A new single-step PCR assay for the detection of the zoonotic malaria parasite Plasmodium knowlesi

BACKGROUND: Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection. METHODOLOGY AND SIGNIFICANT FINDINGS: We have developed a bioinformatics approach to search the available malaria parasite genome database for the identification of suitable DNA sequences relevant for molecular diagnostic tests. Using this approach, we have identified multi-copy DNA sequences distributed in the P. knowlesi genome. We designed and tested several novel primers specific to new target sequences in a single-tube, non-nested PCR assay and identified one set of primers that accurately detects P. knowlesi. We show that this primer set has 100% specificity for the detection of P. knowlesi using three different strains (Nuri, H, and Hackeri), and one human case of malaria caused by P. knowlesi. This test did not show cross reactivity with any of the four human malaria parasite species including 11 different strains of P. vivax as well as 5 additional species of simian malaria parasites. CONCLUSIONS: The new PCR assay based on novel P. knowlesi genomic sequence targets was able to accurately detect P. knowlesi. Additional laboratory and field-based testing of this assay will be necessary to further validate its utility for clinical diagnosis of P. knowlesi.     

Identification of diverse archaeal proteins with class III signal peptides cleaved by distinct archaeal prepilin peptidases

Most secreted archaeal proteins are targeted to the membrane via a tripartite signal composed of a charged N terminus and a hydrophobic domain, followed by a signal peptidase-processing site. Signal peptides of archaeal flagellins, similar to class III signal peptides of bacterial type IV pilins, are distinct in that their processing sites precede the hydrophobic domain, which is crucial for assembly of these extracytoplasmic structures. To identify the complement of archaeal proteins with class III signal sequences, a PERL program (FlaFind) was written. A diverse set of proteins was identified, and many of these FlaFind positives were encoded by genes that were cotranscribed with homologs of pilus assembly genes. Moreover, structural conservation of primary sequences between many FlaFind positives and subunits of bacterial pilus-like structures, which have been shown to be critical for pilin assembly, have been observed. A subset of pilin-like FlaFind positives contained a conserved domain of unknown function (DUF361) within the signal peptide. Many of the genes encoding these proteins were in operons that contained a gene encoding a novel euryarchaeal prepilin-peptidase, EppA, homolog. Heterologous analysis revealed that Methanococcus maripaludis DUF361-containing proteins were specifically processed by the EppA homolog of this archaeon. Conversely, M. maripaludis preflagellins were cleaved only by the archaeal preflagellin peptidase FlaK. Together, the results reveal a diverse set of archaeal proteins with class III signal peptides that might be subunits of as-yet-undescribed cell surface structures, such as archaeal pili.     

Genomics meets transgenics in search of the elusive Cryptosporidium drug target

Cryptosporidium is an important pathogen of humans, and a challenging model for the laboratory. The parasite genome sequence, accessible through a comprehensive database, now provides exciting opportunities for urgently needed advances. Comparative genomics, combined with the genetic system in the related parasite Toxoplasma gondii, outlines a detailed Cryptosporidium parvum metabolic map and facilitates cell biological analyses. New targets for Cryptosporidium drug and vaccine development can be identified and validated based on this approach.     

Detection and identification of sulfhydryl conjugates of p-benzoquinone in microsomal incubations of benzene and phenol

The glutathione and cysteine conjugates of p-benzoquinone are detected and conclusively identified in microsomal incubations of benzene and phenol using liquid chromatography/electrochemistry (LCEC). Identification of the compounds is based on retention time, electrochemical behavior and acid hydrolysis. The fact that both of these compounds can be detected easily in a benzene incubation provides further evidence that p-benzoquinone or the corresponding semiquinone is a product of benzene metabolism in vivo. The conjugation of p-benzoquinone with glutathione is predominantly a nonenzymatic process. This is illustrated by the fact that the addition of cytosolic glutathione-S-transferases do not significantly increase the amount of glutathione conjugate produced in a phenol incubation containing glutathione.The kinetic constants for phenol metabolism to hydroquinone by microsomal protein are calculated. As suspected, the rate of metabolism of phenol is significantly higher than the rate of benzene metabolism. The V_max for phenol metabolism was calculated to be 7.1 nmol/min/mg protein and the K_M was found to be 0.38 mM.The further oxidation of hydroquinone to p-benzoquinone appears to be primarily an enzymatic process. Incubations of just hydroquinone with glutathione at 37°C produced only a small amount of the glutathione conjugate. The addition of cytosolic protein increases the amount of p-benzoquinone produced about 10-fold. This could be due to the peroxidases found in that medium. The addition of microsomal protein and NADPH increases the amount of glutathione conjugate produced to over 100-fold of that produced nonenzymatically. This indicates that a microsomal enzyme is responsible for the oxidation of hydroquinone to p-benzoquinone in vitro and the subsequent covalent binding to macromolecules.     

