scholarly journals Plasmodium falciparum hrp2 and hrp3 gene deletion status in Africa and South America by highly sensitive and specific digital PCR

2021 ◽  
Author(s):  
Claudia A. Vera-Arias ◽  
Aurel Holzschuh ◽  
Colins O. Oduma ◽  
Kingsley Badu ◽  
Mutala Abdul-Hakim ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
South America ◽  
Limit Of Detection ◽  
False Negative ◽  
Digital Pcr ◽  
Wild Type ◽  
Exon 2 ◽  
Negative Results ◽  
Southwestern Ethiopia ◽  
Asymptomatic Individuals

Background: The most commonly used Plasmodium falciparum rapid diagnostic tests target the Histidine-Rich Proteins 2 and 3 (HRP2, HRP3). An increasing number of countries report parasites that carry hpr2 and/or hrp3 gene deletions, resulting in false negative test results. Molecular surveillance of hrp2 and hrp3 deletions is crucial but adequate protocols have been lacking. Methods and Findings: We have developed novel assays for deletion typing based on droplet digital PCR (ddPCR), targeting hrp2 exon1, hrp2 exon 2, and hrp3. In the ddPCR assay, hrp2 or hrp3 and a control gene were quantified with very high accuracy in a single tube. The theoretical limit of detection of the ddPCR assay was 0.33 parasites/uL, and thus well suited for typing of low-density asymptomatic infections. The deletion was reliably detected in mixed infections with wild-type and hrp2-deleted parasites when the proportion of parasites carrying the deletion was >40%. For a side-by-side comparison with the conventional nested PCR (nPCR) assay, 248 samples from asymptomatic individuals from western Kenya were screened in triplicate by ddPCR and nPCR. No deletions were observed by ddPCR, while by nPCR no band for hrp2 was observed in 8% of samples. The ddPCR assay was applied to screen 777 samples from six countries in Africa and South America. No deletions were observed in Kenya (n=241) and Zanzibar/Tanzania (n=91), and very few in Ghana (3/170 hrp3 deletions). In southwestern Ethiopia, 1/47 (2.1%) samples carried hrp2 deletion, and 35/47 (74.5%) hrp3 deletions. In Brazil, 87/187 (46.5%) samples carried hrp2 deletions, and 116/187 (62%) hrp3 deletions. In Ecuador, no hrp2 deletions were observed, but 22/41 (53.7%) samples carried hrp3 deletions. Conclusions: Compared to nPCR, the ddPCR assay minimizes the risk of false-negative results (i.e. hrp2 deletion observed when the sample is wild type), increase sensitivity, and greatly reduces the number of reactions that need to be run. Pronounced differences in the prevalence of deletion were observed among sites, with more hrp3 than hrp2 deletions.

scholarly journals Multiple Hotspot Mutations Scanning by Single Droplet Digital PCR

2018 ◽  
Vol 64 (2) ◽  
pp. 317-328 ◽  
Author(s):  
Charles Decraene ◽  
Amanda B Silveira ◽  
François-Clément Bidard ◽  
Audrey Vallée ◽  
Marc Michel ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Limit Of Detection ◽  
Clinical Importance ◽  
Digital Pcr ◽  
Circulating Tumor Dna ◽  
Droplet Digital Pcr ◽  
Egfr Mutations ◽  
Detection Accuracy ◽  
Exon 2 ◽  
Exon 19

Abstract BACKGROUND Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown a priori remains limited. METHODS We established 2 ddPCR assays detecting all genomic alterations within KRAS exon 2 and EGFR exon 19 mutation hotspots, which are of clinical importance in colorectal and lung cancer, with use of a unique pair of TaqMan® oligoprobes. The KRAS assay scanned for the 7 most common mutations in codons 12/13 but also all other mutations found in that region. The EGFR assay screened for all in-frame deletions of exon 19, which are frequent EGFR-activating events. RESULTS The KRAS and EGFR assays were highly specific and both reached a limit of detection of <0.1% in mutant allele frequency. We further validated their performance on multiple plasma and formalin-fixed and paraffin-embedded tumor samples harboring a panel of different KRAS or EGFR mutations. CONCLUSIONS This method presents the advantage of detecting a higher number of mutations with single-reaction ddPCRs while consuming a minimum of patient sample. This is particularly useful in the context of liquid biopsy because the amount of circulating tumor DNA is often low. This method should be useful as a discovery tool when the tumor tissue is unavailable or to monitor disease during therapy.

