False-negative Polymerase Chain Reaction Research Articles

Direct comparison of clinical diagnostic sensitivity of saliva from buccal swabs versus combined oro-/nasopharyngeal swabs in the detection of SARS-CoV-2 B.1.1.529 Omicron

While current guidelines recommend the use of respiratory tract specimens for the direct detection of SARS-CoV-2 infection, saliva has recently been suggested as preferred sample type for the sensitive detection of SARS-CoV-2 B.1.1.529 (Omicron). By comparing saliva collected using buccal swabs and oro-/nasopharyngeal swabs from patients hospitalized due to COVID-19, we aimed at identifying potential differences in virus detection sensitivity between these sample types. We compare the clinical diagnostic sensitivity of paired buccal swabs and combined oro-/nasopharyngeal swabs from hospitalized, symptomatic COVID-19 patients collected at median six days after symptom onset by real-time polymerase chain reaction (PCR) and antigen test. Of the tested SARS-CoV-2 positive sample pairs, 55.8% were identified as SARS-CoV-2 Omicron BA.1 and 44.2% as Omicron BA.2. Real-time PCR from buccal swabs generated significantly higher quantification cycle (Cq) values compared to those from matched combined oro-/nasopharyngeal swabs and resulted in an increased number of false-negative PCR results. Reduced diagnostic sensitivity of buccal swabs by real-time PCR was observed already at day one after symptom onset. Similarly, antigen test detection rates were reduced in buccal swabs compared to combined oro-/nasopharyngeal swabs. Our results suggest reduced clinical diagnostic sensitivity of saliva collected using buccal swabs when compared to combined oro-/nasopharyngeal swabs in the detection of SARS-CoV-2 Omicron in symptomatic individuals.

Considerations about Reducing False-Negative PCR Test for COVID-19.

Polymerase chain reaction (PCR) for the detection of nucleic acids is the gold standard test for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there is the probability of false-negative results with this test, which poses a threat to public health. Here, we highlight some important factors that should be considered for reducing the false-negative results of the SARS-CoV-2 PCR test.

Assessing the export trade risk of bluetongue virus serotypes 4 and 8 in France.

Bluetongue (BT) causes an economic loss of $3 billion every year in the world. After two serious occurrences of BT (bluetongue virus [BTV] occurrence in 2006 and 2015), France has been controlling for decades, but it has not been eradicated. As the largest live cattle export market in the world, France is also one of the major exporters of breeding animals and genetic materials in the world. The biosafety of its exported cattle and products has always been a concern. The scenario tree quantitative model was used to analyze the risk of BTV release from French exported live cattle and bovine semen. The results showed that with the increase in vaccination coverage rates, the risk decreased. If the vaccine coverage is 0%, the areas with the highest average risk probability of BTV-4 and BTV-8 release from exported live cattle were Haute-Savoie and Puy-de-Dôme, and the risk was 2.96 × 10-4 and 4.25 × 10-4 , respectively. When the vaccine coverage was 90%, the risk probability of BTV-4 and BTV-8 release from exported live cattle was 2.96 × 10-5 and 4.24 × 10-5 , respectively. The average probability of BTV-8 release from bovine semen was 1.09 × 10-10 . Sensitivity analysis showed that the probability of false negative polymerase chain reaction (PCR) test and the probability of BT infection in the bull breeding station had an impact on the model. The identification of high-risk areas and the discovery of key control measures provide a reference for decision makers to assess the risk of French exports of live cattle and bovine semen.

Perioperative Management During the COVID-19 Pandemic: Strategies at Three General Hospitals in Japan and a Narrative Review.

Coronavirus disease 2019 (COVID-19) has rapidly spread globally ever since the virus was first identified in December 2019 in Wuhan, China. Despite efforts to accelerate the supply of COVID-19 vaccines worldwide, the global pandemic has continued. Polymerase chain reaction (PCR) test is currently considered the gold standard for the diagnosis of COVID-19. However, the rate of false-negative PCR for COVID-19 has been reported to be over 10%. Furthermore, an asymptomatic period can last up to 14 days following the infection. Under these circumstances, standard anesthetic practice, surgery scheduling, and approaches to appropriate management of the operating room to protect both patients and medical personnel against COVID-19 transmission need to be reviewed and appropriately modified. In this review, based on our institutional experiences along with the guidelines reported elsewhere, we propose safer and more effective perioperative management amidst the COVID-19 pandemic.

