Ralf Bialek

Evaluation of Two Nested PCR Assays for Detection of Histoplasma capsulatum DNA in Human Tissue

ABSTRACT In order to evaluate the diagnostic relevance of two nested PCR assays for diagnosis of histoplasmosis in clinical specimens, 100 paraffin-embedded biopsy specimens were examined. Upon microscopy of tissue, 50 biopsy specimens were histoplasma positive and 50 were negative. Due to destruction by formalin fixation, successful extraction of amplifiable human DNA was limited to 29 and 33 samples, respectively. A product of the Histoplasma capsulatum nested PCR assay targeting the gene encoding the unique fungal 100-kDa-like protein was detected in 20 histopathologically positive biopsy specimens but in none of the microscopically negative samples. Sequencing revealed that all 20 products of 210 bp were identical to the sequence of H. capsulatum in the GenBank database. In contrast, the nested PCR assay targeting the fungal 18S rRNA genes amplified products in 26 histopathologically positive but also in 18 microscopically negative biopsy specimens. However, sequencing revealed that only 20 of these 44 PCR products (231 bp) were identical to the sequence of H. capsulatum. The remaining 24 sequences were homologous to those of several Euascomycetes. These PCR products were detected only in tissues possibly colonized by nonpathogenic fungi, possibly causing these nonspecific amplifications. The detection limit of both H. capsulatum nested PCR assays was 1 to 5 fungal cells per sample. The two assays were similarly sensitive in identifying H. capsulatum. In this preliminary study, the novel 100-kDa-like-protein gene nested PCR revealed a specificity of 100% without requiring sequencing, which was necessary for identification of the 18S ribosomal DNA nested PCR products in order to avoid a high rate of false-positive results.

Polymerase Chain Reaction Detection of Aspergillus DNA in Experimental Models of Invasive Aspergillosis

To determine the sensitivity of polymerase chain reaction (PCR) assays for the diagnosis of invasive aspergillosis, results of quantitative culture, PCR-ELISA, and a quantitative LightCycler assay (Roche Diagnostics) of blood and organ specimens of experimentally infected mice and rabbits were compared. By PCR-ELISA, 297 of 379 murine lung specimens were positive, but only 235 of 379 were culture positive. Whereas 64 culture-negative lungs were positive by PCR, Aspergillus was grown from only 2 PCR-negative samples. The PCR assay was 19.4 times more sensitive than culture. None of the 68 blood cultures from mice and rabbits were positive for Aspergillus fumigatus, whereas PCR detected Aspergillus DNA in 17 of 68 blood samples. Quantitative PCR analysis of blood samples showed a fungus load of 10(1)-10(2) cfu/mL of blood. The data confirm the superior sensitivity of PCR for the diagnosis of experimental Aspergillus infections.

Molecular Methods To Improve Diagnosis and Identification of Mucormycosis

ABSTRACT Mucormycosis is difficult to diagnose. Samples from suspected cases often fail to grow Mucorales in microbiologic cultures. We identified all hematologic malignancy and stem cell transplant patients diagnosed with proven mucormycosis between 2001 and 2009 at Brigham and Womens Hospital/Dana-Farber Cancer Institute. Seminested PCR targeting Mucorales 18S ribosomal DNA and sequencing were performed on formalin-fixed paraffin-embedded tissue samples. Of 29 cases of mucormycosis, 27 had tissue samples available for PCR and sequencing. Mucorales PCR was positive in 22. Among 12 culture-positive cases, 10 were PCR positive and sequencing was concordant with culture results to the genus level in 9. Among 15 culture-negative cases, PCR was positive and sequencing allowed genus identification in 12. Mucorales PCR is useful for confirmation of the diagnosis of mucormycosis and for further characterization of the infection in cases where cultures are negative.

