Multidrug-Resistant and Virulent Organisms Trauma Infections: Trauma Infectious Disease Outcomes Study Initiative.

INTRODUCTION: During the wars in Iraq and Afghanistan, increased incidence of multidrug-resistant (MDR) organisms, as well as polymicrobial wounds and infections, complicated the management of combat trauma-related infections. Multidrug resistance and wound microbiology are a research focus of the Trauma Infectious Disease Outcomes Study (TIDOS), an Infectious Disease Clinical Research Program, Uniformed Services University, research protocol. To conduct comprehensive microbiological research with the goal of improving the understanding of the complicated etiology of wound infections, the TIDOS MDR and Virulent Organisms Trauma Infections Initiative (MDR/VO Initiative) was established as a collaborative effort with the Brooke Army Medical Center, Naval Medical Research Center, U.S. Army Institute of Surgical Research, and Walter Reed Army Institute of Research. We provide a review of the TIDOS MDR/VO Initiative and summarize published findings. METHODS: Antagonism and biofilm formation of commonly isolated wound bacteria (e.g., ESKAPE pathogens-Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), antimicrobial susceptibility patterns, and clinical outcomes are being examined. Isolates collected from admission surveillance swabs, as part of infection control policy, and clinical infection workups were retained in the TIDOS Microbiological Repository and associated clinical data in the TIDOS database. RESULTS: Over the TIDOS study period (June 2009 to December 2014), more than 8,300 colonizing and infecting isolates were collected from military personnel injured with nearly one-third of isolates classified as MDR. At admission to participating U.S. military hospitals, 12% of wounded warriors were colonized with MDR Gram-negative bacilli. Furthermore, 27% of 913 combat casualties with ≥1 infection during their trauma hospitalization had MDR Gram-negative bacterial infections. Among 335 confirmed combat-related extremity wound infections (2009-2012), 61% were polymicrobial and comprised various combinations of Gram-negative and Gram-positive bacteria, yeast, fungi, and anaerobes. Escherichia coli was the most common Gram-negative bacilli isolated from clinical workups, as well as the most common colonizing MDR secondary to extended-spectrum ß-lactamase resistance. Assessment of 479 E. coli isolates collected from wounded warriors found 188 pulsed-field types (PFTs) from colonizing isolates and 54 PFTs from infecting isolates without significant overlap across combat theaters, military hospitals, and study years. A minority of patients with colonizing E. coli isolates developed subsequent infections with the same E. coli strain. Enterococcus spp. were most commonly isolated from polymicrobial wound infections (53% of 204 polymicrobial cultures). Patients with Enterococcus infections were severely injured with a high proportion of lower extremity amputations and genitourinary injuries. Approximately 65% of polymicrobial Enterococcus infections had other ESKAPE organisms isolated. As biofilms have been suggested as a cause of delayed wound healing, wound infections with persistent recovery of bacteria (isolates of same organism collected ≥14 days apart) and nonrecurrent bacterial isolates were assessed. Biofilm production was significantly associated with recurrent bacteria isolation (97% vs. 59% with nonrecurrent isolates; P < 0.001); however, further analysis is needed to confirm biofilm formation as a predictor of persistent wound infections. CONCLUSIONS: The TIDOS MDR/VO Initiative provides comprehensive and detailed data of major microbial threats associated with combat-related wound infections to further the understanding of wound etiology and potentially identify infectious disease countermeasures, which may lead to improvements in combat casualty care.

Semimechanistic Modeling of the Effects of Blast Overpressure Exposure on Cefazolin Pharmacokinetics in Mice.

Cefazolin is a first-line antibiotic to treat infection related to deployment-associated blast injuries. Prior literature demonstrated a 331% increase cefazolin liver area under the curve (AUC) in mice exposed to a survivable blast compared with controls. We repeated the experiment, validated the findings, and established a semimechanistic two-compartment pharmacokinetic (PK) model with effect compartments representing the liver and skin. We found that blast statistically significantly increased the pseudo-partition coefficient to the liver by 326% (95% confidence interval: 76-737%), which corresponds to the observed 331% increase in cefazolin liver AUC described previously. To a lesser extent, plasma AUC in blasted mice increased 14-45% compared with controls. Nevertheless, the effects of blast on cefazolin PK were transient, normalizing by 10 hours after the dose. It is unclear as to how this blast effect t emporally translates to humans; however, given the short-lived effect on PK, there is insufficient evidence to recommend cefazolin dosing changes based on blast overpressure injury alone. Clinicians should be aware that cefazolin may cause drug-induced liver injury with a single dose and the risk may be higher in patients with blast overpressure injury based on our findings. SIGNIFICANCE STATEMENT: Blast exposure significantly, but transiently, alters cefazolin pharmacokinetics in mice. The questions of whether other medications or potential long-term consequences in humans need further exploration.

