Real-time Reverse Transcriptase-polymerase Chain Reaction Results Research Articles

Low-cost high-throughput targeted sequencing for the accurate detection of respiratory tract pathogens.

The current gold standard for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis by real-time reverse transcriptase polymerase chain reaction (RT-PCR) is limited by the number of genes that can be detected. In this study, we developed a low-cost and high-throughput next-generation sequencing technology that can overcome the limitations of real time RT-PCR. A targeted sequencing panel (TSP) consisting of approximately 500 amplicons was designed. This panel could simultaneously detect a broad range of gene loci of SARS-CoV-2, and genes for the most common infectious viruses that affect the respiratory tract, in a single run and could include up to 96 samples. Four hundred and forty-eight samples and 31 control samples were analyzed independently with both TSP and RT-PCR, and the results were compared for accuracy and other indicators. TSP identified 50 SARS-CoV-2 positive samples with a 99.33% match to RT-PCR results. It is not surprising that TSP also identified multiple infections from the 96 samples, whereas RT-PCR could not. Thus, TSP was able to accurately diagnose the samples which could not be identified based on single RT-PCR test. Our data demonstrated that TSP is a fast and accurate testing method for identifying multiple pathogen infections of the respiratory tract.

Hematological parameters of COVID-19 patients at Jakarta Pondok Kopi Islamic Hospital

Background: The 2019 Corona Virus Disease (Covid-19) has become a pandemic. As of 15 January 2021, 223 countries have been infected, with a total number of 92,262,621 confirmed cases, and 1,995,037 deaths. In Indonesia, as of the same date, there were 896,642 confirmed cases and 25,767 deaths.1 Diagnosis of COVID-19 is based on real-time reverse transcriptase-polymerase chain reaction (RT-PCR) results on oropharyngeal/nasopharyngeal swab samples.2 A basic hematological examination is also conducted on patients with suspected COVID-19. The results of baseline hematological examinations are thought to be a predictor of the patient being infected with COVID-19 before the RT-PCR results are released. The purpose of this study is to identify the hematological parameters of COVID-19 patients at Pondok Kopi Islamic Hospital in Jakarta (RSIJ PK) in order to provide an overview of the hematological parameters that can be used to predict the possibility of a patient being infected with COVID-19. Methods: The study adopted a cross-sectional approach, using the database on COVID-19 patients at Pondok Kopi Islamic Hospital. Data variables were categorized and described by frequency and percentage. Statistical analysis was conducted using the likelihood ratio test and SPSS (statistical package for the social sciences) version 26. Results: The number of research respondents was 250, with 142 males (56.8%). The majority of participants were between the ages of 40 and 59, at 127 (50.8%). Most patients exhibited a moderate level of clinical severity, at 215 (86%). The three most common comorbidities were hypertension in 82 patients (32.8%), diabetes in 68 patients (27.2%), and chronic kidney disease in 27 patients (10.8%). The dominant hematological parameters were normal blood leukocytes in 157 patients (62.8%), increased Neutrophil/Lymphocyte Ratio (NLR) (>3.13) in 148 patients (59.2%), low Absolute Lymphocyte Count (ALC) (<1500/µL) in 133 patients (53.2%), increased C-Reactive Proteins (CRP) (>10 mg/L) in 171 patients (68.4%), and increased d-dimer (>0.5 µg/mL) in 148 patients (59.2%). There was a significant correlation between age (p 0.028), comorbid hypertension (p 0.002), comorbid diabetes (p 0.011), leukocyte levels (p 0.045), and ALC levels (p 0.025), and the severity of the COVID-19 disease. Conclusions: The majority of COVID-19 patients had normal blood leukocyte levels, increased NLR, low ALC, and increased CRP and d-dimer.

Prediction model to identify infectious COVID-19 patients in the emergency department.

