Journal of Clinical Microbiology最新文献

{"title":"Photo Quiz: Asteroid bodies in a skin biopsy of a farmer.","authors":"Xiujiao Xia","doi":"10.1128/jcm.00478-25","DOIUrl":"10.1128/jcm.00478-25","url":null,"abstract":"","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":"63 7","pages":"e0047825"},"PeriodicalIF":6.1,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12239716/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144591366","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An urgent need for diagnostic tools to address global mpox public health emergencies.","authors":"Benjamin M Liu, Zhilong Yang","doi":"10.1128/jcm.01321-24","DOIUrl":"10.1128/jcm.01321-24","url":null,"abstract":"<p><p>Monkeypox virus (MPXV) is the causative agent of mpox, a zoonosis formerly known as monkeypox. MPXV can be divided into clades I and II, which are further divided into subclades Ia, Ib, IIa, and IIb. Since May 2022, subclade IIb MPXV has rapidly spread outside Africa to more than 100 countries due to increased human-to-human transmission. Clade I is a more virulent MPXV endemic in Central Africa with up to 10% mortality in humans. Clade I has recently evolved into a novel subclade Ib and caused outbreaks in non-endemic neighboring countries and other continents. In response to mpox, the World Health Organization has declared Public Health Emergencies of International Concern in July 2022 (subclade IIb) and August 2024 (subclade Ib). The emergence and spread of the more virulent subclade Ib MPXV has caused a significant global public health threat, particularly in low- and middle-income countries (LMICs). The evolution of MPXV has outpaced the development of novel diagnostic assays, hampering the global response. There is an urgent need for additional diagnostic tools for the detection and surveillance of MPXV, especially subclade Ib MPXV, in LMICs. Herein, we provide the current epidemiology of mpox, analyze the diagnostic gaps for mpox, and evaluate the potential of additional detection strategies to be added to the suite of mpox assays. This commentary not only sheds light on the currently available diagnostic tools for mpox but also highlights the urgent need for additional diagnostic tools in response to the new global mpox public health threats.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0132124"},"PeriodicalIF":6.1,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12239723/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144225623","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Accurate and reproducible whole-genome genotyping for bacterial genomic surveillance with Nanopore sequencing data.","authors":"K Prior, K Becker, C Brandt, A Cabal Rosel, J Dabernig-Heinz, C Kohler, M Lohde, W Ruppitsch, F Schuler, G E Wagner, A Mellmann","doi":"10.1128/jcm.00369-25","DOIUrl":"10.1128/jcm.00369-25","url":null,"abstract":"<p><p>Despite recent advances in error rate reduction, until recently, Oxford Nanopore Technologies (ONT) sequences lacked the accuracy required for fine-scale bacterial genomic analysis. Here, recent software improvements of ONT and the ONT-core-genome multilocus sequence typing (cgMLST)-Polisher within the SeqSphere⁺ software were evaluated. We used short-read (Illumina) and long-read ONT sequences of 80 multidrug-resistant organisms (MDROs) for benchmarking. Illumina reads were <i>de novo</i> assembled using SKESA. For ONT, Dorado Super Accurate (SUP) model v.4.3 or v.5.0 basecalled reads were assembled with Flye and then polished with Medaka v.1.12 m4.3 or Medaka v.2.0 bacterial methylation model. In addition, the ONT-cgMLST-Polisher was run over all assemblies. The \"ground truth\" (GT) hybrid assemblies were created using Hybracter v.0.10.0. Sixteen isolates from four species out of the original 80 isolates were sent to six laboratories for a ring trial. The 80 MDROs basecalled with SUP m4.3 had an average cgMLST allele distance (AD) to the GT of 4.94 with Medaka v.1.12 and 1.78 with Medaka v.2.0, respectively. After further polishing the Medaka v.2.0 data with the ONT-cgMLST-Polisher, the AD dropped to 0.09. Using data basecalled with SUP m5.0 with Medaka v.2.0 further reduced the AD significantly to 0.04. While the ring trial data basecalled with Dorado SUP m4.3 showed more variability and insufficient results for some samples, model 5.0 data resulted in average ADs of 0.36 and 0.17 without and with the ONT-cgMLST-Polisher, respectively. In conclusion, recent ONT Dorado and Medaka models combined with the ONT-cgMLST-Polisher improved ONT sequencing accuracy and made it sufficiently reproducible for genomic surveillance of bacteria.IMPORTANCEONT sequencing methodology is especially attractive for small and medium-sized laboratories due to its relatively low capital investment and price per sample consumable costs. However, until recently, it lacked accuracy and reproducibility for bacterial genomic genotyping. Here, we present an evaluation of the most recent ONT bioinformatic (basecalling and polishing of consensus) improvements and a new ONT-cgMLST-Polisher tool. We demonstrate that by applying those procedures, ONT whole-genome genotyping-based surveillance of bacteria is finally accurate and reproducible enough for routine application even in small laboratories.