Juan Sanchez

Next-Generation Sequencing of Plasmodium vivax Patient Samples Shows Evidence of Direct Evolution in Drug-Resistance Genes

Understanding the mechanisms of drug resistance in Plasmodium vivax, the parasite that causes the most widespread form of human malaria, is complicated by the lack of a suitable long-term cell culture system for this parasite. In contrast to P. falciparum, which can be more readily manipulated in the laboratory, insights about parasite biology need to be inferred from human studies. Here we analyze the genomes of parasites within 10 human P. vivax infections from the Peruvian Amazon. Using next-generation sequencing we show that some P. vivax infections analyzed from the region are likely polyclonal. Despite their polyclonality we observe limited parasite genetic diversity by showing that three or fewer haplotypes comprise 94% of the examined genomes, suggesting the recent introduction of parasites into this geographic region. In contrast we find more than three haplotypes in putative drug-resistance genes, including the gene encoding dihydrofolate reductase-thymidylate synthase and the P. vivax multidrug resistance associated transporter, suggesting that resistance mutations have arisen independently. Additionally, several drug-resistance genes are located in genomic regions with evidence of increased copy number. Our data suggest that whole genome sequencing of malaria parasites from patients may provide more insight about the evolution of drug resistance than genetic linkage or association studies, especially in geographical regions with limited parasite genetic diversity.

Understanding the Concept of Health Care-Associated Pneumonia in Lung Transplant Recipients.

BACKGROUND Limited data are available regarding the etiologic impact of health care-associated pneumonia (HCAP) in lung transplant recipients. Therefore, our aim was to evaluate the microbiologic differences between HCAP and hospital-acquired pneumonia (HAP)/ventilator-associated pneumonia (VAP) in lung transplant recipients with a radiographically confirmed diagnosis of pneumonia. METHODS We performed a retrospective cohort study of lung transplant recipients with pneumonia at one transplant center over a 7-year period. Eligible patients included lung transplant recipients who developed a first episode of radiographically confirmed pneumonia ≥ 48 h following transplantation. HCAP, HAP, and VAP were classified according to the American Thoracic Society/Infectious Diseases Society of America 2005 guidelines. χ² and Student t tests were used to compare categorical and continuous variables, respectively. RESULTS Sixty-eight lung transplant recipients developed at least one episode of pneumonia. HCAP (n = 42; 62%) was most common, followed by HAP/VAP (n = 26; 38%) stratified in HAP (n = 20; 77%) and VAP (n = 6; 23%). Pseudomonas aeruginosa was the predominantly isolated organism (n = 22; 32%), whereas invasive aspergillosis was uncommon (< 10%). Multiple-drug resistant (MDR) pathogens were less frequently isolated in patients with HCAP compared with HAP/VAP (5% vs 27%; P = .009). Opportunistic pathogens were less frequently identified in lung transplant recipients with HCAP than in those with HAP/VAP (7% vs 27%; P = .02). Lung transplant recipients with HCAP had a similar mortality at 90 days (n = 9 [21%] vs n = 4 [15%]; P = .3) compared with patients with HAP/VAP. CONCLUSIONS HCAP was the most frequent infection in lung transplant recipients. MDR pathogens and opportunistic pathogens were more frequently isolated in HAP/VAP. There were no differences in 30- and 90-day mortality between lung transplant recipients with HCAP and those with HAP/VAP.

Invasive Diagnostic Strategies in Immunosuppressed Patients with Acute Respiratory Distress Syndrome

Immunosuppression predisposes the host to development of pulmonary infections, which can lead to respiratory failure and the development of acute respiratory distress syndrome (ARDS). There are multiple mechanisms by which a host can be immunosuppressed and each is associated with specific infectious pathogens. Early invasive diagnostic modalities such as fiber-optic bronchoscopy with bronchoalveolar lavage, transbronchial biopsy, and open lung biopsy are complementary to serologic and noninvasive studies and assist in rapidly establishing an accurate diagnosis, which allows initiation of appropriate therapy and may improve outcomes with relative safety.

