Assessing Adequacy: A Meta-Analysis of Rapid Onsite Evaluation of Thyroid Nodules.

INTRODUCTION: Fine-needle aspiration (FNA) is the standard form of preoperative evaluation of thyroid nodule cytological status. A significant number FNAs are classified as inadequate for interpretation, requiring a repeat FNA which is potentially avoidable, costly, and delays treatment. To address these concerns and maximize first-time FNA adequacy, rapid onsite evaluation (ROSE) of FNA specimens was introduced. Our study aims to determine the impact of ROSE on FNA adequacy. METHODS: PubMed, Embase, and Web of Science were searched for primary articles assessing the adequacy of ROSE in thyroid nodules. RESULTS: A total of 17 studies were included for a total of 24,649 thyroid nodes. Thirteen thousand two hundred fifteen (53.6%) thyroid nodules were assessed utilizing ROSE and 11,434 (46.4%) were not. Pooled adequacy increased significantly from 76% without ROSE to 92% with rose (P = 0.001). Use of ROSE increased the odds of adequate FNA by 22% (risk ratio (RR) = 1.22, 95% confidence interval (CI) = 1.12-1.32). At institutions with less than 85% effective diagnostic adequacy without ROSE, the risk for diagnostic adequacy increased by 28% with ROSE implementation (RR = 1.28, 95% CI = 1.20-1.37). In contrast, in studies reported from institutions with an effective diagnostic rate greater than 85% without the use of ROSE, the diagnostic adequacy only increased by 5% with ROSE implementation (RR = 1.05, 95% CI = 1.03-1.06). CONCLUSIONS: The use of ROSE during first-time FNA of thyroid nodules can significantly improve adequacy, especially at institutions with baseline high inadequacy rates. Implementation of ROSE can reduce repeat FNAs and its associated consequences.

Experiences of menopausal transition among populations exposed to chronic psychosocial stress in the United States: a scoping review.

BACKGROUND: The transition to menopause is a significant event affecting health, well-being, and quality of life. Menopause typically occurs between the ages of 44-57, accompanied by symptoms such as hot flashes, mood changes, and sleep disturbances. Being postmenopausal also increases the risk of cardiovascular disease, stroke, and osteoporosis. Despite its importance, menopause is under-researched and under-discussed, particularly concerning the impact of chronic psychosocial stress. METHODS: A scoping review of qualitative, quantitative, and mixed methods research was conducted to map existing literature on the transition to menopause among populations experiencing chronic psychosocial stress in the United States. The review followed the PRISMA-ScR methodology, systematically searching literature in PubMed and SCOPUS databases using MeSH terms. Studies were included which focused on menopausal symptoms and psychosocial stressors. Data extraction and charting were performed using Covidence software. RESULTS: Fifteen studies were included, highlighting relationships between socioeconomic status, intimate partner violence, childhood abuse, and racial disparities which influenced menopausal experiences. Lower- income, higher perceived stress, and negative attitudes towards menopause were associated with increased psychological and somatic symptoms and early onset of menopause (prior to age 45). African American women were found to experience earlier onset and more severe vasomotor symptoms compared to their White counterparts. Women veterans used hormone therapy more frequently than the general population, particularly those with mood or anxiety disorders. The review also identified a geographic bias, with most studies conducted in the Northeast, Midwest, and Western regions of the United States. CONCLUSIONS: This review underscores the necessity of considering social, cultural, and environmental factors in understanding menopausal experiences and addressing health disparities. Future research should aim to include diverse populations and adopt longitudinal and qualitative study designs to capture the dynamic nature of menopausal experiences. Policies and interventions directed at improving the well-being of women experiencing menopause in the context of chronic psychosocial stress are warranted. TRIAL REGISTRATION: N/A.

Bicodon bias can determine the role of synonymous SNPs in human diseases.

