Initial Polymerase Chain Reaction Research Articles

The Next Chapter in Cancer Diagnostics: Advances in HPV-Positive Head and Neck Cancer.

Human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC), particularly oropharyngeal squamous cell carcinoma (OPSCC), is an increasingly prevalent pathology worldwide, especially in developed countries. For diagnosing HPV in HNSCC, the combination of p16 immunohistochemistry (IHC) and polymerase chain reaction (PCR) offers high sensitivity and specificity, with p16 IHC being a reliable initial screen and PCR confirming HPV presence. Advanced techniques like next-generation sequencing (NGS) and RNA-based assays provide detailed insights but are primarily used in research settings. Regardless of HPV status, standard oncological treatments currently include surgery, radiation, and/or chemotherapy. This conventional approach does not account for the typically better prognosis of HPV-positive HNSCC patients, leading to increased chemo/radiation-induced secondary morbidities and reduced quality of life. Therefore, it is crucial to identify and detect HPV positivity and other molecular characteristics of HNSCC to personalize treatment strategies. This comprehensive review aims to summarize current knowledge on various HPV detection techniques and evaluate their advantages and disadvantages, with a focus on developing methodologies to identify new biomarkers in HPV-positive HNSCC. The review discusses direct and indirect HPV examination in tumor tissue, DNA- and RNA-based detection techniques, protein-based markers, liquid biopsy potentials, immune-related markers, epigenetic markers, novel biomarkers, and emerging technologies, providing an overall insight into the current state of knowledge.

Clinician Interpretation and Management of Discordant PCR+/Toxin- Clostridioides difficile Testing Results Post-COVID

Background: Our institution utilizes a two-step algorithm consisting of an initial polymerase chain reaction (PCR) test with positive results reflexed to an enzyme immunoassay (EIA) for toxin. Institutional guidelines implemented during the COVID-19 pandemic recommended applying clinical judgment to patients with PCR+/Toxin- (discordant) results when determining if treatment is indicated. Pre-pandemic, we found that clinicians continued CDI-directed therapy in 56% of patients following the Toxin- result. Our study aims to identify how clinicians interpret PCR+/Toxin- results and reasons for management decisions post-pandemic. Methods: At an academic medical center, we conducted a retrospective cohort study of the first 50 inpatient charts with PCR+/Toxin- results from August to October 2022. Data was abstracted from clinical, pharmacy, and microbiology databases. The primary outcome was the proportion of patients continued on CDI-directed therapy for ≥24 hours after the Toxin- result became available. Secondary outcomes included the proportion of patients prescribed a full treatment course for CDI and the reasons for continuing treatment. Results: There were 37 patients (74%) who started CDI-directed treatment after initial PCR+ Results: Of these patients, 59% (22/37) were continued on treatment for ≥24 hours after the Toxin- result (primary outcome). 77% (17/22) of these patients who met the primary outcome completed full treatment courses. Three patients were transitioned to prophylaxis dosing after the Toxin- result. The most common reason for continuing treatment after discordant results was high clinical suspicion for CDI (Figure). There were no CDI-related complications in this 50-patient cohort. In immunocompromised patients, 70% (16/23) started treatment after initial PCR+ results and 81% (13/16) met the primary outcome. In patients admitted specifically to immunocompromised inpatient services, 90% (9/10) started treatment after initial PCR+ results and 100% (9/9) met the primary outcome. Conclusion: The majority of patients started on treatment after the PCR+ result were continued on treatment following the Toxin- result, though several of these patients did not complete full treatment courses. Treatment rates were similar to our pre-pandemic baseline. When patients were continued on treatment after discordant results, clinicians cited appropriate high clinical suspicion. Notably, every patient admitted to an immunocompromised service was continued on treatment after Toxin- Results: Overall, clinicians are following institutional guidelines by applying clinical judgement when interpreting discordant Results: Further research will help identify what variables affect clinician interpretation and management practices for discordant results, which will help shape institutional guideline updates, clinician education, and additional stewardship interventions.