How Tifacogin Could Not Captivate Severe Community-acquired Pneumonia

Community-acquired pneumonia is the number one cause of severe sepsis and the seventh leading cause of death, with an estimated 1.3 million hospitalizations each year in the United States and an estimated cost of

Update in intensive care medicine: Studies that challenged our practice in the last 5 years

40 billion (1). The mortality rate has not changed significantly over the past 40 years (2, 3). The current definition of severe community-acquired pneumonia (sCAP) is a pneumonia that requires treatment in the ICU beyond the obvious ICU admission criteria such as need for vasopressors support and mechanical ventilation. Multiple severity-of-illness scores have been developed to help physicians identify patients with sCAP that places patients at risk of poor outcomes (3, 4). Over the last two decades, understanding of the coagulation cascade in the pathogenesis of sepsis has led to novel therapeutic targets. Coagulopathy in sepsis is the result of activation of the coagulation process and of diffuse endothelial injury caused by the infecting organism (5). Endothelial injury leads to dysfunction that is associated with activation of the extrinsic and intrinsic coagulation pathways and reduction of anticoagulant activity (6). This leads to capillary thrombosis and hemorrhage that results in severe alterations of the blood flow, contributing to organ dysfunction and death. Based on these alterations of the coagulation process, the use of anticoagulants has been explored in the last two decades as therapy for severe sepsis and septic shock with mixed results (5). Tissue factor (TF) is the primary initiator of the coagulation cascade. Binding to the extracellular domain of TF activates the factor VII, which in activates factor X, leading to generation of thrombin and fibrin. Endotoxins, cytokines, thrombin, and immune complexes can induce the expression of TF on neutrophils, monocytes, and endothelial cells (7). TF pro-coagulant activity is controlled by an endogenous Kunitz-type protease inhibitor tissue factor pathway inhibitor (TFPI). TFPI exists in two forms: TFPI-1 and TFPI-2. TFPI-1 is the main regulator of the tissue factor pathway. TFPI-2 is a strong inhibitor of trypsin, plasmin, plasma kallikrein, and factor Xia. TFPI is predominantly expressed by the microvascular endothelium. The anticoagulant activity of TFPI occurs in a two-stage process. First, TFPI binds and deactivates factor Xa. Second, the TFIP:Xa complex then rapidly binds to TF:VIIa through the first domain, preventing thrombin generation and proinflammatory intracellular signaling through the activation of protease-activated receptor 2 (PAR-2) pathway (8). Secondary to the anticoagulant and antiinflammatory activity of TFPI, a recombinant TFPI (tifacogin) has been trialed in human studies as adjunctive therapy for sepsis (9). Two phase I and II studies revealed a small improvement in the 28-day mortality as compared with placebo, with similar adverse effects in both groups (10). A phase III study revealed no differences in the overall 28-day mortality rate of the treatment and placebo groups (9). However, subgroup analysis showed a trend toward improved survival in patients with sCAP treated with tifacogin. The benefit was documented in patients with a microbiologically identified infection who did not receive heparin prophylaxis. There was also a trend toward benefit from treatment in patients in shock as compared with patients without shock (11). In this issue of the Journal, Wunderink and coworkers (pp. 1561–1568) report a major prospective, blinded, randomized, controlled, multicenter trial that assessed the efficacy of tifacogin in 2,102 patients with sCAP (CAPTIVATE study) (12). This was a well-executed three-arms study; two of the arms compared doses and the third arm received a placebo. The use of heparin was prohibited during the infusion of tifacogin. The primary efficacy endpoint was severity-adjusted, 28-day, all-cause mortality and there were 14 other secondary endpoints. One of the arms of the study, tifacogin given at 0.075 mg/kg/hour, was stopped early for futility. The main result of the study was that the administration of tifacogin did not improve mortality or any of the other secondary outcomes despite evidence of pharmacodynamic effect. Even in well-defined subgroups, such as patients with a defined bacterial etiology, there was no benefit in mortality. There was no difference between the groups in adverse events such as bleeding or venous thromboembolism. What does this negative study mean to the clinical community who takes care of these very ill patients? First, we believe that this study definitely shows that the use of tifacogin in patients with sCAP or in patients with sepsis is not associated with benefits. Second, tifacogin joins the pantheon with other anticoagulants that were not found to be associated with improved outcomes in patients with sepsis, and achieved at best mixed results (5). It will be very difficult to resuscitate it after this well-conducted study. Third, it reminds us of the high mortality of sCAP (about 18% at 28 d), and leaves us for now seeking strategies to optimize the management process of these patients admitted to our hospitals and ICUs (13). We are anxiously waiting for the results of the new trial of the use of drotrecogin in patients in septic shock in the next several weeks. As our understanding of the role of coagulation and fibrinolysis in the pathogenesis of sepsis and sCAP advances, so perhaps will our ability to regulate these pathways to improve outcomes of our patients.

An Unusual Cause of Pulmonary Artery Thrombosis

During the last 5 years, new randomized trials in critically ill patients have challenged a number of traditional treatment strategies in intensive care. The authors review eight studies that helped change their medical practices. Several once-established therapies have failed the test of time, as the result of evidence from clinical trials.