang","email":"","orcid":"","institution":"Jiangsu Institute of Parasitic Disease","correspondingAuthor":false,"prefix":"","firstName":"Jiayao","middleName":"","lastName":"Zhang","suffix":""},{"id":454184861,"identity":"8226022d-992b-4c51-8018-f7d67a205856","order_by":2,"name":"Dan Wang","email":"","orcid":"","institution":"Henan Province CDC: Henan Province Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Wang","suffix":""},{"id":454184862,"identity":"4ca94b7b-15e6-41b0-bc20-95ecbc809a40","order_by":3,"name":"Lidan Cui","email":"","orcid":"","institution":"Henan Children's Hospital: Zhengzhou Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lidan","middleName":"","lastName":"Cui","suffix":""},{"id":454184863,"identity":"0f870fa9-78b9-4447-9b46-a233b473a709","order_by":4,"name":"Qi Sun","email":"","orcid":"","institution":"Henan Children's Hospital: Zhengzhou Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Sun","suffix":""},{"id":454184864,"identity":"28feed1c-c81e-4542-b0fb-998c2921b9a9","order_by":5,"name":"Yanqi Lv","email":"","orcid":"","institution":"Henan Children's Hospital: Zhengzhou Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yanqi","middleName":"","lastName":"Lv","suffix":""},{"id":454184865,"identity":"771b7f68-2760-4862-b2d7-b07faef11475","order_by":6,"name":"Ying Liu","email":"","orcid":"","institution":"Henan Province CDC: Henan Province Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Liu","suffix":""},{"id":454184866,"identity":"acec172e-3c6e-4f6a-8142-8146eaaee33f","order_by":7,"name":"Zhiquan He","email":"","orcid":"","institution":"Henan Province CDC: Henan Province Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Zhiquan","middleName":"","lastName":"He","suffix":""},{"id":454184867,"identity":"d03bbe57-5bbe-4a21-b57d-3cd1ec1b046c","order_by":8,"name":"Yuling Zhao","email":"","orcid":"","institution":"Henan Province CDC: Henan Province Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Yuling","middleName":"","lastName":"Zhao","suffix":""},{"id":454184868,"identity":"947c9e83-8c91-4540-bb50-89ccb8190f43","order_by":9,"name":"Hongwei Zhang","email":"","orcid":"","institution":"Henan Province CDC: Henan Province Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Hongwei","middleName":"","lastName":"Zhang","suffix":""},{"id":454184869,"identity":"6f5aa6a6-9df7-482e-8ace-67559453a144","order_by":10,"name":"Jun Su","email":"","orcid":"","institution":"Henan Children's Hospital: Zhengzhou Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Su","suffix":""},{"id":454184870,"identity":"1129c1ae-2c57-4cc8-950a-072c7d5eed09","order_by":11,"name":"Yaobao Liu","email":"","orcid":"","institution":"Jiangsu Institute of Parasitic Disease","correspondingAuthor":false,"prefix":"","firstName":"Yaobao","middleName":"","lastName":"Liu","suffix":""},{"id":454184871,"identity":"f795e6f6-d4a4-443e-85e5-c14c4f1ca4bd","order_by":12,"name":"Yan Deng","email":"","orcid":"","institution":"Henan Province CDC: Henan Province Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Deng","suffix":""}],"badges":[],"createdAt":"2025-04-28 16:37:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6549687/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6549687/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40249-025-01347-z","type":"published","date":"2025-07-17T16:05:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82715869,"identity":"30edf68a-f4b4-4b40-8c3c-1a8ac50e2768","added_by":"auto","created_at":"2025-05-14 12:09:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":119267,"visible":true,"origin":"","legend":"\u003cp\u003eNeighbor-Joining Tree inferred from partial 18S rRNA sequences (1843bp) from mNGS analysis of \u003cem\u003eN. fowleri\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6549687/v1/272a87b5629c262ed07f1480.jpg"},{"id":82717346,"identity":"379d5e50-08cc-44f8-90b0-77ecb2bc9dad","added_by":"auto","created_at":"2025-05-14 12:25:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":25990,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAgarose gel electrophoresis of PCR amplification products from the CSF sample using species- and genus-specific primers for Naegleria. \u003c/strong\u003eM, DNA marker; lanes 1–4, PCR products from the patient’s CSF sample.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6549687/v1/7532a44b3473d6884bb60669.jpg"},{"id":82716456,"identity":"439d8e4c-9aad-4b22-a195-55b7c9b4e88a","added_by":"auto","created_at":"2025-05-14 12:17:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNaegleria\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e based on ITS gene region. \u003c/strong\u003eThe numbers at the nodes indicate the percentage bootstrap values from 1000 replicates, with only values greater than 50% shown. GenBank accession numbers are indicated before the species names.