Clinical Characteristics and Cycle Thresholds Among Discordant and True Positive Test Results for Clostridiodes difficile

Background: The diagnosis of Clostridioides difficile infection (CDI) is challenging. Despite guideline-directed, multistep testing algorithms and diagnostic stewardship, the treatment of C. difficile colonization persists. The testing algorithm at our system utilizes an initial real-time PCR test (PCR) for Toxin B gene, which if positive, reflexes to an enzyme immunoassay (EIA) for detecting Toxins A and B. Discordant results (PCR +/EIA -) are suggestive of colonization, but the majority of patients with discordant results are treated for CDI. Correlation of C. difficile EIA B polymerase chain reaction (PCR) cycle thresholds (Ct) with the presence of free EIA and disease severity has been observed, but the ability to use Ct in the decision to treat patients with discordant results is unclear. Our study assesses if Ct values and other clinical characteristics favor treatment in select patients with discordant Methods: A retrospective chart review was performed of adult patients (≥ 18-year-old) with positive C. difficile PCR results that were admitted to our health system between June 01 and August 31, 2023. C. difficile PCR and Ct results were obtained by Cepheid GeneXpert and Toxin A and B EIA results were obtained by Meridian Bioscience Immunocard. Patients with discordant (PCR+/EIA) and true positive (PCR+/EIA+) results were compared. We assessed demographics, past medical history, clinical characteristics, severity of illness, PCR Ct values, treatment, and clinical outcomes including: 30-day all-cause mortality and re-admission, and 60-day CDI repeat testing and treatment. Results Of the 122 patients identified, 89 patients had discordant results and 43 had true positive Results: Severity of illness and other clinical and laboratory characteristics were similar between both groups. Mean Ct values were significantly lower for true positive results compared to discordant results, 24.28 vs 29.27, respectively (p26 (p=.08). Of the patients with discordant results, 73 completed treatment for CDI and no difference in clinical outcomes was observed compared to patients with discordant results that were not treated. Conclusion Ct values were lower among patients with true positive results compared to patients with discordant Conclusion: There were no statistically significant different rates of severe or fulminant CDI among patients with discordant results and Ct values < 26, although this finding may be limited by sample size and Ct may be helpful in deciding which discordant patients to treat.

Prevalence of persistent symptoms at least 1 month after SARS-CoV-2 Omicron infection in adults.

Persistent post-COVID-19 symptoms pose an important health care burden. The Omicron variant has rapidly spread across the world and infected millions of people, largely exceeding previous variants. The potential for many of these people to develop persistent symptoms is a major public health concern. The aim of this study was to determine the prevalence and risk factors of post-COVID-19 symptoms associated with Omicron. We conducted a single-centre prospective observational study in Quebec, Canada, between December 2021 and April 2022. Participants were adults enrolled in the Biobanque Québécoise de la COVID-19 (BQC19). Cases were considered Omicron cases as more than 85% were estimated to be attributable to Omicron variant during that period. Adults with polymerase chain reaction (PCR)-confirmed COVID-19 were recruited at least 4 weeks after the onset of infection. Of 1,338 individuals contacted, 290 (21.7%) participants were recruited in BQC19 during that period. Median duration between the initial PCR test and follow-up was 44 days (IQR 31-56 d). A total of 137 (47.2%) participants reported symptoms at least 1-month post-infection. The majority (98.6%) had a history of mild COVID-19 illness. Most common persistent symptoms included fatigue (48.2%), shortness of breath (32.6%), and cough (24.1%). Number of symptoms during acute COVID-19 was identified as a risk factor for post-COVID-19 symptoms (OR 1.07 [95% CI 1.03% to 1.10%] p = 0.009). This is the first study reporting the prevalence of post-COVID-19 symptoms associated with Omicron in Canada. These findings will have important implications for provincial services planning.

