Positive Reverse Transcriptase-polymerase Chain Reaction Results Research Articles

Performance of Rapid Antigen Tests to Detect Symptomatic and Asymptomatic SARS-CoV-2 Infection : A Prospective Cohort Study.

The performance of rapid antigen tests (Ag-RDTs) for screening asymptomatic and symptomatic persons for SARS-CoV-2 is not well established. To evaluate the performance of Ag-RDTs for detection of SARS-CoV-2 among symptomatic and asymptomatic participants. This prospective cohort study enrolled participants between October 2021 and January 2022. Participants completed Ag-RDTs and reverse transcriptase polymerase chain reaction (RT-PCR) testing for SARS-CoV-2 every 48 hours for 15 days. Participants were enrolled digitally throughout the mainland United States. They self-collected anterior nasal swabs for Ag-RDTs and RT-PCR testing. Nasal swabs for RT-PCR were shipped to a central laboratory, whereas Ag-RDTs were done at home. Of 7361 participants in the study, 5353 who were asymptomatic and negative for SARS-CoV-2 on study day 1 were eligible. In total, 154 participants had at least 1 positive RT-PCR result. The sensitivity of Ag-RDTs was measured on the basis of testing once (same-day), twice (after 48 hours), and thrice (after a total of 96 hours). The analysis was repeated for different days past index PCR positivity (DPIPPs) to approximate real-world scenarios where testing initiation may not always coincide with DPIPP 0. Results were stratified by symptom status. Among 154 participants who tested positive for SARS-CoV-2, 97 were asymptomatic and 57 had symptoms at infection onset. Serial testing with Ag-RDTs twice 48 hours apart resulted in an aggregated sensitivity of 93.4% (95% CI, 90.4% to 95.9%) among symptomatic participants on DPIPPs 0 to 6. When singleton positive results were excluded, the aggregated sensitivity on DPIPPs 0 to 6 for 2-time serial testing among asymptomatic participants was lower at 62.7% (CI, 57.0% to 70.5%), but it improved to 79.0% (CI, 70.1% to 87.4%) with testing 3 times at 48-hour intervals. Participants tested every 48 hours; therefore, these data cannot support conclusions about serial testing intervals shorter than 48 hours. The performance of Ag-RDTs was optimized when asymptomatic participants tested 3 times at 48-hour intervals and when symptomatic participants tested 2 times separated by 48 hours. National Institutes of Health RADx Tech program.

AN ANALOGY OF HRCT CHEST AND ANTIGEN ASSAY WITH RT- PCR TEST IN THE DIAGNOSIS OF COVID-19

Introduction We aim to compare Rapid Antigen Test and HRCT chest with Reverse Transcriptase-Polymerase chain reaction (RT-PCR) for diagnosis of COVID-19. MethodsThis observational cross-sectional analytical study included patients tested with RT- PCR and at least one Rapid Antigen test or HRCT chest, which was conducted at ACS Medical college Chennai, from June 2021 to June 2022 after obtaining the ethical clearance. Data analysis was done using statistical packages for social sciences version 16. Results 315 of 755(41.7%) patients had positive RTPCR and 18.4% of 733 patients had positive antigen tests. Of 600 patients, 106(14.0%) were found to have CO-RADS 1,152(20.1%) had CORADS 2, 77(10.2%) had CO-RADS 3, 18(2.4%) had CO-RADS 4 and 247(32.7%) had CO-RADS 5 score on HRCT. Rapid Antigen Test was found to have 43.6% sensitivity, 98.6% specicity, 95.6% Positive predictive value (PPV), and 72.1% negative predictive value (NPV).CO-RADS scoring system was able to distinguish between RT-PCR positive and RT-PCR negative results with an average Area under curve 0.787. CO-RADS 5 had a sensitivity of 71.6%, specicity of 85.1%, PPVof 80.6%, and NPVof 77.6% for a positive RT-PCR result. Acombination of CO-RADS 1 in HRCT chest and a Negative Antigen test can predict PCR negative results with 23.6% sensitivity, 97.3% specicity, 90.5% PPV, and 54.0% NPV. Conclusions Rapid Antigen Test is a better tool for conrmation rather than screening for COVID-19. HRCT chest with higher CO-RADS can be useful to diagnose COVID-19 in suspected patients, even if RT-PCR is negative.