scholarly journals Distribution of SARS-CoV-2 PCR Cycle Threshold Values Provide Practical Insight Into Overall and Target-Specific Sensitivity Among Symptomatic Patients

2020 ◽  
Vol 154 (4) ◽  
pp. 479-485 ◽  
Author(s):  
Blake W Buchan ◽  
Jessica S Hoff ◽  
Cameron G Gmehlin ◽  
Adriana Perez ◽  
Matthew L Faron ◽  
...  
Keyword(s):  
Viral Load ◽  
Limit Of Detection ◽  
False Negative ◽  
Age Group ◽  
Rt Pcr ◽  
Patient Age ◽  
Cycle Threshold ◽  
Negative Results ◽  
Patient Âgé ◽  
False Negative Results

Abstract Objectives We examined the distribution of reverse transcription polymerase chain reaction (RT-PCR) cycle threshold (CT) values obtained from symptomatic patients being evaluated for coronavirus disease 2019 (COVID-19) to determine the proportion of specimens containing a viral load near the assay limit of detection (LoD) to gain practical insight to the risk of false-negative results. We also examined the relationship between CT value and patient age to determine any age-dependent difference in viral load or test sensitivity. Methods We collected CT values obtained from the cobas severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) assay corresponding to 1,213 combined nasopharyngeal-oropharyngeal specimens obtained from symptomatic individuals that were reported as positive or presumptive positive for SARS-CoV-2. CT values were stratified by SARS-CoV target and patient age group. Results In total, 93.3% to 98.4% of specimens demonstrated CT values greater than 3× the assay LoD, at which point false-negative results would not be expected. The mean of CT values between age groups was statistically equivalent with the exception of patients in age group 80 to 89 years, which demonstrated slightly lower CTs. Conclusions Based on the distribution of observed CT values, including the small proportion of specimens with values near the assay LoD, there is a low risk of false-negative RT-PCR results in combined nasopharyngeal-oropharyngeal specimens obtained from symptomatic individuals.

scholarly journals Diagnostic Performance of Hrp2 Based Diagnostic Test Kits and Frequency of Pfhrp2 Gene Deletion in Plasmodium falciparum Isolates of Osogbo, Southwestern Nigeria

2021 ◽  
pp. 20-25
Author(s):  
A. S. Nassar ◽  
A. S. Bakarey ◽  
A. A. Abdulazeez ◽  
O. O. Fayemi
Keyword(s):  
Plasmodium Falciparum ◽  
Diagnostic Test ◽  
Gene Deletion ◽  
False Negative ◽  
University Teaching ◽  
Pcr Analysis ◽  
Southwestern Nigeria ◽  
Blood Samples ◽  
Negative Results ◽  
Test Kit

Introduction: The introduction of P. falciparum encoded HRP-2 based malaria Rapid Diagnostic Test (RDT) kits is widely accepted in Nigeria and worldwide as a simplified form of diagnosis and a cheaper alternative to the microscopy technique (gold standard). However, deletion of Pfhrp2 gene contributes to false negative results and large number of such deletions has been reported in advanced countries thereby highlighting the importance of surveillance to detect such deletions in our local environment. Methodology: Microscopy as well as RDT techniques (using Rapid malaria test kit: SD BIOLINE Malaria Ag P.f/Pv, South Korea) were carried out on the blood samples of three hundred and twenty-three (323) febrile subjects attending Ladoke Akintola University Teaching Hospital, Osogbo, Osun State Nigeria. PCR analysis was also conducted on 50 blood samples that were positive for microscopy but negative for RDT. Results: The results from the study revealed that microscopy had a sensitivity of 99% and specificity of 99.2%. The RDT however had a sensitivity of 100% and a specificity of 60.1%. Fifty (50) samples that were positive for microscopy but negative for RDT were subjected to further PCR examination to detect the possible deletion of the Pfhrp-2 gene and the result revealed that the gene was present in 39 (78%) of the blood samples while remaining 11 (22%) samples lacked the gene which could possibly be the reason for the negative results obtained using the RDT kits. Conclusion: This study provides evidence of low level of presence of Pfhrp-2 gene deletion of Plasmodium falciparum parasites in our healthcare facility setting in Osogbo, Nigeria.

scholarly journals Recurrent SARS-CoV-2 Infection: Case Reports and Review of the Literature

2021 ◽  
Vol 2 (2) ◽  
pp. 1-4
Author(s):  
Brian Conway ◽  
Keyword(s):  
Limit Of Detection ◽  
Case Reports ◽  
False Negative ◽  
Recurrent Infection ◽  
Family Unit ◽  
Her Family ◽  
Negative Results ◽  
False Negative Results ◽  
Clinical Syndromes ◽  
Relapsing Remitting