Repeat virological and serological profiles in hospitalized patients initially tested by nasopharyngeal RT-PCR for SARS-CoV-2.

Real‐time polymerase chain reaction (PCR) for SARS‐CoV‐2 is the mainstay of COVID‐19 diagnosis, yet there are conflicting reports on its diagnostic performance. Wide ranges of false‐negative PCR tests have been reported depending on clinical presentation, the timing of testing, specimens tested, testing method, and reference standard used. We aimed to estimate the frequency of discordance between initial nasopharyngeal (NP) PCR and repeat NP sampling PCR and serology in acutely ill patients admitted to the hospital. Panel diagnosis of COVID‐19 infection is further utilized in discordance analysis. Included in the study were 160 patients initially tested by NP PCR with repeat NP sampling PCR and/or serology performed. The percent agreement between initial and repeat PCR was 96.7%, while the percent agreement between initial PCR and serology was 98.9%. There were 5 (3.1%) cases with discordance on repeat testing. After discordance analysis, 2 (1.4%) true cases tested negative on initial PCR. Using available diagnostic methods, discordance on repeat NP sampling PCR and/or serology is a rare occurrence.

Screening of COVID‐19 polymerase chain reaction tests using saliva for pregnant women and their partners in Himeji city

A screening of coronavirus disease 2019 (COVID-19) polymerase chain reaction (PCR) tests using saliva for pregnant women and their partners was performed at all 12 maternity facilities located in Himeji city between May 29 and September 5, 2020. Pregnant women at 37 or more weeks of gestation or who experienced threatened labor and their partners who cared for an infant underwent a saliva PCR test with informed consent. As a result, all of 1475 pregnant women and 1343 partners tested negative for COVID-19 PCR. There were no cases of false positive or false negative PCR tests. This cohort study revealed for the first time that a screening of COVID-19 PCR tests using saliva may be useful to sustain perinatal medical care during the pandemic period in Japan.

Atypical presentation of COVID-19; an observational retrospective study

BackgroundCOVID-19 infection may present with atypical signs and symptoms and false negative polymerase chain reaction (PCR) tests predisposing healthy people and health care workers to infection. The aim of the current study is to evaluate the features of atypical presentations in COVID-19 infection in a referral center in Tehran, Iran.MethodsHospital database of inpatients admitted to Loghman Hakim hospital between February 20th and May 11th, 2020 was reviewed and all patients with final diagnosis of COVID-19 infection were evaluated for their presenting symptoms. Patients with chief complaints of “fever”, “dyspnea”, and/or “cough” as typical presentations of COVID-19 were excluded and those with other clinical presentations were included.ResultsNineteen patients were included with a mean age of 51 ± 19 years, of whom, 17 were males (89%). Median [IQR] Glasgow coma scale (GCS) was 14 [13, 15]. Almost 10 had referred with chief complaint of methanol poisoning and overdose on substances of abuse. Only 8 cases (42%) had positive COVID-19 test. Nine (47%) needed invasive mechanical ventilation, of whom, two had positive COVID-19 test results (p = ns). Eight patients (42%) died with three of them having positive PCRs.ConclusionsIn patients referring to emergency departments with chief complaint of poisoning (especially poisonings that can result in dyspnea including substances of abuse and toxic alcohols), gastrointestinal, and constitutional respiratory symptoms, attention should be given not to miss possible cases of COVID-19.

