FDA's proposed rule and its regulatory impact on emerging and reemerging neglected tropical diseases in the United States.

Diagnosing infectious diseases significantly influences patient care, aiding in outbreak identification, response, and public health monitoring. However, the range of FDA-approved molecular tests remains notably limited, especially concerning neglected tropical diseases (NTDs). Drawing upon our experience as one of the largest healthcare networks in the greater New York metropolitan area, this viewpoint manuscript aims to spotlight the existing diagnostic landscape and unmet clinical needs for 4 emerging NTDs increasingly prevalent in the United States, additionally, it delves into the possible adverse effects of the FDA's Proposed Rule on Laboratory-Developed Tests for these clinical conditions and the broader spectrum of NTDs.

In vitro diagnostic methods of Chagas disease in the clinical laboratory: a scoping review.

Background: Chagas disease (CD), caused by Trypanosoma cruzi, is a global health concern with expanding geographical reach. Despite improved and accessible test methods, diagnosing CD in its various phases remains complex. The existence of clinical scenarios, including immunosuppressed patients, transplant-related CD reactivation, transfusion-associated cases, and orally transmitted acute infections, adds to the diagnostic challenge. No singular gold standard test exists for all phases, and recommendations from PAHO and the CDC advocate for the use of two serological methods for chronic CD diagnosis, while molecular methods or direct parasite detection are suggested for the acute phase. Given the complexity in the diagnostic landscape of CD, the goal of this scoping review is to characterize available diagnostic tests for CD in the clinical laboratory. Methods: A literature search in PubMed was conducted on studies related to In vitro diagnosis (IVD) in humans published in English, Spanish, or Portuguese language as of 28 August 2023, and extended backward with no predefined time frame. Studies underwent title and abstract screening, followed by full-text review. Studies included were classified based on the diagnostic method used. Test methods were grouped as serological, molecular, and other methods. Performance, availability, and regulatory status were also characterized. Results: Out of 85 studies included in the final review, 115 different tests were identified. These tests comprised 89 serological test types, 21 molecular test types, and 5 other test methods. Predominant serological tests included ELISA (38 studies, 44.70%), Rapid tests (19 studies, 22.35%), and chemiluminescence (10 studies, 11.76%). Among molecular tests, Polymerase Chain Reaction (PCR) assays were notable. Twenty-eight tests were approved globally for IVD or donor testing, all being serological methods. Molecular assays lacked approval for IVD in the United States, with only European and Colombian regulatory acceptance. Discussion and conclusion: Serological tests, specifically ELISAs, remain the most used and commercially available diagnostic methods. This makes sense considering that most Chagas disease diagnoses occur in the chronic phase and that the WHO gold standard relies on 2 serological tests to establish the diagnosis of chronic Chagas. ELISAs are feasible and relatively low-cost, with good performance with sensitivities ranging between 77.4% and 100%, and with specificities ranging between 84.2% and 100%. Molecular methods allow the detection of specific variants but rely on the parasite's presence, which limits their utility to parasitemia levels. Depending on the PCR method and the phase of the disease, the sensitivity ranged from 58.88 to 100% while the mean specificity ranged from 68.8% to 100%. Despite their performance, molecular testing remains mostly unavailable for IVD use. Only 3 molecular tests are approved for IVD, which are available only in Europe. Six commercial serological assays approved by the FDA are available for blood and organ donor screening. Currently, there are no guidelines for testing CD oral outbreaks. Although more evidence is needed on how testing methods should be used in special clinical scenarios, a comprehensive approach of clinical assessment and diagnostics tests, including not IVD methods, is required for an accurate CD diagnosis.

Unmasking the Mechanism behind Miltefosine: Revealing the Disruption of Intracellular Ca2+ Homeostasis as a Rational Therapeutic Target in Leishmaniasis and Chagas Disease.

