High-throughput proteomic analysis reveals systemic dysregulation in virally suppressed people living with HIV.

BACKGROUNDPeople living with HIV (PLHIV) receiving antiretroviral therapy (ART) exhibit persistent immune dysregulation and microbial dysbiosis, leading to development of cardiovascular diseases (CVDs). We initially compared plasma proteomic profiles between 205 PLHIV and 120 healthy control participants (HCs) and validated the results in an independent cohort of 639 PLHIV and 99 HCs. Differentially expressed proteins (DEPs) were then associated to microbiome data. Finally, we assessed which proteins were linked with CVD development in PLHIV.METHODSProximity extension assay technology was used to measure 1,472 plasma proteins. Markers of systemic inflammation (C-reactive protein, D-dimer, IL-6, soluble CD14, and soluble CD163) and microbial translocation (IFABP) were measured by ELISA, and gut bacterial species were identified using shotgun metagenomic sequencing. Baseline CVD data were available for all PLHIV, and 205 PLHIV were recorded for development of CVD during a 5-year follow-up.RESULTSPLHIV receiving ART had systemic dysregulation of protein concentrations, compared with HCs. Most of the DEPs originated from the intestine and lymphoid tissues and were enriched in immune- and lipid metabolism-related pathways. DEPs originating from the intestine were associated with specific gut bacterial species. Finally, we identified upregulated proteins in PLHIV (GDF15, PLAUR, RELT, NEFL, COL6A3, and EDA2R), unlike most markers of systemic inflammation, associated with the presence and risk of developing CVD during 5-year follow-up.CONCLUSIONOur findings suggest a systemic dysregulation of protein concentrations in PLHIV; some proteins were associated with CVD development. Most DEPs originated from the gut and were related to specific gut bacterial species.TRIAL REGISTRATIONClinicalTrials.gov NCT03994835.FUNDINGAIDS-fonds (P-29001), ViiV healthcare grant (A18-1052), Spinoza Prize (NWO SPI94-212), European Research Council (ERC) Advanced grant (grant 833247), and Indonesia Endowment Fund for Education.

Longitudinal proteomic profiling of the inflammatory response in dengue patients.

BACKGROUND: The immunopathogenesis of dengue virus (DENV) infection remains incompletely understood. To increase our understanding of inflammatory response in non-severe dengue, we assessed longitudinal changes in the inflammatory proteome in patients with an acute DENV infection. METHODS: Using a multiplex proximity extension assay (PEA), we measured relative levels of 368 inflammatory markers in plasma samples from hospitalized patients with non-severe DENV infection in the acute (n = 43) and convalescence (n = 35) phase of the infection and samples of healthy controls (n = 10). RESULTS: We identified 203 upregulated and 39 downregulated proteins in acute versus convalescent plasma samples. The upregulated proteins had a strong representation of interferon (IFN) and IFN-inducible effector proteins, cytokines (e.g. IL-10, IL-33) and cytokine receptors, chemokines, pro-apoptotic proteins (e.g. granzymes) and endothelial markers. A number of differentially expressed proteins (DEPs) have not been reported in previous studies. Functional network analysis highlighted a central role for IFNγ, IL-10, IL-33 and chemokines. We identified different novel associations between inflammatory proteins and circulating concentrations of the endothelial glycocalyx disruption surrogate marker syndecan-1. Conclusion: This unbiased proteome analysis provides a comprehensive insight in the inflammatory response in DENV infection and its association with glycocalyx disruption.

Risk Factors for Declined Functional Status within 30 days After Elective Surgeries in Elderly Patients: A Prospective Cohort Study.

