Likelihood Of False-negative Results Research Articles

Clinical Utility of the Molecular Microscope Diagnostic System in a Real-World Transplant Cohort: Moving Towards a New Paradigm.

To evaluate the clinical implications of adjunctive molecular gene expression analysis (MMDx ) of biopsy specimens in heart transplant (HT ) recipients with suspected rejection. Histopathological evaluation remains the standard method for rejection diagnosis in HT. However, the wide interobserver variability combined with a relatively common incidence of "biopsy-negative" rejection has raised concerns about the likelihood of false-negative results. MMDx, which uses gene expression to detect early signs of rejection, is a promising test to further refine the assessment of HT rejection. Single-center prospective study of 418 consecutive for-cause endomyocardial biopsies performed between November 2022 and May 2024. Each biopsy was graded based on histology and assessed for rejection patterns using MMDx. MMDx results were deemed positive if borderline or definitive rejection was present. The impact of MMDx results on clinical management was evaluated. Primary outcomes were 1-year survival and graft dysfunction following MMDx-guided clinical management. Secondary outcomes included changes in donor-specific antibodies, MMDx gene transcripts, and donor-derived cell-free DNA (dd-cfDNA) levels. We analyzed 418 molecular samples from 237 unique patients. Histology identified rejection in 32 cases (7.7%), while MMDx identified rejection in 95 cases (22.7%). Notably, in 79 of the 95 cases where MMDx identified rejection, histology results were negative, with the majority of these cases being antibody-mediated rejection (62.1%). Samples with rejection on MMDx were more likely to show a combined elevation of dd-cfDNA and peripheral blood gene expression profiling than those with borderline or negative MMDx results (36.7% vs 28.0% vs 10.3%; p<0.001). MMDx results led to the implementation of specific antirejection protocols or changes in immunosuppression in 20.4% of cases, and in 73.4% of cases where histology was negative and MMDx showed rejection. 1-year survival was better in the positive MMDx group where clinical management was guided by MMDx results (87.0% vs 78.6%; log rank p=0.0017). In our cohort, MMDx results more frequently indicated rejection than histology, often leading to the initiation of antirejection treatment. Intervention guided by positive MMDx results was associated with improved outcomes.

Performance of Unobserved Self-Collected Nasal Swabs for Detection of SARS-CoV-2 by RT-PCR Utilizing a Remote Specimen Collection Strategy.

BackgroundThe use of a remote specimen collection strategy employing a kit designed for unobserved self-collection for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription polymerase chain reaction (RT-PCR) can decrease the use of personal protective equipment (PPE) and exposure risk. To assess the impact of unobserved specimen self-collection on test performance, we examined results from a SARS-CoV-2 qualitative RT-PCR test for self-collected specimens from participants in a return-to-work screening program and assessed the impact of a pooled testing strategy in this cohort.MethodsSelf-collected anterior nasal swabs from employee return-to-work programs were tested using the Quest Diagnostics Emergency Use Authorization SARS-CoV-2 RT-PCR. The cycle threshold (Ct) values for the N1 and N3 N-gene targets and a human RNase P (RP) gene control target were tabulated. For comparison, we utilized Ct values from a cohort of health care provider–collected specimens from patients with and without coronavirus disease 2019 symptoms.ResultsAmong 47 923 participants, 1.8% were positive. RP failed to amplify for 13/115 435 (0.011%) specimens. The median (interquartile range) Cts were 32.7 (25.0–35.7) for N1 and 31.3 (23.8–34.2) for N3. Median Ct values in the self-collected cohort were significantly higher than those of symptomatic but not asymptomatic patients. Based on Ct values, pooled testing with 4 specimens would have yielded inconclusive results in 67/1268 (5.2%) specimens but only a single false-negative result.ConclusionsUnobserved self-collection of nasal swabs provides adequate sampling for SARS-CoV-2 RT-PCR testing. These findings alleviate concerns of increased false negatives in this context. Specimen pooling could be used for this population, as the likelihood of false-negative results is very low when using a sensitive, dual-target methodology.