Real-time reverse-transcriptase polymerase chain reaction (RT-PCR) has been the gold standard for diagnosing coronavirus disease 2019 (COVID-19) but has a lag time for the results. An effective prediction algorithm for infectious COVID-19, utilized at the emergency department (ED), may reduce the risk of healthcare-associated COVID-19. To develop a prototypic prediction model for infectious COVID-19 at the time of presentation to the ED. Retrospective cohort study of all adult patients admitted to Singapore General Hospital (SGH) through ED between March 15, 2020, and December 31, 2022, with admission of COVID-19 RT-PCR results. Two prediction models were developed and evaluated using area under the curve (AUC) of receiver operating characteristics (ROC) to identify infectious COVID-19 patients (cycle threshold (Ct) of <25). Total of 78,687 patients were admitted to SGH through ED during study period. 6,132 of them tested severe acute respiratory coronavirus 2 positive on RT-PCR. Nearly 70% (4,226 of 6,132) of the patients had infectious COVID-19 (Ct<25). Model that included demographics, clinical history, symptom and laboratory variables had AUROC of 0.85 with sensitivity and specificity of 80.0% & 72.1% respectively. When antigen rapid test results at ED were available and added to the model for a subset of the study population, AUROC reached 0.97 with sensitivity and specificity of 95.0% and 92.8% respectively. Both models maintained respective sensitivity and specificity results when applied to validation data. Clinical predictive models based on available information at ED can be utilized for identification of infectious COVID-19 patients and may enhance infection prevention efforts.

Diagnostic Efficacy of Chest Computed Tomography for Coronavirus Disease 2019.

A significant discrepancy between the results of previous studies is identified regarding the diagnostic efficacy of chest computed tomography (CT) for coronavirus disease 2019 (COVID-19). We aimed to evaluate the diagnostic efficacy of chest CT for COVID-19. Suspected cases of COVID-19 with fever, cough, dyspnea, and evidence of pneumonia on chest CT scan were enrolled in the study. The accuracy, sensitivity, and specificity of chest CT were determined according to real-time reverse transcriptase-polymerase chain reaction (RT-PCR) results as the gold standard method. The study population comprised 356 suspected cases of COVID-19 (174 men and 182 women; age range 3-96 years; mean age ± standard deviation, 55.21 ± 18.38 years). COVID-19 patients were diagnosed using chest CT with 89.8% sensitivity, 78.1% accuracy, 21.3% specificity, 84.7% positive predictive value, and 30.23% negative predictive value. The odds ratio was 2.39 (95% confidence interval, 1.16-4.91). Typical CT manifestations of COVID-19 were observed in 48 (13.5%) patients with negative RT-PCR results and 30 (8.4%) patients with confirmed positive RT-PCR results had no radiological manifestations. Kappa coefficient of chest CT for diagnosis of COVID-19 was 0.78. The results show that when RT-PCR results are negative, chest CT could be considered as a complementary diagnostic method for the diagnosis of COVID-19 patients. A more comprehensive diagnostic method could be established by combining the chest CT examination, clinical symptoms, and RT-PCR assay.

Use of the COVID-19 Reporting and Data System (CO-RADS) classification and chest computed tomography involvement score (CT-IS) in COVID-19 pneumonia.

PurposeThe increasing tendency of chest CT usage throughout the COVID-19 epidemic requires new tools and a systematic scheme for diagnosing and assessing the lung involvement in Coronavirus Disease 2019 (COVID-19). To investigate the use of the COVID-19 Reporting and Data System (CO-RADS) classification and chest CT Involvement Score (CT-IS) in COVID-19 pneumonia.Material and methodsThis retrospective study enrolled 280 hospitalized patients diagnosed with COVID-19 pneumonia in a tertiary hospital in Turkey. All patients underwent non-contrast CT chest imaging. Two radiologists interpreted all CT images according to CO-RADS classification without knowing the clinical features, laboratory findings. We used CT involvement score (CT-IS) for assessing chest CT images of COVID-19 patients. Also, we examined the relationship between CT-IS and clinical outcomes in COVID-19 patients.ResultsOf the patients, 111(39.6%) had positive real-time reverse transcriptase-polymerase chain reaction (RT-PCR) results. CO-RADS 5 group patients had statistically significant positive RT-PCR results than the other groups (P < 0.001). All of the CO-RADS 2 group patients (30) had negative RT-PCR results. The mean total CT-IS in CO-RADS 2 group was 3.4 ± 2.8. The mean total CT-IS in CO-RADS 5 group was 8.2 ± 4.7. Total CT-IS was statistically significantly different among CO-RADS groups (P < 0.001). The mean total CT-IS was statistically significantly different between survivors and patients died of COVID-19 pneumonia (P < 0.001).ConclusionsCO-RADS is useful in detecting COVID-19 disease, even if RT-PCR testing is negative. CT-IS is also helpful as an imaging tool for evaluation of the severity and extent of COVID-19 pneumonia.