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6549687/v1/8a195970d150d393006c568c.jpg"},{"id":82717347,"identity":"5bc70bf8-fc5a-4c8b-8750-448381d8a1b1","added_by":"auto","created_at":"2025-05-14 12:25:17","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":15601,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphology of amoebic cysts in the patient’s CSF. \u003c/strong\u003e400×, Scale Bar = 20μm.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6549687/v1/9ded09aca04b19a207c4f5f4.jpg"},{"id":88506070,"identity":"3bd5bb07-0c6a-4393-b196-718b27d154e5","added_by":"auto","created_at":"2025-08-07 07:30:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":887833,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6549687/v1/8f060cd8-3985-41db-af70-5ba0ef65edfa.pdf"},{"id":82716450,"identity":"c3923885-ee1e-4557-8de4-f751f5f6389b","added_by":"auto","created_at":"2025-05-14 12:17:17","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":119267,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract :\u003c/strong\u003eNeighbor-Joining Tree inferred from partial 18S rRNA sequences (1843bp) from mNGS analysis of \u003cem\u003eN. fowleri\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6549687/v1/fa404fdced3a7f838cb453f4.jpg"},{"id":82717348,"identity":"394e14b0-777a-420e-b1ce-cf448f472c6b","added_by":"auto","created_at":"2025-05-14 12:25:17","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17490,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6549687/v1/bb567d2262dd6ae63414fe2c.docx"}],"financialInterests":"","formattedTitle":"Case Report of Pediatric Naegleria fowleri Meningoencephalitis","fulltext":[{"header":"Background","content":"\u003cp\u003ePathogenic amoebae represent a diverse group of organisms widely distributed in natural and artificial environments, including soil, lakes, rivers, hot springs, inadequately chlorinated swimming pools, and water distribution systems\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Among these, \u003cem\u003eNaegleria fowleri\u003c/em\u003e (\u003cem\u003eN. fowleri\u003c/em\u003e), commonly termed the \"brain-eating amoeba\", is a particularly virulent species\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. This free-living amoeba invades the central nervous system (CNS) by entering through the nasal mucosa, migrating along the olfactory nerve through the cribriform plate into the cranial cavity, and ultimately causing primary amoebic meningoencephalitis (PAM) \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. PAM is a fulminant disease characterized by acute onset, rapid progression, severe clinical manifestations, and an extremely high mortality rate (\u0026gt; 95%), predominantly affecting children and young adults\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Despite its rarity, the aggressive nature of this infection necessitates urgent clinical attention.\u003c/p\u003e \u003cp\u003eCurrently, no rapid and reliable diagnostic methods are universally available for amoebic infections, and early symptoms of PAM—including fever, headache, vomiting, and neck stiffness, overlap significantly with those of other CNS infections\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. This clinical mimicry poses substantial diagnostic challenges, often leading to delayed recognition and suboptimal treatment outcomes. In this case report, we present a detailed analysis of the diagnostic utility of multiple detection techniques applied to cerebrospinal fluid (CSF) samples from a suspected pediatric \u003cem\u003eN. fowleri\u003c/em\u003e infection. Our findings aim to inform clinicians on improving diagnostic accuracy and management strategies for this life-threatening condition.\u003c/p\u003e"},{"header":"Case presentation","content":"\u003ch3\u003e1. Case Summary and History\u003c/h3\u003e\u003cp\u003eThe patient presented with a history of bathing at a local bathhouse 5 days prior to symptom onset, with no reported exposure to rivers, lakes, or environments containing silt or decaying vegetation, as per the mother’s account. On December 5, 2024, the child developed sudden-onset high fever, severe headache, vomiting, and altered mental status. The laboratory findings indicate significant abnormalities in CSF parameters: lactate levels were markedly elevated at 13.51 mmol/L (normal range: 1–2.78 mmol/L), S100 protein showed levels exceeding the reference range at \u0026gt; 39.000 µg/L (normal range: 0–0.105 µg/L), and neuron-specific enolase (NSE) was detected at \u0026gt; 300.000 ng/mL (normal range: 0–16.3 ng/mL). These findings, combined with microbiological analysis confirming Acanthamoeba species infection in the CSF, suggest a severe inflammatory or infectious process affecting the central nervous system. Other clinical findings are included in Supplementary Materials. Initial evaluation and treatment at a local hospital were unsuccessful, prompting transfer to the Eastern District of Henan Children’s Hospital on December 7 for specialized inpatient care.