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0036925"},"PeriodicalIF":6.1,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12239720/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144284492","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of the MIC test strips for antifungal susceptibility testing of <i>Candidozyma auris</i> (<i>Candida auris</i>) using a representative international collection of isolates.","authors":"Maria Siopi, Sevasti Leventaki, Ioannis Pachoulis, Bram Spruijtenburg, Jacques F Meis, Spyros Pournaras, Georgia Vrioni, Athanasios Tsakris, Joseph Meletiadis","doi":"10.1128/jcm.00399-25","DOIUrl":"10.1128/jcm.00399-25","url":null,"abstract":"<p><p>We compared MIC test strips (MTS) with the reference Clinical and Laboratory Standards Institute (CLSI) broth microdilution method using an international panel of 100 <i>Candidozyma auris</i> (<i>Candida auris</i>) isolates belonging to different clades. The agreement (±1 twofold dilution) between the methods and the categorical agreement (CA) based on the Centers for Disease Control and Prevention's (CDC's) tentative resistance breakpoints and MTS-specific wild-type upper limit values (WT-ULVs) were determined. The MTS-CLSI agreement was poor to weak for posaconazole (3%), itraconazole (20%), voriconazole (31%), and 5-flucytosine (37%), and moderate to strong for isavuconazole (58%), anidulafungin (68%), caspofungin (72%), micafungin (77%), and amphotericin B (85%). Most fluconazole MICs were off-scale, precluding a corresponding estimation. Significant interpretation discrepancies were recorded using the CDC's breakpoints for amphotericin B (66% CA, 34% major errors; MaEs), but not for fluconazole (98% CA, 1% MaEs, 1% very major errors; VmEs), anidulafungin (97% CA, 3% MaEs, 0% VmEs), micafungin (99% CA, 1% MaEs, 0% VmEs), and caspofungin (95% CA, 5% MaEs, 0% VmEs). Discrepancies were minimized using the amphotericin B method-specific WT-ULV of 4 mg/L (98% CA, 2% MaEs). The MTS-specific WT-ULVs of echinocandins could help to detect 100% of <i>FKS1</i> mutants. MTS generated higher MICs than the CLSI for azoles and 5-flucytosine. MTS could accurately detect fluconazole and echinocandin resistance among <i>C. auris</i> isolates. Nevertheless, it overestimated amphotericin B resistance as per the CDC's breakpoint of 2 mg/L. This can be improved by using the MTS-specific WT-ULV of 4 mg/L.IMPORTANCE<i>Candidozyma auris</i> (<i>Candida auris</i>) may exhibit resistance to multiple and sometimes even all currently available classes of antifungals. Hence, antifungal susceptibility testing (AFST) is of key importance to guide the clinician in therapeutic decision-making and to detect novel patterns of resistance. Gradient diffusion strips, referred to both Etest and MIC test strip (MTS), are broadly used in laboratory routine for AFST of yeasts. We therefore compared MTS with the reference Clinical and Laboratory Standards Institute (CLSI) broth microdilution method using an international panel of 100 <i>C</i>. <i>auris</i> isolates belonging to different clades. Significant interpretation discrepancies were recorded for amphotericin B (66% categorical agreement, 34% major errors), which could be minimized using the amphotericin B method-specific wild-type upper limit value of 4 mg/L. MTS generated higher MICs than the CLSI for azoles and 5-flucytosine. MTS could accurately detect fluconazole and echinocandin resistance.