Influence of biopsy cores number performed with targeted prostate biopsy on the likelihood of a positive result in patients with clinically significant prostate cancer

Background. Targeted biopsy is proposed as a method of choice in the algorithm of prostate cancer diagnosis, but not all the features of method has been evaluated.Objective: determine the rational number of targeted biopsy samples in patients with clinically significant prostate cancer.Materials and methods. The magnetic resonance imaging and fusion biopsy data of 156 patients with suspected prostate cancer were retrospectively evaluated.Results and conclusion. In the study statistically significant dependence of the positive histological results in patients with clinically significant prostate cancer from the number of biopsy samples was found. The potential probability of a false negative histological examination with an insufficient number of biopsy samples was noted. These results confirm the latest published data of potential targeted biopsy false in true positive patients after multiparametric magnetic resonance imaging. An increase in the number of biopsy samples in the target lesion reduces the likelihood of false-negative results. The main causes of such discrepancy are some technical laxity and the heterogeneous histological structure of prostate cancer. Increase the number of biopsy cores can reduce the likelihood of false-negative results.

A C6orf10/LOC101929163 locus is associated with age of onset in C9orf72 carriers.

The G4C2-repeat expansion in C9orf72 is the most common known cause of amyotrophic lateral sclerosis and frontotemporal dementia. The high phenotypic heterogeneity of C9orf72 patients includes a wide range in age of onset, modifiers of which are largely unknown. Age of onset could be influenced by environmental and genetic factors both of which may trigger DNA methylation changes at CpG sites. We tested the hypothesis that age of onset in C9orf72 patients is associated with some common single nucleotide polymorphisms causing a gain or loss of CpG sites and thus resulting in DNA methylation alterations. Combined analyses of epigenetic and genetic data have the advantage of detecting functional variants with reduced likelihood of false negative results due to excessive correction for multiple testing in genome-wide association studies. First, we estimated the association between age of onset in C9orf72 patients (n = 46) and the DNA methylation levels at all 7603 CpG sites available on the 450 k BeadChip that are mapped to common single nucleotide polymorphisms. This was followed by a genetic association study of the discovery (n = 144) and replication (n = 187) C9orf72 cohorts. We found that age of onset was reproducibly associated with polymorphisms within a 124.7 kb linkage disequilibrium block tagged by top-significant variation, rs9357140, and containing two overlapping genes (LOC101929163 and C6orf10). A meta-analysis of all 331 C9orf72 carriers revealed that every A-allele of rs9357140 reduced hazard by 30% (P = 0.0002); and the median age of onset in AA-carriers was 6 years later than GG-carriers. In addition, we investigated a cohort of C9orf72 negative patients (n = 2634) affected by frontotemporal dementia and/or amyotrophic lateral sclerosis; and also found that the AA-genotype of rs9357140 was associated with a later age of onset (adjusted P = 0.007 for recessive model). Phenotype analyses detected significant association only in the largest subgroup of patients with frontotemporal dementia (n = 2142, adjusted P = 0.01 for recessive model). Gene expression studies of frontal cortex tissues from 25 autopsy cases affected by amyotrophic lateral sclerosis revealed that the G-allele of rs9357140 is associated with increased brain expression of LOC101929163 (a non-coding RNA) and HLA-DRB1 (involved in initiating immune responses), while the A-allele is associated with their reduced expression. Our findings suggest that carriers of the rs9357140 GG-genotype (linked to an earlier age of onset) might be more prone to be in a pro-inflammatory state (e.g. by microglia) than AA-carriers. Further, investigating the functional links within the C6orf10/LOC101929163/HLA-DRB1 pathway will be critical to better define age-dependent pathogenesis of frontotemporal dementia and amyotrophic lateral sclerosis.

Development of a one-step duplex RT-qPCR for the quantification of phocine distemper virus.

