Multidrug resistant P falciparum malaria is common in southeast Asia, but difficult to identify and treat. Genes that encode parasite transport proteins maybe involved in export of drugs and so cause resistance. In this study we show that increase in copy number of pfmdr1, a gene encoding a parasite transport protein, is the best overall predictor of treatment failure with mefloquine. Increase in pfmdr1 copy number predicts failure even after chemotherapy with the highly effective combination of mefloquine and 3 days' artesunate. Monitoring of pfmdr1 copy number will be useful in epidemiological surveys of drug resistance in P falciparum, and potentially for predicting treatment failure in individual patients.

The novel coronavirus SARS-CoV-2 (coronavirus disease 19, or COVID-19) primarily causes pulmonary injury, but has been implicated to cause hepatic injury, both by serum markers and histologic evaluation. The histologic pattern of injury has not been completely described. Studies quantifying viral load in the liver are lacking. Here we report the clinical and histologic findings related to the liver in 40 patients who died of complications of COVID-19. A subset of liver tissue blocks were subjected to polymerase chain reaction (PCR) for viral ribonucleic acid (RNA). Peak levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were elevated; median ALT peak 68 U/l (normal up to 46 U/l) and median AST peak 102 U/l (normal up to 37 U/l). Macrovesicular steatosis was the most common finding, involving 30 patients (75%). Mild lobular necroinflammation and portal inflammation were present in 20 cases each (50%). Vascular pathology, including sinusoidal microthrombi, was infrequent, seen in six cases (15%). PCR of liver tissue was positive in 11 of 20 patients tested (55%). In conclusion, we found patients dying of COVID-19 had biochemical evidence of hepatitis (of variable severity) and demonstrated histologic findings of macrovesicular steatosis and mild acute hepatitis (lobular necroinflammation) and mild portal inflammation. We also identified viral RNA in a sizeable subset of liver tissue samples.

Antimalarial chemotherapy, globally reliant on artemisinin-based combination therapies (ACTs), is threatened by the spread of drug resistance in Plasmodium falciparum parasites. Here we use zinc-finger nucleases to genetically modify the multidrug resistance-1 transporter PfMDR1 at amino acids 86 and 184, and demonstrate that the widely prevalent N86Y mutation augments resistance to the ACT partner drug amodiaquine and the former first-line agent chloroquine. In contrast, N86Y increases parasite susceptibility to the partner drugs lumefantrine and mefloquine, and the active artemisinin metabolite dihydroartemisinin. The PfMDR1 N86 plus Y184F isoform moderately reduces piperaquine potency in strains expressing an Asian/African variant of the chloroquine resistance transporter PfCRT. Mutations in both digestive vacuole-resident transporters are thought to differentially regulate ACT drug interactions with host haem, a product of parasite-mediated haemoglobin degradation. Global mapping of these mutations illustrates where the different ACTs could be selectively deployed to optimize treatment based on regional differences in PfMDR1 haplotypes.

When selection is strong and beneficial alleles have a single origin, local reductions in genetic diversity are expected. However, when beneficial alleles have multiple origins or were segregating in the population prior to a change in selection regime, the impact on genetic diversity may be less clear. We describe an example of such a "soft" selective sweep in the malaria parasite Plasmodium falciparum that involves adaptive genome rearrangements. Amplification in copy number of genome regions containing the pfmdr1 gene on chromosome 5 confer resistance to mefloquine and spread rapidly in the 1990s. Using flanking microsatellite data and real-time polymerase chain reaction determination of copy number, we show that 5-15 independent amplification events have occurred in parasites on the Thailand/Burma border. The amplified genome regions (amplicons) range in size from 14.7 to 49 kb and contain 2-11 genes, with 2-4 copies arranged in tandem. To examine the impact of drug selection on flanking variation, we genotyped 48 microsatellites on chromosome 5 in 326 parasites from a single Thai location. Diversity was reduced in a 170- to 250-kb (10-15 cM) region of chromosomes containing multiple copies of pfmdr1, consistent with hitchhiking resulting from the rapid recent spread of selected chromosomes. However, diversity immediately flanking pfmdr1 is reduced by only 42% on chromosomes bearing multiple amplicons relative to chromosomes carrying a single copy. We highlight 2 features of these results: 1) All amplicon break points occur in monomeric A/T tracts (9-45 bp). Given the abundance of these tracts in P. falciparum, we expect that duplications will occur frequently at multiple genomic locations and have been underestimated as drivers of phenotypic evolution in this pathogen. 2) The signature left by the spread of amplified genome segments is broad, but results in only limited reduction in diversity. If such "soft" sweeps are common in nature, statistical methods based on diversity reduction may be inefficient at detecting evidence for selection in genome-wide marker screens. This may be particularly likely when mutation rate is high, as appears to be the case for gene duplications, and in pathogen populations where effective population sizes are typically very large.

