Decreased Susceptibility to Dihydrofolate Reductase Inhibitors Associated With Genetic Polymorphisms in Ugandan Plasmodium falciparum Isolates.

BACKGROUND: The Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors pyrimethamine and cycloguanil (the active metabolite of proguanil) have important roles in malaria chemoprevention, but drug resistance challenges their efficacies. A new compound, P218, was designed to overcome resistance, but drug-susceptibility data for P falciparum field isolates are limited. METHODS: We studied ex vivo PfDHFR inhibitor susceptibilities of 559 isolates from Tororo and Busia districts, Uganda, from 2016 to 2020, sequenced 383 isolates, and assessed associations between genotypes and drug-susceptibility phenotypes. RESULTS: Median half-maximal inhibitory concentrations (IC50s) were 42 100 nM for pyrimethamine, 1200 nM for cycloguanil, 13000 nM for proguanil, and 0.6 nM for P218. Among sequenced isolates, 3 PfDHFR mutations, 51I (100%), 59R (93.7%), and 108N (100%), were very common, as previously seen in Uganda, and another mutation, 164L (12.8%), had moderate prevalence. Increasing numbers of mutations were associated with decreasing susceptibility to pyrimethamine, cycloguanil, and P218, but not proguanil, which does not act directly against PfDHFR. Differences in P218 susceptibilities were modest, with median IC50s of 1.4 nM for parasites with mixed genotype at position 164 and 5.7 nM for pure quadruple mutant (51I/59R/108N/164L) parasites. CONCLUSIONS: Resistance-mediating PfDHFR mutations were common in Ugandan isolates, but P218 retained excellent activity against mutant parasites.

Associations between Varied Susceptibilities to PfATP4 Inhibitors and Genotypes in Ugandan Plasmodium falciparum Isolates.

Among novel compounds under recent investigation as potential new antimalarial drugs are three independently developed inhibitors of the Plasmodium falciparum P-type ATPase (PfATP4): KAE609 (cipargamin), PA92, and SJ733. We assessed ex vivo susceptibilities to these compounds of 374 fresh P. falciparum isolates collected in Tororo and Busia districts, Uganda, from 2016 to 2019. Median IC50s were 65 nM for SJ733, 9.1 nM for PA92, and 0.5 nM for KAE609. Sequencing of pfatp4 for 218 of these isolates demonstrated many nonsynonymous single nucleotide polymorphisms; the most frequent mutations were G1128R (69% of isolates mixed or mutant), Q1081K/R (68%), G223S (25%), N1045K (16%), and D1116G/N/Y (16%). The G223S mutation was associated with decreased susceptibility to SJ733, PA92, and KAE609. The D1116G/N/Y mutations were associated with decreased susceptibility to SJ733, and the presence of mutations at both codons 223 and 1116 was associated with decreased susceptibility to PA92 and SJ733. In all of these cases, absolute differences in susceptibilities of wild-type (WT) and mutant parasites were modest. Analysis of clones separated from mixed field isolates consistently identified mutant clones as less susceptible than WT. Analysis of isolates from other sites demonstrated the presence of the G223S and D1116G/N/Y mutations across Uganda. Our results indicate that malaria parasites circulating in Uganda have a number of polymorphisms in PfATP4 and that modestly decreased susceptibility to PfATP4 inhibitors is associated with some mutations now present in Ugandan parasites.

CD318 is a ligand for CD6.

It has been proposed that CD6, an important regulator of T cells, functions by interacting with its currently identified ligand, CD166, but studies performed during the treatment of autoimmune conditions suggest that the CD6-CD166 interaction might not account for important functions of CD6 in autoimmune diseases. The antigen recognized by mAb 3A11 has been proposed as a new CD6 ligand distinct from CD166, yet the identity of it is hitherto unknown. We have identified this CD6 ligand as CD318, a cell surface protein previously found to be present on various epithelial cells and many tumor cells. We found that, like CD6 knockout (KO) mice, CD318 KO mice are also protected in experimental autoimmune encephalomyelitis. In humans, we found that CD318 is highly expressed in synovial tissues and participates in CD6-dependent adhesion of T cells to synovial fibroblasts. In addition, soluble CD318 is chemoattractive to T cells and levels of soluble CD318 are selectively and significantly elevated in the synovial fluid from patients with rheumatoid arthritis and juvenile inflammatory arthritis. These results establish CD318 as a ligand of CD6 and a potential target for the diagnosis and treatment of autoimmune diseases such as multiple sclerosis and inflammatory arthritis.