Worldwide, stranded marine mammals and the network personnel who respond to marine mammal mortality have provided much of the information regarding marine morbillivirus infections. An assay to determine the amount of virus present in tissue samples would be useful to assist in routine surveying of animal health and for monitoring large-scale die-off events. False negatives from poor-quality samples prevent determination of the true extent of infection, while only small amounts of tissue samples or archived RNA may be available at the time of collection for future retrospective analysis. We developed a one-step duplex real-time reverse transcriptase-quantitative-PCR assay (RT-qPCR) based on Taqman probe technology to quantify phocine distemper virus (PDV) isolated from an outbreak in harbor (Phoca vitulina concolor) and gray seals (Halichoerus grypus) along the northeast US coast in 2006. The glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene was selected to assess RNA quality. This duplex assay is specific for PDV and sensitive through a range of 10(0) to 10(9) copies ds-plasmid DNA. For the GAPDH target, the reaction in duplex amplified 10(0) to 10(9) copies of ds-plasmid DNA and was detectable in multiple seal species. This assay reduced the likelihood of false negative results due to degradation of tissues and well-to-well variability while providing sensitive and specific detection of PDV, which would be applicable in molecular epidemiologic studies and pathogen detection in field and laboratory investigations involving a variety of seal species.

Comparison of immuno-fluorescence microscopy and optical microscopy after Giemsa staining in the diagnosis of malaria during pregnancy in Buea, Cameroon

Malaria in pregnancy remains a significant threat in sub-Saharan Africa as it is associated with suboptimal pregnancy outcomes. Due to the fact that the current standard diagnostic method is cumbersome, we assessed the diagnostic performance of an alternate method-fluorescent microscopy, compared to optical microscopy after Giemsa staining- the current standard.Venous blood samples were collected from 407 consenting pregnant women and tested by optical microscopy (OM) after Giemsa staining and by fluorescent microscopy (FM) using the Partec-Cyscope (Partec GmbH, Munster, Germany). All participants were asymptomatic at the time of enrollment. Of the 407 samples tested by OM, 255 (62.5%) were plasmodiumpositive. Of the 255 OM positive samples, 207 were also plasmodium-positive by FM, thus a sensitivity of 81.1% (95% confidence interval (CI): 75.8, 85.8%). Of the 152 samples negative by OM, 75 were also negative by FM, thus a specificity of 49.3% (95%CI: 41.1, 57.6%). The positive and negative predictive values of FM were respectively 72.9% (95%CI: 67.3, 78.0%) and 61.0% (95%CI: 51.8, 69.6%). The percentage agreement between both methods was 69.3% (Kappa=0.32, p-value<0.01). There was moderate agreement between FM and OM results. The low specificity and negative predictive value of FM suggest a high likelihood of false negative results if FM is used in place of OM. OM after Giemsa staining remains the gold standard. Keywords : Diagnostic performance, sensitivity, specificity, positive predictive value, negative predictive value, Cameroon.

Axillary Staging by Ultrasound-guided Fine-Needle Aspiration Cytology in Breast Cancer Patients. Still Up to Date?

To investigate whether clinicopathological features of breast cancer patients could predict the likelihood of lymph node metastases and the likelihood of false-negative results of ultrasound-guided fine-needle aspiration cytology of suspicious lymph nodes (US+FNAC). Between 2004 and 2009, US+FNAC was performed in 1,150 axillae (18 bilateral breast carcinomas). Based on final histologic diagnosis, the true- and false-negative group of US+FNAC were defined. Subsequently, 11 clinicopathological factors were compared between these two groups. These factors were also compared between patients with and patients without lymph node metastases. Of 1,150 axillae, 429 had lymph node metastases at final histology. US+FNAC indicated metastases in 107 axillae. 1,043 axillae were negative by US+FNAC. Final histology showed metastases in 323 of these 1,043 axillae, resulting in a false-negative group of US+FNAC of 31%. Both age <60 years and a cT2/cT3 breast carcinoma were significantly associated with lymph node metastases and with false-negative results of US+FNAC. Lymph node metastases were found in 59.6% of patients <60 years with a cT2/cT3 breast carcinoma. In these patients, 52.3% of the negative US+FNAC results were falsely negative. In patients <60 years with a cT2/cT3 breast carcinoma, we recommend to omit US+FNAC preoperatively and perform a SNB directly, because lymph node metastases were found in 59.6% of these patients and 52.3% of negative US+FNAC results were falsely negative.