BACKGROUND: For a long time synonymous single nucleotide polymorphisms were considered as silent mutations. However, nowadays it is well known that they can affect protein conformation and function, leading to altered disease susceptibilities, differential prognosis and/or drug responses, among other clinically relevant genetic traits. This occurs through different mechanisms: by disrupting the splicing signals of precursor mRNAs, affecting regulatory binding-sites of transcription factors and miRNAs, or by modifying the secondary structure of mRNAs. RESULTS: In this paper we considered 22 human genetic diseases or traits, linked to 35 synonymous single nucleotide polymorphisms in 27 different genes. We performed a local sequence context analysis in terms of the ribosomal pause propensity affected by synonymous single nucleotide polymorphisms. We found that synonymous mutations related to the above mentioned mechanisms presented small pause propensity changes, whereas synonymous mutations that were not related to those mechanisms presented large pause propensity changes. On the other hand, we did not observe large variations in the codon usage of codons associated with these mutations. Furthermore, we showed that the changes in the pause propensity associated with benign sSNPs are significantly lower than the pause propensity changes related to sSNPs associated to diseases. CONCLUSIONS: These results suggest that the genetic diseases or traits related to synonymous mutations with large pause propensity changes, could be the consequence of another mechanism underlying non-silent synonymous mutations. Namely, alternative protein configuration related, in turn, to alterations in the ribosome-mediated translational attenuation program encoded by pairs of consecutive codons, not codons. These findings shed light on the latter mechanism based on the perturbation of the co-translational folding process.

NFκB signaling drives pro-granulocytic astroglial responses to neuromyelitis optica patient IgG.

BACKGROUND: Astrocytes expressing the aquaporin-4 water channel are a primary target of pathogenic, disease-specific immunoglobulins (IgG) found in patients with neuromyelitis optica (NMO). Immunopathological analyses of active NMO lesions highlight a unique inflammatory phenotype marked by infiltration of granulocytes. Previous studies characterized this granulocytic infiltrate as a response to vasculocentric complement activation and localized tissue destruction. In contrast, we observe that granulocytic infiltration in NMO lesions occurs independently of complement-mediated tissue destruction or active demyelination. These immunopathological findings led to the hypothesis that NMO IgG stimulates astrocyte signaling that is responsible for granulocytic recruitment in NMO. METHODS: Histopathology was performed on archival formalin-fixed paraffin-embedded autopsy-derived CNS tissue from 23 patients clinically and pathologically diagnosed with NMO or NMO spectrum disorder. Primary murine astroglial cultures were stimulated with IgG isolated from NMO patients or control IgG from healthy donors. Transcriptional responses were assessed by microarray, and translational responses were measured by ELISA. Signaling through the NFκB pathway was measured by western blotting and immunostaining. RESULTS: Stimulation of primary murine astroglial cultures with NMO IgG elicited a reactive and inflammatory transcriptional response that involved signaling through the canonical NFκB pathway. This signaling resulted in the release of pro-granulocytic chemokines and was inhibited by the clinically relevant proteasome inhibitors bortezomib and PR-957. CONCLUSIONS: We propose that the astrocytic NFκB-dependent inflammatory response to stimulation by NMO IgG represents one of the earliest events in NMO pathogenesis, providing a target for therapeutic intervention upstream of irreversible cell death and tissue damage.

Risk Factors for Postoperative Infections Following Appendectomy of Complicated Appendicitis: A Meta-analysis and Retrospective Single-institutional Study.

Patients with complicated appendicitis have an increased risk for postoperative infections. Potential risk factors for postoperative infections through a meta-analysis and retrospective chart review are discussed. A meta-analysis consisting of 35 studies analyzing complicated appendicitis treated with an appendectomy noting at least 1 postoperative infection was performed. A retrospective review was then conducted in patients diagnosed with complicated appendicitis after appendectomy. Of 5326 patients in total, 15.4% developed postoperative infections. Laparoscopic surgery and perioperative hyperoxygenation were found to be protective factors for the development of infection. Retrospectively, 53.2% of patients presented with complicated appendicitis. Patients with complicated appendicitis were more likely to be older in age and have an increased length of stay. Patient demographics, operative time, and comorbid status had no effect on postoperative infection or readmission rate. Physicians should strongly consider minimally invasive techniques to treat all cases of complicated appendicitis irrespective of comorbidities, age, sex, or body mass index.

Safety and Efficacy of Right Axillary Thoracotomy for Repair of Congenital Heart Defects in Children.

