The rapid emergence of diseases and parasites in aquatic wildlife requires improved methodologies to identify and characterize new and future pathogens. While microinjection of pathogens directly into a model organism, such as zebrafish, enables the exploration of infection and immune response in the host, such methodology focuses primarily on identifying causative agents in events of aquatic wildlife mortality due to acute infection. Here, we present an updated protocol of infection by static immersion in larval zebrafish to investigate the possible effect of prolonged environmental exposure to an opportunistic pathogen. By controlling microbial growth and monitoring mortality over 72 h, we show that static immersion can detect minute differences in virulence profiles between and within different strains of Pseudomonas aeruginosa , an important opportunistic pathogen of animals and humans. We then conducted two sets of passive exposure virulence assays in Caenorhabditis elegans , an alternative model. We demonstrated the virulence phenotype, while showing slight differences between experimental models, and showed similar trends. We believe that passive exposure thus offers a practical host–pathogen model that simulates opportunistic infection occurring in the environment and enables the detection of minute changes in virulence between and within bacterial strains.
Introduction. Mycobacterium tuberculosis has held an exceptional presence in medical research, especially in recent years (1). While pulmonary infections largely remain the focus of literature and public health initiatives (2), extrapulmonary tuberculosis (EPTB) infections have been gaining prominence due to their increasing prevalence, particularly in populations characterized by a low incidence of tuberculosis (2–4). Although EPTB infections, such as tuberculous meningitis, carry a high mortality risk (5), delays in diagnosis and treatment persist. These delays are driven by their diverse, non-specific clinical presentations, coupled with the variable performance of diagnostic modalities like the Cartridge-Based Nucleic Acid Amplification Test (CBNAAT) (6–10). While general literature regarding the use and efficacy of CBNAAT in diagnosing EPTB exists (2,11–15), studies focusing on its sensitivity and specificity in paucibacillary specimens are an evolving niche (16). Since there is a greater prevalence of paucibacillary EPTB specimens in our setting, where such an analysis has not been conducted, we performed a retrospective study to understand the overarching diagnostic performance of our existing modalities in EPTB specimens. Methods. An observational retrospective study was conducted with the data collected between 2019 and 2022 at a tertiary care centre in central Kerala, India. A total of 105 specimens – categorized as pleural, lymph node, pus, tissue, cerebrospinal fluid (CSF), peritoneal and others – from presumptive EPTB cases were included in the study. The samples underwent acid-fast bacilli staining microscopy, liquid culture and CBNAAT utilizing the GeneXpert platform. The results were recorded, and the data were analysed with P< 0.05 considered statistically significant. Observations and Results. In the 105 samples collected for the study, 6 (5.7%) were positive on Ziehl–Nielsen staining, 26 (24.8%) samples grew M. tuberculosis complex on liquid culture and CBNAAT detected M. tuberculosis in 16 (15.2%) specimens. The sensitivity and specificity of CBNAAT, with culture as the gold standard, were found to be 50.0 and 96.2%, respectively. The sensitivity and specificity of CBNAAT for pus, peritoneal (including tissue and fluid aspirate), pleural fluids, CSF, lymph nodes and other miscellaneous sample types ranged from 33.3 to 100% and 85.7 to 100%, respectively. Among smear-negative specimens, the sensitivity and specificity of CBNAAT were 42.9 and 96.2%, respectively, whereas in smear-positive specimens, the sensitivity increased to 80.0%. Conclusion. CBNAAT is a viable tool for the detection of M. tuberculosis ; however, in paucibacillary specimens, its use along with other methods like smear and culture could lead to better identification of the mycobacteria.