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0039925"},"PeriodicalIF":6.1,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144553626","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"PCR sensitivity for <i>Mycoplasma pneumoniae</i> detection in nasopharyngeal and oropharyngeal swabs: a comparative study.","authors":"Daisuke Kitagawa, Shin Nishihara, Masayuki Murata, Mai Onishi, Takahiro Mori, Soshi Hachisuka, Tenshin Okubo, Naohiro Yamamoto, Hiroki Nishikawa, Masayuki Onaka, Rika Suzuki, Soma Suzuki, Ayu Yamamoto, Ritsuki Uejima, Fumihiko Nakamura, Sayaka Yoshida, Taito Kitano","doi":"10.1128/jcm.00458-25","DOIUrl":"https://doi.org/10.1128/jcm.00458-25","url":null,"abstract":"<p><p>The differential impact of sample type on polymerase chain reaction (PCR) detection of <i>Mycoplasma pneumoniae</i> (MP) has rarely been investigated. The study aimed to evaluate the diagnostic performance of PCR for the detection of MP and to measure MP DNA load between nasopharyngeal and oropharyngeal swabs. Nasopharyngeal and oropharyngeal samples were obtained simultaneously to evaluate their diagnostic performance in children with suspected MP. Two commercially available PCR tests, multiplex PCR and Smart Gene Myco, were used to analyze the nasopharyngeal and oropharyngeal samples, respectively. Furthermore, real-time PCR (RT-PCR) tests were conducted on both sample residues to validate the results. In total, 422 participants underwent simultaneous PCR testing using nasopharyngeal and oropharyngeal swabs; 139 samples (32.9%) from nasopharyngeal swabs and 176 samples (41.7%) from oropharyngeal samples that tested positive using commercially available tests. RT-PCR tests were positive for 136 (32.2%) nasopharyngeal and 183 (43.4%) oropharyngeal residual samples. With the RT-PCR test of the residual extract from oropharyngeal swabs as a reference, the sensitivity and specificity of detecting MP were 74.9% (95% confidence interval 67.9%-81.0%) and 99.2% (97.0%-99.9%) with the multiplex PCR test on nasopharyngeal swabs, and 96.2% (92.3%-98.4%) and 100.0% (98.5%-100.0%) with the Smart Gene Myco on oropharyngeal samples. A negative correlation was observed between fluoroquinolone use and oropharyngeal DNA loads (<i>P</i> = 0.004). The sensitivity of MP detection was significantly better in oropharyngeal samples than in nasopharyngeal samples. This study indicates that oropharyngeal samples should be used to detect MP rather than nasopharyngeal samples.IMPORTANCEObtaining the best sample is crucial for the accurate diagnosis of <i>Mycoplasma pneumoniae</i> (MP) and timely and appropriate treatment. This study aimed to assess the diagnostic performance of MP detection using polymerase chain reaction (PCR) tests between nasopharyngeal and oropharyngeal samples. This study showed that the sensitivity of detecting MP was 74.9% (95% confidence interval 67.9%-81.0%) with a commercially available PCR test on nasopharyngeal swabs, and 96.2% (92.3%-98.4%) with a commercially available PCR test on oropharyngeal samples. The sensitivity of MP detection was significantly better in oropharyngeal samples than in nasopharyngeal samples. This study supports the idea that oropharyngeal samples should be used to detect MP. The results contribute to guidance in the recommendation regarding sampling methods to detect MP. Accurate identification of MP is crucial not only for timely and appropriate antimicrobial treatment but also for efficient epidemiological surveillance.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0045825"},"PeriodicalIF":6.1,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144553627","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development and evaluation of a duplex RT-qPCR assay for the detection and identification of Mayaro and chikungunya viruses.","authors":"Konrad M Wesselmann, Cécile Baronti, Antoine Nougairède, Laurence Thirion, Xavier de Lamballerie, Remi Charrel, Laura Pezzi","doi":"10.1128/jcm.00420-25","DOIUrl":"https://doi.org/10.1128/jcm.00420-25","url":null,"abstract":"<p><p>Mayaro virus (MAYV) is a mosquito-borne alphavirus that is widespread in the Amazon basin, where it co-circulates with the closely related chikungunya virus (CHIKV). Due to the limited surveillance and technical limitations of diagnostic assays (scarcity of commercial assays, serology cross-reactivity), the true burden of MAYV is uncertain. We designed a new RT-qPCR assay targeting the nsp1 gene for MAYV detection, which can be used in monoplex or duplex format. In the duplex format, the new MAYV assay is combined with a CHIKV assay and a second MAYV assay, both previously published. The lower limit of detection with a 95% positivity rate was determined to be <10 RNA copies/μL in monoplex and duplex formats for both MAYV and CHIKV. Monoplex and duplex assays proved to be linear within the tested range of approximately 10⁸ to 10² RNA copies/μL and showed 100% specificity against a wide panel of arboviruses as well as several other pathogens in clinical samples. The testing of CHIKV-positive sera and MAYV-spiked plasma samples confirmed the suitability of the assays in a clinical setting. These assays offer a reliable tool for detection and differentiation of MAYV and CHIKV in endemic settings.IMPORTANCEMolecular