Originally developed as a chemotherapeutic agent, miltefosine (hexadecylphosphocholine) is an inhibitor of phosphatidylcholine synthesis with proven antiparasitic effects. It is the only oral drug approved for the treatment of Leishmaniasis and American Trypanosomiasis (Chagas disease). Although its precise mechanisms are not yet fully understood, miltefosine exhibits broad-spectrum anti-parasitic effects primarily by disrupting the intracellular Ca2+ homeostasis of the parasites while sparing the human hosts. In addition to its inhibitory effects on phosphatidylcholine synthesis and cytochrome c oxidase, miltefosine has been found to affect the unique giant mitochondria and the acidocalcisomes of parasites. Both of these crucial organelles are involved in Ca2+ regulation. Furthermore, miltefosine has the ability to activate a specific parasite Ca2+ channel that responds to sphingosine, which is different to its L-type VGCC human ortholog. Here, we aimed to provide an overview of recent advancements of the anti-parasitic mechanisms of miltefosine. We also explored its multiple molecular targets and investigated how its pleiotropic effects translate into a rational therapeutic approach for patients afflicted by Leishmaniasis and American Trypanosomiasis. Notably, miltefosine's therapeutic effect extends beyond its impact on the parasite to also positively affect the host's immune system. These findings enhance our understanding on its multi-targeted mechanism of action. Overall, this review sheds light on the intricate molecular actions of miltefosine, highlighting its potential as a promising therapeutic option against these debilitating parasitic diseases.

Reappraisal of p16 for Determining HPV Status of Head and Neck Carcinomas Arising in HPV Hotspots.

In an era of head and neck oncology where HPV status will soon dictate patient management, reliable HPV detection is critical. P16 immunohistochemistry (IHC) is currently recommended as the test of choice for oropharyngeal squamous cell carcinomas (OPSCCs). The purpose of this study was to determine the performance characteristics of p16 IHC based on a large clinical experience of squamous cell carcinomas (SCC) arising from HPV hot-spot regions of the head and neck. Consecutive OPSCCs, sinonasal SCCs, and metastatic SCCs of unknown primary sites were evaluated for the presence of HPV by p16 IHC and PCR-based HPV DNA testing as part of clinical care. For discrepant cases, high-risk HPV E6/E7 mRNA in situ hybridization (ISH) and, when possible, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MassArray) genotyping were performed. 746 cancers underwent HPV testing by p16 IHC and DNA PCR genotyping. There was a 95.6% concordance between the 2 assays. Of the 33 discrepant cases, 32 cases (4.3%) were p16 positive but HPV DNA negative. In these cases, 68% were positive for mRNA ISH, invariably related to a non-16 HPV genotype. P16 IHC had an overall accuracy of 98.8%, a sensitivity of 99.8%, and a specificity of 92.1%. P16 IHC is a sensitive and specific assay for determining HPV status. HPV DNA PCR appears vulnerable to HPV genotype diversity and is prone to missing rare non-16 genotypes. HPV mRNA ISH is a practical and reliable direct measure of HPV that may help eliminate the small number of false-positive p16 cases and avoid potential patient harm related to erroneous HPV classification.

Global and genetic diversity of SARS-CoV-2 in wastewater.

The analysis of SARS-CoV-2 in wastewater has enabled us to better understand the spread and evolution of the virus worldwide. To deepen our understanding of its epidemiological and genomic characteristics, we analyzed 10,147 SARS-CoV-2 sequences from 5 continents and 21 countries that were deposited in the GISAID database up until January 31, 2023. Our results revealed over 100 independent lineages of the virus circulating in water samples from March 2020 to January 2023, including variants of interest and concern. We observed four clearly defined periods of global distribution of these variants over time, with one variant being replaced by another. Interestingly, we found that SARS-CoV-2 water-borne sequences from different countries had a close phylogenetic relationship. Additionally, 40 SARS-CoV-2 water-borne sequences from Europe and the USA did not show any phylogenetic relationship with SARS-CoV-2 human sequences. We also identified a significant number of non-synonymous mutations, some of which were detected in previously reported cryptic lineages. Among the countries analyzed, France and the USA showed the highest degree of sequence diversity, while Austria reported the highest number of genomes (6,296). Our study provides valuable information about the epidemiological and genomic diversity of SARS-CoV-2 in wastewater, which can be employed to support public health initiatives and preparedness.

New daily persistent headache after SARS-CoV-2 infection in Latin America: a cross-sectional study.