We describe a 37-year-old woman who became infected with SARS-CoV-2. Over time, 4 other members in her family unit became infected, with 3/5 developing 2-3 separate clinical syndromes over two months. It is possible that each person had a single prolonged infection, with the literature reporting RNA detection for as long as 83 days in some cases. Syndromes of relapsing/remitting infection have also been well described. Intermittent negative RNA readings may represent “false negative” results with intermittent levels of viremia that occasionally fall below the limit of detection of the assay. An alternative explanation may be multiple episodes of infection, clearance, and re-infection within the family unit. Preliminary reports in the literature suggest onward transmission after recurrent infection in 3 reported cases. An understanding of the prevalence of cases series such as ours and their pathophysiologic and immunologic significance will improve our knowledge about SARS-CoV-2 infection and strategies to control it.

scholarly journals SARS-CoV-2 detection using digital PCR for COVID-19 diagnosis, treatment monitoring and criteria for discharge

2020 ◽  
Author(s):  
Renfei Lu ◽  
Jian Wang ◽  
Min Li ◽  
Yaqi Wang ◽  
Jia Dong ◽  
...  
Keyword(s):  
Viral Load ◽  
Limit Of Detection ◽  
False Negative ◽  
Digital Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pharyngeal Swab ◽  
Clinical Specimens ◽  
Positive Results ◽  
Negative Detection

SummaryBackgroundSARS-CoV-2 nucleic acid detection by RT-PCR is one of the criteria approved by China FDA for diagnosis of COVID-19. However, inaccurate test results (for example, high false negative rate and some false positive rate) were reported in both China and US CDC using RT-PCR method. Inaccurate results are caused by inadequate detection sensitivity of RT-PCR, low viral load in some patients, difficulty to collect samples from COVID-19 patients, insufficient sample loading during RT-PCR tests, and RNA degradation during sample handling process. False negative detection could subject patients to multiple tests before diagnosis can be made, which burdens health care system. Delayed diagnosis could cause infected patients to miss the best treatment time window. False negative detection could also lead to prematurely releasing infected patients who still carry residual SARS-CoV-2 virus. In this case, these patients could infect many others. A high sensitivity RNA detection method to resolve the existing issues of RT-PCR is in need for more accurate COVID-19 diagnosis.MethodsDigital PCR (dPCR) instrument DropX-2000 and assay kits were used to detect SARS-CoV-2 from 108 clinical specimens from 36 patients including pharyngeal swab, stool and blood from different days during hospitalization. Double-blinded experiment data of 108 clinical specimens by dPCR methods were compared with results from officially approved RT-PCR assay. A total of 109 samples including 108 clinical specimens and 1 negative control sample were tested in this study. All of 109 samples, 26 were from 21patients reported as positive by officially approved clinical RT-PCR detection in local CDC and then hospitalized in Nantong Third Hospital. Among the 109 samples, dPCR detected 30 positive samples on ORFA1ab gene, 47 samples with N gene positive, and 30 samples with double positive on ORFA1ab and N genes.ResultsThe lower limit of detection of the optimize dPCR is at least 10-fold lower than that of RT-PCR. The overall accuracy of dPCR for clinical detection is 96.3%. 4 out 4 of (100 %) negative pharyngeal swab samples checked by RT-PCR were positive judged by dPCR based on the follow-up investigation. 2 of 2 samples in the RT-PCR grey area (Ct value > 37) were confirmed by dPCR with positive results. 1 patient being tested positive by RT-PCR was confirmed to be negative by dPCR. The dPCR results show clear viral loading decrease in 12 patients as treatment proceed, which can be a useful tool for monitoring COVID-19 treatment.ConclusionsDigital PCR shows improved lower limit of detection, sensitivity and accuracy, enabling COVID-19 detection with less false negative and false positive results comparing with RT-PCR, especially for the tests with low viral load specimens. We showed evidences that dPCR is powerful in detecting asymptomatic patients and suspected patients. Digital PCR is capable of checking the negative results caused by insufficient sample loading by quantifying internal reference gene from human RNA in the PCR reactions. Multi-channel fluorescence dPCR system (FAM/HEX/CY5/ROX) is able to detect more target genes in a single multiplex assay, providing quantitative count of viral load in specimens, which is a powerful tool for monitoring COVID-19 treatment.

Monitoring tumor specific mutations in plasma during systemic treatment of biliary tract cancer.

