Population Structure Shapes Copy Number Variation in Malaria Parasites.

If copy number variants (CNVs) are predominantly deleterious, we would expect them to be more efficiently purged from populations with a large effective population size (Ne) than from populations with a small Ne. Malaria parasites (Plasmodium falciparum) provide an excellent organism to examine this prediction, because this protozoan shows a broad spectrum of population structures within a single species, with large, stable, outbred populations in Africa, small unstable inbred populations in South America and with intermediate population characteristics in South East Asia. We characterized 122 single-clone parasites, without prior laboratory culture, from malaria-infected patients in seven countries in Africa, South East Asia and South America using a high-density single-nucleotide polymorphism/CNV microarray. We scored 134 high-confidence CNVs across the parasite exome, including 33 deletions and 102 amplifications, which ranged in size from <500 bp to 59 kb, as well as 10,107 flanking, biallelic single-nucleotide polymorphisms. Overall, CNVs were rare, small, and skewed toward low frequency variants, consistent with the deleterious model. Relative to African and South East Asian populations, CNVs were significantly more common in South America, showed significantly less skew in allele frequencies, and were significantly larger. On this background of low frequency CNV, we also identified several high-frequency CNVs under putative positive selection using an FST outlier analysis. These included known adaptive CNVs containing rh2b and pfmdr1, and several other CNVs (e.g., DNA helicase and three conserved proteins) that require further investigation. Our data are consistent with a significant impact of genetic structure on CNV burden in an important human pathogen.

Cow-baited tents are highly effective in sampling diverse Anopheles malaria vectors in Cambodia.

BACKGROUND: The accurate monitoring and evaluation of malaria vectors requires efficient sampling. The objective of this study was to compare methods for sampling outdoor-biting Anopheles mosquitoes in Cambodia. METHODS: In the Cambodian provinces of Pursat, Preah Vihear, and Ratanakiri, six different mosquito trapping methods were evaluated: human landing collection (HLC), human-baited tent (HBT), cow-baited tent (CBT), CDC miniature light trap (LT), CDC miniature light trap baited with molasses and yeast (LT-M), and barrier fence (F) in a Latin square design during four or six consecutive nights at the height of the malaria transmission season. RESULTS: Using all traps, a total of 507, 1175, and 615 anophelines were collected in Pursat, Preah Vihear, and Ratanakiri, respectively. CBTs captured 10- to 20-fold more anophelines per night than the other five sampling methods. All 2297 Anopheles mosquitoes were morphologically identified and molecularly typed using standard morphological keys and sequencing the rDNA ITS2 region to distinguish cryptic species, respectively. Overall, an extremely diverse set of 27 known Anopheles species was sampled. CBTs captured the same molecular species that HLCs and the other four traps did, as well as additional species. Nine specimens representing five Anopheles species (Anopheles hyrcanus, Anopheles barbirostris sensu stricto, Anopheles barbirostris clade III, Anopheles nivipes, and Anopheles peditaeniatus) were infected with Plasmodium falciparum and were exclusively captured in CBTs. CONCLUSIONS: These data indicate that cow-baited tents are highly effective in sampling diverse Anopheles malaria vectors in Cambodia. This sampling method captured high numbers of anophelines with limited sampling effort and greatly reduced human exposure to mosquito bites compared to the gold-standard human landing collection.

A microarray platform and novel SNP calling algorithm to evaluate Plasmodium falciparum field samples of low DNA quantity.

BACKGROUND: Analysis of single nucleotide polymorphisms (SNPs) derived from whole-genome studies allows for rapid evaluation of genome-wide diversity, and genomic epidemiology studies of Plasmodium falciparum provide insights into parasite population structure, gene flow, drug resistance and vaccine development. In areas with adequate cold chain facilities, large volumes of leukocyte-depleted patient blood can be frozen for use in parasite genomic analyses. In more remote endemic areas smaller volumes of infected blood are taken by finger prick, and dried and stored on filter paper. These dried blood spots do not generally yield enough concentrated parasite DNA for whole-genome sequencing. RESULTS: A DNA microarray was designed for use on field samples to type a genome-wide set of SNPs which prior sequencing had shown to be variable in Africa, Southeast Asia, and Papua New Guinea. An algorithm was designed to call SNPs in samples with low parasite DNA. With this new algorithm SNP-calling accuracy of 98% was measured by hybridizing purified DNA from malaria lab strains and comparing calls with SNPs called from full genome sequences. An average accuracy of >98% was likewise obtained for DNA extracted from malaria field samples collected in studies in Southeast Asia, with an average call rate of > 82%. CONCLUSION: This new high-density microarray provided high quality SNP calls from a wide range of parasite DNA quantities, and represents a robust tool for genome-wide analysis of malaria parasites in diverse settings.

