Kathy A. Mangold

Real-Time PCR for Detection and Identification of Plasmodium spp.

ABSTRACT Rapid and accurate detection of malaria parasites in blood is needed to institute proper therapy. We developed and used a real-time PCR assay to detect and distinguish four Plasmodium spp. that cause human disease by using a single amplification reaction and melting curve analysis. Consensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and SYBR Green was used for detection in a LightCycler instrument. Patient specimens infected at 0.01 to 0.02% parasitemia densities were detected, and analytical sensitivity was estimated to be 0.2 genome equivalent per reaction. Melting curve analysis based on nucleotide variations within the amplicons provided a basis for accurate differentiation of Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. For assay validation, 358 patient blood samples from the National University Hospital in Singapore and Evanston Northwestern Healthcare in Illinois were analyzed. Of 76 blinded patient samples with a microscopic diagnosis of P. falciparum, P. vivax, or P. ovale infection, 74 (97.4%) were detected by real-time PCR, including three specimens containing mixed P. falciparum-P. vivax infections. No Plasmodium DNA was amplified in any of the 82 specimens sent for malaria testing but that were microscopically negative for Plasmodium infection. In addition, 200 blood samples from patients whose blood was collected for reasons other than malaria testing were also determined to be negative by real-time PCR. Real-time PCR with melting curve analysis could be a rapid and objective supplement to the examination of Giemsa-stained blood smears and may replace microscopy following further validation.

Species-Level Identification of Staphylococcal Isolates by Real-Time PCR and Melt Curve Analysis

ABSTRACT A real-time PCR assay was developed to identify common staphylococcal species. A single set of consensus primers was designed to amplify a portion of the 16S rRNA gene, and a pair of fluorescence resonance energy transfer probes was used to identify species based on the unique melt properties of the probes resulting from sequence variations in the amplicons from each species. Nine common staphylococcal strains (S. aureus, S. capitis, S. epidermidis, S. haemolyticus, S. hominis, S. lugdunensis, S. schleiferi, S. simulans, and S. warneri) were used for assay development. The species-specific melting profiles were validated by correctly identifying 36 of 37 coagulase-negative staphylococcal (CoNS) isolates identified by ribotyping. In a study of clinical isolates, the PCR/melt curve approach correctly identified 56/56 S. aureus isolates identified by coagulase/protein A latex agglutination. Fifty-four CoNS clinical isolates characterized using the API Staph assay were studied, with the PCR/melt curve approach yielding matching identifications for 32/54 (59%). The API Staph assay was unable to identify 18 CoNS isolates, and differing results were obtained for 4 isolates. Sequencing of the 22 discrepant or unidentified CoNS samples revealed that the PCR/melt curve results were correct for all but one isolate. Thus, PCR/melt curve analysis achieved a nearly 100% accuracy and performed better than biochemical testing. Performance of the PCR/melt curve approach requires less than 2 h after colony selection. This method thus provides a rapid and accurate approach to the identification of staphylococcal species in the clinical laboratory.

Rectal Screening for Klebsiella pneumoniae Carbapenemases: Comparison of Real-Time PCR and Culture Using Two Selective Screening Agar Plates

ABSTRACT Klebsiella pneumoniae carbapenemases (KPCs) have recently been described in Chicago, IL, especially among residents of long-term acute care hospitals (LTACHs). These patients are frequently transferred to local Chicago hospitals for higher acuity of medical care, and rapid detection and isolation of KPC-colonized LTACH residents may interrupt the introduction of KPCs into acute care hospitals. We evaluated the performance of a real-time PCR for bla KPC from enrichment broth versus direct plating of rectal surveillance swabs on two selective culture media, CHROMagar extended-spectrum-β-lactamase (ESBL) and vancomycin, amphotericin B, ceftazidime, and clindamycin (VACC) plates. Rectal surveillance swabs were collected as part of a point prevalence study of KPC carriage rates among 95 residents of two Chicago area LTACHs. Discrepant results between PCR and culture were resolved by subculturing the enrichment broth. Overall, 66 of 95 patients (69.5%) were colonized with KPCs, using the cumulative results of culture as a reference standard. Real-time PCR from enrichment broth was positive in 64 of 66 (97%) colonized patients, including nine surveillance swabs that were missed by both selective culture media. PCR demonstrated higher sensitivity, 97.0%, than culture using either CHROMagar or VACC plates (both with sensitivity of 77.3%). In addition, turnaround time was significantly shorter for the PCR-based method than for culture, with a mean of 24 h versus 64 to 72 h for CHROMagar and VACC plates (P < 0.0001). Overall, PCR for bla KPC represents the best screening test for KPCs with significantly higher sensitivity and with less hands-on time, resulting in a shorter time to results.

