Clinical evaluation and cost analysis of a Trioplex real-time PCR assay for the detection and differentiation of herpes simplex virus 1 and 2 in cutaneous and mucocutaneous lesions.

PURPOSE: Herpes simplex virus (HSV) is a common lifelong sexually transmitted infection. HSV-1 typically manifests as oral cold sores, while HSV-2 is more traditionally associated with sexual transmission and infection. We have developed a real-time PCR (Trioplex) for the simultaneous detection of HSV-1 and -2 and the bacterial phage internal control (IC) MS2. METHODOLOGY: To determine the performance of the Trioplex method and resolve discrepancies, 178 clinical specimens from cutaneous and mucocutaneous sources were tested using 3 different methods; virus culture with direct fluorescent antibody (DFA) immunostaining, Trioplex and a commercially available HSV analyte-specific reagent (ASR). RESULTS: HSV Trioplex was significantly more sensitive than virus culture (89 and 67 % HSV 1/2, respectively) and comparable to the commercial assay (P<0.001). Cost analysis revealed that the Trioplex reduced cost by 80  % compared to cell culture. CONCLUSIONS: The implementation of the HSV Trioplex improved the detection turnaround time from 3-10 days to 2.5 h, thus streamlining Herpes detection, improving sensitivity and reducing laboratory costs.

Plasmodium falciparum Treated with Artemisinin-based Combined Therapy Exhibits Enhanced Mutation, Heightened Cortisol and TNF-α Induction.

The artemisinin-based combined therapy (ACT) post-treatment illness in Plasmodium falciparum-endemic areas is characterized by vague malaria-like symptoms. The roles of treatment modality, persistence of parasites and host proinflammatory response in disease course are unknown. We investigated the hypothesis that ACT post-treatment syndrome is driven by parasite genetic polymorphisms and proinflammatory response to persisting mutant parasites. Patients were categorized as treated, untreated and malaria-negative. Malaria positive samples were analyzed for Pfcrt, Pfmdr1, K13 kelch gene polymorphisms, while all samples were evaluated for cytokines (TNF-α, IL-12p70, IL-10, TGF-ß, IFN-γ) and corticosteroids (cortisol and dexamethasone) levels. The treated patients exhibited higher levels of parasitemia, TNF-α, and cortisol, increased incidence of parasite genetic mutations, and greater number of mutant alleles per patient. In addition, corticosteroid levels declined with increasing number of mutant alleles. TGF-ß levels were negatively correlated with parasitemia, while IL-10 and TGF-ß were negatively correlated with increasing number of mutant alleles. However, IL-12 displayed slight positive correlation and TNF-α exhibited moderate positive correlation with increasing number of mutant alleles. Since post-treatment management ultimately results in patient recovery, the high parasite gene polymorphism may act in concert with induced cortisol and TNF-α to account for ACT post-treatment syndrome.

Surveillance Study of Influenza Occurrence and Immunity in a Wisconsin Cohort During the 2009 Pandemic.

BACKGROUND: Antibody and T-cell immunity to conserved influenza virus antigens can protect animals against infection with diverse influenza strains. Although immunity against conserved antigens occurs in humans, whether such responses provide cross-protection in humans and could be harnessed as the basis for universal influenza vaccines is controversial. The 2009 pandemic provided an opportunity to investigate whether pre-existing cross-reactive immunity affected susceptibility to infection. METHODS: In 2009, we banked sera and peripheral blood mononuclear cells (PBMC) from blood donors, then monitored them for pandemic influenza infection (pH1N1) by polymerase chain reaction or seroconversion. Antibodies to hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix 2 (M2), and HA-pseudotypes were measured in sera. T-cell inteferon-γ enzyme-linked immunospot responses were measured in PBMC. RESULTS: There were 13 infections in 117 evaluable donors. Pre-existing T-cell reactivity to pH1N1 was substantial (of 153 donors tested, 146 had >100 spot-forming cells/106 cells). Antibodies reactive with pH1N1 were common: anti-NP (all donors) and anti-M2 (44% of donors). Pseudotype-neutralizing antibodies to H1 were detected, but not to highly conserved HA epitopes. Unexpectedly, donors with symptomatic pH1N1 infection had sharp rises in HA pseudotype-neutralizing antibodies, not only pH1N1 but also against multiple seasonal H1s. In addition, an exploratory study of a T-cell marker (response to NP418-426) identified probable infection missed by standard criteria. CONCLUSIONS: Although the number of infections was inadequate for conclusions about mechanisms of protection, this study documents the wide variety of pre-existing, cross-reactive, humoral and cellular immune responses to pandemic influenza virus antigens in humans. These responses can be compared with results of other studies and explored in universal influenza vaccine studies.

