Semiquantitative polymerase chain reaction enzyme immunoassay for diagnosis of disseminated Candidiasis

Abstract

A polymerase chain reaction enzyme immunoassay (PCR-EIA) was developed for the semiquantitative of circulating candidal DNA in disseminated candidiasis due to Candida albicans. Polymerase chain reaction was based on primers from the internal transcribed ribosomal region. Binding of the product to a streptavidin-coated microtitration plate was mediated by a biotinylated capture probe. The product was digoxigenylated during PCR; this was the tag to which antibody was bound in the subsequent EIA. The optical density (OD) endpoint was < 0.1 in 15 sera from patients with no evidence of candidal infection (group 1) and in 13 of the 16 sera from colonized patients (group 2); it was > 0.1 in the other three sera from group 2 blood culture-negative patients who required intravenous amphotericin B for cure. The OD was positive in 28 patients with disseminated candidiasis (group 3), defined as positive blood cultures and successful treatment with amphotericin B (n = 11), positive blood culture confirmed at autopsy (n = 11), or negative blood culture first proven at necropsy (n = 6). In patients from whom multiple samples were available, recovery correlated with an optical density of < 0.1 by day 4 in four patients and by day 13 in the rest. In the five patients with fatal outcome from whom multiple samples were available, the mean OD rose from 0.174 to 0.668. Samples seeded with Candida albicans blastoconidia demonstrated that on OD of 0.220 was equivalent to 10 cfu. Assay of the group 3 sera by a commercial antigen detection test gave a corresponding sensitivity of 60% which rose to 67.9% when an in-house reverse passive latex agglutination test was used.

Invasive Fungal Infections and Approaches to Their Diagnosis

“…Under these conditions, the pooled sensitivity and specificity of PCR in diagnosing IC was 95% and 92%, respectively (Avni et al, 2011) (Table 7). Importantly, PCR-based tests for Candida DNA in blood are negative in most subjects with GI colonisation with Candida species and the specificity of these tests is quite high (Avni et al, 2011;Baron, 2006;Burnie, Golband, & Matthews, 1997;Chang et al, 2013;Chryssanthou et al, 1999;Mylonakis et al, 2015;White et al, 2005) (Table 7).…”

Comparison of Different Methods of Isolation of DNA of Commonly Encountered Candida Species and Its Quantitation by Using a Real-Time PCR-Based Assay

“…To date, different procedures have been used to generate candidal template DNA from serum samples for PCR assay (1,(3)(4)(5). However, to our knowledge the efficiencies of these procedures applied simultaneously to the same sera sets have not been compared to same study.…”

“…Several PCR methods have been developed for use either on whole-blood samples (7,11,12) or on serum samples (1,(3)(4)(5). However, in addition to being too time-consuming and laborintensive, protocols for extraction of cellular candidal DNA from blood samples may also (i) show significant amounts of PCR inhibitor compounds, (ii) introduce fungal DNA from most of the commercialized enzymatic preparations used in various phases of sample processing, (iii) amplify human white blood cells DNA when PCR primers based on the rRNA gene are used, and (iv) show different efficiencies of the release of genomic DNA from different Candida species, which may exhibit changes in physical composition of the cell wall and therefore susceptibility to digestion.…”

Current Status of Nonculture Methods for Diagnosis of Invasive Fungal Infections

“…The sensitivity of PCR-EIA to detect candidemia and aspergillosis was higher than that by ethidium bromide staining (68). In addition, the PCR-EIA format provided further amplification without losing the species-specific binding associated with Southern blotting, multiple samples can be assayed in parallel, and semiquantitation of DNA is possible (27,68). The TaqMan PCR (Perkin-Elmer Corp., Applied Biosystems, Foster City, Calif.) is another approach that combines PCR, probe hybridization, and signal generation in one step (15,171).…”

“…Furthermore, in experimentally infected animals and patients, the sensitivity can be higher than those of cultures and serologic tests for diagnosing invasive fungal infections (27,42,56,95,146,159,210,240). DNA results have correlated with clinical improvement and effect of treatment, and these have been demonstrated in patients with invasive aspergillosis (56,126,244), invasive candidiasis (56), and cryptococcal meningitis (165).…”