Molecular detection of bacteria, placental inflammation, and neonatal sepsis risk.

OBJECTIVE: To identify bacteria in umbilical cord tissue and investigate the association with placental inflammation and neonatal sepsis risk score. STUDY DESIGN: Retrospective cohort study from 2017-2019. RNA was extracted from umbilical cord tissue and NanoString nCounter used to identify seven bacteria genera. Sepsis risk score was calculated using the Kaiser sepsis calculator. Placental histopathology was abstracted from medical records. RESULTS: Detection of bacterial RNA in the umbilical cord (n = 96/287) was associated with high-stage maternal and fetal acute placental inflammation (maternal 35.4% vs 22.5%, p = 0.03 and fetal 34.4% vs 19.4%, p < 0.01) and maternal vascular malperfusion (36.5% vs 23.0%, p = 0.02). Detection of Ureaplasma spp. was also associated with increased sepsis risk score (1.5/1000 [0.6, 8.6] vs 0.9/1000 [0.2, 2.9], p = 0.04). CONCLUSION: Umbilical cord bacterial pathogens are linked to fetal and maternal placental inflammation and maternal vascular malperfusion during gestation and associated with increased sepsis risk score in the neonate.

The safety and efficacy of systemic delivery of a new liver-de-targeted TGFß signaling inhibiting adenovirus in an immunocompetent triple negative mouse mammary tumor model.

Aberrant TGFß signaling is linked to metastasis and tumor immune escape of many cancers including metastatic triple negative breast cancer (mTNBC). Previously, we have found that oncolytic adenoviruses expressing a TGFß signaling inhibitory protein (sTGFßRIIFc) induced immune activation in a mouse TNBC (4T1) immunocompetent subcutaneous model with intratumoral injection. Systemic administration of adenoviruses can be a superior route to treat mTNBC but faces the challenges of increased toxicity and viral clearance. Thus, we created a liver-de-targeted sTGFßRIIFc- and LyP-1 peptide-expressing adenovirus (mHAdLyp.sT) with enhanced breast cancer cell tropism. Its safety and immune response features were profiled in the 4T1 model. Our data showed that the systemic administration of mHAdLyp.sT resulted in reduced hepatic and systemic toxicity. mHAdLyp.sT was also effective in increasing Th1 cytokines and anti-tumor cell populations by cytokine analysis, spleen/tumor qRT-PCR, and flow cytometry. We further tested the therapeutic effects of mHAdLyp.sT alone and in combination with immune checkpoint inhibitors (ICIs). mHAdLyp.sT alone and with all ICI combinations elicited significant inhibition of lung metastasis by histological analysis. When mHAdLyp.sT was combined with both anti-PD-1 and anti-CTLA-4 antibodies, primary 4T1 tumor growth was also significantly inhibited. We are confident in advancing this new treatment option for mTNBC.

Multiple broad-spectrum Beta-lactamase targets for comprehensive surveillance.

Real-time PCR testing for blaKPC, blaNDM, blaVIM, blaIMP, and blaCTX-M was performed on rectal swabs obtained from residents of two long-term acute-care facilities. While blaKPC was detected in 69/102 swabs (67.6%), testing for four other targets increased the positivity rate for a broad-spectrum ß-lactamase to 73.5% (McNemar's P = 0.03).

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.

The Prevalence of SARS-CoV-2 in Autopsies Surrounding the Time of Pandemic Onset: A Retrospective Review of Cases.

CONTEXT.­: The first case of COVID-19 in the United States was confirmed in January 2020. Initially, little was known about the epidemiology and clinical course of the disease, and diagnostic testing was limited in the United States until March/April 2020. Since then, many studies have speculated that SARS-CoV-2 may have preexisted undiagnosed outside China before the known outbreak. OBJECTIVE.­: To evaluate the prevalence of SARS-CoV-2 in adult autopsy cases performed just before and during the beginning of the pandemic at our institution, where autopsy was not performed on known COVID-19 cases. DESIGN.­: We included adult autopsies performed in our institution from June 1, 2019, to June 30, 2020. Cases were divided into groups based on the likelihood of cause of death being related to COVID-19, presence of a clinical respiratory illness, and histologic findings of pneumonia. Archived formalin-fixed, paraffin-embedded lung tissue of all COVID-possible cases and COVID-unlikely cases with pneumonia was tested for SARS-CoV-2 RNA, using Centers for Disease Control and Prevention 2019-nCoV quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS.­: Eighty-eight cases were identified, and among those, 42 (48%) were considered COVID-possible cause of death, with 24 of those 42 cases (57%) showing respiratory illness and/or pneumonia. COVID-19 as cause of death was considered unlikely in 46 of 88 cases (52%), with 34 of those 46 cases (74%) showing no respiratory illness or pneumonia. SARS-CoV-2 real-time reverse transcription-polymerase chain reaction was performed on a total of 49 cases, 42 COVID-possible and 7 COVID-unlikely with pneumonia, and all cases were negative (0 of 49). CONCLUSIONS.­: Our data suggest that autopsied patients in our community who died between June 1, 2019, and June 30, 2020, without known COVID-19 were unlikely to have had subclinical and/or undiagnosed COVID-19 infection.

