Misdiagnosis of Bordetella bronchiseptica Respiratory Infection as Bordetella pertussis by Multiplex Molecular Assay.

Multiplex polymerase chain reaction (PCR) tests are useful for the rapid detection of pathogens, though diagnostic challenges may arise. We report 2 immunocompromised patients with Bordetella bronchiseptica respiratory infection misdiagnosed as Bordetella pertussis using PCR, including discussion of transmission, diagnostic testing, clinical implications, and infection control considerations.

Variability in assessing for BK viremia: whole blood is not reliable and plasma is not above reproach - a retrospective analysis.

Polyomavirus nephropathy (PVN) is a major complication of kidney transplantation. Most reports describe polyomavirus viremia either precedes or is detectable at the time of diagnosis of PVN. This association is the basis of current screening recommendations. We retrospectively reviewed the PCR results of blood and urine samples from 29 kidney transplant recipients with biopsy-proven PVN. Biopsies were performed for a rise in serum creatinine or persistent high-level BK viruria. All biopsies showed polyoma virus large T-antigen expression in tubular epithelium using immunohistochemistry. All had viruria preceding or at the time of biopsy (range, 5.2 × 104 to >25 × 106 BKV DNA copies/ml). Twenty (69%) had viremia ranging from 2.5 × 103 to 4.3 × 106 copies/ml at the time of the biopsy. Via blood BK PCR assay, nine (31%) had no BK viremia detected either preceding or at the time of the biopsy. In five recipients where sufficient specimen permitted, additional plasma BK assessment revealed positive detection of viremia. A comparative analysis of assays from two centres was performed with spiked samples. BK DNA may not be detected in the blood of some kidney transplant recipients with histologically confirmed PVN. This may reflect limitation of whole blood as opposed to plasma-based BK DNA assessment.

BioFire FilmArray Respiratory Panel for Detection of Enterovirus D68.

During the enterovirus D68 (EV-D68) outbreak of 2014, the BioFire FilmArray (FA) respiratory panel was used to detect rhinovirus/enterovirus in respiratory specimens; suspected EV-D68-positive specimens were sent to CDC for confirmation. Positive rhinovirus/enterovirus FA targets revealed patterns loosely associated with EV-D68 that may be useful for confirmation triaging.

Comparison of Cepheid Xpert Flu/RSV XC and BioFire FilmArray for Detection of Influenza A, Influenza B, and Respiratory Syncytial Virus.

The Xpert Flu/RSV XC was compared to the FilmArray respiratory panel for detection of influenza (Flu) A, Flu B, and respiratory syncytial virus (RSV), using 128 nasopharyngeal swabs. Positive agreements were 100% for Flu A and RSV and 92.3% for Flu B. The Xpert may be useful in clinical situations when extensive testing is not required and may serve an important role in laboratories already performing broader respiratory panel testing.

Implementation of a Sample Pooling Strategy for the Direct Detection of SARS-CoV-2 by Real-Time Polymerase Chain Reaction During the COVID-19 Pandemic.

OBJECTIVES: To report our institutional experience in devising and implementing a pooling protocol and process for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription polymerase chain reaction (RT-PCR) testing over a 3-month period in the fall of 2020. METHODS: The widespread testing implemented in the United States for detecting SARS-CoV-2 infection in response to the coronavirus disease 2019 pandemic has led to a significant shortage of testing supplies and therefore has become a major impediment to the public health response. To date, several institutions have implemented sample pooling, but publications documenting these experiences are sparse. Nasal and nasopharyngeal samples collected from low-positivity (<5%) areas were tested in pools of five on the Roche cobas 6800 analyzer system. Routine SARS-CoV-2 RT-PCR turnaround times between sample collection to result reporting were monitored and compared before and after sample pooling implementation. RESULTS: A total of 4,131 sample pools were tested over a 3-month period (during which 39,770 RT-PCR results were reported from the Roche system), allowing our laboratory to save 13,824 tests, equivalent to a conservation rate of 35%. A 48-hour or less turnaround time was generally maintained throughout the pooling period. CONCLUSIONS: Sample pooling offers a viable means to mitigate shortfalls of PCR testing supplies in the ongoing pandemic without significantly compromising overall turnaround times.

Sensitive detection and quantification of SARS-CoV-2 in saliva.