\u003c/p\u003e\u003ch2\u003e2. Clinical Course and Outcome\u003c/h2\u003e\u003cp\u003eDespite escalation of therapy with amphotericin B, fluconazole, and rifampicin, the child’s condition deteriorated rapidly. Progressive neurological decline culminated in deep coma by December 9. The family opted to withdraw treatment, and the patient was discharged against medical advice (AMA). The patient succumbed to fulminant PAM-associated multiorgan failure on the afternoon of that day.\u003c/p\u003e\u003ch3\u003e3. Pathogen Genomic Identification\u003c/h3\u003e\u003cp\u003eOn December 10, the Henan Provincial Center for Disease Control and Prevention collected clinical samples from the patient for further analysis. Total nucleic acid was extracted from CSF using the QIAGEN QIAamp DNA Mini Kit with an input volume of 200 µL, followed by quality control checks. Nucleic acid extraction and initial quality assessment were performed by the Jiangsu Institute of Parasitic Diseases, while library preparation and sequencing were conducted by Novogene Co., Ltd. (Beijing). The workflow involved fragmentation of genomic DNA using a Covaris ultrasonicator to generate optimal fragment sizes for Illumina sequencing, followed by end repair, A-tailing, adapter ligation, size selection via AMPure XP beads, PCR amplification, and purification. The finalized libraries were sequenced on the Illumina HiSeq ×10 platform, yielding 10 Gb of paired-end sequencing data. Sequencing reads were analyzed using the Parasite Genome Identification Platform (PGIP), a bioinformatics pipeline developed by the Jiangsu Institute of Parasitic Diseases (accessed via: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pgip.jipd.com:1443/f/login\u003c/span\u003e\u003cspan address=\"https://pgip.jipd.com:1443/f/login\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which enabled taxonomic classification and identification of the parasitic pathogen through alignment and matching against the PGIP-curated parasite genome databases.\u003c/p\u003e\u003ch3\u003eMetagenomic Next-generation Sequencing (mNGS)\u003c/h3\u003e\u003cp\u003eThe metagenomic sequencing results demonstrated high-quality data, with Q20 value of 98.76% and Q30 value of 95.97%. A total of 88,932,436 raw sequencing reads were obtained, of which 2,586,082 clean reads remained after quality control filtering. Taxonomic analysis classified 171,448 reads as parasitic in origin. Subsequent taxonomic analysis identified 50,410 sequencing reads assigned to Naegleria fowleri, accounting for 29.40% relative abundance, providing conclusive evidence of active \u003cem\u003eN. fowleri\u003c/em\u003e infection in the CSF sample (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTop10 species of reads-based metagenomic analysis\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMapped reads\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRelative abundance(%)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNaegleria fowleri\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50410\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e29.40%\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSpirometra erinaceieuropaei\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e33917\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19.78%\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToxoplasma gondii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14393\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.39%\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eWuchereria bancrofti\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9785\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.71%\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePristionchus exspectatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7805\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.55%\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSchmidtea mediterranea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5644\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.29%\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eOnchocerca volvulus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3428\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.00%\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDirofilaria immitis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1807\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.05%\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eParapristionchus giblindavisi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1793\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.05%\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePristionchus pacificus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1698\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.99%\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003eTo