BACKGROUND: Persistent headache is a frequent symptom after coronavirus disease 2019 (COVID-19) and there is currently limited knowledge about its clinical spectrum and predisposing factors. A subset of patients may be experiencing new daily persistent headache (NDPH) after COVID-19, which is among the most treatment-refractory primary headache syndromes. METHODS: We conducted a cross-sectional study in Latin America to characterize individuals with persistent headache after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and to identify factors associated with NDPH. Participants over 18 years old who tested positive for SARS-CoV-2 infection and reported persistent headache among their symptoms completed an online survey that included demographics, past medical history, persistent headache clinical characteristics, and COVID-19 vaccination status. Based on participants' responses, NDPH diagnostic criteria were used to group participants into NDPH and non-NDPH groups. Participant data was summarized by descriptive statistics. Student's t and Mann-Whitney U tests were used according to the distribution of quantitative variables. For categorical variables, Pearson's chi-square and Fisher's exact tests were used according to the size of expected frequencies. Binomial logistic regression using the backward stepwise selection method was performed to identify factors associated with NDPH. RESULTS: Four hundred and twenty-one participants from 11 Latin American countries met the inclusion criteria. One in four participants met the NDPH diagnostic criteria. The mean age was 40 years, with most participants being female (82%). Over 90% of the participants reported having had mild/moderate COVID-19. Most participants had a history of headache before developing COVID-19 (58%), mainly migraine type (32%). The most predominant clinical characteristics in the NDPH group were occipital location, severe/unbearable intensity, burning character, and radiating pain (p < 0.05). A higher proportion of anxiety symptoms, sleep problems, myalgia, mental fog, paresthesia, nausea, sweating of the face or forehead, and ageusia or hypogeusia as concomitant symptoms were reported in participants with NDPH (p < 0.05). Palpebral edema as a concomitant symptom during the acute phase of COVID-19, occipital location, and burning character of the headache were risk factors associated with NDPH. CONCLUSION: This is the first study in Latin America that explored the clinical spectrum of NDPH after SARS-CoV-2 infection and its associated factors. Clinical evaluation of COVID-19 patients presenting with persistent headache should take into consideration NDPH.

Validation of Oxford nanopore sequencing for improved New World Leishmania species identification via analysis of 70-kDA heat shock protein.

BACKGROUND: Leishmaniasis is a parasitic disease caused by obligate intracellular protozoa of the genus Leishmania. This infection is characterized by a wide range of clinical manifestations, with symptoms greatly dependent on the causal parasitic species. Here we present the design and application of a new 70-kDa heat shock protein gene (hsp70)-based marker of 771 bp (HSP70-Long). We evaluated its sensitivity, specificity and diagnostic performance employing an amplicon-based MinION™ DNA sequencing assay to identify different Leishmania species in clinical samples from humans and reservoirs with cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). We also conducted a comparative analysis between our novel marker and a previously published HSP70 marker known as HSP70-Short, which spans 330 bp. METHODS: A dataset of 27 samples from Colombia, Venezuela and the USA was assembled, of which 26 samples were collected from humans, dogs and cats affected by CL and one sample was collected from a dog with VL in the USA (but originally from Greece). DNA was extracted from each sample and underwent conventional PCR amplification utilizing two distinct HSP70 markers: HSP70-Short and HSP70-Long. The subsequent products were then sequenced using the MinION™ sequencing platform. RESULTS: The results highlight the distinct characteristics of the newly devised HSP70-Long primer, showcasing the notable specificity of this primer, although its sensitivity is lower than that of the HSP70-Short marker. Notably, both markers demonstrated strong discriminatory capabilities, not only in distinguishing between different species within the Leishmania genus but also in identifying instances of coinfection. CONCLUSIONS: This study underscores the outstanding specificity and effectiveness of HSP70-based MinION™ sequencing, in successfully discriminating between diverse Leishmania species and identifying coinfection events within samples sourced from leishmaniasis cases.

Human Lobomycosis Caused by Paracoccidioides (Lacazia) loboi, Panama, 2022.

We report a patient from Panama who had lobomycosis caused by Paracoccidioides (Lacazia) loboi. We used combined clinical-epidemiologic and phylogenetic data, including a new gene sequence dataset on this fungus in Panama, for analysis. Findings contribute useful insights to limited knowledge of this fungal infection in the Mesoamerican Biologic Corridor.

Clinical performance of a quantitative pan-genus Leishmania Real-time PCR assay for diagnosis of cutaneous and visceral leishmaniasis.

