J. Alfredo Bonilla

Presence of Pathogens and Indicator Microbes at a Non-Point Source Subtropical Recreational Marine Beach

ABSTRACT Swimming in ocean water, including ocean water at beaches not impacted by known point sources of pollution, is an increasing health concern. This study was an initial evaluation of the presence of indicator microbes and pathogens and the association among the indicator microbes, pathogens, and environmental conditions at a subtropical, recreational marine beach in south Florida impacted by non-point sources of pollution. Twelve water and eight sand samples were collected during four sampling events at high or low tide under elevated or reduced solar insolation conditions. The analyses performed included analyses of fecal indicator bacteria (FIB) (fecal coliforms, Escherichia coli, enterococci, and Clostridium perfringens), human-associated microbial source tracking (MST) markers (human polyomaviruses [HPyVs] and Enterococcus faecium esp gene), and pathogens (Vibrio vulnificus, Staphylococcus aureus, enterovirus, norovirus, hepatitis A virus, Cryptosporidium spp., and Giardia spp.). The enterococcus concentrations in water and sand determined by quantitative PCR were greater than the concentrations determined by membrane filtration measurement. The FIB concentrations in water were below the recreational water quality standards for three of the four sampling events, when pathogens and MST markers were also generally undetectable. The FIB levels exceeded regulatory guidelines during one event, and this was accompanied by detection of HPyVs and pathogens, including detection of the autochthonous bacterium V. vulnificus in sand and water, detection of the allochthonous protozoans Giardia spp. in water, and detection of Cryptosporidium spp. in sand samples. The elevated microbial levels were detected at high tide and under low-solar-insolation conditions. Additional sampling should be conducted to further explore the relationships between tidal and solar insolation conditions and between indicator microbes and pathogens in subtropical recreational marine waters impacted by non-point source pollution.

Critical roles for the digestive vacuole plasmepsins of Plasmodium falciparum in vacuolar function

Knockout mutants of Plasmodium falciparum lacking pfpm1, pfpm2 and pfhap (triple‐PM KO), and mutants lacking all four digestive vacuole (DV) plasmepsins (pfpm4, pfpm1, pfpm2 and pfhap; quadruple‐PM KO), were prepared by double cross‐over integration effecting chromosomal deletions of up to 14.6 kb. The triple‐PM KO was similar to the parental line (3D7) in growth rate, morphology and sensitivity to proteinase inhibitors. The quadruple‐PM KO showed a significantly slower rate of growth in standard medium, which manifested as delayed schizont maturation accompanied by reduced formation of haemozoin. In amino acid‐limited medium, the reduction in growth rate of the quadruple‐PM KO was pronounced. The sensitivity of both the triple‐ and quadruple‐PM KOs to six different HIV aspartic proteinase inhibitors was comparable to that of 3D7, thus establishing that the DV plasmepsins were not the primary targets of the antimalarial activity of these clinically important compounds. Electron microscopic analysis revealed the presence of multilamellar bodies resembling ceroid in the DV of the quadruple‐PM KO, and intermediates of the autophagic pathway accumulated as determined by Western blot analysis. Thus, the DV plasmepsins, although not essential, contribute significantly to the fitness of the parasite and are required for efficient degradation of endosomal vesicles delivered to the DV.

Plasmepsin 4-Deficient Plasmodium berghei Are Virulence Attenuated and Induce Protective Immunity against Experimental Malaria

Plasmodium parasites lacking plasmepsin 4 (PM4), an aspartic protease that functions in the lysosomal compartment and contributes to hemoglobin digestion, have only a modest decrease in the asexual blood-stage growth rate; however, PM4 deficiency in the rodent malaria parasite Plasmodium berghei results in significantly less virulence than that for the parental parasite. P. berghei Deltapm4 parasites failed to induce experimental cerebral malaria (ECM) in ECM-susceptible mice, and ECM-resistant mice were able to clear infections. Furthermore, after a single infection, all convalescent mice were protected against subsequent parasite challenge for at least 1 year. Real-time in vivo parasite imaging and splenectomy experiments demonstrated that protective immunity acted through antibody-mediated parasite clearance in the spleen. This work demonstrates, for the first time, that a single Plasmodium gene disruption can generate virulence-attenuated parasites that do not induce cerebral complications and, moreover, are able to stimulate strong protective immunity against subsequent challenge with wild-type parasites. Parasite blood-stage attenuation should help identify protective immune responses against malaria, unravel parasite-derived factors involved in malarial pathologies, such as cerebral malaria, and potentially pave the way for blood-stage whole organism vaccines.

Cryptosporidium muris, a Rodent Pathogen, Recovered from a Human in Perú

Cryptosporidium muris, predominantly a rodent species of Cryptosporidium, is not normally considered a human pathogen. Recently, isolated human infections have been reported from Indonesia, Thailand, France, and Kenya. We report the first case of C. muris in a human in the Western Hemisphere. This species may be an emerging zoonotic pathogen capable of infecting humans.