further clarify the phylogenetic position of the pathogen, the sequencing data was assembled, and the 18S rRNA gene sequence was extracted for BLAST alignment analysis. Among the alignment results, the longest homologous 18S rRNA gene sequence was selected for phylogenetic tree construction. Phylogenetic analysis revealed that the pathogen in this case showed high consistency with \u003cem\u003eN. fowleri\u003c/em\u003e sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003ch3\u003eTargeted PCR Validation\u003c/h3\u003e\u003cp\u003eTargeted PCR was also carried out for the identification of N. fowleri. Gene primers were synthesized and amplified according to the protocol described in reference\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. For \u003cem\u003eN. fowleri\u003c/em\u003e, the forward primer Fwl (5'-GTGAAAACCTTTTTTCCATTTACA-3') and reverse primer RV1 (5'-AAATAAAAGATTGACCATTTGAAA-3') were used. For the \u003cem\u003eNaegleria\u003c/em\u003e genus, the forward primer Fw2 (5’-GAACCTGCGTAGGGATCATTT-3’) and reverse primer RV2 (5’-TTTCTTTTCCTCCCCTTATTA-3’) were utilized. These primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.\u003c/p\u003e\u003cp\u003eThe PCR reaction mixture consisted of 25 µL of 2× premix, 1.5 µL each of forward and reverse primers (10 µmol/L), 5.0 µL of DNA template, and ddH\u003csub\u003e2\u003c/sub\u003eO to a final volume of 50 µL. The amplification products were identified by 1.5% agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and then sent to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional sequencing.\u003c/p\u003e\u003cp\u003ePCR amplification results showed that the \u003cem\u003eN. fowleri gene\u003c/em\u003e bands were approximately 410 bp and 310 bp in size, which matched their theoretical base pair numbers of 408 bp and 310 bp, respectively, confirming the expected fragment sizes. Sequence analysis of the amplification products via BLAST alignment confirmed both sequences as belonging to the \u003cem\u003eNaegleria\u003c/em\u003e genus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003ch3\u003eMorphological Confirmation\u003c/h3\u003e\u003cp\u003eDefinitive morphological confirmation was achieved through microscopic examination of an iodine-stained CSF smear. At 400× magnification, numerous \u003cem\u003eN. fowleri\u003c/em\u003e cysts were identified, characterized by round morphology with diameters ranging from 15 to 18 µm and smooth, intact cyst walls. The cysts exhibited a homogeneous eosinophilic cytoplasm following iodine staining, consistent with the pathognomonic features of \u003cem\u003eN. fowleri\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion and Conclusions","content":"\u003cp\u003e \u003cem\u003eN. fowleri\u003c/em\u003e is a thermophilic, free-living amoeba with a life cycle that includes three morphological stages: trophozoites, flagellates, and cysts. Trophozoites are active and proliferate in warm water bodies at temperatures ranging from 25 to 42°C. Flagellates can briefly adapt to low-nutrient environments, while cysts are capable of surviving for several months under extreme conditions\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Recent global warming trends have likely expanded the geographic and temperature ranges conducive to \u003cem\u003eN. fowleri\u003c/em\u003e proliferation, thereby amplifying human exposure risks through contaminated water sources\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHuman infection with \u003cem\u003eN. fowleri\u003c/em\u003e typically occurs through direct contact of the nasal mucosa with contaminated water (e.g., during swimming or diving in warm freshwater). Following entry, trophozoites migrate along olfactory nerve axons, penetrating the cribriform plate to invade the CNS. This rapid invasion culminates in the development of PAM, characterized by severe neuronal destruction\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Pathogenic mechanisms include the secretion of extracellular proteases that degrade host tissues, coupled with contact-dependent cytolytic activity, which synergistically trigger massive inflammatory responses, neuronal apoptosis, and life-threatening cerebral edema\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe median incubation period for \u003cem\u003eN. fowleri\u003c/em\u003e infection ranges from 2 to 15 days, with initial symptoms (fever, headache, nausea/vomiting) mimicking bacterial meningitis or viral encephalitis. However, the disease rapidly progresses due to unchecked CNS invasion, often culminating in fatal outcomes within 5–7 days as a result of massive parenchymal destruction, cerebral edema, and brain herniation secondary to intracranial hypertension\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. In this case, the child succumbed to the infection just five days after symptom onset, consistent with the typical clinical trajectory of PAM.