Leishmaniasis is a complex vector-borne disease caused by various Leishmania species, affecting humans and animals. Current diagnostic methods have limitations, leading to potential misdiagnosis. Therefore, there is an urgent need for specific and sensitive diagnostic tools. We evaluated the sensitivity of a quantitative real-time PCR (qPCR) assay targeting the 18S gene in diverse clinical sample matrices. The assay showed a wide dynamic range and a limit of detection (LoD) of 1 parasite equivalent per milliliter (eq-p/mL) for all tested species. It exhibited high specificity for Leishmania DNA, with no amplification against other microorganisms. When applied to samples from patients with visceral and cutaneous leishmaniasis, the qPCR assay provided results that matched the reference methods and allowed estimation of parasite burdens. This assay holds promise for diagnosing and monitoring leishmaniasis by offering high sensitivity, specificity, and the ability to estimate parasitemia. Further studies are needed to enhance Leishmania molecular diagnostics and expand their coverage for improved clinical impact.

Molecular and immune signatures, and pathological trajectories of fatal COVID-19 lungs defined by in situ spatial single-cell transcriptome analysis.

Despite intensive studies during the last 3 years, the pathology and underlying molecular mechanism of coronavirus disease 2019 (COVID-19) remain poorly defined. In this study, we investigated the spatial single-cell molecular and cellular features of postmortem COVID-19 lung tissues using in situ sequencing (ISS). We detected 10 414 863 transcripts of 221 genes in whole-slide tissues and segmented them into 1 719 459 cells that were mapped to 18 major parenchymal and immune cell types, all of which were infected by SARS-CoV-2. Compared with the non-COVID-19 control, COVID-19 lungs exhibited reduced alveolar cells (ACs) and increased innate and adaptive immune cells. We also identified 19 differentially expressed genes in both infected and uninfected cells across the tissues, which reflected the altered cellular compositions. Spatial analysis of local infection rates revealed regions with high infection rates that were correlated with high cell densities (HIHD). The HIHD regions expressed high levels of SARS-CoV-2 entry-related factors including ACE2, FURIN, TMPRSS2 and NRP1, and co-localized with organizing pneumonia (OP) and lymphocytic and immune infiltration, which exhibited increased ACs and fibroblasts but decreased vascular endothelial cells and epithelial cells, mirroring the tissue damage and wound healing processes. Sparse nonnegative matrix factorization (SNMF) analysis of niche features identified seven signatures that captured structure and immune niches in COVID-19 tissues. Trajectory inference based on immune niche signatures defined two pathological routes. Trajectory A primarily progressed with increased NK cells and granulocytes, likely reflecting the complication of microbial infections. Trajectory B was marked by increased HIHD and OP, possibly accounting for the increased immune infiltration. The OP regions were marked by high numbers of fibroblasts expressing extremely high levels of COL1A1 and COL1A2. Examination of single-cell RNA-seq data (scRNA-seq) from COVID-19 lung tissues and idiopathic pulmonary fibrosis (IPF) identified similar cell populations consisting mainly of myofibroblasts. Immunofluorescence staining revealed the activation of IL6-STAT3 and TGF-ß-SMAD2/3 pathways in these cells, likely mediating the upregulation of COL1A1 and COL1A2 and excessive fibrosis in the lung tissues. Together, this study provides a spatial single-cell atlas of cellular and molecular signatures of fatal COVID-19 lungs, which reveals the complex spatial cellular heterogeneity, organization, and interactions that characterized the COVID-19 lung pathology.

Molecular Detection of Candida auris Using DiaSorin Molecular Simplexa® Detection Kit: A Diagnostic Performance Evaluation.