W14-01 Reinventing the wheel in clinical trial design

Recent publications by the Institute of Medicine have unearthed several fundamental flaws in clinical trial methodology that, if corrected by the next generation of clinical investigators, can transform the field of mental health intervention research. Using a clinician-friendly approach, this workshop will succinctly review the essential elements of optimal design and implementation of a randomized controlled clinical study and the strategies to enhance signal detection. These include minimizing bias in the estimate of treatment effect while maintaining a nominal level of type I error (i.e., false positive results) and maintaining sufficient statistical power (i.e. reducing the likelihood of false negative results). Particular attention will be paid to reducing the problems of attrition and the hazards of multiplicity. Methods to examine moderators of the treatment effect will also be explored. Examples from psychopharmacologic, psychotherapy, and vocational rehabilitation trials for the treatment of posttraumatic stress disorder, depression, and panic disorder will be provided to illustrate these issues. Techniques to reduce the study's costs, risks, and participant burden will be described. Following the didactic session, the participants are encouraged to bring forth their own questions regarding clinical trial design for a 45-minute interactive discussion with the presenters. The objectives of the workshop are to improve the participants’ understanding of the goals of clinical trial design and methods to achieve those goals in order to improve their own research techniques, grantsmanship, and abilities to more accurately judge the results of studies presented in the literature.

W14-03 Enhancing signal detection in clinical trials

Dr. Leon will present the biostatistical considerations that contribute to a clinical trial design and the strategies to enhance signal detection. These include minimizing bias in the estimate of treatment effect while maintaining a nominal level of type I error (i.e., false positive results) and maintaining sufficient statistical power (i.e. reducing the likelihood of false negative results). Particular attention will be paid to reducing the problems of attrition and the hazards of multiplicity. Methods to examine moderators of the treatment effect will also be explored. Examples from psychopharmacologic and psychotherapy trials for the treatment of depression and panic disorder will be provided to illustrate these issues. Following the didactic session, the participants will be encouraged to bring forth their own questions regarding clinical trial design for a 45-minute interactive discussion with the presenters. The objectives of the workshop are to improve the participants’ understanding of the goals of clinical trial design and methods to achieve those goals in order to improve their own research techniques, grantsmanship, and abilities to more accurately judge the results of studies presented in the literature.

Conventional Pap smear and liquid based cytology for cervical cancer screening - a comparative study

The study was undertaken to evaluate liquid based cytology (Pap spin) and to compare the sensitivity of Pap spin with conventional Pap smear. Pap smears were taken from 160 patients with gynaecologic complaints with Ayre's spatula and endocervical cytobrush and slides prepared. The residual material on both the spatula and cytobrush were rinsed in Pap spin collection fluid and centrifuged. Direct smears were prepared from the cell button, fixed and stained by Papanicolaou stain. Colposcopy was performed in patients with abnormal smears and biopsy was performed in suspected malignant or dysplastic cases. LSIL and HSIL was diagnosed in 27 (64.4%) cases, mostly between 21-40 years, Commonest presenting complaint was discharge per vaginum seen in 68 (42.5%) cases. Cytological abnormality was found in 42 cases (26.2%) by Pap spin method, whereas conventional Pap smear detected abnormality in only 24 cases (15%). 133 cases (83.1%) were satisfactory for evaluation on Pap spin and 51 cases (31.9%) on conventional Pap smear. The commonest atypical finding on colposcopy was acetowhite area in 14 cases (31.1%). Sensitivity of Pap spin and conventional Pap smear was 97.6% and 53.7% respectively and specificity was 50% in each. Pap spin is strongly advocated in the best interest of public health as it improves the sample quality and reduces the likelihood of false negative results.

Simulating kernel lot sampling: the effect of heterogeneity on the detection of GMO contaminations

Guidelines defining kernel sampling strategies for quality analyses have been provisionally adopted for the detection of genetically modified (GM) contamination in kernel lots. However, these guidelines are not specific for GM material detection and are not intended for the sampling of non-uniform distributions, a probable situation with respect to the presence of GM material in kernel lots. An analysis of the problem of non-random distribution, through the investigation of the effectiveness of different sampling techniques in producing representative bulk samples, is presented. The analysis is based on a two-step modelling procedure: 1) the kernel lot is created and 2) the lot is sampled to produce a bulk sample. This allows the identification of optimal sampling techniques as a function of specific combinations of population characteristics. For each of 5 levels of GM impurity, varying between 0.1% and 2%, we investigated the effect of 5 levels of stratification (lot size=10 7 kernels). Our results indicate: I) For every GM level, the higher the heterogeneity level, the more unstable the GM estimate becomes; even modest levels of stratification affect the stability of GM estimates. 2) As the number of increment samples increases, the coefficient of variation (CV) of the estimate decreases. Although the pattern of decrease remains similar across stratification levels, the estimated CV changes: with low levels of stratification. 50 samples are enough to obtain estimates with CV<10%. In the case of modest levels of stratification even 100 samples are not sufficient to maintain CV<10%. In case of strongly heterogeneous lots estimates based on 100 units have CVs around 50%. At the same time, the likelihood of false negative results increases significantly.

