Role of Burkholderia pseudomallei-Specific IgG2 in Adults with Acute Melioidosis, Thailand.

Melioidosis is a life-threatening infectious disease caused by the gram-negative bacillus Burkholderia pseudomallei. An effective vaccine is needed, but data on protective immune responses in human melioidosis are lacking. We used ELISA and an antibody-dependent cellular phagocytosis assay to identify the major features of protective antibodies in patients with acute melioidosis in Thailand. We found that high levels of B. pseudomallei-specific IgG2 are associated with protection against death in a multivariable logistic regression analysis adjusting for age, diabetes, renal disease, and neutrophil count. Serum from melioidosis survivors enhanced bacteria uptake into human monocytes expressing FcγRIIa-H/R131, an intermediate-affinity IgG2-receptor, compared with serum from nonsurvivors. We did not find this enhancement when using monocytes carrying the low IgG2-affinity FcγRIIa-R131 allele. The findings indicate the importance of IgG2 in protection against death in human melioidosis, a crucial finding for antibody-based therapeutics and vaccine development.

Serum From Melioidosis Survivors Diminished Intracellular Burkholderia pseudomallei Growth in Macrophages: A Brief Research Report.

Melioidosis is a neglected tropical disease with high mortality rate. It is caused by the Gram-negative, CDC category B select agent Burkholderia pseudomallei (B. ps) that is intrinsically resistant to first-line antibiotics. An antibody-based vaccine is likely to be the most effective control measure. Previous studies have demonstrated significant mechanistic roles of antibodies in protection against death in animal models, but data from human melioidosis is scarce. Herein, we used in-vitro antibody-dependent cellular phagocytosis and growth inhibition assays to assess the mechanism of protective antibodies in patients with acute melioidosis. We found that serum from patients who survived the disease enable more live B. ps to be engulfed by THP-1 derived macrophages (median 1.7 × 103 CFU/ml, IQR 1.1 × 103-2.5 × 103 CFU/ml) than serum from patients who did not survive (median 1.2 × 103 CFU/ml, IQR 0.7 × 103-1.8 × 103, p = 0.02). In addition, the intracellular growth rate of B. ps pre-opsonized with serum from survivors (median 7.89, IQR 5.58-10.85) was diminished when compared with those with serum from non-survivors (median 10.88, IQR 5.42-14.88, p = 0.04). However, the difference of intracellular bacterial growth rate failed to reach statistical significance when using purified IgG antibodies (p = 0.09). These results provide new insights into a mechanistic role of serum in protection against death in human melioidosis for antibody-based vaccine development.

Melioidosis DS rapid test: A standardized serological dipstick assay with increased sensitivity and reliability due to multiplex detection.

BACKGROUND: Melioidosis, caused by Burkholderia pseudomallei, is a severe infectious disease with high mortality rates, but is under-recognized worldwide. In endemic areas, there is a great need for simple, low-cost and rapid diagnostic tools. In a previous study we showed, that a protein multiplex array with 20 B. pseudomallei-specific antigens detects antibodies in melioidosis patients with high sensitivity and specificity. In a subsequent study the high potential of anti-B. pseudomallei antibody detection was confirmed using a rapid Hcp1 single protein-based assay. Our protein array also showed that the antibody profile varies between patients, possibly due to a combination of host factors but also antigen variations in the infecting B. pseudomallei strains. The aim of this study was to develop a rapid test, combining Hcp1 and the best performing antigens BPSL2096, BPSL2697 and BPSS0477 from our previous study, to take advantage of simultaneous antibody detection. METHODS AND PRINCIPAL FINDINGS: The 4-plex dipstick was validated with sera from 75 patients on admission plus control groups, achieving 92% sensitivity and 97-100% specificity. We then re-evaluated melioidosis sera with the 4-plex assay that were previously misclassified by the monoplex Hcp1 rapid test. 12 out of 55 (21.8%) false-negative samples were positive in our new dipstick assay. Among those, 4 sera (7.3%) were Hcp1 positive, whereas 8 (14.5%) sera remained Hcp1 negative but gave a positive reaction with our additional antigens. CONCLUSIONS: Our dipstick rapid test represents an inexpensive, standardized and simple diagnostic tool with an improved serodiagnostic performance due to multiplex detection. Each additional band on the test strip makes a false-positive result more unlikely, contributing to its reliability. Future prospective studies will seek to validate the gain in sensitivity and specificity of our multiplex rapid test approach in different melioidosis patient cohorts.

