Lottie Brown

Background: Histoplasmosis is a life-threatening invasive fungal disease with a global distribution. In Southeast Asia, its true burden is unknown due to limited awareness and fungal diagnostics. We conducted a prospective, multi-centre screening study to determine the prevalence, incidence, and outcomes of histoplasmosis among people with advanced HIV disease (AHD) in Vietnam, and to evaluate the diagnostic and predictive performance of the IMMY Clarus urine Histoplasma antigen enzyme immunoassay (HAg EIA). Methods: Adults with AHD (CD4≤100 cells/µL or WHO stage 3/4) hospitalized at three hospitals in Vietnam were consecutively screened for histoplasmosis using the HAg EIA and Myco/F Lytic fungal-mycobacterial blood culture. Participants were followed at least monthly for six months to ascertain proven, probable, or possible histoplasmosis according to European Organization for Research and Treatment of Cancer and Mycoses Study Group 2020 reference standards.Findings: Between February 2021 and August 2023, 901 participants were enrolled. Median CD4 count was 19 cells/mm3 (IQR 9-45), and 747 (82·9%) were male. Histoplasmosis was diagnosed in 27 (3·0%) participants, including 15 proven, four probable, and eight possible cases. The incidence was 12 per 1000 patient-years. Distinguishing clinical features included weight loss (18/27, 66·7%), anaemia (25/27, 92.6%), thrombocytopenia (21/27, 77.8%), and transaminitis (26/27, 96.3%). Co-infections occurred in 22 (81·5%) participants, commonly oral-oesophageal candidiasis (11/27, 40·7%), Pneumocystis pneumonia (10/27, 37·0%), talaromycosis (8/27, 29·6%), and tuberculosis (8/27, 29·6%). Using the data-driven Youden index cut-off (>1·5 EIA units), HAg EIA demonstrated 100% sensitivity, 99·3% specificity, 81·8% positive predictive value, and 100% negative predictive value. Despite appropriate antifungal therapy, the six-month mortality was 29·6%.Interpretation: The HAg EIA demonstrated excellent diagnostic performance as a rapid rule-in and rule-out test for histoplasmosis. Antigen-screening strategy identified a 3·0% prevalence of otherwise undiagnosed histoplasmosis among hospitalized people with AHD in Vietnam. The high mortality despite appropriate treatment highlights the need for earlier screening and treatment.

The dimorphic fungus Talaromyces marneffei causes talaromycosis, a life-threatening fungal disease with limited treatment options. Olorofim, a first-in-class orotomide antifungal that targets pyrimidine synthesis essential for fungal growth, has low minimum inhibitory concentration (MIC) against T. marneffei and clinical efficacy against other invasive fungal diseases. Here, we tested the hypothesis that olorofim synergistically enhances amphotericin B (AmB), a potent membrane-targeting antifungal, against T. marneffei in 55 clinical isolates using a validated colorimetric checkerboard assay. The MIC was defined as the lowest drug concentration inhibiting ≥ 95% of fungal growth. Drug interactions were assessed using the fractional inhibitory concentration index (FICI), which defines ≤0.5 as synergy, 0.5 < FICI ≤ 4.0 as indifference, and FICI > 4 as antagonism. We found that interactions between AmB and olorofim were indifferent across all 55 isolates (0.5 < FICI ≤ 1.03). Time-kill assays showed an expected concentration-dependent fungicidal activity for AmB, but a concentration-independent fungistatic activity for olorofim against T. marneffei. Combinations of AmB and olorofim were also indifferent in time-kill experiments. Although synergy was not observed, and olorofim is unlikely to enhance AmB induction therapy, olorofim may have a role in the consolidation and maintenance therapy of talaromycosis.

Background: The diagnosis of talaromycosis, a life-threatening invasive fungal disease endemic in Southeast Asia, still relies on protracted culture methods. We report the development and clinical evaluation of a novel lateral flow assay (LFA) for the rapid diagnosis of talaromycosis. Methods: The LFA used 2 novel monoclonal antibodies targeting the Talaromyces marneffei-specific protein Mp1p. In a retrospective, case-cohort study, we evaluated the diagnostic performance of the Mp1p LFA against the reference standard of culture-proven talaromycosis from any clinical specimens, and compared to our validated Mp1p enzyme immunoassay (EIA) in paired plasma and urine samples from 239 talaromycosis and 160 non-talaromycosis participants randomly selected from cohorts of hospitalized adults with advanced human immunodeficiency virus (HIV) disease from 5 centers across Vietnam. Results: The Mp1p LFA demonstrated an analytical limit-of-detection of 400 pg/mL, comparable to the EIA. No cross-reactivity with 16 common human fungal pathogens was observed. Clinical sensitivity was higher in urine compared to plasma (95.4% vs 88.7%, P < .01), and clinical specificity was >95% in both specimen types. Clinical performance was similar to the EIA: sensitivity 95.4% versus 97.1%, P = .87 and specificity 95.6% versus 97.5%, P =1.0. Both the LFA and EIA were substantially more sensitive than conventional blood culture collected at the same time (95.4% vs 97.1% vs 77.8%, P < .01). Conclusions: The Mp1p LFA has excellent diagnostic performance, comparable to the EIA, is superior to blood culture, and has the potential to rapidly rule in and rule out talaromycosis at the point-of-care within 15 minutes without need for laboratory infrastructure.

