Neurocryptococcosis: diagnosis by PCR method

Abstract

SUMMARYCryptococcus neoformans detection was optimized using PCR technique with the objective of application in the clinical laboratory diagnosis. The amplification area was ITS and 5,6S which encodes the ribosomal RNA (rRNA). A total of 72 cerebrospinal fluid (CSF) samples were used, obtained from cases with and without AIDS. The patients had cryptococcal meningitis (n = 56) and meningitis caused by other agents (n = 16). The results demonstrated that PCR test had the highest sensitivity rates, superior to culture (85.7%) and to India ink test (76.8%). PCR was found to be sensitive in detecting 1 cell/mL and highly specific since it did not amplify other fungal DNA. The comparative analysis of the methods showed that PCR is more sensitive and specific and is applicable as an important laboratorial resource for neurocryptococcosis diagnosis.

Diagnosis of Invasive Fungal Infections – Current Limitations of Classical and New Diagnostic Methods

“…In general, assays targeting multicopy genes have better detection limits than those targeting single copy genes. 135 Although PCR may become a diagnostic modality to identify Mucorales, [136][137][138] Scedosporium, [139][140][141] and several other fungi, [142][143][144][145][146][147][148][149][150][151] no commercial test is available to date. These techniques hold promise, but they are not yet standardised or readily available in most clinical laboratories.…”

Role of Polymerase Chain reaction (PCR) amplifying conserved 5.8S and ITS regions of the cryptococcal DNA in the diagnosis of Cryptococcal meningitis

“…10 The DNA obtained was amplified using specific primers CN4/CN5. 11 Positive control used was DNA extracted from C. neoformans ATCC strain 32045. C. neoformans specific primers as described by Mitchell et al were used.…”

“…o C for 7min. 11 The final PCR product was analyzed by Gel electrophoresis using 1% agarose, a sample loading dye (bromophenol blue and cyanol). A DNA marker/ladder of 200bp was also set up on the gel.…”

“…Other studies reported sensitivity 86-100% and specificity 100%. 7,11,20,23 Using the same primers, Paschoal et al 11 have detected PCR positive in 52/56 CSF samples from cryptococcal meningitis diagnosed clinically as well as microbiologically. However Saha et al 7 have obtained 100% sensitivity and specificity of PCR by amplifying 278 bp region of 18S rDNA using nested primers for FungusI/II.…”

“…Other reasons for false positivity are presence of rheumatoid factor, agar syneresis fluid, cross reactivity with Capnocytophaga canimorsus DF-2 bacillus), Stomatococcus mucilaginosus or Klebsiella pneumonae, non-specific reactivity in HIV infected patients, interferences starch, disinfectants and soap. 21 Molecular tests have been developed for detection of nucleic acid directly in the biological samples 11 Some of the techniques being used for diagnosis are: PCR, in-situ hybridization technique using DNA probes, micro array technology. 22 The conventional PCR and 33 12,23 In the present study the region ITS and 5.8S rRNA of C. neoformans was amplified as described by Mitchell et al 12 which is species specific that means not amplifying other yeasts.…”