Human Immune Responses to Melioidosis and Cross-Reactivity to Low-Virulence Burkholderia Species, Thailand1.

Melioidosis is a neglected tropical disease with an estimated annual mortality rate of 89,000 in 45 countries across tropical regions. The causative agent is Burkholderia pseudomallei, a gram-negative soil-dwelling bacterium. In Thailand, B. pseudomallei can be found across multiple regions, along with the low-virulence B. thailandensis and the recently discovered B. thailandensis variant (BTCV), which expresses B. pseudomallei-like capsular polysaccharide. Comprehensive studies of human immune responses to B. thailandensis variants and cross-reactivity to B. pseudomallei are not complete. We evaluated human immune responses to B. pseudomallei, B. thailandensis, and BTCV in melioidosis patients and healthy persons in B. pseudomallei-endemic areas using a range of humoral and cellular immune assays. We found immune cross-reactivity to be strong for both humoral and cellular immunity among B. pseudomallei, B. thailandensis, and BTCV. Our findings suggest that environmental exposure to low-virulence strains may build cellular immunity to B. pseudomallei.

Diabetes alters immune response patterns to acute melioidosis in humans.

Diabetes mellitus (DM) is a serious global health problem currently affecting over 450 million people worldwide. Defining its interaction with major global infections is an international public health priority. Melioidosis is caused by Burkholderia pseudomallei, an exemplar pathogen for studying intracellular bacterial infection in the context of DM due to the 12-fold increased risk in this group. We characterized immune correlates of survival in peripheral blood of acute melioidosis patients with and without DM and highlight different immune response patterns. We demonstrate the importance of circulating NK cells and show that CX3CR1 expression on lymphocytes is a novel correlate of survival from acute melioidosis. Furthermore, excessive serum levels of IL-15 and IL-18BP contribute to poor outcome independent of DM comorbidity. CD8+ T cells and granzyme B expression in NK cells are important for survival of non-DM patients, whereas high antibody titers against B. pseudomallei and double-negative T cells are linked to survival of DM patients. Recall responses support a role of γδ T-cell-derived IFN-γ in the establishment of protective immunity in the DM group. Defining the hallmarks of protection in people with DM is crucial for the design of new therapies and vaccines targeting this rapidly expanding risk group.

Antibodies in Melioidosis: The Role of the Indirect Hemagglutination Assay in Evaluating Patients and Exposed Populations.

Melioidosis is a major neglected tropical disease with high mortality, caused by the Gram-negative bacterium Burkholderia pseudomallei (Bp). Microbiological culture remains the gold standard for diagnosis, but a simpler and more readily available test such as an antibody assay is highly desirable. In this study, we conducted a serological survey of blood donors (n = 1,060) and adult melioidosis patients (n = 200) in northeast Thailand to measure the antibody response to Bp using the indirect hemagglutination assay (IHA). We found that 38% of healthy adults (aged 17-59 years) have seropositivity (IHA titer ≥ 1:80). The seropositivity in healthy blood donors was associated with having a declared occupation of rice farmer and with residence in a nonurban area, but not with gender or age. In the melioidosis cohort, the seropositivity rate was higher in adult patients aged between 18 and 45 years (90%, 37/41) compared with those aged ≥ 45 years (68%, 108/159, P = 0.004). The seropositivity rate was significantly higher in people with diabetes (P = 0.008). Seropositivity was associated with decreased mortality on univariable analysis (P = 0.005), but not on multivariable analysis when adjusted for age, diabetes status, preexisting renal disease, and neutrophil count. This study confirms the presence of high background antibodies in an endemic region and demonstrates the limitations of using IHA during acute melioidosis in this population.

Infection with Burkholderia pseudomallei - immune correlates of survival in acute melioidosis.

Melioidosis, caused by Burkholderia pseudomallei, is a potentially lethal infection with no licensed vaccine. There is little understanding of why some exposed individuals have no symptoms, while others rapidly progress to sepsis and death, or why diabetes confers increased susceptibility. We prospectively recruited a cohort of 183 acute melioidosis patients and 21 control subjects from Northeast Thailand and studied immune parameters in the context of survival status and the presence or absence of diabetes. HLA-B*46 (one of the commonest HLA class I alleles in SE Asia) and HLA-C*01 were associated with an increased risk of death (odds ratio 2.8 and 3.1 respectively). Transcriptomic analysis during acute infection in diabetics indicated the importance of interplay between immune pathways including those involved in antigen presentation, chemotaxis, innate and adaptive immunity and their regulation. Survival was associated with enhanced T cell immunity to nine of fifteen immunodominant antigens analysed including AhpC (BPSL2096), BopE (BPSS1525), PilO (BPSS1599), ATP binding protein (BPSS1385) and an uncharacterised protein (BPSL2520). T cell immunity to GroEL (BPSL2697) was specifically impaired in diabetic individuals. This characterization of immunity associated with survival during acute infection offers insights into correlates of protection and a foundation for design of an effective multivalent vaccine.