Background: Talaromyces marneffei causes talaromycosis, a life-threatening fungal disease with limited treatment options. The standard treatment of amphotericin B (AmB) induction followed by itraconazole consolidation still results in 15% to 30% mortality. This study aimed to investigate the potential of AmB and flucytosine (5FC) combination therapy to enhance antifungal activity. Methods: The in vitro antifungal activity of AmB and 5FC alone and in combination against 60 T. marneffei clinical isolates was evaluated using a validated colorimetric antifungal susceptibility assay and the checkerboard method. The minimum inhibitory concentration (MIC) was defined as the lowest drug concentration inhibiting ≥ 95% fungal growth (MIC ₉₅ ) for both AmB and 5FC. The combination effect between AmB and 5FC against T. marneffei was determined using fractional inhibitory concentration index. Combination effects were further tested using a time-kill assay. Results: The MIC ₉₅ was 0.25 – 2 μg/mL (geometric mean [GM] 0.68 μg/mL) for AmB, and 0.03 – 0.5 μg/mL (GM 0.28 μg/mL) for 5FC. Full synergy was observed in 4 isolates (7%), and indifference was observed in the remaining 56 isolates (93%). The time-kill experiments revealed a concentration-dependent fungicidal activity of AmB, and concentration-independent fungistatic effect of 5FC. Synergy between AmB and 5FC was confirmed, showing greater than 2-log ₁₀ reduction in colony forming units when used in combination. No antagonism was observed. Conclusions: Our study demonstrated in vitro evidence of synergistic activity between AmB and 5FC against T. marneffei , providing the evidence to support in vivo and clinical trial testing of AmB and 5FC combination therapy, and dosing reduction strategies of 5FC. Lay Summary: This study demonstrated in vitro synergy between amphotericin B and flucytosine against Talaromyces marneffei , providing the proof of concept to test this antifungal combination in a clinical trial of talaromycosis.

Background: Several antigen-detection assays have been developed for the rapid diagnosis of talaromycosis, but their utility has been limited by a lack of commercial options. The aim of this study was to perform a head-to-head comparison of the performance of our in-house monoclonal antibody-based Mp1p antigen-detecting enzyme immunoassay (EIA) with its recently-developed commercial platform. Methods: In this diagnostic accuracy, retrospective, case-cohort study, we compared the sensitivity, specificity, positive likelihood ratio (LR+) and negative likelihood ratio (LR-) of the commercial Wantai Mp1p EIA versus our in-house Mp1p EIA on paired plasma and urine samples from 424 hospitalized adults with advanced HIV disease, including 224 cases of proven talaromycosis, where Talaromyces marneffei was isolated in culture of blood or other clinical specimens, and 200 controls diagnosed with a range of other opportunistic infections. All participants were randomly selected from prospective cohorts recruited between 2011 and 2019 from five centers in Vietnam. Results: The sensitivity of the Wantai and in-house Mp1p EIAs were comparable in plasma (95.1% vs 92.4%, P = 0.11), in urine (91.5% vs 87.1% P = 0.07), and in combined testing of plasma and urine (96.4% vs 96.0%, P = 1.00), where talaromycosis was diagnosed based on the positivity of either specimen. The specificity of the Wantai and in-house Mp1p EIAs were consistently high in plasma, in urine, and in combined testing (93 – 97%). The Wantai and in-house Mp1p EIAs provided substantially higher sensitivity than blood culture detection (96.4% and 96.0% vs 78.6%, P < 0.001). For both EIAs, LR+ were greater than 10 and LR- were less than 0.1, which increases the confidence to rule in or rule out talaromycosis. Conclusions: The diagnostic performance of the Wantai Mp1p EIA was comparable to our validated in-house Mp1p EIA, and significantly more sensitive than blood culture, offering a standardized tool for the rapid diagnosis of talaromycosis.