REAL-TIME DETECTION of blaKPC IN CLINICAL SAMPLES AND SURVEILLANCE SPECIMENS

ABSTRACT A real-time PCR assay was developed targeting the bla KPC responsible for Klebsiella pneumoniae carbapenemase (KPC)-mediated carbapenem resistance and was validated for testing colonies or enrichment broth cultures. The assay accurately detects KPC-containing strains with high analytical specificity and sensitivity.

Alterations of Adenomatous Polyposis Coli (APC) gene in oral squamous cell carcinoma

The present study assessed the roles of Adenomatous Polyposis Coli (APC) tumor suppressor gene during oral carcinogenesis. Reduction of APC transcript levels and APC loss of heterozygosity (LOH) were found in 39% (7/18) and 29% (10/34) cases of oral squamous cell carcinoma (OSCC), respectively. The apparent APC heterozygosity (27%) in non-cancerous matched oral tissue (NCMOT) adjacent to OSCC at an exon 11 locus was significantly lower than normally found in the Taiwanese population (49%). These findings suggest that the allelic status of APC could indicate a cancer risk. No polymorphism of 11307 allele of APC was identified in NCMOT or OSCC. Our data indicated that alterations of APC are frequent molecular changes of OSCC. Advances in understanding of the APC alterations that accompany OSCC development might provide a means for early diagnosis and possibly new therapeutic strategies.

Lack of an association between Chlamydia psittaci and ocular adnexal lymphoma

The objective of this study was to assess whether there is PCR evidence for C. psittaci DNA in ocular adnexal lymphoma specimens collected in an academic institution in the U.S. This was a retrospective, single-center study of patients from 1994 – 2004. We used 28 ocular adnexal lymphoma biopsy specimens from adult patients, 16 control lymphoma specimens from patients with systemic lymphomas not involving the ocular adnexa, and five control benign adnexal tissue samples. The presence of C. psittaci DNA was investigated by polymerase chain reaction (PCR) in each group. Two different assays were utilized: (1) conventional PCR/gel based assay targeting a 111-bp fragment of the 16S gene and (2) a real-time PCR assay amplifying a 148-bp portion of the 16S gene with detection via a specific fluorescent probe. Amplification was carried out to 60 cycles. Positive controls consisted of isolated DNA from C. psittaci strains VS1, CP3, and FP. A human DNA internal control was used to assess sample DNA quality and amplification success. Mean outcome measure was the presence of C. psittaci DNA. Using both assays, all patient samples in all categories yielded negative results. Both assays detected C. psittaci DNA from isolated strains. Internationally, Chlamydia psittaci has been associated with ocular adnexal lymphomas with great variability. Similar to several other recent studies in the USA, our study could not confirm the presence of C. psittaci in ocular adnexal lymphomas. Differences in the prevalence of C. psittaci infection in various geographic regions or technical differences in the application of the assays may underlie the variability in the association between C. psittaci and ocular adnexal lymphoma.

Neisseria Species Identification Assay for the Confirmation of Neisseria gonorrhoeae-Positive Results of the COBAS Amplicor PCR

ABSTRACT Screening assays for Neisseria gonorrhoeae exhibit low positive predictive values, particularly in low-prevalence populations. A new real-time PCR assay that detects and identifies individual Neisseria spp. using melt curve analysis was compared to two previously published supplementary assays. NsppID, a 16S rRNA real-time PCR/melt curve assay developed to distinguish N. gonorrhoeae from other Neisseria spp., was compared to real-time PCR assays targeting genes reportedly specific for N. gonorrhoeae, the cppB gene and the porA pseudogene. A total of 408 clinical specimens (324 female endocervical swabs and 84 male urine or urogenital swab specimens) were screened using the COBAS Amplicor assay for Chlamydia trachomatis and N. gonorrhoeae (CT/NG) (Roche Diagnostics, Indianapolis, IN) followed by confirmatory testing via real-time PCR. The NsppID assay detected Neisseria spp. in 150/181 COBAS-positive specimens (82%), including six dual infections, and identified N. gonorrhoeae in 102 (56%) specimens. Sixty-nine of 181 (38%) specimens were positive for N. gonorrhoeae by porA pseudogene, and 115/181 (64%) were positive for cppB. However, cppB was also positive in 15% of COBAS-negative specimens, more than either NsppID (4%) or porA pseudogene (2%) assays. The porA pseudogene assay had the highest specificity for both genders but the lowest sensitivity, especially in female specimens. NsppID had a slightly lower specificity but greater sensitivity and overall accuracy. The least desirable confirmatory assay was cppB, due to poor specificity. The NsppID assay is an accurate confirmatory assay for N. gonorrhoeae detection. In addition, the NsppID assay can identify the non-N. gonorrhoeae species responsible for the majority of false-positive results from the COBAS Amplicor CT/NG assay.

