Polymerase Chain Reaction Status Research Articles

Oral Mucosal Manifestations Associated with PCR Positivity in Patients with COVID-19.

To date various oral manifestations in patients with coronavirus disease 2019 (COVID-19) have been reported. In the present study, we investigated the relationship between Polymerase Chain Reaction (PCR) positivity and oral signs in patients with suspected COVID-19. A total of 383 patients who presented to the emergency department for the first time with any symptoms associated with COVID-19 were included in the study. Oral examinations were performed and the findings, PCR status, and thorax computerized tomography (CT) reports were recorded. Oral mucosa was involved in 246 (64.2%) patients. 175 (78,4%) of patients with COVID-19 confirmed the diagnosis with PCR test or CT results had oral manifestation. Dry mouth, microvesicles on the tonsils or pharynx, and petechiae in the oropharynx were significantly higher in patients with positive PCR tests (P = 0.001, P < 0.001, P < 0.001, respectively). The ratio of intact oral mucosa was statistically significantly higher in patients with negative PCR tests compared to those with positive PCR tests (P < 0.001). Microvesicles on the tonsils or pharynx were most associated with PCR positivity in patients without lung involvement (P < 0.001). Dry mouth, erythema of the tonsils and pharynx, petechiae in the oropharynx, and primary/secondary herpes infection are more related to PCR positivity in patients without lung involvement (P < 0.05). Lung involvement in patients with PCR positivity is related to only cheilitis (P = 0.034). Our study revealed that especially microvesicles, petechiae, erythema on the tonsils or pharynx, and some other oral lesions such as dry mouth, oral aphthae, and primary/secondary herpes infection are associated with PCR positivity.

Iris depigmentation and cytomegalovirus in aqueous humor as predictors of uveitic activity and recurrence in chronic and recurrent anterior uveitis.

To examine the clinical outcomes in chronic or recurrent anterior uveitis in the presence or absence of cytomegalovirus (CMV) and investigate the predictive factors for uveitic activity and recurrence. Polymerase chain reaction (PCR) was performed in a prospective cohort of immunocompetent adults with recurrent or chronic anterior uveitis to detect CMV in aqueous humor. The clinical outcomes were compared between eyes with and without CMV DNA. Logistic regression was performed to evaluate associations between iris depigmentation, CMV-PCR status, uveitic activity, and recurrence. Thirty-eight eyes of 38 subjects with a mean age of 61.1 ± 11.2 years old were analyzed. Fifteen eyes were positive for CMV. More eyes with CMV developed recurrences and remained actively inflamed at 6, 12, and 24 weeks though the differences were insignificant. The presence of iris depigmentation was predictive of a greater odd of uveitic recurrences by 12 and 24 weeks (Odds ratio (OR) = 9.17 and 5.72, P = 0.007 and 0.034 respectively), whereas positive CMV-PCR predicts a greater odd of uveitic activity at postoperative 12 and 24 weeks (OR = 13.08, 34.30; P = 0.027, 0.007). Eye with and without detectable CMV behaved similarly in their clinical course. Our findings suggested that iris depigmentation was predictive of more frequent uveitic recurrence, regardless of the PCR status, whereas the presence of CMV in aqueous humor was associated with persistent uveitic activity. Iris changes may be present during the earlier phase of the disease and precede the detection of virus from the aqueous humor at a later stage of CMV infection.

Clinical outcomes associated with blood-culture contamination are not affected by utilization of a rapid blood-culture identification system.

Contaminated blood cultures result in extended hospital stays and extended durations of antibiotic therapy. Rapid molecular-based blood culture testing can speed positive culture detection and improve clinical outcomes, particularly when combined with an antimicrobial stewardship program. We investigated the impact of a multiplex polymerase chain reaction (PCR) FilmArray Blood Culture Identification (BCID) system on clinical outcomes associated with contaminated blood cultures. We conducted a retrospective cohort study involving secondary data analysis at a single institution. In this before-and-after study, patients with contaminated blood cultures in the period before PCR BCID was implemented (ie, the pre-PCR period; n = 305) were compared to patients with contaminated blood cultures during the period after PCR BCID was implemented (ie, the post-PCR implementation period; n = 464). The primary exposure was PCR status and the main outcomes of the study were length of hospital stay and days of antibiotic therapy. We did not detect a significant difference in adjusted mean length of hospital stay before (10.8 days; 95% confidence interval [CI], 9.8-11.9) and after (11.2 days; 95% CI, 10.2-12.3) the implementation of the rapid BCID panel in patients with contaminated blood cultures (P = .413). Likewise, adjusted mean days of antibiotic therapy between patients in pre-PCR group (5.1 days; 95% CI, 4.5-5.7) did not significantly differ from patients in post-PCR group (5.3 days; 95% CI, 4.8-5.9; P = .543). The introduction of a rapid PCR-based blood culture identification system did not improve clinical outcomes, such as length of hospital stay and duration of antibiotic therapy, in patients with contaminated blood cultures.

