The Lancet Microbe

Background: Invasive non-typhoidal Salmonella (iNTS) disease causes an estimated 605,000 cases and 76,000 deaths each year, concentrated in sub-Saharan Africa, where the African Salmonella Typhimurium sequence type 313 (ST313) lineage predominates. Vaccine development is hampered by an absence of efficacy data and undefined correlates of protection. Methods: We conducted a phase 1, randomised, double-blind, dose-escalation controlled human infection model (CHIM) in healthy UK-resident adults, who were randomly assigned 1:1 to oral challenge with S. Typhimurium 4/74 (ST19, associated with gastrointestinal disease) or D23580 (ST313, associated with invasive disease). Dose-escalation was guided by a Bayesian continual reassessment method (CRM). The primary endpoint was Salmonella diagnosis, defined as sustained fever [≥]38{degrees}C on [≥]2 occasions [≥]12 hours apart and/or bacteraemia. Trial registration ClinicalTrials.gov (NCT05870150). Findings: Between August 2023 and December 2024, 50 participants were enrolled (25 per strain). 105 CFU was the maximum feasible dose, with CRM-estimated attack rates of 57.9% (95% credible interval 37.3 - 73.8) for D23580 and 47.4% (26.7 - 65.7) for 4/74. There were no serious adverse events. We found no clinical, microbiological, or immunological difference between the two pathovariants. Higher baseline serum anti-O-antigen IgG was associated with reduced disease (adjusted OR 0.42, 95% CI 0.17 - 0.90) and higher baseline faecal anti-lipopolysaccharide IgA with reduced colonisation (OR 0.12, 95% CI 0.01 - 0.59). Interpretation: This S. Typhimurium CHIM is safe, reproducible, and provides a platform to generate early efficacy signals and candidate correlates of susceptibility, thereby de-risking future iNTS vaccine trials. The absence of a phenotypic difference between the invasive and gastrointestinal pathovariants in immunocompetent adults suggests that host factors, rather than pathogen adaptation alone, shape the invasive phenotype seen in endemic settings. Funding: Wellcome Trust.

BackgroundMelioidosis is a grossly neglected but often-fatal tropical disease. The disease is named "a great mimicker" after its broad clinical manifestations, which makes disease diagnosis challenging and time-consuming. To improve diagnosis, we developed and evaluated the performance of the CRISPR-Cas12a system called "CRISPR-BP34" to detect Burkholderia pseudomallei DNA across clinical specimens from patients suspected to have melioidosis. MethodsWe documented time taken for diagnosis, antibiotics prescribed during the waiting period, and infection outcomes in 875 melioidosis patients treated in a hospital in northeast Thailand between October 2019 and December 2022. In the last six months, we performed CRISPR-BP34 detection on clinical specimens (blood, urine, respiratory secretion, pus and other body fluids) collected from 330 patients with suspected melioidosis and compared its performance to the current gold-standard culture-based method. Discordant results were validated by three independent qPCR tests. FindingsA window of 3-4 days was required for gold-standard culture diagnosis, which resulted in delayed treatment. 199 [22{middle dot}7%] of 875 patients died prior to diagnosis results while 114 [26{middle dot}3%] of 433 follow-up cases had been diagnosed, treated, but died within 28 days of admission. A shorter sample-to-diagnosis time of less than 4 hours offered by CRISPR-BP34 technology could lead to faster administration of correct treatment. We demonstrated an improved sensitivity of CRISPR-BP34 (106 [93{middle dot}0%] of 114 positive cases, 95% CI 86{middle dot}6 - 96{middle dot}9) compared to the culture approach (76 [66{middle dot}7%] of 114 positive cases, 95% CI 57{middle dot}2 - 75{middle dot}2); while maintaining similar specificity (209 [96{middle dot}8%] of 216 negative cases, 95% CI 93{middle dot}4-98{middle dot}7) to the culture (216 [100 %] of 216 negative cases, 95% CI 98{middle dot}3-100{middle dot}0). InterpretationThe sensitivity, specificity, speed, window of clinical intervention, and ease of operation offered by the CRISPR-BP34 support its use as a point-of-care diagnostic for melioidosis. FundingChiang Mai University Thailand and Wellcome Trust UK Research in context Evidence before this studyMelioidosis is an often-severe infectious disease caused by the bacterium Burkholderia pseudomallei. It is estimated to affect 165,000 individuals annually worldwide, of which 89,000 cases are fatal. The disease diagnosis is challenging due to diverse clinical presentations, low awareness, limited diagnostic options, or even a lack of diagnostic tests. A PubMed search conducted from the database inception to 6 May 2023, using the terms "melioidosis" AND "diagnosis test," yielded 207 results, 40 of which presented clinical evaluations of rapid melioidosis diagnostic tests. Antigen-based diagnostic tests, which detect the presence of B. pseudomallei, reported high specificity (median = 98{middle dot}6%, IQR 94{middle dot}0 - 100{middle dot}0), but low sensitivity (median = 57{middle dot}1%, IQR = 44{middle dot}3 - 82{middle dot}5). The test sensitivity suffers from the often-low concentration of the bacterial antigens in patients samples, which can vary by specimen type and stage of infection. Antibody-based diagnostic tests that detect host antibodies against B. pseudomallei typically exhibit satisfactory specificity (median = 94{middle dot}5%, IQR = 88{middle dot}6 - 96{middle dot}2) but poor sensitivity (median = 80{middle dot}2%, IQR = 71{middle dot}0 - 88{middle dot}1). These tests are often impacted by variations in antibody responses to B. pseudomallei and the duration required for antibody production. Furthermore, standardisation remains challenging due to the influence of different serum titres on sensitivity and background of the tests. Likewise, quantitative PCR exhibits a high degree of specificity (median = 99{middle dot}8%, IQR = 91{middle dot}6-100{middle dot}0), but an observed low sensitivity for melioidosis (median = 77{middle dot}1%, IQR = 20{middle dot}8-97{middle dot}8), which is likely attributed to the genetic heterogeneity of B. pseudomallei genomes. Additionally, these studies consistently reported a demand for improved speed and ease of implementation in resource-limited settings where melioidosis is endemic. With the limitations of current diagnostic methods, a culture-confirmed approach with 60% sensitivity, 100% specificity, and a diagnosis time of 2-7 days still stands as the gold standard for melioidosis diagnosis. Added value of this studyTo date, no study has measured the impact of delayed diagnosis on melioidosis. We assessed the number of deaths occurring prior to culture-confirmed diagnosis (22{middle dot}7%) and those after diagnosis but within 28 days post-admission (26{middle dot}3%), highlighting the urgent need for prompt action. To address this, we developed the CRISPR-BP34 test, which utilises isothermal amplification of a nucleic acid target followed by site-specific detection using a CRISPR-Cas12a enzyme. We successfully implemented this assay in a resource-limited setting in northeast Thailand, where the disease prevalence is among the highest in the world. The assay achieved a diagnostic sensitivity and specificity of 93{middle dot}0% and 96{middle dot}8%, respectively, with a limit of detection ranging from 50-250 cfu/mL. Early diagnosis can be achieved within four hours of patient admission, which is significantly faster than the gold-standard test that typically takes several days. Moreover, the ultrasensitivity of the CRISPR-BP34 assay enabled the detection of low levels of B. pseudomallei in hemoculture bottles, which could be missed due to mixed infections, poor aseptic technique, or other causes, leading to undiagnosed melioidosis. Implications of all available evidenceThe CRISPR-BP34 assay holds great promise for the management and control of melioidosis. Its minimal setup and shallow learning curve make it well-suited for resource-limited settings. Additionally, its speed and high sensitivity enable early diagnosis and treatment, which are crucial for saving patients lives.

