BACKGROUND:¹³C-urea breath test (¹³C-UBT) is widely used for Helicobacter pylori (H.pylori) detection but has limitations including inability to distinguish viable from nonviable bacteria, an ambiguous diagnostic gray zone (DOB 4-10), and false-negative results in low bacterial load infections. OBJECTIVES:To compare the diagnostic value of fluorescence rapid on-site evaluation (F-ROSE) and ¹³C-UBT for H. pylori infection, determine the critical viable bacterial count threshold for positive ¹³C-UBT, clarify the real infection status in the ¹³C-UBT gray zone, and reveal the quantitative relationship between DOB values and viable bacterial counts. METHODS:A total of 180 patients undergoing gastroscopy were consecutively enrolled and received synchronous F-ROSE quantitative detection and ¹³C-UBT. F-ROSE used acridine orange-ethidium bromide (AO-EB) dual fluorescent staining to count total, viable, and nonviable bacteria under × 400 high-power field (HPF). ¹³C-UBT results were determined by DOB values. Latent class analysis (LCA) combined with Bayesian modeling was used to infer true infection status. Receiver operating characteristic (ROC) curves were applied to define the critical viable bacterial count for positive ¹³C-UBT and evaluate diagnostic efficacy. RESULTS:Model inference identified 94 true-positive and 86 true-negative cases among 180 patients. The positive rate of F-ROSE was 51.1%, significantly higher than 47.2% of ¹³C-UBT (P < 0.05). Using true infection as reference: F-ROSE showed sensitivity of 93.6%, specificity of 94.2%, and AUC of 0.954; ¹³C-UBT showed sensitivity of 80.9%, specificity of 93.0%, and AUC of 0.878. ROC analysis indicated that a viable bacterial count≥10/HPF was the stable positive threshold for ¹³C-UBT. When viable bacterial count was <10/HPF, the positive rate of ¹³C-UBT was only approximately 20%, with most cases falling into the gray zone or false-negative. There were 32 cases in the ¹³C-UBT gray zone, among which 24 were positive by F-ROSE (positive rate 75.0%), all representing true infections with low viable bacterial load (4-9/HPF). The ¹³C-UBT DOB value was significantly positively correlated with F-ROSE viable bacterial count (r = 0.746, P < 0.001). CONCLUSIONS:¹³C-UBT positivity depends on a viable bacterial count threshold ≥10/HPF. Infections with low viable bacterial count are prone to be classified into the gray zone or missed. The clinical gray zone mostly represents true viable bacterial infection, and the traditional strategy of "observation first" is biased. F-ROSE enables real-time visual identification of viable bacteria and accurate quantification, breaking through the threshold limitation of ¹³C-UBT, making it an optimal technique for accurate diagnosis of H. pylori infections with low bacterial load and in the clinical gray zone.
Cryptococcal meningitis during pregnancy is rare and associated with maternal and fetal morbidity and mortality. Cryptococcus gattii causes disease in individuals without recognized immunosuppression and is associated with severe neurological manifestations. A 27-year-old apparently immunocompetent woman at 11 weeks of gestation, with occupational exposure in animal science and ecotourism, presented with severe headache and progressive neurological deterioration following mild SARS-CoV-2 infection. Initial cerebrospinal fluid (CSF) analysis showed nonspecific lymphomononuclear meningitis, and treatment for herpetic encephalitis was initiated. After 15 days of deterioration, repeat CSF examination revealed encapsulated yeasts, and culture confirmed C. gattii. Amphotericin B deoxycholate and fluconazole were initiated because first-line therapy was unavailable. Despite intensive care, she developed refractory intracranial hypertension associated with cerebral cryptococcoma, fetal demise at 17 weeks, and brain death. This case highlights diagnostic and therapeutic challenges in cryptococcal central nervous system infection during pregnancy and the importance of considering C. gattii in patients without recognized immunosuppression.