Unique finding of a 2009 H1N1 influenza virus-positive clinical sample suggests matrix gene sequence variation.

The 2009 H1N1 influenza virus has rapidly spread all over the world. At this time, regions in the northern hemisphere are at the beginning of the typical annual respiratory virus season, although the 2009 H1N1 virus has been highly prevalent for the last several months. This year, it is likely that

Analytical Performance Determination and Clinical Validation of the Novel Roche RealTime Ready Influenza A/H1N1 Detection Set

ABSTRACT The emergence of a novel pandemic human strain of influenza A (H1N1/09) virus in April 2009 has demonstrated the need for well-validated diagnostic tests that are broadly applicable, rapid, sensitive, and specific. The analytical performance and clinical validity of results generated with the novel Roche RealTime Ready Influenza A/H1N1 Detection Set using the LightCycler 2.0 instrument were characterized. Analytical performance was assessed by processing respiratory samples spiked with H1N1/09 and seasonal influenza A virus, a set of seasonal influenza A virus subtypes, and samples containing common viral and bacterial respiratory pathogens. The clinical validity of results was assessed in comparison to other assays by analyzing 359 specimens at three clinical sites and one reference laboratory. Direct sequencing was used to resolve samples with discrepant results. The assay detected virus concentrations down to <50 RNA copies per reverse transcription (RT)-quantitative PCR (qPCR). Various influenza A virus subtypes were covered. The analytical specificity was 100%. High clinical validity was demonstrated by the 99% positive agreement between seasonal influenza A viruses, 98% positive agreement between H1N1/09 viruses, and 88% agreement between negative results. The analytical sensitivity was compared to those of three other RT-qPCR assays and was found to be equivalent. The novel Roche RealTime Ready Influenza A/H1N1 Detection Set can be utilized on the widely used LightCycler platform. We demonstrate its usefulness for the rapid detection and surveillance of pandemic H1N1/09 influenza A virus infections.

PCR detection of clarithromycin-susceptible and -resistant Helicobacter pylori from formalin-fixed, paraffin-embedded gastric biopsies.

Antimicrobial resistance to clarithromycin is a growing concern in the treatment of Helicobacter pylori and is associated with three major point mutations of the 23S rRNA, A2142C, A2142G, and A2143G. The use of traditional culture-based methods for determination of clarithromycin resistance in H. pylori are time consuming and lack sensitivity. We implemented a real-time PCR with melt curve analysis to detect and characterize H. pylori in formalin-fixed, paraffin-embedded gastric biopsy specimens to assess the frequency of clarithromycin resistance mutations in our study population. One hundred and fifty-three formalin-fixed, paraffin-embedded gastric biopsies were chosen on the basis of positive immunohistochemical staining for H. pylori and an accompanying histopathological diagnosis of Helicobacter-associated gastritis. New adjacent sections were taken for immunohistochemical staining and DNA extraction with subsequent testing by PCR assay and melt curve analysis using a primer and probe combination first described by Oleastro et al.12 One hundred and forty-six samples demonstrated adequate amplification of a human DNA control target. Of these, there were 122 H. pylori immunohistochemistry-positive samples. In all, 103 out of 122 (84%) immunohistochemistry-positive samples demonstrated amplifiable H. pylori 23S rRNA gene target and 19 (16%) demonstrated no amplification of H. pylori. Twenty-two samples were negative for H. pylori by immunohistochemistry and PCR. Two were negative for H. pylori by immunohistochemistry, but were positive for H. pylori by PCR. In all, 52 out of 105 (50%) PCR-positive samples demonstrated resistance mutations, and it was determined that a heterogeneous population of mutated and unmutated organisms was present in 11 out of 52 samples. The use of PCR assays allows for a timely assessment of clarithromycin resistance status without the disadvantages of culture-based methods, and may lead to a decrease in treatment failure rates.