Drug-resistant infections caused by spores of the mould Aspergillus fumigatus pose a major challenge in managing chronic respiratory disease. Evidence shows that a substantial burden of aspergillosis is caused by strains that have evolved resistance to azole antifungal chemicals in the environment, however the contribution of local exposures to the colonisation of patients remains unclear. To investigate routes of acquisition, we whole-genome sequenced A. fumigatus isolates from individuals with chronic pulmonary fungal disease (n=182, 15 individuals), their homes (n=101, 10 homes), and hospital environments (n=102). These data were then integrated with retrospective sequence datasets enabling phylogenetic resolution across 912 genomes of UK A. fumigatus. We found high genetic diversity in clinical isolates, frequent mixed colonisation, and azole resistance in [~]25% of infections, particularly in those patients with cystic fibrosis (CF) and chronic pulmonary aspergillosis (CPA). The TR34/L98H cyp51A resistance allele, a well characterised marker of environmental adaptation to azole agricultural fungicides, was present in 25% of clinical azole-resistant strains. While azole-resistant A. fumigatus was detected in 6/10 homes, phylogenomic analysis revealed no clear genetic link between the home environment and clinical fungal lung isolates. A. fumigatus was prevalent in hospital environments, with azole-resistant isolates comprising 4.5% (n=9/202) of air and 3.4% (n=6/178) of soil isolates, predominantly harbouring the TR34/L98H allele. In contrast to homes, phylogenomic and pairwise SNP analysis revealed numerous clinical isolates with >97% genetic identity when compared to those isolated from the hospital environment and randomly chosen pairs of UK isolates. These findings indicate widespread exposure and potential nosocomial acquisition of drug-resistant genotypes of A. fumigatus, supporting the need for targeted environmental surveillance and mitigation of exposures in healthcare settings.

BackgroundRespiratory metagenomics (RMg) needs evaluation in a pilot service setting to determine utility and inform implementation into clinical practice. MethodsFeasibility, performance and clinical impacts on antimicrobial prescribing and infection control were recorded during a pilot RMg service for patients with suspected lower respiratory tract infection (LRTI) on two general and one specialist respiratory intensive care units (ICU) at Guys & St Thomas NHS foundation Trust, London. ResultsRMg was performed on 128 samples from 87 patients during the first 15-weeks providing same-day results for 110 samples (86%) with median turnaround time of 6.7hrs (IQR 6.1-7.5 hrs). RMg was 92% sensitive and 82% specific for clinically-relevant pathogens compared with routine testing. 48% of RMg results informed antimicrobial prescribing changes (22% escalation; 26% de-escalation) with escalation based on speciation in 20/24 cases and detection of acquired-resistance genes in 4/24 cases. Fastidious or unexpected organisms were reported in 21 samples including anaerobes (n=12), Mycobacterium tuberculosis, Tropheryma whipplei, cytomegalovirus and Legionella pneumophila ST1326, which was subsequently isolated from the bed-side water outlet. Application to consecutive severe community-acquired LRTI cases identified Staphylococcus aureus (two with SCCmec and three with luk F/S virulence determinants), Streptococcus pyogenes (emm1-M1uk clone), S. dysgalactiae subspecies equisimilis (STG62647A) and Aspergillus fumigatus with multiple treatments and public-health impacts. ConclusionsRMg provides frequent diverse benefits for treatment, infection control and public health. The combination of rapid comprehensive results, alongside revealing and characterising a hidden burden of infections makes the case for expediting routine service implementation.

There is an urgent need to improve our understanding of how new antibiotic resistant organisms emerge and spread. A high-priority resistance threat is the ST258 lineage of carbapenem-resistant Klebsiella pneumoniae. Here, we studied resistance to the last-line drug colistin among ST258 by tracking its evolution across 21 U.S. hospitals over the course of a year. Phylogenetic analysis supported a significant fitness cost being associated with resistance, as resistance emergence was common but resistance variants were rarely transmitted. Furthermore, several resistance variants that were transmitted had acquired secondary variants that reverted the strain to susceptible. The exceptions to the general pattern of instability associated with resistance were two large clusters of resistant strains in one sublineage (clade IIB) present across Southern California hospitals. Quantification of transmission fitness in the healthcare environment indicated that, while resistant isolates from other clades were less fit than their susceptible counterparts, clade IIB resistant isolates were more fit, despite having similar resistance variants. Additional analyses supported the increased fitness of colistin-resistant clade IIB isolates being driven by a lineage-defining variant that increased clade IIBs association with patient subpopulations with enhanced colistin exposure. These results show that a favorable genetic background and sustained selective pressure led to the emergence and spread of a colistin-resistant ST258 sublineage across a regional healthcare network. These findings highlight the utility of integrating pathogen genomic and corresponding clinical data from regional healthcare networks to detect emerging antibiotic resistance threats and understand the clinical practices and patient populations that drive their spread. Significance StatementSelective pressure in hospitals leads to frequent antibiotic resistance evolution. However, emergent resistance alleles are often not transmitted to other individuals because of fitness costs associated with resistance. Due to the difficulty of studying pathogen fitness in humans, our understanding of how resistant organisms circumvent these costs is limited. We integrate genomic and clinical data to understand the evolutionary trajectories leading to transmissible resistance for the last-line antibiotic colistin. While colistin resistance is generally associated with a fitness cost that hinders transmission, this cost was mitigated in a sublineage that had previously acquired mutations increasing its association with patient populations more likely to receive colistin, suggesting a key role for historical contingency in the emergence and spread of stable resistance.

