Identification of microbial agents in culture-negative brain abscess samples by 16S/18S rRNA gene PCR and sequencing

@#Despite clinical suspicion of an infection, brain abscess samples are often culture-negative in routine microbiological testing. Direct PCR of such samples enables the identification of microbes that may be fastidious, non-viable, or unculturable. Brain abscess samples (n = 217) from neurosurgical patients were subjected to broad range 16S rRNA gene PCR and sequencing for bacteria. All these samples and seven formalin-fixed paraffin-embedded tissue (FFPE) samples were subjected to species-specific 18S rRNA PCR for neurotropic free-living amoeba that harbour pathogenic bacteria. The concordance between smear and/or culture and PCR was 69%. One-third of the samples were smear- and culture-negative for bacterial agents. However, 88% of these culture-negative samples showed the presence of bacterial 16S rRNA by PCR. Sanger sequencing of 27 selected samples showed anaerobic/fastidious gram negative bacteria (GNB, 38%), facultative Streptococci (35%), and aerobic GNB (27%). Targeted metagenomics sequencing of three samples showed multiple bacterial species, including anaerobic and non-culturable bacteria. One FFPE tissue revealed the presence of Acanthamoeba 18S rRNA. None of the frozen brain abscess samples tested was positive for 18S rRNA of Acanthamoeba or Balamuthia mandrillaris. The microbial 16/18S rRNA PCR and sequencing outperformed culture in detecting anaerobes, facultative Streptococci and FLA in brain abscess samples. Genetic analyses of 16S/18S sequences, either through Sanger or metagenomic sequencing, will be an essential diagnostic technology to be included for diagnosing culture-negative brain abscess samples. Characterizing the microbiome of culture-negative brain abscess samples by molecular methods could enable detection and/or treatment of the source of infection.

Central nervous system infections caused by pathogenic free-living amoebae: An Indian perspective

@#Pathogenic free-living amoebae (FLA), namely Acanthamoeba sp., Naegleria fowleri and Balamuthia mandrillaris are distributed worldwide. These neurotropic amoebae can cause fatal central nervous system (CNS) infections in humans. This review deals with the demographic characteristics, symptoms, diagnosis, and treatment outcomes of patients with CNS infections caused by FLA documented in India. There have been 42, 25, and 4 case reports of Acanthamoeba granulomatous amoebic encephalitis (GAE), N. fowleri primary amoebic meningoencephalitis (PAM), and B. mandrillaris meningoencephalitis (BAE), respectively. Overall, 17% of Acanthamoeba GAE patients and one of the four BAE patients had some form of immunosuppression, and more than half of the N. fowleri PAM cases had history of exposure to freshwater. Acanthamoeba GAE, PAM, and BAE were most commonly seen in males. Fever, headache, vomiting, seizures, and altered sensorium appear to be common symptoms in these patients. Some patients showed multiple lesions with edema, exudates or hydrocephalus in their brain CT/MRI. The cerebrospinal fluid (CSF) of these patients showed elevated protein and WBC levels. Direct microscopy of CSF was positive for amoebic trophozoites in 69% of Acanthamoeba GAE and 96% of PAM patients. One-fourth of the Acanthamoeba GAE and all the BAE patients were diagnosed only by histopathology following autopsy/biopsy samples. Twenty-one Acanthamoeba GAE survivors were treated with cotrimoxazole, rifampicin, and ketoconazole/amphotericin B, and all eleven PAM survivors were treated with amphotericin B alongside other drugs. A thorough search for these organisms in CNS samples is necessary to develop optimum treatment strategies.