Verification of monoplex and multiplex linear-after-the-exponential PCR gene-specific sepsis assays using clinical isolates.

AIMS: To verify monoplex and multiplex gene-specific linear-after-the-exponential polymerase chain reaction (LATE-PCR) assays for identifying 17 microbial pathogens (i.e., Klebsiella sp., Acinetobacter baumannii, Staphylococcus aureus, Enterobacter sp., Pseudomonas aeruginosa, coagulase negative staphylococci, Enterococcus sp., Candida sp.) commonly associated with septicaemia using clinical isolates. METHODS AND RESULTS: Clinical isolates of each target pathogen were collected from the University of California, Davis Medical Center (UCDMC) microbiology laboratory. Five microlitres (µl) of each culture suspension (1 × 10(8) CFU ml(-1) ) were added to 20 µl of monoplex mastermix. DNA extracted from clinical isolates was tested in multiplex. Monoplex assays demonstrated 100% sensitivity at this input level, except Enterobacter cloacae (2.7%), Ac. baumannii (57%) and Ps. aeruginosa (97.8%). All clinical isolates were positive in multiplex, with the exception of two Ac. baumannii, two Klebsiella oxytoca and two Candida parapsilosis isolates. CONCLUSIONS: Sixteen pathogens can be identified by monoplex LATE-PCR assays with sensitivities ≥ 97.8%. The multiplex assay demonstrated 91.4% sensitivity when tested with DNA extracted from 70 different target strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the potential of LATE-PCR to serve as an adjunct to culture if the reagents are optimized for sensitivity. Results warrant further testing through analytical and clinical validation of the multiplex assay.

Structure and content of the Entamoeba histolytica genome.

The intestinal parasite Entamoeba histolytica is one of the first protists for which a draft genome sequence has been published. Although the genome is still incomplete, it is unlikely that many genes are missing from the list of those already identified. In this chapter we summarise the features of the genome as they are currently understood and provide previously unpublished analyses of many of the genes.

Developmental expression of cytochrome P450 aromatase genes (CYP19a and CYP19b) in zebrafish fry (Danio rerio).

Cytochrome P450 aromatase (CYP19) is the terminal enzyme in the steroidogenic pathway that converts androgens (e.g., testosterone) into estrogens (e.g., estradiol). Regulation of this gene dictates the ratio of androgens to estrogens; therefore, appropriate expression of this enzyme is critical for reproduction as well as being pivotal in sex differentiation for most vertebrates. It is assumed that most vertebrates have a single CYP19 gene that is regulated by multiple tissue-specific promoter regions. However, the zebrafish (Danio rerio) has two genes (CYP19a and CYP19b), each encoding a significantly different protein and possessing its own regulatory mechanism. The primary purpose of this study was to determine the pattern of expression of each of the CYP19 genes in the developing zebrafish. A fluorescent-based method of real-time, quantitative RT-PCR provided the sensitivity and specificity to determine transcript abundance in single embryos/juveniles harvested at days 0 through 41 days post-fertilization (dpf), which encompasses the developmental events of sex determination and gonadal differentiation. CYP19 transcripts could be detected as early as 3 or 4 dpf, (CYP19a and CYP19b, respectively) and peak abundance was detected on day five. In general, the CYP19 genes differed significantly in the ontogeny of their expression. In most cases, the gonadal form of CYP19 (CYP19a) was more abundant than the brain form (CYP19b); however, unlike CYP19a, the pattern of CYP19b expression could be clearly segregated into two populations, suggesting an association with sex differentiation. Pharmacological steroids (ethinylestradiol and 17 alpha-methyltestosterone) enhanced the expression of the CYP19b gene at all three days examined (4, 6, and 10 dpf). These data suggest that the timely and appropriate expression of CYP19 is important in development and that the expression of CYP19b (the "extra-gonadal" form) may be associated with sexual differentiation if not sexual determination. J. Exp. Zool. 290:475-483, 2001.

