Absence of chlamydial infection in ocular adnexal MALT lymphoma based on next-generation sequencing in Japan

Absence of chlamydial infection in ocular adnexal MALT lymphoma based on next-generation sequencing in Japan | 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 Short Report Absence of chlamydial infection in ocular adnexal MALT lymphoma based on next-generation sequencing in Japan Yoshihiro Yakushijin, Kazuhiro Horiba, Masanori Hashino, Shintaro Yamanaka, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8898978/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Objectives Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) arises in various extranodal sites and has been linked to chronic inflammation, often triggered by infectious agents. Ocular adnexal (OA) MALT lymphoma has been associated with Chlamydophila psittaci ( C. psittaci ) in some regions. This study aimed to investigate the involvement of pathogenic microorganisms, including C. psittaci , in OA-MALT lymphoma in Japan. Methods DNA was extracted from 17 formalin-fixed, paraffin-embedded OA-MALT lymphoma samples. Next-generation sequencing was performed, and reads were analyzed with bioinformatic pipelines to detect non-human DNA, focusing on complete or partial genomes of known pathogenic microorganisms, including C. psittaci strain 6BC and other bacteria implicated in lymphomagenesis. Results Most sequencing reads mapped to the human genome. No complete or partial genomes of C. psittaci 6BC or other non-human pathogenic microorganisms were detected above background levels, and no consistent enrichment of microbial sequences was observed. Discussion These findings suggest that, unlike in some Western cohorts, infectious agents such as C. psittaci are unlikely to play a major etiologic role in Japanese OA-MALT lymphoma. Conclusion Our analysis strongly suggests that infectious agents, including C. psittaci , are not associated with OA-MALT lymphoma in Japan. Ocular adnexal MALT lymphoma Next-generation sequencing Chlamydial infection Figures Figure 1 Introduction The extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) is a distinct B-cell lymphoma that develops in extranodal sites and usually has an indolent clinical course as a localized disease. This lymphoma was first recognized as a separate clinical pathologic entity by Isaacson and Wright in 1983 ( 1 ) and is presently featured in the World Health Organization (WHO) classification as a distinct lymphoma among the mature B-cell neoplasms ( 2 ). Clinicopathological studies suggest a strong relation between MALT lymphoma and inflammatory diseases of the epithelium, such as autoimmune diseases (Sjogren’s syndrome in the parotid gland and Hashimoto’s thyroiditis in the thyroid) and infections. The well-known example of lymphoid malignancy linked with infections is the connection between MALT lymphoma in the stomach and infection with Helicobacter pylori ( H. pylori ). Since 2004, several studies have reported detection of Chlamydophila psittaci ( C. psittaci ) in ocular adnexal (OA; orbit, conjunctiva, and eyelids) MALT lymphomas using PCR-based techniques ( 3 ). In addition, the therapeutic efficacy of antibiotic eradication of chlamydial infection in patients with OA-MALT lymphoma has been reported. The discussion about C. psittaci infection as a causative factor for OA-MALT lymphoma is still an ongoing pathological topic among researchers of this disease. However, before the discussion about causative agents of OA-MALT lymphoma, accurate diagnosis of this disease should be necessary, because polyclonal lymphoid hyperplasias arising from various inflammations and infections are diseases that also occur in the ocular adnexa at a considerable rate (20–30%). Recently, we analyzed the clinical efficacy of radiotherapy for OA-MALT lymphoma after accurate diagnosis based on molecular/immunological diagnosis in addition to pathological diagnosis ( 4 ). In the current report, we used those well-characterized diagnostic samples as bona fide OA-MALT lymphoma and investigated pathogens potentially involved in OA-MALT onset using next-generation sequencing. Materials & Methods Patients with POA-MALT lymphoma Newly diagnosed patients with POA-MALT (primary disease sites; orbit, lacrimal gland and sac, conjunctiva, and eyelids) lymphoma were retrospectively identified between June 2000 and June 2020 at Ehime University Hospital. All cases were diagnosed by hemato-pathologists. When the tumor samples after surgical resection were large enough for additional procedures to confirm the accurate diagnosis of POA-MALT lymphoma, IgH gene clonality analysis with Southern blot or PCR analysis, and/or flow cytometric analysis of cell surface antigen density (FACS) were employed in cases. In addition, clinical and laboratory data were collected from their medical records. All patients were diagnosed by hemato-pathologists based on their hematoxylin–eosin (HE) staining and immuno-histochemical analysis after the tumor biopsy. Regarding pathological decisions, we have defined the following criteria for microscopic diagnosis. First, infiltrated tumor lymphocytes under the HE staining show small lymphocytes exhibiting plasmacytic and/or monocytoid differentiation. Second, the epithelium (glandular tissue) is invaded by infiltrated tumor lymphocytes resulting in a so-called lymphoepithelial lesion. Third, infiltrated tumor lymphocytes under immuno-histochemical staining show positive expressions of CD20 and CD79a, negative expressions of CD3, CD5, CD10, and cyclinD1, and negative expression of bcl-2 in the germinal center when the tumor shows reactive follicles. Based on above diagnostic criteria and clinical information, final diagnoses were decided after discussions among hemato-pathologists and clinicians. In addition, cryo-preserved primary tumor specimens obtained from patients at the time of tumor biopsy were analyzed with IgH gene clonality analysis and/or FACS to confirm their diagnoses. Finally, we enrolled 17 