Multiplex Identification of Gram-Positive Bacteria and Resistance Determinants Directly from Positive Blood Culture Broths: Evaluation of an Automated Microarray-Based Nucleic Acid Test

Abstract
A multicenter study was conducted to evaluate the diagnostic accuracy (sensitivity and specificity) of the Verigene Gram-Positive Blood Culture Test (BC-GP) test to identify 12 Gram-positive bacterial gene targets and three genetic resistance determinants directly from positive blood culture broths containing Gram-positive bacteria. 1,252 blood cultures containing Gram-positive bacteria were prospectively collected and tested at five clinical centers between April, 2011 and January, 2012. An additional 387 contrived blood cultures containing uncommon targets (e.g., Listeria spp., S. lugdunensis, vanB-positive Enterococci) were included to fully evaluate the performance of the BC-GP test. Sensitivity and specificity for the 12 specific genus or species targets identified by the BC-GP test ranged from 92.6%–100% and 95.4%–100%, respectively. Identification of the mecA gene in 599 cultures containing S. aureus or S. epidermidis was 98.6% sensitive and 94.3% specific compared to cefoxitin disk method. Identification of the vanA gene in 81 cultures containing Enterococcus faecium or E. faecalis was 100% sensitive and specific. Approximately 7.5% (87/1,157) of single-organism cultures contained Gram-positive bacteria not present on the BC-GP test panel. In 95 cultures containing multiple organisms the BC-GP test was in 71.6% (68/95) agreement with culture results. Retrospective analysis of 107 separate blood cultures demonstrated that identification of methicillin resistant S. aureus and vancomycin resistant Enterococcus spp. was completed an average of 41.8 to 42.4 h earlier using the BC-GP test compared to routine culture methods. The BC-GP test was unable to assign mecA to a specific organism in cultures containing more than one Staphylococcus isolate and does not identify common blood culture contaminants such as Micrococcus, Corynebacterium, and Bacillus. The BC-GP test is a multiplex test capable of detecting most leading causes of Gram-positive bacterial blood stream infections as well as genetic markers of methicillin and vancomycin resistance directly from positive blood cultures. Please see later in the article for the Editors' Summary Severe sepsis—a life-threatening condition that is usually is triggered by a bacterial infection of the bloodstream—is a major global cause of illness and death. In the US alone, sepsis causes up to 500,000 hospital admissions and more than 250,000 deaths a year. Normally, when microbes enter the human body, the immune system efficiently kills the invaders. In sepsis, the immune system goes into overdrive and the chemicals it releases into the blood to combat the infection trigger widespread inflammation, which leads to the formation of small blood clots and leaky blood vessels that impair the flow of blood to vital organs. In the most severe cases, multiple organs fail and the patient dies. Anyone can get sepsis but people with weakened immune systems, the elderly, and the very young are most vulnerable. Symptoms of sepsis include fever, chills, rapid breathing, and a fast heart rate. In its early stages, sepsis can be treated with antibiotics alone, but people with severe sepsis need to be admitted to an intensive care unit where their vital organs can be supported while the infection is treated. The outcome of sepsis is affected by many factors, but fast, accurate identification of the bacterial infection and determination of its antibiotic susceptibility is essential to ensure that patients receive appropriate antibiotics. Laboratory diagnosis of bloodstream infections currently requires incubation of blood samples in a liquid medium (broth) followed by growth on solid media to identify the bacteria and to test for antibiotic sensitivity. It takes about 3 days after a positive broth culture is obtained to complete this process during which time patients are treated with broad spectrum antimicrobials, which may be ineffective. In this study, the researchers evaluate the sensitivity (a test's ability to identify patients who are positive for a specific condition) and specificity (a test's ability to identify patients who do not have a specific condition) of the Verigene BC-GP test. This multiplex, automated microarray-based nucleic acid test has been developed to directly and simultaneously identify 12 species of Gram-positive bacteria (the commonest cause of bacterial bloodstream infections) and three antibiotic resistance determinants in blood culture broths that contain Gram-positive bacteria. The researchers used the Verigene BC-GP test and reference culture methods to identify the bacterial species and antibiotic resistance determinants present in 1,252 blood cultures containing Gram-positive bacteria collected at five US clinical centers and in 387 contrived blood cultures that contained bacterial species rarely found in the bloodstream. Compared to the reference culture method, the sensitivity of the Verigene BC-GP test for the bacterial species included in the test ranged from 92.6% to 100%; its specificity was 94.5%–100%. Identification of the mecA gene (a genetic marker for methicillin resistance) in cultures that contained Staphylococcus aureus or S. epidermis (Gram-positive bacteria that are often methicillin resistant) was 98.6% sensitive and 94.3% specific. Identification of the vanA gene (a genetic marker for vancomycin resistance) was 100% sensitive and specific in cultures containing Enterococcus species. Only 7.5% of single-organism cultures contained Gram-positive bacteria not present in the Verigene BC-GP test panel. Importantly, results obtained with the new test agreed those obtained with reference culture methods in three-quarters of cultures that contained more than one bacterial species. Finally, in a retrospective analysis of turn-around-time, identification of methicillin-resistant S. aureus and vancomycin-resistant Enterococcus species with the Verigene BC-GP test was about 42 hours faster than with reference methods. These findings show that the Verigene BC-GP test is capable of accurately identifying most leading causes of bloodstream infection with Gram-positive bacteria. Moreover, they show that the test can detect genetic markers of methicillin and vancomycin resistance directly from positive blood cultures, although they also reveal that the test cannot assign mecA positivity to a specific organism in a mixed culture, a finding that may mean that some patients are treated with unnecessary antibiotics. Overall, the researchers conclude that the Verigene BC-GP test has the potential to markedly reduce the turn-around-time for reporting bacterial identification from positive blood cultures. Its use should, therefore, improve the care of patients with sepsis by allowing physicians to prescribe appropriate antibiotics much earlier than is currently possible. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001478.

This publication has 61 references indexed in Scilit: