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Human African trypanosomiasis, or sleeping sickness, is a neglected tropical disease caused by Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense and is invariably fatal unless treated. Current therapies present limitations in their application, parasite resistance, or require further clinical investigation for wider use. Our work, informed by previous findings, presents novel 4-[4-(4-methylpiperazin-1-yl)phenyl]-6-arylpyrimidine derivatives with promising antitrypanosomal activity. In particular, 32 exhibits an in vitro EC50 value of 0.5 µM against Trypanosoma brucei rhodesiense, and analogues 29, 30 and 33 show antitrypanosomal activities in the

Scrub typhus, a vector-borne bacterial infection, is an important but neglected disease globally. Accurately characterizing the burden is challenging because of nonspecific symptoms and limited diagnostics. Prior seroepidemiology studies have struggled to find consensus cutoffs that permit comparisons of estimates across contexts and time. In this study, we present a novel approach that does not require a cutoff and instead uses information about antibody kinetics after infection to estimate seroincidence. We use data from three cohorts of scrub typhus patients in Chiang Rai, Thailand, and Vellore, India, to characterize antibody kinetics after infection and two population serosurveys in the Kathmandu Valley, Nepal, and Tamil Nadu, India, to estimate seroincidence. The samples were tested for IgM and IgG responses to Orientia tsutsugamushi-derived recombinant 56-kDa antigen using commercial enzyme-linked immunosorbent assay kits. We used Bayesian hierarchical models to characterize antibody responses after scrub typhus infection and used the joint distributions of the peak antibody titers and decay rates to estimate population-level incidence rates in the cross-sectional serosurveys. Median responses persisted above an optical density (OD) of 1.8 for 23.6 months for IgG and an OD of 1 for 4.5 months for IgM. Among 18- to 29-year-olds, the seroincidence was 10 per 1,000 person-years (95% CI, 5-19) in Tamil Nadu, India, and 14 per 1,000 person-years (95% CI: 10-20) in the Kathmandu Valley, Nepal. When seroincidence was calculated with antibody decay ignored, the disease burden was underestimated by more than 50%. The approach can be deployed prospectively, coupled with existing serosurveys, or leverage banked samples to efficiently generate scrub typhus seroincidence estimates.

In the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, epithelial populations in the distal lung expressing Angiotensin-converting enzyme 2 (ACE2) are infrequent, and therefore, the model of viral expansion and immune cell engagement remains incompletely understood. Using human lungs to investigate early host-viral pathogenesis, we found that SARS-CoV-2 had a rapid and specific tropism for myeloid populations. Human alveolar macrophages (AMs) reliably expressed ACE2 allowing both spike-ACE2-dependent viral entry and infection. In contrast to Influenza A virus, SARS-CoV-2 infection of AMs was productive, amplifying viral titers. While AMs generated new viruses, the interferon responses to SARS-CoV-2 were muted, hiding the viral dissemination from specific antiviral immune responses. The reliable and veiled viral depot in myeloid cells in the very early phases of SARS-CoV-2 infection of human lungs enables viral expansion in the distal lung and potentially licenses subsequent immune pathologies.

BackgroundHepatitis-E virus (HEV), an etiologic agent of acute inflammatory liver disease, is a significant cause of morbidity and mortality in South Asia. HEV is considered endemic in Nepal; but data on population-level infection transmission is sparse.MethodsWe conducted a longitudinal serosurvey in central Nepal to assess HEV exposure. At each visit, capillary blood samples were collected and analyzed for the presence of anti-HEV IgG antibodies. The study took place between February 2019 and April 2021, with up to 4 visits per participant approximately 6 months apart.ResultsWe collected 2513 samples from 923 participants aged 0-25 years, finding a seroprevalence of 4.8% and a seroincidence rate of 10.9 per 1000 person-years. Young adults and individuals consuming surface water faced the highest incidence of infection. Geospatial analysis identified potential HEV clusters, suggesting a need for targeted interventions.SignificanceOur findings demonstrate that HEV is endemic in Nepal and that the risk of infection increases with age.