\u003c/p\u003e\u003cp\u003eSince the first reported case of PAM in Australia in 1965, approximately 450 cases have been confirmed globally, with the majority distributed across the United States, Australia, and South Asia\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. In China, following the initial case reported in Zhumadian, Henan Province, in 1978, less than 20 cases have been documented in the public literature to date. Among these, Beijing Friendship Hospital reported one case each in 1991 and 1996. Cases from Hainan Province in 2003, Hebei Province in 2006, and Taiwan in 2011\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e were also associated with a history of freshwater exposure during the summer months.\u003c/p\u003e\u003cp\u003eWith advancements in diagnostic techniques, the number of reported PAM cases in China has increased in recent years. These include one case reported in Zhejiang Province in 2016\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, one case each in Hunan Province\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e and Fujian Province in 2020, one case each in Jiangsu Province and Hubei Province\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e in 2021, and one case each in Zhejiang Province\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e and Henan Province in 2022. However, due to limitations in diagnostic technologies and insufficient clinical awareness, the actual incidence of PAM may be underestimated\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe diagnosis of PAM relies on the identification of cysts via wet mount microscopy of CSF, PCR detection, or histopathological confirmation\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. However, early-stage routine CSF analyses—such as elevated white blood cell counts (predominantly polymorphonuclear cells) and reduced glucose levels, lack diagnostic specificity, often leading to misdiagnosis as viral or tuberculous meningitis\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Further complicating diagnosis is the fact that \u003cem\u003eN. fowleri\u003c/em\u003e does not grow in standard bacterial culture media, and CSF smear microscopy may fail to detect amoebic cysts due to low pathogen concentration or sampling variability. Given the rarity of cases in China, there is a lack of clinical experience in diagnosing PAM, which often results in delayed targeted treatment. In this study, we initially employed metagenomic sequencing to preliminarily identify the pathogen species, which indicated the presence of \u003cem\u003eN. fowleri\u003c/em\u003e infection in the sample. Subsequently, a PCR-based detection method was used to confirm the \u003cem\u003eNaegleria\u003c/em\u003e species infecting the patient. Following the methodology described by Pélandakis et al. \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, we designed two sets of primers: one specific for \u003cem\u003eN. fowleri\u003c/em\u003e and another targeting the genus \u003cem\u003eNaegleria\u003c/em\u003e. These primers were designed based on the ITS1-ITS2 regions. Ribosomal ITS sequences have been reported as a valuable tool for detecting inter- and intraspecific differences among various microorganisms, including \u003cem\u003eCryptosporidium parvum\u003c/em\u003e, \u003cem\u003eNaegleria\u003c/em\u003e spp., and \u003cem\u003eVahlkampfia\u003c/em\u003e spp.\u003csup\u003e[\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e–\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Within the genus \u003cem\u003eNaegleria\u003c/em\u003e, interspecies and intraspecies variations are attributed to sequence polymorphisms in the ITS2 and ITS1 regions. The ITS region has also been utilized for the identification of newly discovered \u003cem\u003eNaegleria\u003c/em\u003e isolates\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Based on the sequencing results of the PCR amplification products, it was determined that the patient had PAM caused by the rare pathogen \u003cem\u003eN. fowleri\u003c/em\u003e. To further confirm the diagnosis, CSF smears were prepared using iodine staining, and microscopic examination revealed amoebic cysts, ultimately confirming the positive diagnosis.\u003c/p\u003e\u003cp\u003eIn terms of treatment, the current mainstream approach involves a combination therapy including liposomal amphotericin B, miltefosine, rifampicin, and dexamethasone\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Despite this multi-drug regimen, the overall survival rate remains below 5%. In this case, although amphotericin B, fluconazole, and rifampicin were added after the CSF examination confirmed the presence of \u003cem\u003eN. fowleri\u003c/em\u003e, the prognosis could not be reversed due to brainstem failure, highlighting the extremely narrow window for effective early intervention.