Malaria rapid diagnostic devices: performance characteristics of the ParaSight F device determined in a multisite field study.

Microscopic detection of parasites has been the reference standard for malaria diagnosis for decades. However, difficulty in maintaining required technical skills and infrastructure has spurred the development of several nonmicroscopic malaria rapid diagnostic devices based on the detection of malaria parasite antigen in whole blood. The ParaSight F test is one such device. It detects the presence of Plasmodium falciparum-specific histidine-rich protein 2 by using an antigen-capture immunochromatographic strip format. The present study was conducted at outpatient malaria clinics in Iquitos, Peru, and Maesod, Thailand. Duplicate, blinded, expert microscopy was employed as the reference standard for evaluating device performance. Of 2,988 eligible patients, microscopy showed that 547 (18%) had P. falciparum, 658 (22%) had P. vivax, 2 (0.07%) had P. malariae, and 1,750 (59%) were negative for Plasmodium. Mixed infections (P. falciparum and P. vivax) were identified in 31 patients (1%). The overall sensitivity of ParaSight F for P. falciparum was 95%. When stratified by magnitude of parasitemia (no. of asexual parasites per microliter of whole blood), sensitivities were 83% (>0 to 500 parasites/microl), 87% (501 to 1,000/microl), 98% (1,001 to 5,000/microl), and 98% (>5,000/microl). Device specificity was 86%.

Comparison of IsoCode STIX and FTA Gene Guard collection matrices as whole-blood storage and processing devices for diagnosis of malaria by PCR.

We compared two collection devices, IsoCode and FTA, with whole blood for the diagnosis of malaria by PCR (n = 100). Using whole blood as the reference standard, both devices were sensitive for the detection of single-species malaria infections by PCR (> or =96%). However, the detection of mixed infections was suboptimal (IsoCode was 42% sensitive, and FTA was 63% sensitive).

Identification of Cryptosporidium parvum dihydrofolate reductase inhibitors by complementation in Saccharomyces cerevisiae.

There is a pressing need for drugs effective against the opportunistic protozoan pathogen Cryptosporidium parvum. Folate metabolic enzymes and enzymes of the thymidylate cycle, particularly dihydrofolate reductase (DHFR), have been widely exploited as chemotherapeutic targets. Although many DHFR inhibitors have been synthesized, only a few have been tested against C. parvum. To expedite and facilitate the discovery of effective anti-Cryptosporidium antifolates, we have developed a rapid and facile method to screen potential inhibitors of C. parvum DHFR using the model eukaryote, Saccharomyces cerevisiae. We expressed the DHFR genes of C. parvum, Plasmodium falciparum, Toxoplasma gondii, Pneumocystis carinii, and humans in the same DHFR-deficient yeast strain and observed that each heterologous enzyme complemented the yeast DHFR deficiency. In this work we describe our use of the complementation system to screen known DHFR inhibitors and our discovery of several compounds that inhibited the growth of yeast reliant on the C. parvum enzyme. These same compounds were also potent or selective inhibitors of the purified recombinant C. parvum DHFR enzyme. Six novel lipophilic DHFR inhibitors potently inhibited the growth of yeast expressing C. parvum DHFR. However, the inhibition was nonselective, as these compounds also strongly inhibited the growth of yeast dependent on the human enzyme. Conversely, the antibacterial DHFR inhibitor trimethoprim and two close structural analogs were highly selective, but weak, inhibitors of yeast complemented by the C. parvum enzyme. Future chemical refinement of the potent and selective lead compounds identified in this study may allow the design of an efficacious antifolate drug for the treatment of cryptosporidiosis.

A role for host phosphoinositide 3-kinase and cytoskeletal remodeling during Cryptosporidium parvum infection.

