Interdomain but not intermolecular interactions observed in CFTR channels.

Gating of the cystic fibrosis transmembrane conductance regulator (CFTR) channels requires interdomain and/or intermolecular interactions involving different parts of the protein, yet the exact nature of those interactions remains unclear. In this study we report that treating wild type CFTR-expressing cells with oxidizing agents results in a significant reduction in the gel mobility of the protein indicative of the formation of disulfide bonds. In contrast, mutant CFTR channels in which cysteine residues in both nucleotide binding domains (NBDs) were mutated to serine, showed little change in gel mobility in oxidizing conditions. Mutation of the two cysteine residues in either the first or the second NBD alone also eliminates the change in gel mobility in oxidizing conditions. Wild type channels treated with oxidizing agents did not appear to form disulfide bonds with other proteins, suggesting that the close association that allows the formation of disulfide bonds occurs only within single proteins and not between separate channels interacting in a multimer.

Expression and subcellular localization of the cyclin-dependent kinase inhibitor p27(Kip1) in epithelial ovarian cancer.

OBJECTIVE: To determine if p27(Kip1) expression was altered in epithelial ovarian cancers as compared to normal ovarian surface epithelial (NOSE) cells and to determine if subcellular localization of p27(Kip1) was an important feature. METHODS: Thirteen tumor samples (1 Stage IC [early] and 12 Stage III/IV [advanced]) from patients with epithelial ovarian cancer and five NOSE samples were evaluated. Samples were surgically dissected to obtain an enriched population (90%) of cancer cells. The level of p27(Kip1) protein expression was determined by Western blot analysis. Actin was used as a loading control, and results were quantified by scanning densitometry using the ratio of the p27(Kip1) signal to the actin signal for comparison. To evaluate the subcellular localization of p27(Kip1), immunocytochemical staining was performed. Clinical pathological parameters were correlated to nuclear p27(Kip1) staining to establish if any association existed. RESULTS: When comparing the expression of p27(Kip1) between NOSE and ovarian cancer samples, only 2 of 13 ovarian cancer samples had altered p27(Kip1) expression. No correlation was found between the expression level of p27(Kip1) on Western blot and clinical pathological correlates. While no correlation between expression level of p27(Kip1) and subcellular localization was found, decreased nuclear staining (1+) was associated with shorter survivals using the log-rank test (P < 0.001). More importantly, in all tumor samples examined under the microscope, no nuclear p27(Kip1) staining was noted in cells that were undergoing mitosis. CONCLUSIONS: p27(Kip1) protein degradation may not be modified in ovarian cancer cells undergoing mitosis. Altered expression of p27(Kip1) is not an overwhelming feature in certain epithelial ovarian cancers. Decreased nuclear staining of p27(Kip1) is associated with poor survival in some epithelial ovarian cancers.

SIMPL is a tumor necrosis factor-specific regulator of nuclear factor-kappaB activity.

The IL-1 receptor-associated kinase (IRAK/mPLK) is linked to the regulation of nuclear factor-kappaB (NF-kappaB)-dependent gene expression. Here we describe a novel binding partner of IRAK/mPLK that we term SIMPL (signaling molecule that associates with the mouse pelle-like kinase). Overexpression of SIMPL leads to the activation of NF-kappaB-dependent promoters, and inactivation of SIMPL inhibits IRAK/mPLK as well as tumor necrosis factor receptor type I-induced NF-kappaB activity. Dominant inhibitory alleles of IkappaB kinase (IKKalpha or IKKbeta) block the activation of NF-kappaB by IRAK/mPLK and SIMPL. Furthermore, SIMPL binds IRAK/mPLK and the IKKs in vitro and in vivo. In the presence of antisense mRNA to SIMPL, the physical association between IRAK/mPLK and IKKbeta but not IRAK/mPLK and IKKalpha is greatly diminished. Moreover, dominant-negative SIMPL blocks IKKalpha- or IKKbeta-induced NF-kappaB activity. These results lead us to propose a model in which SIMPL functions to regulate NF-kappaB activity by linking IRAK/mPLK to IKKbeta/alpha-containing complexes.