Vertical Transmission of Severe Acute Respiratory Syndrome Coronavirus 2: Current Evidence and Perspectives

Vertical Transmission of Severe Acute Respiratory Syndrome Coronavirus 2: Current Evidence and Perspectives

Comparison of Rapid Antigen Tests' Performance Between Delta and Omicron Variants of SARS-CoV-2 : A Secondary Analysis From a Serial Home Self-testing Study.

It is important to document the performance of rapid antigen tests (Ag-RDTs) in detecting SARS-CoV-2 variants. To compare the performance of Ag-RDTs in detecting the Delta (B.1.617.2) and Omicron (B.1.1.529) variants of SARS-CoV-2. Secondary analysis of a prospective cohort study that enrolled participants between 18 October 2021 and 24 January 2022. Participants did Ag-RDTs and collected samples for reverse transcriptase polymerase chain reaction (RT-PCR) testing every 48 hours for 15 days. The parent study enrolled participants throughout the mainland United States through a digital platform. All participants self-collected anterior nasal swabs for rapid antigen testing and RT-PCR testing. All Ag-RDTs were completed at home, whereas nasal swabs for RT-PCR were shipped to a central laboratory. Of 7349 participants enrolled in the parent study, 5779 asymptomatic persons who tested negative for SARS-CoV-2 on day 1 of the study were eligible for this substudy. Sensitivity of Ag-RDTs on the same day as the first positive (index) RT-PCR result and 48 hours after the first positive RT-PCR result. A total of 207 participants were positive on RT-PCR (58 Delta, 149 Omicron). Differences in sensitivity between variants were not statistically significant (same day: Delta, 15.5% [95% CI, 6.2% to 24.8%] vs. Omicron, 22.1% [CI, 15.5% to 28.8%]; at 48 hours: Delta, 44.8% [CI, 32.0% to 57.6%] vs. Omicron, 49.7% [CI, 41.6% to 57.6%]). Among 109 participants who had RT-PCR-positive results for 48 hours, rapid antigen sensitivity did not differ significantly between Delta- and Omicron-infected participants (48-hour sensitivity: Delta, 81.5% [CI, 66.8% to 96.1%] vs. Omicron, 78.0% [CI, 69.1% to 87.0%]). Only 7.2% of the 69 participants with RT-PCR-positive results for shorter than 48 hours tested positive by Ag-RDT within 1 week; those with Delta infections remained consistently negative on Ag-RDTs. A testing frequency of 48 hours does not allow a finer temporal resolution of the analysis of test performance, and the results of Ag-RDTs are based on self-report. The performance of Ag-RDTs in persons infected with the SARS-CoV-2 Omicron variant is not inferior to that in persons with Delta infections. Serial testing improved the sensitivity of Ag-RDTs for both variants. The performance of rapid antigen testing varies on the basis of duration of RT-PCR positivity. National Heart, Lung, and Blood Institute of the National Institutes of Health.

Concordance of SARS-CoV-2 RNA in Aerosols From a Nurses Station and in Nurses and Patients During a Hospital Ward Outbreak

Aerosol-borne SARS-CoV-2 has not been linked specifically to nosocomial outbreaks. To explore the genomic concordance of SARS-CoV-2 from aerosol particles of various sizes and infected nurses and patients during a nosocomial outbreak of COVID-19. This cohort study included patients and nursing staff in a US Department of Veterans Affairs inpatient hospital unit and long-term-care facility during a COVID-19 outbreak between December 27, 2020, and January 8, 2021. Outbreak contact tracing was conducted using exposure histories and screening with reverse transcriptase-polymerase chain reaction (RT-PCR) for SARS-CoV-2. Size-selective particle samplers were deployed in diverse clinical areas of a multicampus health care system from November 2020 to March 2021. Viral genomic sequences from infected nurses and patients were sequenced and compared with ward nurses station aerosol samples. SARS-CoV-2. The primary outcome was positive RT-PCR results and genomic similarity between SARS-CoV-2 RNA in aerosols and human samples. Air samplers were used to detect SARS-CoV-2 RNA in aerosols on hospital units where health care personnel were or were not under routine surveillance for SARS-CoV-2 infection. A total of 510 size-fractionated air particle samples were collected. Samples representing 3 size fractions (>10 μm, 2.5-10 μm, and <2.5 μm) obtained at the nurses station were positive for SARS-CoV-2 during the outbreak (3 of 30 samples [10%]) and negative during 9 other collection periods. SARS-CoV-2 partial genome sequences for the smallest particle fraction were 100% identical with all 3 human samples; the remaining size fractions shared >99.9% sequence identity with the human samples. Fragments of SARS-CoV-2 RNA were detected by RT-PCR in 24 of 300 samples (8.0%) in units where health care personnel were not under surveillance and 7 of 210 samples (3.3%; P = .03) where they were under surveillance. In this cohort study, the finding of genetically identical SARS-CoV-2 RNA fragments in aerosols obtained from a nurses station and in human samples during a nosocomial outbreak suggests that aerosols may have contributed to hospital transmission. Surveillance, along with ventilation, masking, and distancing, may reduce the introduction of community-acquired SARS-CoV-2 into aerosols on hospital wards, thereby reducing the risk of hospital transmission.