Cryptosporidium parvum preferentially infects epithelial cells lining the intestinal mucosa of mammalian hosts. Parasite development and propagation occurs within a unique intracellular but extracytoplasmic parasitophorous vacuole at the apical surface of infected cells. Parasite-induced host cell signaling events and subsequent cytoskeletal remodeling were investigated by using cultured bovine fallopian tube epithelial (BFTE) cells inoculated with C. parvum sporozoites. Indirect-immunofluorescence microscopy detected host tyrosine phosphorylation within 30 s of inoculation. At >30 min postinoculation, actin aggregates were detected at the site of parasite attachment by fluorescein isothiocyanate-conjugated phalloidin staining as well as by indirect immunolabeling with monoclonal anti-actin. The actin-binding protein villin was also detected in focal aggregates at the site of attachment. Host cytoskeletal rearrangement persisted for the duration of the parasitophorous vacuole and contributed to the formation of long, branched microvilli clustered around the cryptosporidial vacuole. The phosphoinositide 3-kinase inhibitor wortmannin significantly inhibited (P < 0.05) C. parvum infection when BFTE cells were pretreated for 60 min at 37 degreesC prior to inoculation. Similarly, treatment of BFTE cells with the protein kinase inhibitors genistein and staurosporine and the cytoskeletally acting compounds 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazapine, cytochalasin D, and 2,3-butanedione monoxime significantly inhibited (P < 0.05) in vitro infection at 24 h postinoculation. These findings demonstrate a prominent role for phosphoinositide 3-kinase activity during the early C. parvum infection process and suggest that manipulation of host signaling pathways results in actin rearrangement at the site of sporozoite attachment.

Actin-dependent motility in Cryptosporidium parvum sporozoites.

The present study investigated the role of actin polymerization and myosin motor protein activity in the gliding motility of Cryptosporidium parvum sporozoites. Short motility trails were detected using an indirect immunofluorescent assay (IFA) with a polyclonal antisporozoite antibody following incubation of sporozoites on poly-L-lysine-coated glass slides. Sporozoite motility was blocked following exposure to cytochalasin D, a myosin light-chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexhydro-1,4-diazapin e, and the myosin ATPase inhibitor 2,3-butanedione monoxime. Sporozoites were observed to form rounded, blunt-ended shapes when exposed to these same inhibitors. Incubation of purified oocysts with these compounds did not significantly inhibit in vitro excystation or subsequent infectivity in cultured epithelial cells. Indirect IFA revealed a uniform distribution of actin protein throughout the body of the sporozoite; immunoelectron microscopy confirmed a diffuse intracellular pattern of gold particles in excysted sporozoites. Collectively, these findings show that sporozoite motility is dependent upon an intact actin-myosin motor system, and the dynamic interaction of F-actin and myosin motor proteins has a further role in maintaining the structural integrity of excysted sporozoites. Further, in vitro excystation and infectivity of C. parvum occurs in the absence of dynamic sporozoite locomotion.

Synergistic anticryptosporidial potential of the combination alpha-1-antitrypsin and paromomycin.

The combined effect of the serine protease inhibitor alpha-1-antitrypsin (AAT) and the aminoglycoside paromomycin on Cryptosporidium parvum infection in vitro was investigated. AAT and paromomycin were mixed with C. parvum oocysts as either single or combined treatments and used to inoculate epithelial cell cultures. Single- and combined-treatment groups had significantly lower (P < 0.01) parasite numbers than untreated controls. The mean fractional inhibitory concentration indices suggested significant synergistic activity.

Antagonistic effect of human alpha-1-antitrypsin on excystation of Cryptosporidium parvum oocysts.

This study evaluated the effects of the human serine protease inhibitor alpha-1-antitrypsin (AAT) on in vitro excystation and infectivity of Cryptosporidium parvum. Excystation was monitored at 37 C in RPMI medium in the presence of 0, 100, 500, or 1,000 micrograms/ml AAT. AAT significantly inhibited (P < 0.05) excystation of bleach-decontaminated oocysts in a concentration-dependent manner at incubation intervals from 15 to 90 min but did not alter the excystation dynamics of unbleached oocysts. Bleach-treated oocysts, suspended in RPMI containing 0, 1, 10, 100, 500, or 1,000 micrograms/ml AAT, were used to inoculate bovine fallopian tube epithelial (BFTE) cell monolayers. Alternately, sporozoites, excysted at 37 degrees C and collected by filtration, were used to inoculate BFTE cells under the same conditions. The mean number of parasites counted in AAT-treated, oocyst-inoculated cells was significantly less (P < 0.01) than control mean values at 24 and 48 hr post-inoculation (PI); longer PI intervals (72-96 hr) exhibited a decreased inhibitory effect. AAT did not inhibit parasite infection when cultures were inoculated with C. parvum sporozoites. The findings of this study show that the anticryptosporidial potential of AAT is primarily associated with an antagonistic effect on oocyst excystation.