Hypoxia activates jun-N-terminal kinase, extracellular signal-regulated protein kinase, and p38 kinase in pulmonary arteries.

Chronic alveolar hypoxia is the major cause of pulmonary hypertension. The cellular mechanisms involved in hypoxia- induced pulmonary arterial remodeling are still poorly understood. Mitogen-activated protein kinase (MAPK) is a key enzyme in the signaling pathway leading to cellular growth and proliferation. The purpose of this investigation was to determine the roles that MAPKs, specifically Jun-N-terminal kinase (JNK), extracellular signal-regulated protein kinase (ERK), and p38 kinase, play in the hypoxia-induced pulmonary arterial remodeling. Rats were exposed to normobaric hypoxia (10% O(2)) for 1, 3, 7, or 14 d. Hypoxia caused significant remodeling in the pulmonary artery characterized by thickening of pulmonary arterial wall and increases in tissue mass and total RNA. JNK, ERK, and p38 kinase tyrosine phosphorylations and their activities were significantly increased by hypoxia. JNK activation peaked at Day 1 and ERK/p38 kinase activation peaked after 7 d of hypoxia. The results from immunohistochemistry show that hypoxia increased phospho-MAPK staining in both large and small intrapulmonary arteries. Hypoxia also upregulated vascular endothelial growth factor messenger RNA (mRNA) and platelet-derived growth factor receptor mRNA levels in pulmonary artery with a time course correlated to the activation of ERK and p38 kinase. The gene expressions of c-jun, c-fos, and egr-1, known as downstream effectors of MAPK, were also investigated. Hypoxia upregulated egr-1 mRNA but downregulated c-jun and c-fos mRNAs. These data suggest that hypoxia-induced activation of JNK is an early response to hypoxic stress and that activation of ERK and p38 kinase appears to be associated with hypoxia-induced pulmonary arterial remodeling.

H(2)O(2)-induced egr-1, fra-1, and c-jun gene expression is mediated by tyrosine kinase in aortic smooth muscle cells.

Hydrogen peroxide (H(2)O(2)) has recently been shown to have a dual effect on cell growth by stimulating proliferation and triggering apoptosis. Apoptosis induced by H(2)O(2) is a direct consequence of oxidant injury, while the proliferative response to H(2)O(2) is thought to be a protective mechanism against oxidant injury. Signaling of the H(2)O(2)-induced proliferative effect has been proposed to occur via the activation of mitogen-activated protein kinase (MAPK) and increase in expression of transcription factors. In the present study, H(2)O(2)-induced mitogenic signaling in aortic smooth muscle cells (ASMC) was investigated with a specific focus on the roles of tyrosine kinase and tyrosine phosphatase in the regulation of the H(2)O(2)-stimulated egr-1, fra-1, and c-jun transcription. The results show that H(2)O(2)-induced increases in egr-1, fra-1, and c-jun mRNA levels, as measured by Northern blot analysis, are time and dose dependent with the peak of the response within 2 h. Tyrosine kinase inhibitors (genistein, amino-genistein, and tyrphostin 51) significantly attenuated H(2)O(2)-induced expression of these genes and a tyrosine phosphatase inhibitor (perox-vanadate) stimulated their expression. H(2)O(2) stimulated tyrosine kinase activities and caused protein tyrosine phosphorylation, which was blocked by tyrphostin 51. H(2)O(2) also caused tyrosine phosphorylation of platelet derived growth factor (PDGF) receptor. These data show that H(2)O(2) increases egr-1, fra-1, and c-jun mRNA levels in vascular smooth muscle cells, and the increase in expression of these genes is mediated by activation of tyrosine kinase. Our data also provide evidence that the H(2)O(2)-induced mitogenic response is, in part, mediated through the receptor tyrosine kinase, PDGF receptor.

Repression of transforming-growth-factor-beta-mediated transcription by nuclear factor kappaB.