Synergistic Killing and Re-Sensitization of Pseudomonas aeruginosa to Antibiotics by Phage-Antibiotic Combination Treatment.

Multidrug-resistant (MDR) Pseudomonas aeruginosa infections pose a serious health threat. Bacteriophage-antibiotic combination therapy is a promising candidate for combating these infections. A 5-phage P. aeruginosa cocktail, PAM2H, was tested in combination with antibiotics (ceftazidime, ciprofloxacin, gentamicin, meropenem) to determine if PAM2H enhances antibiotic activity. Combination treatment in vitro resulted in a significant increase in susceptibility of MDR strains to antibiotics. Treatment with ceftazidime (CAZ), meropenem, gentamicin, or ciprofloxacin in the presence of the phage increased the number of P. aeruginosa strains susceptible to these antibiotics by 63%, 56%, 31%, and 81%, respectively. Additionally, in a mouse dorsal wound model, seven of eight mice treated with a combination of CAZ and PAM2H for three days had no detectable bacteria remaining in their wounds on day 4, while all mice treated with CAZ or PAM2H alone had ~107 colony forming units (CFU) remaining in their wounds. P. aeruginosa recovered from mouse wounds post-treatment showed decreased virulence in a wax worm model, and DNA sequencing indicated that the combination treatment prevented mutations in genes encoding known phage receptors. Treatment with PAM2H in combination with antibiotics resulted in the re-sensitization of P. aeruginosa to antibiotics in vitro and a synergistic reduction in bacterial burden in vivo.

Distribution and Temporal Dynamics of Plasmodium falciparum Chloroquine Resistance Transporter Mutations Associated With Piperaquine Resistance in Northern Cambodia.

BACKGROUND: Newly emerged mutations within the Plasmodium falciparum chloroquine resistance transporter (PfCRT) can confer piperaquine resistance in the absence of amplified plasmepsin II (pfpm2). In this study, we estimated the prevalence of co-circulating piperaquine resistance mutations in P. falciparum isolates collected in northern Cambodia from 2009 to 2017. METHODS: The sequence of pfcrt was determined for 410 P. falciparum isolates using PacBio amplicon sequencing or whole genome sequencing. Quantitative polymerase chain reaction was used to estimate pfpm2 and pfmdr1 copy number. RESULTS: Newly emerged PfCRT mutations increased in prevalence after the change to dihydroartemisinin-piperaquine in 2010, with >98% of parasites harboring these mutations by 2017. After 2014, the prevalence of PfCRT F145I declined, being outcompeted by parasites with less resistant, but more fit PfCRT alleles. After the change to artesunate-mefloquine, the prevalence of parasites with amplified pfpm2 decreased, with nearly half of piperaquine-resistant PfCRT mutants having single-copy pfpm2. CONCLUSIONS: The large proportion of PfCRT mutants that lack pfpm2 amplification emphasizes the importance of including PfCRT mutations as part of molecular surveillance for piperaquine resistance in this region. Likewise, it is critical to monitor for amplified pfmdr1 in these PfCRT mutants, as increased mefloquine pressure could lead to mutants resistant to both drugs.

Comparison of Pseudomonas aeruginosa strains reveals that Exolysin A toxin plays an additive role in virulence.

BACKGROUND: Pseudomonas aeruginosa possesses an array of virulence genes ensuring successful infection development. A two-partner secretion system Exolysin BA (ExlBA) is expressed in the PA7-like genetic outliers consisting of ExlA, a pore-forming toxin and ExlB transporter protein. Presence of exlBA in multidrug-resistant (MDR) strains has not been investigated, particularly in the strains isolated from wounded soldiers. METHODS: We screened whole genome sequences of 2439 MDR- P. aeruginosa strains for the presence of exlBA. We compiled all exlBA positive strains and compared them with a diversity set for demographics, antimicrobial profiles and phenotypic characteristics: surface motility, biofilm formation, pyocyanin production and hemolysis. We compared the virulence of strains with comparable phenotypic characteristics in Galleria mellonella. RESULTS: We identified 33 exlBA-positive strains (1.5%). These strains have increased antibiotic resistance, they are more motile, produce more robust biofilms and have comparable pyocianin production with the diversity set despite the phenotypic differences within the group. In in vivo infection models, these strains were less virulent than Type III Secretion System (T3SS) positive counterparts. CONCLUSIONS: exlBA-positive strains are wide spread among the PA7-like outliers. While not as virulent as strains possessing T3SS, these strains exhibit phenotypic features associated with virulence and are still lethal in vivo.

