BackgroundBlastocystis is a common intestinal parasite with worldwide distribution but the distribution of Blastocystis and its subtypes in East Africa is largely unknown. In this study, we investigate the distribution of Blastocystis subtypes in Zanzibar, Tanzania and report the prevalence of intestinal parasites using both molecular methods and microscopy.MethodsStool samples were collected from both diarrhoeic and non-diarrhoeic outpatients in Zanzibar. In addition to microscopy, real-time PCR for Blastocystis, Entamoeba histolytica and E. dispar, Giardia intestinalis, Cryptosporidium spp., and Dientamoeba fragilis was used. Blastocystis subtypes were determined by a conventional PCR followed by partial sequencing of the SSU-rRNA gene. Genetic assemblages of Giardia were determined by PCR with assemblage specific primers.ResultsIntestinal parasites were detected in 85 % of the 174 participants, with two or more parasites present in 56 %. Blastocystis sp. and Giardia intestinalis were the most common parasites, identified by PCR in 61 and 53 % of the stool samples respectively, but no correlation between carriage of Blastocystis and Giardia was found. The Blastocystis subtype distribution was ST1 34.0 %, ST2 26.4 %, ST3 25.5 %, ST7 0.9 %, and 13.2 % were positive only by qPCR (non-typable). The Giardia genetic assemblages identified were A 6.5 %, B 85 %, A + B 4.3 %, and non-typable 4.3 %. The detection rate with microscopy was substantially lower than with PCR, 20 % for Blastocystis and 13.8 % for Giardia. The prevalence of Blastocystis increased significantly with age while Giardia was most prevalent in children two to five years old. No correlation between diarrhoea and the identification of Giardia, Blastocystis, or their respective genetic subtypes could be shown and, as a possible indication of parasite load, the mean cycle threshold values in the qPCR for Giardia were equal in diarrhoeic and non-diarrhoeic patients.ConclusionsCarriage of intestinal parasites was very common in the studied population in Zanzibar. The most commonly detected parasites, Blastocystis and Giardia, had different age distributions, possibly indicating differences in transmission routes, immunity, and/or other host factors for these two species. In the Blastocystis subtype analysis ST1-3 were common, but ST4, a subtype quite common in Europe, was completely absent, corroborating the geographical differences in subtype distributions previously reported.

The degradation of ornithine decarboxylase (ODC) is mediated by antizyme, a protein regulated by the end-products of ODC activity, the polyamines. High levels of polyamines induce a ϩ1 ribosomal frameshift in the translation of the rat antizyme message leading to the expression of a full-length protein.We have studied whether the regulation of antizyme expression occurs only at the level of translation or whether polyamine levels also affect the transcription of the antizyme gene. Thus, we have cloned and sequenced the mouse homologues of the rat ODC-antizyme gene and cDNA. Northern blot analysis shows that although high concentrations of polyamines do not affect the steady-state levels of antizyme message in L1210 leukemia cells, polyamine depletion using 2-(difluoromethyl)ornithine [Orn(F 2 Me)] leads to a marked decrease in mRNA levels. Results of transient transfections of luciferase-reporter-gene constructs driven by antizyme promoter fragments in untreated and Orn(F 2Me)-treated Balb/C 3T3 cells indicate that the transcription of the antizyme gene is altered upon polyamine depletion. The amount of antizyme protein on Western blots was also altered by polyamine depletion and addition, and the polysomal distribution of antizyme message suggests a general translational increase of the message when polyamine concentrations are high. These results indicate a role for polyamines in the transcriptional and translational regulation of ornithine decarboxylase antizyme.Keywords : polyamine ; ornithine decarboxylase; antizyme; translational frameshifting; transcription.Polyamines are polycations, which bind and interact with protein necessary for making the interaction and the degradation of ODC have been identified, but it is uncertain whether antinumerous molecules within a cell. They are present in all living zyme is degraded at the same time as ODC or whether it is cells, are essential for growth, and play important roles in differrecycled [12Ϫ17]. Recently another factor in the regulation of entiation of eukaryotic cells [1,2]. Cells acquire polyamines by ODC has been described, when the antizyme-inhibitor cDNA uptake from the exterior and/or synthesize them de novo. Orniwas cloned and sequenced [18]. This protein binds to antizyme, thine decarboxylase (ODC) is the key regulatory enzyme and with a much higher affinity than that of ODC [19]. The regulaone of the rate-limiting enzymes in the polyamine-biosynthesis tion of antizyme inhibitor has so far not been investigated. The pathway. It catalyses the conversion of ornithine into putrescine, finding that antizyme plays a role in the regulation of polyamine a diamine that is the precursor of the polyamines spermidine and uptake by an unknown mechanism adds further complexity to spermine [3]. ODC activity is induced by growth factors mostly the tightly regulated polyamine homeostasis [20, 21]. through increased transcription of the ODC gene [4]. ODC isThe level of antizyme mRNA in a cell is usually high even necessary for cell-cycle progression and over expression...

