Advanced Techniques in Diagnostic Microbiology

8. Probe-Based Microbial Detection and Identification 139
ISH is an important technique for identifying and localizing viral nucleic acids
associated with infectious disease and cancer. ISH has been used to determine
the intracellular localization of the hepatitis viruses, human papillomaviruses, and
herpes simplex viruses, and to detect these viruses. ISH has also been used to detect
adenovirus, cytomegalovirus (Wu et al., 1992), JC virus, Epstein–Barr virus
(Prange et al., 1992), and HHV-8 (Li et al., 1996). Human papilloma virus (HPV)
is accepted as the primary causative agent in the development of cervical cancer.
Although there have been approximately 100 HPV genomic types identified, most
of these are not oncogenic and therefore do not lead to the development of cervical
cancer. Those HPV genotypes that have been identified as types that contribute
to the development of cervical cancer are categorized into intermediate and high
risk HPV. ISH has been widely used to detect and differentiate HPV in cervical
specimens. Dako Corporation (Carpinteria, CA, USA) provides biotinylated
DNA probes for HPV ISH, including probes for high-risk group or type-specific
probe.
Peptide–Nucleic Acid Probe
Fluorescence in situ hybridization (FISH) using peptide–nucleic acid (PNA) probes
(PNA FISH) is a novel diagnostic technique combining the simplicity of traditional
staining procedures with the unique performance of PNA probes to provide rapid
and accurate diagnosis of infectious diseases. Peptide nucleic acids are novel synthetic
DNA-like compounds with nucleotide bases attached to a peptide backbone.
PNA probes are DNA probe mimics with an uncharged, neutral backbone that
provides the PNA probes with improved hybridization characteristics such as high
degrees of specificity, strong affinities, and rapid kinetics, as well as an improved
ability to hybridize to highly structured targets such as rRNA. In addition, the
relatively hydrophobic character of PNA probes compared with the character of
DNA enables PNA probes to penetrate the hydrophobic cell wall after preparation
of a standard smear.
PNA FISH probes have been developed and evaluated for S. aureus, C. albicans,
E. faecalis, E. coli, coagulase-negative staphylococci, C. dubliniensis, Klebsiella
pneumoniae, and Pseudomonas aeruginosa. Among these probes, currently the
S. aureus PNA FISH, C. albicans PNA FISH, and E. faecalis PNA FISH are
FDA approved for in vitro diagnosis and are available from AdvanDx, (Woburn
MA, USA). The PNA FISH procedures have been extensively evaluated for rapid
diagnosis of positive blood cultures for S. aureus (Chapin and Musgnug, 2003;
Oliveira et al., 2002, 2003), E. coli, and C. albicans (Oliveira et al., 2001, Rigby
et al., 2002) with high sensitivity and specificity. A recent multicenter evaluation
(Wilson et al., 2005) of the C. albicans PNA FISH assay (AdvanDx) demonstrated
that this method is an accurate means of differentiating C. albicans from non–C.
albicans species present in blood culture bottles. The overall sensitivity, specificity,
positive predictive value, and negative predictive value of the combined routine
screening methods used at the various institutions were 100%, 97.3%, 96.0%,