2017 ◽  
Vol 35 (4_suppl) ◽  
pp. 318-318
Author(s):  
Lars Henrik Jensen ◽  
Rikke Fredslund Andersen ◽  
Anders Kristian Moeller Jakobsen
Keyword(s):  
Biliary Tract ◽  
Biliary Tract Cancer ◽  
Digital Pcr ◽  
Braf V600e ◽  
Wild Type ◽  
Liquid Biopsies ◽  
Exon 2 ◽  
Tract Cancer ◽  
Initial Drop ◽  
Kras Exon

318 Background: We have previously reported a rate of 23% RAS/RAF mutations in plasma from patients with KRAS exon 2 and 3 wild-type, non-resectable biliary tract tumors. We wanted to explore the changes in circulating tumor specific DNA (ctDNA) during systemic chemotherapy in these patients. Methods: Patients with non-resectable biliary tract cancer treated within a phase II trial were included if they had KRAS exon 2 and 3 wild-type tumor tissue, had quantifiable levels of tumor specific DNA in plasma and progressive disease on imaging. They received gemcitabine, oxaliplatin and capecitabine with either bevacizumab or panitumumab. Treatment continued for up to six months until progression. Blood sampling and evaluation according to RECIST 1.1 were done every 12 weeks. Droplet Digital PCR was performed on DNA isolated from 4 ml plasma. A pre-amplification step was done and adequate positive and negative controls were included. The extended RAS and BRAF mutation analysis covered 20 mutations in KRAS exons 3/4, NRAS exon 2/3, PIK3CA and BRAF V600E. The percentage of tumor specific DNA relative to total DNA was reported. Results: The inclusion criteria were met by 13 patients, 10 women and three men. The typical pattern was seen in eight cases, where the percentage of tumor specific DNA dropped at least half during therapy and rose at least two-fold at progression. In three patients, a baseline sample was not available or there was not an initial drop, but the ctDNA rose at progression. One patient had an initial drop, but not a rise a progression based on imaging. The last patient progressed rapidly. Conclusions: This exploratory analysis pointed toward changes in percentage of tumor specific mutations in plasma as a marker of effect and progression. Dynamics of liquid biopsies is a promising tool in monitoring biliary tract cancer patients during systemic therapy. Clinical trial information: NCT01206049.

scholarly journals Mutation Detection in the Menkes Gene ATP7A Using the Protein Truncation Test

2008 ◽  
Vol 1 ◽  
pp. CPath.S565 ◽  
Author(s):  
Lisbeth Birk Møller ◽  
Nina Horn
Keyword(s):  
False Negative ◽  
Copper Metabolism ◽  
Menkes Disease ◽  
Allelic Exclusion ◽  
Missense Mutations ◽  
Coding Region ◽  
Protein Truncation Test ◽  
Exon 2 ◽  
Negative Results ◽  
Protein Truncation

Menkes disease (MD) is a rare recessively inherited lethal disorder of copper metabolism. The gene ATP7A defective in MD consists of 23 exons and the coding region encompasses 4500 bp. About 300 distinct mutations, representing all types, have been identified in ATP7A. However all mutations identified so far in the exon 2 to exon 7, corresponding to 1869 bp of the coding sequence, result in truncated protein products. No missense mutations have been identified in this region. As about 30% of the total number of mutations identified are located in exon 2 to exon 7, we have designed a protein truncation test (PTT) for rapid detecting of mutations in this part of the gene. In order to determine the applicability of the test, we analysed RNA obtained from eleven MD patients with known mutations in this region. As a truncated product could be identified in all the included samples, PTT proves to be a useful technique for rapid detection of mutations in the N-terminal part of the ATP7A gene. Furthermore as MD is a X-linked disease, normally only affecting boys, the risk of false negative results, due to nonsense mediated RNA decay, leading to allelic exclusion, can be left out of account.

scholarly journals To Test, Perchance to Diagnose: Practical Strategies for SARS-CoV-2 Testing

2021 ◽  
Author(s):  
Romney M Humphries ◽  
Marwan M Azar ◽  
Angela M Caliendo ◽  
Andrew Chou ◽  
Robert C Colgrove ◽  
...  
Keyword(s):  
False Negative ◽  
Virus Antigen ◽  
Multifaceted Approach ◽  
Negative Results ◽  
Testing Strategies ◽  
Asymptomatic Patients ◽  
False Negative Results ◽  
Different Populations ◽  
Asymptomatic Individuals ◽  
Professional Guidelines