Personalized medicine in a community health system: the NorthShore experience.

Genomic and personalized medicine implementation efforts have largely centered on specialty care in tertiary health systems. There are few examples of fully integrated care systems that span the healthcare continuum. In 2014, NorthShore University HealthSystem launched the Center for Personalized Medicine to catalyze the delivery of personalized medicine. Successful implementation required the development of a scalable family history collection tool, the Genetic and Wellness Assessment (GWA) and Breast Health Assessment (BHA) tools; integrated pharmacogenomics programming; educational programming; electronic medical record integration; and robust clinical decision support tools. To date, more than 225,000 patients have been screened for increased hereditary conditions, such as cancer risk, through these tools in primary care. More than 35,000 patients completed clinical genetic testing following GWA or BHA completion. An innovative program trained more than 100 primary care providers in genomic medicine, activated with clinical decision support and access to patient genetic counseling services and digital healthcare tools. The development of a novel bioinformatics platform (FLYPE) enabled the incorporation of genomics data into electronic medical records. To date, over 4,000 patients have been identified to have a pathogenic or likely pathogenic variant in a gene with medical management implications. Over 33,000 patients have clinical pharmacogenomics data incorporated into the electronic health record supported by clinical decision support tools. This manuscript describes the evolution, strategy, and successful multispecialty partnerships aligned with health system leadership that enabled the implementation of a comprehensive personalized medicine program with measurable patient outcomes through a genomics-enabled learning health system model that utilizes implementation science frameworks.

Rectal screening for Klebsiella pneumoniae carbapenemases: comparison of real-time PCR and culture using two selective screening agar plates.

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-ß-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.

Germline mutations in high penetrance genes are associated with worse clinical outcomes in patients with non-small cell lung cancer.

Objective: To determine the frequency of pathogenic mutations in high-penetrance genes (HPGs) in patients with non-small cell lung cancer (NSCLC) and identify whether such mutations are associated with clinicopathologic outcomes. Methods: Patients with NSCLC who had consented to participate in a linked clinical database and biorepository underwent germline DNA sequencing using a next-generation sequencing panel that included cancer-associated HPGs and cancer risk-associated single nucleotide polymorphisms (SNPs). These data were linked to the clinical database to assess for associations between germline variants and clinical phenotype using Fisher's exact test and multivariable logistic and Cox regression. Results: We analyzed 151 patients, among whom 33% carried any pathogenic HPG mutation and 23% had a genetic risk score (GRS) >1.5. Among the patients without any pathogenic mutation, 31% were at cancer stage II or higher, compared with 55% of those with 2 types of HPG mutations (P = .0293); 40% of patients with both types of HPG mutations had cancer recurrence, compared with 21% of patients without both types (P = .0644). In multivariable analysis, the presence of 2 types of HPG mutations was associated with higher cancer stage (odds ratio [OR], 3.32; P = .0228), increased recurrence of primary tumor (OR, 2.93; P = .0527), shorter time to recurrence (hazard ratio [HR], 3.03; P = .0119), and decreased cancer-specific (HR, 3.53; P = .0039) and overall survival (HR, 2.44; P = .0114). Conclusions: The presence of mutations in HPGs is associated with higher cancer stage, increased risk of recurrence, and worse cancer-specific and overall survival in patients with NSCLC. Further large studies are needed to better delineate the role of HPGs in cancer recurrence and the potential benefit of adjuvant treatment in patients harboring such mutations.

Real-time detection of blaKPC in clinical samples and surveillance specimens.

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.

Umbilical cord miRNAs to predict neonatal early onset sepsis.