OBJECTIVE:To analyze the clinical characteristics, diagnostic challenges, treatment strategies, and prognosis of patients co-infected with human immunodeficiency virus (HIV) and disseminated Talaromyces marneffei (TM), complicated by Hemophagocytic Lymphohistiocytosis (HLH), with the aim of improving early recognition and comprehensive diagnosis and treatment. METHODS:Three adult inpatients diagnosed with acquired immunodeficiency syndrome (AIDS), TM infection, and HLH between January 2020 and December 2025 at the Fourth People's Hospital of Nanning were enrolled in this study. Demographic characteristics, clinical symptoms, laboratory findings, imaging results, and treatment outcomes were collected from the hospital's electronic medical record system. RESULTS:All three patients (2 males, 1 female) presented with multi-system involvement. Common symptoms included fever, cough, and anemia. All cases met five core diagnostic criteria for HLH. Laboratory tests indicated pancytopenia, elevated inflammatory markers, and multi-organ dysfunction. Chest computed tomography (CT) scans revealed pulmonary infiltrates, lymphadenopathy, and serous cavity effusions in all patients. Treatment consisted of antifungal therapy with amphotericin B combined with itraconazole, and all patients received corticosteroids for HLH management. Two patients improved and were discharged, while one died due to poor baseline condition and multi-organ failure. Hospitalization duration ranged from 23 to 144 days, with no significant adverse drug reactions observed during treatment. CONCLUSION:The clinical manifestations of HIV co-infected with TM and HLH may be atypical. Diagnosis should focus on key indicators such as fever, cytopenia, and hyperferritinemia, with prompt bone marrow examination. Effective treatment requires potent antifungal agents, tailored immunosuppressive therapy, and enhanced organ support.
BACKGROUND:Ureaplasma urealyticum (U. urealyticum, UU) infection in the genital tract can induce severe complications of adverse pregnancy outcomes and infertility. Therefore, the current study aimed to apply and evaluate a rapid SAT method for detecting U. urealyticum. METHODS:This study enrolled 2,728 outpatients with genitourinary complaints. The inclusion criteria were: (1) presence of genitourinary symptoms, and (2) no antibiotic therapy within the 72 h preceding sample collection. Firstly, U. urealyticum identified by culture underwent broth microdilution antimicrobial susceptibility testing. Secondly, a total of 305 UU-positive and 301 UU-negative controls were randomly selected for assessing the sensitivity and specificity of the SAT method. Finally, we compared the sensitivity and specificity of the SAT method with those of real-time fluorescence qPCR. RESULTS:Of the 2,728 samples tested, 1,158 (42.45%) were positive for U. urealyticum by culture assay. The resistance rates against MIN, DOX, JOS, CLA, ROX, AZI, LEV, OFL, and SPA were 1.38%, 2.07%, 5.44%, 12.18%, 17.74%, 17.70%, 16.23%, 27.03%. and 21.59%, respectively. The sensitivity and specificity of the SAT method were 96.39% and 99.00%, respectively. Compared to the real-time fluorescence qPCR, no statistically significant difference was observed in either sensitivity (96.39% vs 96.72%; χ² = 0.0493, p = 0.8243) or specificity (99.00% vs 99.67%; χ² = 0.2517, p = 0.6159). CONCLUSIONS:Although SAT is not superior to qPCR in sensitivity or specificity, it offers comparable performance, along with rapid detection and potential cost-effectiveness. Therefore, SAT represents a promising diagnostic alternative, particularly for rapid detection of U. urealyticum infection.
Recovery of multiple microorganisms from respiratory specimens complicates pathogen attribution in patients with chronic lung disease. A 71-year-old woman with COPD and bronchiectasis presented with severe pneumonia and respiratory failure. She had T-cell lymphopenia (CD3+, CD4+, and CD8+ counts: 189, 147, and 41 cells/μL) and negative HIV testing. BALF Gram staining showed branching Gram-positive rods suspicious for Nocardia spp., and cultures yielded a Nocardia isolate, Aspergillus fumigatus, and Candida albicans. The isolate was presumptively identified as N. terpenica by MALDI-TOF MS (score, 9.534); molecular confirmation and susceptibility testing were unavailable. BALF galactomannan (index, 3.528) supported probable pulmonary aspergillosis. Influenza A virus RNA was detected by qualitative PCR, while C. albicans was considered airway colonization. The patient improved during hospitalization. This case highlights integrated pathogen attribution in complex respiratory microbiology.
