Lymphocytosis and Mycobacteriosis in a 15-Year-Old Mixed-Breed Dog.

The differential impact of sample type on polymerase chain reaction (PCR) detection of Mycoplasma pneumoniae (MP) has rarely been investigated. The study aimed to evaluate the diagnostic performance of PCR for the detection of MP and to measure MP DNA load between nasopharyngeal and oropharyngeal swabs. Nasopharyngeal and oropharyngeal samples were obtained simultaneously to evaluate their diagnostic performance in children with suspected MP. Two commercially available PCR tests, multiplex PCR and Smart Gene Myco, were used to analyze the nasopharyngeal and oropharyngeal samples, respectively. Furthermore, real-time PCR (RT-PCR) tests were conducted on both sample residues to validate the results. In total, 422 participants underwent simultaneous PCR testing using nasopharyngeal and oropharyngeal swabs; 139 samples (32.9%) from nasopharyngeal swabs and 176 samples (41.7%) from oropharyngeal samples that tested positive using commercially available tests. RT-PCR tests were positive for 136 (32.2%) nasopharyngeal and 183 (43.4%) oropharyngeal residual samples. With the RT-PCR test of the residual extract from oropharyngeal swabs as a reference, the sensitivity and specificity of detecting MP were 74.9% (95% confidence interval 67.9%-81.0%) and 99.2% (97.0%-99.9%) with the multiplex PCR test on nasopharyngeal swabs, and 96.2% (92.3%-98.4%) and 100.0% (98.5%-100.0%) with the Smart Gene Myco on oropharyngeal samples. A negative correlation was observed between fluoroquinolone use and oropharyngeal DNA loads (P = 0.004). The sensitivity of MP detection was significantly better in oropharyngeal samples than in nasopharyngeal samples. This study indicates that oropharyngeal samples should be used to detect MP rather than nasopharyngeal samples.IMPORTANCEObtaining the best sample is crucial for the accurate diagnosis of Mycoplasma pneumoniae (MP) and timely and appropriate treatment. This study aimed to assess the diagnostic performance of MP detection using polymerase chain reaction (PCR) tests between nasopharyngeal and oropharyngeal samples. This study showed that the sensitivity of detecting MP was 74.9% (95% confidence interval 67.9%-81.0%) with a commercially available PCR test on nasopharyngeal swabs, and 96.2% (92.3%-98.4%) with a commercially available PCR test on oropharyngeal samples. The sensitivity of MP detection was significantly better in oropharyngeal samples than in nasopharyngeal samples. This study supports the idea that oropharyngeal samples should be used to detect MP. The results contribute to guidance in the recommendation regarding sampling methods to detect MP. Accurate identification of MP is crucial not only for timely and appropriate antimicrobial treatment but also for efficient epidemiological surveillance.

Ideal Timing for Health Care Workers With COVID-19 to Return to Work in the Omicron Variant Pandemic.

To evaluate changing Clostridioides difficile infection (CDI) testing among inpatients with indeterminate enzyme immunoassay (EIA) results (antigen+/toxin-) from reflexive polymerase chain reaction (PCR) testing to clinician-ordered PCR testing. Multicenter, before-and-after, quasi-experimental study. Four large urban tertiary-care hospitals. We evaluated two 6-month periods before and after an intervention. The primary study outcome was the change in the number of CDI diagnoses between periods. Secondary outcomes included the number of PCR tests performed, adverse events, and healthcare cost savings. In total, 500 EIA-indeterminate C. difficile test results were evaluated: 281 before the intervention and 219 thereafter. CDI was diagnosed by PCR among EIA-indeterminate cases in 182 in the preintervention period versus 94 patients in the postintervention period (48% reduction; P < .01). PCR testing was performed in 99.6% of indeterminate cases (280 of 281; 1 not performed due to an inhibitor) in the preintervention period versus 66% (144 of 219) in the postintervention period (34% reduction; P < .01). We observed no differences between study periods in 30-day all-cause (P = .96), GI-related (P = .93), or C. difficile (P = .47) readmissions, nor in 30-day C. difficile infections (P > .99). No patient without a PCR test in the postintervention period and not treated was later diagnosed with CDI. Each reflexive PCR test not performed led to a cost savings of $4,498 per patient. Applying diagnostic stewardship to C. difficile PCR testing in the inpatient setting led to significant reductions in both testing and cases. Changing the C. difficile PCR testing algorithm for EIA-indeterminate cases from reflexive to clinician-required ordering resulted in valuable cost savings without associated adverse events.

