{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,30]],"date-time":"2026-01-30T04:26:54Z","timestamp":1769747214528,"version":"3.49.0"},"reference-count":73,"publisher":"Public Library of Science (PLoS)","issue":"4","license":[{"start":{"date-parts":[[2025,4,24]],"date-time":"2025-04-24T00:00:00Z","timestamp":1745452800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100010221","name":"Higher Education Commision, Pakistan","doi-asserted-by":"publisher","award":["20-16308\/NRPU\/R&D\/HEC\/2021"],"award-info":[{"award-number":["20-16308\/NRPU\/R&D\/HEC\/2021"]}],"id":[{"id":"10.13039\/501100010221","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:sec id=\"sec034\">\n<jats:title>Introduction<\/jats:title>\n<jats:p><jats:italic>Candida auris<\/jats:italic> is an emerging multidrug-resistant pathogen responsible for nosocomial infections worldwide, characterized by high mortality rates and significant challenges in detection due to frequent misidentification. Classified by the WHO as a pathogen of critical importance since it exhibits resistance to multiple antifungal agents, particularly fluconazole, and is highly transmissible in healthcare settings. Conventional detection methods often lack the accuracy required for effective infection control. This study aimed to conduct inferential and molecular analyses of <jats:italic>C. auris<\/jats:italic> and other yeast species infecting immunocompromised patients in the Special and Intensive Care Units (SCU and ICU) of a tertiary care hospital in Quetta, Pakistan. In this region, <jats:italic>C. auris<\/jats:italic> remains rarely studied and is frequently misdiagnosed by clinical staff due to limited awareness and diagnostic challenges. Notably, no prior research has been conducted on <jats:italic>C. auris<\/jats:italic> in Quetta. The study also sought to develop reliable diagnostic methods suitable for resource-limited settings, addressing a critical gap in healthcare infrastructure.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec035\">\n<jats:title>Materials and methods<\/jats:title>\n<jats:p>Samples (150 each) from the ear, axilla, groin, and saliva of SCU\/ICU patients were collected and processed on yeast malt agar, with preliminary identification using Brilliance <jats:italic>Candida<\/jats:italic> Agar (BCA) and CHROMagar <jats:italic>Candida<\/jats:italic> Plus (CCP). Advanced techniques, including PCR amplification of ITS regions, DNA sequencing, RFLP with <jats:italic>Msp<\/jats:italic>1, MALDI-TOF, Vitek 2, and species-specific primers, were used for identification. Antifungal susceptibility to fluconazole, amphotericin B, and voriconazole were also assessed.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec036\">\n<jats:title>Results<\/jats:title>\n<jats:p>The culture test revealed that 42.6% samples were positive for yeast infections. In addition to detecting <jats:italic>Candida auris<\/jats:italic> in 4 cultures, chromogenic media identified 6 other <jats:italic>Candida<\/jats:italic> species: <jats:italic>C. albicans<\/jats:italic>, <jats:italic>C. dubliniensis<\/jats:italic>, <jats:italic>C. glabrata<\/jats:italic>, <jats:italic>C. krusei<\/jats:italic>, <jats:italic>C. parapsilosis<\/jats:italic>, and <jats:italic>C. tropicalis<\/jats:italic>. Further validation through advanced techniques, including molecular diagnostics and MALDI-TOF, enabled the identification of additional species: <jats:italic>C. famata<\/jats:italic>, <jats:italic>C. kefyr<\/jats:italic>, <jats:italic>C. lusitaniae<\/jats:italic>, and <jats:italic>Meyerozyma<\/jats:italic> (<jats:italic>Candida<\/jats:italic>) <jats:italic>guilliermondii<\/jats:italic>. Out of all identified yeast species <jats:italic>C. dubliniensis<\/jats:italic> was the most common, followed by <jats:italic>C. albicans<\/jats:italic> and <jats:italic>C. tropicalis<\/jats:italic>, with the highest infection rates observed in saliva samples. Antifungal Susceptibility Tests (AST) revealed that <jats:italic>C. auris<\/jats:italic> isolates were resistant to Fluconazole, Amphotericin B, and Voriconazole, highlighting multidrug resistance. This study represents the first report of novel multidrug-resistant <jats:italic>C. auris<\/jats:italic> from Quetta, Pakistan, indicating that <jats:italic>C. auris<\/jats:italic> is prevalent among ICU and SCU patients. Novel species specific primers targeting phospholipase B, topoisomerase II, CDR and 18s genes were designed in our laboratory and not previously reported in earlier studies, proved highly effective for the rapid identification of <jats:italic>Candida<\/jats:italic> species. The established protocol using these primers is recommended for implementation in resource-limited laboratory settings. The statistical analysis demonstrated significant correlations between <jats:italic>Candida<\/jats:italic> species infection (dependent variable) and several independent factors (variables) emphasizing the importance of targeted diagnostics and intervention strategies.<\/jats:p>\n<\/jats:sec>","DOI":"10.1371\/journal.pone.0319485","type":"journal-article","created":{"date-parts":[[2025,4,24]],"date-time":"2025-04-24T17:47:51Z","timestamp":1745516871000},"page":"e0319485","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":2,"title":["Investigation of multi-drug resistant Candida auris using species-specific molecular markers in immunocompromised patients from a tertiary care hospital in Quetta, Pakistan"],"prefix":"10.1371","volume":"20","author":[{"given":"Hira","family":"Ejaz","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7585-2644","authenticated-orcid":true,"given":"Muhammad","family":"Mushtaq","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shereen","family":"Khan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nasir","family":"Azim","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Abrar","family":"Hussain","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kaleemullah","family":"Kakar","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Muhammad Zubair","family":"Khan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ayisha","family":"Hafeez","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Syed","family":"Moeezullah","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2025,4,24]]},"reference":[{"issue":"1","key":"pone.0319485.ref001","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1007\/s13225-021-00494-6","article-title":"Trends in yeast diversity discovery","volume":"114","author":"T Boekhout","year":"2021","journal-title":"Fungal Diversity"},{"issue":"9","key":"pone.0319485.ref002","doi-asserted-by":"crossref","first-page":"759","DOI":"10.2174\/1568026618666180528121707","article-title":"Emerging virulence, drug resistance and future anti-fungal drugs for candida pathogens","volume":"18","author":"V Srivastava","year":"2018","journal-title":"Curr Top Med Chem"},{"issue":"10","key":"pone.0319485.ref003","doi-asserted-by":"crossref","first-page":"2724","DOI":"10.1128\/IAI.00469-16","article-title":"Candida albicans pathogenesis: fitting within the host\u2013microbe damage response framework","volume":"84","author":"MA Jabra-Rizk","year":"2016","journal-title":"Infect Immun"},{"issue":"6","key":"pone.0319485.ref004","doi-asserted-by":"crossref","first-page":"919","DOI":"10.1007\/s10096-013-2027-1","article-title":"Multidrug-resistant endemic clonal strain of Candida auris in India","volume":"33","author":"A Chowdhary","year":"2014","journal-title":"Eur J Clin Microbiol Infect Dis"},{"issue":"6","key":"pone.0319485.ref005","doi-asserted-by":"crossref","first-page":"1794","DOI":"10.1093\/jac\/dkx034","article-title":"Candida auris candidaemia in Indian ICUs: analysis of risk factors","volume":"72","author":"SM Rudramurthy","year":"2017","journal-title":"J Antimicrob Chemother"},{"key":"pone.0319485.ref006","unstructured":"Pan American Health Organization, World Health Organization. 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