Changing Antimalarial Drug Sensitivities in Uganda.

Dihydroartemisinin-piperaquine (DP) has demonstrated excellent efficacy for the treatment and prevention of malaria in Uganda. However, resistance to both components of this regimen has emerged in Southeast Asia. The efficacy of artemether-lumefantrine, the first-line regimen to treat malaria in Uganda, has also been excellent, but continued pressure may select for parasites with decreased sensitivity to lumefantrine. To gain insight into current drug sensitivity patterns, ex vivo sensitivities were assessed and genotypes previously associated with altered drug sensitivity were characterized for 58 isolates collected in Tororo, Uganda, from subjects presenting in 2016 with malaria from the community or as part of a clinical trial comparing DP chemoprevention regimens. Compared to community isolates, those from trial subjects had lower sensitivities to the aminoquinolines chloroquine, monodesethyl amodiaquine, and piperaquine and greater sensitivities to lumefantrine and mefloquine, an observation consistent with DP selection pressure. Compared to results for isolates from 2010 to 2013, the sensitivities of 2016 community isolates to chloroquine, amodiaquine, and piperaquine improved (geometric mean 50% inhibitory concentrations [IC50] = 248, 76.9, and 19.1 nM in 2010 to 2013 and 33.4, 14.9, and 7.5 nM in 2016, respectively [P < 0.001 for all comparisons]), the sensitivity to lumefantrine decreased (IC50 = 3.0 nM in 2010 to 2013 and 5.4 nM in 2016 [P < 0.001]), and the sensitivity to dihydroartemisinin was unchanged (IC50 = 1.4 nM). These changes were accompanied by decreased prevalence of transporter mutations associated with aminoquinoline resistance and low prevalence of polymorphisms recently associated with resistance to artemisinins or piperaquine. Antimalarial drug sensitivities are changing in Uganda, but novel genotypes associated with DP treatment failure in Asia are not prevalent.

Duration of upper and lower extremity peripheral nerve blockade is prolonged with dexamethasone when added to ropivacaine: a retrospective database analysis.

BACKGROUND: Dexamethasone, when added to local anesthetics, has been shown to prolong the duration of peripheral nerve blocks; however, there are limited studies utilizing large numbers of patients. The purpose of this study was to examine the effect of adding dexamethasone to ropivacaine on duration of nerve blocks of the upper and lower extremity. METHODS: We reviewed 1,040 patient records collected in an orthopedic outpatient surgery center that had received an upper or lower extremity peripheral nerve block with ropivacaine 0.5% with or without dexamethasone and/or epinephrine. The primary outcome was duration of analgesia in upper or lower extremity blocks containing dexamethasone as an adjunct. Secondary outcomes included postoperative patient pain scores, satisfaction, and the incidence of block related complications. Linear and ordinal logistic regression models were used to examine the independent effect of dexamethasone on outcomes. RESULTS: Dexamethasone was observed to increase median block duration by 37% (95% confidence interval: 31-43%). The increased block duration persisted within body regions (upper and lower) and across a range of block types. Dexamethasone was also observed to reduce pain scores on the day of surgery (P = 0.001) and postoperative day 1 (P < 0.001). There was no significant difference in duration of nerve blocks when epinephrine (1:400,000) was added to 0.5% ropivacaine with or without dexamethasone. CONCLUSION: The addition of dexamethasone to 0.5% ropivacaine prolongs the duration of peripheral nerve blocks of both the upper and lower extremity.

Awareness, Use, and Opinions of Individual Fit Testing of Hearing Protection Devices Among Practicing Michigan Occupational and Environmental Medicine Association Health Care Providers.

OBJECTIVE: The aim of this study is to examine the awareness, opinions, and use of individual fit testing of hearing protection devices (HPDs) among occupational medicine practitioners. METHODS: Members of the Michigan Occupational and Environmental Medicine Association completed a 21-question survey on individual fit testing of HPDs. RESULTS: The survey response rate was 67%, 53% reported having heard of individual fit testing of HPDs, and 24% reported that their clinic/site performed the testing. Major barriers to its use were perceived time to perform (63%), cost (51%), lack of an Occupational Safety and Health Administration requirement (51%), and lack of long-term studies of its effectiveness (20%). CONCLUSIONS: Further work to educate practitioners about the availability, implementation, and potential benefits of fit testing of HPDs is needed if use of this technology is to become more widespread.