Concordance of Preferentially Expressed Antigen in Melanoma by Non-Invasively Collected Polymerase Chain Reaction and Immunohistochemistry on Paraffin Embedded Tissue

Background: ​&#x0D; Pigmented lesion evaluation remains a challenging aspect of dermatology. The DermTech Melanoma Test (DMT) is a non-invasive gene-expression test designed to rule-out melanoma. It consists of the pigmented lesion assay, which detects RNA products of Long Intergenic Non-Coding RNA 00518 (LINC00518) and Preferentially Expressed Antigen in Melanoma (PRAME), and an add-on assay for DNA promoter mutations in telomerase reverse transcriptase (TERT). This registry study examines the concordance of PRAME detection by polymerase chain reaction (PCR) in samples obtained non-invasively prior to biopsy and PRAME detection by immunohistochemistry (IHC) on the same lesions after biopsy.​&#x0D; Methods: ​&#x0D; Between April 2021 and March 2022, multiple geographically diverse sites throughout the US submitted data to a registry to assess real-world use of the DMT. Approximately 8,000 clinically atypical lesions were tested. After receiving the test result, providers followed their clinical judgement for biopsy decision. When lesions expressed genomic markers (LINC, PRAME, and/or TERT) and were biopsied, pathology reports were also submitted to the registry. The presence or absence of PRAME by immunohistochemistry (IHC) was reviewed and compared to the detection of PRAME by PCR from the DMT on the same lesion.​&#x0D; Results: ​&#x0D; At the 1-year mark of the registry, there were roughly 8,000 unique entries. Of those, 1,021 (12.8%) were positive for one or more of the DMT genomic markers. One thousand three lesions (98.2%) had records available. Pathologists used PRAME IHC for 102 lesions (10.2%). Of those, 40 (39.2%) were positive by IHC, and 62 (60.8%) were negative by IHC. PRAME positivity by PCR correlated with PRAME positivity by IHC in 35 of 40 lesions (87.5%). Conversely, PRAME was detected using PCR in 28 of 62 lesions (45.2%) where it was not detected using IHC. ​&#x0D; Conclusions: ​&#x0D; The higher sensitivity of PCR compared to IHC may explain the higher concordance when PRAME is positive by IHC than when it is negative by IHC. In this data set, when PRAME is positive by IHC it is usually also positive by PCR. When PRAME is negative by IHC, it can still be detected by PCR in a substantial percentage of cases. The increased sensitivity of PCR is likely due to several factors, including its detection of the PRAME mRNA and sampling of the entire lesion. As such, PRAME PCR status may aid pathologists in understanding the risk of melanoma even when IHC is negative. Further research is warranted to understand the clinical implications of PRAME PCR versus IHC positivity. ​

CIN2 + detection in high-risk HPV patients with no or minor cervical cytologic abnormalities: a clinical approach validated by machine learning

PurposeTo evaluate the feasibility and diagnostic value of the combination of colposcopy, cytology and hrHPV (high-risk human papilloma virus) PCR (polymerase chain reaction) testing in patients with no or minor cytologic abnormalities and HPV high risk infection and to find the best predictors for the presence of CIN2 + in this patient collective.MethodsThree hundred and thirty-four hrHPV patients with normal cytology or minor cytologic abnormalities who had a colposcopic examination at the center of colposcopy at the university hospital Aachen in 2021 were enrolled in this retrospective cohort analysis. Multivariate logistic regression and a machine-learning technique (random forests, leave-one-out analysis) were used.ResultsThe overall risk for CIN2 + in hrHPV-positive patients with normal cytology was 7.7% (N = 18) (5% for CIN3 +), 18% (N = 16) (10.1% for CIN3 +) in patients with PAP IIp (ASC-US) and 62.5% (N = 5) (25% for CIN3 +) in patients with PAP IIg (AGC). Variables that show a statistically significant influence for the CIN-status are ‘major change’ as the result of colposcopy, transformation zone type T1, PAP IIg upon referral (AGC) and hrHPV category 1a (HPV 16/18) detection. Using machine learning (random forests) techniques, the main influencing variables were confirmed. A monotonously decreasing risk for CIN2 + from hrHPV category 1a to 3 (in accordance to the IACR guidelines) was found.ConclusionIn the collective of hrHPV patients with no or minor cytologic abnormalities, the result of colposcopy and HPV PCR status are key predictors for the detection of CIN2 + with a monotonously decreasing risk for CIN2 + from hrHPV category 1a to 3.