SARS-CoV-2 symptomatic reinfection among patients with primary antibody deficiency

SARS-CoV-2 symptomatic reinfection among patients with primary antibody deficiency

Can naive Bayes classifier predict infection in a close contact of COVID-19? A comparative test for predictability of the predictive model and healthcare workers in Japan

BackgroundThose who are found in close contact with COVID-19 patients and are also negative by polymerase chain reaction (PCR) test may act without waiting for the incubation period to elapse, can become infectious and spread the infection. MethodsA machine learning model that can evaluate the risk of infection in close contact with COVID-19 patients within the incubation period from the contact status reported from the index case was created using posterior probabilities. To confirm actual predictability, a verification test was conducted on 169 new close contacts, and the machine learning model was compared with four experienced healthcare workers for the predictability. ResultsIn a verification test, 33 of the 169 contacts were infected with COVID-19 during the incubation period, and 13 of 33 were negative on initial PCR test, after that the disease developed and their PCR test became positive. The machine learning model predicted the eventual infection in 12 of 13 patients who had negative results on the initial PCR test. In the verification test, the sensitivity of the machine learning model was 0.879 and the specificity was 0.588. The mean−standard deviation of the sensitivity and the specificity of the four health care workers was 0.568 (0.230) for sensitivity and 0.689 (0.103) for specificity. ConclusionIf it is possible to convey that individual risk of infection, the close contact may take suppressive action during the incubation period regardless of the result of the initial PCR test, thereby preventing secondary spread of infection.

Herpes Simplex Virus Encephalitis With Initial Negative Polymerase Chain Reaction in the Cerebrospinal Fluid: Prevalence, Associated Factors, and Clinical Impact.

To describe the prevalence, associated factors, and clinical impact of an initial negative herpes simplex virus (HSV) polymerase chain reaction (PCR) in critically ill patients with PCR-proven HSV encephalitis. Retrospective multicenter study from 2007 to 2017. Forty-seven French ICUs. Critically ill patients admitted to the ICU with possible/probable acute encephalitis and a positive cerebrospinal fluid (CSF) PCR for HSV. None. We included 273 patients with a median Glasgow Coma Scale score of 9 (6-12) at ICU admission. CSF HSV PCR was negative in 11 cases (4%), exclusively in lumbar punctures (LPs) performed less than 4 days after symptoms onset. Patients with an initial negative PCR presented with more frequent focal neurologic signs (4/11 [36.4%] vs 35/256 [13.7%]; p = 0.04) and lower CSF leukocytosis (4 cells/mm3 [3-25 cells/mm3] vs 52 cells/mm3 [12-160 cells/mm3]; p < 0.01). An initial negative PCR was associated with an increased delay between LP and acyclovir treatment (3 d [2-7 ] vs 0 d [0-0 d]; p < 0.01) and was independently associated with a poor neurologic outcome at hospital discharge (modified Rankin Scale score ≥ 4) (adjusted odds ratio, 9.89; 95% CI, 1.18-82.78). In severe herpes simplex encephalitis, initial negative CSF HSV PCR occurred in 4% of cases and was independently associated with worse neurologic outcome at hospital discharge. In these patients, a systematic multimodal diagnostic approach including early brain MRI and EEG will help clinicians avoid delayed acyclovir initiation or early inappropriate discontinuation.

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.

Factors associated with the timely uptake of initial HIV virologic test among HIV-exposed infants attending clinics within a faith-based HIV program in Kenya; a cross-sectional study

BackgroundEarly infant diagnosis (EID) of HIV, followed by effective care including antiretroviral therapy (ART), reduces infant mortality by 76% and HIV progression by 75%. In 2015, 50% of 1.2 million HIV-exposed infants (HEI) in 21 priority countries received a virologic test within the recommended 2 months of birth. We sought to identify factors associated with timely uptake of virologic EID among HEI and gain insight into missed opportunities.MethodsThis was a cross-sectional study that used de-identified data from electronic medical records of 54 health facilities within the Christian Health Association of Kenya (CHAK) HIV Project database. All HEI who had their first HIV virologic test done between January 2015 and December 2017 were included in the study and categorized as either having the test within or after 8 weeks of birth. Multivariate linear mixed effects regression model was used to determine factors associated with uptake of the first HIV EID polymerase chain reaction (PCR). Predictor variables studied include sex, birth weight, the entry point into care, provision of ART prophylaxis for the infant, maternal ART at time of EID, mode of delivery, and place of delivery.ResultsWe included 2020 HEI of whom 1018 (50.4%) were female. A majority, 1596 (79.0%) had their first HIV PCR within 2 months of birth at a median age of 6.4 weeks (interquartile range 6–7.4). Overall, HIV positivity rate at initial test among this cohort was 1.2%. Delayed HIV PCR testing for EID was more likely to yield a positive result [adjusted odds ratio (aOR) = 1.29 (95% confidence interval (CI) 1.09–1.52) p = 0.003]. Infants of mothers not on ART at the time of HIV PCR test and infants who had not received prophylaxis to prevent vertical HIV transmission had significant increased odds of a delayed initial test [aOR = 1.27 (95% CI = 1.18–1.37) p = < 0.0001] and [aOR = 1.43 (95% CI 1.27–1.61) p = < 0.001] respectively.ConclusionAn initial HIV PCR test done after 8 weeks of birth is likely to yield a positive result. Barriers to accessing ART for treatment among HIV-infected pregnant and breastfeeding women, and prophylaxis for the HEI were associated with delayed EID. In order to ensure timely EID, programs need to incorporate both facility and community strategy interventions to ensure all pregnant women seek antenatal care and deliver within health facilities.