Targeted next-generation sequencing (tNGS) enables rapid detection of drug resistance in Mycobacteriumtuberculosis (Mtb), including minor variants that may indicate emerging resistance. We applied tNGS to isolates from 34 (of 49 eligible) participants in a randomized trial of bedaquiline (BDQ) and delamanid (DLM) co-administration for multidrug-resistant TB (MDR-TB). We detected baseline nonsynonymous variants in the nitroimidazole resistance genes fgd2 and ddn (BDQ arm) and fbiB (DLM arm) among 2/12 and 2/12 patients, respectively, and emergent subpopulations with nonsynonymous variants in fbiB, fbiC, and ddn as early as week 1 in 3/34 patients. These findings demonstrate that variants in nitroimidazole resistance genes may be present in the absence of drug exposure, and that tNGS can effectively track variant emergence during MDR-TB treatment.

Monoclonal antibodies (mAbs) are a major class of drugs for treatment and prevention of disease. In early clinical trials, the pharmacokinetics (pK) of mAbs are usually assessed by measuring mAb concentration in serum. However, it is not a given that the mAbs will retain full functionality over time, emphasizing the need for integrated PK and functional assessments. In a re-analysis of data from the CAPRISA 012B trial, a previously published phase 1 study evaluating mAbs CAP256V2LS and VRC07-523LS in HIV-negative women, we report an unexpected disconnect between serum bNAb concentrations and HIV neutralization activity of CAP256V2LS, with implications for ongoing assessment of passive immunization trials.

BackgroundEnteric fever is a serious public health concern. The causative agents, Salmonella enterica serovars Typhi and Paratyphi A, are frequently antimicrobial resistant (AMR), leading to limited treatment options and poorer clinical outcomes. We investigated the genomic epidemiology, resistance mechanisms and transmission dynamics of these pathogens at three urban sites in Africa and Asia. MethodsBacteria isolated from febrile children and adults at study sites in Dhaka, Kathmandu, and Blantyre were sequenced and AMR determinants identified. Phylogenomic analyses incorporating globally-representative genome data, and ancestral state reconstruction, were used to differentiate locally-circulating from imported pathogen variants. FindingsS. Paratyphi A was present in Dhaka and Kathmandu but not Blantyre. S. Typhi genotype 4.3.1 (H58) was common in all sites, but with different dominant variants (4.3.1.1.EA1 in Blantyre; 4.3.1.1 in Dhaka; 4.3.1.2 in Kathmandu). Resistance to first-line antimicrobials was common in Blantyre (98%) and Dhaka (32%) but not Kathmandu (1.4%). Quinolone-resistance mutations were common in Dhaka (99.8%) and Kathmandu (89%) but not Blantyre (2.1%). AcrB azithromycin-resistance mutations were rare (Dhaka only; n=5, 1.1%). Phylogenetic analyses showed that (a) most cases derived from pre-existing, locally- established pathogen variants; (b) nearly all (98%) drug-resistant infections resulted from local circulation of AMR variants, not imported variants or recent de novo emergence; (c) pathogen variants circulated across age groups. Most cases (67%) clustered with others that were indistinguishable by point mutations; individual clusters included multiple age groups and persisted for up to 2.3 years, and AMR determinants were invariant within clusters. InterpretationEnteric fever was associated with locally-established pathogen variants that circulate across age groups. AMR infections resulted from local transmission of resistant strains. These results form a baseline against which to monitor the impacts of control measures. FundingWellcome Trust, Bill & Melinda Gates Foundation, European Unions Horizon 2020, NIHR. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSCurrent knowledge of the enteric fever pathogen populations in Dhaka, Kathmandu, and Blantyre comes from retrospective analysis of isolates captured from routine diagnostics or treatment trials. Due to these study designs, most focus on either adult or paediatric cohorts, which complicates assessment of pathogen variant transmission across age groups. Many studies report prevalence of antimicrobial resistance (AMR) and associated mechanisms amongst enteric fever cases. Genomic studies at these sites and elsewhere have identified the spread of AMR clones, and a recent genomic study quantified the inter- and intra-continental spread of resistant S. Typhi between countries. However, PubMed search of "(typhoid OR (enteric fever)) AND (genom*)" identified no studies quantifying the relative proportion of resistant infections that is attributable to local transmission of resistant variants vs imported strains or de novo emergence of AMR. Added value of this studyWe estimate the vast majority (98%) of drug-resistant enteric fever cases identified in our study resulted from local circulation of resistant variants. Further, we show genetically indistinguishable pathogen variants (either resistant or susceptible) persisting for up to 2.3 years and causing infections across all age groups (under 5 years; 5-15 years; [≥]15 years). Implications of all the available evidenceWhile inter-country transfer of resistant enteric fever pathogens does occur and is concerning, the burden of drug-resistant enteric fever at the study sites is currently caused mainly by transmission of locally-established variants, and transmits across age groups. These data confirm assumptions made in models of vaccine impact regarding heterogeneity of pathogen variants and AMR across age groups, and support that childhood immunisation programmes can be expected to reduce the overall burden of resistant infections in endemic settings.