A Porcine Wound Model of Acinetobacter baumannii Infection.

Objective: To better understand Acinetobacter baumannii pathogenesis and to advance drug discovery against this pathogen, we developed a porcine, full-thickness, excisional, monospecies infection wound model. Approach: The research was facilitated with AB5075, a previously characterized, extensively drug-resistant A. baumannii isolate. The model requires cyclophosphamide-induced neutropenia to establish a skin and soft tissue infection (SSTI) that persists beyond 7 days. Multiple, 12-mm-diameter full-thickness wounds were created in the skin overlying the cervical and thoracic dorsum. Wound beds were inoculated with 5.0 × 104 colony-forming units (CFU) and covered with dressing. Results: A. baumannii was observed in the wound bed and on the dressing in what appeared to be biofilm. When bacterial burdens were measured, proliferation to at least 106 CFU/g (log106) wound tissue was observed. Infection was further characterized by scanning electron microscopy (SEM) and peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) staining. To validate as a treatment model, polymyxin B was applied topically to a subset of infected wounds every 2 days. Then, the treated and untreated wounds were compared using multiple quantitative and qualitative techniques to include gross pathology, CFU burden, histopathology, PNA-FISH, and SEM. Innovation: This is the first study to use A. baumannii in a porcine model as the sole infectious agent. Conclusion: The porcine model allows for an additional preclinical assessment of antibacterial candidates that show promise against A. baumannii in rodent models, further evaluating safety and efficacy, and serve as a large animal in preclinical assessment for the treatment of SSTI.

Association of Enterococcus spp. with Severe Combat Extremity Injury, Intensive Care, and Polymicrobial Wound Infection.

BACKGROUND: Combat-related extremity wound infections can complicate the recovery of injured military personnel. The Enterococcus genus contains both commensal and pathogenic bacteria found in many combat wounds. We describe the patient population susceptible to Enterococcus infection, the characteristics of Enterococcus spp. isolated from combat-related wounds, and the microbiological profile of Enterococcus-positive wounds. METHODS: Patient and culture data were obtained from the Trauma Infectious Disease Outcomes Study. Subjects were divided into a case group with enterococcal extremity wound infections and a comparator group with wound infections caused by other micro-organisms. RESULTS: Case and comparator subjects had similar patterns of injury and infection. Case subjects had higher Injury Severity Scores (33 vs. 30; p < 0.001), longer hospitalization at U.S. facilities (55 vs. 40 days; p = 0.004), and required more large-volume blood transfusions (>20 units) within 24 h post-injury (53% vs. 30%; p < 0.001). Approximately 60% of case subjects had three or more infections, and 91% had one or more polymicrobial infections, compared with 43% and 50%, respectively, in the comparator group. The thigh was the most common site of Enterococcus spp. isolation, contributing 50% of isolates. Enterococcus faecium was the predominant species isolated from case-group infections overall (66%), as well as in polymicrobial infections (74%). Frequent co-colonizing microbes in polymicrobial wound infections with Enterococcus were other ESKAPE pathogens (64%) (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae [and Escherichia coli], Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) and fungi (35%). CONCLUSIONS: The specific pathogenicity of Enterococcus relative to other pathogens in polymicrobial wounds is unknown. Identifying strain-specific outcomes and investigating the interactions of Enterococcus strains with other wound pathogens could provide additional tools and strategies for infection mitigation in combat-related wounds.

SPR741, an Antibiotic Adjuvant, Potentiates the In Vitro and In Vivo Activity of Rifampin against Clinically Relevant Extensively Drug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is responsible for 10% of all nosocomial infections and has >50% mortality rates when causing ventilator-associated pneumonia. In this proof-of-concept study, we evaluated SPR741, an antibiotic adjuvant that permeabilizes the Gram-negative membrane, in combination with rifampin against AB5075, an extensively drug-resistant (XDR) A. baumannii strain. In standard in vitro assays and in a murine pulmonary model, we found that this drug combination can significantly reduce bacterial burden and promote animal survival despite an aggressive infection.

Human Sentinel Surveillance of Influenza and Other Respiratory Viral Pathogens in Border Areas of Western Cambodia.