BACKGROUND: Older adults are at risk of decreasing functional status due to their condition and many factors. Although many studies have been conducted about declining in functional status, based on the author's knowledge, only this study that has conducted about functional status changes in the elderly involving the frailty status which undergoing surgery in Indonesia. There are many factor was postulated, some of that was checked routine and applicable in clinical practice. Furthermore, identification of these risk factors can be used a basis for decision making to perform surgeries in older adults because poor functional status causes declining quality of life in the elderly patients. The aim of this research was to determine the risk factors for declined functional status within 30 days after elective surgeries in elderly patients. METHODS: We conducted a prospective cohort study from July 2021 to December 2021 at Dr. Kariadi Hospital, Semarang, Indonesia. We included patients aged 60 or older who underwent elective surgery under general anesthesia. We excluded those who underwent emergency surgery, day care surgery, or were unwillingness to participate. The functional status were assessed using the ADL (Activity of Daily Living) Barthel index. To identify risk factors of  declined ADL scores, a logistic regression analysis was performed on the age variable, gender, body mass index, frailty status, postoperative complications, as well as haemoglobin, and albumin levels. RESULTS: This study included 191 participants, with 97 women (50.79%) and 94 men (42.21%). Declined in functional status within 30-days after surgery occurred in 54 participants (28.2%). There was a significant changed of functional status before and after surgery. Multivariate analysis showed that independently significant variables for declined functional status were male sex (OR 4.48, p value < 0.001), hypoalbuminemia (OR 2.59, p value 0.02), preoperative functional status (OR 2.37; p value 0.05), and postoperative complications (OR 24.885; p value < 0.001). CONCLUSION: Risk factors for declined functional status within 30 days after elective surgery in older patients are postoperative complications, preoperative functional status, hypoalbuminemia, and male gender.

Hyperresponsive Platelets and a Reduced Platelet Granule Release Capacity Are Associated with Severity and Mortality in COVID-19 Patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is often associated with mild thrombocytopenia and increased platelet reactivity. OBJECTIVE: The aim of the current study was to investigate the adenosine triphosphate (ATP) release kinetics of platelets in hospitalized SARS-CoV-2-infected patients. METHODS: We studied time-dependent platelet activation in whole blood by monitoring the ATP release kinetics upon stimulation with a PAR1 receptor agonist in 41 hospitalized critically ill COVID-19 patients, 47 hospitalized noncritically ill COVID-19 patients, and 30 healthy controls. RESULTS: Our study demonstrated that platelets of critically ill COVID-19 patients were hyper-responsive with a shorter platelet response time (PRT) and a reduced platelet granule release capacity (GRC), probably due to chronic activation. The median PRT of COVID-19 patients admitted to the critical care unit was 10 and 7 seconds shorter than the median PRT in healthy controls and noncritical COVID-19 patients, respectively. Both PRT and GRC were also associated with D-dimer (Spearman r [r s] = -0.51, p < 0.0001 and r s = -0.23, p < 0.05), C-reactive protein (CRP) (r s = -0.59, p < 0.0001 and r s = -0.41, p < 0.01), and neutrophil-to-lymphocyte ratio (NLR) (r s = -0.42, p < 0.0001 and r s = -0.26, p < 0.05). Moreover, an increased PRT and a reduced GRC were associated with an increased mortality (odds ratio [OR]: 18.8, 95% confidence interval [CI]: 6.5-62.8, p < 0.0001 and OR: 4.0; 95% CI: 1.6-10.4, p < 0.01). These relationships remained significant after adjustment for age, sex, D-dimer, CRP, and NLR. CONCLUSION: Using an accessible agonist-induced platelet granule ATP release assay, we show that platelet hyper-responsiveness and reduced platelet GRC in COVID-19 patients were associated with critical illness and mortality.

Microbiome-Related Indole and Serotonin Metabolites are Linked to Inflammation and Psychiatric Symptoms in People Living with HIV.

