Michael L. Wilson

Comparison of Becton Dickinson Directigen EZ Flu A+B Test against the CDC Real-Time PCR Assay for Detection of 2009 Pandemic Influenza A/H1N1 Virus

Pandemic influenza A/H1N1 2009 virus was first reported in Mexico during April 2009, followed by a rapid spread to many countries (10). As of 4 October 2009, >375,000 laboratory-confirmed cases and >4,500 deaths had been reported to the World Health Organization (10). The 2009 influenza season in the southern hemisphere was characterized by a predominance of this strain, which to date is the same pattern observed in the United States during the early 2009-2010 influenza season (3). n nAlthough performance characteristics of several commercial rapid immunodiagnostic tests (RIDT) for the detection of seasonal influenza viruses are known (6), only limited data have been published regarding the ability of RIDT to detect pandemic influenza A/H1N1 2009 virus (1, 4, 5, 7-9). Of these comparisons, two included an evaluation of the Directigen EZ Flu A+B immunoassay (Becton Dickinson, Sparks, MD). In these two reports, the reported sensitivities were 46.7 and 49% (1, 9). To date, there are no reported data comparing the Directigen EZ Flu A+B immunoassay with the real-time PCR (RT-PCR) assay developed by the Centers for Disease Control and Prevention (CDC) to detect pandemic influenza A/H1N1 2009 virus. This communication is a report of such a comparison using fresh, clinical nasopharyngeal wash (NPW) specimens. n nA total of 231 NPW specimens collected using the N-Pak nasopharyngeal aspiration kit (M-Pro, Annandale, MN) were submitted to the Clinical Microbiology Laboratory at Denver Health Medical Center for rapid influenza testing during August, September, and October. Of these, six were found by RT-PCR to be seasonal influenza viruses and were eliminated from the comparison. No swabs were submitted for testing. Directigen EZ Flu A+B RIDTs were performed 7 days per week between the hours of 0700 and 2300. Specimens received after 2300 h were refrigerated, and testing was performed the next morning. All tests were performed according to the manufacturers instructions in the package insert (2). Aliquots of all specimens were refrigerated, saved, and sent daily Monday to Friday via courier to the Laboratory Services Division of the Colorado Department of Public Health and Environment. Testing for pandemic influenza H1N1 2009 virus was performed using the CDC-developed RT-PCR assay. This protocol currently operates under Emergency Use Authorization (EUA) status granted by the FDA and is designed to detect all influenza A viruses, swine strains of influenza A virus, and H1N1 2009. All three targets must yield positive findings for a patient specimen to be considered positive. n nThe results are shown in Table u200bTable1.1. Of 225 NPW specimens, 80 (35.6%) were positive and 145 (64.4%) were negative by RT-PCR. Of the 80 specimens that tested positive by RT-PCR, 39 tested positive by RIDT, for a test sensitivity of 48.7% and a positive predictive value of 88.6%. Of the 145 specimens that tested negative by RT-PCR, 140 tested negative by RIDT, for a test specificity of 96.5% and a negative predictive value of 77.3%. The overall agreement between the two assays was 179/225 (79.5%). n n n nTABLE 1. n nBD Directigen EZ Flu A+B compared to CDC RT-PCR for detecting pandemic influenza A/H1N1 2009 virus n n n nConsiderable attention has been given to clinical laboratory testing for the pandemic influenza A/H1N1 2009 virus. Much of this attention has focused on RIDTs, which have been demonstrated previously to have lower sensitivity than RT-PCR, viral culture, and other methods in the detection of seasonal influenza (6). In published reports regarding the use of RIDT for detecting pandemic influenza A/H1N1 2009 virus, the performance characteristics of several commercial RIDTs have been reported to be low. Of note, however, the multiplicity of methods evaluated, different types of specimens tested, lack of comparison against a consistent gold standard assay, and (in some reports) absence of information regarding the types of RIDTs used make it difficult for potential users to compare these assays. n nTo our knowledge, no study to date has compared the performance characteristics of RIDT with the CDC RT-PCR for the detection of pandemic influenza A/H1N1 2009 virus using exclusively fresh clinical NPW specimens. Our findings are consistent with those reported in the literature, confirming that this immunoassay is specific but insensitive in the detection of this strain of influenza virus. What diagnostic role RIDTs may play during this and subsequent influenza seasons depends on how they are used. For example, when a clearly predominant strain of influenza virus is circulating in a community, these tests may be useful as initial screening tests, although negative test results in cases with a high clinical suspicion of influenza should be followed up by use of a more sensitive test, such as a CDC or other RT-PCR.

Coccidioidomycosis meningitis with massive dural and cerebral venous thrombosis and tissue arthroconidia.