Sensitivity of surveillance testing for multidrug-resistant Gram-negative bacteria in the intensive care unit.

We tested intensive care unit patients for colonization with multidrug-resistant Gram-negative bacilli (MDR GNB) and compared the results with those of concurrent clinical cultures. The sensitivity of the surveillance test for detecting MDR GNB was 58.8% (95% confidence interval, 48.6 to 68.5%). Among 133 patients with positive surveillance tests, 61% had no prior clinical culture with MDR GNB.

Management of non-compressible torso hemorrhage of the abdomen in civilian and military austere environments: a scoping review.

Background: Non-compressible abdominal hemorrhage (NCAH) is the leading cause of potentially preventable deaths in both civilian and military austere environments, and an improvement in mortality due to this problem has not been demonstrated during the past quarter century. Several innovations have been developed to control hemorrhage closer to the point of injury. Objective: This review assessed NCAH interventions in civilian and military settings, focusing on austere environments. It identified innovations, effectiveness, and knowledge gaps for future research. Methodology: The Joanna Briggs Institute for Evidence Synthesis methodology guided this scoping review to completion. Studies evaluating NCAH with human participants in civilian and military austere environments that were eligible for inclusion were limited to English language studies published between December 1990 and January 2023. The PCC (Participant, Concept, Context) framework was used for data synthesis. Deductive and inductive thematic analyses were used to assess the literature that met inclusion criteria, identify patterns/themes to address the research questions and identify common themes within the literature. A stakeholder consultation was conducted to review and provide expert perspectives and opinions on the results of the deductive and inductive thematic analyses. Results: The literature search identified 868 articles; 26 articles met the inclusion criteria. Textual narrative analysis of the 26 articles resulted in the literature addressing four main categories: NCAH, penetrating abdominal trauma, resuscitative endovascular balloon occlusion of the aorta (REBOA), and ResQFoam. The deductive thematic analysis aimed to answer three research questions. Research question 1 addressed the effectiveness of REBOA, damage control resuscitation, and damage control surgery in managing NCAH in austere environments. No effectiveness studies were found on this topic. Research question 2 identified three knowledge gaps in NCAH management in austere environments. The analysis identified early hemorrhage control, prehospital provider decision-making ability, and REBOA implementation as knowledge gaps in NCAH. Research question 3 identified five innovations that may affect the management of NCAH in the future: transport of patients, advanced resuscitative care, expert consultation, REBOA implementation, and self-expanding foam implementation. The inductive thematic analysis resulted in four recurrent themes from the literature: prehospital care, decision-making, hemorrhage control, and mortality in NCAH. During the stakeholders' consultation, the results of the deductive and inductive thematic analyses were reviewed and agreed on by the stakeholders. Special emphasis and discussion were given to prehospital management, expert opinions in the prehospital environment, decision-making in the prehospital environment, transport and resuscitation in the prehospital setting, REBOA, alternative discussion for research, and research gaps. Conclusion: NCAH is still a significant cause of preventable death in both military and civilian austere environments, even with ongoing research and interventions aimed at extending survival in such conditions. This scoping review has identified several potential concepts that could reduce the mortality associated with a preventable cause of death due to hemorrhage in austere environments.

Nontuberculous mycobacterial infection after fractionated CO(2) laser resurfacing.

Nontuberculous mycobacteria are increasingly associated with cutaneous infections after cosmetic procedures. Fractionated CO2 resurfacing, a widely used technique for photorejuvenation, has been associated with a more favorable side effect profile than alternative procedures. We describe 2 cases of nontuberculous mycobacterial infection after treatment with a fractionated CO2 laser at a private clinic. Densely distributed erythematous papules and pustules developed within the treated area within 2 weeks of the laser procedure. Diagnosis was confirmed by histologic analysis and culture. Both infections responded to a 4-month course of a multidrug regimen. An environmental investigation of the clinic was performed, but no source of infection was found. The case isolates differed from each other and from isolates obtained from the clinic, suggesting that the infection was acquired by postprocedure exposure. Papules and pustules after fractionated CO2 resurfacing should raise the suspicion of nontuberculous mycobacterial infection.

COOH-terminal truncation of flightin decreases myofilament lattice organization, cross-bridge binding, and power output in Drosophila indirect flight muscle.