Protease activity associated with excystation of Cryptosporidium parvum oocysts.

A Cryptosporidium parvum homogenate (CPH), prepared from partially excysted oocysts, was examined for proteolytic activity capable of hydrolyzing azocasein. Protease activity, measured at pH 7.0, was not detected in fresh oocysts but increased progressively with incubation at 37 C. Activity peaked after 60 min incubation but progressively decreased with extended incubation intervals. Cryptosporidial protease activity was significantly inhibited (P < 0.01) by the serine protease inhibitors phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DIFP), aprotinin, alpha-1-antitrypsin (AAT), and the cysteine protease inhibitor L-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane (E-64). No single inhibitor completely blocked CPH-associated protease activity; however, the combination of PMSF and E-64 inhibited > 95% of the azocasein hydrolysis measured in untreated control samples. The same group of inhibitors was further evaluated for their ability to inhibit excystation of C. parvum oocysts. PMSF, DIFP, aprotinin, and AAT significantly inhibited (P < 0.05) oocyst excystation at 15-, 30-, and 60-min incubation intervals; E-64 had no significant inhibitory effect on excystation. The results of this study demonstrate proteolytic activity during peak periods of excystation. Further, cryptosporidial protease activity was sensitive to both serine and cysteine protease inhibitors, but only serine protease inhibitors significantly inhibited oocyst excystation. These findings provide preliminary evidence of cryptosporidial proteases of both serine and cysteine protease classes and suggest that serine protease(s) are functionally associated with excystation.

Efficacy of serine protease inhibitors against Cryptosporidium parvum infection in a bovine fallopian tube epithelial cell culture system.

The anticryptosporidial potential of the protease inhibitors alpha-1-antitrypsin (AAT), antipain, aprotinin, leupeptin, methoxysuccinyl-ala-ala-pro-valine chloromethylketone (MAAPVCK), soybean trypsin inhibitor (SBTI), and phenylmethylsulfonyl fluoride (PMSF) was evaluated in a bovine fallopian tube epithelial (BFTE) cell culture system. Protease inhibitor concentrations of 5, 10, 50, 100, and 500 micrograms/ ml (PMSF at 1, 2, and 3 mM) in RPMI medium were mixed with Cryptosporidium parvum oocysts and used to inoculate BFTE cell monolayers. At 24 hr postinoculation (candlejar/37 C), cells were rinsed with RPMI medium, fixed in methanol, and stained with Giemsa. Parasites were enumerated in cell monolayers by brightfield microscopy. The mean number of parasites counted in each protease inhibitor treatment group was expressed as a percentage of the mean number of parasites counted in an infection control group. Leupeptin and SBTI reduced parasite numbers to 40-50% of the control mean at 500 micrograms/ml: AAT, antipain, and aprotinin reduced parasite numbers to 10-15% at the same concentration. PMSF reduced parasite numbers to 40% of the control mean at 3 mM. MAAPVCK did not significantly inhibit cryptosporidial infection. These findings suggest that a protease component of C. parvum may be essential for host cell infection.

Interaction of the human serine protease inhibitor alpha-1-antitrypsin with Cryptosporidium parvum.

The protozoan parasite Cryptosporidium parvum was studied for interaction with a human serine protease inhibitor (serpin), alpha-1-antitrypsin (AAT). A C. parvum homogenate (CPH) prepared from oocysts was incubated with purified human AAT and complexes formed between the serpin and CPH were detected using an enzyme-linked immunosorbent assay (ELISA). The optical density read at 450 nm of AAT:CPH reactivity was significantly increased (P < 0.001) relative to CPH in the absence of AAT treatment. Additionally, ELISA reactivity was blocked by incubating AAT with a cognate target enzyme, porcine pancreatic elastase (PPE), prior to treatment of the CPH. Incubation of a partially excysted sample of C. parvum with AAT (37 C x 60 min) demonstrated preferential fluorescence labeling of sporozoites by indirect immunofluorescence assay; AAT complexes were not detected on intact oocysts. Localization of AAT interactions with C. parvum sporozoites was visualized by transmission immunoelectron microscopy. Collectively, these data suggest that C. parvum sporozoites express a protease-like component that is recognized by human AAT. The ability to block ELISA reactivity with PPE suggests that the AAT interactions we detected are functionally similar to the serpin-enzyme complex AAT forms with a protease target.