Abstract Testing for SARS-CoV-2 in symptomatic and asymptomatic patients is an important component of the multifaceted approach of managing the COVID-19 pandemic. Determining how to best define testing strategies for different populations and incorporating these into broader infection prevention programs can be complex. Many circumstances are not addressed by federal, local or professional guidelines. This commentary describes various scenarios where testing of symptomatic or asymptomatic individuals for SARS-CoV-2 virus (antigen or RNA) can be of potential benefit. Consideration to pre-test probability, risks of testing (impact of false-positive or false-negative results), testing strategy as well as action based on test results are explored. Testing, regardless of setting, must be incorporated into overarching infection control plans which include use of personal protective equipment (e.g., masks), physically distancing, and isolation when exposure is suspected.

scholarly journals High-Throughput Nucleotide Resolution Predictions of Assay Limitations Increase the Reliability and Concordance of Clinical Tests

2021 ◽  
pp. 1085-1095
Author(s):  
Jonathan Bieler ◽  
Christian Pozzorini ◽  
Jessica Garcia ◽  
Alex C. Tuck ◽  
Morgane Macheret ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
False Negative ◽  
Variant Calling ◽  
Biophysical Model ◽  
Sequencing Error ◽  
Genomic Context ◽  
Negative Results ◽  
False Negative Results ◽  
Estimate Reliability ◽  
Nucleotide Resolution

PURPOSE The ability of next-generation sequencing (NGS) assays to interrogate thousands of genomic loci has revolutionized genetic testing. However, translation to the clinic is impeded by false-negative results that pose a risk to patients. In response, regulatory bodies are calling for reliability measures to be reported alongside NGS results. Existing methods to estimate reliability do not account for sample- and position-specific variability, which can be significant. Here, we report an approach that computes reliability metrics for every genomic position and sample interrogated by an NGS assay. METHODS Our approach predicts the limit of detection (LOD), the lowest reliably detectable variant fraction, by taking technical factors into account. We initially explored how LOD is affected by input material amount, library conversion rate, sequencing coverage, and sequencing error rate. This revealed that LOD depends heavily on genomic context and sample properties. Using these insights, we developed a computational approach to predict LOD on the basis of a biophysical model of the NGS workflow. We focused on targeted assays for cell-free DNA, but, in principle, this approach applies to any NGS assay. RESULTS We validated our approach by showing that it accurately predicts LOD and distinguishes reliable from unreliable results when screening 580 lung cancer samples for actionable mutations. Compared with a standard variant calling workflow, our approach avoided most false negatives and improved interassay concordance from 94% to 99%. CONCLUSION Our approach, which we name LAVA (LOD-aware variant analysis), reports the LOD for every position and sample interrogated by an NGS assay. This enables reliable results to be identified and improves the transparency and safety of genetic tests.

scholarly journals Geographic Classification and Identification of SARS-CoV2 From Related Viral Sequences

2021 ◽  
Vol 15 ◽  
pp. 254-259
Author(s):  
Fayroz. F. Sherif ◽  
Khaled. S. Ahmed
Keyword(s):  
South America ◽  
Viral Infections ◽  
Markov Models ◽  
False Negative ◽  
Molecular Tests ◽  
Negative Results ◽  
International Response ◽  
False Negative Results ◽  
Geographical Regions ◽  
Human Infections

The COVID-19 pandemic has introduced to mild the risks of deadly epidemic-prone illnesses sweeping our globalized planet. The pandemic is still going strong, with additional viral variations popping up all the time. For the close to future, the international response will have to continue. The molecular tests for SARS-CoV-2 detection may lead to False-negative results due to their genetic similarity with other coronaviruses, as well as their ability to mutate and evolve. Furthermore, the clinical features caused by SARS-CoV-2 seem to be like the symptoms of other viral infections, making identification even harder. We constructed seven hidden Markov models for each coronavirus family (SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, MERS-CoV, and SARS-CoV), using their complete genome to accurate diagnose human infections. Besides, this study characterized and classified the SARS-CoV2 strains according to their different geographical regions. We built six SARS-CoV2 classifiers for each world's continent (Africa, Asia, Europe, North America, South America, and Australia). The dataset used was retrieved from the NCBI virus database. The classification accuracy of these models achieves 100% in differentiating any virus model among others in the Coronavirus family. However, the accuracy of the continent models showed a variable range of accuracies, sensitivity, and specificity due to heterogeneous evolutional paths among strains from 27 countries. South America model was the highest accurate model compared to the other geographical models. This finding has vital implications for the management of COVID-19 and the improvement of vaccines.

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