Activation of transforming growth factor-beta (TGF-beta) and activin receptors leads to phosphorylation of Sma- and Mad-related protein 2 (Smad2) and Smad3, which function as transcription factors to regulate gene expression. Smad7 is a regulatory protein which is able to inhibit TGF-beta and activin signalling in a negative-feedback loop, mediated by a direct regulation by Smad3 and Smad4 via a Smad-binding element (SBE) in the Smad7 promoter. Interestingly, we found that the Smad7 promoter was also regulated by nuclear factor kappaB (NF-kappaB), a transcription factor which plays an important role in inflammation and the immune response. Expression of NF-kappaB p65 subunit was able to inhibit the Smad7 promoter activity, and this inhibition could be reversed by co-expression of IkappaB, an inhibitor of NF-kappaB. In addition, the inhibitory activity of p65 was observed in a minimal promoter that contained only the Smad7 SBE and a TATA box, without any consensus NF-kappaB binding site. This inhibitory effect appeared to be common to other TGF-beta- and activin-responsive promoters, since p65 also inhibited the forkhead-activin-signal-transducer-2-mediated activation of a Xenopus Mix.2 promoter, as well as the Smad3-mediated activation of 3TP-lux which contains PMA-responsive elements and a plasminogen-activator-inhibitor-1 promoter. Activation of endogenous NF-kappaB by tumour necrosis factor-alpha (TNF-alpha) was also able to inhibit the Smad7 promoter in human embryonic kidney 293 cells. In human hepatoma HepG2 cells, TNF-alpha was able to inhibit TGF-beta- and activin-mediated transcriptional activation. Furthermore, overexpression of the transcription co-activator p300 could abrogate the inhibitory effect of NF-kappaB on the Smad7 promoter. Taken together, these data have indicated a novel mode of crosstalk between the Smad and the NF-kappaB signalling cascades at the transcriptional level by competing for a limiting pool of transcription co-activators.

A comparison of macrophage colony-stimulating factor (M-CSF) gene expression in primary and immortalized endothelial cells.

M-CSF is produced by a wide variety of cell types, including EC, fibroblasts, and monocyte/macrophages, where it functions as a survival factor and a chemotactic agent for monocytes. An early event in the development of atherosclerosis is the infiltration of monocytes into the artery wall. Local expression of M-CSF by EC lining the blood vessels is thought to promote the growth and survival of lesional monocytes and macrophages, thus enhancing lesion development and disease progression. Primary cultures of EC are difficult to maintain for long periods of time, which complicates their use for biochemical and molecular analysis. As a step toward identifying a representative endothelial-like cell line, serum-dependent and IL-1-dependent changes in M-CSF gene expression in two endothelial-like cell lines were compared to that detected in primary EC cultures. The data presented here demonstrate that the two endothelial-like cell lines, like primary cultures of EC, express the M-CSF gene under basal conditions. In both types of cell cultures, IL-1alpha stimulation increased M-CSF mRNA levels 2-7-fold, whereas serum stimulation elicited a more modest effect (2-3-fold increase). The IL-1alpha-induced change in M-CSF gene expression is mediated at the transcriptional level, and M-CSF promoter activity is, in part, dependent on the activity of the NF-kappaB-inducing kinase. Collectively, our results demonstrate that either endothelial-like cell line would be a representative model in which endothelial-specific changes in M-CSF gene expression could be identified.

Molecular Characterization of Vegetative Compatibility Group 4A and 4B Isolates of Verticillium dahliae Associated with Potato Early Dying.

Forty isolates of Verticillium dahliae, collected from potato seed tubers and potato plants from various regions in North America and previously assigned to vegetative compatibility groups (VCGs) 4A or 4B, were characterized using molecular markers. The VCG 4A isolates were previously shown to be a highly virulent pathotype of potato and to interact synergistically with the root-lesion nematode Pratylenchus penetrans to cause potato early dying. All but one of the VCG 4A isolates characterized in this study lacked the subspecies-specific repetitive DNA sequence E18 and could be differentiated from the remaining isolates by restriction fragment length polymorphisms (RFLPs) in the nuclear rDNA and Trp1 loci. The E18 RFLP patterns of several VCG 4B isolates from Maine and New York were highly similar to those of VCG 4B isolates previously collected from potato and tomato fields in Ontario. The data presented here suggest that the molecular markers will be useful for the detection and classification of isolates of V. dahliae associated with potato early dying.