Little is known about circulation of influenza and other respiratory viruses in remote populations along the Thai-Cambodia border in western Cambodia. We screened 586 outpatients (median age 5, range 1-77) presenting with influenza-like-illness (ILI) at 4 sentinel sites in western Cambodia between May 2010 and December 2012. Real-time reverse transcriptase (rRT) PCR for influenza was performed on combined nasal and throat specimens followed by viral culture, antigenic analysis, antiviral susceptibility testing and full genome sequencing for phylogenetic analysis. ILI-specimens negative for influenza were cultured, followed by rRT-PCR for enterovirus and rhinovirus (EV/RV) and EV71. Influenza was found in 168 cases (29%) and occurred almost exclusively in the rainy season from June to November. Isolated influenza strains had close antigenic and phylogenetic relationships, matching vaccine and circulating strains found elsewhere in Cambodia. Influenza vaccination coverage was low (<20%). Western Cambodian H1N1(2009) isolate genomes were more closely related to 10 earlier Cambodia isolates (94.4% genome conservation) than to 13 Thai isolates (75.9% genome conservation), despite sharing the majority of the amino acid changes with the Thai references. Most genes showed signatures of purifying selection. Viral culture detected only adenovirus (5.7%) and parainfluenza virus (3.8%), while non-polio enteroviruses (10.3%) were detected among 164 culture-negative samples including coxsackievirus A4, A6, A8, A9, A12, B3, B4 and echovirus E6 and E9 using nested RT-PCR methods. A single specimen of EV71 was found. Despite proximity to Thailand, influenza epidemiology of these western Cambodian isolates followed patterns observed elsewhere in Cambodia, continuing to support current vaccine and treatment recommendations from the Cambodian National Influenza Center. Amino acid mutations at non-epitope sites, particularly hemagglutinin genes, require further investigation in light of an increasingly important role of permissive mutations in influenza virus evolution. Further research about the burden of adenovirus and non-polio enteroviruses as etiologic agents in acute respiratory infections in Cambodia is also needed.

Attenuation of Plasmodium falciparum in vitro drug resistance phenotype following culture adaptation compared to fresh clinical isolates in Cambodia.

BACKGROUND: There is currently no standardized approach for assessing in vitro anti-malarial drug susceptibility. Potential alterations in drug susceptibility results between fresh immediate ex vivo (IEV) and cryopreserved culture-adapted (CCA) Plasmodium falciparum isolates, as well as changes in parasite genotype during culture adaptation were investigated. METHODS: The 50 % inhibitory concentration (IC50) of 12 P. falciparum isolates from Cambodia against a panel of commonly used drugs were compared using both IEV and CCA. Results were compared using both histidine-rich protein-2 ELISA (HRP-2) and SYBR-Green I fluorescence methods. Molecular genotyping and amplicon deep sequencing were also used to compare multiplicity of infection and genetic polymophisms in fresh versus culture-adapted isolates. RESULTS: IC50 for culture-adapted specimens were significantly lower compared to the original fresh isolates for both HRP-2 and SYBR-Green I assays, with greater than a 50 % decline for the majority of drug-assay combinations. There were correlations between IC50s from IEV and CCA for most drugs assays. Infections were nearly all monoclonal, with little or no change in merozoite surface protein 1 (MSP1), MSP2, glutamate-rich protein (GLURP) or apical membrane antigen 1 (AMA1) polymorphisms, nor differences in P. falciparum multidrug resistance 1 gene (PfMDR1) copy number or single nucleotide polymorphisms following culture adaptation. CONCLUSIONS: The overall IC50 reduction combined with the correlation between fresh isolates and culture-adapted drug susceptibility assays suggests the utility of both approaches, as long as there is consistency of method, and remaining mindful of possible attenuation of resistance phenotype occurring in culture. Further study should be done in higher transmission settings where polyclonal infections are prevalent.

Ex Vivo Drug Susceptibility Testing and Molecular Profiling of Clinical Plasmodium falciparum Isolates from Cambodia from 2008 to 2013 Suggest Emerging Piperaquine Resistance.