BackgroundEffective cholera outbreak response requires accurate bedside rapid diagnostic tests (RDTs) because access to laboratories is often limited. Formative studies suggest cholera diagnostics have multiple vulnerabilities, including antibiotics and predation by bacteriophage ( phage) specific to Vibrio cholerae (Vc). MethodsWe conducted a prospective nationwide study in Bangladesh among over 2000 patients with diarrhoeal disease to characterize how these vulnerabilities impact RDT performance. Assays included culture, qPCR and mass spectrometry. FindingsWith the current gold standard of culture or qPCR Vc positivity, we found no effect of phage on RDT performance. When the diagnostic criteria were expanded to include phage, there was a small decrease in RDT sensitivity. In contrast, large increases in sensitivity and specificity were observed among patients with moderate and severe dehydration. Using the expanded definition, the odds of RDT positivity decreased among cholera patients with phage exposure. The effect was most robust among patients with severe dehydration. Antibiotic were detected in over 80% of samples by LC-MS/MS which limited testing for effects on RDTs. Applying these findings, we estimated that restricting RDT use to severe patients with no reported antibiotic exposure increases sensitivity by 50% compared to unrestricted use. If phage were a diagnostic proxy for Vc, we estimate RDT would miss an additional 17% of cholera cases. InterpretationCholera RDTs have critical limitations that require consideration in global deployments. Inclusion of phage detection in diagnostic criteria may improve case detection which requires further study. The impact of these findings likely extends to other diseases where diagnostics share similar vulnerabilities. RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSWe conducted two Pubmed searches for reports published after Jan 1, 2000, in all languages. The first search terms were [bacteriophage OR phage] AND [pathogen] AND [diagnostic test]. The primary search criteria identified 57 publications. A secondary criterion excluded reviews and papers on phage display technology. Among twenty remaining articles, bacteriophage were used as proxies for pathogens within diverse genera (Yersinia, Mycobacterium, Burkholderia, Staphylococcus, Salmonella, Bacillus, Escherichia, Acinetobacter, Vibrio); phage profiles were also used as biomarkers for infection. The second search terms were [rapid diagnostic test OR RDT] AND [Vibrio]. After excluding reviews, 101 papers were identified that covered immunologic (antibody-based lateral flow assays), molecular (PCR, qPCR, nl-qPCR) and mass spectrometry-based assays; only 5 articles related to vibriophage detection of which three were permutations of the same initiative. Added value of this studyTo our knowledge, this is the first study to investigate the vulnerabilities of RDT performance (e.g., sensitivity and specificity) to virulent bacteriophage and antibiotics in a large multi-site study. The hypothesis that antimicrobials and disease severity might impact RDT performance by decreasing or increasing the target number, respectively, is not novel. However, there is a lack of literature from large clinical studies that rigorously tests this hypothesis. Therefore, the added value of this study is a pragmatic evaluation of this hypothesis and a proof of concept for how to test these complex questions for other less tractable diseases. Implications of all the available evidenceInside the cholera field, we applied the findings by estimating that changing to a mode of restricted RDT use in which RDTs are reserved for severely dehydrated patients who report no antibiotic exposure, significantly improves the sensitivity. Furthermore, we estimate that an additional 17% of cases would be missed by RDT if the case definition were expanded to include phage detection as a proxy for the pathogen. In this context, the RDT remains with limitations that require consideration when optimizing global RDT deployments. Implications likely extend to other diseases where diagnostics share similar vulnerabilities.

Understanding the landscape of resistance to antibiotics before their clinical deployment could inform strategies to slow the development of resistance upon their introduction. We evaluated the associations between bacterial genotypes, patient clinical characteristics, and medical exposures with resistance to two recently approved {beta}-lactam/{beta}-lactamase inhibitor combinations in carbapenem-resistant Klebsiella pneumoniae collected before their clinical deployment. Whole-genome sequencing revealed that even within the clonal sequence type 258, genetic background influenced baseline resistance levels and the propensity for resistance to emerge and spread. Resistance in ST258 clade II was mediated by convergent mutation at a small number of loci, which rarely spread amongst patients. In contrast, resistance in clade I was influenced by a lineage-defining insertion in the OmpK36 porin, which made resistance accessible via the blaKPC transposon moving to a higher copy number plasmid, and was in turn associated with transmissible resistance. Distinguishing patients based on whether resistance was acquired via plasmid-mediated versus mutational resistance revealed that while exposure to carbapenems and cephalosporins were associated with mutational resistance, plasmid-associated resistance in clade I was not associated with antibiotic exposures. These findings underscore how pre-clinical surveillance in clinically relevant populations can identify potential drivers of resistance that could inform molecular surveillance and antibiotic stewardship interventions to prolong the efficacy of novel antibiotics.

Background and Aims The optimal treatment for first episodes and first recurrences of Clostridioides difficile infections (CDI) is unknown and there is emerging evidence for pulse and taper (P-T) regimens. Therefore, we sought to estimate the relative efficacy of treatment options. Methods MEDLINE and CENTRAL were searched from database inception to May 21, 2025 and unpublished conference abstracts were searched from recent infectious disease conferences. RCTs on the treatment of first episodes or first recurrences of CDI comparing fixed-dose or P-T regimens of fidaxomicin or vancomycin were included. The primary and secondary outcomes were 40- and 56-day CDI recurrence, respectively. A random-effects network meta-analysis on the risk ratio (RR) scale was conducted using a standard regimen (10-14 days) of vancomycin as the comparator. Treatments were ranked using the surface under the cumulative ranking curve (SUCRA). Results 8 RCTs were included comprising a total of 2181 patients. For 40-day recurrence, fidaxomicin P-T had the highest probability of ranking best (RR=0.10, 95%Confidence Interval [95%CI]=0.10-0.49, SUCRA=1.00), followed by vancomycin P-T (RR=0.49, 95%CI=0.32-0.76, SUCRA=0.61), fixed-dose fidaxomicin (RR=0.61, 95%CI=0.49-0.76, SUCRA=0.39), and, finally, fixed-dose of vancomycin (SUCRA=0.00). The treatments ranked in the same order for 56-day recurrence, though only 3 RCTs reported on this timepoint. Conclusion Vancomycin P-T, fidaxomicin P-T, and fixed-dose fidaxomicin were all superior to a fixed-dose vancomycin. Head-to-head comparative effectiveness RCTs are needed to quantify their relative effect sizes of and impact on long-term prevention of recurrent CDI.

Nipah virus disease is a bat-borne zoonosis with person-to-person transmission, a case fatality rate of 38-75%, and recognised pandemic potential. The first reported outbreak occurred in Malaysia and Singapore in 1998, since followed by multiple outbreaks in Bangladesh and India. No therapeutics or vaccines have been licensed to date, and only few candidates are in development. This systematic review aimed to assess the evidence for the safety and efficacy of therapeutic options (monoclonal antibodies and small molecules) for Nipah virus and other henipaviral diseases in order to support candidate prioritisation for further evaluation in clinical trials. At present, there is sufficient evidence to trial only m102.4 and remdesivir (singly and/or in combination) for prophylaxis and early treatment of Nipah virus disease. In addition to well-designed clinical efficacy trials, in vivo pharmacokinetic-pharmacodynamic studies to optimise selection and dosing of therapeutic candidates in animal challenge and natural human infection are needed. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSNipah virus infection is a bat-borne zoonosis with person-to-person transmission, a case fatality rate of 38-75%, and recognised pandemic potential. No therapeutics or vaccines have been licensed to date, and only few candidates are in development. We conducted this systematic review to assess the evidence for the safety and efficacy of therapeutic options (monoclonal antibodies and small molecules) for Nipah virus and other henipaviral diseases to support candidate prioritisation for further evaluation in clinical trials. We searched bibliographic databases for journal articles, conference abstracts, and patents: PubMed, Ovid Embase, Ovid CAB Abstracts, Ovid Global Health, Scopus, Web of Science (all databases), and the WHO Global Index Medicus. "Henipavirus" or "Nipah" or "Hendra" along with "therapeutics" or "monoclonal" were the title, abstract, and subject heading keywords, with synonyms and variant spellings as additional search terms. We searched trial registries for clinical trials of Henipavirus, Nipah virus, and Hendra virus at all stages of recruitment: Cochrane Central Register of Controlled Trials, ClinicalTrials.gov, and the WHO International Clinical Trials Registry Platform. We searched the Trip database and WHO website for guidelines and reports. All searches were conducted on 30 May 2022. We did not apply language or publication date limits. Studies were included if they contained primary data on the safety and/or efficacy of monoclonal antibodies (in vivo) or small molecules (in vivo or in vitro) for the treatment and/or prophylaxis of Nipah, Hendra, and related Henipaviridae. Almost all had critical or high risk of bias. Added value of this studyThis is the most detailed systematic review and analysis of the Nipah virus therapeutics landscape to date, including all available in vivo and related in vitro data on the safety, efficacy, and pharmaco-kinetics of monoclonal antibodies and small molecules with the specific aim of supporting prioritisation for clinical trials. We also present a roadmap for how in vivo development of Nipah therapeutics could be strengthened to achieve greater equity, efficiency, and effectiveness. Implications of the available evidenceAt present, there is sufficient evidence to trial only m102.4 and remdesivir for prophylaxis and early treatment of Nipah virus infection. Well-designed clinical efficacy trials as well as in vivo pharmacokinetic-pharmacodynamic studies to optimise selection and dosing of therapeutic candidates in animal challenge and natural human infection are needed.