Oncological Surgery During the COVID-19 Pandemic: Effectiveness of Preoperative Screening and Factors Associated with Postoperative SARS-CoV-2 Infection.

BackgroundRoutine preoperative screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with reverse transcriptase-polymerase chain reaction (RT-PCR) may reduce in-hospital SARS-CoV-2 transmission.MethodsThis was a prospective, observational, cohort study. The endpoints were the incidence of asymptomatic patients with positive preoperative RT-PCR results and the incidence and factors associated with postoperative SARS-CoV-2 infection in patients with cancer referred for elective surgery. Patients with elective surgery between May and October 2020 were included. RT-PCR of nasopharyngeal swabs was performed preoperatively for all patients. Postoperative SARS-CoV-2 infection was assessed within 30 postoperative days.ResultsA total of 1636 preoperative screening RT-PCR tests were performed. Of these, 102 (6.2%) cases were positive, and 1,298 surgical procedures were analyzed. The postoperative SARS-CoV-2 infection rate was 0.9%. The length of stay (odds ratio [OR] 1.08; 95% confidence interval [CI] 1.04–1.11; p < 0.001), surgical time (OR 1.004; 95% CI 1.001–1.008; p = 0.023), intensive care unit admission (OR 7.7; 95% CI 2.03–29.28; p = 0.003), and hospital readmissions (OR 9.56; 95% CI 2.50–36.56; p = 0.001) were associated with postoperative coronavirus disease (COVID-19). Using unadjusted and adjusted logistic regression, length of stay (OR 1.08; 95% CI 1.04–1.11; p < 0.001), and readmission (OR 9.02; 95% CI 2.30–35.48; p = 0.002) were independent factors of postoperative COVID-19.ConclusionsScreening patients preoperatively may reduce in-hospital SARS-CoV-2 transmission. Length of stay and readmission were independently correlated with postoperative COVID-19.

Comparison of COVID-19 Antigen Test and HRCT Chest with RT- PCR Test for Diagnosis of COVID-19

Introduction: We aim to compare Rapid Antigen Test and HRCT chest with Reverse Transcriptase-Polymerase chain reaction (RT-PCR) for diagnosis of COVID-19. Methods: This was a cross-sectional analytical study that included patients tested with RT-PCR and at least one of Rapid Antigen test or HRCT chest, conducted at College of Medical Sciences, Bharatpur, from June 2020 to Feb 2021 after obtaining the ethical clearance. Data analysis was done using statistical packages for social sciences version 16. Results: 315 of 755(41.7%) patients had positive RT-PCR and 18.4% of 733 patients had positive antigen test. Of 600 patients, 106(14.0%) were found to have CO-RADS 1,152(20.1%) had CO-RADS 2, 77(10.2%) had CO-RADS 3, 18(2.4%) had CO-RADS 4 and 247(32.7%) had CO-RADS 5 score on HRCT. Rapid Antigen Test was found to have 43.6% sensitivity, 98.6% specificity, 95.6% Positive predictive value (PPV) and 72.1% negative predictive value (NPV).CO-RADS scoring system was able to distinguish between RT-PCR positive and RT-PCR negative results with an average Area under curve 0.787. CO-RADS 5 had a sensitivity of 71.6%, specificity of 85.1%, PPV of 80.6% and NPV of 77.6% for a positive RT-PCR result. A combination of CO-RADS 1 in HRCT chest and Negative Antigen test can predict PCR negative result with 23.6% sensitivity, 97.3% specificity, 90.5% PPV and 54.0% NPV. Conclusions: Rapid Antigen Test is a better tool for confirmation rather than screening of COVID-19. HRCT chest with higher CO-RADS can be useful to diagnose COVID-19 in suspected patients, even if RT-PCR is negative.