Whole-Genome Sequence for Methicillin-Resistant Staphylococcus aureus Strain ATCC BAA-1680.

We report here the whole-genome sequence of the USA300 strain of methicillin-resistant Staphylococcus aureus (MRSA), designated ATCC BAA-1680, and commonly referred to as community-associated MRSA (CA-MRSA). This clinical MRSA isolate is commercially available from the American Type Culture Collection (ATCC) and is widely utilized as a control strain for research applications and clinical diagnosis. The isolate was propagated in ATCC medium 18, tryptic soy agar, and has been utilized as a model S. aureus strain in several studies, including MRSA genetic analysis after irradiation with 470-nm blue light.

Disproportional effects in populations of concern for pandemic influenza: insights from seasonal epidemics in Wisconsin, 1967-2004.

BACKGROUND: Influenza infections pose a serious burden of illness in the United States. We explored age, influenza strains, and seasonal epidemic curves in relation to influenza-associated mortality. METHODS: The state of Wisconsin death records for the years 1967-2004 were analyzed for three distinct populations: children, general population, and elderly. Yearly parameters of duration, intensity, and peak timing were obtained from Annual Harmonic Regression coefficients. RESULTS: Overall, elderly had the highest rate and intensity of influenza mortality. The children and infant subpopulations showed an earlier and wider range in duration of peak timing than elderly. During A/Hong Kong/1/68 pandemic years, the elderly subpopulation showed no change in mortality rates while a sharp increase was observed for the children and infant subpopulations. In epidemic years such as 1966-1969, children and infants showed a dramatic decrease in the severity of influenza outbreaks over time. The elderly had increased baseline mortality in years (1986-1987) where predominant strain was characterized as A/Singapore/6/86. CONCLUSIONS: Our findings indicate that the younger populations may have benefited from the lack of a major shift in viral strains for a number of decades. Furthermore, we demonstrate considerable heterogeneity in the spread of seasonal influenza across age categories, with implications both for the modeling of influenza seasonality, risk assessment, and effective distribution and timing of vaccine and prophylactic interventions.

Identification of human enteric pathogens in gull feces at Southwestern Lake Michigan bathing beaches.

Ring-billed (Larus delawarensis Ord, 1815) and herring (Larus argentatus Pontoppidan, 1763) gulls are predominant species of shorebirds in coastal areas. Gulls contribute to the fecal indicator burden in beach sands, which, once transported to bathing waters, may result in water quality failures. The importance of these contamination sources must not be overlooked when considering the impact of poor bathing water quality on human health. This study examined the occurrence of human enteric pathogens in gull populations at Racine, Wisconsin. For 12 weeks in 2004 and 2005, and 7 weeks in 2006, 724 gull fecal samples were examined for pathogen occurrence on traditional selective media (BBL CHROMagar-Salmonella, Remel Campy-BAP, 7% horse blood agar) or through the use of novel isolation techniques (Campylobacter, EC FP5-funded CAMPYCHECK Project), and confirmed using polymerase chain reaction (PCR) for pathogens commonly harbored in gulls. An additional 226 gull fecal samples, collected in the same 12-week period in 2004, from a beach in Milwaukee, Wisconsin, were evaluated with standard microbiological methods and PCR. Five isolates of Salmonella (0.7%), 162 (22.7%) isolates of Campylobacter, 3 isolates of Aeromonas hydrophila group 2 (0.4%), and 28 isolates of Plesiomonas shigelloides (3.9%) were noted from the Racine beach. No occurrences of Salmonella and 3 isolates of Campylobacter (0.4%) were found at the Milwaukee beach. A subset of the 2004 samples was also examined for Giardia and Cryptosporidium and was found to be negative. Information as to the occurrence of human pathogens in beach ecosystems is essential to design further studies assessing human health risk and to determine the parameters influencing the fate and transport of pathogens in the nearshore environment.