The clinical impact of pneumocystis and viral PCR testing on bronchoalveolar lavage in immunosuppressed patients.

BackgroundThe temporal dynamics of SARS-CoV-2 infectivity in immunocompromised children (IC) are unknown but may have important infection control implications. We evaluated SARS-CoV-2 viral persistence and assessed factors associated with viral persistence and cycle threshold (CT) values as a surrogate of viral load for IC. MethodsWe conducted a retrospective cohort study of SARS-CoV-2-positive IC at a large quaternary pediatric hospital from March 2020-2021. Immunocompromised status was defined as primary or secondary/acquired immunodeficiencies due to comorbidities or immunosuppressive treatment. The primary outcome was time to first-of-two consecutively negative SARS-CoV-2 PCR tests ≥ 24 hours apart. Polymerase chain reaction (PCR) testing of sequential patient samples was conducted using the Centers for Disease Control 2019-nCoV Real-Time RT-PCR Diagnostic Panel (CDC assay). Chi-square, Fisher exact, and Wilcoxon tests were used to compare demographic and clinical characteristics. Kaplan-Meier curve median event times and log-rank tests were used to compare outcomes. Subjects without 2 consecutive negative tests censored at the last test. Analyses were conducted using SAS v 9.4.ResultsNinety-one children met inclusion criteria, and 67 children had more than 1 test (Figure 1). Median age was 15.5 years (IQR 8-18 yrs), 64% were male, 58% of children were white, and 43% were Latinx. Most (67%) were tested in outpatient settings, and 58% of children were asymptomatic. The median time to two negative tests was 42 days (IQR 25.0,55.0), with no difference in duration of positivity with specific diagnoses, degree of lymphopenia, or symptomatic vs asymptomatic illness. Five of 7 (71%) children with samples available for repeat testing had initial CT values < 30, indicating a moderate to high viral load, and of these, 4 (57%) had repeat testing 21 to 30 days later with CT values < 30 (Figure 2), suggesting persistence of moderate to high viral loads. Figure 1. Plot of immunocompromised children in cohort with positive SARS CoV2 PCR and subsequent testing (n = 67).Timelines of immunocompromised children in cohort with positive SARS CoV2 PCR and subsequent testing, grouped by immunocompromising condition. Each line represents an individual patient. Positive results are shown in light grey, negative results are shown in black.Figure 2. Plot of CT values from SARS-CoV-2 PCR testing over time among children with sequential samples available for retesting (n = 7)Plot of CT values (y axis) from SARS-CoV-2 PCR testing on the CDC assay over time (x axis) in days from initial positive test. Repeated testing which yielded a negative result on the CDC assay or intermittent negative results on clinical testing represented as CT value of 40. Each line represents a unique patient.ConclusionThe median duration of viral persistence among IC with SARS-CoV-2 infection was 6 weeks, with no significant difference in immunocompromised diagnoses or clinical presentation, with over half of children with testing on the same platform having moderate to high viral loads after 3 weeks, suggesting potential transmission risk. DisclosuresSamuel R. Dominguez, MD, PhD, BioFire Diagnostics (Consultant, Research Grant or Support)DiaSorin Molecular (Consultant)Pfizer (Grant/Research Support) Samuel R. Dominguez, MD, PhD, BioFire (Individual(s) Involved: Self): Consultant, Research Grant or Support; DiaSorin Molecular (Individual(s) Involved: Self): Consultant; Pfizer (Individual(s) Involved: Self): Grant/Research Support Suchitra Rao, MBBS, MSCS, BioFire (Research Grant or Support)

