Evaluation of a laboratory-developed multiplex real-time PCR assay for diagnosis of syphilis, herpes and chancroid genital ulcers in four public health laboratories in the USA.

OBJECTIVE: To evaluate the field performance of a multiplex PCR (M-PCR) assay for detection of herpes simplex virus (HSV)-1 and HSV-2, Treponema pallidum (T. pallidum) and Haemophilus ducreyi (H. ducreyi) in genital ulcer disease (GUD) specimens. METHODS: GUD M-PCR was performed on 186 remnant specimens, previously collected for HSV testing, by four public health laboratories (PHLs) and the Laboratory Reference and Research Branch (LRRB) at the Centers for Disease Control and Prevention. The results from the PHLs were compared with those of LRRB, which served as the reference testing method, and percentage agreement was calculated. RESULTS: HSV was detected in 31 of 52 (59.6%), 20 of 40 (50%), 43 of 44 (97.7%) and 19 of 50 (38.0%) specimens from PHL1, PHL2, PHL3 and PHL4, respectively. There were seven discrepant results for HSV, and the overall percent agreement between the PHLs and the LRRB was 94%-100%, with a kappa value of 0.922, which demonstrates high agreement. T. pallidum was identified in 7 of 51 (13.7%) specimens from PHL1 with 94.1% agreement and in 2 of 40 (5.0%) specimens from PHL2 with 100% agreement. The LRRB identified three additional T. pallidum-positive specimens from PHL1. The kappa value (0.849) for T. pallidum testing suggests good agreement. Consistent with the LRRB results, no T. pallidum was detected in specimens from PHL3 and PHL4, and H. ducreyi was not detected at any of the study sites. CONCLUSIONS: The GUD M-PCR assay performed well in four independent PHLs and 12 suspected syphilis cases were identified in this study. The M-PCR assay could provide improved diagnostic options for GUD infections in state and local PHLs.

Innate Immune Activation by cGMP-AMP Nanoparticles Leads to Potent and Long-Acting Antiretroviral Response against HIV-1.

HIV-1 evades immune detection by the cGAS-STING cytosolic DNA-sensing pathway during acute infection. STING is a critical mediator of type I IFN production, and STING agonists such as cGMP-AMP (cGAMP) and other cyclic dinucleotides elicit potent immune and antitumor response. In this article, we show that administration of cGAMP, delivered by an ultra-pH-sensitive nanoparticle (NP; PC7A), in human PBMCs induces potent and long-acting antiretroviral response against several laboratory-adapted and clinical HIV-1 isolates. cGAMP-PC7A NP requires endocytosis for intracellular delivery and immune signaling activation. cGAMP-PC7A NP-induced protection is mediated through type I IFN signaling and requires monocytes in PBMCs. cGAMP-PC7A NPs also inhibit HIV-1 replication in HIV+ patient PBMCs after ex vivo reactivation. Because pattern recognition receptor agonists continue to show more clinical benefits than the traditional IFN therapy, our data present important evidence for potentially developing cGAMP or other STING agonists as a new class of immune-stimulating long-acting antiretroviral agents.

Co-circulation dynamics and persistence of newly introduced clades of 2012 outbreak associated West Nile Virus in Texas, 2012-2015.

The second largest outbreak of West Nile encephalitis and West Nile fever ever recorded occurred in the United States (U.S) in the summer of 2012. The outbreak was related to the widespread circulation of closely related clades, or groups, of West Nile virus (WNV) into multiple states where they were not previously found. Whether the invading 2012 strains were able to circulate and overwinter in states with their own endemic population of WNV is unknown and the effect of viral genetics on adaptation and persistence in a new ecological niche is unclear. In this study, we sequenced 70 mosquito isolates from multiple counties throughout Texas in 2012-2015. We identified isolates representative of previously described 2012 WNV groups (Groups 8-10) and discovered a novel group which we called Group 11. Although we identified isolates representative of WNV endemic (2/70) to Texas, most isolates (68/70) were related to the invading 2012 strains, and of these Group 10 (45/68) was predominant. We also observed differences among the 2012 WNV groups correlating to their genotype. Group 10 WNV in Texas, which carry two putative positively selected variants, had limited introductions into Texas, wide circulation, and strong evidence of continued persistence perhaps indicative of overwintering. In contrast, Groups 8 and 11, without positively selected variants, had multiple introductions into Texas, limited circulation, and limited persistence. Lastly, we identified a potential transmission source in New York for incoming Group 8 WNV into Texas. Altogether our study suggests that mutations in the WNV genome may influence the range and dynamics of WNV circulation, and the ability of different strains to persist in new ecological niches.

Genomic Heterogeneity of Methicillin Resistant Staphylococcus aureus Associated with Variation in Severity of Illness among Children with Acute Hematogenous Osteomyelitis.

INTRODUCTION: The association between severity of illness of children with osteomyelitis caused by Methicillin-resistant Staphylococcus aureus (MRSA) and genomic variation of the causative organism has not been previously investigated. The purpose of this study is to assess genomic heterogeneity among MRSA isolates from children with osteomyelitis who have diverse severity of illness. MATERIALS AND METHODS: Children with osteomyelitis were prospectively studied between 2010 and 2011. Severity of illness of the affected children was determined from clinical and laboratory parameters. MRSA isolates were analyzed with next generation sequencing (NGS) and optical mapping. Sequence data was used for multi-locus sequence typing (MLST), phylogenetic analysis by maximum likelihood (PAML), and identification of virulence genes and single nucleotide polymorphisms (SNP) relative to reference strains. RESULTS: The twelve children studied demonstrated severity of illness scores ranging from 0 (mild) to 9 (severe). All isolates were USA300, ST 8, SCC mec IVa MRSA by MLST. The isolates differed from reference strains by 2 insertions (40 Kb each) and 2 deletions (10 and 25 Kb) but had no rearrangements or copy number variations. There was a higher occurrence of virulence genes among study isolates when compared to the reference strains (p = 0.0124). There were an average of 11 nonsynonymous SNPs per strain. PAML demonstrated heterogeneity of study isolates from each other and from the reference strains. DISCUSSION: Genomic heterogeneity exists among MRSA isolates causing osteomyelitis among children in a single community. These variations may play a role in the pathogenesis of variation in clinical severity among these children.

Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses.

Retroviruses, including HIV, can activate innate immune responses, but the host sensors for retroviruses are largely unknown. Here we show that HIV infection activates cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) to produce cGAMP, which binds to and activates the adaptor protein STING to induce type I interferons and other cytokines. Inhibitors of HIV reverse transcriptase, but not integrase, abrogated interferon-ß induction by the virus, suggesting that the reverse-transcribed HIV DNA triggers the innate immune response. Knockout or knockdown of cGAS in mouse or human cell lines blocked cytokine induction by HIV, murine leukemia virus, and simian immunodeficiency virus. These results indicate that cGAS is an innate immune sensor of HIV and other retroviruses.