Outcome of COVID-19 infection in cancer patients during active systemic anticancer treatment. Single-institution experience. A retrospective analysis.

IntroductionPatients with cancer undergoing active systemic anticancer treatment (chemotherapy, immunotherapy, targeted, or combination therapy) are at greater risk of COVID-19 infection than persons without cancer. In this paper, the authors analyse the spread of the coronavirus among cancer patients undergoing systemic therapy, and the impact of COVID-19 infection on the continuation of cancer treatment and its outcome at one community hospital in a mid-sized city in the south of Poland.Material and methodsNasopharyngeal swab was the only collection method used to obtain specimens for testing via real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Only those with positive RT-PCR results were considered as confirmed SARS-CoV-2 cases. We analysed the medical records of patients quarantined in a hospital clinical oncology ward due to confirmed COVID-19 infection in one member of the group. Qualitative measures are presented as the percentage of their occurrence, and these were evaluated with Fisher’s test. Differences were considered significant at p < 0.05.ResultsCancer patients had more frequent confirmed COVID-19 infection than other patients (3.7% vs. 1.2%). Among cancer patients COVID-19 infection was significantly more frequent in women than in men, p = 0.005. The fatality rate was 27.3% in cancer patients undergoing active anticancer therapy, compared to 3% in the general Polish population. Neither heparin nor G-CSF use had any influence on COVID-19 infection.ConclusionsIn this analysis, the only significant negative factor for COVID-19 infection was female sex, RR (95% CI) = 4.5 (1.3–15.8), (p = 0.005), and this was attributable to individual behaviour.

Prolonged SARS-CoV-2 detection and reversed RT-PCR results in mild or asymptomatic patients

Background The clinical course and viral detection period in mild or asymptomatic coronavirus disease 2019 (COVID-19) patients are not yet known. The presumed low diagnostic sensitivity of upper respiratory specimens for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) makes it difficult to confirm infection and recommend de-isolation. Methods We retrospectively reviewed real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test results of mild or asymptomatic COVID-19 patients who were admitted at the Daegu-Gyeongbuk 7th community treatment centre in Korea between 9 March 2020 and 10 April 2020. Patients underwent an upper respiratory RT-PCR test every week until discharge. From the RT-PCR results, we evaluated the rate of prolonged (>3 weeks) SARS-CoV-2 RNA positivity. We analysed the proportion of reversed results, defined as a positive or indeterminate result one day after a negative RT-PCR result, according to time (<14, 15–21, 22–28, >28 days) from the initial positive RT-PCR result. Results In 23% (69/300) of patients, SARS-CoV-2 was detected more than 3 weeks after the initial positive RT-PCR. In 14% (42/300) of patients, the RT-PCR results were positive for more than 4 weeks. For 37.5% (152/405) of negative RT-PCR results, the results were reversed in the next day’s test. And 43.5% (123/283) of negative RT-PCR results were reversed within 3 weeks of diagnosis. Conclusions The detection of SARS-CoV-2 lasting more than 3 weeks was common in mild or asymptomatic patients. Upper respiratory RT-PCR results were frequently reversed from negative to positive.

Nitric Oxide Synthesis Inhibition and Anti-Inflammatory Effect of Polypeptide Isolated from Chicken Feather Meal in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages.