OBJECTIVE: To determine if miRNA (miR) expression in umbilical cord blood and umbilical cord tissue differs between neonates with early onset sepsis (EOS) versus neonates without true infection. METHODS: Retrospective case-control study design of human patients with EOS (n = 8), presumed sepsis (N = 12) and non-infected control patients (N = 21). Differential expression of >300 miRs was examined using the MIHS-3001ZE-miScript miRNA PCR Array Human miFinder 384HC. Expression levels of miRs were normalized using the global Ct mean of expressed miR and compared between groups. Data analysis was performed using GeneGlobe data analysis software. Ratios of over and under-expressed miRs were calculated and compared between groups using receiver operating characteristic (ROC) curves. RESULTS: Both umbilical cord plasma and umbilical cord tissue revealed several miRs with differential expression with little overlap between the two specimen types. The most overexpressed miR in plasma of EOS patients was miR-211-5p and the most overexpressed in EOS cord tissue was miR-223-5p. ROC curves comparing the ratios of over and under-expressed miRs for EOS patients and controls resulted in an area under the curve of 0.787 for cord plasma (miR-211-5p/miR-142-3p) and 0.988 for umbilical cord tissue (miR-223-5p/miR-22-3p), indicating good discrimination. CONCLUSIONS: miRs show differential expression in EOS versus non-infected controls and presumed sepsis. A ratio of over and under-expressed miRs can provide a potentially sensitive and specific diagnostic test for EOS.

Analytical performance determination and clinical validation of the novel Roche RealTime Ready Influenza A/H1N1 Detection Set.

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.

Performance and Utilization of a Laboratory-Developed Nucleic Acid Amplification Test (NAAT) for the Diagnosis of Pulmonary and Extrapulmonary Tuberculosis in a Low-Prevalence Area.

OBJECTIVES: Tuberculosis (TB) is a significant global health problem. In low-prevalence areas and low clinical suspicion, nucleic acid amplification tests (NAAT) for direct detection of Mycobacterium tuberculosis complex (MTBC) can speed therapy initiation and infection control. An NAAT assay (TBPCR) targeting MTBC IS6110 is used for detecting MTBC in our low-prevalence population. METHODS: Fifteen-year review of patient records identified 146 patients with culture-positive pulmonary tuberculosis (PTB) or extrapulmonary tuberculosis (EPTB). Laboratory-developed TBPCR was retrospectively compared with standard stain and cultures for PTB and EPTB diagnoses. RESULTS: TBPCR assay was used in 57% of patients with PTB and 33% of patients with EPTB. TBPCR detected 88.4% of all TB (smear-positive, 97%; smear-negative, 79%) with 100% specificity. Low bacterial load was indicated in TBPCR-negative PTB (P = .002) and EPTB (P < .008). CONCLUSIONS: TBPCR performance was optimum but significantly underused. Guidelines are proposed for mandated use of TBPCR that capture patients with clinically suspected PTB. Focused TBPCR use in low prevalence populations will benefit patient care, infection prevention, and public health.

LyP-1-Modified Oncolytic Adenoviruses Targeting Transforming Growth Factor ß Inhibit Tumor Growth and Metastases and Augment Immune Checkpoint Inhibitor Therapy in Breast Cancer Mouse Models.

We report here the development of oncolytic adenoviruses (Ads) that have reduced toxicity, enhanced tumor tropism, produce strong antitumor response, and can overcome resistance to immune checkpoint inhibitor therapy in breast cancer. We have shown that LyP-1 receptor (p32) is highly expressed on the surface of breast cancer cells and tumors from cancer patients, and that increased stromal expression of transforming growth factor ß-1 (TGFß-1) is associated with triple-negative breast cancer. Therefore, we constructed oncolytic Ads, AdLyp.sT and mHAdLyp.sT, in which the p32-binding LyP-1 peptide was genetically inserted into the adenoviral fiber protein. Both AdLyp.sT and mHAdLyp.sT express sTGFßRIIFc, a TGFß decoy that can inhibit TGFß pathways. mHAdLyp.sT is an Ad5/48 chimeric hexon virus in which hypervariable regions (HVRs 1-7) of Ad5 are replaced with the corresponding Ad48 HVRs. AdLyp.sT and mHAdLyp.sT exhibited better binding, replication, and produced higher sTGFßRIIFc protein levels in breast cancer cell lines compared with Ad.sT or mHAd.sT control viruses without LyP-1 peptide modification. Systemic delivery of mHAdLyp.sT in mice resulted in reduced hepatic/systemic toxicity compared with Ad.sT and AdLyp.sT. Intravenous delivery of AdLyp.sT and mHAdLyp.sT elicited a strong antitumor response in a human MDA-MB-231 bone metastasis model in mice, as indicated by bioluminescence imaging, radiographic tumor burden, serum TRACP 5b and calcium, and body weight analyses. Furthermore, intratumoral delivery of AdLyp.sT in 4T1 model in immunocompetent mice inhibited tumor growth and metastases, and augmented anti-PD-1 and anti-CTLA-4 therapy. Based on these studies, we believe that AdLyp.sT and mHAdLyp.sT can be developed as potential targeted immunotherapy agents for the treatment of breast cancer.