MO846THE SPECTRUM OF CLINICAL AND SEROLOGICAL FEATURES OF COVID-19 IN URBAN HEMODIALYSIS PATIENTS

Background and AimsThe inherent immunosuppression of uremia increases the susceptibility of hemodialysis patients to infection. There is still limited evidence on hemodialysis patients and COVID-19. The clinical and analytical spectrum and treatment responses and mortality are poorly characterized.MethodClinical and analytical features, chest X-ray, polymerase chain reaction (PCR) and antibodies for SARS-CoV-2, treatment and outcomes were analyzed in 48 patients diagnosed with COVID-19 during March and April 2020 in two coordinated Spanish hemodialysis units.ResultsIn 200 haemodialysis patients, COVID-19 was diagnosed in 48, of whom 22 were PCR positive, eight PCR negative but seroconverted and two were diagnosed on typical clinical grounds. Despite a mean age of 72.6 years, the overall mortality rate was 5/48 (10%). Among the PCR positive patients, 21 (55%) required admission and five (13%) died. PCR positive patients were more often symptomatic and hospitalized and had higher troponin I levels than PCR negative patients, but did not differ in lymphocyte counts, D-dimer or interleukin-6 (IL-6) levels. Among PCR negative COVID-19 patients, three out of 10 (30%) required admission, and none died. The most frequent symptom among the 48 patients was fever (31%), followed by asymptomatic patients (23%). A low number of lymphocytes was the only parameter significantly different between hospitalized and ambulatory COVID-19 patients, independently of PCR status.ConclusionCOVID-19 hemodialysis patients are frequently asymptomatic, and mortality may be lower than previously reported. Diagnosis may be retrospective, based on seroconversion, as PCR may be negative. This information should guide preventive and patient isolation strategies.

The Spectrum of Clinical and Serological Features of COVID-19 in Urban Hemodialysis Patients.

Introduction: The inherent immunosuppression of uremia increases the susceptibility of hemodialysis patients to infection. There is still limited evidence on hemodialysis patients and COVID-19. The clinical and analytical spectrum and treatment responses and mortality are poorly characterized. Material and Methods: Clinical and analytical features, chest X-ray, polymerase chain reaction (PCR) and antibodies for SARS-CoV-2, treatment and outcomes were analyzed in 48 patients diagnosed with COVID-19 during March and April 2020 in two coordinated Spanish hemodialysis units. Results: In 200 haemodialysis patients, COVID-19 was diagnosed in 48, of whom 22 were PCR positive, eight PCR negative but seroconverted and two were diagnosed on typical clinical grounds. Despite a mean age of 72.6 years, the overall mortality rate was 5/48 (10%). Among the PCR positive patients, 21 (55%) required admission and five (13%) died. PCR positive patients were more often symptomatic and hospitalized and had higher troponin I levels than PCR negative patients, but did not differ in lymphocyte counts, D-dimer or interleukin-6 (IL-6) levels. Among PCR negative COVID-19 patients, three out of 10 (30%) required admission, and none died. The most frequent symptom among the 48 patients was fever (31%), followed by asymptomatic patients (23%). A low number of lymphocytes was the only parameter significantly different between hospitalized and ambulatory COVID-19 patients, independently of PCR status. Conclusions: COVID-19 hemodialysis patients are frequently asymptomatic, and mortality may be lower than previously reported. Diagnosis may be retrospective, based on seroconversion, as PCR may be negative. This information should guide preventive and patient isolation strategies.

