- Volume 74, Issue 7, 2025

Introduction. Fungal infections are increasingly concerning, particularly in immunocompromised patients. These patients often suffer from comorbidities and receive multiple, non-antifungal medications.

Gap Statement. The effects of these co-administered medications on fungal cells – and their potential to influence antifungal drug efficacy – are poorly understood.

Aim. This study investigates non-antifungal medications commonly administered in parallel to antifungals and evaluates their impact on fungal susceptibility.

Methodology. We systematically reviewed clinical guidelines to identify non-antifungal medications frequently co-prescribed with antifungals. Focusing on Candida albicans, the most prevalent fungal pathogen, we examined whether the presence of these drugs influences antifungal responses of C. albicans. First, we tested the selected compounds together with antifungals in combination assays. Interactions were then characterized using checkerboard assays, and the impact on antifungal resistance and tolerance was evaluated through disc diffusion assays. To further explore these effects in vivo, the influence of selected antagonistic interactions on treatment efficacy was assessed using a Galleria mellonella model of disseminated candidiasis.

Results. From 119 medications used to manage 40 conditions linked to a high risk of fungal infections, we identified 34 compounds that altered the effectiveness of the antifungals fluconazole (FLC) and/or anidulafungin. Most of these compounds reduced or antagonized antifungal efficacy, often due to increased resistance or tolerance. Validation in a G. mellonella infection model confirmed that compounds antagonistic to FLC, including loperamide, estradiol and levothyroxine, interfere with antifungal treatment efficacy in this in vivo model.

Conclusion. Our findings highlight that medications frequently used by patients at risk for fungal infections can inadvertently increase fungal pathogen drug tolerance or resistance. We suggest that drugs targeting non-fungal conditions yet affecting fungal pathogens might represent an underestimated factor contributing to rising antifungal resistance and tolerance.

Introduction. Healthcare-associated infections (HAIs) significantly contribute to the burden of antimicrobial resistance. A major factor in HAIs is the colonization of indwelling medical devices by biofilm-forming opportunistic pathogens such as Candida albicans and Staphylococcus aureus. These organisms frequently co-infect, resulting in synergistic interactions with enhanced virulence and resistance to treatment.

Hypothesis/Gap statement. C. albicans and S. aureus readily form dual-species biofilms on silicone elastomers, a commonly used medical device material, yet the colonization phenotypes of these organisms on such surfaces remain poorly understood.

Aim. We aimed to develop a simple, optically tractable model to mimic the colonization of indwelling medical devices to investigate C. albicans and S. aureus biofilm formation.

Methodology. The system utilizes discs of a silicone elastomer embedded in agar, reflecting device-associated conditions and enabling high-resolution imaging of biofilms formed by C. albicans and S. aureus co-cultures.

Results. Initial results using the silicone elastomer colonization model reveal robust biofilm formation. These biofilms exhibited morphological differences between dual-species biofilms formed by S. aureus co-cultures with either yeast- or hyphal-form C. albicans, indicating the impact of differing C. albicans cell morphotypes in biofilm-associated medical device colonization on silicone elastomers. Quantification of biofilm formation by crystal violet staining provided further validation of the system.

Conclusion. These findings underscore the importance of developing tools for biofilm study which more closely resemble the infectious microenvironment, with our work detailing such a system which can be employed in further study to improve strategies against device-related HAIs.

Introduction. Pneumocystis jirovecii pneumonia (PJP, formerly known as Pneumocystis carinii pneumonia), an opportunistic fungal infection caused by the fungus P. jirovecii, is a severe pulmonary infection that primarily affects immunocompromised patients, including those with lung cancer. Traditional diagnostic methods for PJP, such as Grocott–Gomori’s methenamine silver staining and real-time PCR, have limitations, including low positivity and high missed diagnosis rates.

Gap Statement. Despite the critical need for accurate and sensitive diagnostic tools for PJP, especially in immunocompromised populations, existing methods fall short in providing the necessary reliability and efficiency.

Aim. This study aims to evaluate the efficacy of nanopore-based metagenomic third-generation sequencing in diagnosing P. jirovecii infection in lung cancer patients, hypothesizing that this approach may offer superior sensitivity and specificity.