Recurrent and More Severe COVID-19 Infection: Two Elderly Case Reports.

Although the sensitivity of reverse transcriptase-polymerase chain reaction (RT-PCR) is low in the diagnosis of coronavirus disease 2019 (COVID-19), it is the gold standard. Clinical improvement is prioritized in the follow-up of patients with COVID-19 who are followed as possible or definitive cases. Although the priority in the discharge decision is the resolution of complaints, it is also important to see radiological improvement and RT-PCR negativity. A total of 2 of our patients who were hospitalized and treated in our clinic with a diagnosis of COVID-19 were discharged after their complaints were resolved and their treatment was completed. The patients had 2 negative RT-PCR results at discharge. Both of them presented to the hospital with symptoms such as fever, cough, and shortness of breath after the discharge, and both showed positive RT-PCR results. Considering recurrent COVID-19 infection, we aimed to present treatment and the 2 cases we followed.

Detection of SARS-CoV-2 in Simultaneously Collected Tear and Throat Swab Samples from the Patients with 2019- new SARS-CoV-2 Infection Disease: A Single Center Cross-sectional Study

ABSTRACT Objecive : This study aimed to evaluate whether Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) can be detected in the tears in the eyes of Coronavirus Disease 2019 (COVID-2019) infected patients and compare the detection consistency of two simultaneously collected samples, from the tears and throat swabs. Methods : A total of 35 COVID-2019 patients were included in this cross-sectional case series study. Throat samples from all enrolled patients were collected with sampling swab, and simultaneously, tear samples were collected with sampling swab from 9 patients (No.1–9) and with Schirmer’s strip from the remaining patients (No.10–35) (bilateral eyes for all patients). Sample collecting and testing were performed in three separate time points: first from patients No.1–9, second from patients No.10–29, and third from patients No. 30–35. Reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay was performed. Results : Among enrolled patients, 29 (No.1–29) had mild or moderate clinical symptoms and 6 (No.30–35) had severe symptoms. The mean time interval from the sample collection day to diagnosis confirmation day was 9.71 ± 6.50 days (ranged from 3 to 29 days). None of the patients had conjunctivitis. Nineteen out of 35 (54.3%) throat samples presented positive Rt-PCR results. Three (no.13,21,31) out of 35 (8.6%) tear samples presented positive RT-PCR results. Two (no.21, 31) of these three patients were throat swab positive and one (No. 13) was negative. The consistency analysis indicated that tears and throat samples showed poor consistency (Kappa = −0.009, P = .9).The cycle threshold value (Ct-value) of tear samples collected by sampling swab was significantly higher than that by Schirmer’s strip (t = 2.288, P = .03). Conclusion : In spite of the low SARS-CoV-2 positive detection rate of tear samples from COVID-2019 patients, we cannot fully rule out the transmission by ocular surface. Whether tear testing can be used as an aid in judging of SARS-CoV-2 infection need further investigation.

The Role of the Computed Tomography (CT) Thorax in the Diagnosis of COVID-19 for Patients Presenting with Acute Surgical Emergencies. A Single Institute Experience.