samples which were diagnosed for OA-MALT lymphoma in Ehime University Hospital ( 4 ) ( 5 ). next-generation sequencing (NGS) The DNA extracted from each sample of OA-MALT lymphoma was analyzed using next-generation sequencing (NGS) to identify the genome derived from pathogenic microorganisms ( 6 ). Sequencing libraries were prepared using a QIAseq FX DNA library kit (Qiagen, Hilden, Germany) for DNA sequencing according to the manufacturer’s instructions. Illumina sequencing was performed using a NextSeq 1000 system (Illumina, San Diego, CA, USA) with a 2 × 150 bp paired-end protocol. The NGS data generated by the Illumina sequencing platform was processed in the metagenomic pipeline PATHDET for the detection of pathogen-derived sequences (The PATHDET pipeline comprises several steps conducted by different open-source software programs) ( 7 ). First, the sequence reads mapped to the human genome database by Bowtie2 and Kraken2 were removed as human genome subtraction. Second, MegaBLAST was used to search for highly similar species in the National Center for Biotechnology Information nt database from the human genome subtracted sequences. The PATHDET pipeline ultimately compiled all studied microorganisms and discussed potential pathogen candidates. Results First, we used NGS to examine whether the 17 OA-MALT specimens contained sequences from C. psittaci (6BC complete genome; NC_017287.1). We performed 2 × 150 bp paired-end sequencing, enabling more accurate read alignment and detection of insertion–deletion variants. Mapping of sequence reads to the C. psittaci reference did not yield sufficient evidence for the presence of C. psittaci . Aligned segments did not reach the read length (150 bp) and did not indicate C. psittaci -specific sequences (Table 1 ). These findings confirm the absence of the C. psittaci 6BC complete genome. Table 1 Mapping status to the reference sequence. Case Average Coverage (fold) Mapping Coverage (%) Mapping bases (bases) Mapping sequencing reads (reads) 1 0 0 0 0 2 0 0 0 0 3 0 0 0 0 4 0 0 0 0 5 0 0.0029 34 1 6 0.0001 0.0043 50 2 7 0 0 0 0 8 0 0 0 0 9 0.0001 0.0026 31 2 10 0 0 0 0 11 0.0001 0.0026 31 2 12 0 0 0 0 13 0 0 0 0 14 0 0 0 0 15 0.0001 0.006 70 1 16 0 0 0 0 17 0 0.0024 28 2 Mapping statistics of NGS reads from ocular adnexal MALT lymphoma (OA-MALT) specimens (n = 17) to the Chlamydia psittaci strain 6BC genome (NC_017287.1). Average coverage (fold) represents mean depth across the entire reference genome; mapping coverage (%) indicates the proportion of reference bases covered by ≥ 1 read. Mapping bases and reads indicate the total number of aligned bases and aligned reads, respectively. Only 1–2 reads were aligned in a small subset of cases (28–70 total aligned bases per case), yielding negligible breadth and depth of coverage. These findings are consistent with the absence of C. psittaci genomic sequences in these Japanese samples. We next examined, within these specimens (n = 17), potential infectious agents responsible for chronic inflammation that may contribute to OA-MALT development. Bacterial reads from the metagenomic NGS analysis of biopsy samples were counted and normalized to reads per million (RPM). We define detected genes as pathogen candidates if they have sequences of 200 RPM or higher ( 7 ). All bacterial sequencing reads were less than 2.0 RPM. In total, 80 bacterial species were detected at minimal levels and were not considered significant. In addition, using MegaBLAST against the nt database, no reads derived from Chlamydiaceae , including the suspected C. psittaci , were identified (Fig. 1 ). These results indicate the absence of pathogenic bacterial DNA contamination in the 17 OA-MALT lymphoma samples. Discussion The association between chlamydial infection and OA-MALT lymphoma has been extensively debated and remains unresolved. A primary challenge is the diagnosis of OA-MALT lymphoma itself. Among lymphoid tumors of the ocular adnexa, MALT lymphoma is the most common; however, a substantial proportion (20–30%) represent polyclonal lymphoid hyperplasia ( 8 ). To exclude these possibilities and achieve an accurate diagnosis, ancillary studies such as flow cytometry and molecular testing, in addition to expert hematopathologic evaluation, are necessary. Obtaining sufficient tissue for these studies can be difficult given the orbital location. Obtaining sufficient tissue for these studies is difficult due to the orbital site. We also consider the relatively frequent t(14;18)(q32;q21) translocation (IGH–MALT1), detected by PCR or FISH ( 9 ) ( 10 ), although it is not feasible to perform this in all cases. These circumstances may complicate the proposed relationship between OA-MALT and Chlamydia; that is, one must carefully assess whether benign disease caused by chlamydial infection has been adequately excluded from clinical series. Second, regarding chlamydial genetic polymorphism, while genes of C. pneumoniae are relatively conserved, those of C. psittaci and C. trachomatis exhibit substantial strain-to-strain variability ( 11 ). Therefore, PCR-based detection of Chlamydia requires cautious interpretation that considers primer design and strain specificity. Finally, the prevalence of the putative pathogens in the general population should be considered. Clear geographic differences between Europe and North America have been noted in the association of chronic Hepatitis C with malignant lymphoma ( 12 ) ( 13 ) ( 14 ) ( 15 ) ( 16 ), and similar regional variation has been suggested for Borrelia burgdorferi and lymphoma ( 17 ). Such regional differences may introduce noise into amplification-based detection of chlamydial genes, which can be detected relatively easily even in blood. In our series, we found no evidence supporting an association between chlamydial infection ( C. psittaci , C. trachomatis , C. pneumoniae ) and OA-MALT lymphoma in our region. Conclusion Our analysis suggests that C. psittaci infection may not be associated with OA-MALT lymphoma in Japan. Abbreviations DNA Deoxyribonucleic acid FACS Flow cytometric analysis of cell surface antigen density FISH Fluorescence in situ hybridization HE Hematoxylin–eosin IGH Immunoglobulin heavy chain IRB Institutional Review Board MALT Mucosa-associated lymphoid tissue NGS Next-generation sequencing OA Ocular adnexal OA-MALT Ocular adnexal mucosa-associated lymphoid tissue PATHDET Metagenomic pipeline for pathogen detection PCR Polymerase chain reaction POA-MALT Primary ocular adnexal mucosa-associated lymphoid tissue RPM Reads per million WHO World Health Organization Declarations Ethics approval and consent to participate This study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board (IRB) of Ehime University Hospital (approval No: Ehime2406005; Date: June 9, 2024). Owing to the retrospective design and use of anonymized data, the requirement for written informed consent was waived by the IRB. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Author Contribution YY conceived and designed the study and drafted the manuscript. KH and MH performed the NGS analyses. SY (Shintaro Yamanaka), SH, SY (Shoichiro Yamamoto), TF, KT, and MY contributed to the study design and critically verified the integrity and reliability of the data. All authors read and approved the final manuscript. Acknowledgements Not applicable. Data Availability The datasets used and analysed during the current study are available from the corresponding author on reasonable request. References Isaacson PG, Wright DH. Malignant lymphoma of mucosa-associated lymphoid tissue. A distinctive type of B-cell lymphoma. Cancer. 1983;52(8):1410–6. 10.1002/1097-0142(19831015)52:8%3C1410::aid-cncr2820520813%3E3.0.co;2-3 . Isaacson PG, Berger F, Muller-Hermelink HK, et al. Extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). In: Jaffe ES, Harris NL, Stein H, Vardiman JW, editors. Pathology and genetics of tumours of haematopoietic and lymphoid tissues. Volume 3. Lyon (France): IARC; 2001. pp. 157–60. Ferreri AJM, Guidoboni M, Ponzoni M, De Conciliis C, Dell'Oro S, Fleischhauer K, et al. Evidence for an association between Chlamydia psittaci and ocular adnexal lymphomas. J Natl Cancer Inst. 2004;96(8):586–94. 10.1093/jnci/djh102 . Masuda Y, Takeuchi K, Kodama T, Fujisaki T, Imaizumi Y, Otsuka E, et al. Treatment-associated outcomes of patients with primary ocular adnexal MALT lymphoma after accurate diagnosis. Int J Clin Oncol. 2019;24(12):1620–8. 10.1007/s10147-019-01481-3 . Yakushijin Y, Kodama T, Takaoka I, Tanimoto K, Bessho H, Sakai I, et al. Absence of chlamydial infection in Japanese patients with ocular adnexal lymphoma of mucosa-associated lymphoid tissue. Int J Hematol. 2007;85(3):223–30. 10.1532/IJH97.06065 . Kong HH, Oh J, Deming C, Conlan S, Grice EA, Beatson MA, et al. Temporal shifts in the skin microbiome associated with disease flares and treatment in children with atopic dermatitis. Genome Res. 2012;22(5):850–9. 10.1101/gr.131029.111 . Horiba K, Torii Y, Aizawa Y, Yamaguchi M, Haruta K, Okumura T, et al. Performance of Nanopore and Illumina metagenomic sequencing for pathogen detection and transcriptome analysis in infantile central nervous system infections. Open Forum Infect Dis. 2022;9(10):ofac504. 10.1093/ofid/ofac504 . Knowles DM, Jakobiec FA, McNally L, Burke JS. Lymphoid hyperplasia and malignant lymphoma occurring in the ocular adnexa (orbit, conjunctiva, and eyelids): a prospective multiparametric analysis of 108 cases during 1977 to 1987. Hum Pathol. 1990;21(9):959–73. 10.1016/0046-8177(90)90181-4 . Streubel B, Lamprecht A, Dierlamm J, Cerroni L, Stolte M, Ott G, et al. T(14;18)(q32;q21) involving IGH and MALT1 is a frequent chromosomal aberration in MALT lymphoma. Blood. 2003;101(6):2335–9. 10.1182/blood-2002-09-2963 . Adachi A, Tamaru J, Kaneko K, Kuroda H, Miura I, Kojima T, et al. No evidence of a correlation between BCL10 expression and API2-MALT1 gene rearrangement in ocular adnexal MALT lymphoma. Pathol Int. 2004;54(1):16–25. 10.1111/j.1440-1827.2004.01580.x . Meijer A, Morré SA, van den Brule AJC, Savelkoul PHM, Ossewaarde JM. Genomic relatedness of Chlamydia isolates determined by amplified fragment length polymorphism analysis. J Bacteriol. 1999;181(15):4469–75. 10.1128/JB.181.15.4469-4475.1999 . Ferri C, Caracciolo F, Zignego AL, La Civita L, Monti M, Longombardo G, et al. Hepatitis C virus infection in patients with non-Hodgkin's lymphoma. Br J Haematol. 1994;88(2):392–4. 10.1111/j.1365-2141.1994.tb05036.x . Mazzaro C, Zagonel V, Monfardini S, Tulissi P, Pussini E, Fanni M, et al. Hepatitis C virus and non-Hodgkin's lymphomas. Br J Haematol. 1996;94(3):544–50. 10.1046/j.1365-2141.1996.6912313.x . Sève P, Renaudier P, Sasco AJ, Trépo C, Dumontet C, Moullet I, et al. Hepatitis C virus infection and B-cell non-Hodgkin's lymphoma: a cross-sectional study in Lyon, France. Eur J Gastroenterol Hepatol. 2004;16(12):1361–5. 10.1097/00042737-200412000-00021 . Morgensztern D, Rosado M, Silva O, Santos E, Abdullah S, Goodman M, et al. Prevalence of hepatitis C infection in patients with non-Hodgkin's lymphoma in South Florida and review of the literature. Leuk Lymphoma. 2004;45(12):2459–64. 10.1080/10428190400007771 . Waters L, Stebbing J, Mandalia S, Young AM, Nelson M, Gazzard B, et al. Hepatitis C infection is not associated with systemic HIV-associated non-Hodgkin's lymphoma: a cohort study. Int J Cancer. 2005;116(1):161–3. 10.1002/ijc.20988 . Roggero E, Zucca E, Mainetti C, Bertoni F, Valsangiacomo C, Pedrinis E, et al. Eradication of Borrelia burgdorferi infection in primary marginal zone B-cell lymphoma of the skin. Hum Pathol. 2000;31(2):263–8. 