Candida auris is a globally emerging fungal pathogen that is associated with healthcare-related infections. The accurate and rapid detection of C. auris is crucial for effective infection prevention, control, and patient management. This study aimed to validate the analytical and diagnostic performance of the DiaSorin Molecular C. auris Detection Kit. The analytical specificity, sensitivity, and reproducibility of the assay were evaluated. The limit of detection (LOD) was determined to be 266 CFU/µL using the ZeptoMetrix Candida auris Z485 strain and standard calibration curves. The assay demonstrated high analytical specificity and showed no amplification against a diverse panel of bacteria and fungi. Clinical validation was conducted using deidentified residual axillary/groin surveillance culture specimens from C. auris culture-positive and culture-negative patients. The DiaSorin Molecular Detection Kit exhibited 100% agreement in sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) when compared to cultures coupled with MALDI-TOF identification. Intra- and inter-reproducibility testing demonstrated consistent and reliable diagnostic performance. This validated assay offers rapid and accurate detection of C. auris, facilitating timely implementation of infection control measures and appropriate patient care. The DiaSorin Molecular C. auris Detection Kit has the potential to aid in controlling the outbreaks caused by this emerging fungal pathogen. Providing a reliable diagnostic tool can contribute to the effective management and containment of C. auris infections in healthcare settings and ultimately improve patient outcomes.

First case of rapidly fatal mpox from secondary (household) transmission in a kidney transplant recipient.

In 2022, a global outbreak of mpox (formerly known as monkeypox) was reported outside of areas considered endemic for the disease, predominantly in persons identifying as men who have sex with men, gay, or bisexual. To date, 3 cases of mpox in solid organ transplant recipients have been published; however, no instances of secondary transmission among solid organ transplant recipients have been reported. Here, we report a case of a 53-year-old male kidney transplant recipient who contracted mpox while caring for a household contact with the disease. The patient's clinical and laboratory findings during his rapidly fatal course, autopsy findings, and genomics analysis are presented, emphasizing the need for expanded pre-exposure prophylaxis efforts in patient populations without the risk factors prioritized by public health authorities for vaccination.

Genomic epidemiology of SARS-CoV-2 variants during the first two years of the pandemic in Colombia.

BACKGROUND: The emergence of highly transmissible SARS-CoV-2 variants has led to surges in cases and the need for global genomic surveillance. While some variants rapidly spread worldwide, other variants only persist nationally. There is a need for more fine-scale analysis to understand transmission dynamics at a country scale. For instance, the Mu variant of interest, also known as lineage B.1.621, was first detected in Colombia and was responsible for a large local wave but only a few sporadic cases elsewhere. METHODS: To better understand the epidemiology of SARS-Cov-2 variants in Colombia, we used 14,049 complete SARS-CoV-2 genomes from the 32 states of Colombia. We performed Bayesian phylodynamic analyses to estimate the time of variants' introduction, their respective effective reproductive number, and effective population size, and the impact of disease control measures. RESULTS: Here, we detect a total of 188 SARS-CoV-2 Pango lineages circulating in Colombia since the pandemic's start. We show that the effective reproduction number oscillated drastically throughout the first two years of the pandemic, with Mu showing the highest transmissibility (Re and growth rate estimation). CONCLUSIONS: Our results reinforce that genomic surveillance programs are essential for countries to make evidence-driven interventions toward the emergence and circulation of novel SARS-CoV-2 variants.

Colombia reported its first COVID-19 case on 6th March 2020. By April 2022, the country had reported over 6 million infections and over 135,000 deaths. Here, we aim to understand how SARS-CoV-2, the virus that causes COVID-19, spread through Colombia over this time and how the predominant version of the virus (variant) changed over time. We found that there were multiple introductions of different variants from other countries into Colombia during the first two years of the pandemic. The Gamma variant was dominant earlier in 2021 but was replaced by the Delta variant. The Mu variant had the highest potential to be transmitted. Our findings provide valuable insights into the pandemic in Colombia and highlight the importance of continued surveillance of the virus to guide the public health response.

Characterizing the blood microbiota of omnivorous and frugivorous bats (Chiroptera: Phyllostomidae) in Casanare, eastern Colombia.

Bats are known reservoirs of seemingly-innocuous pathogenic microorganisms (including viruses, bacteria, fungi, and protozoa), which are associated with triggering disease in other zoonotic groups. The taxonomic diversity of the bats' microbiome is likely associated with species-specific phenotypic, metabolic, and immunogenic capacities. To date, few studies have described the diversity of bat blood microbial communities. Then, this study used amplicon-based next generation sequencing of the V4 hypervariable region of the 16S-rRNA gene in blood samples from omnivorous (n = 16) and frugivorous (n = 9) bats from the department of Casanare in eastern Colombia. We found the blood microbiota in bats to be composed of, among others, Bartonella and Mycoplasma bacterial genera which are associated with various disease phenotypes in other mammals. Furthermore, our results suggest that the bats' dietary habits might determine the composition and the persistence of some pathogens over others in their bloodstream. This study is among the first to describe the blood microbiota in bats, to reflect on co-infection rates of multiple pathogens in the same individual, and to consider the influence of diet as a factor affecting the animal's endogenous microbial community.