OBJECTIVES:The molecular detection of toxigenic Clostridioides difficile (C. difficile) infections mostly relies on the use of unpreserved stool specimens. However, recently the feasibility of sampling systems for stool pathogen detection comprising flocked swabs and preservation media has been highlighted. Hence, we evaluated the Copan FecalSwab stool sampling systems for the detection of toxigenic C. difficile by the Cepheid Xpert C. difficile BT PCR assay. METHODS:Unpreserved stool samples from patients with suspected toxigenic C. difficile infections were tested for C. difficile using the Xpert C. diff BT PCR assay on a Cepheid GeneXpert IV device (Cepheid, Sunnyvale, CA, USA). In parallel, from this stool sample a flocked swab sample was taken and transferred into a FecalSwab preservation tube. Subsequently, 400 µL of the preservation media was utilized for performing the Xpert C. difficile BT PCR. From the same FecalSwab sample an immunological rapid test for the detection of C. difficile (C. DIFF QUIK CHEK COMPLETE, Techlab, Blacksburg, VA, USA) was performed, representing the current analytical standard in our hospital. The qualitative results of all tests and the cycle threshold (Ct) values from the PCR runs were compared. RESULTS:Fortytwo unpreserved stool samples were included, of whom 16 were positive for toxigenic C. difficile. Fifteen were also detected positive using the FecalSwabs. Hence, the qualitative agreement was almost perfect (Cohen's kappa 0.949, p < 0.001). The Ct values compared between the sampling materials correlated well (Spearman's Rho 0.893, p < 0.001). However, in comparison the median Ct value obtained from the unpreserved stool samples was lower (23.6 versus 25.6). A median Ct value difference of 1.3 was observed. CONCLUSIONS:The FecalSwab is suitable for preanalytical stool collection for the detection of toxigenic C. difficile with the Xpert C. difficile PCR test. However, to reach optimal sensitivity the preanalytical processing may need further investigations.
Lipoarabinomannan (LAM), recognized as a characteristic antigen within the cell wall of Mycobacterium tuberculosis (MTB), can be detected in the urine of patients suffering from active tuberculosis, which provides an important biomarker for non-invasive diagnosis of tuberculosis. This paper systematically reviews the structure, immunomodulatory function and application value of LAM in the diagnosis of tuberculosis, focusing on the progress and performance comparison of various detection methods from traditional ELISA to emerging quantum dot microsphere technology. Studies have indicated that LAM test has high sensitivity and specificity in HIV-positive patients, especially in CD4 low patients, but the diagnostic efficiency in HIV-negative people still needs to be improved. Nanotechnology such as quantum dots has significantly improved the detection sensitivity (up to fg/mL) through signal amplification and multimodal output, and has shown good potential for point-of-care application. In the future, the development of LAM detection technology should focus on platform integration and intelligence, joint detection of multiple markers, population stratification optimization and standardization system construction. Together, these efforts will make it an important diagnostic tool in the global TB system.
Distinguishing between histoplasmosis and sarcoidosis can be challenging as both can present with non-specific clinical findings. We present a 73-year-old immunocompetent Caucasian male who was recently diagnosed with sarcoidosis via left upper lobe biopsy who presented with lethargy and poor oral intake. The patient was found to have fever, splenomegaly, pancytopenia, hypofibrinogenemia, and elevated inflammatory markers. After bone marrow biopsy, he was diagnosed with disseminated histoplasmosis and secondary hemophagocytic lymphohistiocytosis (HLH). The patient was treated with liposomal amphotericin B and a dexamethasone taper followed by itraconazole for 1 year. After the patient was found to have disseminated histoplasmosis, his original diagnosis of sarcoidosis was reconsidered. Disseminated histoplasmosis is a rare cause of secondary HLH in an immunocompetent adult. This case highlights the difficulties distinguishing between histoplasmosis and other causes of granulomatous disease and identifies histoplasmosis as a rare cause of HLH.