Absence of lipophosphoglycan-like glycoconjugates in Entamoeba dispar.

Invasive amoebiasis is the result of infection of Entamoeba histolytica. The closely related Entamoeba dispar can colonize the human gut but does not cause invasive disease. In this study, E. dispar was analysed for the presence of the lipophosphoglycan-like (LPG) glycoconjugate known to be present on the cell surface of E. histolytica. E. dispar cells were radio-isotope labelled with [3H]galactose or [3H]inositol. The acidic glycoconjugates were extracted and analysed by hydrophobic chromatography over phenyl-Sepharose and by sodium dodecyl sulphate polyacrylamide gel electrophoresis. No LPG-like molecules could be identified in E. dispar in contrast to E. histolytica, suggesting that these molecules may be absent in the non-pathogenic species.

Immunodetection of hepatic peroxisomal PMP70 as an indicator of peroxisomal proliferation in the mummichog, Fundulus heteroclitus.

Peroxisomes are important sites for beta-oxidative fatty acid metabolism and peroxidative detoxification. Agents causing peroxisomal proliferation have been associated with reproductive and developmental toxicity and hepatocarcinogenesis. Female mummichog (Fundulus heteroclitus) were exposed to waterborne 2,4-dichlorophenoxyacetic acid (2,4-D), a model peroxisome proliferator, at sublethal concentrations of 0.01, 0.10, and 1.00 ppm or dimethyl sulfoxide (DMSO) vehicle for 7, 14, or 21 days. A polyclonal antibody to rat PMP70 protein (70 kDa peroxisomal membrane protein, a major component of peroxisomes and member of the ABC transporter superfamily) was used for Western blotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine possible peroxisome proliferation. Significant increases of an approximately 70-kDa protein band recognized by anti-PMP70 were observed on all days, especially at the highest exposure concentration. The data suggest that immunoassay of PMP70 is a useful biomarker assay for peroxisome proliferation, and may be applicable to a wide range of species. The response also suggests that this assay could be used for measuring chronic exposures to environmental peroxisome proliferating agents.

The effects of Entamoeba histolytica lysates on human colonic mucins.

Detergent lysates of Entamoeba histolytica trophozoites contained high levels of beta-N acetyl-D-glucosaminidase, beta-N acetyl-D-galactosaminidase and alpha-D-galactosidase activity, and lower but significant levels of five other glycosidases. Although these activities should have been capable of largely degrading the oligosaccharide side-chains of human colonic mucin, in fact only about one third of high MW mucin was degraded in 72 h and trypsin alone produced a similar effect. There was no evidence that these glycosidases were excreted and we conclude that they are unlikely to represent significant virulence factors for E. histolytica.

Activity of disulfiram (bis(diethylthiocarbamoyl)disulphide) and ditiocarb (diethyldithiocarbamate) against metronidazole-sensitive and -resistant Trichomonas vaginalis and Tritrichomonas foetus.

Clinical resistance of Trichomonas vaginalis to metronidazole is best correlated with MIC values measured under aerobic conditions. Under these conditions both disulfiram (bis(diethylthiocarbamoyl)disulphide), and its first mammalian metabolite, ditiocarb (diethyldithiocarbamate), showed high levels of activity against metronidazole-sensitive (disulfiram MIC, 0.1-0.7 microM; ditiocarb MIC, 0.3-9 microM) and -resistant (MICs 0.2-1.3 microM and 1.2-9 microM respectively) isolates. Tritrichomonas foetus was also sensitive-the MICs for seven metronidazole-sensitive isolates were 0.1-1.0 microM for disulfiram and 1.0-6.9 microM for ditiocarb; those for two highly metronidazole-resistant strains were 0.3-1.3 microM and 0.6-6 microM respectively. Under anerobic conditions most strains became highly resistant to both compounds. Surprisingly, disulfiram was consistently more active than ditiocarb.