A nonsense mutation in TLR5 is associated with survival and reduced IL-10 and TNF-α levels in human melioidosis.

BACKGROUND: Melioidosis, caused by the flagellated bacterium Burkholderia pseudomallei, is a life-threatening and increasingly recognized emerging disease. Toll-like receptor (TLR) 5 is a germline-encoded pattern recognition receptor to bacterial flagellin. We evaluated the association of a nonsense TLR5 genetic variant that truncates the receptor with clinical outcomes and with immune responses in melioidosis. METHODOLOGY/PRINCIPAL FINDINGS: We genotyped TLR5 c.1174C>T in 194 acute melioidosis patients in Thailand. Twenty-six (13%) were genotype CT or TT. In univariable analysis, carriage of the c.1174C>T variant was associated with lower 28-day mortality (odds ratio (OR) 0.21, 95% confidence interval (CI) 0.05-0.94, P = 0.04) and with lower 90-day mortality (OR 0.25, 95% CI 0.07-086, P = 0.03). In multivariable analysis adjusting for age, sex, diabetes and renal disease, the adjusted OR for 28-day mortality in carriers of the variant was 0.24 (95% CI 0.05-1.08, P = 0.06); and the adjusted OR for 90-day mortality was 0.27 (95% CI 0.08-0.97, P = 0.04). c.1174C>T was associated with a lower rate of bacteremia (P = 0.04) and reduced plasma levels of IL-10 (P = 0.049) and TNF-α (P < 0.0001). We did not find an association between c.1174C>T and IFN-γ ELISPOT (T-cell) responses (P = 0.49), indirect haemagglutination titers or IgG antibodies to bacterial flagellin during acute melioidosis (P = 0.30 and 0.1, respectively). CONCLUSIONS/SIGNIFICANCE: This study independently confirms the association of TLR5 c.1174C>T with protection against death in melioidosis, identifies lower bacteremia, IL-10 and TNF-α production in carriers of the variant with melioidosis, but does not demonstrate an association of the variant with acute T-cell IFN-γ response, indirect haemagglutination antibody titer, or anti-flagellin IgG antibodies.

Detection of chikungunya virus antigen by a novel rapid immunochromatographic test.

Chikungunya fever is a mosquito-borne disease of key public health importance in tropical and subtropical countries. Although severe joint pain is the most distinguishing feature of chikungunya fever, diagnosis remains difficult because the symptoms of chikungunya fever are shared by many pathogens, including dengue fever. The present study aimed to develop a new immunochromatographic diagnosis test for the detection of chikungunya virus antigen in serum. Mice were immunized with isolates from patients with Thai chikungunya fever, East/Central/South African genotype, to produce mouse monoclonal antibodies against chikungunya virus. Using these monoclonal antibodies, a new diagnostic test was developed and evaluated for the detection of chikungunya virus. The newly developed diagnostic test reacted with not only the East/Central/South African genotype but also with the Asian and West African genotypes of chikungunya virus. Testing of sera from patients suspected to have chikungunya fever in Thailand (n = 50), Laos (n = 54), Indonesia (n = 2), and Senegal (n = 6) revealed sensitivity, specificity, and real-time PCR (RT-PCR) agreement values of 89.4%, 94.4%, and 91.1%, respectively. In our study using serial samples, a new diagnostic test showed high agreement with the RT-PCR within the first 5 days after onset. A rapid diagnostic test was developed using mouse monoclonal antibodies that react with chikungunya virus envelope proteins. The diagnostic accuracy of our test is clinically acceptable for chikungunya fever in the acute phase.

Chikungunya virus was isolated in Thailand, 2010.