Cambodia's first-line artemisinin combination therapy, dihydroartemisinin-piperaquine (DHA-PPQ), is no longer sufficiently curative against multidrug-resistant Plasmodium falciparum malaria at some Thai-Cambodian border regions. We report recent (2008 to 2013) drug resistance trends in 753 isolates from northern, western, and southern Cambodia by surveying for ex vivo drug susceptibility and molecular drug resistance markers to guide the selection of an effective alternative to DHA-PPQ. Over the last 3 study years, PPQ susceptibility declined dramatically (geomean 50% inhibitory concentration [IC50] increased from 12.8 to 29.6 nM), while mefloquine (MQ) sensitivity doubled (67.1 to 26 nM) in northern Cambodia. These changes in drug susceptibility were significantly associated with a decreased prevalence of P. falciparum multidrug resistance 1 gene (Pfmdr1) multiple copy isolates and coincided with the timing of replacing artesunate-mefloquine (AS-MQ) with DHA-PPQ as the first-line therapy. Widespread chloroquine resistance was suggested by all isolates being of the P. falciparum chloroquine resistance transporter gene CVIET haplotype. Nearly all isolates collected from the most recent years had P. falciparum kelch13 mutations, indicative of artemisinin resistance. Ex vivo bioassay measurements of antimalarial activity in plasma indicated 20% of patients recently took antimalarials, and their plasma had activity (median of 49.8 nM DHA equivalents) suggestive of substantial in vivo drug pressure. Overall, our findings suggest DHA-PPQ failures are associated with emerging PPQ resistance in a background of artemisinin resistance. The observed connection between drug policy changes and significant reduction in PPQ susceptibility with mitigation of MQ resistance supports reintroduction of AS-MQ, in conjunction with monitoring of the P. falciparum mdr1 copy number, as a stop-gap measure in areas of DHA-PPQ failure.

Pathogens present in acute mangled extremities from Afghanistan and subsequent pathogen recovery.

Given the changing epidemiology of infecting pathogens in combat casualties, we evaluated bacteria and fungi in acute traumatic wounds from Afghanistan. From January 2013 to February 2014, 14 mangled lower extremities from 10 explosive-device injured casualties were swabbed for culture at Role 3 facilities. Bacteria were recovered from all patients on the date of injury. Pathogens recovered during routine patient care were recorded. The median injury severity score was 29, median initial Role 3/4 blood product support was 32 units, and median evacuation time was 42 minutes to first surgical care. Gram-positive bacteria were found in some wounds but not methicillin-resistant Staphylococcus aureus or vancomycin-resistant Enterococcus. Most wounds were colonized with low-virulence, environmental gram-negative bacteria, and not recovered again during therapy, reflecting wound contamination. Only one wound had the same bacteria (E. cloacae) throughout care at the Role 3, 4, and 5 facilities. Three cultures from two patients had multidrug-resistant bacteria (E. cloacae, E. coli), all detected at Role 5 facilities. Molds were not detected at Role 3, whereas one patient had a mold at Role 4 and 5. Mangled lower extremity injuries have a high contamination rate with environmental organisms, which are not typically associated with infections during the course of the patient's care.

Independent emergence of artemisinin resistance mutations among Plasmodium falciparum in Southeast Asia.

BACKGROUND: The emergence of artemisinin-resistant Plasmodium falciparum in Southeast Asia threatens malaria treatment efficacy. Mutations in a kelch protein encoded on P. falciparum chromosome 13 (K13) have been associated with resistance in vitro and in field samples from Cambodia. METHODS: P. falciparum infections from artesunate efficacy trials in Bangladesh, Cambodia, Laos, Myanmar, and Vietnam were genotyped at 33 716 genome-wide single-nucleotide polymorphisms (SNPs). Linear mixed models were used to test associations between parasite genotypes and parasite clearance half-lives following artesunate treatment. K13 mutations were tested for association with artemisinin resistance, and extended haplotypes on chromosome 13 were examined to determine whether mutations arose focally and spread or whether they emerged independently. RESULTS: The presence of nonreference K13 alleles was associated with prolonged parasite clearance half-life (P = 1.97 × 10(-12)). Parasites with a mutation in any of the K13 kelch domains displayed longer parasite clearance half-lives than parasites with wild-type alleles. Haplotype analysis revealed both population-specific emergence of mutations and independent emergence of the same mutation in different geographic areas. CONCLUSIONS: K13 appears to be a major determinant of artemisinin resistance throughout Southeast Asia. While we found some evidence of spreading resistance, there was no evidence of resistance moving westward from Cambodia into Myanmar.

Utility of a sports medicine model in military combat concussion and musculoskeletal restoration care.

Combat-related concussions are significant sources of injury and morbidity among deployed military service members. Musculoskeletal injury also is one of the most prevalent battle and nonbattle-related deployed injury types. Both injuries threaten the service member's physical condition as well as unit and mission readiness due to reduced duty status or evacuation from military theater of operations. In August 2010, the Concussion Restoration Care Center (CRCC) was established at Camp Leatherneck, Afghanistan, to address the need for consistent and specialized evaluation and care of concussion and musculoskeletal injury. This performance improvement effort examined evaluation and treatment of concussion and musculoskeletal injury at the CRCC. Among 4,947 military personnel evaluated at the CRCC between August 2010 and May 2013, 97.9% were returned to duty and retained in theater. Members averaged 10 to 12 days of limited duty status to achieve complete recovery. Concussion injury was secondary to blast injury in 90% of cases. Sport/recreation, occupational, and other accidental injuries each represented 30% of the musculoskeletal injuries with only 10% reported as result of combat. The utilization patterns and outcome measures demonstrate the success and utility of a multidisciplinary clinical model of care for these two types of injuries in the far-forward deployed setting.

