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.

Disseminated crytococcosis with extensive cutaneous involvement in AIDS.

Cutaneous infections is observed in 15% of patients with disseminated cryptococcosis with AIDS. We present here a case of a 34 years old female with AIDS. She presented with multiple skin coloured umbilicated over face, neck, trunk and limbs, which mimicked molluscum contagiosum and kaposi sarcoma. The tissue from cutaneous lesions was collected by excision biopsy and processed by standard mycological methods. Cryptococcus neoformans was isolated and identified. Cerebrospinal fluid (CSF) also yielded the growth of C. neoformans . Cryptococcal antigen was detected with a titre of 1024 by Latex agglutination, is serum and CSF. Her serum was reactive for HIVI and 2 antibodies. The CD4 lymphocytes count was 80/cmm. The HIV viral load was 2,48,084 copies/mL. She was treated with amphotericin B injectable and oral fluconazole. She responded well and lesions regressed.

Preceding infection as a risk factor of stroke in the young.

INTRODUCTION: The cause of stroke in the young remains unknown in 20-50% of the patients. Infections preceding stroke have been recently recognised to be an independent risk factor of stroke. MATERIAL AND METHODS: Sixty consecutive patients aged 40 years or less presenting with ischaemic completed stroke are taken up for the study. Patients with neurological deficit of less than 24 hours, evidence of haemorrhage on CT scan, infection occurring after the onset of stroke were excluded. Controls consisted of age and sex matched persons residing in the same area. Both the groups were enquired about preceding fever and infections and were examined for evidence of infections. Serum was examined for antibodies against measles, herpes simplex, and Japanese B encephalitis viruses. Cultures were put up from appropriate samples and CSF examined in patients only. RESULTS: Evidence of infection was noted in 26 (43.3%) of patients and 6 controls (p < 0.001). History of fever was elicited in 23 patients and 3 controls while 15 patients were febrile on examination at admission. Signs of local infection was observed in 14 patients and one control. The commonest site of infection was respiratory tract. Cultures were positive in 11 patients, commonest being beta haemolytic streptococci in six from throat. Conventional risk factors were identical in both groups of patients with and without evidence of preceding infection. Smoking and alcoholism were significantly higher in patients with preceding infection. CONCLUSION: Preceding infection is an important risk factor of stroke in the young. Smoking and alcoholism are more frequent in patients with preceding infection. Whether they predispose the individual for infection or infection increases the stroke risk in them needs to be examined.

Histological and immunological correlates of suspected leprosy lesions.

Thirty-two subjects with suspected leprosy lesions were investigated to assess various modalities of sensibility and sweat function and these were correlated with immunological and histological parameters. It was found that pain and temperature, mediated by small unmyelinated fibres were impaired in the early lesions. Impairment of sweat function was seen only when one of the modalities of sensibility was also affected. Antibodies specific to a protein (35 kDa) antigen and phenolic glycolipid 1 of Mycobacterium leprae were positive in nine and 12 cases respectively, while 15 of the 31 biopsies revealed the presence of mycobacterial antigens in these lesions. The implications of these findings are discussed.

Studies on serum proteins in leprosy by polyacrylamide gel electrophoresis (page)--I.

Serum protein pattern was studied in the leprosy spectrum, their contacts and in normal individuals by employing polyacrylamide gel electrophoresis. Sera from 80% of untreated BL/LL, 70% of untreated TT/BT patients and 67% of contacts have shown dysproteinaemia either for 232 kD or for 175 kD or for both these proteins together. Tendency of these proteins to return to normal levels was observed after treatment. But both these proteins come back to normal levels only after subsidence of the disease.

Effect of leprosy sera and foetal calf serum (FCS) on the T cell number of peripheral blood of leprosy patients.

Study was conducted in 24 cases of various types of leprosy and 10 healthy controls to find out the effect of various sera on the T cell count of peripheral blood lymphocytes by sheep erythrocyte rosetting method. The percentage of T lymphocytes in lepromatous and tuberculoid cases were significantly lower compared to that in normal healthy controls. All sera except FCS had a stimulatory effect on the number of T cells. The cells incubated for 24 hours in FCS did not show any stimulatory effect on the number of T cells, however, these FCS incubated cells showed a significant elevation in the number of T cells when further incubated in sera either from leprosy cases or from healthy subjects.