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Purpose To identify factors influencing patient’s availability to re-schedule primary total knee replacement (TKR) or revision (RKR) surgery after the lockdown (March–May 2020) during the COVID-19 pandemic. Methods A prospective cohort study through a telephone survey was performed in 156 patients (143 for primary and 13 for revision) included in the TKR and RKR surgical waiting list before March 2020. Contact of each patient with COVID-19, stress and anxiety, perceived pain, and function were obtained in the interviews, and also the preference of each patient to have re-scheduled surgery (early or late). Finally, we registered their response (acceptance or refusal) when surgery was effectively re-scheduled. Results 88 out of 156 patients waiting for knee replacement (76/143 of those waiting for TKR, 12/13 of those waiting for RKR) declared themselves ready for surgery in less than 1 month. When re-scheduled, 115 patients underwent surgery and 41 refused. Significantly different preferences were found for age (more prone to surgery if under 65), revision surgery (more readily available), pain (7.9 ± 1.7/10 in NRS in those undergoing surgery, 5.6 ± 2.3/10 in those refusing, p  = 0.000), or COVID-19 diagnosis, but not other close contact with COVID-19, comorbidities, stress, or anxiety. A logistic regression model confirmed that revision surgery (OR 9.33), perceived severe pain (OR 5.21), and age under 65 years (OR 5.82) were significantly associated with patient preference. The probability of patients over 65 to prefer early surgery reached 60% only with pain at or above 9/10. Conclusions Surgical timing preferences for knee replacement vary between patients older than 65 years (immediate surgery only when pain is intense) and younger patients (immediate surgery no matter the amount of pain). Even if COVID-19 severely stroke our population, the need for knee replacement stood in the young population and even in the aged population at risk for COVID when pain was important.

Decades of unmanaged insecticide use and routine exposure to agrochemicals have left many populations of malaria vectors in the Americas resistant to multiple classes of insecticides, including pyrethroids. The molecular basis of pyrethroid resistance is relatively uncharacterised in American malaria vectors, preventing the design of suitable resistance management strategies. Using whole transcriptome sequencing, we characterized the mechanisms of pyrethroid resistance in Anopheles albimanus from Peru and Guatemala. An. albimanus were phenotyped as either deltamethrin or alpha-cypermethrin resistant. RNA from 1) resistant, 2) unexposed, and 3) a susceptible laboratory strain of An. albimanus was sequenced and analyzed using RNA-Seq. Expression profiles of the three groups were compared based on the current annotation of the An. albimanus reference genome. Several candidate genes associated with pyrethroid resistance in other malaria vectors were found to be overexpressed in resistant An. albimanus. In addition, gene ontology terms related to serine-type endopeptidase activity, extracellular activity and chitin metabolic process were also commonly overexpressed in the field caught resistant and unexposed samples from both Peru and Guatemala when compared to the susceptible strain. The cytochrome P450 CYP9K1 was overexpressed 14x in deltamethrin and 8x in alpha-cypermethrin-resistant samples from Peru and 2x in deltamethrin-resistant samples from Guatemala, relative to the susceptible laboratory strain. CYP6P5 was overexpressed 68x in deltamethrin-resistant samples from Peru but not in deltamethrin-resistant samples from Guatemala. When comparing overexpressed genes between deltamethrin-resistant and alpha-cypermethrin-resistant samples from Peru, a single P450 gene, CYP4C26, was overexpressed 9.8x (p<0.05) in alpha-cypermethrin-resistant samples. In Peruvian deltamethrin-resistant samples, the knockdown resistance mutation (kdr) variant alleles at position 1014 were rare, with approximately 5% frequency, but in the alpha-cypermethrin-resistant samples, the frequency of these alleles was approximately 15-30%. Functional validation of the candidate genes and the kdr mutation as a resistance marker for alpha-cypermethrin will confirm the role of these mechanisms in conferring pyrethroid resistance.