Polymerase Chain Reaction Multiplication for the Detection of Bacterial Isolates Causing Neonatal Sepsis

BACKGROUND: Neonatal sepsis is a clinical syndrome caused by the presence of bacteria in the blood accompanied by symptoms and systemic signs of infection that occurs in the first 4 weeks of life after birth. The process of identifying pathogenic microorganisms is essential in determining the clinical condition in neonatal sepsis.&#x0D; AIM: The study was aimed to develop a multiplex polymerase chain reaction (PCR) method to identify bacterial isolates that cause neonatal sepsis in Indonesia with the main target of optimization of an initial design and PCR optimization.&#x0D; METHODS: This research is an explorative in vitro study for the optimization of an initial design and PCR methods for the detection of the main bacteria that cause sepsis neonatorum in Indonesia, namely, bacteria Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, and Pseudomonas aeruginosa. The study was conducted at the Biomolecular Laboratory of the Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia.&#x0D; RESULTS: Sequencing carried out and continued with Basic Local Alignment Search Tool (BLAST) sequencing results, it appears that the PCR product that has been produced conforms with the optimization targets that were previously set when doing the primer design. The level of homology found in all four species based on the results of BLAST in a sequence is as follows: K. pneumoniae 94%, P. aeruginosa 99%, E. cloacae 100%, and E. coli 100%.&#x0D; CONCLUSION: PCR multiplex method using design primers and conventional PCR analysis methods (using agarose gel) can be used to detect four species of bacteria that cause neonatal sepsis, namely, K. pneumoniae, P. aeruginosa, E. cloacae, and E. coli.

Immunocompromised Child on Infliximab: A Case Report of Listeria monocytogenes Meningitis.

IntroductionPatients with naturally occurring, impaired cell-mediated immunity secondary to age and pregnancy are known to be at risk of developing severe and invasive Listeria monocytogenes infections. Immunosuppressant medications, particularly infliximab, are also known to increase this risk.Case ReportWe present the case of a seven-year-old female on infliximab who was diagnosed with culture positive L. monocytogenes meningitis after a negative cerebral spinal fluid polymerase chain reaction (PCR).ConclusionPatients receiving infliximab who display signs of central nervous system infection should be suspected to have L. monocytogenes as an infecting agent, and empiric addition of ampicillin to their antibiotic regimen should be considered, with substitution of trimethoprim-sulfamethoxazole in cases of penicillin allergy, regardless of initial PCR results.

Association of fecal sample collection technique and treatment history with Tritrichomonas foetus polymerase chain reaction test results in 1717 cats.

BackgroundFecal polymerase chain reaction (PCR) testing for Tritrichomonas foetus is considered the most sensitive means for diagnosis of infection but results could be influenced by fecal collection technique and prior use of antimicrobial drugs.ObjectivesTo establish any association between fecal collection technique or treatment history and results of fecal PCR testing for T. foetus.AnimalsFecal samples from 1717 cats submitted by veterinarians between January 2012 and December 2017.MethodsThis study used a retrospective analysis. T. foetus PCR test results from 1808 fecal samples submitted for diagnostic testing were examined for their association with method of fecal collection and prior antimicrobial treatments. Data were collected from sample submission form.ResultsPositive T. foetus PCR test results were obtained for 274 (16%) cats. Fecal samples collected via fecal loop had increased probability of positive PCR test results (odds ratio [OR] 2.04, 95% confidence interval [CI] 1.31‐3.17, P = .002) compared to samples collected by colonic flush. There was no association between PCR test results and treatment history, treatment type, or prior treatment with ronidazole. After an initial positive PCR test, 4/19 (21%; 95% CI 2.7%‐39.4%) cats treated with ronidazole had a second positive test result.Conclusions and Clinical ImportanceResults of this study support that fecal samples collected by loop might be better for PCR diagnosis of T. foetus infection. Lack of association of ronidazole with PCR test results and a 21% all‐potential‐causes failure rate of ronidazole in cats with preconfirmed infection are important limitations to use of this drug.