Methodology. A prospective observational study was conducted on 118 lung cancer patients with suspected pulmonary P. jirovecii infection at the Sixth Hospital of Nantong City, China, from January 2021 to December 2023. The identification of pathogens in bronchoalveolar lavage fluid samples was performed using both metagenomics and traditional tests.

Results. Metagenomics showed a significantly higher detection rate of P. jirovecii (33.0%) compared to methenamine silver staining (4.2%) and real-time PCR (30.5%). The sensitivity, specificity and accuracy of metagenomics detection were all 100%, which is markedly superior to traditional methods. Furthermore, metagenomics also identified mixed infections with other pathogens, such as Cytomegalovirus and Epstein–Barr virus.

Conclusion. Metagenomics technology demonstrates high sensitivity and specificity in diagnosing P. jirovecii infection, including mixed infections with other pathogens, in lung cancer patients. It provides a clear direction for clinical treatment and is a powerful tool for diagnosing PJP, contributing to improved diagnostic efficiency and accuracy, reducing misdiagnosis and missed diagnosis rates and improving clinical outcomes in these patients.

Introduction. Mycoplasma genitalium, a small and slow-growing bacterium, has gained notoriety due to rapidly increasing rates of antibiotic resistance.

Gap Statement. M. genitalium is difficult to culture, limiting efforts to understand its biology and the mechanisms of antimicrobial resistance.

Aim. To understand factors that influence success in primary isolation of M. genitalium from clinical samples.

Methodology. Neat urine or swabs (high vaginal and anal, in universal transport medium) were collected from patients with confirmed or suspected M. genitalium infections attending the Melbourne Sexual Health Centre. The specimens were stored at −80 °C prior to laboratory analysis. Initial diagnosis was by transcription-mediated amplification (TMA) assay, and samples subsequently testing positive by quantitative PCR (qPCR) were washed twice and inoculated into Vero cell monolayers with a selective antibiotic mixture (cycloheximide and Thayer-Martin Medium I). Cultures were incubated at 37 °C with 5% CO2 for 8 weeks and observed daily, with qPCR used to monitor growth.

Results. In total, 127 TMA-positive samples were subjected to qPCR, and M. genitalium genomic DNA (gDNA) was detected in 53.5% (68/127) of these samples. An isolate was obtained from 26.5% (18/68) of the gDNA-positive samples following co-culture with Vero cells. The isolation rate varied between sample types, with growth detected in 12.5% (3/24) of the high vaginal swabs and 37.5% (15/40) of the urine samples. No isolates were obtained from anal swabs. The proportion with a successful culture was influenced by the initial M. genitalium load in the sample, which translated into the inoculum size for the Vero cell monolayer. Isolation was unsuccessful with low inoculum (<2,000 genome equivalents, geq), partially successful (13.3%) with a moderate inoculum (2,500–9,500 geq) and highly successful (100%) in samples with a high inoculum (>10,000 geq).

Conclusion. The initial bacterial load emerged as a critical determinant of isolation success. This emphasizes the importance of optimizing sample collection and M. genitalium isolation procedures.

Introduction. The microbiota, which has a major impact on both health and illness, has recently become one of the most popular research topics.

Hypothesis/Gap statement. To the best of our knowledge, no research has undertaken a bibliometric analysis of publications examining the connection between microbiome and urological cancer to date. In this respect, it is thought that our study will contribute to the literature.

Aim. The purpose of this study is to raise awareness of the topic by performing a bibliometric analysis of the publications examining the connection between the microbiota and the most common urological cancers, including bladder, prostate, and kidney cancers.

Methodology. All publications about prostate, renal and bladder cancers and microbiota indexed in Web of Science between 2000 and 2024 were included in the study.

Results. A total of 310 publications were obtained. Before 2018, there were only three or fewer publications annually; however, following 2018, the number of publications increased rapidly, reaching a peak of 77 in 2024. The USA led with 98 (31.61%) documents, followed by China (60, 19.35%) and Italy (31, 10%). With 19 publications, Hirotsugu Uemura is the most contributing author, followed by Norio Nonomura with 17. Prostate cancer accounted for 45.48% of the publications, bladder cancer for 36.77% and kidney malignancies for 17.64%.