2790. Respiratory Viral Testing and Antimicrobial De-escalation Among Hospitalized Patients at a Tertiary Care Facility, 2015–2016: A Matched Cohort Study Series

BackgroundThe use of multiplex respiratory viral panels (RVP) is increasing. They have the potential to reduce unnecessary antibiotic use, but data are limited on their clinical effectiveness. Our objective was to estimate risk differences for antimicrobial de-escalation (discontinuation, intravenous to oral, or spectrum narrowing) between different sequences and results of RVP and rapid polymerase chain reaction (PCR) tests for influenza +/− respiratory syncytial virus.MethodsWe conducted a retrospective chart review of adults (age ≥18 years) admitted to a floor or stepdown unit at University of North Carolina Hospitals who had a respiratory viral test (RVT) within 48 hours of admission between September 2015 and April 2016. We estimated 3-day RDs for the relation between RVT and antimicrobial de-escalation. To control confounding and account for the 37-hour mean lag between PCR (faster) and RVP (slower) tests resulting, we leveraged the treatment decision design over a series of 1:1 matched cohort studies. Each targeted a clinically relevant scenario: (1) ordering RVP test (vs no RVP order) after learning PCR status; (2) learning RVP+ result (vs. no RVP result) after knowing PCR status; (3) learning RVP+ result (vs. RVP–) after knowing PCR–status; and (4) learning RVP+ result (vs. RVP–) given no prior PCR. For each subcohort, referent patients were matched to index patients by race, gender, RVT in prior month (y/n), age (±10 years), and season (±1.7 months).ResultsThe overall cohort (n = 1,342) was 61% White, 29% African American, and 51% female. Median age was 56 years (IQR 39–69). Across all matched subcohorts (Figure 1), the matching success rate was 79–88% and referent frequency of antimicrobial de-escalation ranged 0.6%–1.9%. In scenario 1, ordering RVP results was associated with higher de-escalation (3-day RD 7.6%; 95% confidence intervals [CI] 3.2%, 12.1%). In scenarios 2–4, learning RVP+ results was associated with more frequent de-escalation (3-day RDs 14.8%, 13.8%, and 15.4%).ConclusionRVP testing and positive RVP results were associated with increased antimicrobial de-escalation, although de-escalation was overall infrequent. Future research should assess effect modification across subgroups and evaluate cost-effectiveness. Disclosures All authors: No reported disclosures.

Revealing the secrets of PCR

The polymerase chain reaction (PCR) method and its quantitative variant (qPCR) were a landmark discovery for detection and quantification of small amounts of unambiguous deoxyribonucleic acids (DNA) due to its enormous sensitivity and specificity. The current methods for qPCR protocol optimization provide no information of the PCR propagation during the cycles as only single data point is extracted at the end of each thermal cycles, limiting a thorough understanding of reaction details. In this study, we utilized the continuous fluorescence monitoring method to observe progress of the reaction in real time with over 100 data points per each cycle, thus gaining a profound insight into the PCR itself. This provided information about the real-time PCR status, dominating reactions and their completion/incompletion during each cycle as well as their reaction kinetics. We then adjusted the duration of either annealing or elongation steps to ensure their completion within each cycle, resulting in the protocol optimization with complete amplification, enhancing PCR efficiency and taking < 20 min to obtain maximum product amount. The proposed method was verified using DNA with lengths of 177 base pairs (bp), 250 bp, and 400 bp. It can also be adopted for helping with qPCR troubleshooting as well as protocol optimizing just by reprogramming commercial real-time PCR cyclers.

BRAF V600 mutation detection in melanoma: a comparison of two laboratory testing methods

AimsThe assessment of B-raf proto-oncogene, serine/threonine kinase (BRAF) gene status is now standard practice in patients diagnosed with metastatic melanoma with its presence predicting a clinical response to treatment with...

Candidatus Liberibacter asiaticus causes damage to citrus fibrous roots before visual decline of Huanglongbing-infected citrus trees