Susceptibility of Ugandan Plasmodium falciparum Isolates to the Antimalarial Drug Pipeline.

Malaria, especially Plasmodium falciparum infection, remains an enormous problem, and its treatment and control are seriously challenged by drug resistance. New antimalarial drugs are needed. To characterize the Medicines for Malaria Venture pipeline of antimalarials under development, we assessed the ex vivo drug susceptibilities to 19 compounds targeting or potentially impacted by mutations in P. falciparum ABC transporter I family member 1, acetyl-CoA synthetase, cytochrome b, dihydroorotate dehydrogenase, elongation factor 2, lysyl-tRNA synthetase, phenylalanyl-tRNA synthetase, plasmepsin X, prodrug activation and resistance esterase, and V-type H+ ATPase of 998 fresh P. falciparum clinical isolates collected in eastern Uganda from 2015 to 2022. Drug susceptibilities were assessed by 72-h growth inhibition (half-maximum inhibitory concentration [IC50]) assays using SYBR green. Field isolates were highly susceptible to lead antimalarials, with low- to midnanomolar median IC50s, near values previously reported for laboratory strains, for all tested compounds. However, outliers with decreased susceptibilities were identified. Positive correlations between IC50 results were seen for compounds with shared targets. We sequenced genes encoding presumed targets to characterize sequence diversity, search for polymorphisms previously selected with in vitro drug pressure, and determine genotype-phenotype associations. We identified many polymorphisms in target genes, generally in <10% of isolates, but none were those previously selected in vitro with drug pressure, and none were associated with significantly decreased ex vivo drug susceptibility. Overall, Ugandan P. falciparum isolates were highly susceptible to 19 compounds under development as next-generation antimalarials, consistent with a lack of preexisting or novel resistance-conferring mutations in circulating Ugandan parasites. IMPORTANCE Drug resistance necessitates the development of new antimalarial drugs. It is important to assess the activities of compounds under development against parasites now causing disease in Africa, where most malaria cases occur, and to determine if mutations in these parasites may limit the efficacies of new agents. We found that African isolates were generally highly susceptible to the 19 studied lead antimalarials. Sequencing of the presumed drug targets identified multiple mutations in these genes, but these mutations were generally not associated with decreased antimalarial activity. These results offer confidence that the activities of the tested antimalarial compounds now under development will not be limited by preexisting resistance-mediating mutations in African malaria parasites.

The current role of ultrasound use in teaching regional anesthesia: a survey of residency programs in the United States.

BACKGROUND AND OBJECTIVES: The purpose of this survey was to determine the current teaching practices of regional anesthesia and the prevalence of ultrasound use in guiding peripheral nerve blocks in the academic institutions across the United States. METHODS: A survey was distributed to all American Board of Anesthesiology-accredited residency programs via email and/or the U.S. postal service. The survey was designed to determine the number of peripheral nerve blocks (PNBs) performed, the role of the ultrasound guidance, the barriers to its use, and the methods by which teaching physicians acquired their ultrasound skills. RESULTS: We received 82 responses (62%) of the 132 programs surveyed. Eighty-eight percent of the responding programs performed more than 20 PNBs/week and 46% performed more than 40 PNBs/week. Three-fourths of the respondents relied on ultrasound to guide the majority of single injection and continuous PNBs. When using ultrasound, most programs (79%) used real-time ultrasound without nerve stimulator. Most teaching physicians supervising ultrasound-guided PNBs received their training via workshops and/or from other colleagues. The three main reasons for using ultrasound were to 1) achieve a higher success rate; 2) improve safety; and 3) teach anesthesia trainees. However, the three main barriers to using ultrasound were 1) lack of training; 2) perceived decreased efficiency; and 3) the lack of immediate availability of equipment. Overall, ultrasound was less utilized to guide lower extremity vs upper extremity PNBs. CONCLUSIONS: Ultrasound-guided PNBs are universally taught across residency programs in the United States. Most teaching physicians believe that ultrasound increases PNB's success and improves safety of regional anesthesia. Barriers to ultrasound use are lack of faculty training and unavailability of ultrasound equipment.