RationalePneumococcal pneumonia remains a global health problem. Pneumococcal colonisation increases local and systemic protective immunity, suggesting nasal administration of live attenuated S. pneumoniae strains could help prevent infections. ObjectivesWe used a controlled human infection model to investigate whether nasopharyngeal colonisation with attenuated S. pneumoniae strains protected against re-colonisation with wild-type (WT) S. pneumoniae (Spn). MethodsHealthy adults aged 18-50 years were randomised (1:1:1:1) for nasal administration twice (two weeks interval) with saline, WT Spn6B (BHN418) or one of two genetically modified Spn6B strains - SpnA1 ({Delta}fhs/piaA) or SpnA3 ({Delta}proABC/piaA) (Stage I). After 6 months, participants were challenged with SpnWT to assess protection against re-colonisation (Stage II). Measurements and Main Results125 participants completed both study stages as per intention to treat. No Serious Adverse Events were reported. In Stage I, colonisation rates were similar amongst groups: SpnWT 58.1% (18/31), SpnA1 60% (18/30) and SpnA3 59.4% (19/32). Anti-Spn nasal IgG levels post-colonisation were similar in all groups whilst serum IgG responses were higher in the SpnWT and SpnA1 groups than the SpnA3 group. In colonised individuals, increases in IgG responses were identified against 197 Spn protein antigens and serotype 6 capsular polysaccharide using a pangenome array. Participants given SpnWT or SpnA1 but not SpnA3 in phase 1 were partially protected against re-colonisation with SpnWT (recolonisation rates of 29% versus 30% respectively). ConclusionNasal colonisation with genetically modified live attenuated Spn was safe and induced protection against recolonisation, suggesting nasal adminstration of live attenuated Spn could be an effective stategy for preventing pneumococcal infections.

BackgroundClassified by the WHO as one of the 19 most dangerous fungal pathogens, Pneumocystis jirovecii has been associated with increasing outbreaks of Pneumocystis pneumonia (PCP) among solid organ transplant (SOT) recipients worldwide. Mycophenolic acid (MPA), an inosine monophosphate dehydrogenase (IMPDH) inhibitor commonly used as an immunosuppressant to prevent organ rejection, is a risk factor for PCP. However, MPA also displays antifungal activity, potentially protecting against PCP, despite not being used to treat it. Therefore the underlying factors driving these outbreaks remain undefined. MethodsIn this international multicenter retrospective observational study, P. jirovecii samples were collected from 96 SOT patients (including 94 from nine separate outbreaks and 84 on MPA therapy) and 67 non-transplant controls (none on MPA), between 1986 and 2020 across six countries in Europe, North America and Asia. All samples underwent extensive targeted sequencing of the P. jirovecii inosine monophosphate dehydrogenase (impdh) gene and multiple genetic markers, with selected samples further analyzed for complete mitogenome and restriction fragment length polymorphisms. Computational modeling was employed to predict the effects of IMPDH mutations on protein structure and MPA binding. ResultsSix impdh mutations (including one previously reported) were identified, with frequencies of 4-21% each in SOT patients and 0-1% in controls. These mutations were strongly associated with prior MPA exposure and showed marked geographic segregation and temporal shifts. Four mutations were each linked to multiple distinct genotype profiles, representing separate P. jirovecii strains. Structure modeling predicted that these four mutations reduced protein stability and binding affinity to MPA. ConclusionsThis study suggests that the widespread use of MPA in SOT recipients has unexpectedly driven the emergence of multiple impdh mutations in P. jirovecii, each presumably arising independently in multiple strains worldwide. These mutations likely confer drug resistance and provide a selective survival advantage to P. jirovecii in SOT recipients exposed to MPA, thereby facilitating transmission and outbreaks. These findings have significant implications for the prevention and clinical management of PCP in SOT recipients, highlighting a rare example of how antimicrobial resistance can emerge through unexpected pathways, transcending conventional antimicrobial use and emphasizing the need for increased vigilance and strategic adaptation in clinical practice.