Huanglongbing (HLB), associated with Candidatus Liberibacter asiaticus (Las), was first detected in Florida in late 2005 and is now widely distributed throughout the commercial citrus-growing regions. In recent seasons, concurrent with freeze and drought episodes, symptomatic HLBinfected trees were much more affected by the extremes of temperature and moisture than trees without HLB. Symptoms exhibited by the stressed trees were excessive leaf loss and premature fruit drop even when HLB-infected trees were managed with good nutritional and irrigation practices recommended to support health of HLB-affected trees. This stress intolerance may be due to a loss of fibrous roots. To assess root status of HLB-infected trees on Swingle citrumelo rootstock (Citrus paradisi × Poncirus trifoliata), blocks of 2,307 three-yr-old Hamlin orange trees and 2,693 four-yr-old Valencia orange trees were surveyed visually and with a real time polymerase chain reaction (PCR) assay to determine Las infection status. The incidence of Las-infected trees (pre-symptomatic: Las+, visually negative, and symptomatic: Las+, visually positive) trees was 89% for the Hamlin block and 88% for the Valencia block. Las+ trees had 30 and 37% lower fibrous root mass density for pre-symptomatic and symptomatic trees, respectively, compared to Las- trees. In a second survey, 10- to 25-yr-old Valencia trees on ‘Swingle’ citrumelo or ‘Carrizo’ citrange (C. sinensis (L.) × P. trifoliata) rootstock were sampled within 3-6 months after identification of visual HLB status as symptomatic (Las+, visually positive) or non-symptomatic (Las-, visually negative) in orchards located in the central ridge, south-central and southwest flatwoods. Pairs of HLB symptomatic and non-symptomatic trees were evaluated for PCR status, fibrous root mass density and Phytophthora nicotianae progagules in the rhizosphere soil. Las+ trees had 27-40% lower fibrous root mass density and in one location higher P. nicotianae per root but Phytophthora populations per cm 3 soil were high on both Las+ and Las- trees. Fibrous root loss from HLB damage interacted with P. nicotianae depending on orchard location and time of year.

Presymptomatic Fibrous Root Decline in Citrus Trees Caused by Huanglongbing and Potential Interaction with Phytophthora spp.

Huanglongbing (HLB), associated with 'Candidatus Liberibacter asiaticus', was first detected in Florida in late 2005 and is now widely distributed throughout the commercial citrus-growing regions. In recent seasons, concurrent with freeze and drought episodes, symptomatic HLB-infected trees were much more affected by the extremes of temperature and moisture than trees without HLB. Symptoms exhibited by the stressed trees were excessive leaf loss and premature fruit drop even when HLB-infected trees were managed with good nutritional and irrigation practices recommended to support health of HLB-affected trees. This stress intolerance may be due to a loss of fibrous roots. To assess root status of HLB-infected trees on 'Swingle' citrumelo rootstock (Citrus paradisi × Poncirus trifoliata), blocks of 2,307 3-year-old 'Hamlin' orange trees and 2,693 4-year-old 'Valencia' orange trees were surveyed visually and with a real-time polymerase chain reaction (PCR) assay to determine 'Ca. L. asiaticus' infection status. The incidence of 'Ca. L. asiaticus'-infected trees (presymptomatic: 'Ca. L. asiaticus'+, visually negative; and symptomatic: 'Ca. L. asiaticus'+, visually positive) trees was 89% for the Hamlin block and 88% for the Valencia block. 'Ca. L. asiaticus'+ trees had 30 and 37% lower fibrous root mass density for presymptomatic and symptomatic trees, respectively, compared with 'Ca. L. asiaticus'- trees. In a second survey, 10- to 25-year-old Valencia trees on Swingle citrumelo or 'Carrizo' citrange (C. sinensis (L.) × P. trifoliata) rootstock were sampled within 3 to 6 months after identification of visual HLB status as symptomatic ('Ca. L. asiaticus'+, visually positive) or nonsymptomatic ('Ca. L. asiaticus'-, visually negative) in orchards located in the central ridge, south-central, and southwest flatwoods. Pairs of HLB symptomatic and nonsymptomatic trees were evaluated for PCR status, fibrous root mass density, and Phytophthora nicotianae propagules in the rhizosphere soil. 'Ca. L. asiaticus'+ trees had 27 to 40% lower fibrous root mass density and, in one location, higher P. nicotianae per root but Phytophthora populations per cubic centimeter of soil were high on both 'Ca. L. asiaticus'+ and 'Ca. L. asiaticus'- trees. Fibrous root loss from HLB damage interacted with P. nicotianae depending on orchard location and time of year.