10.1016/s0046-8177(00)80233-6 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 25 Mar, 2026 Reviews received at journal 16 Mar, 2026 Reviewers agreed at journal 09 Mar, 2026 Reviewers invited by journal 06 Mar, 2026 Editor assigned by journal 19 Feb, 2026 Submission checks completed at journal 19 Feb, 2026 First submitted to journal 17 Feb, 2026 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-8898978","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":603174172,"identity":"f713aabc-9f25-4f4f-ac99-479a82f667c6","order_by":0,"name":"Yoshihiro Yakushijin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYHACNoYEBgYDfiCLmcEASZSgFskGkrQAgYHBAZAWYoDB8fZnDx622RkbXzvA+LigYJscg0QC44cfDHx5OLWcOWNukNiWbGZ2O4HZeIbBbWOgFmbJHga2YpxabuSwSSS2MdsAtbBJ8xjcTtx/I4FBGujcxAacWtKfAbXU2xjPTmD/DdRS3wC05Td+LQlmQC2HzQykE9iYgVoSgA5jw2uL5JkzZhIJ544bS9xObAY5zLCB52GbZY8Bbr/wAUNM8kdZtWH/7OSDn3n+3JZnYE8+fONHxTGcIaZwAEgwgqOGEeYSEMPgWAIuLfJgdX8wJWpwahkFo2AUjIIRBwAqWVIWJfUVUgAAAABJRU5ErkJggg==","orcid":"","institution":"Ehime University Graduate School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Yoshihiro","middleName":"","lastName":"Yakushijin","suffix":""},{"id":603174173,"identity":"d4efc7c8-0a42-434b-83b5-b2bac80996f7","order_by":1,"name":"Kazuhiro Horiba","email":"","orcid":"","institution":"National Institute of Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Kazuhiro","middleName":"","lastName":"Horiba","suffix":""},{"id":603174174,"identity":"ecd4332a-034e-4a50-b928-869540b12fdf","order_by":2,"name":"Masanori Hashino","email":"","orcid":"","institution":"National Institute of Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Masanori","middleName":"","lastName":"Hashino","suffix":""},{"id":603174175,"identity":"39feaaa9-70fe-4efe-9d27-406af6c91ea3","order_by":3,"name":"Shintaro Yamanaka","email":"","orcid":"","institution":"Ehime University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shintaro","middleName":"","lastName":"Yamanaka","suffix":""},{"id":603174176,"identity":"8f226d09-0043-4391-b482-33c636daa97b","order_by":4,"name":"Shinji Hasebe","email":"","orcid":"","institution":"Ehime University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shinji","middleName":"","lastName":"Hasebe","suffix":""},{"id":603174177,"identity":"a4edfe3e-2556-4013-b482-05d33f4e9444","order_by":5,"name":"Shoichiro Yamamoto","email":"","orcid":"","institution":"Ehime University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shoichiro","middleName":"","lastName":"Yamamoto","suffix":""},{"id":603174178,"identity":"5457be4e-5d18-4b8b-af5c-0a286337273d","order_by":6,"name":"Tomomi Fujii","email":"","orcid":"","institution":"Ehime University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tomomi","middleName":"","lastName":"Fujii","suffix":""},{"id":603174179,"identity":"9467b412-df02-4ca8-946a-276043916d2a","order_by":7,"name":"Katsuto Takenaka","email":"","orcid":"","institution":"Ehime University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Katsuto","middleName":"","lastName":"Takenaka","suffix":""},{"id":603174180,"identity":"5fcfefcb-20d3-4f62-b6ef-1bcd7893bf84","order_by":8,"name":"Masaki Yasukawa","email":"","orcid":"","institution":"Ehime University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Masaki","middleName":"","lastName":"Yasukawa","suffix":""}],"badges":[],"createdAt":"2026-02-17 08:23:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8898978/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8898978/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104504233,"identity":"d9a35f82-e546-4e17-abaf-2e99491c7522","added_by":"auto","created_at":"2026-03-12 14:32:45","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":200617,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDNA sequences derived from bacteria detected in patients with ocular adnexal MALT lymphomas.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacterial reads were counted from the metagenomic NGS analysis of the samples. The read counts were normalized to reads per million (RPM).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8898978/v1/a6a528a42a122d93103c4319.jpeg"},{"id":104781000,"identity":"d5bc332c-80c8-4f8d-93b0-8e4a9f8b25d0","added_by":"auto","created_at":"2026-03-17 07:54:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":806831,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8898978/v1/537ad5b7-33f3-484b-a432-94f21c52e8f1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Absence of chlamydial infection in ocular adnexal MALT lymphoma based on next-generation sequencing in Japan","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) is a distinct B-cell lymphoma that develops in extranodal sites and usually has an indolent clinical course as a localized disease. This lymphoma was first recognized as a separate clinical pathologic entity by Isaacson and Wright in 1983 (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) and is presently featured in the World Health Organization (WHO) classification as a distinct lymphoma among the mature B-cell neoplasms (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Clinicopathological studies suggest a strong relation between MALT lymphoma and inflammatory diseases of the epithelium, such as autoimmune diseases (Sjogren\u0026rsquo;s syndrome in the parotid gland and Hashimoto\u0026rsquo;s thyroiditis in the thyroid) and infections. The well-known example of lymphoid malignancy linked with infections is the connection between MALT lymphoma in the stomach and infection with \u003cem\u003eHelicobacter pylori\u003c/em\u003e (\u003cem\u003eH. pylori\u003c/em\u003e). Since 2004, several studies have reported detection of \u003cem\u003eChlamydophila psittaci\u003c/em\u003e (\u003cem\u003eC. psittaci\u003c/em\u003e) in ocular adnexal (OA; orbit, conjunctiva, and eyelids) MALT lymphomas using PCR-based techniques (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). In addition, the therapeutic efficacy of antibiotic eradication of chlamydial infection in patients with OA-MALT lymphoma has been reported. The discussion about \u003cem\u003eC. psittaci\u003c/em\u003e infection as a causative factor for OA-MALT lymphoma is still an ongoing pathological topic among researchers of this disease. However, before the discussion about causative agents of OA-MALT lymphoma, accurate diagnosis of this disease should be necessary, because polyclonal lymphoid hyperplasias arising from various inflammations and infections are diseases that also occur in the ocular adnexa at a considerable rate (20\u0026ndash;30%).