BackgroundMetagenomic sequencing has the potential to transform clinical microbiology by enabling rapid pathogen identification and antimicrobial resistance (AMR) prediction in critically ill patients with bloodstream infections (BSIs). However, its clinical implementation has been hindered by challenges in speed, accuracy, and technical feasibility. We present a direct from positive blood culture workflow using Oxford Nanopore sequencing that overcomes these limitations, delivering results rapidly and accurately. MethodsWe developed and evaluated a direct-from-blood culture metagenomic sequencing method for rapid pathogen and AMR prediction using Oxford Nanopore Sequencing. Species prediction was performed using Kraken2 with a comprehensive standard database, employing heuristic and random forest classification models. Additionally, we benchmarked AMR classification tools and databases, including ResFinder, CARD, and NCBI AMRFinderPlus. We processed 273 randomly selected blood cultures (211 positive, 62 negative) from hospitalised patients in real time, comparing species identification, AMR detection and time-to-result against standard culture-based diagnostics performed by the routine microbiology laboratory. FindingsOur method achieved 97% sensitivity and 94% specificity (both improving to 100% after accounting for plausible additional infections) for species identification compared to established diagnostic methods. We detected 18 additional infections--13 polymicrobial and 5 previously unidentifiable--and delivered findings in 3.5 hours, nearly a third of time taken by routine methods. For the top ten pathogens, our method produced AMR results 20 hours faster than current antimicrobial susceptibility testing, with 88% sensitivity and 93% specificity. For Staphylococcus aureus and Escherichia coli, AMR prediction sensitivity was 100% and 91%, and specificity 99% and 94% respectively. InterpretationThese findings highlight the potential of metagenomic sequencing to improve BSI diagnosis by providing rapid and comprehensive pathogen and AMR detection. Integrating this approach into routine clinical workflows could bridge critical diagnostic gaps in sepsis care, reduce empirical antibiotic use, and inform targeted treatment within hours rather than days. FundingNational Institute for Health Research (NIHR) Biomedical Research Centre, Oxford. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSBloodstream infections (BSIs) remain a critical global health burden, affecting over 30 million people annually with mortality rates reaching up to 30%. The urgency of early diagnosis is well-established -delays in appropriate antimicrobial therapy, even by one hour, can significantly reduce survival in severe cases. Conventional blood culture, the current diagnostic standard, is hampered by long turnaround times (24-72 hours), reduced sensitivity in patients already on antibiotics, and its inability to detect fastidious, slow-growing, or unculturable organisms. To explore current advancements in the field, we conducted a search on PubMed and Google Scholar on 6th April 2025 using a combination of relevant medical subject heading terms: "infection," "diagnosis," "metagenomic," and "bloodstream." This search yielded 59 results, from which we excluded pilot studies, those with fewer than 50 samples and studies focused on diagnosing specific pathogens. After applying these exclusions, we found four that contributed valuable insights into the application of metagenomics in BSIs. Blauwkamp and colleagues demonstrated strong concordance between cell-free DNA sequencing and blood culture, although the technique showed limited utility in predicting antimicrobial resistance. Similarly, Rossoff and colleagues used a cell-free DNA assay at a CLIA-certified laboratory demonstrating performance with >90% with turnaround times within 48 hours. Anson and colleagues developed a method to extract bacterial DNA from positive blood cultures for whole-genome sequencing, successfully identifying Staphylococcus and Gram-negative species, though antimicrobial resistance prediction was limited with Illumina data alone. A more recent study by Harris and colleagues demonstrated that direct-from-blood culture methods can accurately identify causative pathogens at the species level in ICU patients, particularly in monomicrobial infections, approximately 9-17 hours after a positive blood culture result. To date, no study, to our knowledge, at scale has systematically evaluated the diagnostic yield, speed, and practical clinical utility of metagenomic sequencing applied directly to randomised routine blood culture samples. Added value of this studyThis study presents the first clinical implementation of metagenomic sequencing using Oxford Nanopore technology directly on routine blood cultures at scale, with a focus on real-time diagnosis of bloodstream infections. It uniquely assesses the ability of metagenomics to detect not only common but polymicrobial, uncultivable, or previously undetected pathogens, and to predict antimicrobial resistance within clinically actionable timeframes. The study advances the field by demonstrating how accurate and rapid pathogen identification and resistance profiling can be integrated into existing diagnostic workflows, thereby reducing diagnostic delay and enabling early, targeted antimicrobial therapy. Implications of all the available evidenceThe collective evidence indicates that clinical metagenomics has reached a stage of maturity where we now see workflows ready for implementation in clinical settings. This study adds to the growing recognition that, when applied to positive blood cultures, metagenomic sequencing can bridge critical diagnostic gaps in sepsis care, reduce empirical antibiotic use, and inform precision treatment within hours rather than days. Nonetheless, widespread clinical adoption will require further standardisation of laboratory protocols, development of robust interpretive frameworks, and evidence of cost-effectiveness in real-world settings. As antimicrobial resistance continues to rise globally, metagenomics offers a robust and strategic approach to meet one of the most pressing challenges in infectious diseases.