To our knowledge we report the first case of meningitis from Coccidioides immitis associated with massive dural and cerebral venous thrombosis and with mycelial forms of the organism in brain tissue. The patient was a 43-year-old man with late-stage acquired immunodeficiency syndrome (AIDS) whose premortem and postmortem cultures confirmed C immitis as the only central nervous system pathogenic organism. Death was attributable to multiple hemorrhagic venous infarctions with cerebral edema and herniation. Although phlebitis has been noted parenthetically to occur in C immitis meningitis in the past, it has been overshadowed by the arteritic complications of the disease. This patients severe C immitis ventriculitis with adjacent venulitis appeared to be the cause of the widespread venous thrombosis. AIDS-related coagulation defects may have contributed to his thrombotic tendency.

Contemporary tetracycline susceptibility testing: doxycycline MIC methods and interpretive criteria (CLSI and EUCAST) performance when testing Gram-positive pathogens☆

International susceptibility testing breakpoint organizations and regulatory agencies have markedly differing interpretive criteria for the tetracycline class. Here we examined the magnitude of these differences for doxycycline and tetracycline hydrochloride (HCL) when tested against a collection of 13,176 Gram-positive cocci from a worldwide surveillance network (SENTRY Antimicrobial Surveillance Program, 2010). Clinical and Laboratory Standards Institute (CLSI) breakpoints are routinely higher, usually 4-fold, compared to those of the European Committee on Antimicrobial Susceptibility Testing (EUCAST); however, CLSI recently (2013) modified Streptococcus pneumoniae breakpoints (≤ 2 μg/mL in 2012) to ≤ 0.25 and ≤ 1 μg/mL for doxycycline and tetracycline HCL, respectively. We report that these changes are a promising step toward international breakpoint harmonization, but lack a comprehensive approach needed for testing tetracyclines against all Gram-positive cocci. Generally, EUCAST breakpoint criteria showed i) lower spectrums (reduced susceptibility rates) for the tetracyclines, but highest for doxycycline versus all species examined; ii) greater test accuracy (lower predictive categorical errors), especially for tetracycline to predict doxycycline susceptibility (99.91%); and iii) zone diameter correlate breakpoints which are generally available online. Molecular tests for tet resistance genes demonstrate that tet (K) and tet (M) containing strains can occur in the susceptible population of MIC results by both CLSI and EUCAST breakpoint criteria. In summary, doxycycline continues to show greater comparative potency versus tetracycline HCL against all monitored Gram-positive species and the international harmonization of tetracycline breakpoints should be a priority, as the most recent CLSI update only addressed 1 streptococcal species and 2 tetracycline agents.

Contemporary potencies of minocycline and tetracycline HCL tested against Gram-positive pathogens: SENTRY Program results using CLSI and EUCAST breakpoint criteria

Tetracycline class agents vary widely in their activity against emerging important antimicrobial-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter spp. Also, published susceptibility breakpoints are discordant between the Clinical and Laboratory Standards Institute (CLSI), the European Committee on Antimicrobial Susceptibility Testing (EUCAST), and regulatory-approved documents. We have assessed the impact of these differences for tetracycline HCL and minocycline when tested against contemporary Gram-positive pathogens. The SENTRY Antimicrobial Surveillance Program (2011) compared minocycline and tetracycline HCL activity via reference methods (M07-A9) using a worldwide collection of S. aureus (SA; 4917 strains with 1955 MRSA), Streptococcus pneumoniae (SPN; 1899), S. pyogenes (GRA; 246), and S. agalactiae (GRB; 217). Regardless of applied categorical breakpoints, minocycline exhibited wider coverage (% susceptible) than tetracycline HCL of 4.5-11.8/0.5-2.6/1.4-2.3/0.4-0.4% for MRSA/SPN/GRB/GRA, respectively. Lower EUCAST susceptible breakpoints produced reduced susceptibility rates for minocycline ranging from no difference (≤0.5 μg/mL) for GRA to -8.9% (≤1 μg/mL) for MRSA (97.2% susceptible by CLSI; 88.3% by EUCAST). Use of tetracycline HCL-susceptible results to predict minocycline susceptibility was very accurate (99.0-100.0%), with absolute categorical agreement rates ranging from 92.1% to 98.4% (CLSI) to 98.4% to 99.6% (EUCAST) for streptococci; greatest predictive error was noted using the CLSI breakpoints (14.7%) compared to EUCAST criteria (only 5.0%; acceptable), both for MRSA testing dominated by false-resistant results for minocycline. In conclusion, minocycline demonstrates continued superior in vitro activity compared to tetracycline HCL when testing SA (especially MRSA) and pathogenic streptococci. When testing tetracyclines, laboratories must recognize the expanded spectrum of minocycline against certain pathogens and utilize methods minimizing interpretive error. We conclude that EUCAST breakpoint criteria (≤0.5 or ≤1 μg/mL) represent the most conservative (better recognize strains with tet resistance mechanisms) and accurate tetracycline breakpoint guidelines for testing contemporary isolates of Gram-positive cocci.