The current Coronavirus disease 2019 (COVID-19) pandemic has had a huge impact on emergency surgical services in the UK. The Royal College of Surgeons (RCS) published guidelines about COVID-19 pandemic in March, 2020 to aid decision making for the surgeons. These guidelines recommended that all patients requiring urgent surgery should have reverse transcriptase polymerase chain reaction (RT-PCR) and/or computed tomography (CT) thorax pre-operatively. However, it is currently unclear whether the use of CT thorax is a sensitive and specific diagnostic test. The objective of this study was to find out whether CT thorax is a reliable and accurate test in the diagnosis of COVID-19 compared to RT-PCR. This is particularly important in surgical patients where there is no time to wait for RT-PCR results. A prospective cohort study of patients presented with acute surgical emergencies at a University Teaching Hospital was conducted. Data was collected from March 23, to May 15, 2020, during the peak of the crisis in the UK. All adult patients presented with operable general surgical emergencies were considered eligible. Another group of patients, admitted with acute medical emergencies but with suspected COVID-19 infection, was used for comparison. Data was manually collected, and sensitivity, specificity and predictive value were calculated using the MedCalc statistical software version 19.2.6. Standard reporting for COVID-19 infection for CT chest based on guidelines from British Society of Thoracic Imaging (BSTI) and Radiological Society of North America (RSNA) was used. Patients who had their CT thorax reported as typical or classic of COVID 19 (high probability) were treated as infected cases with extra precautions in the wards and surgical theatres as suggested by health and safety executive (HSE). These patients had serial RT-PCR during their admissions or in the post-operative phase, if the first swab was negative. For the study, 259 patients were considered eligible for inclusion from both groups. Patients admitted for acute surgical emergencies were treated according to RCS guidelines and subjected to RT-PCR test and/or CT scan of the thorax. There were 207 patients with high clinical suspicion of COVID-19. Of those 207 patients, 77 patients had CT thorax with radiographic features consistent with COVID-19 pneumonia. However, only 40 patients had a positive RT-PCR result. CT thorax was normal in 130 patients, out of which 29 patients were found to have COVID-19 diagnosis after swab test. Sensitivity of CT scan to diagnose COVID-19 infection was found to be 58% (95% CI; 45.48% to 69.76%) whilst specificity was 73% (95% CI; 64.99% to 80.37%) with a negative predictive value of 77.69% (95% CI; 72.17% to 82.39%). CT scan was found to be a reliable tool in the diagnosis of COVID-19. With a negative predictive value of up to 82.4%, CT thorax can play an important role to help surgeons in their decision making for asymptomatic suspected cases of COVID-19. However, over-reliance on CT scan which also has a high false positive rate for diagnosis of COVID-19 infections can lead to overtreatment, overuse of resources and delays in decision-making process. Hence, results should be interpreted with caution and correlated with clinical presentation and swab test results.

Guillain–Barré Syndrome Associated with Zika Virus Infection in Colombia

BackgroundZika virus (ZIKV) infection has been linked to the Guillain–Barré syndrome. From November 2015 through March 2016, clusters of cases of the Guillain–Barré syndrome were observed during the outbreak of ZIKV infection in Colombia. We characterized the clinical features of cases of Guillain–Barré syndrome in the context of this ZIKV infection outbreak and investigated their relationship with ZIKV infection.MethodsA total of 68 patients with the Guillain–Barré syndrome at six Colombian hospitals were evaluated clinically, and virologic studies were completed for 42 of the patients. We performed reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assays for ZIKV in blood, cerebrospinal fluid, and urine, as well as antiflavivirus antibody assays.ResultsA total of 66 patients (97%) had symptoms compatible with ZIKV infection before the onset of the Guillain–Barré syndrome. The median period between the onset of symptoms of ZIKV infection and symptoms of the Guillain–Barré syndrome was 7 days (interquartile range, 3 to 10). Among the 68 patients with the Guillain–Barré syndrome, 50% were found to have bilateral facial paralysis on examination. Among 46 patients in whom nerve-conduction studies and electromyography were performed, the results in 36 patients (78%) were consistent with the acute inflammatory demyelinating polyneuropathy subtype of the Guillain–Barré syndrome. Among the 42 patients who had samples tested for ZIKV by RT-PCR, the results were positive in 17 patients (40%). Most of the positive RT-PCR results were in urine samples (in 16 of the 17 patients with positive RT-PCR results), although 3 samples of cerebrospinal fluid were also positive. In 18 of 42 patients (43%) with the Guillain–Barré syndrome who underwent laboratory testing, the presence of ZIKV infection was supported by clinical and immunologic findings. In 20 of these 42 patients (48%), the Guillain–Barré syndrome had a parainfectious onset. All patients tested were negative for dengue virus infection as assessed by RT-PCR.ConclusionsThe evidence of ZIKV infection documented by RT-PCR among patients with the Guillain–Barré syndrome during the outbreak of ZIKV infection in Colombia lends support to the role of the infection in the development of the Guillain–Barré syndrome. (Funded by the Bart McLean Fund for Neuroimmunology Research and others.)