An Oncolytic Adenovirus Targeting Transforming Growth Factor ß Inhibits Protumorigenic Signals and Produces Immune Activation: A Novel Approach to Enhance Anti-PD-1 and Anti-CTLA-4 Therapy.

In an effort to develop a new therapy for cancer and to improve antiprogrammed death inhibitor-1 (anti-PD-1) and anticytotoxic T lymphocyte-associated protein (anti-CTLA-4) responses, we have created a telomerase reverse transcriptase promoter-regulated oncolytic adenovirus rAd.sT containing a soluble transforming growth factor receptor II fused with human IgG Fc fragment (sTGFßRIIFc) gene. Infection of breast and renal tumor cells with rAd.sT produced sTGFßRIIFc protein with dose-dependent cytotoxicity. In immunocompetent mouse 4T1 breast tumor model, intratumoral delivery of rAd.sT inhibited both tumor growth and lung metastases. rAd.sT downregulated the expression of several transforming growth factor ß (TGFß) target genes involved in tumor growth and metastases, inhibited Th2 cytokine expression, and induced Th1 cytokines and chemokines, and granzyme B and perforin expression. rAd.sT treatment also increased the percentage of CD8+ T lymphocytes, promoted the generation of CD4+ T memory cells, reduced regulatory T lymphocytes (Tregs), and reduced bone marrow-derived suppressor cells. Importantly, rAd.sT treatment increased the percentage of CD4+ T lymphocytes, and promoted differentiation and maturation of antigen-presenting dendritic cells in the spleen. In the immunocompetent mouse Renca renal tumor model, similar therapeutic effects and immune activation results were observed. In the 4T1 mammary tumor model, rAd.sT improved the inhibition of tumor growth and lung and liver metastases by anti-PD-1 and anti-CTLA-4 antibodies. Analysis of the human breast and kidney tumors showed that a significant number of tumor tissues expressed high levels of TGFß and TGFß-inducible genes. Therefore, rAd.sT could be a potential enhancer of anti-PD-1 and anti-CTLA-4 therapy for treating breast and kidney cancers.

Integrity and amplification of nucleic acids from snap-frozen prostate tissues from robotic-assisted laparoscopic and open prostatectomies.

CONTEXT: Recently, robotic-assisted laparoscopic prostatectomy has replaced open retropubic radical prostatectomy as the surgical procedure of choice. This less-invasive approach offers many advantages but exposes prostate tissue to longer periods of warm ischemia that may affect subsequent analysis of biomarkers. OBJECTIVE: To analyze the nucleic acid quality and quantity isolated from open versus laparoscopic prostatectomies. DESIGN: Nucleic acids were isolated from 10 open-obtained and 10 laparoscopic-obtained tissues stored in our prostate sample repository. Nucleic acid integrity was assessed via electrophoresis and polymerase chain reaction amplification of RNA and DNA targets ranging in size from 125 to 939 base pairs. RESULTS: The DNA yield, integrity, and polymerase chain reaction amplification were identical between samples obtained from both surgical approaches. The RNA integrity number and yield were similar, as was ß-2 microglobulin mRNA amplification up to 652 base pairs. However, 2 of 10 samples (20%) collected robotically showed decreased real-time reverse transcriptase-polymerase chain reaction amplification of prostate-specific antigen messenger RNA, especially with targets larger than 300 base pairs. CONCLUSIONS: Generally, the quality and quantity of nucleic acids isolated from prostate tissue obtained via open or laparoscopic approaches are equivalent, suggesting that procurement of tissues is appropriate from either procedure. However, some loss of reverse transcriptase-polymerase chain reaction amplification of larger RNA targets was noted in the laparoscopic samples; appropriate design of assays to keep amplicon sizes small and the use of internal controls to assess RNA integrity is recommended.

Detection of BRCA1 and BRCA2 Ashkenazi Jewish founder mutations in formalin-fixed paraffin-embedded tissues using conventional PCR and heteroduplex/amplicon size differences.