SUMMARYNitric oxide (NO) plays a key role in the pathogenesis of inflammation and has been implicated in endotoxin-induced tissue injury. Chicken feather meal is a rich source of amino acids that may serve as a peptide hydrolysate to inhibit NO activity. Anti-inflammatory hydrolysates of chicken feather meal were prepared and fractionated into five samples based on molecular mass. The smallest fraction (<0.65 kDa) exhibited the highest NO inhibitory activity without cytotoxicity towards macrophage RAW 264.7 cells. Further subfractions were sufficient to obtain amino acid sequences by Q-TOF LC-MS/MS ESI analysis. Of these, the SNPSVAGVR (885.97 Da) peptide and its corresponding pure synthetic peptide have inhibitory activity against NO production by RAW 264.7 cells (IC50=(55.2±0.2) mM) without cytotoxicity. Reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR results revealed that the peptide of the obtained fraction reduced transcript expression levels of the pro-inflammatory cytokines iNOS, TNF-α, COX-2 and IL-6 in lipopolysaccharide-stimulated RAW 264.7 cells. These results suggest that the peptides derived from the chicken feather meal protein could potentially be used as a promising ingredient in functional foods or nutraceuticals against inflammatory diseases.

Investigation of avian influenza infection in wild birds in Ismailia and Damietta cities, Egypt.

Aim:This study was carried out to monitor avian influenza (AI) infection in wild birds in Egypt.Materials and Methods:A total of 135 wild birds were examined for the presence of H5, H7, and H9 hemagglutination inhibition antibodies. Organs and swab samples of 75 birds were screened by multiplex real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) to detect AI subtypes H5, H7, and H9 matrix genes.Results:The highest seropositive result was recorded in cattle egrets (90.9%) followed by crows (88.6%), semi-captive pigeons (44.8%), and moorhens (39.1%). In cattle egrets, semi-captive pigeons and moorhens, H5 antibodies predominated. In crows, H9 antibodies predominated. Multiple infections with two or three virus subtypes were highest in crows (6/39, 15.4%) followed by cattle egrets (3/30, 10%) and moorhens’ (1/9, 11.1%) positive samples. Multiplex RRT-PCR results revealed two positive samples in cattle egrets and moorhens.Conclusion:The results indicated high seropositive rates against AI virus subtypes H5 and H9 in the examined wild birds. Multiple infections with more than one AI virus (AIV) subtypes were detected in some birds. This requires a collaboration of efforts to monitor AIV infection in wild birds and implement suitable early intervention measures.

Prevalence and molecular characterization of avian infectious bronchitis virus in poultry flocks in Morocco from 2010 to 2014 and first detection of Italy 02 in Africa

The aim of this study was to investigate the prevalence and diversity of infectious bronchitis virus (IBV) genotypes in poultry flocks in 16 areas of Morocco between 2010 and 2014. A total of 360 chicken flocks suspected of being infected by IBV were screened for the IBV N gene using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Flocks were classified into four groups according to their IBV vaccination programme. Group 1 contained unvaccinated birds. Group 2 received a single application of live H120 vaccine. Groups 3 and 4 birds received one or two booster vaccination(s), respectively, mostly using the H120 vaccine. The real-time RT-PCR results showed that 51.7% of the flocks were positive for the IBV genome with geographical disparities. Molecular characterization of IBV was performed on 50 RT-PCR positive samples by partially sequencing the S1 gene, including the hypervariable regions (nucleotides 705–1097). Two predominant genotypes were detected, with the Massachusetts type dominating (66%), among which 25% of the samples were identical to the H120 vaccine. The second most common genotype (present in 32% of the flocks) was surprisingly Italy 02, revealing the first detection of this genotype in Morocco and also in Africa. 793B, the predominant genotype in the late 1990s in Morocco, was only detected on one occasion and was identical to the 4/91 vaccine strain. This study highlights the high prevalence of IBV in poultry farms in Morocco and confirms its continuous dynamic changes and evolution.

