William Lainhart

Shiga Toxin as a Bacterial Defense against a Eukaryotic Predator, Tetrahymena thermophila

Bacterially derived exotoxins kill eukaryotic cells by inactivating factors and/or pathways that are universally conserved among eukaryotic organisms. The genes that encode these exotoxins are commonly found in bacterial viruses (bacteriophages). In the context of mammals, these toxins cause diseases ranging from cholera to diphtheria to enterohemorrhagic diarrhea. Phage-carried exotoxin genes are widespread in the environment and are found with unexpectedly high frequency in regions lacking the presumed mammalian targets, suggesting that mammals are not the primary targets of these exotoxins. We suggest that such exotoxins may have evolved for the purpose of bacterial antipredator defense. We show here that Tetrahymena thermophila, a bacterivorous predator, is killed when cocultured with bacteria bearing a Shiga toxin (Stx)-encoding temperate bacteriophage. In cocultures with Tetrahymena, the Stx-encoding bacteria display a growth advantage over those that do not produce Stx. Tetrahymena is also killed by purified Stx. Disruption of the gene encoding the StxB subunit or addition of an excess of the nontoxic StxB subunit substantially reduced Stx holotoxin toxicity, suggesting that this subunit mediates intake and/or trafficking of Stx by Tetrahymena. Bacterially mediated Tetrahymena killing was blocked by mutations that prevented the bacterial SOS response (recA mutations) or by enzymes that breakdown H(2)O(2) (catalase), suggesting that the production of H(2)O(2) by Tetrahymena signals its presence to the bacteria, leading to bacteriophage induction and production of Stx.

Implications for changes in Anopheles darlingi biting behaviour in three communities in the peri-Iquitos region of Amazonian Peru

AbstractBackgroundMalaria transmission in the peri-Iquitos region of Amazonian Peru has been designated as seasonal and hypo-endemic with recently described hyper-endemicn hotspots. Despite relatively recent distribution of long-lasting insecticidal bed nets (LLINs), malaria in Amazonian Peru persists and increased substantially in 2014 compared to previous years. Anopheles darlingi, identified as the main malaria vector, is known for its variable behaviour depending on locality and environment.MethodsTo evaluate vector biology metrics in relation to seasonality and malaria transmission, mosquito collections were carried out in three localities in the peri-Iquitos region, Loreto, Peru in 2011–2012. Human landing catch (HLC) collection method, Shannon (SHA) and CDC trap types were compared for effectiveness in a neotropical setting. Abundance, human biting rate and entomological inoculation rate (EIR) were measured to provide an updated view of transmission patterns post-LLIN distribution.ResultsHLC collected significantly more anopheline mosquitoes than SHA and CDC light traps. Anopheles darlingi was the most prevalent species in all three villages (84% overall). Biting patterns varied depending on trap type, season and village. EIR varied temporally (monthly) and spatially and the highest (2.52) occurred during the 2012 malaria outbreak in Cahuide. Unexpectedly there was a high infection rate (1.47 and 1.75) outside the normal malaria transmission season, coincident with a second local outbreak in Cahuide. The first identification of Anopheles dunhami and Anopheles oswaldoi C in Peru, using molecular markers, is also reported in this study.ConclusionThese data underscore the importance of HLC as the most meaningful collection method for measuring vector biology indices in this region. The highest monthly EIR provides additional evidence of seasonal transmission in riverine localities correlated with high river levels, and An. darlingi as the only contributor to transmission. The trend of an increase in outdoor-biting together with early-evening infected mosquitoes may undermine the effectiveness of LLINs as a primary malaria intervention.

Evidence for temporal population replacement and the signature of ecological adaptation in a major Neotropical malaria vector in Amazonian Peru

