Rapid IDH1-R132 genotyping panel utilizing locked nucleic acid loop-mediated isothermal amplification.

While the detection of single-nucleotide variants (SNVs) is important for evaluating human health and disease, most genotyping methods require a nucleic acid extraction step and lengthy analytical times. Here, we present a protocol which utilizes the integration of locked nucleic acids (LNAs) into self-annealing loop primers for the allelic discrimination of five isocitrate dehydrogenase 1 R132 (IDH1-R132) variants using loop-mediated isothermal amplification (LAMP). This genotyping panel was initially evaluated using purified synthetic DNA to show proof of specific SNV discrimination. Additional evaluation using glioma tumor lysates with known IDH1-R132 mutational status demonstrated specificity in approximately 35 min without the need for a nucleic acid extraction purification step. This LNA-LAMP-based genotyping assay can detect single base differences in purified nucleic acids or tissue homogenates, including instances where the variant of interest is present in an excess of background wild-type DNA. The pH-based colorimetric indicator of LNA-LAMP facilitates convenient visual interpretation of reactions, and we demonstrate successful translation to an end-point format using absorbance ratio, allowing for an alternative and objective approach for differentiating between positive and negative reactions. Importantly, the LNA-LAMP genotyping panel is highly reproducible, with no false-positive or false-negative results observed.

Rapid extraction-free detection of the R132H isocitrate dehydrogenase mutation in glioma using colorimetric peptide nucleic acid-loop mediated isothermal amplification (CPNA-LAMP).

The R132H isocitrate dehydrogenase one (IDH1) mutation is a prognostic biomarker present in a subset of gliomas and is associated with heightened survival when paired with aggressive surgical resection. In this study, we establish proof-of-principle for rapid colorimetric detection of the IDH1-R132H mutation in tumor samples in under 1 hour without the need for a nucleic acid extraction. Colorimetric peptide nucleic acid loop-mediated isothermal amplification (CPNA-LAMP) utilizes 4 conventional LAMP primers, a blocking PNA probe complementary to the wild-type sequence, and a self-annealing loop primer complementary to the single nucleotide variant to only amplify the DNA sequence containing the mutation. This assay was evaluated using IDH1-WT or IDH1-R132H mutant synthetic DNA, wild-type or IDH1-R132H mutant U87MG cell lysates, and tumor lysates from archived patient samples in which the IDH1 status was previously determined using immunohistochemistry (IHC). Reactions were performed using a hot water bath and visually interpreted as positive by a pink-to-yellow color change. Results were subsequently verified using agarose gel electrophoresis. CPNA-LAMP successfully detected the R132H single nucleotide variant, and results from tumor lysates yielded 100% concordance with IHC results, including instances when the single nucleotide variant was limited to a portion of the tumor. Importantly, when testing the tumor lysates, there were no false positive or false negative results.

Association of bacterial colonization at the time of presentation to a combat support hospital in a combat zone with subsequent 30-day colonization or infection.

U.S. casualties have developed multidrug-resistant (MDR) bacterial infections. A surveillance project to evaluate U.S. military patients for the presence of MDR pathogens from wounding through the first 30 days of care in the military healthcare system (MHS) was performed. U.S. military patients admitted to a single combat support hospital in Iraq during June-July of 2007 had screening swabs obtained for the detection of MDR bacteria and a subsequent retrospective electronic medical records review for presence of colonization or infection in the subsequent 30 days. Screening of 74 U.S. military patients in Iraq found one colonized with methicillin-resistant Staphylococcus aureus. Fifty-six patients of these were screened for Acinetobacter in Germany and one found colonized. Of patients evacuated to the U.S., 9 developed infections. Carefully obtained screening cultures immediately after injury combined with look-back monitoring supports the role of nosocomial transmission. Consistent infection control strategies are needed for the entire MHS.

IEIIS Meeting minireview: Bordetella evolution: lipid A and Toll-like receptor 4.