PCR Assays for Identification of Coccidioides posadasii Based on the Nucleotide Sequence of the Antigen 2/Proline-Rich Antigen

ABSTRACT A conventional nested PCR and a real-time LightCycler PCR assay for detection of Coccidioides posadasii DNA were designed and tested in 120 clinical strains. These had been isolated from 114 patients within 10 years in Monterrey, Nuevo Leon, Mexico, known to be endemic for coccidioidomycosis. The gene encoding the specific antigen 2/proline-rich antigen (Ag2/PRA) was used as a target. All strains were correctly identified, whereas DNA from related members of the family Onygenaceae remained negative. Melting curve analysis by LightCycler and sequencing of the 526-bp product of the first PCR demonstrated either 100% identity to the GenBank sequence of the Silveira strain, now known to be C. posadasii (accession number AF013256 ), or a single silent mutation at position 1228. Length determination of two microsatellite-containing loci (GAC and 621) identified all 120 isolates as C. posadasii. Specific DNA was amplified by conventional nested PCR from three microscopically spherule-positive paraffin-embedded tissue samples, whereas 20 human tissue samples positive for other dimorphic fungi remained negative. Additionally, the safety of each step of a modified commercially available DNA extraction procedure was evaluated by using 10 strains. At least three steps of the protocol were demonstrated to sufficiently kill arthroconidia. This safe procedure is applicable to cultures and to clinical specimens.

Comparison of fluorescence, antigen and PCR assays to detect Cryptosporidium parvum in fecal specimens

To optimize routine screening for cryptosporidiosis, 198 stool samples from patients at risk and from calves were examined by enzyme immunoassay (EIA), a direct fluorescent-antibody (DFA) and a modified immunofluorescence assay. Ninety-nine samples were positive in at least one assay, whereas 99 were negative in all three assays. Sensitivity of antigen EIA and DFA were similar (94%, 95% CI: 88-98%, and 91%, 95% CI: 84-95%). The modified immunofluorescence was significantly less sensitive (64%, 95% CI: 55-74%). 149 samples were also examined by two nested PCR assays targeting either the 18S rRNA or Cryptosporidium outer wall protein (COWP) gene. A PCR product was amplified from 86 out of 89 samples being positive in at least one other assay (sensitivity 97%, 95% CI: 91-99%). None was obtained from 60 samples negative in the three other assays. PCR assays did not increase the detection rate. Antigen EIA or DFA appear sufficient for routine Cryptosporidium screening of fecal samples.

Detection of Cryptococcus neoformans DNA in Tissue Samples by Nested and Real-Time PCR Assays

ABSTRACT Two PCR protocols targeting the 18S rRNA gene of Cryptococcus neoformans were established, compared, and evaluated in murine cryptococcal meningitis. One protocol was designed as a nested PCR to be performed in conventional block thermal cyclers. The other protocol was designed as a quantitative single-round PCR adapted to LightCycler technology. One hundred brain homogenates and dilutions originating from 20 ICR mice treated with different azoles were examined. A fungal burden of 3 × 101 to 2.9 × 104 CFU per mg of brain tissue was determined by quantitative culture. Specific PCR products were amplified by the conventional and the LightCycler methods in 86 and 87 samples, respectively, with products identified by DNA sequencing and real-time fluorescence detection. An analytical sensitivity of 1 CFU of C. neoformans per mg of brain tissue and less than 10 CFU per volume used for extraction was observed for both PCR protocols, while homogenates of 70 organs from mice infected with other fungi were PCR negative. Specificity testing was performed with genomic DNA from 31 hymenomycetous fungal species and from the ustilaginomycetous yeast Malassezia furfur, which are phylogenetically related to C. neoformans. Twenty-four strains, including species of human skin flora like M. furfur and Trichosporon spp., were PCR negative. Amplification was observed with Cryptococcus amylolentus, Filobasidiella depauperata, Cryptococcus laurentii, and five species unrelated to clinical specimens. LightCycler PCR products from F. depauperata and Trichosporon faecale could be clearly discriminated by melting curve analysis. The sensitive and specific nested PCR assay as well as the rapid and quantitative LightCycler PCR assay might be useful for the diagnosis and monitoring of human cryptococcal infections.