Although the New York/New Jersey (NY/NJ) area is an epicenter for carbapenem-resistant Enterobacteriaceae (CRE), there are few multicenter studies of CRE from this region. We characterized patients with CRE bacteremia in 2013 at eight NY/NJ medical centers and determined the prevalence of carbapenem resistance among Enterobacteriaceae bloodstream isolates and CRE resistance mechanisms, genetic backgrounds, capsular types (cps), and antimicrobial susceptibilities. Of 121 patients with CRE bacteremia, 50% had cancer or had undergone transplantation. The prevalences of carbapenem resistance among Klebsiella pneumoniae, Enterobacter spp., and Escherichia coli bacteremias were 9.7%, 2.2%, and 0.1%, respectively. Ninety percent of CRE were K. pneumoniae and 92% produced K. pneumoniae carbapenemase (KPC-3, 48%; KPC-2, 44%). Two CRE produced NDM-1 and OXA-48 carbapenemases. Sequence type 258 (ST258) predominated among KPC-producing K. pneumoniae (KPC-Kp). The wzi154 allele, corresponding to cps-2, was present in 93% of KPC-3-Kp, whereas KPC-2-Kp had greater cps diversity. Ninety-nine percent of CRE were ceftazidime-avibactam (CAZ-AVI)-susceptible, although 42% of KPC-3-Kp had an CAZ-AVI MIC of Ն4/4 g/ml. There was a median of 47 h from bacteremia onset until active antimicrobial therapy, 38% of patients had septic shock, and 49% died within 30 days. KPC-3-Kp bacteremia (adjusted odds ratio [aOR], 2.58; P ϭ 0.045), cancer (aOR, 3.61, P ϭ 0.01), and bacteremia onset in the intensive care unit (aOR, 3.79; P ϭ 0.03) were independently associated with mortality. Active empirical ther-

We document the neuropathologic findings of a 73-year old man who died from acute cerebellar hemorrhage in the context of relatively mild SARS-CoV2 infection. The patient developed sudden onset of headache, nausea, and vomiting, immediately followed by loss of consciousness on the day of admission. Emergency medical services found him severely hypoxemic at home, and the patient suffered a cardiac arrest during transport to the emergency department. The emergency team achieved return of spontaneous circulation after over 17 min of resuscitation. A chest radiograph revealed hazy bilateral opacities; and real-time-PCR for SARS-CoV-2 on the nasopharyngeal swab was positive. Computed tomography of the head showed a large right cerebellar hemorrhage, with tonsillar herniation and intraventricular hemorrhage. One day after presentation, he was transitioned to comfort care and died shortly after palliative extubation. Autopsy performed 3 h after death showed cerebellar hemorrhage and acute infarcts in the dorsal pons and medulla. Remarkably, there were microglial nodules and neuronophagia bilaterally in the inferior olives and multifocally in the cerebellar dentate nuclei. This constellation of findings has not been reported thus far in the context of SARS-CoV-2 infection.

BackgroundCarbapenem-resistant Enterobacteriaceae (CRE) have emerged as an urgent public health threat. Intestinal colonization with CRE has been identified as a risk factor for the development of systemic CRE infection, but has not been compared to colonization with third and/or fourth generation cephalosporin-resistant (Ceph-R) Enterobacteriaceae. Moreover, the risk conferred by colonization on adverse outcomes is less clear, particularly in critically ill patients admitted to the intensive care unit (ICU).MethodsWe carried out a cohort study of consecutive adult patients screened for rectal colonization with CRE or Ceph-R upon ICU entry between April and July 2013. We identified clinical variables and assessed the relationship between CRE or Ceph-R colonization and subsequent systemic CRE infection within 30 days (primary outcome) and all-cause mortality within 90 days (secondary outcome).ResultsAmong 338 ICU patients, 94 (28%) were colonized with either Ceph-R or CRE. 26 patients developed CRE infection within 30 days of swab collection; 47% (N = 17/36) of CRE-colonized and 3% (N = 2/58) of Ceph-R colonized patients. 36% (N = 13/36) of CRE-colonized patients died within 90 days compared to 31% (N = 18/58) of Ceph-R-colonized and 15% (N = 37/244) of non-colonized patients. In a multivariable analysis, CRE colonization independently predicted development of a systemic CRE infection at 30 days (aOR 10.8, 95% CI2.8–41.9, p = 0.0006); Ceph-R colonization did not (aOR 0.5, 95% CI0.1–3.3, p = 0.5). CRE colonization was associated with increased 90-day mortality in a univariable analysis (p-value 0.001), in a multivariable model, previous hospitalization and medical ICU admission were independent predictors of 90-day mortality whereas CRE colonization approached significance (aOR 2.3, 95% CI1.0–5.3, p = 0.056).ConclusionsOur study highlights the increased risk of CRE infection and mortality in patients with CRE colonization at the time of ICU admission. Future studies are needed to assess how CRE colonization can guide empiric antibiotic choices and to develop novel decolonization strategies.

Summary The search for antimalarial chemotypes with modes of action unrelated to existing drugs has intensified with the recent failure of first-line therapies across Southeast Asia. Here, we show that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by Plasmodium sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies, validated with conditional knockdown parasites, molecular docking, and recombinant kinase assays, identified cGMP-dependent protein kinase (PKG) as the primary target of MMV030084. PKG is known to play essential roles in Plasmodium invasion of and egress from host cells, matching MMV030084's activity profile. Resistance selections and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under pressure. These studies highlight PKG as a resistance-refractory antimalarial target throughout the Plasmodium life cycle and promote MMV030084 as a promising Plasmodium PKG-targeting chemotype.