Saliva has significant advantages as a test medium for detection of SARS-CoV-2 infection in patients, such as ease of collection, minimal requirement of supplies and trained personnel, and safety. Comprehensive validation in a large cohort of prospectively collected specimens with unknown SARS-CoV-2 status should be performed to evaluate the potential and limitations of saliva-based testing. We developed a saliva-based testing pipeline for detection of SARS-CoV-2 nucleic acids using real-time reverse transcription PCR (RT-PCR) and droplet digital PCR (ddPCR) readouts, and measured samples from 137 outpatients tested at a curbside testing facility and 29 inpatients hospitalized for COVID-19. These measurements were compared to the nasal swab results for each patient performed by a certified microbiology laboratory. We found that our saliva testing positively detects 100% (RT-PCR) and 93.75% (ddPCR) of curbside patients that were identified as SARS-CoV-2 positive by the Emergency Use Authorization (EUA) certified nasal swab testing assay. Quantification of viral loads by ddPCR revealed an extremely wide range, with 1 million-fold difference between individual patients. Our results demonstrate for both community screening and hospital settings that saliva testing reliability is on par with that of the nasal swabs in detecting infected cases, and has potential for higher sensitivity when combined with ddPCR in detecting low-abundance viral loads that evade traditional testing methods.

Evaluation of the EUROIMMUN Anti-SARS-CoV-2 ELISA Assay for detection of IgA and IgG antibodies.

As the Coronavirus 2019 (COVID-19) pandemic evolves, the development of immunoassays to help determine exposure and potentially predict immunity has become a pressing priority. In this report we present the performance of the EUROIMMUN enzyme-linked immunosorbent assay (ELISA) for semi-quantitative detection of IgA and IgG antibodies in serum and plasma samples using recombinant S1 domain of the SARS-CoV-2 spike protein as antigen. Specimens from patients, with and without COVID-19 infection, were tested at the University of Chicago Clinical Microbiology and Immunology Laboratory. Of 86 samples from SARS-CoV-2 PCR-negative patients, including 28 samples positive for common human coronavirus strains, 76 tested negative and 10 tested positive for IgA (88.4% agreement, 95% CI: 79.9-93.6) while 84 tested negative and 2 tested positive for IgG (97.7% agreement, 95% CI: 91.9-99.6). Of 82 samples from SARS-CoV-2 PCR-positive patients, 14 tested negative and 68 tested positive for IgA (82.9% agreement, 95% CI: 73.4-89.5) while 27 tested negative and 55 tested positive for IgG (67.1% agreement, 95% CI: 56.3-76.3). Of samples collected ≥4 days after positive PCR, 38 of 42 (90.5% agreement, 95% CI: 77.9-96.2) were positive for IgA, and 42 of 42 (100% agreement, 95% CI: 91.6-100) were positive for IgG, respectively. The EUROIMMUN Anti-SARS-CoV-2 ELISA Assay demonstrated good sensitivity for detection of IgA and excellent sensitivity for detection of IgG antibodies from samples collected ≥4 days, after COVID-19 diagnosis by PCR. This assay demonstrated good specificity for IgA and excellent specificity for IgG and demonstrated only borderline cross reaction in 2 of the 28 samples from patients with common human coronaviruses infection, types NL63 and OC43.

Sensitive detection and quantification of SARS-CoV-2 in saliva.

Saliva has significant advantages as a test medium for detection of SARS-CoV-2 infection in patients, such as ease of collection, minimal requirement of supplies and trained personnel, and safety. Comprehensive validation in a large cohort of prospectively collected specimens with unknown SARS-CoV-2 status should be performed to evaluate the potential and limitations of saliva-based testing. We developed a saliva-based testing pipeline for detection of SARS-CoV-2 nucleic acids using real-time reverse transcription PCR (RT-PCR) and droplet digital PCR (ddPCR) readouts, and measured samples from 137 outpatients tested at a curbside testing facility and 29 inpatients hospitalized for COVID-19. These measurements were compared to the nasal swab results for each patient performed by a certified microbiology laboratory. We found that our saliva testing positively detects 100% (RT-PCR) and 93.75% (ddPCR) of curbside patients that were identified as SARS-CoV-2 positive by the Emergency Use Authorization (EUA) certified nasal swab testing assay. Quantification of viral loads by ddPCR revealed an extremely wide range, with 1 million-fold difference between individual patients. Our results demonstrate for both community screening and hospital settings that saliva testing reliability is on par with that of the nasal swabs in detecting infected cases, and has potential for higher sensitivity when combined with ddPCR in detecting low-abundance viral loads that evade traditional testing methods.