1675MO Screening of COVID-19 disease based on chest CT and PCR for cancer patients undergoing radiotherapy in a French coronavirus hotspot

The coronavirus disease (COVID-19) pandemic has caused 180,000 confirmed cases in France with more than 28, 000 deaths as of May 19. A large part of COVID-19 patients seem asymptomatic and cancer patients may be more vulnerable. We evaluated a screening strategy combining chest computed tomography (CT) and PCR for patients treated with radiotherapy (RT). A screening strategy was organized from March 18, in our RT department. An inspiratory breath hold chest acquisition was proposed during the CT simulation for RT. Images was reviewed by a radiologist according to the CO-RADS classification. A nasal swab with a polymerase chain reaction (PCR) assay was proposed by the radiation oncologist in case of evocative imaging or clinical context. For patients who were already undergoing RT at this time, a PCR was proposed in case of evocative symptoms and before concomitant chemotherapy. From March 18 to May 1, 2020, 507 CT simulation were performed for 449 patients, including 445 chest acquisition. 237 of the chest CT (53%) showed lung abnormalities, of which 34 (8%) were COVID-19 compatible (CO-RADS ≥ 3). 102 patients were tested by PCR after the chest CT. 24 of the 449 (5.3%) patients were considered as COVID-19 patients: 19 had positive PCR, and five were considered positive on the basis of imaging despite PCR-negative PCR. Four of the patients (17%) were diagnosed during RT: 3 on routine screening before chemoradiotherapy, and one on symptoms. Four patients needed several PCR for the diagnosis of COVID-19 with six confirmed false negative PCR (Sensitivity (Se)= 76 % (19/25)). Three PCR positive patients had no evocative lung images (Se = 84%). During this period, an additional 169 patients whose CT simulation was prior to March 18, were also undergoing RT. Among them, six patients (3.6%) were diagnosed with COVID-19 by PCR during RT, performed for symptoms in 4 cases and on screening for the other 2. Of the 30 COVID-19 patients, only 8 (27%) had symptoms at the time of diagnosis. Twelve patients (40%) reported no symptoms and benefited from screening. This study confirms the high proportion of asymptomatic patients with COVID-19 and suggests the value of screening by CT and PCR during COVID-19 pandemics.

Why the United States failed to contain COVID-19.

Why the United States failed to contain COVID-19.

Clinical value of polymerase chain reaction in detecting group B streptococcus during labor.

To reduce the intrapartum use of antibiotics in women with prolonged rupture of the membranes (PROM) by restriction of antibiotics to women who are colonized with group B streptococci (GBS), as identified with the Cepheid Gene Xpert polymerase chain reaction (PCR) for detecting GBS. We conducted a randomized controlled trial among full-term delivering women with PROM. Fifty-four women were enrolled, based on a power calculation with a significance level of 5% and a power of 95%. Twenty-seven women received the standard treatment (rectovaginal swab [RVS] for bacterial culture and antibiotics). For another 27 women PCR was performed on the RVS and antibiotics were used only when the PCR was positive. The primary outcome was reduction in antibiotic use, defined as the percentage of women who received antibiotics during labor. 54 Women were enrolled in the study between 1 May and 18 November 2014. There were no significant differences in baseline characteristics. In total, 10 of the 54 women were GBS positive (18.5%). Of those 10 women, three were identified on bacterial culture and seven on PCR. In the bacterial culture group all the women received antibiotics. In the PCR group 10 women (37%) received antibiotics (P = 0.002). Two false-positive PCR tests were identified. There were no false-negative PCR tests. Real-time identification of GBS on PCR reduces the intrapartum use of antibiotics in women with PROM.

Nitazoxanide Is an Ineffective Treatment of Chronic Norovirus in Patients With X-Linked Agammaglobulinemia and May Yield False-Negative Polymerase Chain Reaction Findings in Stool Specimens.

Nitazoxanide Is an Ineffective Treatment of Chronic Norovirus in Patients With X-Linked Agammaglobulinemia and May Yield False-Negative Polymerase Chain Reaction Findings in Stool Specimens.

A misleading false-negative result of Pneumocystis real-time PCR assay due to a rare punctual mutation: A French multicenter study.