We applied a high-resolution PCR-based typing method, multiple-locus variable-number tandem repeat analysis (MLVA), for discrimination of 30 multidrug-resistant clinical isolates of Staphylococcus epidermidis. The results of MLVA were congruent with results obtained by pulsed-field gel electrophoresis (PFGE). MLVA generated discrete character data, and its discriminatory capacity was comparable to that of PFGE.The clinical significance of Staphylococcus epidermidis is increasingly recognized. The bacterium is primarily associated with nosocomial infections such as prosthesis-and intravascular-catheter-related infections and a variety of postoperative infections (17). Moreover, nosocomial isolates of S. epidermidis frequently exhibit multidrug resistance, which limits the therapeutic arsenal (5). Despite the growing importance of S. epidermidis as a cause of hospital-acquired infections, there is still limited information available regarding the epidemiology of S. epidermidis in the hospital setting. Methods that may distinguish clinically significant strains from contaminant strains are lacking. For epidemiological studies, there is also a lack of easy-to-use, rapid typing methods with high reproducibility. Since the 1990s, pulsed-field gel electrophoresis (PFGE) of whole-genome SmaI digests has been considered the "gold standard" for molecular typing of S. epidermidis (13,14). The method has high discriminatory power but is laborious and therefore costly. Interpretation and exchange of PFGE-typing data are not uncomplicated because they depend on banding patterns and subjective decisions regarding the true existence of discrete bands. There is a consensus that exchange of typing data between laboratories is highly desirable, and systems with a binary output (numbers or characters), rather than methods generating banding patterns such as PFGE, are therefore preferred. Multiple-locus variable-number tandem repeat analysis (MLVA) has proved efficient for high-resolution typing of several bacterial species (4, 6, 16). The method targets multiple genomic loci and relies on the detection of different copy numbers of short repeated sequences that are arranged in tandem arrays. MLVA is an easy-to-use, PCR-based method that provides simple numerical data by identifying the copy numbers of tandem repeats that are present at the assayed genomic loci. The aim of the present study was to develop and evaluate an MLVA system for molecular typing of S. epidermidis.

Two divergently transcribed open reading frames: cpsX and cpsY separated by a common regulatory region was identified upstream of the cpsA^D genes involved in polysaccharide capsule biosynthesis in group B streptococci (GBS). We suggest that these genes are involved in the regulation of capsule expression in GBS, since the CpsX protein shares sequence similarities with LytR of Bacillus subtilis, an attenuator of transcription while CpsY has similarity to a wide variety of members of the LysR family of transcriptional regulators. No deletions, insertions, DNA rearrangements, or apparent differences were discovered in the postulated regulatory genes when the gene region was compared in GBS with different capsule phenotypes. Thus, other yet unidentified gene loci may control capsule phase variation in GBS. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Community-acquired pneumonia may present with similar clinical symptoms, regardless of viral or bacterial cause. Diagnostic assays are needed to rapidly discriminate between causes, because this will guide decisions on appropriate treatment. Therefore, a quantitative real-time PCR (qPCR) assay with duplex reactions targeting eight bacteria and six viruses was developed. Technical performance was examined with linear plasmids. Upper and lower respiratory tract specimens were used to compare the qPCR assay with standard microbiological methods. The limit of detection was 5 to 20 DNA template copies with approximately 1000-fold differences in concentrations of the two competing templates. SDs for positive controls were <5%. The use of the qPCR assay resulted in 113 positive identifications in 94 respiratory specimens compared with 38 by using standard diagnostics. Diagnostic accuracy of the qPCR assay varied between 60% positive agreement with standard tests for Streptococcus pneumoniae and 100% for Mycoplasma pneumoniae, Moraxella catarrhalis, and Staphylococcus aureus. Negative percentage of agreement was >95% for M. pneumoniae, Streptococcus pyogenes, respiratory syncytial virus, and influenza A virus; whereas it was only 56% for Haemophilus influenzae. Multiple microbial agents were identified in 19 of 44 sputum and 19 of 50 nasopharynx specimens. We conclude that in parallel qPCR detection of the targeted respiratory bacteria and viruses is feasible. The results indicate good technical performance of the assay in clinical specimens.

Although PCR offers the potential for sensitive detection of parasites there are several pitfalls for optimal performance, especially when DNA is extracted from a complex sample material such as stool. With the aid of a sensitive inhibitor control in a duplex real-time PCR (qPCR) for identification of Entamoeba histolytica and Entamoeba dispar we have evaluated factors that influenced the performance of the qPCR and have suggested a rationale to be used in the analysis of clinical samples. Pre-PCR processing was found to be of outmost importance for an optimal amplification since inhibitors caused false-negative results when higher amounts of sample were used. Stool sampling with a flocked swab (ESwab, Copan), yielding on average 173 mg, gave positive qPCR results in samples with cysts of E. dispar that were negative in serially diluted stool samples. The degree of inhibition found varied between samples and was not an on-off phenomenon. Even low-grade inhibition, shown as an increase of two cycles in the qPCR for the inhibitor control, could lead to false negativity in samples with low amounts of parasites. Lack of amplification in the qPCR due to inhibition could be overcome by dilution of the extracted DNA by 1/10-1/20. We also describe the use of guanidinium thiocyanate buffer for transport and storage of samples as well as a time-saving semi-automated DNA extraction method in an Arrow instrument (Nordiag) preceded by bead beating.

Two divergently transcribed open reading frames: cpsX and cpsY separated by a common regulatory region was identified upstream of the cpsA-D genes involved in polysaccharide capsule biosynthesis in group B streptococci (GBS). We suggest that these genes are involved in the regulation of capsule expression in GBS, since the CpsX protein shares sequence similarities with LytR of Bacillus subtilis, an attenuator of transcription while CpsY has similarity to a wide variety of members of the LysR family of transcriptional regulators. No deletions, insertions, DNA rearrangements, or apparent differences were discovered in the postulated regulatory genes when the gene region was compared in GBS with different capsule phenotypes. Thus, other yet unidentified gene loci may control capsule phase variation in GBS.