Influences of Cropping Practices on Verticillium dahliae Populations in Commercial Processing Tomato Fields in Ontario.

ABSTRACT The abundance of Verticillium dahliae in the soil and the incidence of V. dahliae-infected plants were determined for 12 commercial processing tomato fields in Kent County, Ontario. Comparison of the data with those from a previous survey of fields in adjacent Essex County showed that soil inoculum levels and incidence of infection were generally lower in Kent County fields and that race 2 V. dahliae was not common in Kent County. From the two counties, 128 isolates were characterized by restriction fragment length polymorphism (RFLP) analysis, using the subspecies-specific repetitive DNA sequence E18. A subset of these isolates was also characterized by vegetative compatibility and DNA hybridization analysis with a second subspecies-specific DNA sequence. Isolates with E18 RFLP profiles highly similar to those of isolates previously collected from potato fields in North America were prevalent in Essex County tomato fields but not common in Kent County fields. The data are consistent with the hypothesis that the group I isolates were introduced into southwestern Ontario with potato and that the different cultural practices in Essex County and Kent County have contributed to the differences in the accumulation of these isolates in the two regions.

Mouse receptor interacting protein 3 does not contain a caspase-recruiting or a death domain but induces apoptosis and activates NF-kappaB.

The death domain-containing receptor superfamily and their respective downstream mediators control whether or not cells initiate apoptosis or activate NF-kappaB, events critical for proper immune system function. A screen for upstream activators of NF-kappaB identified a novel serine-threonine kinase capable of activating NF-kappaB and inducing apoptosis. Based upon domain organization and sequence similarity, this novel kinase, named mRIP3 (mouse receptor interacting protein 3), appears to be a new RIP family member. RIP, RIP2, and mRIP3 contain an N-terminal kinase domain that share 30 to 40% homology. In contrast to the C-terminal death domain found in RIP or the C-terminal caspase-recruiting domain found in RIP2, the C-terminal tail of mRIP3 contains neither motif and is unique. Despite this feature, overexpression of the mRIP3 C terminus is sufficient to induce apoptosis, suggesting that mRIP3 uses a novel mechanism to induce death. mRIP3 also induced NF-kappaB activity which was inhibited by overexpression of either dominant-negative NIK or dominant-negative TRAF2. In vitro kinase assays demonstrate that mRIP3 is catalytically active and has autophosphorylation site(s) in the C-terminal domain, but the mRIP3 catalytic activity is not required for mRIP3 induced apoptosis and NF-kappaB activation. Unlike RIP and RIP2, mRIP3 mRNA is expressed in a subset of adult tissues and is thus likely to be a tissue-specific regulator of apoptosis and NF-kappaB activity. While the lack of a dominant-negative mutant precludes linking mRIP3 to a known upstream regulator, characterizing the expression pattern and the in vitro functions of mRIP3 provides insight into the mechanism(s) by which cells modulate the balance between survival and death in a cell-type-specific manner.

Redox reagents and divalent cations alter the kinetics of cystic fibrosis transmembrane conductance regulator channel gating.