Chikungunya fever (CHIKF) is an acute febrile illness caused by a mosquito-borne alphavirus, chikungunya virus (CHIKV). This disease re-emerged in Kenya in 2004, and spread to the countries in and around the Indian Ocean. The re-emerging epidemics rapidly spread to regions like India and Southeast Asia, and it was subsequently identified in Europe in 2007, probably as a result of importation of chikungunya cases. On the one hand, chikungunya is one of the neglected diseases and has only attracted strong attention during large outbreaks. In 2008-2009, there was a major outbreak of chikungunya fever in Thailand, resulting in the highest number of infections in any country in the region. However, no update of CHIKV circulating in Thailand has been published since 2009. In this study, we examined the viral growth kinetics and sequences of the structural genes derived from CHIKV clinical isolates obtained from the serum specimens of CHIKF-suspected patients in Central Thailand in 2010. We identified the CHIKV harboring two mutations E1-A226V and E2-I211T, indicating that the East, Central, and South African lineage of CHIKV was continuously circulating as an indigenous population in Thailand.

Monoclonal antibody targeting chikungunya virus envelope 1 protein inhibits virus release.

Chikungunya virus (CHIKV) causes an acute clinical illness characterized by sudden high fever, intense joint pain, and skin rash. Recent outbreaks of chikungunya disease in Africa and Asia are a major public health concern; however, there is currently no effective licensed vaccine or specific treatment. This study reported the development of a mouse monoclonal antibody (MAb), CK47, which recognizes domain III within the viral envelope 1 protein and inhibited the viral release process, thereby preventing the production of progeny virus. The MAb had no effect on virus entry and replication processes. Thus, CK47 may be a useful tool for studying the mechanisms underlying CHIKV release and may show potential as a therapeutic agent.

Sequence variation of dengue type 2 virus isolated from clinical cases in Thailand.

Dengue fever (DF) and dengue hemorrhagic fever (DHF) are caused by mosquito-borne dengue virus (DENV) infection leading to death in tropical and subtropical countries. In Thailand, all 4 serotypes of DENV are circulating. The most severe cases of DF and DHF are primarily introduced by secondary infections. Epidemiological studies have demonstrated that approximately 20% of the primary infection cases were caused by DENV-1 and -3, while the cases of DENV-2 or -4 accounted for less than 3%. For this reason, DENV-2 and -4 from primary infections have not been well studied. In this study, the sequence diversity of the envelope gene of 8 DENV-2 clinical isolates from primary/secondary infections was analyzed. DENV-2 from primary infections were highly heterogeneous in individual patients, whereas those from secondary infections were homogeneous. Phylogenetic analysis demonstrated that the heterogeneous population of DENV-2 from primary infections was composed of closely related quasispecies. Homogenous DENV-2 could be derived from selection of a particular viral population in secondary infections. The degree of sequence diversity of DENV-2 varied, and thus quasispecies may be involved in the progression of DENV infection.

Low levels of antibody-dependent enhancement in vitro using viruses and plasma from dengue patients.

BACKGROUND: The majority of dengue patients infected with any serotype of dengue virus (DENV) are asymptomatic, but the remainder may develop a wide spectrum of clinical symptoms, ranging from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF). Severe cases occur more often in patients who experience a secondary infection with a different virus serotype. A phenomenon called antibody-dependent enhancement (ADE) has been proposed to explain the onset of these severe cases, but the exact mechanism of ADE remains unclear. METHODOLOGY/PRINCIPAL FINDING: Virus neutralization and ADE assays were performed using ultracentrifugation supernatants of acute-phase sera from patients with secondary infections or human monoclonal antibodies (HuMAbs) as anti-DENV antibodies. Virus sources included infectious serum-derived viruses from the ultracentrifugation precipitates, laboratory-culture adapted DENV, or recombinant DENVs derived from patient sera. In contrast to the high levels of ADE observed with laboratory virus strains, low ADE was observed with autologous patient-derived viruses, when patient sera were used to provide the antibody component in the ADE assays. Similar results were obtained using samples from DF and DHF patients. Recombinant-viruses derived from DHF patients showed only minor differences in neutralization and ADE activity in the presence of HuMAbs or plasma derived from the same DHF patient. CONCLUSION/SIGNIFICANCE: Serum or plasma taken from patients during the acute phase of a secondary infection showed high levels of ADE, but no neutralization activity, when assayed in the presence of laboratory-adapted virus strains. By contrast, serum or plasma from the same patient showed high levels of neutralization activity but failed to induce significant ADE when the assays were performed with autologous virus. These results demonstrate the significance of the virus source when measuring ADE. They also suggest that repeated passage of DENV in cell culture has endowed it with the capacity to induce high levels of ADE.

Cross-reactivity of human monoclonal antibodies generated with peripheral blood lymphocytes from dengue patients with Japanese encephalitis virus.