Monitoring diagnosis, retention in care and viral load suppression in children testing HIV polymerase chain reaction-positive in two districts in South Africa.

Retention in care is associated with improved virological control and survival among HIV-infected children. However, retention of children in HIV care remains a challenge. To describe, using routine laboratory HIV test data, the retention-in-care and virological outcomes of HIV-infected children aged &lt;18 months in two districts in South Africa. HIV polymerase chain reaction (PCR)-positive results of children from uMkhanyakude and Tshwane districts in KwaZulu-Natal and Gauteng provinces, respectively, tested between April 2015 and May 2016, were extracted from the National Health Laboratory Service's Corporate Data Warehouse (CDW). HIV-related tests (PCR, viral load (VL), CD4+) were documented longitudinally for each child for ≥13 months after the first positive PCR result by manually searching demographics within the CDW, supplemented by an automated patient-linking algorithm. Test sets were linked if two or more demographics (surname, name, date of birth, folder number) matched exactly. Programmatic indicators assessed included age at first positive PCR test, presumed confirmatory test rates, retention in care, and VL suppression at 6 and 12 months. Ninety-four and 304 children tested HIV PCR-positive in uMkhanyakude and Tshwane, respectively. The median age at diagnosis was 3.6 months (interquartile range (IQR) 1.4 - 7.1) for uMkhanyakude and 2.3 months (IQR 0.1 - 6.7) for Tshwane. In uMkhanyakude, confirmed in utero infections accounted for 18.1% of transmissions (n=17), compared with 29.6% (n=90) in Tshwane. Presumed confirmatory test rates following an initial positive PCR result were 77.7% and 71.7% for uMkhanyakude and Tshwane, respectively. Within 6 months of starting antiretroviral therapy, 43 children (58.9%) were lost to follow-up in uMkhanyakude compared with 160 (73.4%) in Tshwane. Of those retained in care at 6 months with a VL measurement, 15 (60.0%) from uMkhanyakude had a VL &lt;1 000 copies/mL, compared with 24 (48.0%) in Tshwane. For both districts, a third of all HIV PCR-positive children were retained in care at the end of follow-up, with 29 (30.9%) in uMkhanyakude and 99 (32.5%) in Tshwane. Of these, 12 (41.4%) had a VL &lt;1 000 copies/mL in uMkhanyakude compared with 28 (28.3%) in Tshwane. We demonstrate the value of routine laboratory data in monitoring diagnosis, retention and VL suppression in HIV-infected children. This approach is scalable, can be reported near real-time, is relatively inexpensive to implement, and provides a tool for improving paediatric HIV services until clinical databases can assume this role.