Diagnosis and Monitoring of Murine Histoplasmosis by a Nested PCR Assay

ABSTRACT A newly developed nested PCR assay was applied to murine models of histoplasmosis. ICR and BALB/c mice were intravenously infected withHistoplasma capsulatum and sacrificed up to 29 days later. Samples of blood, spleen, and lung homogenates were cultured and examined by the PCR assay. In the ICR mouse model, 265 of 319 organ samples showed concordant results. With 7 samples, the culture was positive and the PCR assay was negative whereas a positive PCR but a negative culture were obtained with 47 samples (P < 0.0001 according to McNemars test). Organ homogenates and blood samples of either spontaneously cured or treated BALB/c mice were PCR negative. The nested PCR assay performs excellently in the monitoring of spontaneously and treatment-cured murine histoplasmosis. It limits the infection risks of the laboratory staff and might be of diagnostic value for humans.

Autofluorescence microscopy for the detection of nematode eggs and protozoa, in particular Isospora suis, in swine faeces.

Parasites from swine faeces were examined for autofluorescence. Oocysts of Eimeria polita, E. scabra and Isospora suis, cysts of Balantidium coli and eggs of Oesophagostomum dentatum, Strongyloides ransomi and Trichuris suis (but not those of Ascaris suum) emitted light after excitation with UV light. I. suis oocyst counts in McMaster chambers utilising autofluorescence were compared to those from conventional bright field microscopy. Similarly, faecal smears containing I. suis were examined using the same techniques. Autofluorescence was superior to bright field microscopy in detecting oocysts after flotation and was highly significantly more sensitive when direct smears were examined.

Nested PCR Assays for Detection of Blastomyces dermatitidis DNA in Paraffin-Embedded Canine Tissue

ABSTRACT A Blastomyces dermatitidis nested PCR assay targeting the gene encoding the Wisconsin 1 (WI-1) adhesin was developed and compared with a nested PCR targeting the 18S rRNA gene (rDNA) of members of the family Onygenaceae. We examined 73 paraffin-embedded tissue samples obtained from nine dogs which died of blastomycosis and nine dogs which succumbed to lymphosarcoma according to autopsy findings; amplifiable canine DNA was extracted from 25 and 33 specimens from the two groups, respectively. The B. dermatitidis PCR amplified DNA from 8 of 13 tissue samples in which yeast cells were detected by microscopy. Sequencing revealed that all PCR products were homologous to the B. dermatitidis WI-1 adhesin gene. No PCR product was amplified from 12 microscopically negative biopsy specimens from dogs with blastomycosis or from 33 biopsy specimens from dogs with lymphosarcoma. The 18S rDNA PCR amplified DNA from 10 and 9 tissue samples taken from dogs which died of blastomycosis and lymphosarcoma, respectively. Only six products were identified as being identical to B. dermatitidis 18S rDNA; they were exclusively obtained from specimens positive by the B. dermatitidis nested PCR. For specificity testing, 20 human biopsy specimens proven to have histoplasmosis were examined, and a specific H. capsulatum product was amplified by the 18S rDNA PCR from all specimens, whereas no product was obtained from any of the 20 samples by the B. dermatitidis PCR assay. In conclusion, the PCR targeting a gene encoding the unique WI-1 adhesin is as sensitive as but more specific than the PCR targeting the 18S rDNA for detection of B. dermatitidis in canine tissue.

Comparison of viability assays for Cryptosporidium parvum oocysts after disinfection

In order to test various viability assays for Cryptosporidium parvum oocysts were used to infect HCT-8 cells in vitro or baby mice. Infected cells were either stained with fluorescent anti-Cryptosporidium-antibody or lysed and subjected to C. parvum-specific PCR after 48 h. Titrations with infective oocysts were performed and compared to oocysts disinfected with Neopredisan for 2 h at varying concentrations. Caecal smears and histological sections from infected animals were examined in parallel. The number of foci of parasite development in vitro after immunofluorescent staining correlated well with the infection dose. PCR was less quantifiable and the results were not always reproducible, especially when low infection doses were used. Disinfection resulted in a dose-dependent reduction of oocyst infectivity when compared to the controls in all three assays. The infection of cells cultured in vitro with oocysts of C. parvum provides a suitable tool for the estimation of viability after treatment with chemical disinfectants. Immunofluorescence is easy to perform and gives quantitative results, while PCR-based detection of parasite DNA, although possible, requires the use of more sophisticated tools for quantification.