BACKGROUND: Hybridomas that produce human monoclonal antibodies (HuMAbs) against Dengue virus (DV) had been prepared previously using peripheral blood lymphocytes from patients with DV during the acute and convalescent phases of a secondary infection. Anti-DV envelope glycoprotein (E) 99 clones, anti-DV premembrane protein (prM) 8 clones, and anti-DV nonstructural protein 1 (NS1) 4 clones were derived from four acute-phase patients, and anti-DV E 2 clones, anti-DV prM 2 clones, and anti-DV NS1 8 clones were derived from five convalescent-phase patients. METHODS AND RESULTS: In the present study, we examined whether these clones cross-reacted with Japanese encephalitis virus (JEV), which belongs to the same virus family. Forty-six of the above-described 99 (46/99) anti-E, 0/8 anti-prM, and 2/4 anti-NS1 HuMAbs from acute-phase, and 0/2 anti-E, 0/2 anti-prM, and 5/8 anti-NS1 HuMAbs from convalescent-phase showed neutralizing activity against JEV. Thus, most of the anti-E and anti-NS1 (but not the anti-prM) antibodies cross-reacted with JEV and neutralized this virus. Interestingly, 3/46 anti-E HuMAbs derived from acute-phase patients and 3/5 anti-NS1 HuMAbs from convalescent-phase patients showed particularly high neutralizing activity against JEV. Consequently, the HuMAbs showing neutralization against JEV mostly consisted of two populations: one was HuMAbs recognizing DV E and showing neutralization activity against all four DV serotypes (complex-type) and the other was HuMAbs recognizing DV NS1 and showing subcomplex-type cross-reaction with DV. CONCLUSION: Anti-DV E from acute phase (46/99) and anti-DV NS1 (7/12) indicate neutralizing activity against JEV. In particular, three of 46 anti-DV E clones from acute phase and three of five anti-NS1 clones from convalescent phase showed strong neutralizing activity against JEV.

Characterization of chikungunya virus infection of a human keratinocyte cell line: role of mosquito salivary gland protein in suppressing the host immune response.

The chikungunya virus (CHIKV) is a mosquito-borne virus that has recently re-emerged in several countries. On infection, the first vertebrate cells to come into contact with CHIKV are skin cells; mosquitoes inoculate the virus together with salivary gland protein into host skin while probing and feeding on blood. However, there is little known about the susceptibility of human skin cells to CHIKV infection. To clarify this, we investigated the kinetics of CHIKV in the human keratinocyte cell line, HaCaT. CHIKV actively replicated in HaCaT cells, with virus titers in the supernatant increasing to 2.8 × 10(4) plaque-forming units (PFU) ml(-1) 24h post infection. CHIKV infection suppressed production of interleukin-8 (IL-8) in HaCaT cells. The function of IL-8 is to recruit immune cells to virus-infected sites, a process known as chemotaxis. Furthermore, we assessed the role of mosquito salivary gland protein in CHIKV infections by comparing the levels of CHIKV gene expression and chemokine production in HaCaT cells with and without salivary gland extract (SGE). SGE enhanced both the expression of the CHIKV gene and the suppression effect of CHIKV on IL-8 production. Our data suggest that the HaCaT cell line represents an effective tool for investigating the mechanism of CHIKV transmission and spread in skin cells. At the mosquito bite site, CHIKV works together with SGE to ensure the virus replicates in skin cells and escapes the host immune system by suppression of IL-8 production.

Dengue virus neutralization and antibody-dependent enhancement activities of human monoclonal antibodies derived from dengue patients at acute phase of secondary infection.

Public health concern about dengue diseases, caused by mosquito-borne infections with four serotypes of dengue virus (DENV-1-DENV-4), is escalating in tropical and subtropical countries. Most of the severe dengue cases occur in patients experiencing a secondary infection with a serotype that is different from the first infection. This is believed to be due to antibody-dependent enhancement (ADE), by which one DENV serotype uses pre-existing anti-DENV antibodies elicited in the primary infection to facilitate entry of a different DENV serotype into the Fc receptor-positive macrophages. Recently, we prepared a number of hybridomas producing human monoclonal antibodies (HuMAbs) by using peripheral blood lymphocytes from Thai patients at acute phase of secondary infection with DENV-2. Here, we characterized 17 HuMAbs prepared from two patients with dengue fever (DF) and one patient with dengue hemorrhagic fever (DHF) that were selected as antibodies recognizing viral envelope protein and showing higher neutralization activity to all serotypes. In vivo evaluation using suckling mice revealed near perfect activity to prevent mouse lethality following intracerebral DENV-2 inoculation. In a THP-1 cell assay, these HuMAbs showed ADE activities against DENV-2 at similar levels between HuMAbs derived from DF and DHF patients. However, the F(ab')2 fragment of the HuMAb showed a similar virus neutralization activity as original, with no ADE activity. Thus, these HuMAbs could be one of the therapeutic candidates against DENV infection.