Objective The spread of carbapenemase-producing Enterobacteriaceae (CPE) has become a global concern. Therefore, reliable methods for rapid carbapenemase detection are needed. Molecular methods are faster and offer higher sensitivity and specificity than phenotypic methods. However, conventional PCR assays often target partial sequences, which may miss variants or subtypes. This study aimed to develop a new full-sequence gene fluorescent quantitative PCR system for the detection of blaKPC and blaNDM genes in CPE. Methods We first designed full-sequence amplification primers targeting the blaKPC and blaNDM genes and established a full-sequence fluorescence quantitative PCR system. The specificity, sensitivity, and limit of detection of the system were subsequently evaluated. Finally, the performance of the system was assessed using 94 clinical carbapenem-resistant Enterobacterales (CRE) isolates (including 69 Klebsiella pneumoniae and 17 Escherichia coli). Results The full-sequence fluorescence quantitative PCR system specifically amplified the blaKPC and blaNDM genes, with a limit of detection of 10²CFU/mL for both genes. The system exhibited strong robustness against interference. The sensitivity was 100.00% for both genes; the specificity was 91.30% for blaKPC and 98.41% for blaNDM. The Kappa coefficients for blaKPC and blaNDM were 0.9147 and 0.9761, respectively. The system was also capable of detecting strains harboring the blaKPC-33 and blaKPC-71 variants. Conclusions We developed a novel full-sequence fluorescence quantitative PCR detection system for the blaKPC and blaNDM genes, which are the carbapenemase genes with the highest detection rates in clinical settings. This detection method demonstrates high sensitivity, high specificity, and the ability to detect mutant genes.
Accurate species-level identification within the genus Serratia remains challenging due to extensive phenotypic overlap and high genomic relatedness among closely related and recently described taxa. This study presents an evaluation of routine and genome-based identification approaches applied to clinical Serratia isolates, integrating phenotypic assays, MALDI-TOF MS (Bruker Daltonics), 16S rRNA gene sequencing, and Whole-Genome Sequencing (WGS). A total of 103 isolates collected from a teaching hospital were analyzed. WGS was performed on a subset of isolates. Conventional biochemical methods classified all isolates as Serratia marcescens, whereas MALDI-TOF MS identified 60.1% as S. marcescens, 11.6% as S. ureilytica, and 28.1% just at the genus level. Peak analysis from MALDI-TOF MS revealed specific peaks associated with S. marcescens and S. ureilytica, but limited discriminatory power. WGS of six isolates initially identified as S. ureilytica by MALDI-TOF MS revealed reclassification as Serratia sarumanii (n = 5) and Serratia montpellierensis (n = 1), supported by Average Nucleotide Identity (ANI), Average Amino Acid Identity (AAI), and Digital DNA-DNA Hybridization (dDDH) thresholds. In contrast, 16S rRNA analysis showed limited species-level resolution. Phylogenomic and SNP-based analyses confirmed these classifications with strong support. Overall, this study underscores the critical role of high-resolution genomic approaches for precise species identification and highlights the need for continuous expansion and curation of MALDI-TOF MS reference databases to support reliable clinical diagnostics and epidemiological surveillance of emerging Serratia species.
Blastocystis is a ubiquitous intestinal microeukaryote with controversial pathogenic potential. Epidemiological data on Blastocystis in patients with gastrointestinal neoplasms remain limited, especially in tropical regions of China. This study aimed to determine the prevalence and molecular subtype distribution of Blastocystis in hospitalized patients with gastrointestinal cancer or polyps in Hainan Province. A descriptive cross-sectional study was conducted at a tertiary hospital in Hainan. A total of 509 consecutive patients were enrolled, including 357 with pathologically confirmed gastrointestinal cancer and 152 with gastrointestinal polyps. Blastocystis was detected by nested PCR targeting the SSU rRNA gene, and positive amplicons were sequenced for subtype identification. The overall detection rate of Blastocystis was 2.2% (11/509) in gastrointestinal tumor patients, with 1.7% (6/357) and 3.3% (5/152) in gastrointestinal cancer and polyp patients, respectively. Three subtypes (STs) were identified: ST3 (n = 7, 63.6%), ST1 (n = 2, 18.2%), and ST7 (n = 2, 18.2%). ST1 and ST3 are known to circulate widely in human populations, while ST7 is primarily an avian-adapted subtype with occasional zoonotic potential. An exploratory analysis suggested a possible age-dependent colonization pattern in polyp patients (uncorrected P = 0.003), but this finding requires validation in larger cohorts due to the small sample size. This study provides baseline data on Blastocystis subtype distribution in gastrointestinal disease patients in Hainan. Further well-designed studies with appropriate control groups and rigorous control for confounding factors are required to clarify the clinical significance of Blastocystis colonization in this patient population.