Assessment of PCR status of vector-borne pathogens in dogs presenting arrhythmias

Arrhythmias are frequent and can be caused by many diseases. Vector-borne diseases are increasing in importance as a cause of arrhythmias in veterinary medicine. The objective of the study was to evaluate dogs presenting arrhythmias of any aetiology and to assess the influence of polymerase chain reaction (PCR) status for vector-borne pathogens in the Mediterranean area of Spain. A total of 120 dogs presenting arrhythmias were prospectively selected, and PCR for several vector-borne pathogens (Leishmania infantum, Ehrlichia/Anaplasma spp., Babesia spp., Rickettsia spp., Mycoplasma spp., Hepatozoon canis and Bartonella spp.) were analysed in blood samples. Twenty-four (20 %) dogs had positive results for PCR. Found pathogens were Ehrlichia/Anaplasma spp. (n = 10, 8.3 %), Babesia spp. (n = 7, 5.8 %), Rickettsia spp. (n = 5, 4.2 %), Leishmania spp. (n = 4, 3.3 %), Mycoplasma spp. (n = 4, 3.3 %), and H. canis (n = 1, 0.8 %). No significant differences were found in relation to PCR status and type or cause of the arrhythmia. Although there is a clear association between some infectious diseases and cardiac diseases, this study could not find evidence of the role of infectious agents as triggers for arrhythmias in dogs. More studies are needed to further explore this association.

Reply to V. Procházka et al

In their letter, Prochazka et al raise several issues as to the validity of our conclusion that in patients with relapsed or refractory follicular lymphoma, the presence of tumor cells in blood or bone marrow—as detected by BCL2/IgH major break point region polymerase chain reaction (PCR) at the end of rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone induction therapy—is not predictive of progression-free survival (PFS). First, they suggest that we should have analyzed separately patients with clinical complete (CR) and partial remission (PR). We agree that this would have been a relevant subgroup analysis. Unfortunately, the four subgroups (PCRpositive CR, PCR-negative CR, PCR-positive PR, and PCR-negative PR) were too small to allow for a meaningful statistical comparison. However, as mentioned in our report, there was no difference between patients in clinical CR or PR with regard to the proportion of bone marrow (BM) or peripheral blood PCR negativity at the end of induction treatment (50% v 55%). This is clearly indicative of the compartmentalization effect and at least partly explains the lack of prognostic value of PCR status post induction treatment. Second, the authors state that our finding that PFS was not different between patients who converted to PCR negativity and those who did not is in contrast with previous publications by others. We agree and consider this to be an important message of our report. Prochazka et al mention two observations in our article that they consider to be supportive of the predictive value of PCR status. Their first consideration is based on our finding that there was highly significant improved PFS by rituximab maintenance in patients with postinduction PCR-negative BM, whereas the improvement was not significant in patients with PCR-positive BM (Figs 4A, 4B). In fact, there was a clear difference in the PCR-positive BM group, but this did not reach statistical significance because of limited numbers. However, highly significant improvement in PFS by rituximab maintenance was observed in both patients with postinduction PCR-negative blood and those with postinduction PCR-positive blood (Figs 4C, 4D). These observations do not change our conclusion that benefit from rituximab maintenance is independent of postinduction PCR status. Second, they mention our observation that patients who were still PCR positive at the end of maintenance treatment progressed rapidly after stopping maintenance therapy. In our opinion, this observation is not indicative of the predictive value of postinduction PCR status, but it is a strong argument for prolonging maintenance treatment.

EFFECTS OF MYCOPLASMAL UPPER RESPIRATORY TRACT DISEASE ON MORBIDITY AND MORTALITY OF GOPHER TORTOISES IN NORTHERN AND CENTRAL FLORIDA

Gopher tortoise (Gopherus polyphemus) populations on four tracts of public lands in northern and central Florida were studied from 1998 to 2001 to assess the effects of mycoplasmal upper respiratory tract disease (URTD). Adult gopher tortoises (n=205) were marked for identification, serum and nasal flush samples were obtained for mycoplasmal diagnostic assays, and clinical signs of URTD (nasal discharge, ocular discharge, palpebral edema, and conjunctivitis) were evaluated. A subset of tortoises (n=68) was radio-instrumented to facilitate repeated sampling and document potential mortality. Presence of serum antibody to Mycoplasma agassizii was determined by enzyme-linked immunosorbent assay (ELISA), and mollicutes species were detected in nasal flushes by polymerase chain reaction (PCR). Antibody prevalence varied among sites and years but was highest in 1998, exceeding 70% at two sites. Only 11 tortoises (5%) were positive by PCR, and three species (M. agassizii, M. testudineum, and a nonpathogenic Acholeplasma) were identified in nasal flush specimens. Nasal discharge, though rare (6% of tortoises), was significantly correlated with higher ELISA ratios, study site, and positive PCR status. Mortality events (n=11) occurred on two of the three M. agassizii-positive sites; no mortality was observed on the M. agassizii-negative control site. However, none of the tested variables (ELISA result, study site, year, sex, presence of clinical signs, or carapace length) showed significant ability to predict the odds of death. Mycoplasmal URTD is believed to be a chronic disease with high morbidity but low mortality, and follow-up studies are needed to detect long-term effects.