Tranexamic acid administration to pediatric trauma patients in a combat setting: the pediatric trauma and tranexamic acid study (PED-TRAX).

BACKGROUND: Early administration of tranexamic acid (TXA) has been associated with a reduction in mortality and blood product requirements in severely injured adults. It has also shown significantly reduced blood loss and transfusion requirements in major elective pediatric surgery, but no published data have examined the use of TXA in pediatric trauma. METHODS: This is a retrospective review of all pediatric trauma admissions to the North Atlantic Treaty Organization Role 3 hospital, Camp Bastion, Afghanistan, from 2008 to 2012. Univariate and logistic regression analyses of all patients and select subgroups were performed to identify factors associated with TXA use and mortality. Standard adult dosing of TXA was used in all patients. RESULTS: There were 766 injured patients 18 years or younger (mean [SD] age, 11 [5] years; 88% male; 73% penetrating injury; mean [SD], Injury Severity Score [ISS], 10 [9]; mean [SD] Glasgow Coma Scale [GCS] score, 12 [4]). Of these patients, 35% required transfusion in the first 24 hours, 10% received massive transfusion, and 76% required surgery. Overall mortality was 9%. Of the 766 patients, 66 (9%) received TXA. The only independent predictors of TXA use were severe abdominal or extremity injury (Abbreviated Injury Scale [AIS] score ≥ 3) and a base deficit of greater than 5 (all p < 0.05). Patients who received TXA had greater injury severity, hypotension, acidosis, and coagulopathy versus the patients in the no-TXA group. After correction for demographics, injury type and severity, vitals, and laboratory parameters, TXA use was independently associated with decreased mortality among all patients (odds ratio, 0.3; p = 0.03) and showed similar trends for subgroups of severely injured (ISS > 15) and transfused patients. There was no significant difference in thromboembolic complications or other cardiovascular events. Propensity analysis confirmed the TXA-associated survival advantage and suggested significant improvements in discharge neurologic status as well as decreased ventilator dependence. CONCLUSION: TXA was used in approximately 10% of pediatric combat trauma patients, typically in the setting of severe abdominal or extremity trauma and metabolic acidosis. TXA administration was independently associated with decreased mortality. There were no adverse safety- or medication-related complications identified. LEVEL OF EVIDENCE: Therapeutic study, level IV.

Ex vivo activity of endoperoxide antimalarials, including artemisone and arterolane, against multidrug-resistant Plasmodium falciparum isolates from Cambodia.

Novel synthetic endoperoxides are being evaluated as new components of artemisinin combination therapies (ACTs) to treat artemisinin-resistant Plasmodium falciparum malaria. We conducted blinded ex vivo activity testing of fully synthetic (OZ78 and OZ277) and semisynthetic (artemisone, artemiside, artesunate, and dihydroartemisinin) endoperoxides in the histidine-rich protein 2 enzyme-linked immunosorbent assay against 200 P. falciparum isolates from areas of artemisinin-resistant malaria in western and northern Cambodia in 2009 and 2010. The order of potency and geometric mean (GM) 50% inhibitory concentrations (IC50s) were as follows: artemisone (2.40 nM) > artesunate (8.49 nM) > dihydroartemisinin (11.26 nM) > artemiside (15.28 nM) > OZ277 (31.25 nM) > OZ78 (755.27 nM). Ex vivo activities of test endoperoxides positively correlated with dihydroartemisinin and artesunate. The isolates were over 2-fold less susceptible to dihydroartemisinin than the artemisinin-sensitive P. falciparum W2 clone and showed sensitivity comparable to those with test endoperoxides and artesunate, with isolate/W2 IC50 susceptibility ratios of <2.0. All isolates had P. falciparum chloroquine resistance transporter mutations, with negative correlations in sensitivity to endoperoxides and chloroquine. The activities of endoperoxides (artesunate, dihydroartemisinin, OZ277, and artemisone) significantly correlated with that of the ACT partner drug, mefloquine. Isolates had mutations associated with clinical resistance to mefloquine, with 35% prevalence of P. falciparum multidrug resistance gene 1 (pfmdr1) amplification and 84.5% occurrence of the pfmdr1 Y184F mutation. GM IC50s for mefloquine, lumefantrine, and endoperoxides (artesunate, dihydroartemisinin, OZ277, OZ78, and artemisone) correlated with pfmdr1 copy number. Given that current ACTs are failing potentially from reduced sensitivity to artemisinins and partner drugs, newly identified mutations associated with artemisinin resistance reported in the literature and pfmdr1 mutations should be examined for their combined contributions to emerging ACT resistance.