Polymerase chain reaction testing for Clostridium difficile

Evaluation of polymerase chain reaction and dot blot hybridisation tests in the diagnosis of pigeon circovirus infections

Older age is associated with sustained detection of SARS-CoV-2 in nasopharyngeal swab samples

BackgroundCytomegalovirus (CMV) Polymerase chain reaction (PCR) test is a valuable tool for diagnosis and therapeutic monitoring of CMV infection. Academic medical centers with a high volume of organ transplant and immunosuppressed patients often utilize CMV quantitative and qualitative PCR testing. CMV qualitative PCR typically does not alter clinical management, and positive CMV qualitative test often needs follow up quantitative test. Despite its lack of usefulness, CMV qualitative PCR is often over-ordered and may unnecessarily raise the cost of hospitalization. We evaluated utility of CMV PCR testing at a tertiary care medical center.MethodsWe reviewed CMV PCR testing done from June 2015-November 2016 at Hahnemann University Hospital in Philadelphia, PA. CMV qualitative test was performed at LabCorp and CMV quantitative test was done at Focus Labs. Data collected included demographics, length of stay, and immunosuppression. Selected patients had either CMV qualitative PCR positive without follow up quantitative PCR, or negative CMV qualitative PCR with unnecessary CMV quantitative PCR ordered.ResultsWe evaluated 226 CMV PCR test results including 162 qualitative and 64 quantitative CMV PCR in 139 patients. 39 (28%) patients had superfluous CMV testing. Mean age was 52.6 years, 61% were male, 46% were African American. Mean length of stay was 24 days. A half (N = 19,49%) were immunocompromised. 28 (17.2%) results were positive for CMV qualitative PCR, 7 (25%) of whom follow up CMV quantitative PCR were not sent. Thirty-two patients had negative CMV qualitative testing yet had CMV quantitative PCR sent. Six had redundant CMV quantitative PCR tests within 7 days likely resulting from delayed result report from send out. After performing cost analysis, these unnecessary tests would have saved $3930.ConclusionIn our cohort, significant unnecessary CMV testing resulted in increased health care cost and patient discomfort. Positive Qualitative CMV PCR without Quantitative testing impairs diagnosis and treatment follow up. Given complicated testing algorithm and limited value of CMV qualitative PCR testing in the adult population, we plan to simplify CMV testing to quantitative only and perform in house testing to shorten result time.DisclosuresAll authors: No reported disclosures.

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

BackgroundIn the last decade, multi-drug resistant (MDR) and extensively drug-resistant (XDR) microorganisms have been an emerging global health problem. The antimicrobial resistance surveillance of the Department of Health in 2019 reported 3 cases of resistant Salmonella typhi to ciprofloxacin. Due to the long turn-around time for blood cultures, management may be delayed for patients affected by MDR S. typhi. As such, there is a need to explore new methods for early detection of MDR S. typhi. The primary objective of this research is to determine the diagnostic application of Polymerase Chain Reaction (PCR) in early detection of MDR S. typhi among patients in the San Lazaro Hospital in the past 5 years.Table 1.Demographic profile of respondents using archived data from SLH-Community Acquired Bacteremia studyMethodsClinical data were extracted from San Lazaro Hospital, PCR test results at SLH-Nagasaki Molecular Laboratory, Microbiology Laboratory and the medical records of San Lazaro Hospital. Data were analyzed using descriptive statistics, comparative analysis, and phi coefficient test.Table 2.Clinical profile of respondents using archived data from SLH-Community Acquired Bacteremia studyResultsOut of 84 admitted patients diagnosed of typhoid fever, seven (8.3%) were tested positive by blood culture while 15 (17.9%) were tested positive using Nested PCR method. Considering blood culture as standard, the Nested PCR method had sensitivity, specificity and accuracy of 14.29%, 81.82% and 76.19%; respectively, in detecting S. typhi. In addition, one sample from gryA gene group was tested resistant to Ciprofloxacin and two samples of CatP gene group were resistant to Chloramphenicol out of the 15 samples tested for AMR using Monoplex PCR.Table 3.Sensitivity, Specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV), and Likelihood Ratios (LR) of the Nested Polymerase Chain Reaction (PCR) Test in Detecting Salmonella typhi (S. typhi) against Blood Culture Test among the Respondents (n = 84)Table 4. Genotypic identification of S. typhi drug resistant gene using Monoplex PCR (n = 15)ConclusionThe results of the PCR test and gene-specific PCR tests were not associated with those of the culture and sensitivity test. Due to the lack of MDR S. typhi cases detected via culture-sensitivity test necessary to examine the diagnostic capacity of PCR test, the ability of PCR in detecting MDR S. typhi remains unclear. Its utility as a reliable diagnostic test for the detection of MDR S. typhi requires further exploration and analyses.Table 6.7.8. Sensitivity, Specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV), and Likelihood Ratios (LR) of the Monoplex Polymerase Chain Reaction (PCR-sul2) Test in Detecting Antibiotic Resistant Salmonella typhi (S. typhi) against Blood Culture Test Ciprofloxacin, Trimethoprime-Sulfamethoxazole and AmpicillinDisclosuresAll Authors: No reported disclosures