diagnostic capabilities for detecting alphaviruses other than chikungunya virus (CHIKV) remain limited. Mayaro virus (MAYV), an emerging mosquito-borne alphavirus, co-circulates with CHIKV in the Americas, making clinical differentiation between the two viruses challenging. To address this, we developed a novel RT-qPCR assay specifically for the detection of MAYV, which can also be used in a duplex format to simultaneously detect and distinguish CHIKV. The assay was thoroughly evaluated in both monoplex and duplex formats and demonstrated high sensitivity and specificity. This new tool is particularly valuable for the detection of MAYV, especially in resource-limited settings, where its duplex format offers efficient and accurate differentiation of acute infections.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0042025"},"PeriodicalIF":6.1,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144553625","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A multicenter study to assess the performance of the point-of-care RT-PCR Cobas SARS-CoV-2 & Influenza A/B nucleic acid test for use on the Cobas Liat system in comparison with centralized assays across healthcare facilities in the United States.","authors":"Elissa M Robbins, Rasa Bertuzis, Ho-Chen Chiu, Lupe Miller, Christopher Noutsios","doi":"10.1128/jcm.01459-24","DOIUrl":"https://doi.org/10.1128/jcm.01459-24","url":null,"abstract":"<p><p>Respiratory diseases can share many of the same symptoms, highlighting the need for timely and accurate differentiation to facilitate effective clinical management and reduce transmission. Compared with centralized testing, molecular point-of-care tests (POCTs) can provide a faster time to result. We evaluated the RT-PCR POCT Cobas® SARS-CoV-2 & Influenza A/B qualitative assay for use on the Cobas Liat system (the POC SARS-CoV-2 & Influenza A/B test) in nasal and nasopharyngeal swab samples from 10 diverse healthcare facilities in the United States. A composite comparator design consisting of three centralized tests was used to analyze severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), while performance vs a single centralized test was used for analysis of influenza A and B. Evaluations included performance stratified by sample type (prospective/retrospective and nasal/nasopharyngeal [paired by subject]), collection method (self/healthcare worker-collected [alternated and approximately balanced]), symptom status (symptomatic/asymptomatic), and SARS-CoV-2 vaccination status, as well as assay inclusivity and system ease of use. A total of 2,247 samples were tested. For SARS-CoV-2, the overall percent agreement (OPA) was 98.8% (95% confidence interval [CI]: 97.9, 99.3) in nasal swab samples and 99.0% (95% CI: 98.2, 99.4) in nasopharyngeal swab samples. Regression analysis showed that cycle threshold values from paired nasal and nasopharyngeal swab samples were highly correlated (correlation coefficient 0.83). The OPA was ≥99.5% (sample type dependent) and 100.0% for influenza A and B, respectively. The POC SARS-CoV-2 & Influenza A/B test was easy to use. These results support the use of the POCT in various sample types and by various operators in the intended-use setting.</p><p><strong>Importance: </strong>This study highlights the benefits of RT-PCR point-of-care tests, namely comparable performance to centralized testing in multiple sample types and ease of use. Utilizing assays such as the POC Cobas SARS-CoV-2 & Influenza A/B test may improve the timely differentiation of respiratory diseases that share similar symptoms.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0145924"},"PeriodicalIF":6.1,"publicationDate":"2025-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144540374","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Waste to worth: diagnostic accuracy of Xpert MTB/XDR on contaminated liquid cultures to salvage the detection of drug-resistant tuberculosis.","authors":"Yonas Ghebrekristos, Erick Auma, Zama Mahlobo, Rouxjeane Venter, Natalie Beylis, Jay Achar, Brigitta Derendinger, Sarishna Singh, Megan Burger, Christoffel Opperman, Robin Warren, Grant Theron","doi":"10.1128/jcm.00580-25","DOIUrl":"10.1128/jcm.00580-25","url":null,"abstract":"<p><p>Mycobacterium Growth Indicator Tube (MGIT) 960 culture is critical for tuberculosis (TB) drug susceptibility testing (DST) but is vulnerable to contamination. We evaluated the accuracy of Xpert MTB/XDR, a molecular DST for isoniazid, fluoroquinolone, amikacin, and ethionamide, on to-be-discarded contaminated growth. Xpert MTB/XDR was applied to acid-fast-bacilli-negative, contaminated cultures from sputum from people