The effect of peptidoglycan on the secretion of pro-inflammatory cytokines by dendritic cells and the regulation of T helper 17 responses in experimental autoimmune uveitis

Objective To investigate the effect of peptidoglycan (PGN) on the secretion of proinflammatory cytokines by dendritic cells (DCs) and the regulation of T helper 17 (Th17) responses in experimental autoimmune uveitis.Methods Bone marrow cells from naive mice were cultured with granulocyte macrophage-colony-stimulating factor and interleukin (IL)-4 to induce DCs.DCs cultured for six days were randomly divided into two groups:PGN-treated group and control group.The DCs in PGN-treated group were stimulated with PGN and the same volume of phosphate buffered saline was added to the DCs as control group.The relative mRNA expression levels of IL-23,tumor necrotic factor α (TNF-α),IL-6,IL-1βwere measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR).Peptide fragment of interphotoreceptor retinoid-binding protein (IRBP1-20)-specific T cells,which were isolated from the spleen and draining lymph nodes of C57BL/6 mice immunized with IRBP1-20 peptide fragments 13 days earlier,were co-cultured with PGN-treated or untreated DCs,respectively.Total RNA from T cells cocultured for two days were isolated and the relative expression of retinoic acid receptor-related orphan receptor γt (ROR-γt),IL-17,T-box expression in T cells (T-bet),interferon γ (IFN-γ) mRNA were detected by real-time RT-PCR.On the second,the fifth and the seventh day,the cocultured T cells were analyzed by flow cytometry to detect the percentages of IFN-γ,IL-17 positive cells.Results The real-time RT-PCR results revealed that the level of IL-23,IL-1β,IL-6,TNF-α mRNA from PGN-stimulated DCs were significantly increased compared to the control group (t =-14.363,-5.627,-3.85,-28.151 ;P＜0.05).The level of RORγt,IL-17 mRNA from the T cells cocultured with PGN-stimulated DCs were greatly increased compared with the control group (t=-5.601,-19.76; P＜0.05).However,the level of T-bet,IFN-γmRNA from the T cells cocultured with PGN-stimulated DCs were significantly decreased compared with the control group (t=4.717,11.207; P＜0.05).Data of flow cytometry showed that at two days,five days,seven days after cocultured with PGN-treated DCs,the percentages of IL-17 positive T cells were increased compared to the control group (t=-2.944,-3.03,-4.81; P＜0.05),and the percentages of IFN-γpositive T cells had no remarkable change (t=-1.25,-0.18,-2.16; P＞0.05).Conclusion PGN can promote the secretion of Th17-related cytokines by DCs,which favors proliferation and differentiation of Th17 in experimental autoimmune uveitis. Key words: Peptidoglycan/pharmacology; Uveitis/physiopathology; Toll-like receptor 2; T-cell antigen receptor specificity; Th17 cells

Pre-emptive Therapy for the Cytomegalovirus Infection After Liver Transplantation in Endemic Areas and Its Optimal Diagnostic Method

BackgroundThe incidence of positive cytomegalovirus (CMV) IgG tests among Asian populations is high. Both universal prophylaxis and pre-emptive therapy (PT) have been recommended for the moderate-risk group (D+/R+), whose incidence of CMV infection has been reported variously, and for whom the optimal diagnostic method has not been firmly established. Herein, we sought to analyze our experience with CMV infections using PT and to discuss the optimal diagnostic method. MethodsWe retrospectively, analyzed 32 consecutive liver transplant recipients between December 2009 and April 2012 for clinicopathologic data including mortality and rejection rates, comparing 2 diagnostic tools for CMV: pp65 antigen assay and real-time reverse-transcriptase polymerase chain reaction (RT-PCR). ResultsTwenty-one patients (65.6%) were positive for the CMV antigen assay, and 13 (40.6%) had positive RT-PCR results. There were no cases of CMV disease during the follow-up and no difference in rejection (P = .529) or mortality (P = .471) rates with regard to PCR positivity. The mean diagnosis time was 26.5 days postoperative. Among the patients who exhibited negative RT-PCR results, 7 (41.18%) were positive on the pp65 antigen assay. ConclusionCMV infection rates were higher when compared to same-risk population from Western countries. As a diagnostic tool for CMV infection, screening with the pp65 antigen assay and confirmation with real-time RT-PCR seemed to provide an optimal diagnostic tool.