This article describes a previously unreported mutation at position 210 (C210T) of the mitochondrial large subunit ribosomal RNA (mtLSUrRNA) gene of Pneumocystis jirovecii, which led to a false-negative result of a real-time polymerase chain reaction (PCR) assay. Since the aforementioned real-time PCR assay is widely used in France, a French multicenter study was conducted to estimate the mutation frequency and its potential impact on the routine diagnosis of Pneumocystis pneumonia (PCP). Through analysis of data obtained from eight centers, the mutation frequency was estimated at 0.28%. This low frequency should not call into question the routine use of this PCR assay. Nonetheless, the occurrence of the false-negative PCR result provides arguments for maintaining microscopic techniques combined to PCR assays to achieve PCP diagnosis.

Description of Haemoproteus ciconiae sp. nov. (Haemoproteidae, Haemosporida) from the white stork Ciconia ciconia, with remarks on insensitivity of established polymerase chain reaction assays to detect this infection

Haemoproteus ciconiae sp. nov. (Haemosporida, Haemoproteidae) was found in the white stork Ciconia ciconia (Ciconiiformes, Ciconiidae) after spring migration in Lithuania. This organism is illustrated and described based on the morphology of its blood stages. The new species can be readily distinguished from all other haemoproteids parasitizing ciconiiform birds due to the presence of large number (approximately 20 on average) small (<1μm) pigment granules in its mature gametocytes. Growing and mature gametocytes of H. ciconiae were readily visible in all blood films (parasitemia of 0.001%). However, experienced researchers were unable to detect sequences of its mitochondrial cytochrome b (cyt b) or apicoplast genes from the microscopically positive sample by using five established assays for polymerase chain reaction (PCR)-based detection of avian haemosporidian parasites. The white stork cyt b sequence was readily detectable, indicating the well-optimised PCR protocols and the good quality of total DNA in the sample containing the new species. The failure to amplify this parasite DNA indicates insufficient sensitivity of the currently used PCR-based assays in diagnostics of avian haemosporidian infections. We suggest possible explanations of this observation. To minimize number of the false negative PCR reports, we call for the continued use of optical microscopy in parallel with molecular diagnostics in studies of haemosporidian parasites, particularly in wildlife.

False-negative HIV-1 polymerase chain reaction in a 15-month-old boy with HIV-1 subtype C infection.

Polymerase chain reaction (PCR) testing is the gold standard for determining the HIV status in children <18 months of age. However, when clinical manifestations are not consistent with laboratory results, additional investigation is required. We report a 15-month-old HIV-exposed boy referred to our hospital after he had been admitted several times for infectious diseases. A rapid antibody test on the child was positive, while routine diagnostic HIV PCRs using the Roche COBAS Ampliprep/COBAS TaqMan HIV Qual Test were negative at 6 weeks, 6 months, 7 months and 15 months. In addition, the same PCR test performed on the HIV-infected mother was also negative. Alternative PCR and viral load assays using different primer sets detected HIV RNA or proviral DNA in both child and mother. Gag sequences from the child and his mother classified both infections as HIV-1 subtype C, with very rare mutations that may have resulted in PCR assay primer/probe mismatch. Consequently, the child was commenced on antiretroviral therapy and made a remarkable recovery. These findings indicate that more reliable PCR assays capable of detecting a wide range of HIV subtypes are desirable to circumvent the clinical problems created by false-negative PCR results.

Acid-fast Smear and Histopathology Results Provide Guidance for the Appropriate Use of Broad-Range Polymerase Chain Reaction and Sequencing for Mycobacteria.

New molecular diagnostic tests are attractive because of the potential they hold for improving diagnostics in microbiology. The value of these tests, which is often assumed, should be investigated to determine the best use of these potentially powerful tools. To investigate the usefulness of broad-range polymerase chain reaction (PCR), followed by sequencing, in mycobacterial infections. We reviewed the test performance of acid-fast bacilli (AFB) PCR and traditional diagnostic methods (histopathology, AFB smear, and culture). We assessed the diagnostic effect and cost of the unrestricted ordering of broad-range PCR for the detection and identification of mycobacteria in clinical specimens. The AFB PCR was less sensitive than culture and histopathology and was less specific than culture, AFB smear, and histopathology. During 18 months, $93 063 was spent on 183 patient specimens for broad-range PCR and DNA sequencing for mycobacteria to confirm one culture-proven Mycobacterium tuberculosis infection that was also known to be positive by AFB smear and histopathology. In this cohort, there was a false-negative AFB PCR for M tuberculosis and a false-positive AFB PCR for Mycobacterium lentiflavum . Testing of AFB smear-negative specimens from patients without an inflammatory response supportive of a mycobacterial infection is costly and has not been proven to improve patient care. Traditional diagnostics (histopathology, AFB smear, and culture) should remain the primary methods for the detection of mycobacteria in clinical specimens.