\u003c/p\u003e \u003cp\u003eRecently, we analyzed the clinical efficacy of radiotherapy for OA-MALT lymphoma after accurate diagnosis based on molecular/immunological diagnosis in addition to pathological diagnosis (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). In the current report, we used those well-characterized diagnostic samples as bona fide OA-MALT lymphoma and investigated pathogens potentially involved in OA-MALT onset using next-generation sequencing.\u003c/p\u003e"},{"header":"Materials \u0026 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients with POA-MALT lymphoma\u003c/h2\u003e \u003cp\u003eNewly diagnosed patients with POA-MALT (primary disease sites; orbit, lacrimal gland and sac, conjunctiva, and eyelids) lymphoma were retrospectively identified between June 2000 and June 2020 at Ehime University Hospital. All cases were diagnosed by hemato-pathologists. When the tumor samples after surgical resection were large enough for additional procedures to confirm the accurate diagnosis of POA-MALT lymphoma, IgH gene clonality analysis with Southern blot or PCR analysis, and/or flow cytometric analysis of cell surface antigen density (FACS) were employed in cases. In addition, clinical and laboratory data were collected from their medical records. All patients were diagnosed by hemato-pathologists based on their hematoxylin\u0026ndash;eosin (HE) staining and immuno-histochemical analysis after the tumor biopsy. Regarding pathological decisions, we have defined the following criteria for microscopic diagnosis. First, infiltrated tumor lymphocytes under the HE staining show small lymphocytes exhibiting plasmacytic and/or monocytoid differentiation. Second, the epithelium (glandular tissue) is invaded by infiltrated tumor lymphocytes resulting in a so-called lymphoepithelial lesion. Third, infiltrated tumor lymphocytes under immuno-histochemical staining show positive expressions of CD20 and CD79a, negative expressions of CD3, CD5, CD10, and cyclinD1, and negative expression of bcl-2 in the germinal center when the tumor shows reactive follicles. Based on above diagnostic criteria and clinical information, final diagnoses were decided after discussions among hemato-pathologists and clinicians. In addition, cryo-preserved primary tumor specimens obtained from patients at the time of tumor biopsy were analyzed with IgH gene clonality analysis and/or FACS to confirm their diagnoses. Finally, we enrolled 17 samples which were diagnosed for OA-MALT lymphoma in Ehime University Hospital (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003enext-generation sequencing (NGS)\u003c/h3\u003e\n\u003cp\u003eThe DNA extracted from each sample of OA-MALT lymphoma was analyzed using next-generation sequencing (NGS) to identify the genome derived from pathogenic microorganisms (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Sequencing libraries were prepared using a QIAseq FX DNA library kit (Qiagen, Hilden, Germany) for DNA sequencing according to the manufacturer\u0026rsquo;s instructions. Illumina sequencing was performed using a NextSeq 1000 system (Illumina, San Diego, CA, USA) with a 2 \u0026times; 150 bp paired-end protocol. The NGS data generated by the Illumina sequencing platform was processed in the metagenomic pipeline PATHDET for the detection of pathogen-derived sequences (The PATHDET pipeline comprises several steps conducted by different open-source software programs) (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). First, the sequence reads mapped to the human genome database by Bowtie2 and Kraken2 were removed as human genome subtraction. Second, MegaBLAST was used to search for highly similar species in the National Center for Biotechnology Information nt database from the human genome subtracted sequences. The PATHDET pipeline ultimately compiled all studied microorganisms and discussed potential pathogen candidates.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFirst, we used NGS to examine whether the 17 OA-MALT specimens contained sequences from \u003cem\u003eC. psittaci\u003c/em\u003e (6BC complete genome; NC_017287.1). We performed 2 \u0026times; 150 bp paired-end sequencing, enabling more accurate read alignment and detection of insertion\u0026ndash;deletion variants. Mapping of sequence reads to the \u003cem\u003eC. psittaci\u003c/em\u003e reference did not yield sufficient evidence for the presence of \u003cem\u003eC. psittaci\u003c/em\u003e. Aligned segments did not reach the read length (150 bp) and did not indicate \u003cem\u003eC. psittaci\u003c/em\u003e-specific sequences (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These findings confirm the absence of the \u003cem\u003eC. psittaci\u003c/em\u003e 6BC complete genome.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\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\u003eMapping status to the reference sequence.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCase\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAverage Coverage\u003c/p\u003e \u003cp\u003e(fold)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMapping Coverage\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMapping bases\u003c/p\u003e \u003cp\u003e(bases)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMapping sequencing\u003c/p\u003e \u003cp\u003ereads (reads)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0043\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0026\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0026\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eMapping statistics of NGS reads from ocular adnexal MALT lymphoma (OA-MALT) specimens (n\u0026thinsp;=\u0026thinsp;17) to the \u003cem\u003eChlamydia psittaci\u003c/em\u003e strain 6BC genome (NC_017287.1). Average coverage (fold) represents mean depth across the entire reference genome; mapping coverage (%) indicates the proportion of reference bases covered by \u0026ge;\u0026thinsp;1 read. Mapping bases and reads indicate the total number of aligned bases and aligned reads, respectively. Only 1\u0026ndash;2 reads were aligned in a small subset of cases (28\u0026ndash;70 total aligned bases per case), yielding negligible breadth and depth of coverage. These findings are consistent with the absence of \u003cem\u003eC. psittaci\u003c/em\u003e genomic sequences in these Japanese samples.