Background: Polymerase chain reaction (PCR) is highly sensitive and specific for the rapid diagnosis of invasive fungal disease (IFD) but is not yet widely implemented due to concerns regarding limited standardisation between assays, the lack of commercial options and the absence of clear guidance on interpreting results. Objectives and Methods: This review provides an update on technical and clinical aspects of PCR for the diagnosis of the most pertinent fungal pathogens, including Aspergillus, Candida, Pneumocystis jirovecii, Mucorales spp., and endemic mycoses. Summary: Recent meta-analyses have demonstrated that quantitative PCR (qPCR) offers high sensitivity for diagnosing IFD, surpassing conventional microscopy, culture and most serological tests. The reported specificity of qPCR is likely underestimated due to comparison with imperfect reference standards with variable sensitivity. Although the very low limit of detection of qPCR can generate false positive results due to procedural contamination or patient colonisation (particularly in pulmonary specimens), the rates are comparable to those observed for biomarker testing. When interpreting qPCR results, it is essential to consider the pre-test probability, determined by the patient population, host factors, clinical presentation and risk factors. For patients with low to moderate pre-test probability, the use of sensitive molecular tests, often in conjunction with serological testing or biomarkers, can effectively exclude IFD when all tests return negative results, reducing the need for empirical antifungal therapy. Conversely, for patients with high pre-test probability and clinical features of IFD, qPCR testing on invasive specimens from the site of infection (such as tissue or bronchoalveolar lavage fluid) can confidently rule in the disease. The development of next-generation sequencing methods to detect fungal infection has the potential to enhance the diagnosis of IFD, but standardisation and optimisation are essential, with improved accessibility underpinning clinical utility.

Exposure to tropical plants and farmed animals and proximity to highland regions are linked to risk for infection.

Talaromycosis is an invasive fungal disease endemic to Southeast Asia. While culture is essential in identification, susceptibility testing, and typing, the low sensitivity and long turnaround times limit its clinical utility. Several promising monoclonal antibody–based (MAb) antigen-detection assays have been evaluated in large patient cohorts. This includes the MAb-Mp1p and MAb-4D1 enzyme immunoassays and their point-of-care platforms. Quantitative polymerase chain reaction (qPCR) assays targeting the 5.8S or 18S region of ribosomal RNA have been developed. These antigen and qPCR assays are highly specific and more sensitive than blood culture, making them excellent rapid rule-in tests for talaromycosis in people with a compatible clinical syndrome. Metagenomic next-generation sequencing is emerging as a promising tool for non-bias detection of talaromycosis. Host-based diagnostics targeting antibodies, interferon-gamma release, and transcriptomics are being actively developed. This review summarizes recent advances in the diagnosis of talaromycosis and provides expert recommendations on the application of these novel tests to improve the diagnostic algorithm and management of talaromycosis.

Talaromyces marneffei is a dimorphic fungus endemic in Southeast Asia that causes the invasive fungal disease talaromycosis in immunocompromised individuals. Detection of T. marneffei nucleic acid in blood by PCR has demonstrated potential as a diagnostic tool for talaromycosis, but previously developed assays have limited sensitivity. This study described the optimization of a quantitative PCR (qPCR) assay for the diagnosis of talaromycosis. Our assay performance was maximized by testing different primers, methods of cell lysis and DNA extraction, whole blood vs. plasma, and methods of specimen treatment, using mean quantification cycle (Cq) values to compare performance. Our qPCR assay achieved the highest analytical sensitivity of 1 yeast cell per mL of whole blood, using primers targeting the 5.8S ribosomal DNA, cell lysis by bead beating, and DNA extraction by the MasterPure Yeast Purification Kit. There was no cross-reactivity observed with six Penicillium species and nine clinically related fungal isolates. In a case-control, diagnostic validation study of 138 cases of talaromycosis and 30 controls with other invasive fungal diseases and opportunistic infections, our 5.8S qPCR assay detected T. marneffei in 99.0% (101/102, 95% CI: 94.6%–99.9%) of blood culture-positive and 55.6% (20/37, 95% CI: 38.1%–72%) of blood culture-negative patients. Overall, our 5.8S qPCR assay had significantly higher sensitivity compared to conventional BACTEC blood culture, 87.7% (95% CI: 80.7%–92.5%) vs. 73.9% (95% CI: 65.6%–80.8%, P < .001), and the specificity was 96.7% (95% CI: 80.9%–99.8%). Our 5.8S qPCR assay has potential as a non-invasive and rapid rule-in test for talaromycosis.