with rifampicin-resistant TB when Xpert MTB/XDR on sputum was unsuccessful (not resistant or susceptible for all drugs), either at diagnosis (Cohort A) or during treatment monitoring (Cohort B). Future DSTs within 3 months served as a reference standard. We determined potential care cascade improvements. In Cohort A, 10% (66/650) of people had a contaminated culture; 89% (59/66) of contaminated growths were Xpert MTB/XDR TB-positive. Sensitivity and specificity for isoniazid, fluoroquinolone, amikacin, and ethionamide resistance were 100% (95% confidence interval [CI] 85, 100) and 100% (79, 100); 100% (59, 100) and 100% (89, 100); 100% (16, 100) and 100% (91, 100); and 100% (72, 100) and 96% (78, 100), respectively. In Cohort B, 22% (28/129) of people with a contaminated culture were Xpert MTB/XDR TB-positive. Of these, 57% (16/28), 7% (2/28), and 43% (12/28) were isoniazid-, fluoroquinolone-, and ethionamide-resistant (in two, one, and four people, respectively, this would be the first resistant result). In both cohorts, time-to-DST could improve by a median (IQR) of 22 (12-42) days. Xpert MTB/XDR on contaminated MGIT960 cultures had high sensitivity and specificity for DST. This approach could mitigate culture contamination's negative effects and improve gaps in the drug-resistant TB diagnostic cascade.</p><p><strong>Importance: </strong>Culture contamination is a common impediment to drug susceptibility testing for tuberculosis, the single biggest infectious cause of death globally. Xpert MTB/XDR is a World Health Organization-recommended rapid molecular test for second-line drug resistance. We evaluated Xpert MTB/XDR on contaminated liquid culture growth that would otherwise be discarded, with the people who provided these specimens potentially lost from care cascades. By applying Xpert MTB/XDR to contaminated growth in a high-volume programmatic laboratory, we found the number of people who had second-line DST improved, as did the number of resistant cases diagnosed and time to diagnosis. Furthermore, DST information was generated in people who otherwise would have had none. This approach can therefore reduce the effect of culture contamination on tuberculosis DST, permitting earlier diagnosis and effective treatment initiation and potentially ultimately contributing to improving clinical outcomes and reducing transmission of drug-resistant TB.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0058025"},"PeriodicalIF":6.1,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144540423","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Validation and multi-site deployment of a lyophilized qRT-PCR reagent for the molecular diagnosis of avian influenza and rabies in Sub-Saharan African regions.","authors":"Petra Drzewnioková, Irene Brian, Marzia Mancin, Andrea Fortin, Morgane Gourlaouen, Angélique Angot, Mamadou Niang, Isaac Dah, Kouramoudou Berete, Adama Diakite, Fatou Tall Lo, Clement Meseko, Emilie Go-Maro, Valeria D'Amico, Viviana Valastro, Baba Soumare, Paola De Benedictis, Isabella Monne, Valentina Panzarin","doi":"10.1128/jcm.00080-25","DOIUrl":"https://doi.org/10.1128/jcm.00080-25","url":null,"abstract":"<p><p>Molecular methods are widely accepted as gold-standard techniques for the laboratory diagnosis of most human and animal pathogens. However, most molecular protocols rely on reagents that need to be transported and stored at a freezing temperature, a requirement that might affect their reliability in areas where the cold chain cannot be guaranteed. Over the years, several lyophilized molecular products have been marketed to circumvent this issue. We therefore evaluated the feasibility of replacing liquid reagents with freeze-dried formulations for the molecular diagnosis of avian influenza (AIV) and rabies (RABV) viruses, two priority zoonotic pathogens widely spread in Sub-Saharan Africa. Among six different commercial freeze-dried kits, we selected one reagent due to its easy-to-use features, single-reaction format, and preliminary performance assessment. Through a more in-depth evaluation, we determined its analytical and diagnostic performance and formulation stability and obtained results comparable to those of standard liquid master mixes. However, for the detection of divergent lyssaviruses, the lyophilized reagent's sensitivity was affected by suboptimal complementarity between the oligonucleotides and target sequences. Finally, a multi-site evaluation in four veterinary diagnostic laboratories located in Sub-Saharan Africa demonstrated the successful deployment of AIV and RABV assays utilizing the freeze-dried reagent, which can interchangeably replace liquid qRT-PCR/RT-PCR