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWe next examined, within these specimens (n\u0026thinsp;=\u0026thinsp;17), potential infectious agents responsible for chronic inflammation that may contribute to OA-MALT development. Bacterial reads from the metagenomic NGS analysis of biopsy samples were counted and normalized to reads per million (RPM). We define detected genes as pathogen candidates if they have sequences of 200 RPM or higher (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). All bacterial sequencing reads were less than 2.0 RPM. In total, 80 bacterial species were detected at minimal levels and were not considered significant. In addition, using MegaBLAST against the nt database, no reads derived from \u003cem\u003eChlamydiaceae\u003c/em\u003e, including the suspected \u003cem\u003eC. psittaci\u003c/em\u003e, were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These results indicate the absence of pathogenic bacterial DNA contamination in the 17 OA-MALT lymphoma samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe association between chlamydial infection and OA-MALT lymphoma has been extensively debated and remains unresolved. A primary challenge is the diagnosis of OA-MALT lymphoma itself. Among lymphoid tumors of the ocular adnexa, MALT lymphoma is the most common; however, a substantial proportion (20\u0026ndash;30%) represent polyclonal lymphoid hyperplasia (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). To exclude these possibilities and achieve an accurate diagnosis, ancillary studies such as flow cytometry and molecular testing, in addition to expert hematopathologic evaluation, are necessary. Obtaining sufficient tissue for these studies can be difficult given the orbital location. Obtaining sufficient tissue for these studies is difficult due to the orbital site. We also consider the relatively frequent t(14;18)(q32;q21) translocation (IGH\u0026ndash;MALT1), detected by PCR or FISH (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), although it is not feasible to perform this in all cases. These circumstances may complicate the proposed relationship between OA-MALT and Chlamydia; that is, one must carefully assess whether benign disease caused by chlamydial infection has been adequately excluded from clinical series.\u003c/p\u003e \u003cp\u003eSecond, regarding chlamydial genetic polymorphism, while genes of \u003cem\u003eC. pneumoniae\u003c/em\u003e are relatively conserved, those of \u003cem\u003eC. psittaci\u003c/em\u003e and \u003cem\u003eC. trachomatis\u003c/em\u003e exhibit substantial strain-to-strain variability (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Therefore, PCR-based detection of Chlamydia requires cautious interpretation that considers primer design and strain specificity.\u003c/p\u003e \u003cp\u003eFinally, the prevalence of the putative pathogens in the general population should be considered. Clear geographic differences between Europe and North America have been noted in the association of chronic \u003cem\u003eHepatitis C\u003c/em\u003e with malignant lymphoma (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), and similar regional variation has been suggested for \u003cem\u003eBorrelia burgdorferi\u003c/em\u003e and lymphoma (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Such regional differences may introduce noise into amplification-based detection of chlamydial genes, which can be detected relatively easily even in blood. In our series, we found no evidence supporting an association between chlamydial infection (\u003cem\u003eC. psittaci\u003c/em\u003e, \u003cem\u003eC. trachomatis\u003c/em\u003e, \u003cem\u003eC. pneumoniae\u003c/em\u003e) and OA-MALT lymphoma in our region.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur analysis suggests that \u003cem\u003eC. psittaci\u003c/em\u003e infection may not be associated with OA-MALT lymphoma in Japan.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDeoxyribonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFACS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFlow cytometric analysis of cell surface antigen density\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFISH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFluorescence in situ hybridization\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHematoxylin\u0026ndash;eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIGH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunoglobulin heavy chain\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIRB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInstitutional Review Board\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMALT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMucosa-associated lymphoid tissue\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNGS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNext-generation sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOcular adnexal\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOA-MALT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOcular adnexal mucosa-associated lymphoid tissue\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePATHDET\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMetagenomic pipeline for pathogen detection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePOA-MALT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePrimary ocular adnexal mucosa-associated lymphoid tissue\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRPM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReads per million\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWHO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWorld Health Organization\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eThis study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board (IRB) of Ehime University Hospital (approval No: Ehime2406005; Date: June 9, 2024). Owing to the retrospective design and use of anonymized data, the requirement for written informed consent was waived by the IRB.