Prevalence and Correlates of Cryptosporidium Infections in Kenyan Children With Diarrhea and Their Primary Caregivers.

BACKGROUND: Cryptosporidium is a leading cause of diarrhea in Sub-Saharan Africa and is associated with substantial morbidity and mortality in young children. METHODS: We analyzed data from children aged 6-71 months presenting to 2 public hospitals in Western Kenya with acute diarrhea and their primary caregivers, including detection of Cryptosporidium by quantitative polymerase chain reaction (PCR) and immunoassay analysis in stool samples from both children and their caregivers. Associations between potential transmission sources and child/caregiver Cryptosporidium infection were evaluated using prevalence ratios (PRs). Secondary analyses evaluated host and clinical risk factors of child/caregiver Cryptosporidium infection. RESULTS: Among 243 child-caregiver pairs enrolled, 77 children (32%) and 57 caregivers (23%) had Cryptosporidium identified by either immunoassay or PCR. Twenty-six of the 243 child-caregiver pairs (11%) had concordant detection of Cryptosporidium. Cryptosporidium infection in children was associated with detection of Cryptosporidium in caregivers (adjusted PR [aPR], 1.8; 95% CI, 1.2 to 2.6; P = .002) and unprotected water source (aPR, 2.0; 95% CI, 1.3 to 3.2; P = .003). Risk factors for Cryptosporidium detection in caregivers included child Cryptosporidium infection (aPR, 2.0; 95% CI, 1.3 to 3.0; P = .002) as well as cow (aPR, 3.1; 95% CI, 1.4 to 7.0; P = .02) and other livestock ownership (aPR, 2.6; 95% CI, 1.1 to 6.3; P = .03) vs no livestock ownership. Recent diarrhea in caregivers and children was independently associated with child and caregiver Cryptosporidium infections, respectively. CONCLUSIONS: Our results are consistent with the hypothesis that Cryptosporidium transmission can occur directly between child-caregiver dyads as well as through other pathways involving water and livestock. Additional research into caregivers as a source of childhood Cryptosporidium infection is warranted.

Biochemical Screening of Five Protein Kinases from Plasmodium falciparum against 14,000 Cell-Active Compounds.

In 2010 the identities of thousands of anti-Plasmodium compounds were released publicly to facilitate malaria drug development. Understanding these compounds' mechanisms of action--i.e., the specific molecular targets by which they kill the parasite--would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children's Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC50 < 1 µM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple Plasmodium kinase targets without harming human cells is challenging but feasible.

The gatekeeper residue and beyond: homologous calcium-dependent protein kinases as drug development targets for veterinarian Apicomplexa parasites.

Specific roles of individual CDPKs vary, but in general they mediate essential biological functions necessary for parasite survival. A comparative analysis of the structure-activity relationships (SAR) of Neospora caninum, Eimeria tenella and Babesia bovis calcium-dependent protein kinases (CDPKs) together with those of Plasmodium falciparum, Cryptosporidium parvum and Toxoplasma gondii was performed by screening against 333 bumped kinase inhibitors (BKIs). Structural modelling and experimental data revealed that residues other than the gatekeeper influence compound-protein interactions resulting in distinct sensitivity profiles. We subsequently defined potential amino-acid structural influences within the ATP-binding cavity for each orthologue necessary for consideration in the development of broad-spectrum apicomplexan CDPK inhibitors. Although the BKI library was developed for specific inhibition of glycine gatekeeper CDPKs combined with low inhibition of threonine gatekeeper human SRC kinase, some library compounds exhibit activity against serine- or threonine-containing CDPKs. Divergent BKI sensitivity of CDPK homologues could be explained on the basis of differences in the size and orientation of the hydrophobic pocket and specific variation at other amino-acid positions within the ATP-binding cavity. In particular, BbCDPK4 and PfCDPK1 are sensitive to a larger fraction of compounds than EtCDPK1 despite the presence of a threonine gatekeeper in all three CDPKs.