Determination of hydroxylated aromatic compounds produced via superoxide-dependent formation of hydroxyl radicals by liquid chromatography/electrochemistry

Hydroxyl radicals may be formed in a xanthine oxidase/hypoxanthine system, where the superoxide anion radical O-.2 and H2O2 are produced. The superoxide-dependent production of the OH. radicals may be monitored by determining the amount of hydroxylated aromatic compounds formed in such a system. Liquid chromatography/electrochemistry is a powerful tool for the determination of hydroxylated aromatic compounds. A technique is presented in which aniline and phenol are hydroxylated in xanthine oxidase/hypoxanthine incubations. No sample derivatization is needed for the determinations which can be accomplished by direct injection of the incubation mixture. Detection limits for 1,2- and 1,4-hydroxylated compounds are in the picomole range.     

Survival of Salmonellae During Pepperoni Manufacture

Survival of salmonellae in artificially contaminated beef-pork mixtures (approximately 10(4) salmonellae/g) was studied in pepperoni prepared by either a natural flora or lactic starter culture fermentation or in nonfermented sausages. The pepperoni did not become salmonellae free during the usual commercial 15 to 30-day drying period. Salmonella dublin was present in all products, fermented or unfermented, after 42 to 43 days of drying. At a lower level of contamination, 10(3)/g, S. dublin could not be recovered from starter culture-fermented pepperoni after 14 days of drying but persisted in the natural flora-fermented sausage. S. typhimurium (initial count, 10(4)/g) was absent after 42 days of drying when starter culture was used to ferment the pepperoni, but was still present in the natural flora-fermented and unfermented products. S. dublin, host adapted to cattle, or S. choleraesuis, host adapted to swine, had similar survival patterns in beef pork, or beef-pork pepperoni. Heating salmonellae contaminated beef-pork pepperoni (after fermantation but before drying) to an internal temperature of 60 C (trichinae inactivating) eliminated the food-borne pathogen from the sausage product.     

Rapid evolution in a conserved gene family. Evolution of the actin gene family in the sea urchin genus Heliocidaris and related genera

Camarodont sea urchins possess a rapidly evolving actin gene family whose members are expressed in distinct cell lineages in a developmentally regulated fashion. Evolutionary changes in the actin gene family of echinoids include alterations in number of family members, site of expression, and gene linkage, and a dichotomy between rapidly and slowly evolving isoform-specific 3' untranslated regions. We present sequence comparisons and an analysis of the actin gene family in two congeneric sea urchins that develop in radically different modes, Heliocidaris erythrogramma and H. tuberculata. The sequences of several actin genes from the related species Lytechinus variegatus are also presented. We compare the features of the Heliocidaris and Lytechinus actin genes to those of the the actin gene families of other closely related sea urchins and discuss the nature of the evolutionary changes among sea urchin actins and their relationship to developmental mode.      

Destruction of Staphylococcus aureus during frankfurter processing.

We studied the thermal resistance of Staphylococcus aureus during frankfurter processing in respect to whether staphylococci are killed by the heating step of the process and whether heat injury interferes with the quantitative estimation of the survivors. With S. aureus 198E, heat injury could be demonstrated only when large numbers of cells (10(8)/g) were present and at a product temperature of 140 degrees F (60 degrees C). On tryptic soy agar and tryptic soy agar plus 7% NaCl media, at temperatures less than 140 degrees F, the counts were virtually identical; above 140 degrees F, the counts converged, with the organisms dying so rapidly that heat injury was not demonstrable. Heat injury was thus judged not to interfere with the quantitative estimation of staphylococci surviving the normal commercial heating given frankfurters. By using a combination of direct plating on tryptic soy agar and a most-probable-number technique, we detected no viable cells (less than 0.3/g) of several strains of S. aureus in frankfurters heated to 160 degrees F (71.1 degrees C). This temperature is compatible with the normal final temperature to which federally inspected processors heat their frankfurters and with the temperature needed to destroy salmonellae.     

Structure-based design, synthesis, and biological evaluation of 1,1-dioxoisothiazole and benzo[b]thiophene-1,1-dioxide derivatives as novel inhibitors of hepatitis C virus NS5B polymerase

A novel series of HCV NS5B polymerase inhibitors comprising 1,1-dioxoisothiazoles and benzo[b]thiophene-1,1-dioxides were designed, synthesized, and evaluated. SAR studies guided by structure-based design led to the identification of a number of potent NS5B inhibitors with nanomolar IC₅₀ values. The most potent compound exhibited IC₅₀ less than 10 nM against the genotype 1b HCV polymerase and EC₅₀ of 70 nM against a genotype 1b replicon in cell culture. The DMPK properties of selected compounds were also evaluated.