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Microscopic diagnosis of malaria using Giemsa-stained blood smears is the standard of care in resource-limited settings. These smears represent a potential source of DNA for PCR testing to confirm Plasmodium infections or for epidemiological studies of archived samples. Therefore, we assessed the use of DNA extracts from stained blood smears for the detection of Plasmodium species using real-time PCR. We extracted DNA from archived blood smears and corresponding red blood cell pellets collected from asymptomatic children in southwestern Uganda in 2010. We then performed real-time PCR followed by high-resolution melting (HRM) to identify Plasmodium species, and we compared our results to those of microscopy. We analyzed a total of 367 blood smears and corresponding red blood cell pellets, including 185 smears (50.4%) that were positive by microscopy. Compared to microscopy, PCR-HRM analysis of smear DNA had a sensitivity of 93.0% (95% confidence interval [CI], 88.2 to 96.2%) and a specificity of 96.7% (95% CI, 93.0 to 98.8%), and PCR-HRM analysis of pellet DNA had a sensitivity of 100.0% (95% CI, 98.0 to 100.0%) and a specificity of 94.0% (95% CI, 89.4 to 96.9%). Identification of positive PCR-HRM results to the species level revealed Plasmodium falciparum (92.0%), Plasmodium ovale (5.6%), and Plasmodium malariae (2.4%). PCR-HRM analysis of DNA extracts from Giemsa-stained thick blood smears or corresponding blood pellets had high sensitivity and specificity for malaria diagnosis, compared to microscopy. Therefore, blood smears can provide an adequate source of DNA for confirmation of Plasmodium species infections and can be used for retrospective genetic studies., (Copyright © 2017 American Society for Microbiology.)

We report a case of hemolytic uremic syndrome in a 69-year-old woman due to Shiga toxin-producing Escherichia coli, possibly serotype O111, to illustrate the potentially deleterious implications of a Campylobacter enzyme immunoassay (EIA) result and the increasing importance of molecular testing when conventional methods are limited.

A C-to-T transition mutation in the neuraminidase gene from seasonal A/H1N1 causes a His-to-Tyr mutation at amino acid position 275 (H274Y, universal N2 numbering), conferring resistance against oseltamivir (Tamiflu). This mutation was first detected in clinical samples in Europe during the 2007-2008 influenza season. Viruses with this mutation reached a prevalence of ∼11% by the end of the season in North American isolates tested by the CDC. We developed a highly sensitive and specific quantitative real-time reverse transcriptase PCR assay to detect the H274Y mutation. This assay utilizes a 5'-methyl-isocytosine (isoC) residue and fluorescent reporters on genotype-specific primers. During PCR, a quencher coupled to isoguanine (isoG) is site-specifically incorporated complementary to the isoC/dye, resulting in loss of fluorescence. Optimization of primers and assay conditions produced a limit of detection of 100 gene copies per reaction for both wild-type and H274Y genotypes. In samples with mixed populations, it can reliably detect as little as a 1% wild-type or 0.1% H274Y component. This high sensitivity makes the assay usable on samples with viral loads too low for dideoxy or pyrosequencing analysis. Additionally, the assay distinguishes seasonal A/H1N1 from A/H3N2, influenza B, or 2009 pandemic A/H1N1, making it useful for influenza virus subtyping as well as for drug resistance detection. We probed seasonal A/H1N1 samples from the 2005-2006, 2006-2007, and 2007-2008 influenza seasons. Data from the new assay closely matched available drug resistance genotype data previously determined by dideoxy sequencing. The H274Y mutation was only found in samples from the 2007-2008 season.