BackgroundFeline immunodeficiency virus (FIV) infection is an important cause of disease of cats worldwide. Initial screening is commonly performed by commercially available point‐of‐care (POC) ELISA tests. Confirmatory testing for positive POC test results is recommended. Polymerase chain reaction (PCR) tests for FIV are commonly used additional testing methods; however, reported measures of diagnostic accuracy vary widely between PCR tests, making interpretation of results difficult.Hypothesis/ObjectiveThere is very good agreement between results of a commercially available PCR test and a POC ELISA test for FIV for specimens collected from owned and shelter‐housed cats.AnimalsBlood samples from 168 cats from 2 adoption guarantee shelters, an FIV Sanctuary, and 64 private homes were used.MethodsThis was a prospective study. Whole blood samples were collected in K2‐EDTA, divided, and submitted for PCR and ELISA testing. Follow‐up whole blood samples were collected in lithium heparin from cats with discordant results and submitted for virus isolation (VI).ResultsThere was very good agreement between ELISA and PCR (kappa 0.87; P < .001; 95% CI 0.79, 0.95). Of 168 cats, eleven had discordant ELISA/PCR results: 7 ELISA+/PCR‐ and 4 ELISA‐/PCR+. Using VI as a reference standard, there were 4 false‐positive PCR results, 5 false‐positive ELISA results, and 1 false‐negative PCR result (1 cat lost to follow‐up).Conclusions and Clinical ImportanceWhile there was good agreement between the POC ELISA and PCR tests, the discordant results highlight the importance of cautious interpretation of test results and the necessity of confirmatory testing.