Background: People living with HIV (PLHIV) exhibit dysregulation of tryptophan metabolism. Altered gut microbiome composition in PLHIV might be involved. Mechanistic consequences within the 3 major tryptophan metabolism pathways (serotonin, kynurenine, and indoles), and functional consequences for platelet, immune and behavioral functions are unknown. We investigated plasma tryptophan metabolites, gut microbiome composition, and their association with platelet function, inflammation, and psychiatric symptoms. Methods: This study included 211 PLHIV on long-term antiretroviral treatment (ART). Plasma tryptophan pathway metabolites were measured using time-of-flight mass spectrometry. Bacterial composition was profiled using metagenomic sequencing. Platelet reactivity and serotonin levels were quantified by flowcytometry and ELISA, respectively. Circulating inflammatory markers were determined using ELISA. Symptoms of depression and impulsivity were measured by DASS-42 and BIS-11 self-report questionnaires, respectively. Results: Plasma serotonin and indole metabolites were associated with gut bacterial composition. Notably, species enriched in PLHIV were associated with 3-methyldioxyindole. Platelet serotonin concentrations were elevated in PLHIV, without effects on platelet reactivity. Plasma serotonin and indole metabolites were positively associated with plasma IL-10 and TNF-α concentrations. Finally, higher tryptophan, serotonin, and indole metabolites were associated with lower depression and anxiety, whereas higher kynurenine metabolites were associated with increased impulsivity. Conclusion: Our results suggest that gut bacterial composition and dysbiosis in PLHIV on ART contribute to tryptophan metabolism, which may have clinical consequences for immune function and behavior.

The characteristics of bacteremia among patients with acute febrile illness requiring hospitalization in Indonesia.

Blood culturing remains the "gold standard" for bloodstream infection (BSI) diagnosis, but the method is inaccessible to many developing countries due to high costs and insufficient resources. To better understand the utility of blood cultures among patients in Indonesia, a country where blood cultures are not routinely performed, we evaluated data from a previous cohort study that included blood cultures for all participants. An acute febrile illness study was conducted from July 2013 to June 2016 at eight major hospitals in seven provincial capitals in Indonesia. All participants presented with a fever, and two-sided aerobic blood cultures were performed within 48 hours of hospital admission. Positive cultures were further assessed for antimicrobial resistance (AMR) patterns. Specimens from participants with negative culture results were screened by advanced molecular and serological methods for evidence of causal pathogens. Blood cultures were performed for 1,459 of 1,464 participants, and the 70.6% (1,030) participants that were negative by dengue NS1 antigen test were included in further analysis. Bacteremia was observed in 8.9% (92) participants, with the most frequent pathogens being Salmonella enterica serovar Typhi (41) and Paratyphi A (10), Escherichia coli (14), and Staphylococcus aureus (10). Two S. Paratyphi A cases had evidence of AMR, and several E. coli cases were multidrug resistant (42.9%, 6/14) or monoresistant (14.3%, 2/14). Culture contamination was observed in 3.6% (37) cases. Molecular and serological assays identified etiological agents in participants having negative cultures, with 23.1% to 90% of cases being missed by blood cultures. Blood cultures are a valuable diagnostic tool for hospitalized patients presenting with fever. In Indonesia, pre-screening patients for the most common viral infections, such as dengue, influenza, and chikungunya viruses, would maximize the benefit to the patient while also conserving resources. Blood cultures should also be supplemented with advanced laboratory tests when available.

Correction: An observational prospective cohort study of the epidemiology of hospitalized patients with acute febrile illness in Indonesia.

[This corrects the article DOI: 10.1371/journal.pntd.0007927.].

Effect of oseltamivir phosphate versus placebo on platelet recovery and plasma leakage in adults with dengue and thrombocytopenia; a phase 2, multicenter, double-blind, randomized trial.

