{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,2]],"date-time":"2025-10-02T00:55:25Z","timestamp":1759366525860,"version":"build-2065373602"},"reference-count":31,"publisher":"American Society of Tropical Medicine and Hygiene","issue":"4","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2025,10,1]]},"abstract":"<jats:title>ABSTRACT.<\/jats:title>\n<jats:p>In this study, we aimed to assess the efficacy of polymerase chain reaction (PCR) targeting <jats:italic>Leishmania<\/jats:italic> kinetoplast minicircle DNA (<jats:italic>kDNA<\/jats:italic>) for diagnosing visceral leishmaniasis-associated hemophagocytic lymphohistiocytosis (VL-HLH). Three pediatric patients diagnosed with VL-HLH via the microscopic examination of bone marrow smears were selected to explore the diagnostic value of <jats:italic>kDNA<\/jats:italic> PCR in <jats:italic>Leishmania<\/jats:italic> species identification and parasite load quantification in three cases of visceral leishmaniasis. Total DNA was extracted from the bone marrow samples, and PCR testing targeting the <jats:italic>Leishmania<\/jats:italic> <jats:italic>kDNA<\/jats:italic> was conducted. The amplified PCR products were sequenced and analyzed by using National Center for Biotechnology Information Basic Local Alignment Search Tool for <jats:italic>Leishmania<\/jats:italic> species identification and homology cluster analysis. Furthermore, universal primers and probes were designed to quantify the <jats:italic>kDNA<\/jats:italic> load in patient samples through real-time quantitative PCR (qPCR) testing. Polymerase chain reaction testing targeting the <jats:italic>kDNA<\/jats:italic> gene yielded a specific 139 base pairs band in all samples. Real-time qPCR testing targeting the <jats:italic>kDNA<\/jats:italic> gene revealed significant amplification in all patients. The <jats:italic>kDNA<\/jats:italic> copy numbers for the three patients were 3,830\/\u00b5L, 150\/\u00b5L, and 77,168\/\u00b5L, respectively. Homology analysis revealed 97.8% similarity between Patients 1 and 2, as well as 87.1% and 87.8% similarity between Patients 1 and 3 and Patients 2 and 3, respectively. All sequences were identified as belonging to the genus <jats:italic>Leishmania<\/jats:italic>, with Patients 1 and 2 clustering with <jats:italic>Leishmania donovani<\/jats:italic> sequences and Patient 3 clustering with <jats:italic>Leishmania infantum<\/jats:italic>. Polymerase chain reaction testing targeting the <jats:italic>kDNA<\/jats:italic> gene is a rapid, accurate, and sensitive method for detecting and quantifying <jats:italic>Leishmania<\/jats:italic> in VL-HLH patients. It also facilitates species identification through sequencing, demonstrating its potential as a valuable tool in clinical diagnostics.<\/jats:p>","DOI":"10.4269\/ajtmh.24-0792","type":"journal-article","created":{"date-parts":[[2025,8,7]],"date-time":"2025-08-07T13:41:02Z","timestamp":1754574062000},"page":"799-805","source":"Crossref","is-referenced-by-count":0,"title":["Polymerase Chain Reaction-Based Diagnosis of Leishmaniasis Targeting Kinetoplast Minicircle DNA in Infants with Hemophagocytic Lymphohistiocytosis"],"prefix":"10.4269","volume":"113","author":[{"given":"Heng","family":"Zhao","sequence":"first","affiliation":[{"name":"Key Laboratory of Systemic Innovative Research on Virus Vaccine, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming City, China;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shulan","family":"Shi","sequence":"additional","affiliation":[{"name":"Clinical Laboratory, Kunming Children\u2019s Hospital, Kunming City, China;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ying","family":"Zhang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Systemic Innovative Research on Virus Vaccine, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming City, China;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Haijing","family":"Shi","sequence":"additional","affiliation":[{"name":"Key Laboratory of Systemic Innovative Research on Virus Vaccine, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming City, China;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guorun","family":"Jiang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Systemic Innovative Research on Virus Vaccine, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming City, China;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lilian","family":"Yang","sequence":"additional","affiliation":[{"name":"Kunming Third People\u2019s Hospital, Kunming City, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Heng","family":"Li","sequence":"additional","affiliation":[{"name":"Key Laboratory of Systemic Innovative Research on Virus Vaccine, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming City, China;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Huiwen","family":"Zheng","sequence":"additional","affiliation":[{"name":"Key Laboratory of Systemic Innovative Research on Virus Vaccine, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming City, China;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Li","family":"Yu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Systemic Innovative Research on Virus Vaccine, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming City, China;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yun","family":"Liao","sequence":"additional","affiliation":[{"name":"Key Laboratory of Systemic Innovative Research on Virus Vaccine, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming City, China;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xin","family":"Zhao","sequence":"additional","affiliation":[{"name":"Key Laboratory of Systemic Innovative Research on Virus Vaccine, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming City, China;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mei","family":"Yuan","sequence":"additional","affiliation":[{"name":"Key Laboratory of Systemic Innovative Research on Virus Vaccine, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming City, China;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Na","family":"Li","sequence":"additional","affiliation":[{"name":"Key Laboratory of Systemic Innovative Research on Virus Vaccine, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming City, China;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Longding","family":"Liu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Systemic Innovative Research on Virus Vaccine, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming City, China;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dandan","family":"Li","sequence":"additional","affiliation":[{"name":"Key Laboratory of Systemic Innovative Research on Virus Vaccine, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming City, China;"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"2795","reference":[{"key":"b1","series-title":"BMC Infect Dis","first-page":"464","article-title":"Genetic diversity analysis of Chinese Leishmania isolates and development of L. donovani complex-specific markers by RAPD","volume":"21","author":"Yuan","year":"2021"},{"year":"2010","key":"b2"},{"key":"b3","series-title":"PLoS Negl Trop Dis","first-page":"e0004349","article-title":"A historical overview of the classification, evolution, and dispersion of Leishmania parasites and sandflies","volume":"10","author":"Akhoundi","year":"2016"},{"key":"b4","series-title":"PLoS One","first-page":"e35671","article-title":"Leishmaniasis worldwide and global estimates of its incidence","volume":"7","author":"Alvar","year":"2012"},{"key":"b5","series-title":"Semin Immunopathol","first-page":"231","article-title":"Implications 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