BackgroundSalmonella Isangi is an under-characterised serovar repeatedly associated with antimicrobial resistant hospital infections. Outbreaks of extensively drug-resistant (XDR) Salmonella Isangi occurred in close succession within hospitals in Malawi and South Africa, prompting us to characterise the serovar using epidemiologic, phenotypic, and genomic perspectives. MethodsIn Malawi, we integrated hospital blood culture surveillance with environmental sampling from neonatal wards and urban waterways. In South Africa, we analysed isolates from five hospitals involved in a regional outbreak. We used whole genome sequencing (Illumina and MinION) to characterise AMR genes and plasmids, assessed biofilm formation, disinfectant susceptibility, in vivo virulence, and analysed all publicly available Salmonella Isangi genomes. Findings224 / 345 (65%) of genomes in the global collection belonged to Salmonella Isangi sequence type (ST) 335. Of these, 221 (99%) originated from Malawi and South Africa, including the isolates recovered from both outbreaks. 199 (89%) ST335 genomes carried determinants of resistance to fluoroquinolones and third-generation cephalosporins, consistent with an XDR profile. In Malawi, a single ST335 clade caused the outbreak and was simultaneously present in both the hospital environment and nearby rivers. Inter-hospital transmission of a separate ST335 clade sustained the outbreak in South Africa. Closely related Malawian and South African isolates carried distinct plasmids encoding similar resistance determinants; evidence from our study and public databases suggests gene transfer via a cointegrate intermediate. Five non-outbreak South African ST335 isolates harboured additional carbapenem and macrolide resistance genes. Phenotypically, Salmonella Isangi ST335 resembled Salmonella Typhimurium in biofilm formation and disinfectant tolerance but was less virulent in mice. InterpretationSalmonella Isangi ST335 combines a locally untreatable XDR profile with nosocomial transmission and environmental persistence, suggesting a high potential for future outbreaks. A distinct and potentially greater threat lies in the horizontal spread of its resistance determinants to Salmonella Typhimurium and Salmonella Enteritidis, the two dominant invasive serovars in the region. Strengthened surveillance, integrating phenotypic testing with targeted genomics, is urgently needed. Its absence in Malawi, in contrast to South Africa, underscores inequities in preparedness for emerging AMR threats. FundingThis work was supported by the Wellcome Trust through the Core Grant (206545/Z/17/Z) and the COVID-19 Sequencing Grant (220757/Z/20/Z) awarded to MLW. Additional support was provided by the Global Health Research Professorship to Melita Gordon from the UK National Institute for Health and Care Research (NIHR) (NIHR300039). Peter Johnston is funded by the Liverpool Clinical PhD Programme for Health Priorities in the Global South, supported by the Wellcome Trust (223502/Z/21/Z). For open access, the author has applied a CC BY public copyright license to any author-accepted manuscript version arising from this submission. Whole-genome sequencing of Salmonella isolates from South Africa was made possible by support from the SEQAFRICA project which is funded by the Department of Health and Social Cares Fleming Fund using UK aid. The views expressed in this publication are those of the authors and not necessarily those of the UK Department of Health and Social Care or its Management Agent, Mott MacDonald. Analyses in this study were supported in part through use of software and workflows developed under National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH) grant R21AI178369. The NIH had no role in study design, data collection, analysis/interpretation, or publication decisions. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSSalmonella Isangi is a recurrent cause of antimicrobial resistant hospital outbreaks. We searched PubMed for Salmonella Isangi and related synonyms (to February 23rd, 2026) and identified 39 articles. No prior studies have examined transmission routes or provided phenotypic characterisation beyond antimicrobial resistance testing. Outbreaks have been reported from five hospitals on three continents, as well as a foodborne outbreak in China. Two major sequence types (STs) consistently appeared: ST335 and ST216. ST216 was widely geographically distributed and recovered from a variety of animal, meat, and environmental sources. ST335 was primarily associated with human clinical cases. Added value of this studyThis investigation was motivated by an outbreak of extensively drug-resistant (XDR) Salmonella Isangi at a hospital in Malawi. The Malawian outbreak occurred shortly before a multi-centre nosocomial outbreak in South Africa, and we provide insights from both in our analysis. We combined local epidemiology, phenotypic analyses, and global genomic characterisation to deliver a comprehensive description of the serovar. Both outbreaks were caused by ST335, which is the dominant sequence type in South Africa and Malawi, but by distinguishable clades in each country. In Malawi, genetically indistinguishable isolates were simultaneously circulating among patients, the hospital environment, and rivers throughout Blantyre City. Transfer of patients between hospitals is likely to have sustained the outbreak in South Africa. Recombination through a cointegrate intermediate may explain why the same resistance determinants are carried on distinct plasmid backbones within the Malawian and South African ST335 clades. We identify five ST335 isolates in South Africa that were not related to either outbreak and which harbour carbapenem and macrolide resistance genes in addition to an XDR genotype. Implications of all available evidenceXDR Salmonella Isangi ST335 is a major threat in Malawi because effective therapy requires antibiotics that are seldom accessible in routine care. The ability of ST335 to transmit in hospitals and to persist in the environment may increase the risk of future outbreaks. Salmonella Isangi readily acquires and maintains antimicrobial resistance determinants through diverse plasmid backbones and recombination, raising concern for transfer to locally prevalent invasive Salmonella serovars. National genomic surveillance of the kind that exists in South Africa is essential to track and contain further resistance emergence, but such surveillance does not exist in Malawi. There is an urgent need to expand genomic surveillance in low-income countries if the threat posed by Salmonella Isangi and other pathogens that drive antimicrobial resistance is to be recognised early and effectively contained.

Background: Vancomycin-resistant Enterococcus (VRE) species are common healthcare-associated pathogens that cause difficult-to-treat infections. Whole genome sequencing of patients has revealed a substantial burden of patient-to-patient VRE transmission in hospitals, with patients in intensive care units (ICUs) at particularly high risk of acquisition. However, few studies adequately characterize the pathways of VRE transmission between patients in acute care settings, a necessary step to identify current gaps in infection prevention practices. By harnessing genomic clustering analyses of whole genome sequences of VRE isolates from patients, environmental surfaces, and healthcare providers (HCP) in ICUs, we aim to reconstruct indirect pathways of pathogen movement to identify patterns of VRE spread and opportunities for transmission prevention. Methods and Findings: We collected daily samples (N = 6848) from ICUs in two hospitals over 13 weeks from four main sampling sources: patients, HCP hands, patient rooms, and shared surfaces. Samples were cultured on selective media and sent for whole genome sequencing (WGS). We used genomic thresholds to identify clusters of related VRE isolates and distinguish unrelated isolates. VRE was detected in samples from 20 out of 322 unique occupant-stays (6.22%). VRE isolates were detected from all sampling sources except for shared surfaces. A total of 44 unique VRE isolates were identified, 43 Enterococcus faecium (VREfm) and one Enterococcus faecalis (VREf). Two distinct patterns of VREfm spread were observed: 1) an outbreak setting with observed patient-to-patient transmission and low VRE diversity, and 2) high VRE diversity and pathogen movement between occupant-stays facilitated by persistent HCP and environmental contamination, but no observed transmission events. VRE detection probabilities were not significantly different between occupant-stays in outbreak and non-outbreak settings (OR = 0.63, 95% CI (0.23, 1.83), p = 0.32). However, inclusion of VRE isolated from non-patient samples increased the number of occupant-stays with VRE detection from 6 to 20, a 3.3-fold increase, as compared to patient samples alone. Inclusion of non-patient samples also increased the number of VRE multi-isolate genomic clusters detected by 7-fold. Our findings are limited because sampling was primarily conducted in ICUs. Due to the combination of short ICU stay durations and imperfect test sensitivity, VRE transmission events were probably underdetected. Conclusions: Our findings characterize the complex nature of VRE transmission pathways in ICU settings. Even without an ongoing outbreak, we found substantial evidence of VRE movement between occupant-stays, facilitated by a combination of HCP hands and environmental surfaces. This study highlights the importance of environmental sampling for understanding VRE transmission potential, which is likely to be underestimated using patient sampling alone. We recommend that future studies incorporate follow-up sampling after discharge to better understand the true burden of transmission.

Identification of antigens targeted by a protective response is a central quest in malaria vaccinology. Whole-genome sieve analysis (SAWG) in samples collected from placebo-controlled field trials of Plasmodium falciparum (Pf) sporozoite (SPZ) vaccines may enable identification of Pf pre-erythrocytic antigens. We applied SAWG to genomic data generated from Pf isolates collected during two field trials measuring the efficacy, in malaria-exposed African adults, of two PfSPZ vaccines. These randomized, double-blind, placebo-controlled trials were conducted in regions of Mali and Burkina Faso characterized by high seasonal transmission, where parasite genetic diversity is high. Genomic sites in which the vaccine allelic state was significantly underrepresented among breakthrough infections in vaccinees relative to placebo recipients were termed "target sites". Protein-coding loci containing target sites that changed amino acids were termed "target loci". The SAWG conducted on clinical trial samples from the Burkina Faso and Mali trials identified 138 and 80 single-copy protein-coding target loci in the Burkinabe and Malian data sets, respectively, with twelve common to both, a number significantly higher than expected (E = 3.9; 99%CI = [0, 9]). Among these was the thrombospondin-related anonymous protein locus, which encodes PfSSP2|TRAP, one of the most abundant and well-characterized pre-erythrocytic stage antigen as well as other genes encoding membrane-associated proteins of unknown function. These results identify SAWG as a potentially powerful tool for identifying protective vaccine antigens in recombining pathogens with large genome size and reveals potential new protective Pf antigens.