\u003c/p\u003e\u003cp\u003eGiven the 95% mortality rate associated with \u003cem\u003eN. fowleri\u003c/em\u003e infections and the potential risks of environmental exposure, it is imperative to implement multi-level preventive and control measures. For instance, the public should avoid activities such as diving or jumping into warm freshwater bodies that are not adequately disinfected, including lakes, hot springs, and slow-flowing rivers. Municipal authorities must strengthen water quality monitoring and chlorine disinfection management, especially during summer when water temperatures exceed 30°C. Clear warning signs should be posted in recreational water areas, and real-time microbial risk data should be made publicly available.\u003c/p\u003e\u003cp\u003ePrimary healthcare facilities must enhance diagnostic vigilance for \u003cem\u003eN. fowleri\u003c/em\u003e-associated PAM, prioritizing patients with recent freshwater exposure who present with the characteristic triad of sudden-onset high fever, severe frontal headache, and meningeal signs\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. For such cases, immediate action is critical: rapid diagnostic testing via CSF PCR for \u003cem\u003eN. fowleri\u003c/em\u003e DNA and iodine-stained microscopy for cyst/trophozoite identification\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e must be paired with empiric anti-amoebic therapy initiated within the critical 24-hour window to counteract the disease’s fulminant progression, even prior to confirmatory results\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Concurrently, multidisciplinary consultation involving infectious disease specialists, neurologists, and critical care teams is essential to optimize treatment strategies. Clinicians must also recognize the pathognomonic symptom progression of PAM, such as rapid evolution from meningeal signs to altered mental status within 3–5 days, and integrate public health reporting mechanisms to enable timely environmental risk assessments and case tracking\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. These measures are imperative given the disease’s \u0026lt; 5% survival rate and narrow therapeutic window, underscoring the need for heightened diagnostic preparedness and system-wide collaboration in regions with endemic risk\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePAM: Primary amoebic meningoencephalitis\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eN. fowleri\u003c/em\u003e: \u003cem\u003eNaegleria fowleri\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCNS: Central nervous system\u003c/p\u003e\n\u003cp\u003eCSF: Cerebrospinal fluid\u003c/p\u003e\n\u003cp\u003eIVIG: Intravenous immunoglobulin\u003c/p\u003e\n\u003cp\u003eNSE: Neuron-specific enolase\u003c/p\u003e\n\u003cp\u003ePCR: Polymerase chain reaction\u003c/p\u003e\n\u003cp\u003ePGIP: Parasite Genome Identification Platform\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Review Committee of the Henan Provincial Center for Disease Control and Prevention (Ethics Approval No. 2021-KY-010-02).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from the families of the participants for procedures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYan Deng designed the study.Yuanjing Kou and Jiayao Zhang conducted the experimental analysis and drafted the manuscript. Lidan Cui , Qi Sun,Yanqi L\u0026uuml;, and Jun Su recruited patients and collected samples/data. Wang Dan performed lab tests. Ying Liu and Zhiquan He managed data. \u0026nbsp;Yuling Zhao, Hongwei Zhang, and Yaobao Liu edited the manuscript. All authors reviewed/revised the manuscript and approved the final version for submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank all staffs of Eastern District of Zhengzhou Children\u0026rsquo;s Hospital.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMessenger L A, Bern C. Congenital Chagas disease: current diagnostics, limitations and future perspectives[J]. Curr Opin Infect Dis, 2018, 31(5): 415-421.\u003c/li\u003e\n\u003cli\u003eGrace E, Asbill S, Virga K. Naegleria fowleri: pathogenesis, diagnosis, and treatment options[J]. Antimicrob Agents Chemother, 2015, 59(11): 6677-81.\u003c/li\u003e\n\u003cli\u003eCHEN baojian, XIE hanguo, LIN wanhui. Clinical analysis of the first Naegleria fowleri infection in Fujian Province [J]. Chinese Journal of Zoonoses, 2022, 38(01): 89-92. (In Chinese)\u003c/li\u003e\n\u003cli\u003eJahangeer M, Mahmood Z, Munir N, et al. Naegleria fowleri: Sources of infection, pathophysiology, diagnosis, and management; a review[J]. Clin Exp Pharmacol Physiol, 2020, 47(2): 199-212.\u003c/li\u003e\n\u003cli\u003eFong H, Leid Z H, Debnath A. Approaches for Targeting Naegleria fowleri Using Nanoparticles and Artificial Peptides[J]. Pathogens, 2024, 13(8).