In many families with histories suggestive of BRCA1- or BRCA2-related disease, the proband is deceased. Reliable assessment of archived tissue blocks not amenable to full gene sequencing would be helpful. In this study, a polymerase chain reaction (PCR) assay using primers that bracket the BRCA mutation site and microfluidics-based detection of heteroduplex/amplicon size differences was developed to circumvent artifacts associated with low quality DNA from formalin-fixed paraffin-embedded (FFPE) tissue. Genomic DNA was extracted from 100 FFPE specimens from patients that had previously undergone BRCA gene sequence analysis on blood specimens. Conventional PCR amplification products were differentiated using the Agilent 2100 Bioanalyzer. One FFPE specimen failed to amplify the wild-type alleles for all three sites and was therefore called indeterminate. All 62 FFPE specimens with known Ashkenazi Jewish founder mutations had both the wild-type and the correct mutated allele amplified, including one specimen that failed to amplify the mutant allele in other real-time PCR assays. Appropriately, 21 FFPE specimens known to have other BRCA1/2 mutations and 16 without any mutation had only the wild-type allele correctly amplified for each target. Therefore, by changing the primer location and detecting amplicons via heteroduplexes formed by size differences, we identified mutations from FFPE tissues missed using real-time methods.

Influenza A subtyping: seasonal H1N1, H3N2, and the appearance of novel H1N1.

Influenza virus subtyping has emerged as a critical tool in the diagnosis of influenza. Antiviral resistance is present in the majority of seasonal H1N1 influenza A infections, with association of viral strain type and antiviral resistance. Influenza A virus subtypes can be reliably distinguished by examining conserved sequences in the matrix protein gene. We describe our experience with an assay for influenza A subtyping based on matrix gene sequences. Viral RNA was prepared from nasopharyngeal swab samples, and real-time RT-PCR detection of influenza A and B was performed using a laboratory developed analyte-specific reagent-based assay that targets a conserved region of the influenza A matrix protein gene. FluA-positive samples were analyzed using a second RT-PCR assay targeting the matrix protein gene to distinguish seasonal influenza subtypes based on differential melting of fluorescence resonance energy transfer probes. The novel H1N1 influenza strain responsible for the 2009 pandemic showed a melting profile distinct from that of seasonal H1N1 or H3N2 and compatible with the predicted melting temperature based on the published novel H1N1 matrix gene sequence. Validation by comparison with the Centers for Disease Control and Prevention real-time RT-PCR for swine influenza A (novel H1N1) test showed this assay to be both rapid and reliable (>99% sensitive and specific) in the identification of the novel H1N1 influenza A virus strain.

Neisseria species identification assay for the confirmation of Neisseria gonorrhoeae-positive results of the COBAS Amplicor PCR.

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.

Prostate cancer cells regulate growth and differentiation of bone marrow endothelial cells through TGFbeta and its receptor, TGFbetaRII.

BACKGROUND: The underlying mechanisms permitting prostate cancer bone metastasis are poorly understood. We previously showed that the highly metastatic prostate cancer cell line, PC-3, inhibits bone marrow endothelial (HBME-1) cell growth in collagen gels and induces them to differentiate into cords, resembling angiogenesis in vivo. METHODS: cDNA microarray analysis was performed to identify cytokines responsible for the effects of PC-3 cells on HBME-1 cells. Cytokine and neutralizing antibody studies were done to further investigate specific angiogenic factors, such as transforming growth factor beta (TGFbeta). TGFbeta RNA and protein were detected by real-time RT-PCR and enzyme-linked immunosorbent assay (ELISA) analysis to measure their production by prostate cancer cell lines. Conditioned media experiments using TGFbeta neutralizing antibodies were used to analyze TGFbeta activation by prostate cancer cells. RESULTS: PC-3 conditioned media altered the expression of several TGFbeta-regulated or -associated genes in HBME-1 cells. Low concentrations of TGFbeta cytokines inhibited HBME-1 cell growth to a similar level as PC-3 conditioned media and partially induced differentiation. Inhibitors and neutralizing antibodies directed against TGFbeta isoforms and TGFbeta receptor type 2 (TGFbetaRII) reversed the growth inhibition of HBME-1 cells conferred by PC-3 conditioned media. Yet, only TGFbetaRII neutralizing antibodies significantly inhibited HBME-1 differentiation. Also, prostate cancer cell lines produced low levels of TGFbeta RNA and protein, and were shown to activate serum-derived TGFbeta. CONCLUSIONS: These results suggest that prostate cancer cells mediate growth inhibition and differentiation of bone marrow endothelial cells both through production and activation of TGFbeta as well as alteration of TGFbetaRII-mediated signal transduction. This could contribute to the establishment and growth of bone metastases.