Mayaro virus detection by integrating sample preparation with isothermal amplification in portable devices.

Mayaro virus (MAYV) is an emerging mosquito-borne alphavirus that causes clinical symptoms similar to those caused by Chikungunya virus (CHIKV), Dengue virus (DENV), and Zika virus (ZIKV). To differentiate MAYV from these viruses diagnostically, we have developed a portable device that integrates sample preparation with real-time, reverse-transcription, loop-mediated isothermal amplification (rRT-LAMP). First, we designed a rRT-LAMP assay targeting MAYV's non-structural protein (NS1) gene and determined the limit of detection of at least 10 viral genome equivalents per reaction. The assay was specific for MAYV, without cross-reactions with CHIKV, DENV, or ZIKV. The rRT-LAMP assay was integrated with a sample preparation device (SPD) wherein virus lysis and RNA enrichment/purification were carried out on the spot, without requiring pipetting, while subsequent real-time amplification device (RAD) enables virus detection at the point of care (POC). The functions of our platform were demonstrated using purified MAYV RNA or blood samples containing viable viruses. We have used the devices for detection of MAYV in as short as 13 min, with limit of detection to as low as 10 GEs/reaction.

Sequential intrahost evolution and onward transmission of SARS-CoV-2 variants.

Persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been reported in immune-compromised individuals and people undergoing immune-modulatory treatments. Although intrahost evolution has been documented, direct evidence of subsequent transmission and continued stepwise adaptation is lacking. Here we describe sequential persistent SARS-CoV-2 infections in three individuals that led to the emergence, forward transmission, and continued evolution of a new Omicron sublineage, BA.1.23, over an eight-month period. The initially transmitted BA.1.23 variant encoded seven additional amino acid substitutions within the spike protein (E96D, R346T, L455W, K458M, A484V, H681R, A688V), and displayed substantial resistance to neutralization by sera from boosted and/or Omicron BA.1-infected study participants. Subsequent continued BA.1.23 replication resulted in additional substitutions in the spike protein (S254F, N448S, F456L, M458K, F981L, S982L) as well as in five other virus proteins. Our findings demonstrate not only that the Omicron BA.1 lineage can diverge further from its already exceptionally mutated genome but also that patients with persistent infections can transmit these viral variants. Thus, there is, an urgent need to implement strategies to prevent prolonged SARS-CoV-2 replication and to limit the spread of newly emerging, neutralization-resistant variants in vulnerable patients.

Genomic and ultrastructural analysis of monkeypox virus in skin lesions and in human/animal infected cells reveals further morphofunctional insights into viral pathogenicity.

Monkeypox (MPOX) is a zoonotic disease that affects humans and other primates, resulting in a smallpox-like illness. It is caused by monkeypox virus (MPXV), which belongs to the Poxviridae family. Clinically manifested by a range of cutaneous and systemic findings, as well as variable disease severity phenotypes based on the genetic makeup of the virus, the cutaneous niche and respiratory mucosa are the epicenters of MPXV pathogenicity. Herein, we describe the ultrastructural features of MPXV infection in both human cultured cells and cutaneous clinical specimens collected during the 2022-2023 MPOX outbreak in New York City that were revealed through electron microscopy. We observed typical enveloped virions with brick-shaped morphologies that contained surface protrusions, consistent with the classic ultrastructural features of MPXV. In addition, we describe morpho-functional evidence that point to roles of distinct cellular organelles in viral assembly during clinical MPXV infection. Interestingly, in skin lesions, we found abundant melanosomes near viral assembly sites, particularly in the vicinity of mature virions, which provides further insight into virus-host interactions at the subcellular level that contribute to MPXV pathogenesis. These findings not only highlight the importance of electron microscopic studies for further investigation of this emerging pathogen but also in characterizing MPXV pathogenesis during human infection.