Gating of the cystic fibrosis Cl(-) channel requires hydrolysis of ATP by its nucleotide binding folds, but how this process controls the kinetics of channel gating is poorly understood. In the present work we show that the kinetics of channel gating and presumably the rate of ATP hydrolysis depends on the species of divalent cation present and the oxidation state of the protein. With Ca(2+) as the dominant divalent cation instead of Mg(2+), the open burst duration of the channel is increased approximately 20-fold, and this change is reversible upon washout of Ca(2+). In contrast, "soft" divalent cations such as Cd(2+) interact covalently with cystic fibrosis transmembrane conductance regulator (CFTR). These metals decrease both opening and closing rates of the channel, and the effects are not reversed by washout. Oxidation of CFTR channels with a variety of oxidants resulted in a similar slowing of channel gating. In contrast, reducing agents had the opposite effect, increasing both opening and closing rates of the channel. In cell-attached patches, CFTR channels exhibit both oxidized and reduced types of gating, raising the possibility that regulation of the redox state of the channel may be a physiological mode of control of CFTR channel activity.

Modulation of tumor necrosis factor and interleukin-1-dependent NF-kappaB activity by mPLK/IRAK.

The innate immune response is an important defense against pathogenic agents. A component of this response is the NF-kappaB-dependent activation of genes encoding inflammatory cytokines such as interleukin-8 (IL-8) and cell adhesion molecules like E-selectin. Members of the serine/threonine innate immune kinase family of proteins have been proposed to mediate the innate immune response. One serine/threonine innate immune kinase family member, the mouse Pelle-like kinase/human interleukin-1 receptor-associated kinase (mPLK/IRAK), has been proposed to play an obligate role in promoting IL-1-mediated inflammation. However, it is currently unknown whether mPLK/IRAK catalytic activity is required for IL-1-dependent NF-kappaB activation. The present study demonstrates that mPLK/IRAK catalytic activity is not required for IL-1-mediated activation of an NF-kappaB-dependent signal. Intriguingly, catalytically inactive mPLK/IRAK inhibits type 1 tumor necrosis factor (TNF) receptor-dependent NF-kappaB activation. The pathway through which mPLK/IRAK mediates this TNF response is TRADD- and TRAF2-independent. Our data suggest that in addition to its role in IL-1 signaling, mPLK/IRAK is a component of a novel signal transduction pathway through which TNF R1 activates NF-kappaB-dependent gene expression.

Regulation of interleukin-1-stimulated GMCSF mRNA levels in human endothelium.

The regulation of interleukin-1 (IL-1)-mediated increases in GMCSF mRNA levels in human endothelium was examined and determined to occur in a time- and protein kinase C (PKC)-dependent manner. IL-1beta induced the early activation and translocation of PKC isotypes alpha and beta2 to the nucleus and PKC inhibition attenuated the IL-1-mediated increase in GMCSF mRNA levels. PKC activation by PMA alone, in the absence of IL-1beta activation, however, was insufficient to allow GMCSF mRNA detection. Increasing cyclic adenosine nucleotide (cAMP) levels suppressed IL-1beta-induced increases in GMCSF mRNA levels. In contrast, botulinum toxin C, which mediates the ADP ribosylation of a 21 kD ras-related G protein, augmented IL-1beta-induced GMCSF mRNA expression. Inhibition of protein synthesis (with cycloheximide) raised basal GMCSF mRNA transcripts to detectable levels, augmented IL-1-induced increases in GMCSF mRNA levels, and exhibited negative regulation by cAMP. Finally, disruption of either microtubules (with colchicine) or microfilaments (with cytochalasin B) resulted in reduced GMCSF mRNA expression in response to IL-1beta. These results are compatible with a model wherein IL-1-mediated increases in human endothelial cell GMCSF mRNA may be linked to both nuclear protein kinase C activation and activation of a low molecular weight G-protein, although neither activity alone is sufficient to increase the levels of GMCSF mRNA.

Comparison of the ParaSight-F test and the ICT Malaria Pf test with the polymerase chain reaction for the diagnosis of Plasmodium falciparum malaria in travellers.