Penta-o-galloyl-beta-d-Glucose (PGG) inhibits inflammation in human rheumatoid arthritis synovial fibroblasts and rat adjuvant-induced arthritis model.

O-GlcNAcylation is a reversible post-translational modification that regulates numerous cellular processes, including embryonic development as well as immune responses. However, its role in inflammation remains ambiguous. This study was designed to examine the role of O-GlcNAcylation in rheumatoid arthritis (RA) and its regulation using human RA patient-derived synovial fibroblasts (RASFs). The efficacy of penta-O-galloyl-beta-D-glucose (PGG), a potent anti-inflammatory molecule, in regulating inflammatory processes in human RASFs was also evaluated. Human synovial tissues and RASFs exhibited higher expression of O-GlcNAcylation compared to their non-diseased counterparts. Pretreatment of RASFs with Thiamet G, an inhibitor of O-GlcNAcase, markedly increased the O-GlcNAc-modified proteins and concomitantly inhibited the IL-1ß-induced IL-6 and IL-8 production in human RASFs in vitro. Pretreatment of human RASFs with PGG (0.5-10 µM) abrogated IL-1ß-induced IL-6 and IL-8 production in a dose-dependent manner. Immunoprecipitation analysis showed that PGG inhibited O-GlcNAcylation of TAB1 to reduce its association with TGF ß-activated kinase 1 (TAK1) and its autophosphorylation, an essential signaling step in IL-1ß-induced signaling pathways. Molecular docking in silico studies shows that PGG occupies the C174 position, an ATP-binding site in the kinase domain to inhibit TAK1 kinase activity. Oral administration of PGG (25 mg/kg/day) for 10 days from disease onset significantly ameliorated rat adjuvant-induced (AIA) in rats. PGG treatment reduced the phosphorylation of TAK1 in the treated joints compared to AIA joints, which correlated with the reduced disease severity and suppressed levels of serum IL-1ß, GM-CSF, TNF-α, and RANKL. These findings suggest O-GlcNAcylation as a potential therapeutic target and provide the rationale for testing PGG or structurally similar molecule for their therapeutic efficacy.

Face Burns: A 4-Year Experience.

Burns on the face pose unique management challenges because they are in a place that is constantly visible, so scars are hard to hide. The goal of this study was to review our experience of adult patients who had face burns. We performed a retrospective review of adult patients (≥18 years old) who were admitted to a regional burn center from July 2015 to June 2019 with face burns. Sex, age, ethnicity, burn etiology, burn size, and discharge status were collected from electronic medical records of the patients who met study criteria. Descriptive statistics, Student's t-tests, and chi-square tests were performed in Stata/SE 16.1. Significance was defined as a P-value < .05. In 4 years, 595/1705 patients (~35% of admissions) were admitted with face burns. The mean age was 44.9 ± 17.0 (mean ± SD) years, with the majority being men (475, 80%). The mean burn size was 19.8 ± 20.9% TBSA with 10.1 ± 19.8% TBSA being third degree. The mean head burn size for any face burn was 2.8 ± 1.8% TBSA. The majority of burns were due to flames (478, 80%) and of those 122 (21%) were from accelerant use and 43 (7%) resulted from propane or butane use. Scalds caused 53 (9%), electric 25 (4%), hot tar 5 (1%), and chemical 5 (1%). Overall, 208 (35%) patients had grafting of some portion of their body, but only 31 patients (5.2%) had face grafting. The mean age of those with face grafting compared with patients who did not need grafting was 45.9 ± 13.8 and 44.9 ± 17.2 years, respectively. Patients who needed grafting had a mean third-degree burn size of 31.7 ± 25.4% TBSA and a mean head (including face) burn size of 4.7 ± 2.0% TBSA, whereas patients who did not need grafting had a mean third-degree burn size of 8.9 ± 18.7% TBSA and a mean head burn size of 2.7 ± 1.8% head TBSA. Patients requiring face grafts had longer lengths of stay, intensive unit stays, ventilator days, and mortality than those whose face burns healed spontaneously. Overall, head burns in adults were common within the 4-year time span we studied, but only a small fraction (5%) had face grafts. The patients who needed grafting for their head burns had significantly larger total body and face burns and had a 2.4-fold higher mortality rate compared to patients who did not need grafting. Most face burns were caused by flame, especially the use of accelerants or flammable gases. Prevention efforts should focus on avoiding the use of accelerants and being careful with flammable gases.