The evolution of Bordetella pertussis and Bordetella parapertussis from Bordetella bronchiseptica involved changes in host range and pathogenicity. Recent data suggest that the human-adapted Bordetella modified their interaction with host immune systems to effect these changes and that decreased stimulation of Toll-like receptor 4 (TLR4) by lipid A is central to this. We discuss Bordetella lipid A structure and genetics within the context of evolution and host immunity.

Delayed role of tumor necrosis factor- alpha in overcoming the effects of pertussis toxin.

Bordetella pertussis causes whooping cough, an endemic respiratory disease that is increasing in prevalence despite vaccination efforts. Although host immunity is modulated by virulence factors of this pathogen, it is unclear what host factors are required to overcome their effects. Here, we investigate an apparent relationship between the effects of pertussis toxin and tumor necrosis factor (TNF)- alpha . B. pertussis grew efficiently and caused moderate pathology in wild-type mice, whereas TNF- alpha (-/-) mice had higher numbers of bacteria and leukocytes in lungs, experienced more airway resistance, and died of the infection. Interestingly, an isogenic B. pertussis strain lacking pertussis toxin did not induce these effects in TNF- alpha (-/-) mice and behaved similarly in wild-type and TNF- alpha -deficient hosts. Together, these results indicate that TNF- alpha is essential for the host to overcome the effects of pertussis toxin, allowing both control of B. pertussis numbers and regulation of the inflammation induced by infection.

Acinetobacter skin carriage among US army soldiers deployed in Iraq.

Skin carriage of Acinetobacter calcoaceticus-baumannii complex was not detected among a representative sample of 102 US Army soldiers stationed in Iraq. This observation refutes the hypothesis that preinjury skin carriage serves as the reservoir for the Acinetobacter infections seen in US military combat casualties.

The complex mechanism of antibody-mediated clearance of Bordetella from the lungs requires TLR4.

Although the antibacterial effects of Abs are well studied in in vitro systems, the in vivo effects of Abs cannot always be accurately predicted. Complicated cross-talk between different effector functions of Abs and various arms of the immune system can affect their activities in vivo. Using the mouse respiratory pathogen Bordetella bronchiseptica, we examined the mechanisms of Ab-mediated clearance of bacteria from the respiratory tract. Interestingly, although TLR4 was not necessary for protective immunity following infection, it was required for rapid bacterial clearance in mice that were vaccinated or adoptively transferred Abs. TLR4 was important for the rapid recruitment of neutrophils that are necessary for Ab-mediated bacterial clearance via a mechanism that requires both FcgammaR and CR3. These data are consistent with a model in which TLR4-mediated inflammatory responses aid in the recruitment of neutrophils, which phagocytose Ab- and complement-opsonized bacteria via FcgammaRs and CR3. Although pattern recognition receptors are known to be involved in innate immunity and the generation of adaptive immunity, their contributions to specific adaptive immune functions should be considered in ongoing efforts to improve vaccine-induced protective immunity.

Comparative toll-like receptor 4-mediated innate host defense to Bordetella infection.

Bordetella pertussis, B. parapertussis, and B. bronchiseptica are closely related species associated with respiratory disease in humans and other mammals. While B. bronchiseptica has a wide host range, B. pertussis and B. parapertussis evolved separately from a B. bronchiseptica-like progenitor to naturally infect only humans. Despite very different doubling times in vitro, all three establish similar levels of infection in the mouse lung within 72 h. Recent work has revealed separate roles for Toll-like receptor 4 (TLR4) in immunity to B. pertussis and B. bronchiseptica, while no role for TLR4 during B. parapertussis infection has been described. Here we compared the requirement for TLR4 in innate host defense to these organisms using the same mouse infection model. While B. bronchiseptica causes lethal disease in TLR4-deficient mice, B. pertussis and B. parapertussis do not. Correspondingly, TLR4 is critical in limiting B. bronchiseptica but not B. pertussis or B. parapertussis bacterial numbers during the first 72 h. Interestingly, B. bronchiseptica induces a TLR4-dependent cytokine response that is considerably larger than that induced by B. pertussis or B. parapertussis. Analysis of their endotoxins using RAW cells suggests that B. bronchiseptica lipopolysaccharide (LPS) is 10- and 100-fold more stimulatory than B. pertussis or B. parapertussis LPS, respectively. The difference in LPS stimulus is more pronounced when using HEK293 cells expressing human TLR4. Thus, it appears that in adapting to infect humans, B. pertussis and B. parapertussis independently modified their LPS to reduce TLR4-mediated responses, which may compensate for slower growth rates and facilitate host colonization.