Identification of novel candidate tumour marker genes for intrahepatic cholangiocarcinoma

Specific markers are required for early detection and diagnosis of intrahepatic cholangiocarcinoma (ICC); however, the tumour markers currently in use are not specific for ICC. We compared an ICC cDNA library with that of hepatocellular carcinoma (HCC) by serial analysis of gene expression (SAGE). The expression patterns in each were confirmed by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and immunohistochemical analysis of 74 samples including 16 ICC samples. A comparison of the two libraries revealed distinct gene expression patterns for each type of liver cancer. In addition to the known tumour markers, we detected nine novel genes associated with ICC. By comparing the mean transcript abundance in the ICC library with those in other libraries, including gastric, colon, prostate and breast cancer, together with our RT-PCR results, we identified three genes as specific markers of ICC: biglycan, insulin-like growth factor-binding protein 5 and claudin-4. Immunoblotting and immunohistochemical analyses showed that claudin-4 was highly expressed in ICC. Moreover, discrimination analysis revealed that a combination of these genes could be used to distinguish ICC from HCC or metastatic adenocarcinoma. We identified novel marker genes of ICC that are potentially useful for the diagnosis of liver cancer.

Distribution of free cancer cells in the abdominal cavity suggests limitations of bursectomy as an essential component of radical surgery for gastric carcinoma

Bursectomy, which has been performed so as to resect peritoneal deposits disseminated within the omental bursa, is considered as an essential component of radical surgery for gastric carcinoma in Japan. Bursectomy has also been described in the Japanese Treatment Guidelines for Gastric Carcinoma as a mandatory procedure for the treatment of serosa-positive cancer. However, no evidence to support the prognostic significance of this procedure has been reported to date. Cytologic examination and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of the peritoneal washes obtained from the Douglas pouch, left subphrenic cavity, and inside the omental bursa were performed for 136 patients who underwent potentially curative surgery for gastric carcinoma. Carcinoembryonic antigen (CEA) or cytokeratin (CK) 20 mRNA was detected in one or more samples from the three different sites of peritoneal washes in 43 of the 136 patients. In 14 patients, the mRNAs were detected in samples obtained from the bursa omentalis (10.3% of all patients and 32.6% of patients with positive RT-PCR results). In 12 of these 14 patients, the mRNAs were also detected in samples taken from either or both of the remaining two sites. Only in the 2 other patients was the sample only from inside the omental bursa positive for CEA. It is unlikely that viable cancer cells disseminated into the bursa remain restricted to this cavity without migrating into the free abdominal cavity. Routine bursectomy may not be an essential procedure for resecting gastric cancer, from the viewpoint of eliminating microscopic peritoneal deposits within the omental bursa.

Leptin Receptor Isoforms mRNA Expression in Peripheral Blood Mononuclear Cells from Patients with Chronic Viral Hepatitis

There is accumulating evidence that leptin has a pleiotropic role in hematopoiesis, immune response, fibrogenesis, and hepatocarcinogenesis. We investigated the expression of leptin and leptin receptor (OB-R) at the protein level by flow cytometry and also quantified by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) the two major leptin receptor isoforms (OB-Rl, OB-Rs) in peripheral blood mononuclear cells (PBMCs) of patients with hepatitis B (HBV; n = 31), hepatitis C (HCV; n = 34), and nonviral liver disease (n = 25), and healthy controls (n = 36), as well as in liver tissues of HBV (n = 8), HCV (n = 7), and healthy individuals (n = 6). Serum leptin levels were measured in all participants (N = 126). We observed significantly lower OB-Rl and OB-Rs mRNA levels in PBMCs of HBV and HCV patients compared with healthy controls and nonviral liver disease patients (P < 0.05). Flow cytometry analysis confirmed the real-time RT-PCR results. Expression of leptin and OB-Rl was significantly increased in viral hepatitis liver tissues compared with healthy tissues (P < 0.01). OB-Rl mRNA levels were not associated with hepatitis patients' clinical status (inactive, chronic hepatitis, or cirrhosis). We also found decreased serum leptin in HBV and HCV patients compared with healthy individuals and the nonviral liver disease group. Leptin was expressed in 3 of 34 HCV (8.8%) and 19 of 25 (76%) nonviral liver disease patients. Moreover, expression of OB-Rl and OB-Rs were associated when all individuals were grouped together (r = 0.78, P < 0.001). In conclusion, our findings may suggest the involvement of the leptin system in the immunopathology of chronic viral hepatitis.