BACKGROUND: Minimally invasive repair of congenital heart defects in children has not gained wide popularity yet compared to minimally invasive approaches in adults. We sought to review our experience with this approach in children. METHODS: This study included a total of 37 children (24 girls, 64.9%) with a mean age of 6.5 ± 5.1 years, who underwent vertical axillary right minithoracotomy for repair of a variety of congenital heart defects between May 2020 and June 2022. RESULTS: The mean weight of these children was 25.66 ± 18.3 kg. Trisomy 21 syndrome was present in 3 patients (8.1%). The most common congenital heart defects that were repaired via this approach were atrial septal defects (secundum in 11 patients, 29.7%; primum in 5, 13.5%; and unroofed coronary sinus in 1, 2.7%). Twelve patients (32.4%) underwent repair of partial anomalous pulmonary venous connections with or without sinus venosus defects, while 4 patients (10.8%) underwent closure of membranous ventricular septal defects. Mitral valve repair, resection of cor triatriatum dexter, epicardial pacemaker placement, and myxoma resection occurred in 1 patient (2.7%) each. No early mortality or reoperations. All patients were extubated in the operating room, and the mean length of hospital stay was 3.3 ± 2.04 days. Follow-up was complete (mean 7 ± 5 months). No late mortality or reoperations. One patient required epicardial pacemaker placement due to sinus node dysfunction 5 months after surgery. CONCLUSIONS: Vertical axillary right thoracotomy is a cosmetically superior approach that is safe and effective for repair of a variety of congenital heart defects in children.

Comparison of Medical Management versus Parathyroidectomy in Patients with Mild Primary Hyperparathyroidism: A Meta-Analysis.

BACKGROUND: Parathyroidectomy is the definitive cure for patients with primary hyperparathyroidism (pHPT) and has an annual prevalence of 0.2-1% in the United States. Some patients with mild disease are medically managed effectively using calcium-lowering medications and drugs against complications such as osteoporosis; however, many maintain a persistently high calcium level that negatively impacts their skeletal, renal, and psychogenic systems over the long term. This meta-analysis aims to compare the outcomes of medical management versus parathyroidectomy in patients with mild pHPT. STUDY DESIGN: This meta-analysis was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines using PubMed, Embase, and Web of Science by two teams of investigators. Analysis was run using R packages. RESULTS: A total of 12 publications including seven randomized control, two prospective, and three retrospective trials with a total of 1346 patients were included for analysis. The average follow-up for all patients was 41 ± 23.8 months. Demographics, pre-treatment calcium, PTH, and bone mineral density (BMD) were similar between the medical (N = 632) and surgical (N = 714) cohorts. Post-treatment calcium and PTH levels were significantly higher in the medical cohort (10.46 vs. 9.39, p < 0.01), (106.14 vs. 43.25, p = 0.001), respectively. Interestingly, the post-treatment PTH in the medical cohort increased when compared to pre-treatment (83.84 to 106.14). Patients in the medical cohort had lower BMD in lumbar (0.48 g/cm2; OR = 0.42, 95% CI = 0.21, 0.83), femoral (0.48; OR = 0.42, 95% CI = 0.29, 0.61), and hip (0.61; OR = 0.33, 95% CI = 0.13, 0.85). Incidences of fracture, nephrolithiasis, cardiovascular death, or overall mortality were not significantly different between the cohorts. CONCLUSIONS: The present study is the most comprehensive meta-analysis on mild pHPT to date. Our findings reflect that parathyroidectomy is the superior option in the treatment of mild pHPT patients as opposed to medical management.

Differences in synovial fibrosis relative to range of motion in knee osteoarthritis patients.

This study tests if differences exist in the severity of synovial fibrosis between patients undergoing total knee arthroplasty (TKA) for osteoarthritis (OA) to help explain disparate deficits in pre- and postoperative range of motion (ROM) between patient groups. 117 knee OA patients were grouped by women (n = 74) and men (n = 43) or those who self-reported as Black (n = 48) or White (n = 69). ROM was measured pre- and post-TKA. Condyles and synovium collected during TKA were scored histologically for OA severity and synovitis. Fibrosis was measured from picrosirius-stained sections of the synovium. Data were analyzed using Mann-Whitney, parametric, and Spearman's rho tests with alpha at 0.05. We found no significant differences between patient age, BMI, radiographic scores, or deformity type when grouped by sex or race, or between metrics or OA severity when grouped by sex. Notably, higher synovitis was measured in women (p = .039) than men. White patients had greater ROM before (p = 0.46) and after surgery (p = .021) relative to Black patients. Fibrosis, but not OA severity and synovitis scores, for the total patient sample negatively correlated with preoperative (r s = -0.330; p = .0003) but not postoperative (rs = -0.032; p = .7627) ROM. Black patients manifested more fibrosis than White patients (p = <.0001), without significant differences between sexes. Statement of Clinical Significance: Coupled with histological scoring, measuring perioperative differences in synovial fibrosis against ROM may refine OA classification and justify the in-depth preoperative assessment of the knee as a whole. Such individualized analyses could guide personalized strategies to relieve symptomatic OA when TKA is not readily accessible and promote equitable TKA outcomes.