The indirect flight muscle (IFM) of insects is characterized by a near crystalline myofilament lattice structure that likely evolved to achieve high power output. In Drosophila IFM, the myosin rod binding protein flightin plays a crucial role in thick filament organization and sarcomere integrity. Here we investigate the extent to which the COOH terminus of flightin contributes to IFM structure and mechanical performance using transgenic Drosophila expressing a truncated flightin lacking the 44 COOH-terminal amino acids (fln(ΔC44)). Electron microscopy and X-ray diffraction measurements show decreased myofilament lattice order in the fln(ΔC44) line compared with control, a transgenic flightin-null rescued line (fln(+)). fln(ΔC44) fibers produced roughly 1/3 the oscillatory work and power of fln(+), with reduced frequencies of maximum work (123 Hz vs. 154 Hz) and power (139 Hz vs. 187 Hz) output, indicating slower myosin cycling kinetics. These reductions in work and power stem from a slower rate of cross-bridge recruitment and decreased cross-bridge binding in fln(ΔC44) fibers, although the mean duration of cross-bridge attachment was not different between both lines. The decreases in lattice order and myosin kinetics resulted in fln(ΔC44) flies being unable to beat their wings. These results indicate that the COOH terminus of flightin is necessary for normal myofilament lattice organization, thereby facilitating the cross-bridge binding required to achieve high power output for flight.

Adaptive copy number evolution in malaria parasites.

Copy number polymorphism (CNP) is ubiquitous in eukaryotic genomes, but the degree to which this reflects the action of positive selection is poorly understood. The first gene in the Plasmodium folate biosynthesis pathway, GTP-cyclohydrolase I (gch1), shows extensive CNP. We provide compelling evidence that gch1 CNP is an adaptive consequence of selection by antifolate drugs, which target enzymes downstream in this pathway. (1) We compared gch1 CNP in parasites from Thailand (strong historical antifolate selection) with those from neighboring Laos (weak antifolate selection). Two percent of chromosomes had amplified copy number in Laos, while 72% carried multiple (2-11) copies in Thailand, and differentiation exceeded that observed at 73 synonymous SNPs. (2) We found five amplicon types containing one to greater than six genes and spanning 1 to >11 kb, consistent with parallel evolution and strong selection for this gene amplification. gch1 was the only gene occurring in all amplicons suggesting that this locus is the target of selection. (3) We observed reduced microsatellite variation and increased linkage disequilibrium (LD) in a 900-kb region flanking gch1 in parasites from Thailand, consistent with rapid recent spread of chromosomes carrying multiple copies of gch1. (4) We found that parasites bearing dhfr-164L, which causes high-level resistance to antifolate drugs, carry significantly (p = 0.00003) higher copy numbers of gch1 than parasites bearing 164I, indicating functional association between genes located on different chromosomes but linked in the same biochemical pathway. These results demonstrate that CNP at gch1 is adaptive and the associations with dhfr-164L strongly suggest a compensatory function. More generally, these data demonstrate how selection affects multiple enzymes in a single biochemical pathway, and suggest that investigation of structural variation may provide a fast-track to locating genes underlying adaptation.

Acute eosinophilic pneumonia secondary to daptomycin: a report of three cases.

We describe 3 cases of daptomycin-induced pulmonary toxic effects that are consistent with drug-induced acute eosinophilic pneumonia. Patients presented similarly with dyspnea, cough, hypoxia, and diffuse ground-glass opacities at chest computed tomography. Clinical suspicion for this adverse drug event and cessation of daptomycin until definitive diagnosis can be made is crucial.

Influencing leadership perceptions of patient safety through just culture training.

There are differences in perceptions of safety culture between healthcare leaders and staff. Evidence suggests that an organization's actual safety performance is more closely reflected in staff perceptions suggesting that frontline staff may be more aware than the leadership of actual patient safety challenges within their organization. Closing the perception gap between healthcare leaders and staff is critical to aligning the resources and strategies required to create a true culture of safety.

A variable domain near the ATP-binding site in Drosophila muscle myosin is part of the communication pathway between the nucleotide and actin-binding sites.