Editor, Herpes simplex keratitis (HSK) is a major cause of unilateral blindness in developed countries (Guess et al. 2007). A fast and highly sensitive test could assist in the diagnosis to guide adequate antiviral therapy. We aimed to report the use of herpes simplex virus (HSV) polymerase chain reaction (PCR) test for the diagnosis of HSK and its relationship to the outcomes in these patients over a 2-year period at the Sydney Eye Hospital, Australia. A retrospective case review of all patients who received antiviral medications for HSK was conducted. Cases were identified from HSV PCR swab results, pharmacy records and hospital coding data from 2012 to 2013. Clinical details were collated from the medical records. Herpes simplex virus type 1 and type 2 were detected using the HSV-1 HSV-2 VZV R-gene® kit, a real-time PCR on DNA extracted from human clinical samples (Argene, bioMérieux SA, Marcy-l'Etoile, France) according to manufacturer’s instructions. The tests were performed using a Roche LC480 thermal cycler for real-time amplification. The R-gene kit’s sensitivity ranged between 91% and 100% and specificity 95% and 100% depending on the study (Biomerieux 2016). Patients were sub-grouped depending on the clinical features: epithelial, stromal with ulceration (SHSK+U), stromal without ulceration (SHSK−U), endothelial, keratouveitis and active HSK with prior corneal graft. Outcome was determined when the initial antiviral therapy was stopped or changed and classified as clinically improved or worsened. Two hundred and fifty-two patients were included with HSV PCR performed in 166 (66%) at presentation. The HSV-1 PCR test was positive in 32/94 (34%) patients with epithelial HSK, 4/14 (29%) patients with SHSK+U, 3/11 (27%) patients with active HSK and corneal graft, and 6/33 (18%) patients with keratouveitis. Overall, the positivity rate was 27% (45/166). Of the patients with prior corneal graft, two were diagnosed with epithelial HSK and one with SHSK+U. A clinically improved or worsened outcome was determined for 112 of 166 patients (67%) tested with HSV PCR (Table 1). There was no statistical association between the type of outcome and HSV PCR result (p = 0.5). Polymerase chain reaction test has been reported as a highly sensitive diagnostic test for HSK (Subhan et al. 2004; Azher et al. 2017). Its diagnostic power varies depending on the type of HSK, previous acyclovir therapy, use of topical anaesthetics or dyes, and viral load in the specimen (Shoji et al. 2016). The PCR test most likely identifies patients with a dendritic ulcer as it is a manifestation of active viral infection (Azher et al. 2017). Consequently, clinicians requested the test to patients with a presence of a epithelial defect and provisional diagnosis of epithelial HSK [94/123, (76%)], SHSK+U [14/19, (74%)], keratouveitis [33/49, (67%)] or active HSK with corneal graft [11/25, (44%)]. Stromal HSK is thought to occur due to an immune response to the virus rather than via viral infection (Azher et al. 2017). It is also assumed that stromal and endothelial HSK are recurrent episodes (Guess et al. 2007) and the patients may have a previous positive PCR result. Due to the absence of an epithelial defect, it is not possible to access the stroma and therefore to obtain a sample of the area affected. This may explain the negative PCR results for this type of HSK (Azher et al. 2017). This may also be the reason why clinicians did not request as many PCR tests on patients with SHSK−U [11/22, (50%)] and endothelial HSK [3/14, (21%)] compared with the epithelial HSK (76%) and SHSK+U (74%) groups. The positivity rate for both, SHSK−U and endothelial HSK, was 0% supporting the above hypothesis. In conclusion, the HSV PCR test had an overall low positive rate. Regardless of the PCR result, most patients improved with the initial antiviral therapy, especially for epithelial HSK. A clinical diagnosis of HSK may therefore be sufficient to guide treatment.

Reports on the sensitivity of polymerase chain reaction (PCR) for the diagnosis of lymph node tuberculosis (TB) show divergent results. We evaluated the accuracy of the Roche Amplicor Mycobacterium tuberculosis PCR test with lymph node aspirate and biopsy samples. The study was conducted at a public reference hospital in Lima, Peru. From the period of January 2003 to January 2004, we included patients who had lymphadenopathy and in whom the attending physician suspected TB. Aspirate and biopsy samples were submitted for culturing in Lowenstein-Jensen medium, for histopathologic testing, and for PCR. The sensitivity and specificity of PCR were calculated against a reference standard based on histopathologic findings and culture. Our study included 154 patients. Median age was 29 years (interquartile range, 21-40 years); 97 patients (62.9%) were men. Twenty-nine patients (18.8%) had acid fast bacilli-positive histopathologic findings, and 44 (28.6%) had a positive culture result. Using the combination of histopathologic findings and culture as reference standard, 55 patients (35.7%) had a diagnosis of tuberculous lymphadenitis. The sensitivity of the PCR test was 58.2%, and the specificity was 93.9%. For biopsy tissue only, the sensitivity of PCR was 52.7%, and the specificity was 97.0%. For aspirate samples only, the sensitivity of PCR was 47.3%, and the specificity was 96.0%. The Amplicor PCR test revealed low sensitivity and high specificity for the diagnosis of lymph node TB. The sensitivity was higher in cases in which the bacillary load was high--in acid fast bacilli-positive samples and among HIV-infected patients. Considering the results of microbiological and PCR tests together, there was still a patient group in whom no final diagnosis could be established.

Three SARS-CoV-2 PCR-negative cases of COVID-19 diagnosed using isothermal amplification methods