Comparison of different diagnostic tools for the detection of Anaplasma phagocytophilum in dogs

Anaplasma phagocytophilum is common in dogs, but the best way to diagnose an infection is still not determined. Antibody detection assays are frequently used in veterinary practice. Additionally, polymerase chain reaction (PCR) is available for detection of A phagocytophilum DNA. It is still unknown, how well different diagnostic methods correlate. The aim of the study was to compare 2 antibody detection assays, an immunofluorescence assay (IFA) and the ELISA SNAP4Dx, and to determine the correlation of these assays by evaluating the sensitivity and specificity compared with PCR as a direct detection method of the organism. Sera of 200 prospectively included dogs were tested for antibodies to A phagocytophilum using IFA and SNAP4Dx. DNA of the organism was detected by PCR on whole blood. Sensitivity, specificity, and predictive values, including their 95% confidence intervals, were calculated for IFA and SNAP4Dx in relation to PCR. Four of 200 animals were PCR-positive. Sensitivity of IFA and SNAP4Dx was 100% (95% CI 51.01-100). Specificity of IFA was 52.9% (95% CI 50.42-64.17) and that of SNAP4Dx, 57.4% (95% CI 45.83-59.70). Agreement of the 2 antibody tests was fair (κ 0.334). Immunofluorescence assay and SNAP4Dx were very sensitive and therefore can be useful as screening tests for A phagocytophilum infection. However, the specificity was low, and agreement between both antibody tests was insufficient. This could be due to either false-positive antibody test results, or false-negative PCR results in dogs that were actually infected.

Influence of Parasite Density and Sample Storage Time on the Reliability of Entamoeba histolytica-specific PCR From Formalin-fixed and Paraffin-embedded Tissues

We report on the reliability of polymerase chain reaction (PCR) for the detection of Entamoeba histolytica from formalin-fixed, paraffin-embedded tissue in comparison with microscopy and have determined predictors that may influence PCR results. E. histolytica-specific and Entamoeba dispar-specific real-time PCR and microscopy from adjacent histologic sections were performed using a collection of formalin-fixed, paraffin-embedded tissue specimens obtained from patients with invasive amebiasis. Specimens had been collected during the previous 4 decades. Association of sample age, parasite density, and reliability of PCR was analyzed. E. histolytica PCR was positive in 20 of 34 biopsies (58.8%); 2 of these 20 were microscopically negative for amebae in neighboring tissue sections. PCR was negative in 9 samples with visible amebae in neighboring sections and in 5 samples without visible parasites in neighboring sections. PCR was negative in all specimens that were older than 3 decades. Low parasite counts and sample ages older than 20 years were predictors for false-negative PCR results. All samples were negative for E. dispar DNA. PCR is suitable for the detection of E. histolytica in formalin-fixed, paraffin-embedded tissue samples that are younger than 2 decades and that contain intermediate to high parasite numbers. Negative results in older samples were due to progressive degradation of DNA over time as indicated by control PCRs targeting the human 18S rRNA gene. Moreover, our findings support previous suggestions that only E. histolytica but not E. dispar is responsible for invasive amebiasis.

Does Empirical Clostridium difficile Infection (CDI) Therapy Result in False-Negative CDI Diagnostic Test Results?