BackgroundThe major Neotropical malaria vector, Anopheles darlingi, was reintroduced into the Iquitos, Loreto, Peru area during the early 1990s, where it displaced other anophelines and caused a major malaria epidemic. Since then, case numbers in Loreto have fluctuated, but annual increases have been reported since 2012.MethodsThe population genetic structure of An. darlingi sampled before and after the introduction of long-lasting insecticidal nets (LLINs) was investigated to test the hypothesis of temporal population change (2006 vs. 2012). Current samples of An. darlingi were used to test the hypothesis of ecological adaptation to human modified (highway) compared with wild (riverine) habitat, linked to forest cover. In total, 693 An. darlingi from nine localities in Loreto, Peru area were genotyped using 13 microsatellite loci. To test the hypothesis of habitat differentiation in An. darlingi biting time patterns, HBR and EIR, four collections of An. darlingi from five localities (two riverine and three highway) were analysed.ResultsAnalyses of microsatellite loci from seven (2006) and nine settlements (2012–2014) in the Iquitos area detected two distinctive populations with little overlap, although it is unclear whether this population replacement event is associated with LLIN distribution or climate. Within the 2012–2014 population two admixed subpopulations, A and B, were differentiated by habitat, with B significantly overrepresented in highway, and both in near-equal proportions in riverine. Both subpopulations had a signature of expansion and there was moderate genetic differentiation between them. Habitat and forest cover level had significant effects on HBR, such that Plasmodium transmission risk, as measured by EIR, in peridomestic riverine settlements was threefold higher than in peridomestic highway settlements. HBR was directly associated with available host biomass rather than forest cover.ConclusionsA population replacement event occurred between 2006 and 2012–2014, concurrently with LLIN distribution and a moderate El Niño event, and prior to an increase in malaria incidence. The likely drivers of this replacement cannot be determined with current data. The present-day An. darlingi population is composed of two highly admixed subpopulations, which appear to be in an early stage of differentiation, triggered by anthropogenic alterations to local habitat.

A sensitive, specific and reproducible real-time polymerase chain reaction method for detection of Plasmodium vivax and Plasmodium falciparum infection in field-collected anophelines.

We describe a simple method for detection of Plasmodium vivax and Plasmodium falciparum infection in anophelines using a triplex TaqMan real-time polymerase chain reaction (PCR) assay (18S rRNA). We tested the assay on Anopheles darlingi and Anopheles stephensi colony mosquitoes fed with Plasmodium-infected blood meals and in duplicate on field collected An. darlingi. We compared the real-time PCR results of colony-infected and field collected An. darlingi, separately, to a conventional PCR method. We determined that a cytochrome b-PCR method was only 3.33% as sensitive and 93.38% as specific as our real-time PCR assay with field-collected samples. We demonstrate that this assay is sensitive, specific and reproducible.

The association of depressive symptoms and pulmonary function in healthy adults.

Objective Chronic lung disease is exacerbated by comorbid psychiatric issues and treatment of depression may improve disease symptoms. We sought to add to the literature as to whether depression is associated with pulmonary function in healthy adults. Methods In 2551 healthy adults from New York State, we studied the association of depression via the Center for Epidemiologic Studies Depression scale (CES-D) scale score and forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) using general linear models and a cross-sectional design. Results We identified statistically significant inverse trends in FEV1, FVC, FEV1%, and FVC% by CES-D category, especially in ever-smokers and men. When adjusted for covariates, the difference in FEV1 and FEV1% for smokers with more than 18.5 lifetime pack-years from CES-D scores 0 to 3 to 16 or more (depressed) is approximately 0.25 l and 5.0% (adjusted p values for trend are <.001 and .019, respectively). In men, we also observed statistically significant inverse trends in pulmonary function with increasing CES-D. Conclusions We identified an inverse association of depressive symptoms and pulmonary function in healthy adults, especially in men and individuals with a heavy smoking history. Further studies of these associations are essential for the development and tailoring of interventions for the prevention and treatment of chronic lung disease.

Identification of Nocardia, Streptomyces, and Tsukamurella using MALDI-TOF MS with the Bruker Biotyper

Nocardia species are the most commonly isolated aerobic actinomycetes from human clinical specimens. Our objective was to assess the identification of clinically relevant actinomycetes using the Bruker Biotyper MALDI-TOF system, including comparison of extraction methods, Biotyper library versions, score cutoffs, and media. Banked Streptomyces (n=10), Tsukamurella (n=2), and Nocardia isolates (n=60) were cultured and extracted using three methods: mycobacterial extraction, ethanol formic acid extraction, or direct on-target extraction. Following MALDI-TOF analysis, spectra were analyzed using versions 5 and 6 of the BDAL Biotyper library. Optimal species-level identifications for Nocardia were achieved using BDAL v6 at a score cutoff of ≥1.8 after direct extraction (49/60, 82%). Overall, the Biotyper platform with BDAL v6 accurately identified 12/16 species of Nocardia, demonstrating the utility of MALDI-TOF for identification of clinically relevant actinomycetes without the need for supplementation of the database.