Paratransgenic control of vector borne diseases.

Conventional methodologies to control vector borne diseases with chemical pesticides are often associated with environmental toxicity, adverse effects on human health and the emergence of insect resistance. In the paratransgenic strategy, symbiotic or commensal microbes of host insects are transformed to express gene products that interfere with pathogen transmission. These genetically altered microbes are re-introduced back to the insect where expression of the engineered molecules decreases the host's ability to transmit the pathogen. We have successfully utilized this strategy to reduce carriage rates of Trypanosoma cruzi, the causative agent of Chagas disease, in the triatomine bug, Rhodnius prolixus, and are currently developing this methodology to control the transmission of Leishmania donovani by the sand fly Phlebotomus argentipes. Several effector molecules, including antimicrobial peptides and highly specific single chain antibodies, are currently being explored for their anti-parasite activities in these two systems. In preparation for eventual field use, we are actively engaged in risk assessment studies addressing the issue of horizontal gene transfer from the modified bacteria to environmental microbes.

The paratransgenic sand fly: a platform for control of Leishmania transmission.

BACKGROUND: Leishmania donovani is transmitted by the bite of the sand fly, Phlebotomus argentipes. This parasite is the agent of visceral leishmaniasis (VL), an endemic disease in Bihar, India, where prevention has relied mainly on DDT spraying. Pesticide resistance in sand fly populations, environmental toxicity, and limited resources confound this approach. A novel paratransgenic strategy aimed at control of vectorial transmission of L. donovani is presented using Bacillus subtilis, a commensal bacterium isolated from the sand fly gut. In this work, B. subtilis expressing Green Fluorescent Protein (GFP) was added to sterilized larval chow. Control pots contained larval chow spiked either with untransformed B. subtilis or phosphate-buffered saline. Fourth-instar P. argentipes larvae were transferred into the media and allowed to mature. The number of bacterial colony forming units, relative abundance and the mean microbial load were determined per developmental stage. RESULTS: Addition of B. subtilis to larval chow did not affect sand fly emergence rates. B. cereus and Lys fusiformis were identified at each developmental stage, revealing transstadial passage of endogenous microbes. Larvae exposed to an exogenous bolus of B. subtilis harbored significantly larger numbers of bacteria. Bacterial load decreased to a range comparable to sand flies from control pots, suggesting an upper limit to the number of bacteria harbored. Emerging flies reared in larval chow containing transformed B. subtilis carried large numbers of these bacteria in their gut lumens. Strong GFP expression was detected in these paratransgenic flies with no spread of transformed bacteria to other compartments of the insects. This is the first demonstration of paratransgenic manipulation of P. argentipes. CONCLUSIONS: Paratransgenic manipulation of P. argentipes appears feasible. Expression of leishmanicidal molecules via commensal bacteria commonly found at breeding sites of P. argentipes could render adult sand flies refractory to L. donovani infection.

Identification of aerobic gut bacteria from the kala azar vector, Phlebotomus argentipes: a platform for potential paratransgenic manipulation of sand flies.

Visceral leishmaniasis is an understudied parasitic disease responsible for significant global morbidity and mortality. We are presently investigating a method of disease prevention termed paratransgenesis. In this approach, symbiotic or commensal bacteria are transformed to produce anti-Leishmania molecules. The transformed bacteria are delivered back to sand flies to inactivate the parasite within the vector itself. In this study, we identified 28 distinct gut microorganisms from Phlebotomus argentipes trapped from four visceral leishmaniasis-endemic sites in India. A significant percent of Staphylococcus spp., environmental bacteria, and Enterobacteriaceae were identified. Two non-pathogenic organisms, Bacillus megaterium and Brevibacterium linens, were also isolated. Both organisms are also used extensively in industry. Our results indicate that B. megaterium and B. linens are possible candidates for use in a model of paratransgenesis to prevent transmission of Leishmania.