Efficacy of two versus three-day regimens of dihydroartemisinin-piperaquine for uncomplicated malaria in military personnel in northern Cambodia: an open-label randomized trial.

INTRODUCTION: Emerging antimalarial drug resistance in mobile populations remains a significant public health concern. We compared two regimens of dihydroartemisinin-piperaquine in military and civilians on the Thai-Cambodian border to evaluate national treatment policy. METHODS: Efficacy and safety of two and three-day regimens of dihydroartemisinin-piperaquine were compared as a nested open-label evaluation within a malaria cohort study in 222 otherwise healthy volunteers (18% malaria-infected at baseline). The first 80 volunteers with slide-confirmed Plasmodium falciparum or vivax malaria were randomized 1:1 to receive either regimen (total dose 360 mg dihydroartemisinin and 2880 mg piperaquine) and followed weekly for up to 6 months. The primary endpoint was malaria recurrence by day 42. Volunteers with vivax infection received primaquine at study discharge with six months follow-up. RESULTS: Eighty patients (60 vivax, 15 falciparum, and 5 mixed) were randomized to dihydroartemisinin-piperaquine. Intention-to-treat all-species efficacy at Day 42 was 85% for the two-day regimen (95% CI 69-94) and 90% for the three-day regimen (95% CI 75-97). PCR-adjusted falciparum efficacy was 75% in both groups with nearly half (45%) still parasitemic at Day 3. Plasma piperaquine levels were comparable to prior published reports, but on the day of recrudescence were below measurable in vitro piperaquine IC50 levels in all falciparum treatment failures. CONCLUSIONS: In the brief period since introduction of dihydroartemisinin-piperaquine, there is early evidence suggesting declining efficacy relative to previous reports. Parasite IC50 levels in excess of plasma piperaquine levels seen only in treatment failures raises concern for clinically significant piperaquine resistance in Cambodia. These findings warrant improved monitoring of clinical outcomes and follow-up, given few available alternative drugs. TRIAL REGISTRATION: ClinicalTrials.gov NCT01280162.

Evaluation of parasite subpopulations and genetic diversity of the msp1, msp2 and glurp genes during and following artesunate monotherapy treatment of Plasmodium falciparum malaria in Western Cambodia.

BACKGROUND: Despite widespread coverage of the emergence of artemisinin resistance, relatively little is known about the parasite populations responsible. The use of PCR genotyping around the highly polymorphic Plasmodium falciparum msp1, msp2 and glurp genes has become well established both to describe variability in alleles within a population of parasites, as well as classify treatment outcome in cases of recurrent disease. The primary objective was to assess the emergence of minority parasite clones during seven days of artesunate (AS) treatment in a location with established artemisinin resistance. An additional objective was to investigate whether the classification of clinical outcomes remained valid when additional genotyping was performed. METHODS: Blood for parasite genotyping was collected from 143 adult patients presenting with uncomplicated falciparum malaria during a clinical trial of AS monotherapy in Western Cambodia. Nested allelic type-specific amplification of the genes encoding the merozoite surface proteins 1 and 2 (msp1 and msp2) and the glutamate-rich protein (glurp) was performed at baseline, daily during seven days of treatment, and again at failure. Allelic variants were analysed with respect to the size of polymorphisms using Quantity One software to enable identification of polyclonal infections. RESULTS: Considerable variation of msp2 alleles but well-conserved msp1 and glurp were identified. At baseline, 31% of infections were polyclonal for one or more genes. Patients with recurrent malaria were significantly more likely to have polyclonal infections than patients without recurrence (seven of nine versus 36 of 127, p = 0.004). Emergence of minority alleles during treatment was detected in only one of twenty-three cases defined as being artemisinin resistant. Moreover, daily genotyping did not alter the final outcome classification in any recurrent cases. CONCLUSIONS: The parasites responsible for artemisinin-resistant malaria in a clinical trial in Western Cambodia comprise the dominant clones of acute malaria infections rather than minority clones emerging during treatment. Additional genotyping during therapy was not beneficial. Disproportionately high rates of polyclonal infections in cases of recurrence suggest complex infections lead to poor treatment outcomes. Current research objectives should be broadened to include identification and follow-up of recurrent polyclonal infections so as to define their role as potential agents of emerging resistance.