ImportanceMost research to understand postacute sequelae of SARS-CoV-2 infection (PASC), or long COVID, has focused on adults, with less known about this complex condition in children. Research is needed to characterize pediatric PASC to enable studies of underlying mechanisms that will guide future treatment.ObjectiveTo identify the most common prolonged symptoms experienced by children (aged 6 to 17 years) after SARS-CoV-2 infection, how these symptoms differ by age (school-age [6-11 years] vs adolescents [12-17 years]), how they cluster into distinct phenotypes, and what symptoms in combination could be used as an empirically derived index to assist researchers to study the likely presence of PASC.Design, setting, and participantsMulticenter longitudinal observational cohort study with participants recruited from more than 60 US health care and community settings between March 2022 and December 2023, including school-age children and adolescents with and without SARS-CoV-2 infection history.ExposureSARS-CoV-2 infection.Main outcomes and measuresPASC and 89 prolonged symptoms across 9 symptom domains.ResultsA total of 898 school-age children (751 with previous SARS-CoV-2 infection [referred to as infected] and 147 without [referred to as uninfected]; mean age, 8.6 years; 49% female; 11% were Black or African American, 34% were Hispanic, Latino, or Spanish, and 60% were White) and 4469 adolescents (3109 infected and 1360 uninfected; mean age, 14.8 years; 48% female; 13% were Black or African American, 21% were Hispanic, Latino, or Spanish, and 73% were White) were included. Median time between first infection and symptom survey was 506 days for school-age children and 556 days for adolescents. In models adjusted for sex and race and ethnicity, 14 symptoms in both school-age children and adolescents were more common in those with SARS-CoV-2 infection history compared with those without infection history, with 4 additional symptoms in school-age children only and 3 in adolescents only. These symptoms affected almost every organ system. Combinations of symptoms most associated with infection history were identified to form a PASC research index for each age group; these indices correlated with poorer overall health and quality of life. The index emphasizes neurocognitive, pain, and gastrointestinal symptoms in school-age children but change or loss in smell or taste, pain, and fatigue/malaise-related symptoms in adolescents. Clustering analyses identified 4 PASC symptom phenotypes in school-age children and 3 in adolescents.Conclusions and relevanceThis study developed research indices for characterizing PASC in children and adolescents. Symptom patterns were similar but distinguishable between the 2 groups, highlighting the importance of characterizing PASC separately for these age ranges.

BackgroundSmall incentives could improve engagement in HIV care. We evaluated the short-term and longer-term effects of financial incentives for visit attendance on viral suppression among adults initiating antiretroviral therapy (ART) in Tanzania.MethodsIn a type 1 hybrid effectiveness-implementation study, we randomised (1:1) 32 primary care HIV clinics in four Tanzanian regions to usual care (control group) or the intervention (usual care plus ≤6 monthly incentives [22 500 Tanzanian Shillings, about US$10, each], conditional on visit attendance). Adults (aged ≥18 years) initiating ART (