BACKGROUND: Thrombocytopenia, bleeding and plasma leakage are major complications of dengue. Activation of endogenous sialidases with desialylation of platelets and endothelial cells may underlie these complications. We aimed to assess the effects of the neuraminidase inhibitor oseltamivir on platelet recovery and plasma leakage in dengue. METHODS: We performed a phase 2, double-blind, multicenter, randomized trial in adult dengue patients with thrombocytopenia (<70,000/µl) and a duration of illness ≤ 6 days. Oseltamivir phosphate 75mg BID or placebo were given for a maximum of five days. Primary outcomes were the time to platelet recovery (≥ 100,000/µl) or discharge from hospital and the course of measures of plasma leakage. RESULTS: A total of 70 patients were enrolled; the primary outcome could be assessed in 64 patients (31 oseltamivir; 33 placebo). Time to platelet count ≥100,000/µl (n = 55) or discharge (n = 9) were similar in the oseltamivir and placebo group (3.0 days [95% confidence interval, 2.7 to 3.3] vs. 2.9 days [2.5 to 3.3], P = 0.055). The kinetics of platelet count and parameters of plasma leakage (gall bladder thickness, hematocrit, plasma albumin, syndecan-1) were also similar between the groups. DISCUSSION: In this trial, adjunctive therapy with oseltamivir phosphate had no effect on platelet recovery or plasma leakage parameters. TRIAL REGISTRATION: ISRCTN35227717.

Prevalence of HIV Infection and Resistance Mutations in Patients Hospitalized for Febrile Illness in Indonesia.

HIV prevalence in Indonesia is increasing, and only 64% of infected individuals know their status. In a prospective cohort of 1,453 hospitalized patients with unexplained fever, 46 (3.2%) had HIV, including 15 (1.1%) patients without a prior HIV diagnosis. Among 31 subjects previously known to have HIV, 21 (68%) had been receiving combination antiretroviral therapy (cART) at the time of enrollment. Of 39 HIV cases with HIV RNA levels ≥ 100 copies/mL, sequencing for genotype analysis and resistance testing was successful in 30 (77%) subjects. The most common HIV subtypes were AE (90%) and B (10%). Five (16.7%) subjects had resistance mutations to nucleoside and non-nucleoside reverse transcriptase inhibitors, and all of them were on cART. No evidence of transmitted drug resistance was found in newly diagnosed individuals. Hospital-based screening may be an efficient method to expand HIV testing and identify a significant number of new cases. Access to care, close monitoring, expansion of anti-retroviral options, and ensuring availability of CD4 determinations, viral load testing, and genotyping are crucial to control of the epidemic in Indonesia.

Increased Plasma Heparanase Activity and Endothelial Glycocalyx Degradation in Dengue Patients Is Associated With Plasma Leakage.

Background: Endothelial hyper-permeability with plasma leakage and thrombocytopenia are predominant features of severe dengue virus infection. It is well established that heparanase, the endothelial glycocalyx degrading enzyme, plays a major role in various diseases with vascular leakage. It is yet to be elucidated whether heparanase activity plays a major role in dengue-associated plasma leakage. Moreover, the major source of heparanase secretion and activation in dengue remains elusive. Since a relatively high amount of heparanase is stored in platelets, we postulate that heparanase released by activated platelets contributes to the increased plasma heparanase activity during dengue virus infection. Methods: Heparanase activity (plasma and urine), and heparan sulfate and syndecan-1 (plasma levels) were measured in dengue patients with thrombocytopenia in acute phase (n=30), during course of disease (n=10) and in convalescent phase (n=25). Associations with clinical parameters and plasma leakage markers were explored. Platelets from healthy donors were stimulated with dengue non-structural protein-1, DENV2 virus and thrombin to evaluate heparanase release and activity ex vivo. Results: Heparanase activity was elevated in acute dengue and normalized during convalescence. Similarly, glycocalyx components, such as heparan sulfate and syndecan-1, were increased in acute dengue and restored during convalescence. Increased heparanase activity correlated with the endothelial dysfunction markers heparan sulfate and syndecan-1, as well as clinical markers of plasma leakage such as ascites, hematocrit concentration and gall-bladder wall thickening. Notably, platelet number inversely correlated with heparanase activity. Ex vivo incubation of platelets with thrombin and live DENV2 virus, but not dengue virus-2-derived non-structural protein 1 induced heparanase release from platelets. Conclusion: Taken together, our findings suggest that the increase of heparanase activity in dengue patients is associated with endothelial glycocalyx degradation and plasma leakage. Furthermore, thrombin or DENV2 activated platelets may be considered as a potential source of heparanase.