Prenatal diagnosis of the Rhesus D fetal blood type on amniotic fluid in daily practice

To retrospectively examine the diagnostic accuracy of prenatal RhD blood type genotyping on amniotic fluid, using a combination of two polymerase chain reaction (PCR) methods in daily practice. Amniotic fluid was obtained from women undergoing amniocentesis. Two PCR protocols were carried out in two different laboratories. We obtained the postnatal serological RhD status. In cases with differing prenatal and postnatal test results, we investigated the possible error source by different methods. Sensitivity, specificity and the predictive values were calculated. Prenatal RhD genotyping was applied in 1,640 cases, of which the postnatal serologic RhD status was obtained in 927. No discordance between both PCR methods occurred. In nine out of 927 cases differing results between PCR and serologic status were encountered. The sensitivity was 99.5%, the specificity 98.6%, and both positive and negative predictive values 99.1%. Prenatal diagnosis of the fetal RhD blood type with PCR from amniotic fluid is highly accurate in daily practice and associated with a minimal sensitivity of 99.5% and a minimal specificity of 98.6%.

Sentinel Lymph Node Molecular Ultrastaging in Patients With Melanoma: A Systematic Review and Meta-Analysis of Prognosis

Molecular biology-based ultrastaging of cancer is already part of the standard management of patients with hematologic malignancies, whereas the evidence for solid tumors is much more debated. Polymerase chain reaction (PCR) -based detection of melanoma cells in sentinel lymph nodes (SLN) of patients with melanoma represents an appealing prognostic tool. However, no consensus exists on the clinical implementation of this prognostic indicator for the management of these patients. Twenty-two studies enrolling 4,019 patients who underwent SLN biopsy for clinical stage I to II cutaneous melanoma were reviewed. Correlation of PCR status with TNM stage, disease recurrence rates, and survival was assessed by means of association statistics and formal meta-analysis, respectively. PCR status correlated with both TNM stage (stage I to II v III; PCR positivity, 95.1% v 46.6%; P < .0001) and disease recurrence (PCR positive v negative; relapse rate, 16.8% v 8.7%; P < .0001). PCR positivity was also associated with worse overall (hazard ratio [HR], 5.08; 95% CI, 1.83 to 14.08; P = .002) and disease-free (HR, 3.41; 95% CI, 1.86 to 6.24; P < .0001) survival. Statistical heterogeneity was significant, underscoring the variability among overall effect estimates across studies; metaregression and subgroup analysis did not identify clear-cut sources of heterogeneity, although some study design variables were suggested as potential causes. PCR status of SLN appears to have a clinically valuable prognostic power in patients with melanoma. Although the heterogeneity of the studies so far published warrants caution to avoid overestimating the favorable results of pooled data, our findings strongly support additional investigation in this field.

High-dose therapy with autologous bone marrow support as consolidation of remission in follicular lymphoma: long-term clinical and molecular follow-up.

To evaluate the long-term results of high-dose therapy (HDT) in follicular lymphoma, with specific emphasis on the prognostic significance of polymerase chain reaction (PCR)-detectable Bcl-2/IgH rearrangements. Between June 1985 and October 1995, 99 patients with follicular lymphoma received HDT as consolidation of second or subsequent remission. Bone marrow was treated in vitro with anti-B-cell antibodies and complement. Sixty-five patients remained alive, 49 treatment-failure free, with a median follow-up of 5.5 years (range, 1.5 to 12.5 years). Four "early" and 10 "late" deaths occurred from treatment-related causes; seven of the latter were due to secondary myelodysplasia (s-MDS) or secondary acute myeloblastic leukemia. Overall, 12 (12%) of the 99 patients developed s-MDS or acute myeloblastic leukemia. Kaplan-Meier estimates of freedom from recurrence (FFR) and survival rates at 5 years were 63% (95% confidence interval [CI], 52% to 72%) and 69% (95% CI, 58% to 78%), respectively. For all 99 patients, in multivariate analysis, absence of the Bcl-2/IgH rearrangement at the time of diagnosis (hazards ratio [HR], 0.39; P =.04) and three or fewer treatment episodes before HDT (HR, 0.03; P =.001) were significant prognostic factors for improved survival. For patients bearing Bcl-2/IgH rearrangements, in univariate and multivariate analyses, absence of a PCR-detectable Bcl-2/IgH rearrangement during follow-up was associated with a significantly lower risk of recurrence (adjusted HR, 0.13; P <.001) and death (HR, 0.25; P =.02), whereas the PCR status of the reinfused bone marrow did not correlate with outcome. Prolonged FFR can be achieved in patients with follicular lymphoma after HDT, but as yet there is no survival advantage compared with conventional treatment. These results confirm that elimination of cells bearing the Bcl-2/IgH rearrangement is highly desirable and should be attempted. The incidence of s-MDS is of increasing concern in this setting.