Drosophila expresses several muscle myosin isoforms from a single gene by alternatively splicing six of the 19 exons. Here we investigate exon 7, which codes for a region in the upper 50 kDa domain near the nucleotide-binding pocket. This region is of interest because it is also the place where a large insert is found in myosin VI and where several cardiomyopathy mutations have been identified in human cardiac myosin. We expressed and purified chimeric muscle myosins from Drosophila, each varying at exon 7. Two chimeras exchanged the entire exon 7 domain between the indirect flight muscle (IFI, normally containing exon 7d) and embryonic body wall muscle (EMB, normally containing exon 7a) isoforms to create IFI-7a and EMB-7d. The second two chimeras replaced each half of the exon 7a domain in EMB with the corresponding portion of exon 7d to create EMB-7a/7d and EMB-7d/7a. Transient kinetic studies of the motor domain from these myosin isoforms revealed changes in several kinetic parameters between the IFI or EMB isoforms and the chimeras. Of significance were changes in nucleotide binding, which differed in the presence and absence of actin, consistent with a model in which the exon 7 domain is part of the communication pathway between the nucleotide and actin-binding sites. Homology models of the structures suggest how the exon 7 domain might modulate this pathway.

An alternative domain near the nucleotide-binding site of Drosophila muscle myosin affects ATPase kinetics.

In Drosophila melanogaster expression of muscle myosin heavy chain isoforms occurs by alternative splicing of transcripts from a single gene. The exon 7 domain is one of four variable regions in the catalytic head and is located near the nucleotide-binding site. To ascribe a functional role to this domain, we created two chimeric myosin isoforms (indirect flight isoform-exon 7a and embryonic-exon 7d) that differ from the native indirect flight muscle and embryonic body-wall muscle isoforms only in the exon 7 region. Germline transformation and subsequent expression of the chimeric myosins in the indirect flight muscle of myosin-null Drosophila allowed us to purify the myosin for in vitro studies and to assess in vivo structure and function of transgenic muscles. Intriguingly, in vitro experiments show the exon 7 domain modulates myosin ATPase activity but has no effect on actin filament velocity, a novel result compared to similar studies with other Drosophila variable exons. Transgenic flies expressing the indirect flight isoform-exon 7a have normal indirect flight muscle structure, and flight and jump ability. However, expression of the embryonic-exon 7d chimeric isoform yields flightless flies that show improvements in both the structural stability of the indirect flight muscle and in locomotor abilities as compared to flies expressing the embryonic isoform. Overall, our results suggest the exon 7 domain participates in the regulation of the attachment of myosin to actin in order to fine-tune the physiological properties of Drosophila myosin isoforms.

Artemisinin-resistant Plasmodium falciparum clinical isolates can infect diverse mosquito vectors of Southeast Asia and Africa.

Artemisinin-resistant Plasmodium falciparum parasites are rapidly spreading in Southeast Asia, yet nothing is known about their transmission. This knowledge gap and the possibility that these parasites will spread to Africa endanger global efforts to eliminate malaria. Here we produce gametocytes from parasite clinical isolates that displayed artemisinin resistance in patients and in vitro, and use them to infect native and non-native mosquito vectors. We show that contemporary artemisinin-resistant isolates from Cambodia develop and produce sporozoites in two Southeast Asian vectors, Anopheles dirus and Anopheles minimus, and the major African vector, Anopheles coluzzii (formerly Anopheles gambiae M). The ability of artemisinin-resistant parasites to infect such highly diverse Anopheles species, combined with their higher gametocyte prevalence in patients, may explain the rapid expansion of these parasites in Cambodia and neighbouring countries, and further compromise efforts to prevent their global spread.

Central line-associated infections as defined by the Centers for Medicare and Medicaid Services' Hospital-acquired condition versus standard infection control surveillance: why hospital compare seems conflicted.

OBJECTIVE: To evaluate the concordance of case-finding methods for central line-associated infection as defined by Centers for Medicare and Medicaid Services (CMS) hospital-acquired condition (HAC) compared with traditional infection control (IC) methods. SETTING: One tertiary care and 2 community hospitals in North Carolina. PATIENTS: Adult and pediatric hospitalized patients determined to have central line infection by either case-finding method. METHODS: We performed a retrospective comparative analysis of infection detected using HAC versus standard IC central line-associated bloodstream infection surveillance from October 1, 2007, through December 31, 2009. One billing and 2 IC databases were queried and matched to determine the number and concordance of cases identified by each method. Manual review of 25 cases from each discordant category was performed. Sensitivity and positive predictive value (PPV) were calculated using IC as criterion standard. RESULTS: A total of 1,505 cases were identified: 844 by International Classification of Diseases, Ninth Revision (ICD-9), and 798 by IC. A total of 204 cases (24%) identified by ICD-9 were deemed not present at hospital admission by coders. Only 112 cases (13%) were concordant. HAC sensitivity was 14% and PPV was 55% compared with IC. Concordance was low regardless of hospital type. Primary reasons for discordance included differences in surveillance and clinical definitions, clinical uncertainty, and poor documentation. CONCLUSIONS: The case-finding method used by CMS HAC and the methods used for traditional IC surveillance frequently do not agree. This can lead to conflicting results when these 2 measures are used as hospital quality metrics.

