Rapid semiautomated subtyping of influenza virus species during the 2009 swine origin influenza A H1N1 virus epidemic in Milwaukee, Wisconsin.

In the spring of 2009, a novel influenza A (H1N1) virus (swine origin influenza virus [S-OIV]) emerged and began causing a large outbreak of illness in Milwaukee, WI. Our group at the Midwest Respiratory Virus Program laboratory developed a semiautomated real-time multiplex reverse transcription-PCR assay (Seasonal), employing the NucliSENS easyMAG system (bioMérieux, Durham, NC) and a Raider thermocycler (HandyLab Inc., Ann Arbor, MI), that typed influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) and subtyped influenza A virus into the currently circulating H1 and H3 subtypes, as well as a similar assay that identified H1 of S-OIV. The Seasonal and H1 S-OIV assays demonstrated analytical limits of detection of <50 50% tissue culture infective doses/ml and 3 to 30 input copies, respectively. Testing of the analytical specificities revealed no cross-reactivity with 41 and 26 different common organisms and demonstrated outstanding reproducibility of results. Clinical testing showed 95% sensitivity for influenza A virus and influenza B virus and 95 and 97% specificity compared to tissue culture. Comparisons of results from other molecular tests showed levels of positive agreement with the Seasonal and H1 S-OIV assay results of 99 and 100% and levels of negative agreement of 98 and 100%. This study has demonstrated the use of a semiautomated system for sensitive, specific, and rapid detection of influenza A virus, influenza B virus, and RSV and subtyping of influenza A virus into human H1 and H3 and S-OIV strains. This assay/system performed well in clinical testing of regular seasonal influenza virus subtypes and was outstanding during the 2009 Milwaukee S-OIV infection outbreak. This recent outbreak of infection with a novel influenza A (H1N1) virus also demonstrates the importance of quickly distributing information on new agents and of having rapid influenza virus subtyping assays widely available for clinical and public health decisions.

Clostridium difficile: epidemiology, pathogenesis, management, and prevention of a recalcitrant healthcare-associated pathogen.

Clostridium difficile is the leading cause of healthcare-associated infectious diarrhea. Although C difficile is part of normal flora in some healthy individuals, patients with selective risk factors are often vulnerable to the toxigenic potential of this virulent healthcare pathogen. The spectrum of C difficile infection (CDI) is highly variable, ranging from mild to severe illness, presenting with single to multiple disease recurrences. Current approaches to treatment are based on severity of illness, number of recurrences, and clinical presentation. Oral vancomycin and metronidazole have formed the foundation for treatment of CDI, but therapeutic failures are commonly reported, especially involving hypervirulent clones. Alternative therapies, including newer antimicrobials, probiotics, immunotherapy, and fecal transplantation, have all met with varying degrees of efficacy. Although toxigenic culture (TC) testing from anaerobic culture remains the gold standard, newer technologies, including enzyme immunoassay, common antigen (glutamate dehydrogenase) testing, and real-time polymerase chain reaction (PCR) are less time-consuming and rapid. However, TC and PCR have reported high specificity and sensitivity when compared with other laboratory tests. Because of the significant morbidity and mortality associated with CDI, a high index of suspicion is warranted. Prevention and eradication of CDI require a multidisciplinary approach, including early disease recognition through appropriate surveillance, implementation of effective contact isolation strategies, adherence to environmental controls, judicious hand hygiene, evidence-based treatment, and management that includes antibiotic stewardship, continuous education of healthcare workers, and administrative support.