Etiologies of severe acute respiratory infection (SARI) and misdiagnosis of influenza in Indonesia, 2013-2016.

BACKGROUND: Severe acute respiratory infection (SARI) accounts for a large burden of illness in Indonesia. However, epidemiology of SARI in tertiary hospitals in Indonesia is unknown. This study sought to assess the burden, clinical characteristics, and etiologies of SARI and concordance of clinical diagnosis with confirmed etiology. METHODS: Data and samples were collected from subjects presenting with SARI as part of the acute febrile Illness requiring hospitalization study (AFIRE). In tertiary hospitals, clinical diagnosis was ascertained from chart review. Samples were analyzed to determine the "true" etiology of SARI at hospitals and Indonesia Research Partnership on Infectious Diseases (INA-RESPOND) laboratory. Distribution and characteristics of SARI by true etiology and accuracy of clinical diagnosis were assessed. RESULTS: Four hundred and twenty of 1464 AFIRE subjects presented with SARI; etiology was identified in 242 (57.6%), including 121 (28.8%) viruses and bacteria associated with systemic infections, 70 (16.7%) respiratory bacteria and viruses other than influenza virus, and 51 (12.1%) influenza virus cases. None of these influenza patients were accurately diagnosed as having influenza during hospitalization. CONCLUSIONS: Influenza was misdiagnosed among all patients presenting with SARI to Indonesian tertiary hospitals in the AFIRE study. Diagnostic approaches and empiric management should be guided by known epidemiology. Public health strategies to address the high burden of influenza should include broad implementation of SARI screening, vaccination programs, clinician education and awareness campaigns, improved diagnostic capacity, and support for effective point-of-care tests.

Chikungunya in Indonesia: Epidemiology and diagnostic challenges.

BACKGROUND: Chikungunya virus (CHIKV) is often overlooked as an etiology of fever in tropical and sub-tropical regions. Lack of diagnostic testing capacity in these areas combined with co-circulation of clinically similar pathogens such as dengue virus (DENV), hinders CHIKV diagnosis. To better address CHIKV in Indonesia, an improved understanding of epidemiology, clinical presentation, and diagnostic approaches is needed. METHODOLOGY/PRINCIPAL FINDINGS: Acutely hospitalized febrile patients ≥1-year-old were enrolled in a multi-site observational cohort study conducted in Indonesia from 2013 to 2016. Demographic and clinical data were collected at enrollment; blood specimens were collected at enrollment, once during days 14 to 28, and three months after enrollment. Plasma samples negative for DENV by serology and/or molecular assays were screened for evidence of acute CHIKV infection (ACI) by serology and molecular assays. To address the co-infection of DENV and CHIKV, DENV cases were selected randomly to be screened for evidence of ACI. ACI was confirmed in 40/1,089 (3.7%) screened subjects, all of whom were DENV negative. All 40 cases initially received other diagnoses, most commonly dengue fever, typhoid fever, and leptospirosis. ACI was found at five of the seven study cities, though evidence of prior CHIKV exposure was observed in 25.2% to 45.9% of subjects across sites. All subjects were assessed during hospitalization as mildly or moderately ill, consistent with the Asian genotype of CHIKV. Subjects with ACI had clinical presentations that overlapped with other common syndromes, atypical manifestations of disease, or persistent or false-positive IgM against Salmonella Typhi. Two of the 40 cases were possibly secondary ACI. CONCLUSIONS/SIGNIFICANCE: CHIKV remains an underdiagnosed acute febrile illness in Indonesia. Public health measures should support development of CHIKV diagnostic capacity. Improved access to point-of-care diagnostic tests and clinical training on presentations of ACI will facilitate appropriate case management such as avoiding unneccessary treatments or antibiotics, early response to control mosquito population and eventually reducing disease transmission.

Underdiagnoses of Rickettsia in patients hospitalized with acute fever in Indonesia: observational study results.