First Report of Phytoplasma Infecting Pterocarpus indicus in China

Pterocarpus indicus Willd., an adaptable and fast-growing landscape tree, is commonly cultivated in landscaping in southern China. In April 2018, a P. indicus tree with symptoms of flat branches and typical fasciation, which had the same symptoms of phytoplasma infection, was observed in Haikou, Hainan province, China. To determine the presence of phytoplasma, stem samples of symptomatic and asymptomatic P. indicus were subjected to total DNA extraction using the plant Genomic DNA Kit (Tiangen, Beijing, China), followed by nested polymerase chain reaction (PCR) detection of phytoplasma (Jomantiene et al. 1998; Lee et al. 1998). Briefly, the universal primer pair R16mF2/R16mR1 was employed for the initial PCR amplification of 16SrDNA fragment of phytoplasma and primer set R16F2n/R16R2 (Lee et al. 1998) for nested PCR amplification. As expected, the PCR products were generated from the samples of the symptomatic plant but not from the symptomless control samples. The anticipated amplicon was approximately 1.2 kb in size. The PCR products were gel purified using TIANgel Midi Purification Kit (Tiangen, Beijing, China) and cloned into pMD-18T vector (Takara Bio, Dalian, China). Three independent positive clones were subjected to Sanger sequencing. The obtained sequence was deposited into NCBI GenBank database with GenBank accession no. MH727702. A BLASTn search against the nonredundant nucleotide database revealed high sequence identities with the corresponding regions of phytoplasmas of aster yellows phytoplasma group (16SrI). A maximum of 99.7% nucleotide sequence identity with Malvastrum coromandelianum phyllody phytoplasma (GenBank accession no. MF490802) was observed. Moreover, an iPhyClassifier (Zhao et al. 2013) operation was used to perform sequence comparison and generate a virtual restriction fragment length polymorphism (RFLP) profile for the sequence derived from the symptomatic P. indicus sample, which showed the most similarity with that of the reference strain (GenBank accession no. AP006628) belonging to 16SrI group. However, this strain may represent a new subgroup because the similarity coefficient was only 0.91, which is within the values set for a new subgroup (Biswas et al. 2013). To our best knowledge, this is the first report of phytoplasma infecting P. indicus in China.

Early diagnostic value of the antimycoplasma antibody (IgM) inMycoplasma pneumoniaepneumonia: A single-center study in 2015

Purpose: Recently, the incidence of refractory Mycoplasma pneumoniae (MP) pneumonia has increased in Korea. Given that its early diagnosis is helpful in selection of the treatment, this study aimed at investigating the value of the antimycoplasma antibody (IgM) for early diagnosis of MP pneumonia. Methods: A total of 315 children admitted with MP pneumonia from September 2015 to May 2016 were investigated with the IgM and polymerase chain reaction (PCR) for the diagnosis of MP pneumonia. Specifically, patients were grouped into nonrefractory re spiratory MP and refractory MP groups according to their response to macrolide therapy. Results: In the 44 PCR-negative seroconversed children, seroconversed IgM was more frequent in the refractory MP group com pared with the nonrefractory respiratory MP group with statistical significance (P<0.001). In the 264 IgM-positive children, the time of antibody reaction was more delayed in the refractory MP group compared to the nonrefractory respiratory MP group with statis tical significance (P<0.001). Conclusion: This study showed that there was a higher incidence of seroconversed IgM and delayed antibody reaction in the refrac tory MP group. In children with suspect MP pneumonia, follow-up studies of antibody are necessary, even through initial antibody and PCR showed negative findings. In addition, this result may suggest that the diagnosis of refractory MP pneunomia will be help ful in establishing the strategy of the treatment. (Allergy Asthma Respir Dis 2019;7:129-136)

High concordance of BRAF mutational status in matched primary and metastatic melanoma.

Techniques for the accurate identification of activating mutations of BRAF in metastatic melanoma are of great clinical importance, due to the availability of targeted therapies for these tumors. There is uncertainty regarding the frequency with which BRAF status differs between primary and metastatic sites. Between 2011 and 2016, 219 melanoma cases underwent BRAF testing in our institution. In 53 of these cases, paired primary and metastatic specimens were available for polymerase chain reaction (PCR) and immunohistochemical evaluation. Fifty-two out of 53 cases (98%) showed concordant BRAF status between primary and metastatic site by immunohistochemistry (IHC). In one case, a metastasis and its matched primary were positive by IHC, but the metastasis was negative on PCR. On further investigation, PCR was positive in the primary, and repeat PCR in the metastasis was positive, following macrodissection. Our results suggest that discordance of BRAF mutational status between primaries and metastases is a rare occurrence. In one case, IHC provided strong evidence that initial PCR testing had provided a false-negative result due to low tumor volume. Thus, in cases where tissue is difficult to obtain from a metastasis or unavailable, the primary tumor can be used with confidence.

Prognostic Value of Low PCR Cytomegalovirus Load in Kidney Transplant Recipients.