Epidemic and Non-Epidemic Hot Spots of Malaria Transmission Occur in Indigenous Comarcas of Panama

From 2002–2005, Panama experienced a malaria epidemic that has been associated with El Niño Southern Oscillation weather patterns, decreased funding for malaria control, and landscape modification. Case numbers quickly decreased afterward, and Panama is now in the pre-elimination stage of malaria eradication. To achieve this new goal, the characterization of epidemiological risk factors, foci of transmission, and important anopheline vectors is needed. Of the 24,681 reported cases in these analyses (2000–2014), ~62% occurred in epidemic years and ~44% in indigenous comarcas (5.9% of Panama’s population). Sub-analyses comparing overall numbers of cases in epidemic and non-epidemic years identified females, comarcas and some 5-year age categories as those disproportionately affected by malaria during epidemic years. Annual parasites indices (APIs; number of cases per 1,000 persons) for Plasmodium vivax were higher in comarcas compared to provinces for all study years, though P. falciparum APIs were only higher in comarcas during epidemic years. Interestingly, two comarcas report increasing numbers of cases annually, despite national annual decreases. Inclusion of these comarcas within identified foci of malaria transmission confirmed their roles in continued transmission. Comparison of species distribution models for two important anophelines with Plasmodium case distribution suggest An. albimanus is the primary malaria vector in Panama, confirmed by identification of nine P. vivax-infected specimen pools. Future malaria eradication strategies in Panama should focus on indigenous comarcas and include both active surveillance for cases and comprehensive anopheline vector surveys.

Changes in Genetic Diversity from Field to Laboratory During Colonization of Anopheles darlingi Root (Diptera: Culicidae).

The process of colonizing any arthropod species, including vector mosquitoes, necessarily involves adaptation to laboratory conditions. The adaptation and evolution of colonized mosquito populations needs consideration when such colonies are used as representative models for pathogen transmission dynamics. A recently established colony of Anopheles darlingi, the primary malaria vector in Amazonian South America, was tested for genetic diversity and bottleneck after 21 generations, using microsatellites. As expected, laboratory An. darlingi had fewer private and rare alleles (frequency < 0.05), decreased observed heterozygosity, and more common alleles (frequency > 0.50), but no significant evidence of a bottleneck, decrease in total alleles, or increase in inbreeding compared with field specimens (founder population). Low-moderate differentiation between field and laboratory populations was detected. With these findings, and the documented inherent differences between laboratory and field populations, results of pathogen transmission studies using this An. darlingi colony need to be interpreted cautiously.

First published record of urban malaria in Puerto Gaitan, Meta, Colombia

Patterns of malaria cases were compared between the department of Meta and the municipality of Puerto Gaitán, Colombia, to examine temporal change in malaria from 2005-2010. During this time frame in Meta the mean ratio was 2.53; in contrast, in Puerto Gaitán it was 1.41, meaning that a surprisingly high proportion of Plasmodium falciparum cases were reported from this municipality. A detailed analysis of data from Puerto Gaitán for 2009 and 2010 detected a significant difference (χ2, p < 0.001) in the distribution of plasmodia, with Plasmodium vivax more prevalent in 2009 and P. falciparum in 2010. Males had the highest number of cases but there was no difference in the distribution of cases between sexes and years. In both years, for both sexes, people 16-40 accounted for the majority of cases (58.9% in 2009; 60.4% in 2010). There were significant differences in the distribution of both P. vivax (χ2, p < 0.01) and P. falciparum cases (χ2, p < 0.05) by geographic setting (urban vs. non-urban) between years. Urban cases of both P. vivax and P. falciparum are recorded in this study for the first time in Puerto Gaitán, possibly the result of area wide displacement and migration due to armed conflict.

Pitfalls associated with the use of molecular diagnostic panels in the diagnosis of cryptococcal meningitis

Abstract We report the case of a kidney transplantation patient on chronic immunosuppressive therapy presenting with subacute meningitis. The final diagnosis of cryptococcal meningitis was delayed due to 2 false-negative cryptococcal results on a molecular diagnostic panel. Caution with such platforms in suspected cryptococcal meningitis is needed.