BackgroundThere is concerted effort to reduce the burden of 6 months antimicrobial treatment for tuberculosis (TB). Early treatment cessation at 8 weeks is effective for most but incurs increased risk of disease relapse. We tested the hypothesis that blood RNA signatures of TB disease or C-reactive protein (CRP) measurements discriminate microbiological cure after 8 weeks of treatment, as a pre-requisite for a biomarker to stratify risk of relapse. MethodsWe identified blood RNA signatures of TB disease or cure by systematic review. We evaluated CRP measurements and blood RNA signatures that could be reproduced in genome-wide transcriptomic data from a previously reported longitudinal dataset in pulmonary TB, spanning samples collected pre-treatment, at 2 and 8 weeks of treatment, and after 2 years of follow up. In our primary analysis, we tested discrimination of sputum culture positivity at 8 weeks by contemporary blood RNA and CRP measurements using area under the receiver operating characteristic curve (AUROC) analysis. In secondary analyses, we tested the relationship between biomarker measurements and time to culture positivity as a surrogate for bacterial load in sputum culture positive cases at 8 weeks, and discrimination of sputum culture status at 8 weeks by biomarker measurements at any other time point. FindingsWe evaluated 12 blood RNA signatures. Blood RNA signature scores normalised over time from TB treatment initiation. 11/44 cases with available blood RNA, CRP and sputum culture results, were sputum culture positive at 8 weeks of treatment. None of the 12 blood RNA signature scores tested achieved statistically significant discrimination between sputum culture-positive vs. negative patients at this time point, with AUROC point estimates of 0.48-0.61. CRP achieved the best AUROC of 0.69 (95% confidence interval 0.52-0.87). None of the contemporary biomarker measurements correlated with bacterial load, and no measurements pre-treatment or at 2 weeks discriminated sputum culture status at 8 weeks. InterpretationThe current repertoire of blood RNA signatures of TB and CRP will not provide host response surrogates of microbiological cure to support cessation of TB treatment at 8 weeks. Decoupling of blood transcriptional host-response from the presence of viable bacteria is indicative of subpopulations of Mycobacterium tuberculosis able to colonise the respiratory tract without triggering a detectable immune response. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe performed a systematic review, using comprehensive terms for "tuberculosis", "transcriptional" and "biomarker" with no language or date restrictions in Medline on October 4, 2023. Many studies have described normalisation of blood RNA signatures during the course of tuberculosis treatment. Five studies have evaluated blood RNA signatures as a test of microbiological cure after completion of 6 months of treatment. However, there is growing interest in their application as a test of cure to support shortened treatment regimens. The performance of one blood RNA signature has been reported to provide modest discrimination of contemporary sputum culture status at 8 weeks of treatment among HIV co-infected patients with recurrent tuberculosis. We found no reports of whether these findings are generalisable to other blood RNA signatures or to HIV negative patients with their first episode of tuberculosis, who are most likely to be candidates for shortened treatment regimens. Added value of this studyTo our knowledge, we provide the first evaluation and comparison of multiple blood RNA signatures of tuberculosis for discrimination of microbiological cure after 8 weeks of tuberculosis therapy among HIV negative patients. 12 previously validated blood RNA signatures of tuberculosis identified by systematic review underwent head-to-head evaluation, alongside blood C-reactive protein measurement as an alternative biomarker of disease, to determine whether they discriminated contemporary sputum culture status after 8 weeks of treatment among 44 HIV negative patients with smear-positive drug-sensitive tuberculosis enrolled to a previously reported randomised controlled trial of adjunctive vitamin D therapy. None of the blood RNA signatures showed statistically significant discrimination of contemporary sputum culture status after 8 weeks tuberculosis treatment, or quantitative relationships with sputum bacterial load among sputum culture positive cases. Importantly, most sputum culture positive cases at this time point, showed contemporary blood RNA signature scores within the normal range. Implications of all the available evidenceAssuming that microbiological cure is a pre-requisite for early cessation of antimicrobial treatment for tuberculosis, the current repertoire of blood RNA signatures of tuberculosis does not provide host response surrogates of microbiological cure to support introduction of 8-week treatment regimens. Therefore, there is a need for further discovery and validation of new biomarkers to support risk stratification for truncated therapy both for research and clinical practice applications. The lack of association between blood transcriptomic signatures and sputum culture status after 8 weeks of treatment suggests the existence of microbial sub-populations that do not trigger a host response. Whether this reflects a state of latency or bacterial persistence in immune privileged compartments requires further investigation.

Invasive fungal infections are a global threat; early diagnosis is critical for patient outcomes, but current diagnostic measures remain notoriously slow. Here we extend our multiplexed, hybridization-based rRNA-targeted strategy for rapid, sensitive pathogen identification, previously designed for diverse bacteria and Candida species, to identify diverse fungal pathogens. We created a set of 91 probes targeting 86 medically relevant fungal species, designed to recognize regions of differential conservation across taxonomic groupings, from class- to species-specific probes. We assessed assay performance across a Training Set of 93 clinical isolates spanning 32 species of common fungal pathogens across 18 genera, with Pearson correlations of probeset reactivity profiles (PSRPs) identifying the pathogen at the species, genus, and family level with 83%, 94%, and 95% accuracy, respectively, in a leave-one-out analysis. We developed a more sophisticated classifier on this Training Set, using taxonomic categories to select progressively more informative probes at each taxonomic level. After optimization, we assessed performance on an independent Validation Set of 54 clinical isolates spanning the same species as the Training Set, with 91%, 94%, and 98% at the species, genus, and family levels, respectively. We piloted our assay on formalin-fixed paraffin-embedded (FFPE) tissue, demonstrating rapid, culture-independent fungal identification from this high-value clinical sample type, often the sole specimen available. The assay requires <30 minutes hands-on time (or <65 minutes from FFPE tissue), returning results in <8 hours from cultured specimen or FFPE tissue to answer on an RNA detection platform available in clinical laboratories. ImportanceTimely identification of fungal pathogens is critical for patient outcomes. A multiplexed hybridization-based assay targeting rRNA enables accurate identification of >50 species of pathogenic fungi from crude lysates of cultured clinical isolates.