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYY conceived and designed the study and drafted the manuscript. KH and MH performed the NGS analyses. SY (Shintaro Yamanaka), SH, SY (Shoichiro Yamamoto), TF, KT, and MY contributed to the study design and critically verified the integrity and reliability of the data. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eIsaacson PG, Wright DH. Malignant lymphoma of mucosa-associated lymphoid tissue. A distinctive type of B-cell lymphoma. Cancer. 1983;52(8):1410\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/1097-0142(19831015)52:8%3C1410::aid-cncr2820520813%3E3.0.co;2-3\u003c/span\u003e\u003cspan address=\"10.1002/1097-0142(19831015)52:8%3C1410::aid-cncr2820520813%3E3.0.co;2-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIsaacson PG, Berger F, Muller-Hermelink HK, et al. Extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). In: Jaffe ES, Harris NL, Stein H, Vardiman JW, editors. Pathology and genetics of tumours of haematopoietic and lymphoid tissues. Volume 3. Lyon (France): IARC; 2001. pp. 157\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerreri AJM, Guidoboni M, Ponzoni M, De Conciliis C, Dell'Oro S, Fleischhauer K, et al. Evidence for an association between Chlamydia psittaci and ocular adnexal lymphomas. J Natl Cancer Inst. 2004;96(8):586\u0026ndash;94. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jnci/djh102\u003c/span\u003e\u003cspan address=\"10.1093/jnci/djh102\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMasuda Y, Takeuchi K, Kodama T, Fujisaki T, Imaizumi Y, Otsuka E, et al. Treatment-associated outcomes of patients with primary ocular adnexal MALT lymphoma after accurate diagnosis. Int J Clin Oncol. 2019;24(12):1620\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10147-019-01481-3\u003c/span\u003e\u003cspan address=\"10.1007/s10147-019-01481-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYakushijin Y, Kodama T, Takaoka I, Tanimoto K, Bessho H, Sakai I, et al. Absence of chlamydial infection in Japanese patients with ocular adnexal lymphoma of mucosa-associated lymphoid tissue. Int J Hematol. 2007;85(3):223\u0026ndash;30. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1532/IJH97.06065\u003c/span\u003e\u003cspan address=\"10.1532/IJH97.06065\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKong HH, Oh J, Deming C, Conlan S, Grice EA, Beatson MA, et al. Temporal shifts in the skin microbiome associated with disease flares and treatment in children with atopic dermatitis. Genome Res. 2012;22(5):850\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/gr.131029.111\u003c/span\u003e\u003cspan address=\"10.1101/gr.131029.111\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoriba K, Torii Y, Aizawa Y, Yamaguchi M, Haruta K, Okumura T, et al. Performance of Nanopore and Illumina metagenomic sequencing for pathogen detection and transcriptome analysis in infantile central nervous system infections. Open Forum Infect Dis. 2022;9(10):ofac504. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/ofid/ofac504\u003c/span\u003e\u003cspan address=\"10.1093/ofid/ofac504\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKnowles DM, Jakobiec FA, McNally L, Burke JS. Lymphoid hyperplasia and malignant lymphoma occurring in the ocular adnexa (orbit, conjunctiva, and eyelids): a prospective multiparametric analysis of 108 cases during 1977 to 1987. Hum Pathol. 1990;21(9):959\u0026ndash;73. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/0046-8177(90)90181-4\u003c/span\u003e\u003cspan address=\"10.1016/0046-8177(90)90181-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStreubel B, Lamprecht A, Dierlamm J, Cerroni L, Stolte M, Ott G, et al. T(14;18)(q32;q21) involving IGH and MALT1 is a frequent chromosomal aberration in MALT lymphoma. Blood. 2003;101(6):2335\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1182/blood-2002-09-2963\u003c/span\u003e\u003cspan address=\"10.1182/blood-2002-09-2963\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdachi A, Tamaru J, Kaneko K, Kuroda H, Miura I, Kojima T, et al. No evidence of a correlation between BCL10 expression and API2-MALT1 gene rearrangement in ocular adnexal MALT lymphoma. Pathol Int. 2004;54(1):16\u0026ndash;25. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1440-1827.2004.01580.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1440-1827.2004.01580.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeijer A, Morr\u0026eacute; SA, van den Brule AJC, Savelkoul PHM, Ossewaarde JM. Genomic relatedness of Chlamydia isolates determined by amplified fragment length polymorphism analysis. J Bacteriol. 1999;181(15):4469\u0026ndash;75. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/JB.181.15.4469-4475.1999\u003c/span\u003e\u003cspan address=\"10.1128/JB.181.15.4469-4475.1999\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerri C, Caracciolo F, Zignego AL, La Civita L, Monti M, Longombardo G, et al. Hepatitis C virus infection in patients with non-Hodgkin's lymphoma. Br J Haematol. 1994;88(2):392\u0026ndash;4. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1365-2141.1994.tb05036.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2141.1994.tb05036.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMazzaro C, Zagonel V, Monfardini S, Tulissi P, Pussini E, Fanni M, et al. Hepatitis C virus and non-Hodgkin's lymphomas. Br J Haematol. 1996;94(3):544\u0026ndash;50. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1046/j.1365-2141.1996.6912313.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1365-2141.1996.6912313.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS\u0026egrave;ve P, Renaudier P, Sasco AJ, Tr\u0026eacute;po C, Dumontet C, Moullet I, et al. Hepatitis C virus infection and B-cell non-Hodgkin's lymphoma: a cross-sectional study in Lyon, France. Eur J Gastroenterol Hepatol. 2004;16(12):1361\u0026ndash;5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1097/00042737-200412000-00021\u003c/span\u003e\u003cspan address=\"10.1097/00042737-200412000-00021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorgensztern D, Rosado M, Silva O, Santos E, Abdullah S, Goodman M, et al. Prevalence of hepatitis C infection in patients with non-Hodgkin's lymphoma in South Florida and review of the literature. Leuk Lymphoma. 2004;45(12):2459\u0026ndash;64. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/10428190400007771\u003c/span\u003e\u003cspan address=\"10.1080/10428190400007771\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaters L, Stebbing J, Mandalia S, Young AM, Nelson M, Gazzard B, et al. Hepatitis C infection is not associated with systemic HIV-associated non-Hodgkin's lymphoma: a cohort study. Int J Cancer. 2005;116(1):161\u0026ndash;3. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/ijc.20988\u003c/span\u003e\u003cspan address=\"10.1002/ijc.20988\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoggero E, Zucca E, Mainetti C, Bertoni F, Valsangiacomo C, Pedrinis E, et al. Eradication of Borrelia burgdorferi infection in primary marginal zone B-cell lymphoma of the skin. Hum Pathol. 2000;31(2):263\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s0046-8177(00)80233-6\u003c/span\u003e\u003cspan address=\"10.1016/s0046-8177(00)80233-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\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-agents-and-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"iaac","sideBox":"Learn more about [Infectious Agents and Cancer](http://infectagentscancer.biomedcentral.com/)","snPcode":"13027","submissionUrl":"https://submission.nature.com/new-submission/13027/3","title":"Infectious Agents and Cancer","twitterHandle":"@IAC_journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Ocular adnexal MALT lymphoma, Next-generation sequencing, Chlamydial infection","lastPublishedDoi":"10.21203/rs.3.rs-8898978/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8898978/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eObjectives\u003c/b\u003e\u003c/p\u003e \u003cp\u003eExtranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) arises in various extranodal sites and has been linked to chronic inflammation, often triggered by infectious agents. Ocular adnexal (OA) MALT lymphoma has been associated with \u003cem\u003eChlamydophila psittaci\u003c/em\u003e (\u003cem\u003eC. psittaci\u003c/em\u003e) in some regions. This study aimed to investigate the involvement of pathogenic microorganisms, including \u003cem\u003eC. psittaci\u003c/em\u003e, in OA-MALT lymphoma in Japan.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDNA was extracted from 17 formalin-fixed, paraffin-embedded OA-MALT lymphoma samples. Next-generation sequencing was performed, and reads were analyzed with bioinformatic pipelines to detect non-human DNA, focusing on complete or partial genomes of known pathogenic microorganisms, including \u003cem\u003eC. psittaci\u003c/em\u003e strain 6BC and other bacteria implicated in lymphomagenesis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMost sequencing reads mapped to the human genome. No complete or partial genomes of \u003cem\u003eC. psittaci\u003c/em\u003e 6BC or other non-human pathogenic microorganisms were detected above background levels, and no consistent enrichment of microbial sequences was observed.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDiscussion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThese findings suggest that, unlike in some Western cohorts, infectious agents such as \u003cem\u003eC. psittaci\u003c/em\u003e are unlikely to play a major etiologic role in Japanese OA-MALT lymphoma.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur analysis strongly suggests that infectious agents, including \u003cem\u003eC. psittaci\u003c/em\u003e, are not associated with OA-MALT lymphoma in Japan.\u003c/p\u003e","manuscriptTitle":"Absence of chlamydial infection in ocular adnexal MALT lymphoma based on next-generation sequencing in Japan","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-12 14:32:37","doi":"10.21203/rs.3.rs-8898978/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-25T20:09:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-16T15:40:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"125375292746865128139419669432940380375","date":"2026-03-09T13:44:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-06T16:49:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-19T09:24:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-19T09:23:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Infectious Agents and Cancer","date":"2026-02-17T08:07:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"infectious-agents-and-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"iaac","sideBox":"Learn more about [Infectious Agents and Cancer](http://infectagentscancer.biomedcentral.com/)","snPcode":"13027","submissionUrl":"https://submission.nature.com/new-submission/13027/3","title":"Infectious Agents and Cancer","twitterHandle":"@IAC_journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"95dc5a3d-8fc5-4e45-b5be-6f744960be07","owner":[],"postedDate":"March 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-24T18:38:57+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-12 14:32:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8898978","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8898978","identity":"rs-8898978","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}