Observing and improving hand hygiene compliance: implementation and refinement of an electronic-assisted direct-observer hand hygiene audit program.

We implemented a direct-observer hand hygiene audit program that used trained observers, wireless data entry devices, and an intranet portal. We improved the reliability and utility of the data by standardizing audit processes, regularly retraining auditors, developing an audit guidance tool, and reporting weighted composite hand hygiene compliance scores.

Ability of an antibiogram to predict Pseudomonas aeruginosa susceptibility to targeted antimicrobials based on hospital day of isolation.

OBJECTIVE: To determine the utility of an antibiogram in predicting the susceptibility of Pseudomonas aeruginosa isolates to targeted antimicrobial agents based on the day of hospitalization the specimen was collected. DESIGN: Single-center retrospective cohort study. SETTING: A 750-bed tertiary care medical center. PATIENTS AND METHODS: Isolates from consecutive patients with at least 1 clinical culture positive for P. aeruginosa from January 1, 2000, to June 30, 2007, were included. A study antibiogram was created by determining the overall percentages of P. aeruginosa isolates susceptible to amikacin, ceftazidime, ciprofloxacin, gentamicin, imipenem-cilastin, piperacillin-tazobactam, and tobramycin during the study period. Individual logistic regression models were created to determine the day of infection after which the study antibiogram no longer predicted susceptibility to each antibiotic. RESULTS: A total of 3,393 isolates were included. The antibiogram became unreliable as a predictor of susceptibility to ceftazidime, imipenem-cilastin, piperacillin-tazobactam, and tobramycin after day 10 and ciprofloxacin after day 15 but longer for gentamicin (day 21) and amikacin (day 28). Time to unreliability of the antibiogram varied for antibiotics based on location of isolation. For example, the time to unreliability of the antibiogram for ceftazidime was 5 days (95% confidence interval [CI], <1-8) in the intensive care unit (ICU) and 12 days (95% CI, 7-21) in non-ICU hospital wards (P = .003). CONCLUSIONS: The ability of the antibiogram to predict susceptibility of P. aeruginosa decreases as duration of hospitalization increases.

The impact of depth of infection and postdischarge surveillance on rate of surgical-site infections in a network of community hospitals.

OBJECTIVE: To describe the epidemiology of surgical-site infections (SSIs) in community hospitals and to explore the impact of depth of SSI, healthcare location at the time of diagnosis, and variations in surveillance practices on the overall rate of SSI. DESIGN: Retrospective cohort study. SETTING: Thirty-seven community hospitals in the southeastern United States. PATIENTS: Consecutive sample of patients undergoing surgical procedures between July 1, 2007, and December 31, 2008. METHODS: ANOVA was used to compare rates of SSIs, and the F test was used to compare the distribution of rates of SSIs. Wilcoxon Signed Rank test [corrected] was used to test for differences in performance rankings of hospitals. RESULTS: Following 177,706 surgical procedures, 1,919 SSIs were identified (incidence, 1.08 per 100 procedures). Sixty-four percent (1,223 of 1,919) of these were identified as complex SSIs; 87% of the complex SSIs were diagnosed in inpatient settings. The median proportion of superficial-incisional SSIs was 37% (interquartile range, 29.6%-49.5%). Postdischarge SSI surveillance was variable, with 58% of responding hospitals using surgeon letters. As reporting focus was narrowed from all SSIs to complex SSIs (incidence, 0.69 per 100 procedures) and, finally, to complex SSIs diagnosed in the inpatient setting (incidence, 0.51 per 100 procedures), variance in rates changed significantly ([Formula: see text]). Performance ranking of individual hospitals, based on rates of SSIs, differed significantly, depending on the reporting method utilized ([Formula: see text]). CONCLUSIONS: Inconsistent reporting methods focused on variable depths of infection and healthcare location at time of diagnosis significantly impact rates of SSI, distribution of rates of SSI, and hospital comparative-performance rankings. We believe that public reporting of SSI rates should be limited to complex SSIs diagnosed in the inpatient setting.