The remission status before and the PCR status after high-dose therapy with peripheral blood stem cell support are prognostic factors for relapse-free survival in patients with follicular non-Hodgkin's lymphoma.

It was the aim of our study to examine the clinical significance of t(14;18)-positive cells in samples from 47 patients with follicular non-Hodgkin's lymphoma (NHL) who underwent high-dose therapy with autologous peripheral blood stem cell (PBSC) transplantation. At the time of PBSC mobilization, 25 patients were in first remission, while 22 patients had a history of previous treatment failure. At the same time, 43 patients had polymerase chain reaction (PCR)-positive cells in samples from bone marrow (BM) and/or peripheral blood (PB). Independent of the remission status, high-dose cytarabine and mitoxantrone with granulocyte colony-stimulating factor (G-CSF) support were administered for PBSC mobilization. Following high-dose conditioning therapy which consisted of cyclophosphamide (200 mg/kg) and hyperfractionated total body irradiation (TBI, 14.4 Gy) or BEAM (carmustine, etoposide, cytarabine, melphalan), 34 patients received PCR-positive and 13 patients received PCR-negative autografts. After a median follow-up time of 20 months (range, 6-50) post-transplantation, 33 patients were in remission, while 14 patients had relapsed after a median time of 14.5 months (range, 10-42). Using the Andersen-Gill proportional hazards regression model for the analysis of relapse-free survival, we found that PCR-positive findings in samples from BM and/or PB at any given time-point after transplantation were associated with an increased estimated hazard ratio of 4.5 in comparison with a PCR-negative finding (P=0.013). On the other hand, patients included while they were in first remission had a smaller estimated hazard ratio of 0.3 when compared with patients with a history of previous treatment failure (P=0.048). For the latter group of patients, this translates into a significantly smaller probability of relapse-free survival in comparison to patients who were in first remission at the time of PBSC-mobilization (P=0.012). In conclusion, the remission status of the patients before autografting and the PCR status as assessed on the occasion of follow-up examinations are significant prognostic parameters for relapse-free survival in patients with follicular lymphoma undergoing high-dose therapy with PBSC autografting.

Conversion of interferon-induced, long-term cytogenetic remissions in chronic myelogenous leukemia to polymerase chain reaction negativity.

Interferon alfa (IFN-alpha) induces complete cytogenetic remission in a significant minority of patients with chronic myelogenous leukemia (CML). To date, use of the polymerase chain reaction (PCR) has suggested that residual disease is present in virtually all these individuals at median follow-up intervals of approximately 1 year after initial achievement of cytogenetic remission. We sought to determine the PCR status of CML patients with very long continuous complete cytogenetic remissions on IFN-alpha. Blood and/or bone marrow samples from 18 CML patients maintained in continuous cytogenetic remission by IFN-alpha for at least 1 year were studied for residual BCR-ABL mRNA by single-step reverse-transcriptase (RT)-PCR methodology, followed by detection of the amplification product by the hybridization protection assay (HPA). Ten of 18 patients showed RT-PCR negativity for BCR-ABL. The median duration of continuous complete cytogenetic remission at the time of sample collection for the negative patients was 42 months versus 21 months for the patients whose tests remained positive (P = .05). Very long-term follow-up of IFN-alpha-treated CML patients in continuous complete cytogenetic remission is associated with a decrease in the number of BCR-ABL transcripts to a level below that detectable by the RT-PCR assay used in this study.