The development of a competitive PCR–ELISA for the detection of equine herpesvirus-1

Equine herpesvirus-1 (EHV-1) infection is of significant animal welfare and economic importance. Yet, no standardised molecular techniques are available for diagnosis or confirmation of viral infection. The purpose of this study was to develop a standardised and quantitative assay system for the reliable detection of EHV-1 infection which was capable of eliminating the likelihood of false negative results. A region within the EHV-1 glycoprotein B gene was amplified by polymerase chain reaction (PCR), cloned and subjected to site-directed mutagenesis to generate a control plasmid, amplifiable by identical primers to wild type EHV-1, yet capable of detection by an alternate dinitrophenylated oligonucleotide probe in a PCR–ELISA system. A competitive PCR–ELISA system which can control for the presence of PCR inhibitors and which is capable of detecting 63 genome equivalents of EHV-1 has been developed. EHV-1 presence in infected equine tissue and cell culture material was demonstrated using this system. The entire assay can be completed within one working day and facilitates multiple sample analysis. The availability of a robust, competitive PCR–ELISA system for the detection of EHV-1 will facilitate the rapid and sensitive detection of EHV-1 and offers the potential for eliminating the occurrence of abortion storms in stud farms.

Investigation of 9-O-acetylated sialoglycoconjugates in childhood acute lymphoblastic leukaemia.

Modern treatment protocols of childhood acute lymphoblastic leukaemia (ALL) utilize the biological and clinical features of the disease to tailor the intensity of therapy to the risk of relapse by evaluation of minimal residual disease (MRD) using several molecular techniques. To minimize the likelihood of false negative results, tandem application of at least two methods for detection of MRD is currently recommended. To address this question, the present review deals with the recently evolved information reported by our group regarding the presence of O-acetylated sialoglycoconjugates (O-AcSGs) in childhood ALL and their potential as novel biomarkers for monitoring the disease.

Nested primers improve sensitivity in the detection of Helicobacter pylori by the polymerase chain reaction

To investigate potential routes of spread of infection by the polymerase chain reaction (PCR) it is important that the technique is effective in the types of specimen to be investigated. To establish the limits of detection of Helicobacter pylori by PCR in clinical material from the gastric mucosa, faeces, dental plaque and oral rinses, samples were seeded with known numbers of bacteria. DNA extraction was followed by amplification with primers from the urease C gene. Nested primers were used to amplify the PCR product which was detected using a digoxigenin-labelled probe. Faeces or plaque inhibited the single reaction 10(2)-10(6) fold. A second amplification using nested primers and probing increased the sensitivity to a level similar to that obtained with pure culture. This method is potentially useful with less likelihood of false negative results when trying to detect H. pylori by PCR in highly contaminated, clinical material.

Variability in lesion depth on prone and supine CT scans of the chest: implications for the accuracy of transthoracic needle aspiration biopsy.

Transthoracic needle biopsy of the lung is often performed fluoroscopically in the prone position; nodule depth measurements are made from accompanying computed tomographic (CT) scans performed supine. We evaluated the effect of prone and supine positioning on the effect of nodule depth as measured from the skin surface. Twenty consecutive patients having CT-guided biopsy performed in the prone position were assessed. Nodule depth from posterior skin surface to nodule was compared with prebiopsy supine CT scan. Nodules above the carina showed minimal change in depth. Those below the carina showed considerable variability, with depth changes < or = 4.0 cm. Awareness of the magnitude of the potential effect of patient position on lesion depth should be helpful in reducing the likelihood of false negative results in fluoroscopically guided biopsy.