Conclusion. Despite the fact that microbiota has been known for 80 years, research on the connection between microbiota and cancer accelerated after the completion of the Human Microbiome Project. The number of studies examining the connection between urological cancer and microbiota peaked in 2024 and is probably going to rise. More research is required on this topic, since the correlation between microbiota and especially prostate and bladder malignancies raises the possibility that variations in microbiota may be utilized in diagnosis, treatment and prognosis.

Introduction. Sexually transmitted infections (STIs) are a worldwide health issue with a high number of asymptomatic cases and the possibility of multiple infections.

Gap statement. New multiplex real-time PCR kits targeting pathogens involved in nonviral STIs are regularly launched, but only some of them have been evaluated in comparative studies.

Aim. This study evaluated the clinical performance of two multiplex real-time PCR commercial kits for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis: the Bosphore STD Urethritis Mini Bundle Kit (BK; Anatolia Geneworks) and the Viasure Sexually Transmitted Disease Real-Time PCR Detection Kit (VK; CerTest BIOTEC).

Methodology. A total of 240 clinical specimens were evaluated. The results were compared with those of the Cobas CT/NG and TV/MG kits (Roche Diagnostics), used as reference methods.

Results. Positive agreement ranged between 83.3% and 87.8% for the detection of C. trachomatis, N. gonorrhoeae and T. vaginalis using validated specimen types. For M. genitalium detection, positive agreement was 83.0% for the BK and 68.1% for the VK, which missed 31.9% of M. genitalium-positive specimens. Negative agreement ranged between 98.4% and 100% across the targeted micro-organisms. Both kits were easy to use and compatible with several DNA extraction and PCR thermal cyclers. The VK also detected the genital commensal bacteria Ureaplasma spp. and Mycoplasma hominis, which should not be targeted in STI detection kits.

Conclusion. Both kits are convenient methods and showed good performance for the detection of nonviral STIs, but users should be aware of a lower sensitivity of the VK for the detection of M. genitalium.

Introduction. This study underscores the critical role of identifying heteroresistant infections of Mycobacterium tuberculosis (Mtb) in enhancing the diagnostics of tuberculosis (TB). These conditions complicate diagnostics and treatment, underlining the need for advanced techniques to detect and characterize resistant populations effectively.

Hypothesis/Gap statement. Current diagnostics may fail to identify heteroresistance and mixed infections, limiting the understanding of their impact on treatment outcomes.

Aim. This pilot study aimed to phenotypically and genotypically characterize rifampicin-heteroresistant clinical isolates and assess their genetic diversity and resistance patterns.

Methodology. A retrospective analysis of 2,917 Mtb genomes from Peru (1999–2020) was conducted using MTBseq and TB-Profiler. Techniques included indirect microscopic observation drug susceptibility, MIC determination via tetrazolium microplate assay, agar proportion method and sequencing. From each clinical isolate, three colonies were isolated from both rifampicin-supplemented (1 µg mL−1) and drug-free media for subsequent phenotypic and genotypic characterization, including rpoB sequencing.

Results. Of the 2,917 genomes analysed, 14.6% were classified as mixed infections, 3.8% exhibited heteroresistance to at least 1 drug between 21 antibiotics analysed and 0.79% were rifampicin-heteroresistant. Colonies from rifampicin-supplemented media displayed high resistance (MIC >1 µg mL−1) with mutations such as S450L in the RpoB protein. In contrast, those from drug-free media exhibited sensitivity to rifampicin (MIC <1 µg ml−1), harbouring other RpoB mutations including D435Y, L452P and L430P. Notably, some colonies retained WT RpoB sequences, suggesting a diversity of subpopulations within isolates.

Conclusion. Whole-genome sequencing and phenotypic analysis confirmed the coexistence of rifampicin-susceptible and rifampicin-resistant Mtb populations within single clinical isolates. Subculturing in drug-free media favoured the selection of sensitive strains, emphasizing the critical need for advanced diagnostic tools to accurately detect and characterize heteroresistant and mixed infections. These findings pave the way for more targeted treatment strategies to combat antimicrobial resistance in TB.