BACKGROUND: Reports of human rickettsial infection in Indonesia are limited. This study sought to characterize the epidemiology of human rickettsioses amongst patients hospitalized with fever at 8 tertiary hospitals in Indonesia. METHODS: Acute and convalescent blood from 975 hospitalized non-dengue patients was tested for Rickettsia IgM and IgG by ELISA. Specimens from cases with seroconversion or increasing IgM and/or IgG titers were tested for Rickettsia IgM and IgG by IFA and Rickettsia genomes using primers for Rickettsia (R.) sp, R. typhi, and Orientia tsutsugamushi. Testing was performed retrospectively on stored specimens; results did not inform patient management. RESULTS: R. typhi, R. rickettsii, and O. tsutsugamushi IgG antibodies were identified in 269/872 (30.8%), 36/634 (5.7%), and 19/504 (3.8%) of samples, respectively. For the 103/975 (10.6%) non-dengue patients diagnosed with acute rickettsial infection, presenting symptoms included nausea (72%), headache (69%), vomiting (43%), lethargy (33%), anorexia (32%), arthralgia (30%), myalgia (28%), chills (28%), epigastric pain (28%), and rash (17%). No acute rickettsioses cases were suspected during hospitalization. Discharge diagnoses included typhoid fever (44), dengue fever (20), respiratory infections (7), leptospirosis (6), unknown fever (6), sepsis (5), hepatobiliary infections (3), UTI (3), and others (9). Fatalities occurred in 7 (6.8%) patients, mostly with co-morbidities. CONCLUSIONS: Rickettsial infections are consistently misdiagnosed, often as leptospirosis, dengue, or Salmonella typhi infection. Clinicians should include rickettsioses in their differential diagnosis of fever to guide empiric management; laboratories should support evaluation for rickettsial etiologies; and public policy should be implemented to reduce burden of disease.

Leptospirosis in Indonesia: diagnostic challenges associated with atypical clinical manifestations and limited laboratory capacity.

BACKGROUND: The burden of leptospirosis in Indonesia is poorly understood. Data from an observational study conducted from 2013 to 2016 in seven cities across Indonesia was used to estimate the incidence of leptospirosis and document its clinical manifestations in patients requiring hospitalization. METHODS: Specimens from patients hospitalized with acute fever were collected at enrollment, 14-28 days, and 3 months. Demographic and clinical information were collected during study visits and/or retrieved from medical records and double-entered into clinical report forms. After initially screening for dengue virus and other pathogens, specimens were tested at a central Reference Laboratory for anti-Leptospira IgM using commercial ELISA kits and for Leptospira DNA using an in-house quantitative real-time PCR assay. RESULTS: Of 1464 patients enrolled, 45 (3.1%) confirmed cases (by PCR and/or sero-coversion or four-fold increase of IgM) and 6 (0.4%) probable cases (by high titer IgM) of leptospirosis were identified by the Reference Laboratory. Disease incidence at sites ranged from 0 (0%) cases in Denpasar to 17 (8.9%) cases in Semarang. The median age of patients was 41.2 years (range of 5.3 to 85.0 years), and 67% of patients were male. Twenty-two patients (43.1%) were accurately diagnosed at sites, and 29 patients (56.9%) were clinically misdiagnosed as having another infection, most commonly dengue fever (11, 37.9%). Clinically, 20 patients (39.2%) did not present with hyperbilirubinemia or increased creatinine levels. Two patients (3.9%) died, both from respiratory failure. Fifteen patients (29.4%) clinically diagnosed with leptospirosis at sites were negative based on IgM ELISA and/or PCR at the Reference Laboratory. CONCLUSIONS: Leptospirosis remains an important cause of hospitalization in Indonesia. It can have diverse clinical presentations, making it difficult to differentiate from other common tropical infections. PCR combined with ELISA is a powerful alternative to the cumbersome gold-standard microscopic agglutination test, particularly in resource-limited settings.

An observational prospective cohort study of the epidemiology of hospitalized patients with acute febrile illness in Indonesia.