Direct comparison of the histidine-rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR green I fluorescence (MSF) drug sensitivity tests in Plasmodium falciparum reference clones and fresh ex vivo field isolates from Cambodia.

BACKGROUND: Performance of the histidine-rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR Green I fluorescence (MSF) drug sensitivity tests were directly compared using Plasmodium falciparum reference strains and fresh ex vivo isolates from Cambodia against a panel of standard anti-malarials. The objective was to determine which of these two common assays is more appropriate for studying drug susceptibility of "immediate ex vivo" (IEV) isolates, analysed without culture adaption, in a region of relatively low malaria transmission. METHODS: Using the HRP-2 and MSF methods, the 50% inhibitory concentration (IC50) values against a panel of malaria drugs were determined for P. falciparum reference clones (W2, D6, 3D7 and K1) and 41 IEV clinical isolates from an area of multidrug resistance in Cambodia. Comparison of the IC50 values from the two methods was made using Wilcoxon matched pair tests and Pearson's correlation. The lower limit of parasitaemia detection for both methods was determined for reference clones and IEV isolates. Since human white blood cell (WBC) DNA in clinical samples is known to reduce MSF assay sensitivity, SYBR Green I fluorescence linearity of P. falciparum samples spiked with WBCs was evaluated to assess the relative degree to which MSF sensitivity is reduced in clinical samples. RESULTS: IC50 values correlated well between the HRP-2 and MSF methods when testing either P. falciparum reference clones or IEV isolates against 4-aminoquinolines (chloroquine, piperaquine and quinine) and the quinoline methanol mefloquine (Pearson r = 0.85-0.99 for reference clones and 0.56-0.84 for IEV isolates), whereas a weaker IC50 value correlation between methods was noted when testing artemisinins against reference clones and lack of correlation when testing IEV isolates. The HRP-2 ELISA produced a higher overall success rate (90% for producing IC50 best-fit sigmoidal curves), relative to only a 40% success rate for the MSF assay, when evaluating ex vivo Cambodian isolates. Reduced sensitivity of the MSF assay is likely due to an interference of WBCs in clinical samples. CONCLUSIONS: For clinical samples not depleted of WBCs, HRP-2 ELISA is superior to the MSF assay at evaluating fresh P. falciparum field isolates with low parasitaemia (<0.2%) generally observed in Southeast Asia.

Evaluation of in vitro cross-reactivity to avian H5N1 and pandemic H1N1 2009 influenza following prime boost regimens of seasonal influenza vaccination in healthy human subjects: a randomised trial.

INTRODUCTION: Recent studies have demonstrated that inactivated seasonal influenza vaccines (IIV) may elicit production of heterosubtypic antibodies, which can neutralize avian H5N1 virus in a small proportion of subjects. We hypothesized that prime boost regimens of live and inactivated trivalent seasonal influenza vaccines (LAIV and IIV) would enhance production of heterosubtypic immunity and provide evidence of cross-protection against other influenza viruses. METHODS: In an open-label study, 26 adult volunteers were randomized to receive one of four vaccine regimens containing two doses of 2009-10 seasonal influenza vaccines administered 8 (±1) weeks apart: 2 doses of LAIV; 2 doses of IIV; LAIV then IIV; IIV then LAIV. Humoral immunity assays for avian H5N1, 2009 pandemic H1N1 (pH1N1), and seasonal vaccine strains were performed on blood collected pre-vaccine and 2 and 4 weeks later. The percentage of cytokine-producing T-cells was compared with baseline 14 days after each dose. RESULTS: Subjects receiving IIV had prompt serological responses to vaccine strains. Two subjects receiving heterologous prime boost regimens had enhanced haemagglutination inhibition (HI) and neutralization (NT) titres against pH1N1, and one subject against avian H5N1; all three had pre-existing cross-reactive antibodies detected at baseline. Significantly elevated titres to H5N1 and pH1N1 by neuraminidase inhibition (NI) assay were observed following LAIV-IIV administration. Both vaccines elicited cross-reactive CD4+ T-cell responses to nucleoprotein of avian H5N1 and pH1N1. All regimens were safe and well tolerated. CONCLUSION: Neither homologous nor heterologous prime boost immunization enhanced serum HI and NT titres to 2009 pH1N1 or avian H5N1 compared to single dose vaccine. However heterologous prime-boost vaccination did lead to in vitro evidence of cross-reactivity by NI; the significance of this finding is unclear. These data support the strategy of administering single dose trivalent seasonal influenza vaccine at the outset of an influenza pandemic while a specific vaccine is being developed. TRIAL REGISTRATION: ClinicalTrials.gov NCT01044095.