Toll-like receptor 4-dependent early elicited tumor necrosis factor alpha expression is critical for innate host defense against Bordetella bronchiseptica.

Toll-like receptor 4 (TLR4) mediates the response to lipopolysaccharide, and its activation induces the expression of a large number of inflammatory genes, many of which are also induced by other pathogen-associated molecular patterns. Interestingly, the subset of genes that are dependent on TLR4 for optimal expression during gram-negative bacterial infection has not been determined. We have previously shown that TLR4-deficient mice rapidly develop acute pneumonia after inoculation with Bordetella bronchiseptica, suggesting that TLR4 is required for expression of early elicited gene products in this model. Microarray analysis with macrophages derived from wild-type and TLR4-deficient mice was used to identify genes whose expression, within 1 h of bacterial exposure, is dependent on TLR4. The results of this investigation suggest that TLR4 is not required for the majority of the transcriptional response to B. bronchiseptica. However, early tumor necrosis factor alpha (TNF-alpha) mRNA expression is primarily dependent on TLR4 and in vitro and in vivo protein levels substantiate this finding. TLR4-deficient mice and TNF-alpha-/- mice are similarly susceptible to infection with relatively low doses of B. bronchiseptica and in vivo neutralization studies indicate that it is the TLR4-dependent early elicited TNF-alpha response that is critical for preventing severe pneumonia and limiting bacterial growth. These results suggest that one critical role for TLR4 is the generation of a robust but transient TNF-alpha response that is critical to innate host defense during acute gram-negative respiratory infection.

Toll-like receptor 4 is critical to innate host defense in a murine model of bordetellosis.

Bordetellae are important respiratory pathogens that cause pertussis (whooping cough) in humans and analogous diseases in domestic and wild animals. Immunity to Bordetella is poorly understood, in particular the early innate immune responses that contribute to inflammation, pathology, and the subsequent generation of adaptive immunity. Using B. bronchiseptica, which naturally infects mice, we show that Toll-like receptor-4 (TLR4) is required for cytokine responses to this pathogen's lipopolysaccharide (LPS) and that TLR4 deficiency results in impaired cytokine responses in vitro and in vivo. TLR4-deficient mice rapidly succumb following inoculation with as few as 1000 organisms, indicating that TLR4 is critical to innate host defense against bordetellosis.

Role of antibodies in immunity to Bordetella infections.

The persistence of Bordetella pertussis and B. parapertussis within vaccinated populations and the reemergence of associated disease highlight the need to better understand protective immunity. The present study examined host immunity to bordetellae and addressed potential concerns about the mouse model by using a comparative approach including the closely related mouse pathogen B. bronchiseptica. As previously observed with B. pertussis, all three organisms persisted throughout the respiratory tracts of B-cell-deficient mice, indicating that B cells are required for bacterial clearance. However, adoptively transferred antibodies rapidly cleared B. bronchiseptica but not human pathogens. These results obtained with the mouse model are consistent with human clinical observations, including the lack of correlation between antibody titers and protection, as well as the limited efficacy of intravenous immunoglobulin treatments against human disease. Together, this evidence suggests that the mouse model accurately reflects substantial differences between immunities to these organisms. Although both B. pertussis and B. parapertussis are more closely related to B. bronchiseptica than they are to each other, they share the ability to resist rapid clearance from the lower respiratory tract by adoptively transferred antibodies, an adaptation that correlates with their emergence as human pathogens that circulate within vaccinated populations.