BACKGROUND: The epidemiology of acute febrile illness, a common cause of hospitalization in Indonesia, has not been systematically studied. METHODOLOGY/PRINCIPAL FINDINGS: This prospective observational study enrolled febrile patients (temperature ≥38°C) aged ≥1 year from July 2013 until June 2016 at eight government referral teaching hospitals in seven provincial capitals in Indonesia. Patients were managed according to the hospital standard-of-care (SOC), and blood samples were drawn for molecular and serological assays. Clinical data, laboratory results, and specimens for additional tests were collected at enrollment, days 14-28, and at three months. Regular follow-up visits were then scheduled for every three months either until symptoms resolved or until one year. In total, this study included 1,486 adult and pediatric patients presenting with multi-organ (768, 51.7%), gastrointestinal (497, 33.0%), respiratory (114, 7.7%), constitutional (62, 4.2%), skin and soft-tissue (24, 1.6%), central nervous system (17, 1.1%), or genitourinary (4, 0.3%) manifestations. Microbiological diagnoses were found in 1,003/1,486 (67.5%) participants, of which 351/1,003 (35.0%) were not diagnosed during hospitalization using SOC diagnostic tests. Missed diagnoses included all cases caused by Rickettsia spp., chikungunya, influenza, and Seoul virus. The most common etiologic agents identified were dengue virus (467, 46.6%), Salmonella spp. (103, 10.3%), and Rickettsia spp. (103, 10.3%). The overall mortality was 89 (5.9%). CONCLUSIONS/SIGNIFICANCE: Febrile illness in Indonesia has various microbiologic etiologies and substantial overall mortality. Diagnostic limitations and lack of epidemiologic data resulted in potentially treatable, and at times fatal, diseases being missed.

Platelet dysfunction contributes to bleeding complications in patients with probable leptospirosis.

BACKGROUND: Severe leptospirosis is frequently complicated by a hemorrhagic diathesis, of which the pathogenesis is still largely unknown. Thrombocytopenia is common, but often not to the degree that spontaneous bleeding is expected. We hypothesized that the hemorrhagic complications are not only related to thrombocytopenia, but also to platelet dysfunction, and that increased binding of von Willebrand factor (VWF) to platelets is involved in both platelet dysfunction and increased platelet clearance. METHODOLOGY/PRINCIPAL FINDINGS: A prospective study was carried out in Semarang, Indonesia, enrolling 33 hospitalized patients with probable leptospirosis, of whom 15 developed clinical bleeding, and 25 healthy controls. Platelet activation and reactivity were determined using flow cytometry by measuring the expression of P-selectin and activation of the αIIbß3 integrin by the binding of fibrinogen in unstimulated samples and after ex vivo stimulation by the platelet agonists adenosine-diphosphate (ADP) and thrombin-receptor activating peptide (TRAP). Platelet-VWF binding, before and after VWF stimulation by ristocetin, as well as plasma levels of VWF, active VWF, the VWF-inactivating enzyme ADAMTS13, thrombin-antithrombin complexes (TAT) and P-selectin were also measured. Bleeding complications were graded using the WHO bleeding scale. Our study revealed that platelet activation, with a secondary platelet dysfunction, is a feature of patients with probable leptospirosis, especially in those with bleeding manifestations. There was a significant inverse correlation of bleeding score with TRAP-stimulated P-selectin and platelet-fibrinogen binding (R = -0.72, P = 0.003 and R = -0.66, P = 0.01, respectively) but not with platelet count. Patients with bleeding also had a significantly higher platelet-VWF binding. Platelet counts were inversely correlated with platelet-VWF binding (R = -0.74; P = 0.0009. There were no correlations between platelet-VWF binding and the degree of platelet dysfunction, suggesting that increased platelet-VWF binding does not directly interfere with the platelet αIIbß3 signaling pathway in patients with probable leptospirosis. CONCLUSION/SIGNIFICANCE: Platelet dysfunction is common in probable leptospirosis patients with manifest bleeding. Increased VWF-platelet binding may contribute to the activation and clearance of platelets.