Quantifying Mediators of Racial Disparities in Knee Osteoarthritis Outcome Scores: A Cross-Sectional Analysis.

Studies on symptomatic osteoarthritis suggest that Black patients report worse pain and symptoms compared with White patients with osteoarthritis. In this study, we aimed to quantify the relationship among variables such as overall health and socioeconomic status that may contribute to disparities in patient-reported outcomes. METHODS: A total of 223 patients were enrolled. A mediation analysis was used to evaluate cross-sectional associations between race and the Knee injury and Osteoarthritis Outcome Score (KOOS) questionnaire, which was administered to patients prior to undergoing primary total knee arthroplasty. RESULTS: Black patients had worse KOOS pain, symptoms, and activities of daily living subscale scores than White patients. In our cohort, Black patients were younger, more likely to be female, and more likely to report lower educational status. We identified age, sex, Charlson Comorbidity Index, and education as partial mediators of racial disparities in KOOS subscale scores. Insurance status, deformity, radiographic (Kellgren-Lawrence) grade, C-reactive protein level, marital status, body mass index, and income did not show mediating effects. We found that, if age and sex were equal in both cohorts, the racial disparity in KOOS symptom scores would be reduced by 20.7% and 9.1%, respectively (95% confidence intervals [CIs], -5.1% to 47% and -5.5% to 26.3%). For KOOS pain scores, age and education level explained 18.9% and 5.1% of the racial disparity (95% CIs, -0.6% to 37% and -10.8% to 22.9%). Finally, for KOOS activities of daily living scores, education level explained 3.2% of the disparity (95% CI, -19.4% to 26.6%). CONCLUSIONS: No single factor in our study completely explained the racial disparity in KOOS scores, but our findings did suggest that several factors can combine to mediate this disparity in outcome scores. Quantification of variables that mediate racial disparity can help to build models for risk adjustment, pinpoint vulnerable populations, and identify primary points of intervention. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

HoSeIn: A Workflow for Integrating Various Homology Search Results from Metagenomic and Metatranscriptomic Sequence Datasets.

Data generated by metagenomic and metatranscriptomic experiments is both enormous and inherently noisy. When using taxonomy-dependent alignment-based methods to classify and label reads, the first step consists in performing homology searches against sequence databases. To obtain the most information from the samples, nucleotide sequences are usually compared to various databases (nucleotide and protein) using local sequence aligners such as BLASTN and BLASTX. Nevertheless, the analysis and integration of these results can be problematic because the outputs from these searches usually show inconsistencies, which can be notorious when working with RNA-seq. Moreover, and to the best of our knowledge, existing tools do not criss-cross and integrate information from the different homology searches, but provide the results of each analysis separately. We developed the HoSeIn workflow to intersect the information from these homology searches, and then determine the taxonomic and functional profile of the sample using this integrated information. The workflow is based on the assumption that the sequences that correspond to a certain taxon are composed of: sequences that were assigned to the same taxon by both homology searches; sequences that were assigned to that taxon by one of the homology searches but returned no hits in the other one.

Signatures of cell stress and altered bioenergetics in skin fibroblasts from patients with multiple sclerosis.