\u003c/li\u003e\n\u003cli\u003eG\u0026uuml;\u0026eacute;mez A, Garc\u0026iacute;a E. Primary Amoebic Meningoencephalitis by Naegleria fowleri: Pathogenesis and Treatments[J]. Biomolecules, 2021, 11(9).\u003c/li\u003e\n\u003cli\u003ePanda A, Khalil S, Mirdha B R, et al. Prevalence of Naegleria fowleri in Environmental Samples from Northern Part of India[J]. PLoS One, 2015, 10(10): e0137736.\u003c/li\u003e\n\u003cli\u003eSiddiqui R, Ali I K M, Cope J R, et al. Biology and pathogenesis of Naegleria fowleri[J]. Acta Trop, 2016, 164: 375-394.\u003c/li\u003e\n\u003cli\u003eStahl L M, Olson J B. Environmental abiotic and biotic factors affecting the distribution and abundance of Naegleria fowleri[J]. FEMS Microbiol Ecol, 2020, 97(1).\u003c/li\u003e\n\u003cli\u003eCabanes P A, Wallet F, Pringuez E, et al. Assessing the risk of primary amoebic meningoencephalitis from swimming in the presence of environmental Naegleria fowleri[J]. Appl Environ Microbiol, 2001, 67(7): 2927-31.\u003c/li\u003e\n\u003cli\u003eRojo J U, Rajendran R, Salazar J H. Laboratory Diagnosis of Primary Amoebic Meningoencephalitis[J]. Lab Med, 2023, 54(5): e124-e132.\u003c/li\u003e\n\u003cli\u003eOkuda D T, Hanna H J, Coons S W, et al. Naegleria fowleri hemorrhagic meningoencephalitis: report of two fatalities in children[J]. J Child Neurol, 2004, 19(3): 231-3.\u003c/li\u003e\n\u003cli\u003eMukhtar F, Wazir M S. NAEGLARIA FOWLERI: THE BRAIN EATING AMOEBA OR AN ENIGMA?[J]. J Ayub Med Coll Abbottabad, 2015, 27(3): 735-6.\u003c/li\u003e\n\u003cli\u003eTung M C, Hsu B M, Tao C W, et al. Identification and significance of Naegleria fowleri isolated from the hot spring which related to the first primary amebic meningoencephalitis (PAM) patient in Taiwan[J]. Int J Parasitol, 2013, 43(9): 691-6.\u003c/li\u003e\n\u003cli\u003eZhang L L, Wu M, Hu B C, et al. Identification and molecular typing of Naegleria fowleri from a patient with primary amebic meningoencephalitis in China[J]. Int J Infect Dis, 2018, 72: 28-33.\u003c/li\u003e\n\u003cli\u003eZhou W, Ouyang Y, Zhang D, et al. Case Report and Literature Review: Bacterial Meningoencephalitis or Not? Naegleria fowleri Related Primary Amoebic Meningoencephalitis in China[J]. Front Pediatr, 2022, 10: 785735.\u003c/li\u003e\n\u003cli\u003eTian Keqing, Wang Yi, He Liangcai, et al. An investigation of one death case probably caused by Naegleria fowleri infection in Hubei, 2021 [J]. Disease Surveillance, 2023, 38(01): 124-127. (In Chinese)\u003c/li\u003e\n\u003cli\u003eCHEN Rui, YUAN Qiong-hui, XIA Wan-bao. Laboratory diagnosis of a rare case of primary amebic meningoencephalitis [J]. Chinese Journal of Parasitology and Parasitic Diseases, 2022, 40(05): 616-621. (In Chinese)\u003c/li\u003e\n\u003cli\u003eHuang S, Liang X, Han Y, et al. A pediatric case of primary amoebic meningoencephalitis due to Naegleria fowleri diagnosed by next-generation sequencing of cerebrospinal fluid and blood samples[J]. BMC Infect Dis, 2021, 21(1): 1251.\u003c/li\u003e\n\u003cli\u003eKang H, Seong G S, Sohn H J, et al. Effective PCR-based detection of Naegleria fowleri from cultured sample and PAM-developed mouse[J]. Eur J Protistol, 2015, 51(5): 401-8.\u003c/li\u003e\n\u003cli\u003eHall A D, Kumar J E, Golba C E, et al. Primary amebic meningoencephalitis: a review of Naegleria fowleri and analysis of successfully treated cases[J]. Parasitol Res, 2024, 123(1): 84.\u003c/li\u003e\n\u003cli\u003eP\u0026eacute;landakis M, Serre S, Pernin P. Analysis of the 5.8S rRNA gene and the internal transcribed spacers in Naegleria spp. and in N. fowleri[J]. J Eukaryot Microbiol, 2000, 47(2): 116-21.\u003c/li\u003e\n\u003cli\u003eAurongzeb M, Fatima S Z, Hussain S I, et al. Detection and identification of Naegleria species along with Naegleria fowleri in the tap water samples[J]. BMC Med Genomics, 2025, 18(1): 6.\u003c/li\u003e\n\u003cli\u003eTiewcharoen S, Junnu V, Sassa-Deepaeng T, et al. Analysis of the 5.8S rRNA and internal transcribed spacers regions of the variant Naegleria fowleri Thai strain[J]. Parasitol Res, 2007, 101(1): 139-43.\u003c/li\u003e\n\u003cli\u003eGao Z, Jousset A, Kowalchuk G A, et al. Five Groups in the Genus Allovahlkampfia and the Description of the New Species Vahlkampfia bulbosis n.sp[J]. Protist, 2022, 173(3): 125870.\u003c/li\u003e\n\u003cli\u003eSheehan K B, Fagg J A, Ferris M J, et al. PCR detection and analysis of the free-living amoeba Naegleria in hot springs in Yellowstone and Grand Teton National Parks[J]. Appl Environ Microbiol, 2003, 69(10): 5914-8.\u003c/li\u003e\n\u003cli\u003eJhulki S, Bhowmik B, Pal A. Enlightening the promising role of nanoparticle-based treatments against Naegleria fowleri-induced primary amoebic meningoencephalitis: A brain-eating disease[J]. Microb Pathog, 2025, 199: 107234.