Detection rate and clinical impact of respiratory viruses in children with Kawasaki disease

PurposeThe purpose of this prospective case-control study was to survey the detection rate of respiratory viruses in children with Kawasaki disease (KD) by using multiplex reverse transcriptase-polymerase chain reaction (RT-PCR), and to investigate the clinical implications of the prevalence of respiratory viruses during the acute phase of KD.MethodsRT-PCR assays were carried out to screen for the presence of respiratory syncytial virus A and B, adenovirus, rhinovirus, parainfluenza viruses 1 to 4, influenza virus A and B, metapneumovirus, bocavirus, coronavirus OC43/229E and NL63, and enterovirus in nasopharyngeal secretions of 55 KD patients and 78 control subjects.ResultsVirus detection rates in KD patients and control subjects were 32.7% and 30.8%, respectively (P=0.811). However, there was no significant association between the presence of any of the 15 viruses and the incidence of KD. Comparisons between the 18 patients with positive RT-PCR results and the other 37 KD patients revealed no significant differences in terms of clinical findings (including the prevalence of incomplete presentation of the disease) and coronary artery diameter.ConclusionA positive RT-PCR for currently epidemic respiratory viruses should not be used as an evidence against the diagnosis of KD. These viruses were not associated with the incomplete presentation of KD and coronary artery dilatation.

Transcription of thyroid autoantigens in non-expressing tissues.

Reverse transcriptase- polymerase chain reaction (RT-PCR) enhances the probability of detecting rare transcripts in complex mixtures of mRNA. Using thyroid autoantigens and the controversy about the role of the TSH-receptor (TSH-R) in thyroid-associated ophthalmopathy as an example, this study demonstrates the problems of interpreting RT-PCR results in typically non-expressing tissues resulting from the extremely high sensitivity of the method. Unexpected transcripts for thyroperoxidase, thyroglobulin, TSH-R (exon 1-4, 354 bp), FSH-receptor, or insulin fragments were demonstrated in a number of thyroid or orbit-derived as well as unrelated tissues or cell types. Unexpected transcripts were most prevalent in fibroblasts, irrespective of the tissue of origin and most likely caused by ectopic transcription. To establish a physiological significance of rare transcripts such as the TSH-R in orbital tissues, demonstration of the protein in addition to the positive RT-PCR results is needed.

Molecular and biochemical testing in stage III melanoma: multimarker reverse transcriptase-polymerase chain reaction assay of lymph fluid after lymph node dissection and preoperative serum lactate dehydrogenase level

There is a need for biomarkers to identify patients at risk for disease progression after resection of melanoma regional lymph node metastasis. This study assessed the prognostic value of multimarker reverse transcriptase-polymerase chain reaction (RT-PCR) assay in lymphatic drainage (LY) after lymph node dissection (LND) and of preoperative serum lactate dehydrogenase (LDH) levels in American Joint Committee on Cancer (AJCC) stage III melanoma patients. We collected 24-h LY from 255 stage III melanoma patients after radical LND [114, completion LND after positive sentinel node biopsy (CLND); 141, therapeutic LND for clinically/cytologically detected regional nodal metastases (TLND)]. For detection of melanoma cells, RT-PCR assays with primers specific for tyrosinase, MART1 (MelanA) and uMAGE mRNA were conducted. The LY sample was deemed positive if at least one marker was detected. In 244 patients, the preoperative serum LDH level was available. Median follow-up time was 25 months (range 5-60). The LY multimarker RT-PCR assay results were positive in 82 of 255 patients (32%). A significantly higher rate of melanoma recurrence was observed in patients with positive LY multimarker RT-PCR results (P = 0.01). Significant relationships were observed between positive LY multimarker RT-PCR results and shorter 3-year overall survival (OS) and disease-free survival (DFS), both in univariate and multivariate analyses (P = 0.007). Preoperative serum LDH level was increased in 79 of 244 patients (32%) [40.5% in TLND group and 23.0% in CLND group (P = 0.003)]. There were significant differences in OS between patients with normal and high preoperative LDH levels (P = 0.007), and these differences were seen mainly in patients in the TLND group. The multimarker RT-PCR assay detected melanoma cells in approximately 32% of LY after LND, which correlated significantly with early melanoma recurrence and shorter survival. Increased pre-LND serum LDH level had an additional negative impact on OS of melanoma patients with palpable nodal metastases after TLND.