Multicenter Study To Evaluate the OptiMAL Test for Rapid Diagnosis of Malaria in U.S. Hospitals

ABSTRACT More than 1,000 cases of malaria are diagnosed each year in the United States. Reported numbers, however, may be artificially low because many clinicians fail to consider the diagnosis on presentation, U.S. hospital laboratory technologists have very limited experience in detecting and identifying malaria parasites, and reporting of malaria to state health departments is sporadic in many states. In this study, a rapid malaria diagnostic test, the OptiMAL test (DiaMed; under license from Flow Inc., Portland, Oreg.) was evaluated in six U.S. hospitals and compared with results of microscopy. The OptiMAL test is a 15-min rapid immunochromatographic test that both identifies and differentiates Plasmodium falciparum from non-P. falciparum malaria parasites on the basis of the detection of parasite lactate dehydrogenase in a drop of patient blood. A total of 216 specimens from patients suspected of having malaria were tested. Results indicated that 43 samples (20%) were positive for malaria parasites by microscopy (32 P. falciparum, 11 non-P. falciparum) while 42 (19%) were positive by OptiMAL (31 P. falciparum, 11 non-P. falciparum). The sensitivity of the OptiMAL test was 98%; its specificity was 100%, with positive and negative predictive values of 100 and 99%, respectively. Participating hospital physicians and laboratory directors independently reported that the OptiMAL rapid malaria test was accurate, easy to use, and well accepted by those working in their diagnostic laboratories. The overall conclusion was that integration of the OptiMAL rapid malaria test into the U.S. health care infrastructure would provide an important and easy-to-use tool for the timely diagnosis of malaria.

Daily measures of microbes and human health at a non-point source marine beach

Studies evaluating the relationship between microbes and human health at non-point source beaches are necessary for establishing criteria which would protect public health while minimizing economic burdens. The objective of this study was to evaluate water quality and daily cumulative health effects (gastrointestinal, skin, and respiratory illnesses) for bathers at a non-point source subtropical marine recreational beach in order to better understand the inter-relationships between these factors and hence improve monitoring and pollution prevention techniques. Daily composite samples were collected, during the Oceans and Human Health Beach Exposure Assessment and Characterization Health Epidemiologic Study conducted in Miami (Florida, USA) at a non-point source beach, and analyzed for several pathogens, microbial source tracking markers, indicator microbes, and environmental parameters. Analysis demonstrated that rainfall and tide were more influential, when compared to other environmental factors and source tracking markers, in determining the presence of both indicator microbes and pathogens. Antecedent rainfall and F+ coliphage detection in water should be further assessed to confirm their possible association with skin and gastrointestinal (GI) illness outcomes, respectively. The results of this research illustrate the potential complexity of beach systems characterized by non-point sources, and how more novel and comprehensive approaches are needed to assess beach water quality for the purpose of protecting bather health.

Identification of Leishmania Proteins Preferentially Released in Infected Cells Using Change Mediated Antigen Technology (CMAT)

Although Leishmania parasites have been shown to modulate their host cells responses to multiple stimuli, there is limited evidence that parasite molecules are released into infected cells. In this study, we present an implementation of the change mediated antigen technology (CMAT) to identify parasite molecules that are preferentially expressed in infected cells. Sera from mice immunized with cell lysates prepared from L. donovani or L. pifanoi-infected macrophages were adsorbed with lysates of axenically grown amastigotes of L. donovani or L. pifanoi, respectively, as well as uninfected macrophages. The sera were then used to screen inducible parasite expression libraries constructed with genomic DNA. Eleven clones from the L. pifanoi and the L. donovani screen were selected to evaluate the characteristics of the molecules identified by this approach. The CMAT screen identified genes whose homologs encode molecules with unknown function as well as genes that had previously been shown to be preferentially expressed in the amastigote form of the parasite. In addition a variant of Tryparedoxin peroxidase that is preferentially expressed within infected cells was identified. Antisera that were then raised to recombinant products of the clones were used to validate that the endogenous molecules are preferentially expressed in infected cells. Evaluation of the distribution of the endogenous molecules in infected cells showed that some of these molecules are secreted into parasitophorous vacuoles (PVs) and that they then traffic out of PVs in vesicles with distinct morphologies. This study is a proof of concept study that the CMAT approach can be applied to identify putative Leishmania parasite effectors molecules that are preferentially expressed in infected cells. In addition we provide evidence that Leishmania molecules traffic out of the PV into the host cell cytosol and nucleus.

Quantification of Protozoa and Viruses from Small Water Volumes.

Large sample volumes are traditionally required for the analysis of waterborne pathogens. The need for large volumes greatly limits the number of samples that can be processed. The goals of this study were to compare extraction and detection procedures for quantifying protozoan parasites and viruses from small volumes of marine water. The intent was to evaluate a logistically simpler method of sample collection and processing that would facilitate direct pathogen measures as part of routine monitoring programs. Samples were collected simultaneously using a bilayer device with protozoa capture by size (top filter) and viruses capture by charge (bottom filter). Protozoan detection technologies utilized for recovery of Cryptosporidium spp. and Giardia spp. were qPCR and the more traditional immunomagnetic separation—IFA-microscopy, while virus (poliovirus) detection was based upon qPCR versus plaque assay. Filters were eluted using reagents consistent with the downstream detection technologies. Results showed higher mean recoveries using traditional detection methods over qPCR for Cryptosporidium (91% vs. 45%) and poliovirus (67% vs. 55%) whereas for Giardia the qPCR-based methods were characterized by higher mean recoveries (41% vs. 28%). Overall mean recoveries are considered high for all detection technologies. Results suggest that simultaneous filtration may be suitable for isolating different classes of pathogens from small marine water volumes. More research is needed to evaluate the suitability of this method for detecting pathogens at low ambient concentration levels.