kits. Altogether, our results indicate that the investigated lyophilized master mix represents a valid alternative to liquid reagents for the molecular diagnosis of avian influenza and rabies and has the potential for broader applications to other relevant infectious diseases upon proper validation.IMPORTANCEMolecular diagnostic protocols rely on reagents that need to be transported and stored at freezing temperatures. Meeting this requirement can be challenging in areas where the maintenance of the cold chain is not guaranteed, such as in Sub-Saharan Africa. Our study aimed to assess the feasibility of using lyophilized reagents as a replacement for liquid reagents in the molecular diagnosis of two widespread zoonotic pathogens in Sub-Saharan Africa, namely, avian influenza and rabies. To accomplish this, we selected a commercially available lyophilized reagent based on its format and performance characteristics. We conducted a laboratory validation to assess the use of the lyophilized reagent throughout the entire diagnostic process. We also conducted a reproducibility test involving African laboratories as potential end-users. Our findings confirm that the lyophilized reagent can replace traditional liquid reagents to diagnose rabies and avian influenza and suggest its possible use for a wider range of infectious diseases after undergoing appropriate validation.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0008025"},"PeriodicalIF":6.1,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144540377","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development and validation of next-generation sequencing-based clinical test for triazole resistance prediction in <i>Aspergillus fumigatus</i>.","authors":"J R Caldera, Ashley Dayo, Nathan Wiederhold, Shangxin Yang","doi":"10.1128/jcm.00291-25","DOIUrl":"https://doi.org/10.1128/jcm.00291-25","url":null,"abstract":"<p><p>The rising rates of triazole drug resistance in <i>Aspergillus fumigatus</i> have placed greater reliance on antifungal susceptibility testing (AFST) to guide therapeutic management, particularly in medically complicated patients. Current methods, however, utilize conventional phenotypic assays that may pose significant challenges in performance, result interpretation, and time to reporting. Herein, we developed and validated a next-generation sequencing-based clinical test to predict the AFST phenotype of <i>A. fumigatus</i> against voriconazole, posaconazole, isavuconazole, and itraconazole using the <i>cyp51A</i> genotype as a marker for susceptibility. We sequenced 109 isolates comprising reference and clinical samples from the University of California, Los Angeles, Clinical Microbiology Lab, University of Texas Health Science Center at San Antonio Fungus Testing Laboratory, and the Centers for Disease Control and Prevention and Food and Drug Administration Antimicrobial Resistance Isolate Bank. Additionally, we integrated data from 14 previously published studies to produce comprehensive and robust interpretive criteria for the assay. Despite the complex association between <i>cyp51A</i> mutations and drug resistance, our triazole resistance assay produced a remarkable negative percent agreement (specificity) of ≥95%, thus offering presumptive, yet clinically actionable identification of triazole-wild-type isolates. Clinically, this allows for the rapid discrimination between acquired microbiological resistance in <i>A. fumigatus</i> and observed clinical resistance due to patients' declining health to help guide the most effective therapeutic management.IMPORTANCEThe rising rates of antifungal resistance have been expressed by many as \"the silent pandemic,\" profoundly reshaping the landscape of fungal disease management. Innovations in clinical mycology, however, have remained limited, particularly in comparison to the significant advances seen in the greater field of microbiology. Here, we sought to capitalize upon the expanding utility of next-generation sequencing to address a gap in clinical mycology diagnostics and antifungal susceptibility testing. We developed a whole-genome sequencing protocol to evaluate <i>Aspergillus fumigatus cyp51A</i> genotype to predict phenotypic susceptibility to triazole drugs. Our triazole resistance assay offers clinically actionable identification of triazole-wild-type isolates of <i>A. fumigatus</i> in a much more expeditious timeline than traditional phenotypic susceptibility testing.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0029125"},"PeriodicalIF":6.1,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144540376","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}