Optimizing the HRP-2 in vitro malaria drug susceptibility assay using a reference clone to improve comparisons of Plasmodium falciparum field isolates.

BACKGROUND: Apparent emerging artemisinin-resistant Plasmodium falciparum malaria in Southeast Asia requires development of practical tools to monitor for resistant parasites. Although in vitro anti-malarial susceptibility tests are widely used, uncertainties remain regarding interpretation of P. falciparum field isolate values. METHODS: Performance parameters of the W2 P. falciparum clone (considered artemisinin "sensitive") were evaluated as a reference for the HRP-2 immediate ex vivo assay. Variability in W2 IC50s was assessed, including intra- and inter-assay variability among and between technicians in multiple experiments, over five freeze-thaw cycles, over five months of continuous culture, and before and after transport of drug-coated plates to remote field sites. Nominal drug plate concentrations of artesunate (AS) and dihydroartemisinin (DHA) were verified by LC-MS analysis. Plasmodium falciparum field isolate IC50s for DHA from subjects in an artemisinin-resistant area in Cambodia were compared with W2 susceptibility. RESULTS: Plate drug concentrations and day-to-day technical assay performance among technicians were important sources of variability for W2 IC50s within and between assays. Freeze-thaw cycles, long-term continuous culture, and transport to and from remote sites had less influence. Despite variability in W2 susceptibility, the median IC50s for DHA for Cambodian field isolates were higher (p <0.0001) than the W2 clone (3.9 nM), both for subjects with expected (less than 72 hours; 6.3 nM) and prolonged (greater or equal to 72 hours; 9.6 nM) parasite clearance times during treatment with artesunate monotherapy. CONCLUSION: The W2 reference clone improved the interpretability of field isolate susceptibility from the immediate ex vivo HRP-2 assay from areas of artemisinin resistance. Methods to increase the reproducibility of plate coating may improve overall assay interpretability and utility.

Ex vivo drug sensitivity profiles of Plasmodium falciparum field isolates from Cambodia and Thailand, 2005 to 2010, determined by a histidine-rich protein-2 assay.

BACKGROUND: In vitro drug susceptibility assay of Plasmodium falciparum field isolates processed "immediate ex vivo" (IEV), without culture adaption, and tested using histidine-rich protein-2 (HRP-2) detection as an assay, is an expedient way to track drug resistance. METHODS: From 2005 to 2010, a HRP-2 in vitro assay assessed 451 P. falciparum field isolates obtained from subjects with malaria in western and northern Cambodia, and eastern Thailand, processed IEV, for 50% inhibitory concentrations (IC50) against seven anti-malarial drugs, including artesunate (AS), dihydroartemisinin (DHA), and piperaquine. RESULTS: In western Cambodia, from 2006 to 2010, geometric mean (GM) IC50 values for chloroquine, mefloquine, quinine, AS, DHA, and lumefantrine increased. In northern Cambodia, from 2009-2010, GM IC50 values for most drugs approximated the highest western Cambodia GM IC50 values in 2009 or 2010. CONCLUSIONS: Western Cambodia is associated with sustained reductions in anti-malarial drug susceptibility, including the artemisinins, with possible emergence, or spread, to northern Cambodia. This potential public health crisis supports continued in vitro drug IC50 monitoring of P. falciparum isolates at key locations in the region.

Secreted complement regulatory protein clusterin interacts with dengue virus nonstructural protein 1.

Vascular leakage and shock are the major causes of death in patients with dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). It has been suggested that patients with an elevated level of the free soluble form of dengue virus (DV) nonstructural protein 1 (sNS1) are at risk of developing DHF. To understand the role of sNS1 in blood, we searched for the host molecule with which NS1 interacts in human plasma by affinity purification using a GST-fused NS1. Complement inhibitory factor clusterin (Clu), which naturally inhibits the formation of terminal complement complex (TCC), was identified by mass spectrometry. A recombinant sNS1 produced from 293T cells and sNS1 from DV-infected Vero cells interacted with human Clu. Since an activated complement system reportedly causes vascular leakage, the interaction between sNS1 and Clu may contribute to the progression of DHF.