Multiple sclerosis (MS) is a central nervous system inflammatory demyelinating disease and the most common cause of non-traumatic disability in young adults. Despite progress in the treatment of the active relapsing disease, therapeutic options targeting irreversible progressive decline remain limited. Studies using skin fibroblasts derived from patients with neurodegenerative disorders demonstrate that cell stress pathways and bioenergetics are altered when compared to healthy individuals. However, findings in MS skin fibroblasts are limited. Here, we collected skin fibroblasts from 24 healthy control individuals, 30 patients with MS, and ten with amyotrophic lateral sclerosis (ALS) to investigate altered cell stress profiles. We observed endoplasmic reticulum swelling in MS skin fibroblasts, and increased gene expression of cell stress markers including BIP, ATF4, CHOP, GRP94, P53, and P21. When challenged against hydrogen peroxide, MS skin fibroblasts had reduced resiliency compared to ALS and controls. Mitochondrial and glycolytic functions were perturbed in MS skin fibroblasts while exhibiting a significant increase in lactate production over ALS and controls. Our results suggest that MS skin fibroblasts have an underlying stress phenotype, which may be disease specific. Interrogating MS skin fibroblasts may provide patient specific molecular insights and aid in prognosis, diagnosis, and therapeutic testing enhancing individualized medicine.

Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification.

Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca2+ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.

Exercise and metformin counteract altered mitochondrial function in the insulin-resistant brain.

Insulin resistance associates with increased risk for cognitive decline and dementia; however, the underpinning mechanisms for this increased risk remain to be fully defined. As insulin resistance impairs mitochondrial oxidative metabolism and increases ROS in skeletal muscle, we considered whether similar events occur in the brain, which - like muscle - is rich in insulin receptors and mitochondria. We show that high-fat diet-induced (HFD-induced) brain insulin resistance in mice decreased mitochondrial ATP production rate and oxidative enzyme activities in brain regions rich in insulin receptors. HFD increased ROS emission and reduced antioxidant enzyme activities, with the concurrent accumulation of oxidatively damaged mitochondrial proteins and increased mitochondrial fission. Improvement of insulin sensitivity by both aerobic exercise and metformin ameliorated HFD-induced abnormalities. Moreover, insulin-induced enhancement of ATP production in primary cortical neurons and astrocytes was counteracted by the insulin receptor antagonist S961, demonstrating a direct effect of insulin resistance on brain mitochondria. Further, intranasal S961 administration prevented exercise-induced improvements in ATP production and ROS emission during HFD, supporting that exercise enhances brain mitochondrial function by improving insulin action. These results support that insulin sensitizing by exercise and metformin restores brain mitochondrial function in insulin-resistant states.

A simplified method for the extraction of baculoviral DNA for PCR analysis: a practical application.

There are two major strategies to genetically modify baculoviruses. One uses a bacmid-based system which replicates in Escherichia coli using a bacterial origin of replication. The other employs a transfer vector and viral DNA which are co-transfected into insect cells and utilise host enzyme-mediated homologous recombination. Putative recombinants are then typically screened by plaque assay. The bacmid system is more convenient, but it requires a number of complex construction and isolation steps to obtain the correct bacmid genome. Generally, the transfer vector method is preferable when only a small number of genetic modifications are required. In this study a rapid and reliable method was developed to extract baculovirus DNA for PCR analysis from cultured insect cells. Briefly, viral DNA was isolated in three steps: SDS lysis, chloroform extraction and ethanol precipitation. The method was tested for direct screening of recombinant viruses in plaque assays. Contrary to previous reports, baculovirus DNA was isolated directly from viral plaques and successfully analysed by PCR. No prior amplification of the virus by passage in tissue culture was necessary. The major advantage of this method was a reduction in assay times from a few days to a few hours. Moreover, this method is very convenient for detecting baculoviruses in cell culture: cross-contamination within viral stocks, monitoring mixed viral infection and confirmation of viral genomic integrity.

Inflammatory cytokine-induced changes in neural network activity measured by waveform analysis of high-content calcium imaging in murine cortical neurons.