INTRODUCTION:Cervical cancer is a health issue in the Amazon region. Human papillomavirus (HPV) is the primary cause of cervical cancer. Oxidative stress has been shown to play a role in the HPV-associated carcinogenesis process. OBJECTIVES:To evaluate the prevalence of HPV infection and its correlation with oxidative stress biomarkers in women from Belém, Pará. METHODS:This was a cross-sectional pilot study involving 71 women conducted between March 2021 and March 2022. Endocervical samples were analyzed for HPV DNA using PCR, and the oxidative stress biomarkers malondialdehyde (MDA) and glutathione (GSH) were measured in blood samples. Statistical analyses were performed using SPSS software, version 21.0. RESULTS:The prevalence of HPV was 28.2% (20/71). Participants aged 39 to 48 years showed significantly higher levels of MDA (≥3.54 nmol/mL), with 34% exhibiting elevated levels (p-value = 0.036). HPV-positive women tended to have lower levels of GSH (p-value = 0.090). DISCUSSION:There is likely an imbalance between oxidative damage and antioxidant defense mechanisms in HPV pathogenesis; furthermore, low levels of the antioxidant GSH indicate that the studied population may be exposed to the harmful effects of reactive oxygen species (ROS). Oxidative stress may play a role in HPV persistence and cervical carcinogenesis. CONCLUSION:The high prevalence of sexually transmitted HPV infection and the presence of oxidative stress among women in the Brazilian Amazon may indicate a limited impact of cervical cancer prevention measures. Oxidative stress biomarkers (MDA and GSH) could potentially serve as low-cost diagnostic options, offering an alternative for resource-poor areas in the Brazilian Amazon.
Objectives Interleukin-32 (IL-32) is a pro-inflammatory cytokine implicated in the pathogenesis of multiple inflammatory diseases, but its expression pattern and clinical significance in syphilis have not been fully elucidated. This study aimed to determine serum IL-32 levels in different stages of syphilis and evaluate its potential value in clinical staging and diagnosis. Methods A total of 166 patients with syphilis (secondary syphilis, late latent syphilis, sero-resistant syphilis, and neurosyphilis) and 30 healthy controls were enrolled. Serum IL-32 concentrations were detected using enzyme-linked immunosorbent assay (ELISA). Clinical and laboratory parameters were measured by standardized protocols, and correlation analysis was performed. Results Serum IL-32 levels were significantly lower in secondary syphilis patients than in healthy controls (34.83 vs. 75.93 pg/mL, p < 0.0001) and late latent syphilis patients (34.83 vs. 156.55 pg/mL, p < 0.0001). IL-32 was also decreased in neurosyphilis compared with controls (p = 0.0277), while no significant difference was observed in sero-resistant patients (p > 0.05). In secondary syphilis, IL-32 was negatively correlated with A:G ratio and apolipoprotein A (APOA), and positively correlated with total cholesterol (CHOL) and non-high-density lipoprotein cholesterol (non-HDL-C). Conclusions Serum IL-32 is dynamically altered in syphilis and exhibits stage-specific changes, especially in secondary syphilis. Its correlations with lipid-related parameters suggest a possible association between host inflammatory responses and metabolic disturbance during Treponema pallidum infection. These findings suggest that IL-32 may provide exploratory adjunctive information for stage-related immune assessment in syphilis, but independent validation is required before clinical application.