An improved colorimetric PCR-based method for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in feces.

The epidemiological implications of the recent separation of "Entamoeba histolytica" into two separate species, pathogenic E. histolytica sensu stricto and commensal E. dispar, will not become apparent without methods of distinguishing between them which are applicable to large numbers of specimens. We have modified a PCR-based method to produce such a technique which may be completed in 1 day while still identifying 10(-1) E. histolytica and 1 to 10 E. dispar trophozoites per g of feces when present separately and 10 E. histolytica and 100 E. dispar trophozoites per g in the presence of 10(6) trophozoites per g of the other species. Applied to fecal specimens from 18 patients from which E. histolytica or E. dispar had been grown and identified to the species level by hexokinase isoenzyme analysis, the method in every case yielded the correct result. Positive and negative results are easily distinguished by eye, and we are now applying this technique to a large-scale epidemiological study of amebiasis in the eastern Mediterranean region.

Gut Coccidia--Isospora, Cryptosporidium, Cyclospora and Sarcocystis.

The gut Coccidia are members of a large, varied, and exclusively intracellular group of protozoan parasites, four species of which (Isospora, Cryptosporidium, Cyclospora, and Sarcocystis) are human pathogens. The first three, but particularly Cryptosporidium parvum, have moved from medical curiosities to major problems with the coming of the acquired immunodeficiency syndrome (AIDS) epidemic, but are now known to also cause disease in the immunocompetent patient. They are easy to acquire and difficult to remove from the environment and, in the case of cryptosporidiosis, impossible to treat properly. Further research into many aspects of the biology of these organisms is urgently needed.

Field evaluation of an improved Kato-Katz thick smear technique for quantitative determination of helminth eggs in faeces.

A new method for the quantification of helminth eggs in faeces was developed, in which 7.5% nigrosin in 10% formaldehyde mixed with 5% eosin yellow in 10% formaldehyde was substituted for the malachite green solution used in the standard Kato-Katz method. This modification revealed the eggs of parasites like Schistosoma mansoni, Ascaris lumbricoides, Trichuris trichiura and hookworms distinctly. The slides made with this new technique could be accurately read within one hour. Faecal smears from 100 pupils in Kigungu, Entebbe, Uganda, were studied with both methods. The egg counts of S. mansoni, A. lumbricoides and T. trichiura by both methods were equal. The modified method, however, showed significantly higher hookworm egg counts (p < 0.001). Hookworm eggs were equal one hour after preparation of the slides as 16 hours after preparation. The intensity of infection detected was higher with the modified method for both S. mansoni and hookworms.

A novel transcribed repeat element from Entamoeba histolytica.

We have identified an unusual 0.55-kb DNA repeat element specific to Entamoeba histolytica (Eh) which we call interspersed element (IE). The IE is a common feature in independently isolated genomic and cDNA fragments. Hybridization of labeled IE sequences to trophozoite DNA, RNA and first-strand cDNA prepared from poly(A)-enriched mRNA indicate that the IE are reiterated about 500 times per Eh trophozoite and that one or more can be found as RNA transcripts. These features and the degree of conservation of IE suggest a possible role for these sequences.

Transient expression of luciferase in Entamoeba histolytica driven by the ferredoxin gene 5' and 3' regions.

We have successfully transfected Entamoeba histolytica trophozoites with constructs containing ferredoxin sequences fused to the reporter gene luciferase. We have determined the conditions and parameters necessary to maximise transient luciferase expression in our system. Our optimal construct gave values of 268 x 10(3) relative light units per second (RLU s-1) when assayed representing a stimulation of 18,000-fold over the control vector. Comparison of differing constructs allowed us to conclude that the 5' and 3' ferredoxin sequences are both necessary for optimal luciferase expression from our vectors. Transcriptional initiation occurs within the consensus sequence ATTCA in both construct and chromosomal ferredoxin promoters.