INTEGRATION OF MOLECULAR M ICROBIOLOGY AND HISTOPATHOLOGY FOR THE DIAGNOSIS OF CULTURE NEGATIVE INFECTIONS: CLINICAL UTILITY AND DIAGNOSTIC LIMITATIONS—A SYSTEMATIC REVIEW

Abstract

Background: Culture-negative infections remain a major diagnostic challenge in clinical microbiology and histopathology. Prior antimicrobial administration, fastidious organisms, low microbial burden, biofilm formation, and inadequate specimen collection can limit conventional microbiological recovery. Molecular techniques, including broad-range 16S ribosomal RNA gene polymerase chain reaction (PCR), fungal 18S ribosomal RNA/internal transcribed spacer (ITS) sequencing, pathogen-specific PCR, and metagenomic next generation sequencing (mNGS), offer additional approaches to pathogen identification. Histopathology provides complementary evidence by demonstrating tissue inflammation, necrosis, microorganisms, and invasive disease. Objective: To examine the complementary diagnostic contributions, clinical utility, and limitations of integrating molecular microbiology with histopathological examination in suspected culture-negative infections. Methods: A structured literature assessment was undertaken using accessible PubMed/MEDLINE records, journal publications, and supplementary citation identification for studies available through October 21, 2026. Nineteen original clinical investigations involving infective endocarditis, osteoarticular infection, spinal infection, and invasive fungal disease were mapped into a qualitative evidence synthesis. Diagnostic yield, sensitivity, specificity, incremental etiological identification, treatment implications, and methodological limitations were examined. The review framework follows PRISMA 2020 and diagnostic test accuracy reporting principles; formal exhaustive screening and independent risk-of-bias adjudication remain outstanding. Results: Across the examined studies, molecular assays frequently detected additional organisms in culture negative specimens, particularly following antimicrobial exposure. In a 266-patient infective endocarditis investigation, valve-tissue 16S PCR identified microorganisms in 36% of blood-culture-negative cases. Another valve study demonstrated a diagnostic benefit in 75% of blood-culture-negative episodes. A prospective periprosthetic joint infection study reported sensitivities of 63.2% for mNGS, 68.4% for culture, and 52.6% for histopathology, indicating that molecular testing did not invariably outperform conventional investigations. In spinal infections, molecular sequencing generally increased organism-detection rates, although findings varied with clinical selection and diagnostic reference standards. In invasive fungal disease, histopathology-directed molecular testing improved organism identification, whereas testing histologically negative formalin-fixed tissue frequently produced limited clinically significant yield. Contamination, nonviable microbial DNA, specimen processing, variable assay sensitivity, and the absence of susceptibility data were recurring limitations. Conclusion: Molecular microbiology and histopathology provide complementary rather than interchangeable diagnostic information. An integrated, specimen-directed approach can improve etiological recognition and diagnostic confidence in selected culture-negative infections. Reliable implementation requires rigorous contamination control, appropriate specimen allocation, validated molecular assays, clinical correlation, and multidisciplinary interpretation.