During acute neuroinflammation, increased levels of cytokines within the brain may contribute to synaptic reorganization that results in long-term changes in network hyperexcitability. Indeed, inflammatory cytokines are implicated in synaptic dysfunction in epilepsy and in an array of degenerative and autoimmune diseases of the central nervous system. Current tools for studying the impact of inflammatory factors on neural networks are either insufficiently fast and sensitive or require complicated and costly experimental rigs. Calcium imaging offers a reasonable surrogate for direct measurement of neuronal network activity, but traditional imaging paradigms are confounded by cellular heterogeneity and cannot readily distinguish between glial and neuronal calcium transients. While the establishment of pure neuron cultures is possible, the removal of glial cells ignores physiologically relevant cell-cell interactions that may be critical for circuit level disruptions induced by inflammatory factors. To overcome these issues, we provide techniques and algorithms for image processing and waveform feature extraction using automated analysis of spontaneous and evoked calcium transients in primary murine cortical neuron cultures transduced with an adeno-associated viral vector driving the GCaMP6f reporter behind a synapsin promoter. Using this system, we provide evidence of network perturbations induced by the inflammatory cytokines TNFα, IL1ß, and IFNγ.

Soil Metagenomes from Different Pristine Environments of Northwest Argentina.

This is the first study to use a high-throughput metagenomic shotgun approach to explore the biosynthetic potential of soil metagenomes from different pristine environments of northwest Argentina. Our data sets characterize these metagenomes and provide information on the possible effect these ecosystems have on their diversity and biosynthetic potential.

Metatranscriptomic Analysis of Larval Guts from Field-Collected and Laboratory-Reared Spodoptera frugiperda from the South American Subtropical Region.

This is the first study to report a high-throughput approach integrating gene expression data from Spodoptera frugiperda guts and their associated metatranscriptomes. Our datasets provide information on the potential effects of environmental conditions on the expression profile of S. frugiperda larval guts, their associated metatranscriptome, and putative interactions between them.

Development of a recombination system for the generation of occlusion positive genetically modified Anticarsia gemmatalis multiple nucleopolyhedrovirus.

Anticarsia gemmatalis is an important pest in legume crops in South America and it has been successfully controlled using Anticarsia gemmatalis Multiple Nucleopolyhedrovirus (AgMNPV) in subtropical climate zones. Nevertheless, in temperate climates its speed of kill is too slow. Taking this into account, genetic modification of AgMNPV could lead to improvements of its biopesticidal properties. Here we report the generation of a two-component system that allows the production of recombinant AgMNPV. This system is based on a parental AgMNPV in which the polyhedrin gene (polh) was replaced by a bacterial ß-galactosidase (lacZ) gene flanked by two target sites for the homing endonuclease I-PpoI. Co-transfection of insect cells with linearized (I-PpoI-digested) parental genome and a transfer vector allowed the restitution of polh and the expression of a heterologous gene upon homologous recombination, with a low background of non-recombinant AgMNPV. The system was validated by constructing a recombinant occlusion-positive (polh+) AgMNPV expressing the green fluorescent protein gene (gfp). This recombinant virus infected larvae normally per os and led to the expression of GFP in cell culture as well as in A. gemmatalis larvae. These results demonstrate that the system is an efficient method for the generation of recombinant AgMNPV expressing heterologous genes, which can be used for manifold purposes, including biotechnological and pharmaceutical applications and the production of orally infectious recombinants with improved biopesticidal properties.

Polymerase chain reaction-based assay for the detection and identification of sand fly gregarines in Lutzomyia longipalpis, a vector of visceral leishmaniasis.

Gregarines that parasitise phlebotomine sand flies belong to the genus Psychodiella and, even though they are highly host-specific, only five species have been described to date. Their most outstanding features include the unique localisation of the oocysts in the accessory glands of the female host, which ensures contamination of the egg surface during oviposition, and the fact that they naturally parasitise the vectors of Leishmania, causal agent of leishmaniasis. The type species, Ps. chagasi, was first described in Lutzomyia longipalpis, vector of visceral leishmaniasis (VL), from Brazil. We recently reported Ps. chagasi sequences in Lu. longipalpis from Posadas (Misiones, Argentina), an endemic VL location where this gregarine had not been previously recorded. In order to analyse the incidence of Ps. chagasi infections in Lu. longipalpis from this location, the aim of this study was to develop a diagnostic assay for sand fly gregarine parasites in Lu. longipalpis. For this, we designed primers using the Ps. chagasi sequences we previously identified and performed an in vitro validation by PCR amplification of the original sand fly samples. Their specificity and sensitivity as diagnostic primers were subsequently confirmed by PCR reactions using total DNA extracted from naturally infected Lu. longipalpis from the same location (Posadas, Argentina).