Background Malaria remains an important public health problem in Latin America, and the development of insecticide resistance in malaria vectors poses a major threat to malaria elimination efforts. Monitoring of insecticide susceptibility and the determination of the mechanisms involved in insecticide resistance are needed to effectively guide the deployment of appropriate vector control measures. Here, molecular assays have been developed to screen for mutations associated with insecticide resistance on the voltage-gated sodium channel ( VGSC ) and acetylcholinesterase-1 ( Ace - 1 ) genes in four malaria vectors from Latin America. Methods Degenerate primers were designed to amplify a partial fragment on the VGSC and Ace - 1 genes. Wild-caught individuals for Anopheles albimanus (also historical samples and individuals from a laboratory strain), Anopheles darlingi , Anopheles vestitipennis and Anopheles pseudopunctipennis were used to optimize the PCR assays. All samples were sequenced to validate the PCR results and DNA alignments were constructed for each gene using the unique haplotypes observed. Results Primers designed successfully amplified the VGSC gene in An. albimanus , An. darlingi, An. vestitipennis and An. pseudopunctipennis , and the Ace - 1 gene in both An. albimanus and An. darlingi . DNA sequencing revealed that compared with Anopheles gambiae , there were a total of 29, 28, 21 and 24 single nucleotide polymorphisms (SNPs) on the VGSC gene for An. albimanus (308 bp), An. darlingi (311 bp), An. pseudopunctipennis (263 bp) and An. vestitipennis (254 bp), respectively. On the 459 bp fragment of the Ace - 1 gene, a total of 70 SNPs were detected in An. darlingi and 59 SNPs were detected in An. albimanus compared with An. gambiae . The SNPs detected on the VGSC gene were all synonymous. On the Ace - 1 gene, non-synonymous substitutions were identified on three different codons. All species showed the homozygous wild-type kdr allele (coding for leucine) at codon 995 (formerly reported as codon 1014) on the VGSC gene, but one sample was heterozygous at codon 280 (formerly reported as codon 119) on the Ace - 1 gene, coding for both the resistant (serine) and susceptible (glycine) amino acids. Conclusions New molecular assays to amplify and screen the regions of the VGSC and Ace - 1 genes associated with insecticide resistance are reported for An. albimanus , An. darlingi , An. vestitipennis , and An. pseudopunctipennis . The development of these PCR assays presents an important advance in the analysis of target-site resistance in malaria vectors in the Americas, and will further facilitate the characterization of insecticide resistance mechanisms in these species.

Background Research on mosquito-microbe interactions may lead to new tools for mosquito and mosquito-borne disease control. To date, such research has largely utilized laboratory-reared mosquitoes that typically lack the microbial diversity of wild populations. A logical progression in this area involves working under controlled settings using field-collected mosquitoes or, in most cases, their progeny. Thus, an understanding of how laboratory colonization affects the assemblage of mosquito microbiota would aid in advancing mosquito microbiome studies and their applications beyond laboratory settings. Methods Using high throughput 16S rRNA amplicon sequencing, the internal and cuticle surface microbiota of F1 progeny of wild-caught adult Anopheles albimanus from four locations in Guatemala were characterized. A total of 132 late instar larvae and 135 2–5 day-old, non-blood-fed virgin adult females that were reared under identical laboratory conditions, were pooled (3 individuals/pool) and analysed. Results Results showed location-associated heterogeneity in both F1 larval internal (p = 0.001; pseudo-F = 9.53) and cuticle surface (p = 0.001; pseudo-F = 8.51) microbiota, and only F1 adult cuticle surface (p = 0.001; pseudo-F = 4.5) microbiota, with a more homogenous adult internal microbiota (p = 0.12; pseudo-F = 1.6) across collection sites. Overall, ASVs assigned to Leucobacter, Thorsellia, Chryseobacterium and uncharacterized Enterobacteriaceae, dominated F1 larval internal microbiota, while Acidovorax, Paucibacter, and uncharacterized Comamonadaceae, dominated the larval cuticle surface. F1 adults comprised a less diverse microbiota compared to larvae, with ASVs assigned to the genus Asaia dominating both internal and cuticle surface microbiota, and constituting at least 70% of taxa in each microbial niche. Conclusions These results suggest that location-specific heterogeneity in filed mosquito microbiota can be transferred to F1 progeny under normal laboratory conditions, but this may not last beyond the F1 larval stage without adjustments to maintain field-derived microbiota. These findings provide the first comprehensive characterization of laboratory-colonized F1 An. albimanus progeny from field-derived mothers. This provides a background for studying how parentage and environmental conditions differentially or concomitantly affect mosquito microbiome composition, and how this can be exploited in advancing mosquito microbiome studies and their applications beyond laboratory settings.