Molecular Detection and Prevalence of Citrus Viroids in Texas

Viroids are graft- or mechanically transmissible agents, disseminated through budding. Biological indexing of commercially important citrus cultivars grown in the Lower Rio Grande Valley of Texas showed that many are infected with citrus viroids. Most of these trees carried more than one viroid. In most cases, the infected trees are asymptomatic carriers because sour orange, the predominant rootstock used in Texas, does not show symptoms of viroid infection. Detection of viroids through biological indexing on sensitive indicator plants followed by sequential polyacrylamide gel electrophoresis (sPAGE) is the gold standard but is time-consuming and requires plants to be kept at optimum conditions. A conditional use of reverse transcriptase–polymerase chain reaction (RT-PCR) provides an efficient and alternative detection of viroids for use in the Texas virus-free citrus budwood certification program. RT-PCR could be useful in Texas to help expedite the evaluation for the presence of viroids before conducting the final biologic indexing. Using RT-PCR, we could detect, clone, and sequence full-length viroids of Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd) (both cachexia and noncachexia variants), Citrus viroid-III (Citrus dwarfing viroid), and Citrus viroid-IV (Citrus bark cracking viroid) from a collection of viroid-inoculated grapefruit plants. The source plants were previously shown to be viroid-infected by biological indexing on Etrog citron plants. Based on our results, RT-PCR can be a conditional substitute for biological indexing of mother trees in foundation blocks and shoot tip-grafted trees in the virus-free budwood program. A positive RT-PCR result has a serendipitous value because those trees can be discarded from the pool before expensive biological indexing.

1195 POSTER The role of adjuvant radiation therapy in uterine sarcoma

Melanoma frequently metastasizes to the central nervous system (CNS). The diagnosis of CNS metastases typically is made following the onset of clinical symptoms. Thus, more sensitive diagnostic approaches are needed to identify subclinical CNS metastases. Currently, standard cytologic analysis of the cerebrospinal fluid (CSF) is limited by its poor sensitivity. A more sensitive assay was therefore developed using multiple reverse transcriptase-polymerase chain reaction (RT-PCR) markers. CSF was collected and assessed by RT-PCR for three known melanoma-associated markers (MAGE-3, MART-1, and tyrosinase) to detect occult metastatic melanoma cells in the CSF of 37 American Joint Committee on Cancer (AJCC) stage IV melanoma patients. Cytologic analysis of CSF was performed on all patients, and immunohistochemistry (IHC) analysis was performed on 33 CSF samples using anti-S100 and anti-HMB-45 antibodies. Only one patient (3%) had tumor-positive CSF cytology and IHC upon entry into the study, whereas 12 patients (32%) were positive for at least one RT-PCR marker. The correlation between CSF RT-PCR positivity of MART-1 and/or MAGE-3 and the development of CNS metastases at 3 mo was significant (p = 0.04). Fifteen of 37 patients (41%) had either positive MRI and/or positive RT-PCR results. Multimarker RT-PCR is more informative and sensitive than cytology/IHC in assessing the CSF of melanoma patients.

Detection of Melanoma Cells in the Lymphatic Drainage after Lymph Node Dissection in Melanoma Patients by Using Two-Marker Reverse Transcriptase-Polymerase Chain Reaction Assay

The aim of this study was to evaluate the role of melanoma gene expression as a marker of the presence of melanoma cells in lymphatic drainage routinely collected after lymphadenectomy and to correlate reverse transcriptase-polymerase chain reaction (RT-PCR) assay results with recurrence, survival, and prognostic factors. We collected 24-hour postoperative lymphatic drainage samples (between days 2 and 4) from 93 patients with stage III melanoma who underwent radical lymphadenectomy between May 2002 and November 2003. We used RT-PCR assays with primers specific for the tyrosinase and MART-1 (Melan-A) genes. The samples were considered positive if at least one marker was expressed. Median follow-up time was 12.8 months. In 18 (19.4%) of 93 patients, the RT-PCR assay results were positive: in 8 of 18 for tyrosinase only, in 7 of 18 for MART-1 only, and in 3 of 18 for both markers. We observed a significantly higher recurrence rate in patients with positive RT-PCR results (15 of 18; 83%) than negative results (26 of 75; 35%; P = .0001). Positive results of RT-PCR correlated with the number of involved lymph nodes (P = .0001) and extracapsular extension of nodal metastases (P = .03). We observed significant differences in overall and disease-free survival for RT-PCR-positive and -negative patients in univariate and multivariate analyses. We observed positive RT-PCR assay results for melanoma cells in the lymphatic drainages of approximately 20% of patients after lymphadenectomy. This correlated significantly with early recurrence and shorter survival. These results may suggest that the RT-PCR assay could be useful for routinely analyzing postoperatively collected lymphatic drainage in stage III melanoma patients and for predicting disease progression.