Rapid and accurate methods are needed for the diagnosis of imported malaria. The ParaSight-F test and the ICT Malaria Pf test are commercially available kits marketed for the diagnosis of Plasmodium falciparum malaria. Both tests are antigen-capture assays based on the detection of P. falciparum histidine-rich protein 2 in peripheral blood. Using microscopy and a polymerase chain reaction (PCR)-based method as reference standards, we performed a 'blinded' comparison of these assays for the detection of P. falciparum infection in 200 febrile travellers returning from malaria-endemic areas. As determined by PCR and microscopy, 148 travellers had malaria and, of these patients, 54.7% (81/148) were infected with P. vivax only, 31.1% (46/148) with P. falciparum only, 9.5% (14/148) with P. ovale, 0.7% (1/148) with P. malariae, and 4.1% (6/148) had mixed infections. Compared to PCR, the ParaSight-F and ICT Malaria Pf tests had initial sensitivities of 94% and 90% and specificities of 95% and 97%, respectively, for the detection of P. falciparum malaria. When discrepant samples were retested with day 0 and day 1 bloods, the sensitivities improved to 96% and 94%, respectively. The 2 remaining false negative results with the Para-Sight-F test and 2 of the 3 false negative results with the ICT Malaria Pf test occurred in samples with < 100 parasites/microL. The performance of these kits was not significantly different (P = 0.75) and both are simple, rapid, and accurate tests for the detection of P. falciparum infection in the returned traveller.

Regulation of mouse colony-stimulating factor-1 gene promoter activity by AP1 and cellular nucleic acid-binding protein.

Macrophage colony-stimulating factor (M-CSF; CSF-1) is a member of a complex network of cytokines that regulate monocytic cell development and activity. It is produced in nearly all organs by cell types commonly found in connective tissue, including fibroblasts and monocytes. Whether different cell types share common or have divergent mechanisms for regulating CSF-1 gene expression is not known. To address this question, the identity of cis-acting elements and cognate trans-acting factors was characterized in a region of the CSF-1 promoter known to be more active in monocytes than in fibroblasts. The results of DNase I protection assays performed with fibroblast- or monocyte-derived nuclear extracts revealed a difference in the pattern of DNA-binding proteins. One protected region, common to both fibroblasts and monocytes, spans a putative phorbol ester-responsive element (TRE), and binding to the TRE by AP1 was verified with antibodies directed against c-fos and c-jun family members. Mutational analysis revealed that the TRE is required for CSF-1 gene expression in proliferating fibroblasts and monocytes. Binding of a second putative trans-acting factor, preferentially expressed in fibroblasts, to the region immediately upstream of the TRE was also detected. Screening a mouse expression library with oligonucleotides spanning the putative cis-acting element identified cellular nucleic acid-binding protein (CNBP) as the cognate binding activity, and antiserum to CNBP disrupted the electromobility shift assay complex. Mutational analysis revealed that loss of CNBP binding leads to a decrease in CSF-1 promoter activity in fibroblasts but has no effect on CSF-1 promoter activity in monocytes. Our results demonstrate that control of CSF-1 gene expression in monocytes and fibroblasts is mediated by common and cell type-specific trans-acting factors.

Phosphorylation of nuclear MyoD is required for its rapid degradation.

MyoD is a basic helix-loop-helix transcription factor involved in the activation of genes encoding skeletal muscle-specific proteins. Independent of its ability to transactivate muscle-specific genes, MyoD can also act as a cell cycle inhibitor. MyoD activity is regulated by transcriptional and posttranscriptional mechanisms. While MyoD can be found phosphorylated, the functional significance of this posttranslation modification has not been established. MyoD contains several consensus cyclin-dependent kinase (CDK) phosphorylation sites. In these studies, we examined whether a link could be established between MyoD activity and phosphorylation at putative CDK sites. Site-directed mutagenesis of potential CDK phosphorylation sites in MyoD revealed that S200 is required for MyoD hyperphosphorylation as well as the normally short half-life of the MyoD protein. Additionally, we determined that turnover of the MyoD protein requires the proteasome and Cdc34 ubiquitin-conjugating enzyme activity. Results of these studies demonstrate that hyperphosphorylated MyoD is targeted for rapid degradation by the ubiquitin pathway. The targeted degradation of MyoD following CDK phosphorylation identifies a mechanism through which MyoD activity can be regulated coordinately with the cell cycle machinery (CDK2 and CDK4) and/or coordinately with the cellular transcriptional machinery (CDK7, CDK8, and CDK9).