\u003c/li\u003e\n\u003cli\u003eVargas-Zepeda J, G\u0026oacute;mez-Alcal\u0026aacute; A V, V\u0026aacute;squez-Morales J A, et al. Successful treatment of Naegleria fowleri meningoencephalitis by using intravenous amphotericin B, fluconazole and rifampicin[J]. Arch Med Res, 2005, 36(1): 83-6.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"infectious-diseases-of-poverty","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"idop","sideBox":"Learn more about [Infectious Diseases of Poverty](http://idpjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/idop/default.aspx","title":"Infectious Diseases of Poverty","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Primary amoebic meningoencephalitis, Naegleria fowleri, Genetic analysis, Metagenomic sequencing","lastPublishedDoi":"10.21203/rs.3.rs-6549687/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6549687/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNaegleria fowleri\u003c/em\u003e, a pathogenic free-living amoeba, is the causative agent of primary amoebic meningoencephalitis (PAM), a rare but devastating disease. PAM is characterized by its acute onset, rapid progression, and severe clinical manifestations, with mortality rates exceeding 95%. Despite its rarity, the catastrophic outcomes associated with this infection underscore the critical importance of prevention. In this report, we present a rare pediatric fatality caused by PAM in China, highlighting the challenges of diagnosis and treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCase presentation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe report a pediatric case of PAM that occurred in Lushan County, Pingdingshan City, Henan Province, China. The patient, a 6-year-old child, initially presented with persistent high fever on December 5, 2024, accompanied by headache, vomiting, and altered mental status. After receiving ineffective treatment at a local hospital, the child was transferred to the Eastern District of Henan Children’s Hospital on December 7 for further evaluation and management. Upon admission, cerebrospinal fluid was collected for laboratory analysis, and antimicrobial therapy, including amphotericin B, fluconazole, and rifampicin, was promptly initiated. Despite these interventions, the patient’s condition deteriorated rapidly, and the child succumbed to the infection on December 9.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe clinical presentation and laboratory findings strongly suggest that the child was infected with \u003cem\u003eNaegleria fowleri\u003c/em\u003e, resulting in PAM. Epidemiological investigation suggests that the infection may have been acquired during bathing at a public bathhouse. Given the survival characteristics of the amoeba and the accelerating effects of global warming, which may expand its habitat, this sporadic case serves as a stark reminder of the lethal potential of PAM. With a mortality rate exceeding 95%, early recognition and prompt intervention are crucial. Clinicians should maintain a high index of suspicion and consider PAM in the differential diagnosis of patients presenting with similar symptoms, particularly in regions where exposure to warm freshwater environments is common.\u003c/p\u003e","manuscriptTitle":"Case Report of Pediatric Naegleria fowleri Meningoencephalitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-14 12:09:12","doi":"10.21203/rs.3.rs-6549687/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-05-11T10:49:12+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-09T08:09:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-09T02:15:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Infectious Diseases of Poverty","date":"2025-05-08T03:39:16+00:00","index":"","fulltext":""},{"type":"decision","content":"Minor revision","date":"2025-05-06T04:43:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"infectious-diseases-of-poverty","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"idop","sideBox":"Learn more about [Infectious Diseases of Poverty](http://idpjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/idop/default.aspx","title":"Infectious Diseases of Poverty","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"05f02fe8-af26-4598-9e3d-745917f178f1","owner":[],"postedDate":"May 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-07T07:12:53+00:00","versionOfRecord":{"articleIdentity":"rs-6549687","link":"https://doi.org/10.1186/s40249-025-01347-z","journal":{"identity":"infectious-diseases-of-poverty","isVorOnly":false,"title":"Infectious Diseases of Poverty"},"publishedOn":"2025-07-17 16:05:04","publishedOnDateReadable":"July 17th, 2025"},"versionCreatedAt":"2025-05-14 12:09:12","video":"","vorDoi":"10.1186/s40249-025-01347-z","vorDoiUrl":"https://doi.org/10.1186/s40249-025-01347-z","workflowStages":[]},"version":"v1","identity":"rs-6549687","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6549687","identity":"rs-6549687","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}