RT-PCR for PSA as a prognostic factor for patients with clinically localized prostate cancer treated with radiotherapy

Objectives To study prospectively the prognostic significance of the reverse transcriptase-polymerase chain reaction (RT-PCR) assay for patients with prostate cancer treated definitively with external beam radiotherapy. The RT-PCR assay for prostate-specific antigen (PSA) has been used to detect circulating prostate cancer cells in the serum of patients with prostate cancer. Methods In prospective fashion, serum RT-PCR testing was performed before and/or after definitive therapy, with the results scored as positive or negative. The results were analyzed for 161 patients, and the RT-PCR result was correlated with the treatment outcome. Results The median follow-up was 29 months. The pretreatment RT-PCR result was not predictive of biochemical relapse-free survival (bRFS) or clinical disease-free survival (DFS). Of 25 patients with T3-T4 tumors, those with a negative pretreatment RT-PCR result had better bRFS and a trend was noted toward better DFS compared with those with a positive result. Among patients with Gleason score 8 to 10 tumor who underwent pretreatment testing (n = 19), those with a negative RT-PCR result had better bRFS and DFS compared with those with a positive result. A trend toward better bRFS was seen for patients with negative versus positive post-treatment RT-PCR results. The DFS was better for patients with negative versus positive post-treatment RT-PCR results. Conclusions RT-PCR, when obtained before radiotherapy, may be predictive of outcome for patients with more advanced stages or higher Gleason scores. Post-treatment testing predicted for clinical relapse. Additional study is needed before RT-PCR is used regularly in clinical practice.

Tyrosinase mRNA in blood of patients with melanoma treated with adjuvant interferon.

To evaluate the clinical significance of the detection of circulating melanoma cells in patients treated with adjuvant interferon and to determine their potential value as a marker of interferon response. We prospectively analyzed 616 peripheral-blood samples from 120 melanoma patients with stage IIA (n = 33), IIB (n = 22), III (n = 50), or IV (surgically resected) (n = 15) disease receiving adjuvant interferon alfa-2b therapy. Tyrosinase mRNA was assayed by reverse transcriptase polymerase chain reaction (RT-PCR) as a marker of circulating melanoma cells before the start of interferon and every 2 to 3 months thereafter. With a median follow-up time of 32.3 months (range, 7.1 to 77.5 months), 47 patients (39.8%) relapsed and 31 (26%) died. During adjuvant interferon treatment, 76 patients (64%) had undetected circulating melanoma cells and 44 patients (36%) had a positive RT-PCR result in at least one sample. Actuarial 5-year disease-free survival was 62% in patients with persistently negative RT-PCR during interferon treatment and 38% for patients with positive RT-PCR during interferon (P =.02). Actuarial 5-year overall survival was 75% and 50%, respectively (P =.03). Patients with melanoma and tyrosinase mRNA detected in the blood during adjuvant interferon therapy had a worse prognosis compared with patients with undetected tyrosinase mRNA during treatment. Further investigation into the detection of circulating melanoma cells as a surrogate marker of response to adjuvant interferon therapy is warranted.

Molecular prediction of progression in patients with conservatively managed prostate cancer

Objectives. To report our results with the use of reverse transcriptase-polymerase chain reaction (RT-PCR) as a potential predictor of prostate cancer (CaP) progression in patients managed with watchful waiting. There has been much recent debate about the safety of treating older patients with localized CaP with watchful waiting. The RT-PCR is an assay that can detect small numbers of prostate cells in circulating blood. Methods. Blood samples were taken from male and female control patients and from patients with advanced, hormone-treated and untreated localized (watchful-waiting) CaP. Sensitive nested RT-PCR assays were carried out on these samples using primers for both prostate-specific antigen (PSA) and prostate-specific membrane antigen mRNA. Results. Fifty-one blood samples were taken from patients managed with watchful waiting. Fourteen of these had positive RT-PCR results. These patients had a significantly higher PSA velocity than did the patients with negative RT-PCR results. Circulating prostate cells were detected in 18 of 24 patients with advanced CaP, 2 of 34 patients with stable, hormone-treated CaP, and in none of the negative controls. The assay was able to detect 20 LNCaP cells reliably when added to a 5-mL volunteer blood sample. Conclusions. A significant minority (27%) of patients with untreated localized CaP had detectable circulating prostate cells, and these patients tended to have a progressively rising serum PSA level. Despite low-grade disease and sometimes low serum PSA values, these patients may be at risk of early metastatic progression. RT-PCR, in conjunction with existing prognostic tests, may be of use in predicting which “watchful-waiting” patients are at risk of early progression.