Isolation by size of epithelial tumor cells in peripheral blood of patients with breast cancer: correlation with real-time reverse transcriptase–polymerase chain reaction results and feasibility of molecular analysis by laser microdissection

The aim of this study is the counting and the immunomorphological and molecular characterization of circulating tumor cells (CTCs) by the isolation by size of epithelial tumor cells (ISET) method in the peripheral blood of patients with breast cancer. An evaluation of the method's ability to reveal the presence of occult carcinoma cells in blood of a patient with breast cancer was performed and the results compared with those obtained by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay for the evaluation of cytokeratin-19 (CK-19) mRNA expression. The feasibility of molecular analysis of CTCs after laser microdissection of filters used in ISET was illustrated, referring to HER-2 amplification. Blood samples drawn from 44 patients with breast cancer were preoperatively analyzed by ISET. From the same samples, total RNA was extracted and submitted to quantitative real-time RT-PCR for the detection of CK-19 mRNA-positive cells using TaqMan technology. HER-2 amplification was measured by real-time RT-PCR on DNA extracted from cells recovered by laser microdissection from 7 selected ISET-positive filters. Of 44 samples, 12 (27%) showed the presence of epithelial cells on the filter (mean +/- SE: 8.5 +/- 2.4 cells per milliliter of blood). A statistically significant agreement (P = .001) was observed between real-time RT-PCR results and those obtained by ISET. With regard to HER-2 amplification, a good correspondence was found between the results obtained from microdissected CTCs and those obtained using DNA extracted from the primary tumor (R = 0.918; P < .01), as well as the immunohistochemistry results. The ISET method allows for the collection of breast carcinoma cells by filtration despite their smaller dimension relative to other carcinoma cell types. The sensitivity and specificity of the method is comparable with those obtained using the quantitative real-time RT-PCR assay for the evaluation of CK-19 mRNA expression. Moreover, the laser microdissection technique allows for the recovery of nucleic acids for further molecular analysis and CTC characterization.

Reliable Transcript Quantification by Real-Time Reverse Transcriptase-Polymerase Chain Reaction in Primary Neuroblastoma Using Normalization to Averaged Expression Levels of the Control Genes HPRT1 and SDHA

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) represents a sensitive and efficient technique to determine expression levels of target genes in multiple samples and is increasingly used in clinical oncology to evaluate the patient's outcome or to detect minimal residual disease. Normalization of raw data are required to obtain comparable results between different specimens and is usually achieved by correlating transcript abundances of target genes with those of a single control gene with putatively stable expression levels. In this study, expression stability of six supposed control genes was evaluated in 64 samples of primary neuroblastoma and HPRT1 and SDHA mRNA levels were shown to exhibit the least expression variability among the samples. Because application of more than one control gene may enhance reliability of real-time RT-PCR results, various normalization factors consisting of the geometrical mean of multiple control gene expression values were calculated and evaluated by mRNA quantification of 14 target genes. Comparison with transcript levels determined by oligonucleotide-array expression analysis revealed that target gene mRNA quantification became most consistent after normalization to averaged expression levels of HPRT1 and SDHA. This normalization factor was in addition demonstrated to be not associated with stage of disease or MYCN amplification status of the tumor. Thus, these data indicate that the geometrical mean of HPRT1 and SDHA transcript levels represents a suitable internal control for biological and clinical studies investigating differential gene expression in primary neuroblastoma by real-time RT-PCR.