Bone forage to treat early osteonecrosis of the femoral head (ONFH) has evolved as the channel to percutaneously deliver cell therapy into the femoral head. However, its efficacy is variable and the drivers towards higher efficacy are currently unknown. The aim of this study was to evaluate the forage technique and correlate it with the efficacy to heal ONFH in a multicentric, multinational clinical trial to implant autologous mesenchymal stromal cells expanded from bone marrow (BM-hMSCs). Methods: In the context of EudraCT 2012-002010-39, patients with small and medium-sized (mean volume = 13.3%, range: 5.4 to 32.2) ONFH stage II (Ficat, ARCO, Steinberg) C1 and C2 (Japanese Investigation Committee (JIC)) were treated with percutaneous forage and implantation of 140 million BM-hMSCs in a standardized manner. Postoperative hip radiographs (AP—anteroposterior and lateral), and MRI sections (coronal and transverse) were retrospectively evaluated in 22 patients to assess the femoral head drilling orientation in both planes, and its relation to the necrotic area. Results: Treatment efficacy was similar in C1 and C2 (coronal plane) and in anterior to posterior (transverse plane) osteonecrotic lesions. The drill crossed the sclerotic rim in all cases. The forage was placed slightly valgus, at 139.3 ± 8.4 grades (range, 125.5–159.3) with higher dispersion (f = 2.6; p = 0.034) than the anatomical cervicodiaphyseal angle. Bonferroni’s correlation between both angles was 0.50 (p = 0.028). More failures were seen with a varus drill positioning, aiming at the central area of the femoral head, outside the weight-bearing area (WBA) (p = 0.049). In the transverse plane, the anterior positioning of the drill did not result in better outcomes (p = 0.477). Conclusion: The forage drilling to deliver cells should be positioned within the WBA in the coronal plane, avoiding varus positioning, and central to anterior in the transverse plane. The efficacy of delivered MSCs to regenerate bone in ONFH could be influenced by the drilling direction. Standardization of this surgical technique is desirable.

Severe osteonecrosis of the femoral head (ONFH) secondary to corticosteroid therapy in symptomatic, hematological young patients currently has no therapeutic alternative, and early total hip replacement (THR) is a high-risk intervention in those patients. To evaluate feasibility, safety, and early efficacy of allogeneic expanded mesenchymal stromal cells (MSCs) in a pilot clinical trial. Pilot phase 1 open, noncontrolled, nonrandomized clinical trial evaluating the bone regeneration capacity in seven hips from four patients (young females 11-19 year old) with symptomatic, severe bilateral femoral head osteonecrosis (secondary to corticosteroid therapy), 1 year after being surgically treated with 140 × 10 allogenic MSC plus forage. The proposed therapy proved feasibility, safety at 1 and 4 years (no related serious adverse events [SAEs]), and early efficacy (nonsignificant) in the case analysis (5/7 hips avoiding THR at 4 years). The implantation of expanded allogeneic MSC in young patients to prevent conversion to a THR or collapse of the femoral head due to severe osteonecrosis is feasible without safety concerns in the longer-term follow-up (FU) upto 4 years. EudraCT number: 2018-000886-35.

Eukaryotic 5S rRNA, synthesized by RNA polymerase III (Pol III), is an essential component of the large ribosomal subunit. Most organisms contain hundreds of 5S rRNA genes organized into tandem arrays. However, the genome of the protozoan parasite Leishmania major contains only 11 copies of the 5S rRNA gene, which are interspersed and associated with other Pol III-transcribed genes. Here we report that, in general, the number and order of the 5S rRNA genes is conserved between different species of Leishmania. While in most organisms 5S rRNA genes are normally associated with the nucleolus, combined fluorescent in situ hybridization and indirect immunofluorescence experiments showed that 5S rRNA genes are mainly located at the nuclear periphery in L. major. Similarly, the tandemly repeated 5S rRNA genes in Trypanosoma cruzi are dispersed throughout the nucleus. In contrast, 5S rRNA transcripts in L. major were localized within the nucleolus, and scattered throughout the cytoplasm, where mature ribosomes are located. Unlike other rRNA species, stable antisense RNA complementary to 5S rRNA is not detected in L. major.

Background Insecticide-treated bed nets (ITNs) are widely used for the prevention and control of malaria. In Guatemala, since 2006, ITNs have been distributed free of charge in the highest risk malaria-endemic areas and constitute one of the primary vector control measures in the country. Despite relying on ITNs for almost 15 years, there is a lack of data to inform the timely replacement of ITNs whose effectiveness becomes diminished by routine use. Methods The survivorship, physical integrity, insecticide content and bio-efficacy of ITNs were assessed through cross-sectional surveys conducted at 18, 24 and 32 months after a 2012 distribution of PermaNet® 2.0 in a malaria focus in Guatemala. A working definition of ‘LLIN providing adequate protection’ was developed based on the combination of the previous parameters and usage of the net. A total of 988 ITNs were analysed (290 at 18 months, 349 at 24 months and 349 at 32 months). Results The functional survivorship of bed nets decreased over time, from 92% at 18 months, to 81% at 24 months and 69% at 32 months. Independent of the time of the survey, less than 80% of the bed nets that were still present in the household were reported to have been used the night before. The proportion of bed nets categorized as “in good condition” per World Health Organization (WHO) guidelines of the total hole surface area, diminished from 77% to 18 months to 58% at 32 months. The portion of ITNs with deltamethrin concentration less than 10 mg/m 2 increased over time. Among the bed nets for which bioassays were conducted, the percentage that met WHO criteria for efficacy dropped from 90% to 18 months to 52% at 32 months. The proportion of long-lasting insecticidal nets (LLINs) providing adequate protection was 38% at 24 months and 21% at 32 months. Conclusions At 32 months, only one in five of the LLINs distributed in the campaign provided adequate protection in terms of survivorship, physical integrity, bio-efficacy and usage. Efforts to encourage the community to retain, use, and properly care for the LLINs may improve their impact. Durability assessments should be included in future campaigns.