Cytomegalovirus (CMV) infection after kidney transplantation is associated with significant morbidity and mortality. Use of Polymerase Chain Reaction (PCR) is recommended for diagnosis and/or monitoring of CMV infection. Nevertheless the PCR value threshold to start treatment has not yet been defined. Material and methods: In this retrospective monocentric study, we included all kidney recipients between 05/2012 and 07/2013. CMV PCR (whole blood, detection threshold = 1,79 log, Abbott® RealTime CMV) was performed weekly during the first four months after transplantation and then monthly during one year except for low risk patients (D-/R-). Prophylaxis (valganciclovir) was given for 3 months for R+ and 6 months for D+/R-. All patients with at least one positive PCR were included in our analysis. Curative treatment (ganciclovir iv or oral valganciclovir) was started in case of high CMV load (> 4 log) and/or clinical/biological abnormalities associated with positive CMV load (whatever the level). We assessed indications and incidence of curative treatment. Results were expressed as mean ±SD. Results: During the 14-month period, a first positive CMV PCR occurred in 59 /140 patients after 6,4 ± 5,2 months (5 patients under prophylaxis). Twenty seven (45%) were treated (55,5% D+/R-; 33,3% D-/R+; 11,1% D+/R+). Initial PCR level was similar between treated and non-treated patients (2,9 ± 0,99 log and 2,8 ± 0,96 log respectively). All patients with a PCR level higher than 4 log initially or during the follow-up were treated (n=16, 59%). Thirty two patients (55%) were not treated (50% D+/R+; 40,6% D-/R+; 6,25% D+/R-; 3,12% D-/R-). Interestingly, 26 non-treated patients (81%) had spontaneous undetectable CMV load after two controls and no further positive CMV PCR load was observed during the follow-up. Conclusion: We suggest that a “wait-and-see” attitude may be indicated for patients with low positive CMV load (threshold <4log on whole blood) in the absence of clinical or biological abnormalities.

Progressive Diplopia and Facial Weakness in a 62-Year-Old Woman

Progressive Diplopia and Facial Weakness in a 62-Year-Old Woman

Unnecessary repeat Clostridium difficile PCR testing in hospitalized adults with C. difficile-negative diarrhea

The aim of this study was to determine the extent and associated costs of repeat Clostridium difficile stool polymerase chain reaction (PCR) assays in patients with initially negative PCRs. C. difficile stool PCRs were done on adult hospitalized patients with diarrhea. The number/time course of repeat PCRs on initially negative PCR patients was determined. Of 5,027 C. difficile stool PCRs, 814 (16.2 %) were positive and 4,213 (83.8 %) were negative. Ninety-seven of the initially PCR-negative patients had >2 repeat tests 1-59 days after the initial negative stool PCR. Repeat negative PCR testing rarely resulted in a subsequent positive result (0.05 %). The unnecessary costs of 97 repeat PCRs was $32,658.00. Many of these patients were originally given empiric oral anti-C. difficile therapy, in spite of repeatedly negative PCRs.

Toward Optimal Detection of the Common Prenatal Aneuploidies by Quantitative Fluorescent–Polymerase Chain Reaction: Comparison of Two Commercial Assays

To evaluate and compare the performance of the recently released Aneufast™ v2 (MolgentixSL) and QST*RplusV2 commercial assays (Gen-Probe), both designed for the quantitative fluorescent-polymerase chain reaction (PCR) detection of the common aneuploidies during pregnancy. A series of 160 consecutive fetal samples referred for rapid aneuploidy detection testing and an additional 25 samples enriched for the presence of an abnormality were selected for comparison. To confidently rule out a chromosome abnormality, a second round of short tandem repeat typing was required for 14.1% (26) and 9.7% (18) of the specimens analyzed with Aneufast v2 and QST*RplusV2, respectively. Reflex testing was required for 7.6% (14) and 5.9% (11) of the specimens analyzed with respective assays to confidently rule out an autosomal trisomy. For the sex chromosomes, the difference in the amount of follow-up testing is greater between the assays, as a result of the inclusion in the initial PCR of the TAF9L paralogous marker in the QST*RplusV2 assay. Overall, both assays performed similarly in the detection of aneuploidies. In this sample set, the QST*RplusV2 kit required less frequent reflex testing, which translates into shorter turnaround time and cost savings. The incorporation of the TAF9L paralogous sequence in the initial PCR is advantageous for diagnostic use.