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Molecular diagnostic tests based on the PCR or alternative nucleic acid amplification technologies are commonly used for pathogen screening at blood drawing centers. Contrived process surveillance using test-specific external and internal controls is critical for the efficient leverage of PCR power. We describe here novel control constructs for use in nucleic acid amplification assays for pathogens with a single-stranded DNA genome, e.g., parvovirus B19. These controls are derived from a deletion mutant of the filamentous phage fd-tet, fKN16, and consist of single-stranded DNA packaged in a protein coat. They are essentially noninfectious to Escherichia coli and highly resistant to nuclease degradation. fKN16 based controls can be readily manufactured and highly purified. Despite their confirmed filamentous morphology, they can be precisely and accurately diluted over a wide range. Stability studies reveal that the novel control constructs are highly resistant to temperature stress, regardless of whether they are tested as concentrated stocks in storage buffer or diluted in buffer or human plasma. Real-time amplification curves derived from recombinant control constructs containing a parvovirus B19 specific sequence fragment match those derived from native virus. In summary, our data demonstrate the feasibility of novel nuclease-resistant single-stranded DNA controls as surrogates for parvovirus B19 and their applicability in routine molecular diagnostics.

The COBAS AmpliPrep instrument (Roche Diagnostics GmbH, D-68305 Mannheim, Germany) automates the entire sample preparation process of nucleic acid isolation from serum or plasma for polymerase chain reaction analysis. We report the analytical performance of the LightCycler Parvovirus B19 Quantification Kit (Roche Diagnostics) using nucleic acids isolated with the COBAS AmpliPrep instrument. Nucleic acids were extracted using the Total Nucleic Acid Isolation Kit (Roche Diagnostics) and amplified with the LightCycler Parvovirus B19 Quantification Kit. The kit combination processes 72 samples per 8-hour shift. The lower detection limit is 234 IU/ml at a 95% hit-rate, linear range approximately 10(4)-10(10) IU/ml, and overall precision 16 to 40%. Relative sensitivity and specificity in routine samples from pregnant women are 100% and 93%, respectively. Identification of a persistent parvovirus B19-infected individual by the polymerase chain reaction among 51 anti-parvovirus B19 IgM-negative samples underlines the importance of additional nucleic acid testing in pregnancy and its superiority to serology in identifying the risk of parvovirus B19 transmission via blood or blood products. Combination of the Total Nucleic Acid Isolation Kit on the COBAS AmpliPrep instrument with the LightCycler Parvovirus B19 Quantification Kit provides a reliable and time-saving tool for sensitive and accurate detection of parvovirus B19 DNA.

Lag1p and Lac1p are two homologous transmembrane proteins of the endoplasmic reticulum in Saccharomyces cerevisiae. Homologous genes have been found in a wide variety of eukaryotes. In yeast, both genes, LAC1 and LAG1, are required for efficient endoplasmic reticulum-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins. In this study, we show that lag1 Delta lac1 Delta cells have reduced sphingolipid levels due to a block of the fumonisin B1-sensitive and acyl-CoA-dependent ceramide synthase reaction. The sphingolipid synthesis defect in lag1 Delta lac1 Delta cells can be partially corrected by overexpression of YPC1 or YDC1, encoding ceramidases that have been reported to have acyl-CoA-independent ceramide synthesis activity. Quadruple mutant cells (lag1 Delta lac1 Delta ypc1 Delta ydc1 Delta) do not make any sphingolipids, but are still viable probably because they produce novel lipids. Moreover, lag1 Delta lac1 Delta cells are resistant to aureobasidin A, an inhibitor of the inositolphosphorylceramide synthase, suggesting that aureobasidin A may be toxic because it leads to increased ceramide levels. Based on these data, LAG1 and LAC1 are the first genes to be identified that are required for the fumonisin B1-sensitive and acyl-CoA-dependent ceramide synthase reaction.

Multiple or pleiotropic drug resistance often occurs in the yeast Saccharomyces cerevisiae through genetic activation of the Cys(6)-Zn(II) transcription factors Pdr1p and Pdr3p. Hyperactive alleles of these proteins cause overproduction of target genes that include drug efflux pumps, which in turn confer high level drug resistance. Here we provide evidence that both Pdr1p and Pdr3p act to regulate production of an enzyme involved in sphingolipid biosynthesis in S. cerevisiae. The last step in formation of the major sphingolipid in the yeast plasma membrane, mannosyldiinositol phosphorylceramide, is catalyzed by the product of the IPT1 gene, inositol phosphotransferase (Ipt1p). Transcription of the IPT1 gene is responsive to changes in activity of Pdr1p and Pdr3p. A single Pdr1p/Pdr3p response element is present in the IPT1 promoter and is required for regulation by these factors. Loss of IPT1 has complex effects on drug resistance of the resulting strain, consistent with an important role for mannosyldiinositol phosphorylceramide in normal plasma membrane function. Direct assay for lipid contents of cells demonstrates that changes in sphingolipid composition correlate with changes in the activity of Pdr3p. These data suggest that Pdr1p and Pdr3p may act to modulate the lipid composition of membranes in S. cerevisiae through activation of sphingolipid biosynthesis along with other target genes.

There is an increasing interest in non-albicans Candida species because of the increasing number of fungal infections they cause. Most of these infections can be found in immunocompromised individuals, especially in those infected with human immunodeficiency virus (HIV). Candida dubliniensis is a recently identified yeast, mostly isolated in HIV-positive individuals with oral candidiasis. Candida dubliniensis is a germ tube- and chlamydospore-form yeast. Thus, it shares diagnostic characteristics with Candida albicans. Probably, Candida dubliniensis has been present in the community for a long time and has been misidentified as Candida albicans. Significant phenotypic characteristics of Candida dubliniensis (difference in the carbohydrate assimilation profile, difference in colony color on CHROMagar Candida, and positive tetrazolium test, etc.) have been found, but none of them seem to be sufficient alone for the definitive identification of the species. Recently, PCR tests were developed to discriminate Candida albicans from Candida dubliniensis. However, these prove difficult in the context of routine mycological diagnostics. Moreover, an increased resistance to antifungal drugs has been described. This shows the importance of identification of Candida dubliniensis. To elucidate the current insight into Candida dubliniensis, the phenotypic and genotypic characteristics as well as the prevalence and the antifungal drug susceptibilities of this species are discussed from a clinical standpoint.

Persistent diarrhea is a major health problem among children in developing areas of the world. Since few community-based studies have addressed the epidemiology or etiology of this condition, we undertook prospective diarrheal surveillance among a cohort of 175 children less than 5 years of age over a 28-month period in an urban slum in northeastern Brazil. Very high diarrhea illness burdens were found. The children in this cohort had an average of 11 episodes per year and spent 82 days per year with diarrhea. A total of 65% of children had at least one episode of persistent diarrhea (greater than or equal to 14 days duration). These episodes accounted for 50% of all days of diarrhea and 11% of all episodes. The occurrence of at least one episode of persistent diarrhea identified all children who spent at least 15% percent of days with diarrhea. Among children with and without diarrhea, rotavirus was the agent isolated most frequently, followed by Giardia lamblia and enterotoxigenic coliforms. The agents isolated from children with acute and persistent diarrhea were similar, which suggests that other factors must be operative in the development of persistent diarrhea.