BackgroundTrypanosoma cruzi, the causative agent of Chagas disease, is primarily transmitted by triatomine insects, including Triatoma dimidiata. In Central America, vector control programs have significantly reduced transmission; however, certain regions, such as Comapa municipality, department of Jutiapa, Guatemala, continue to experience persistent T. dimidiata infestation. This study presents a 10-year follow-up assessment of triatomine infestation, T. cruzi infection and acute Chagas disease cases after an eco-bio-social intervention.MethodsBetween June and August 2022, entomological surveys were conducted in four communities of Comapa municipality. Seventy-six households were systematically searched for triatomines using the one-person hour method. Triatomines were collected and processed for T. cruzi detection using real-time PCR (qPCR), and blood meal analysis was performed to assess host feeding patterns. Dog samples and environmental DNA from household surfaces were also processed for T. cruzi detection. Additionally, surveillance for acute Chagas disease cases was carried out in collaboration with the Ministry of Health.ResultsPersistent household infestation by T. dimidiata was observed across all four communities, with infestation rates ranging from 17% to 38% and colonization levels ranging from 9% to 29%. The mean household triatomine density remained low, suggesting a possible reduction in transmission risk. A total of 86 triatomines were collected, of which 26% tested positive for T. cruzi (all TcI strain). Amplicon deep-sequencing analysis of the blood meals from triatomines identified seven vertebrate species and one insect family as hosts upon which triatomines had previously fed, with chickens and dogs being the most common blood meal sources (occurring in 85% of triatomines). Of the 132 dogs processed, 22% were positive for T. cruzi (all TcI strain). Two acute Chagas disease cases in children were detected in the surveillance period, including one child in 2015 who remained seropositive in 2022, emphasizing the need for continued surveillance.ConclusionsDespite the multiple interventions that have been carried out for over a decade in Comapa municipality, T. dimidiata infestation remains high in the area, with sustained evidence of acute Chagas disease in humans, necessitating continued vector control efforts. The persistence of T. cruzi transmission among triatomines and dogs and the predominant role of chickens and dogs in supporting the vector population highlight the need for innovative control strategies, including those that target domestic animals, to mitigate Chagas disease risk.

Background: Osteonecrosis (ON) of the femoral head represents a potentially severe disease of the hip where the lack of bone regeneration may lead to femoral head collapse and secondary osteoarthritis, with serious pain and disability. The aim of this European, multicentric clinical trial was to prove safety and early efficacy to heal early femoral head ON in patients through minimally invasive surgical implantation of autologous mesenchymal stromal cells (MSC) expanded from bone marrow (BM) under good manufacturing practices (GMP). Methods: Twenty-two patients with femoral head ON (up to ARCO 2C) were recruited and surgically treated in France, Germany, Italy and Spain with BM-derived, expanded autologous MSC (total dose 140 million MSC in 7 mL). The investigational advanced therapy medicinal product (ATMP) was expanded from BM under the same protocol in all four countries and approved by each National Competent Authority. Patients were followed during two years for safety, based on adverse events, and for efficacy, based on clinical assessment (pain and hip score) and imaging (X-rays and MRIs). Patients were also reviewed after 5 to 6 years at latest follow-up for final outcome. Results: No severe adverse event was recalled as related to the ATMP. At 12 months, 16/20 per protocol and 16/22 under intention-to-treat (2 drop-out at 3 and 5 months) maintained head sphericity and showed bone regeneration. Of the 4 hips with ON progression, 3 required total hip replacement (THR). At 5 years, one patient (healed at 2 years visit) was not located, and 16/21 showed no progression or THR, 4/21 had received THR (all in the first year) and 1 had progressed one stage without THR. Conclusions: Expanded MSCs implantation was safe. Early efficacy was confirmed in 80% of cases under protocol at 2 years. At 5 years, the overall results were maintained and 19% converted to THR, all in the first year.