Comparison of 3 Nucleic Acid Amplification Tests and a Rapid Antigen Test with Culture for the Detection of Group A Streptococci from Throat Swabs.

BACKGROUND: Recently, the US Food and Drug Administration cleared 3 nucleic acid amplification test (NAAT) assays for detection of Streptococcus pyogenes [group A Streptococcus (GAS)] in pharyngeal specimens. However, there are limited studies evaluating the performance of these NAAT assays. METHODS: We compared the results of 3 NAATs (cobas Liat, Luminex Aries, and Cepheid Xpert Xpress) and a rapid antigen assay (Quidel QuickVue in-line strep A) with the accepted gold standard method, bacterial culture. RESULTS: Sixty-eight throat swab specimens collected between August and October 2017 were tested. Compared to bacterial culture, the sensitivities, specificities, positive predictive value, and negative predictive value for detecting GAS were as follows: cobas Liat: 100%, 97.4%, 96.7%, and 100%; Cepheid Xpert: 100%, 97.4%, 96.7%, and 100%; Luminex Aries: 95.2%, 100%, 100%, and 95.5%. The Quidel QuickVue in-line strep A assay showed poor sensitivity, detecting only 5.2% of culture-positive specimens. CONCLUSION: The 3 NAATs have high sensitivity when compared with bacterial culture for detection of GAS. With rapid turnaround time and ease of use, these tests can be considered as reliable point-of-care tests for the diagnosis of GAS, replacing the need for back-up culture.

Optimizing empirical antimicrobial therapy for infection due to gram-negative pathogens in the intensive care unit: utility of a combination antibiogram.

OBJECTIVE: To determine whether the use of dual antimicrobial therapy based on the results of a combination antibiotic susceptibility report (antibiogram) increases the likelihood of selecting adequate empirical coverage in critically ill patients with infection due to potentially resistant gram-negative pathogens. DESIGN: Retrospective data analysis. SETTING: Urban academic medical center. METHODS: An analysis of culture results and susceptibility data from intensive care unit patients determined by the clinical microbiology laboratory was performed. The proportion of 5 common gram-negative pathogens susceptible to monotherapy with 1 of 3 antipseudomonal antibiotics (piperacillin-tazobactam, ceftazidime, or imipenem) was compared with the proportion susceptible to each of these 3 "backbone" agents plus 1 of 4 additional antimicrobial agents used in combination. RESULTS: More than 5,000 clinical isolates were examined. When all isolates recovered during the entire study period were included, the addition of any of the second antibiotics studied to each of the 3 backbone agents significantly increased the likelihood of covering the causative pathogen (P < .01 for each). The benefit of combination therapy was variable when results for each of the 5 organisms were examined individually. When temporal trends in susceptibility were examined, the decrease in the proportion of organisms susceptible to monotherapy was statistically significant for both imipenem and ceftazidime (P < .01). CONCLUSIONS: Reporting antibiotic susceptibility data in the form of a combination antibiogram may be useful to clinicians who are considering empirical antimicrobial therapy in the intensive care unit.

Pneumonia and empyema caused by penicillin-resistant Neisseria meningitidis: a case report and literature review.

Pneumonia is an uncommon manifestation of Neisseria meningitidis infection, and empyema is rarely reported. Uniform penicillin susceptibility has been assumed for meningococcal infections for many years, but decreased penicillin susceptibility has been recognized recently with increasing frequency. Breakpoints to define different categories of susceptibility were published recently by the Clinical and Laboratory Standards Institute. We report the case of a teenage girl with sepsis and extensive bilateral pneumonia with empyema caused by an N meningitidis isolate that was resistant to penicillin. Her protracted clinical course suggested that penicillin resistance contributed to her delayed recovery. Our experience with this patient suggests that susceptibility testing should be performed in every case of N meningitidis isolation, and treatment with a third-generation cephalosporin should be provided until the susceptibility results are known. Clinical suspicion of N meningitidis as a possible cause of respiratory symptoms accompanied by hypotension, even in the absence of a rash, may aid in diagnosis and therefore in the treatment and provision of prophylaxis to contacts of patients with meningococcal disease.