PLA2-independent induction of CSF-1 gene expression in growth-arrested fibroblasts.

At the site of a wound or an infection, localized production of colony-stimulating factor-1 (CSF-1) by resident macrophages is chemotactic for circulating monocytes. Several intracellular signaling pathways, including those initiated in response to activation of phospholipase A2 (PLA2) have been proposed to play a role in the regulation of CSF-1 gene expression. The goal of these studies was to determine whether PLA2 is required for the initial increase in CSF-1 gene expression in serum- or IL-1 alpha-stimulated growth-arrested fibroblasts. IL-1 alpha- or serum-stimulation of growth-arrested fibroblasts had no effect on PLA2 enzyme activity and inhibitors of cytosolic or Ca(2+)-independent PLA2 activity had no effect on IL-1 alpha- or serum-mediated increases in CSF-1 mRNA levels. High concentrations of the PLA2 inhibitors, 4-bromophenacyl lactone and quinacrine, resulted in a generalized decrease in cellular mRNA levels. Our results, obtained in fibroblasts, suggest treatment with 4-bromophenacyl lactone or quinacrine, instead of inhibiting PLA2 activity specifically, results in a generalized depression of cellular mRNA levels. These data demonstrate that the initial increase in CSF-1 gene expression in growth-arrested fibroblasts treated with serum or IL-1 alpha occurs through a PLA2-independent mechanism.

Imported malaria: prospective analysis of problems in diagnosis and management.

Imported malaria is an increasing problem in many countries. The objective of this study was to prospectively evaluate the diagnosis and treatment of imported malaria cases identified by active surveillance. Microscopic diagnosis at the community level was also compared to reference microscopic and blinded molecular diagnostic methods. Most travelers who acquire malaria had sought pretravel advice from a physician; however, only 11% used recommended chemoprophylaxis and only 17% used insect protection measures. The diagnosis of malaria was initially missed in 59% of cases. Community-based microscopic diagnosis provided incorrect species identification in 64% of cases. After presentation, the average delay before treatment was 7.6 days for falciparum malaria and 5.1 days for vivax malaria. Overall, 7.5% of Plasmodium falciparum-infected patients developed severe malaria, and in 11% of all cases therapy failed. Patients who present to a center without expertise in tropical medicine receive suboptimal treatment. Improvements in recognition, diagnosis, and treatment of malaria are essential to prevent morbidity and death among travelers.

Evaluation of a non-isotopic polymerase chain reaction-based assay to detect and predict treatment failure of Plasmodium vivax malaria in travellers.

With the emergence of chloroquine-resistant Plasmodium vivax (CRPV), new tests to detect P. vivax and predict response to therapy would be useful for clinical and research applications. We performed a 'blinded' evaluation of a non-isotopic (colourimetric) polymerase chain reaction (PCR) based assay (Digene SHARP Signal System) compared with microscopy and PCR/radiometric probe hybridization of ribosomal ribonucleic acid genes (RPH) for the detection of P. vivax malaria in 182 febrile travellers. Compared with PCR/RPH as the reference standard, the colourimetric assay had a sensitivity of 100% and specificity of 98%. Using microscopy as the reference standard, 84 of 87 patients with P. vivax infection had a positive colourimetric assay. The 3 patients with a negative assay were subsequently shown to be infected with P. ovale as determined by PCR/RPH. In a subset of patients followed longitudinally, the colourimetric assay was positive in 5 of 13 patients 6 or more days after initiation of therapy. Of these 5 patients, 4 were subsequently demonstrated to be infected with CRPV as determined by treatment failure in vivo and/or chloroquine blood levels. A positive assay result 6 or more days after initiation of therapy was associated with subsequent treatment failure (P < 0.01). This non-isotopic assay is a sensitive, specific, and rapid method for the detection of P. vivax PCR products and may prove useful in predicting treatment failure.