Patients with suspected Clostridium difficile infection (CDI) often receive empirical therapy prior to collection of stool specimens for diagnostic testing. The likelihood that such empirical therapy might result in false-negative CDI test results is unknown. We conducted a prospective study of CDI patients to determine the time to conversion of CDI test results, including polymerase chain reaction (PCR) for toxin B genes, glutamate dehydrogenase, and toxigenic culture, from positive to negative during CDI therapy. We evaluated the frequency of and risk factors for persistence of positive CDI tests. For patients receiving empirical therapy, we assessed the frequency of conversion of positive CDI test results at the time of the test order to negative by the time clinical staff collected stool specimens for testing. For 51 CDI patients, PCR, glutamate dehydrogenase, and toxigenic culture results converted to negative at similar rates. For PCR, 14%, 35%, and 45% of positive CDI tests converted to negative after 1, 2, and 3 days of treatment, respectively. Increased age and infection with North American pulsed-field gel electrophoresis strains were associated with persistent positive PCR results. For CDI patients diagnosed at the time of the test order, conversion to negative PCR results by the time clinical stool specimens were collected occurred in 4 of 9 (44%) patients who were prescribed empirical CDI therapy versus 0 of 23 (0%) who were not (P = .004). Empirical treatment for suspected CDI cases may result in false-negative PCR results if there are delays in stool specimen collection.

Decapitation Improves Detection of &lt;I&gt;Wolbachia pipientis&lt;/I&gt; (Rickettsiales: Anaplasmataceae) in &lt;I&gt;Culex pipiens&lt;/I&gt; (Diptera: Culicidae) Mosquitoes by the Polymerase Chain Reaction

Polymerase chain reaction (PCR) is often used to detect microorganisms, pathogens, or both, including the reproductive parasite Wolbachia pipientis (Rickettsiales: Anaplasmataceae), in mosquitoes. Natural populations of Culex pipiens L. (Diptera: Culicidae) mosquitoes are infected with one or more strains of W. pipientis, and crosses between mosquitoes harboring different Wolbachia strains provide one of the best-known examples of cytoplasmic incompatibililty (CI). When we used PCR to monitor Wolbachia in the Buckeye strain of Culex pipiens, and in a Wolbachia-cured sister colony obtained by tetracycline treatment, we noted false negative PCR reactions with DNA samples from infected mosquitoes; these results were inconsistent with direct microscopic observation of Wolbachia-like particles in gonads dissected from mosquitoes in the same population. Assays with diluted template often improved detection of positive samples, suggesting that DNA prepared from whole mosquitoes contained an inhibitor of the PCR reaction. We reconciled discrepancies between PCR and microscopy by systematic measurement of the PCR reaction in the presence of an internal standard. Mosquito decapitation before DNA extraction restored the reliability of the PCR reaction, allowing accurate determination of Wolbachia infection status in infected and tetracycline-cured mosquito populations, consistent with microscopic examination. Using PCR primers based on the Tr1 gene, we confirmed that the Wolbachia infection in the Buckeye strain of Culex pipiens belongs to the genotype designated wPip1. Finally, to explore more widely the distribution of PCR inhibitors, we demonstrated that DNA isolated from the cricket, Acheta domesticus (L.); the beetle, Tenebrio molitor L.; the honey bee, Apis mellifera L.; and the mosquito, Anopheles punctipennis Say also contained PCR inhibitors. These results underscore the importance of measuring the presence of inhibitors in PCR templates by using a known positive standard, and provide an approach that will facilitate use of PCR to monitor environmental samples of mosquitoes that harbor endosymbionts or pathogenic organisms.

Ataxia and Progressive Encephalopathy in a 4-Year-Old Girl

The spinocerebellar ataxias (SCAs) are a rare group of neurodegenerative disorders with progressive cerebellar ataxia as the primary feature. These disorders are phenotypically and genetically variable, both between and within subtypes. Seven of the SCA subtypes are caused by CAG trinucleotide repeats within the respective genes, and clinically most of these diseases demonstrate anticipation. Testing for these disorders typically relies upon conventional polymerase chain reaction (PCR) and fragment analysis. However, conventional PCR may give false-negative results in cases in which the CAG expansion is unusually long. We report a case of spinocerebellar ataxia type 2 (SCA2) in a 4-year-old girl with false-negative conventional PCR results. Specifically, the SCA2 disorder is caused by a CAG repeat within the ATXN2 gene on chromosome 12. Subsequent confirmatory testing using modified PCR and primers specific for the CAG repeat were performed and revealed an expanded allele with 109 repeats in our patient.