Background. Cervical screening with high-precision assays such as human papillomavirus (HPV) DNA testing is essential for the detection and treatment of precancerous lesions. HPV genotypes have different oncogenic potential and require different clinical management, illustrating the importance of extended genotyping. HPV quantification has demonstrated clinical relevance in both diagnosis and treatment. Objective. To develop a droplet digital PCR assay for the detection and quantification of 16 HPV genotypes, with comparison to a commercial test and validation on clinical samples. Methods. Primers and probes were designed to target the E6 region of 16 HPV genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73 and 82. Each target was evaluated to assess performance and reliability using synthetic DNA constructs, quantified international reference standards and clinical screening samples (n=303) genotyped using the Seegene Anyplex II HR HPV Detection assay. Results. Each assay demonstrated high target specificity, without cross-reactivity observed among the HPV genotypes selected. Using international molecular standards, the assay reliably detected high-risk genotypes across serial dilutions, with detection down to the level of one international unit or genome equivalent per microlitre. When applied to clinical samples with and without a histological diagnosis of cervical intraepithelial neoplasia 2 or worse (CIN2+), the assay reliably detected key HPV genotypes, with the exception of 7 of 234 (3%) of HPV-positive samples, all of which exhibited very low viral load. Conclusion. Although previous studies have described digital PCR methods targeting HPV E6, they have typically focused on a limited number of genotypes. This study expands upon existing methodology by introducing a sensitive and specific method for detection and quantification of 16 high-risk and potentially high-risk HPV genotypes.
Introduction. Gram staining is a fundamental and widely used technique in clinical microbiology because of its simplicity and diagnostic value, particularly in the diagnosis of bacterial meningitis. However, its reliability may be compromised by contamination and staining artefacts. Case Presentation. This report describes a case of pseudobacterial meningitis in a previously healthy 25-year-old woman who presented with fever, headache and nuchal rigidity. Cerebrospinal fluid (CSF) analysis revealed pleocytosis with a predominance of mononuclear cells. Gram staining revealed findings consistent with Gram-negative bacilli, prompting empiric antibacterial therapy with concurrent antiviral treatment. Comprehensive microbiological investigations, including CSF culture and broad-range 16S rRNA PCR testing, yielded negative results. Residual material or microscopic debris on supposedly ‘clean’ slides was suspected to produce artefact-like appearances that mimicked micro-organisms, leading to false-positive interpretations. Conclusion. This case highlights a potential laboratory pitfall in the interpretation of CSF Gram stains and underscores the importance of considering staining artefacts, particularly when Gram stain findings are discordant with the overall clinical and laboratory findings.
Background. Syphilis remains a diagnostic challenge due to its wide range of clinical manifestations, particularly in immunocompromised patients. Secondary syphilis may present with atypical cutaneous and neurological features, complicating timely recognition and management. High clinical suspicion is essential in such scenarios. Case Report. We describe the case of a 62-year-old man with a 6-month history of palmoplantar keratoderma, nail dystrophy, bilateral hearing loss and symmetrical proximal myopathy of the lower limbs. A comprehensive diagnostic approach, including clinical, histopathological, serological and cerebrospinal fluid (CSF) analyses, was undertaken. Skin biopsy revealed vacuolar interface dermatitis with a superficial perivascular inflammatory infiltrate, and immunohistochemistry confirmed the presence of spirochetes. Serological tests were positive for Treponema pallidum , and newly diagnosed human immunodeficiency virus (HIV) coinfection was identified. Despite a negative CSF Venereal Disease Research Laboratory (VDRL), the presence of pleocytosis, elevated protein concentration and hypoglycorrhachia supported a diagnosis of probable neurosyphilis. The patient was treated with intravenous crystalline penicillin G and initiated on antiretroviral therapy, resulting in full resolution of cutaneous and neurological symptoms. Conclusion. This case underscores the polymorphic nature of syphilis in immunocompromised individuals, particularly those with HIV infection. Clinicians should maintain a high index of suspicion for neurosyphilis even when CSF VDRL results are negative, provided that clinical and laboratory findings are supportive. Early diagnosis and appropriate treatment are critical for favourable outcomes.