ABSTRACT : Introduction:: Gastrointestinal (GI) parasites are major health concerns in both humans and domestic animals. Livestock farming is one of the common livelihood practices in rural Nepal. The proximity at human and domestic animal interface increases the chances of dissemination of enteric parasites, especially those of zoonotic importance. This study was aimed at finding the parasite prevalence and risk factors in both humans and their domestic animals in Jirel community. Materials and Methods:: A field survey was conducted on the Jirel ethnic people and their domestic animals in Dolakha district, where a total of 152 fresh fecal samples from humans and domestic animals (cow, pigs, goats, chickens, ducks, and pigeons) were collected. The feces were examined by wet mounts and concentration techniques. A structured questionnaire survey was carried out among the local people and owners of the domestic animals to gather sociodemographic information, awareness, and hygienic practices in relation to parasite transmission Results:: The enteric parasite prevalence was found to be highest in goats (80.0%;12/15), followed by pigs (55.55%;5/9), cows (45.45%;6/11), chickens (11.7%;4/34), and humans (1.41%;1/71), while the fecal samples of ducks and pigeons did not contain any parasites. The only parasite identified in humans was Ascaris lumbricoides. Similarly, three genera of GI parasites (Eimeria sp., Strongyloides sp, and Trichuris sp.) from goats, two genera each from cow (Eimeria sp. and Strongyloides sp.), pigs (Entamoeba sp. and A. suum), and chickens (Eimeria sp. and Ascaridia galli), were detected Conclusions:: Based on the direct field observation, questionnaire survey and laboratory analysis, it is concluded that the Jirel community people are aware of health and hygiene; however, intervention measures are necessary to prevent parasitic infection in their domestic animals.

BackgroundHIV testing rates among South African men lag behind rates for women and national targets. Community-based HIV self-screening (HIVSS) distribution and follow-up by community health workers (CHWs) is a scalable option to increase testing coverage, diagnosis, and treatment initiation. We provided HIVSS and assisted linkage to care to men not recently tested (within the past 12 months) residing in high-HIV-burden areas of Johannesburg.MethodsCHWs distributed HIVSS in 6 clinic catchment areas. Follow-up to encourage confirmatory testing and antiretroviral therapy initiation was conducted through personal support (PS) or an automated short message service (SMS) follow-up and linkage system in 3 clinic areas each. Using a quasi-experimental pre-post design, we compared differences in the proportion of men testing in the clinic catchment areas during the HIVSS campaign (June-August 2019) to the 3 months prior (March-May 2019) and compared treatment initiations by assisted linkage strategy.ResultsAmong 4793 participants accepting HIVSS, 62% had never tested. Among 3993 participants with follow-up data, 90.6% reported using their HIVSS kit. Testing coverage among men increased by 156%, from under 4% when only clinic-based HIV testing services were available to 9.5% when HIVSS and HIV testing services were available (z = -11.6; P < 0.01). Reported test use was higher for men followed through PS (99% vs. 68% in SMS); however, significantly more men reported reactive self-test results in the SMS group compared with PS (6.4% vs. 2.0%), resulting in more antiretroviral therapy initiations in the SMS group compared with PS (23 vs. 9; P < 0.01).ConclusionsCHW HIVSS distribution significantly increases testing among men. While PS enabled personalized follow-up, reporting differences indicate SMS is more acceptable and better aligned with expectations of privacy associated with HIVSS.

As a proposed mediator between stigma-related stressors and negative mental health outcomes, HIV-related shame has been predictive of increased rates of substance use and difficulties adhering to antiretroviral treatment among people with HIV. These downstream manifestations have ultimately impeded progress toward national goals to End the HIV Epidemic, in part due to limited success of conventional psychotherapies in addressing HIV-related shame. In a pilot clinical trial (N = 12), receipt of psilocybin-assisted group therapy was associated with a large pre-post decrease in HIV-related shame as measured by the HIV and Abuse Related Shame Inventory, with a median (IQR) change of - 5.5 (- 6.5, - 3.5) points from baseline to 3-months follow-up (Z = - 2.6, p = 0.009, r = - 0.75). A paradoxical exacerbation of sexual abuse-related shame experienced by two participants following receipt of psilocybin raises critical questions regarding the use of psilocybin therapy among patients with trauma. These preliminary findings carry potential significance for the future of HIV care.

Language barriers are major obstacles that Asian American immigrants face when accessing health care in the USA. This study was conducted to explore the impact of language barriers and facilitators on the health care of Asian Americans. Qualitative, in-depth interviews and quantitative surveys were conducted with 69 Asian Americans (Chinese, Filipino, Japanese, Malaysian, Indonesian, Vietnamese, and mixed Asian backgrounds) living with HIV (AALWH) in three urban areas (New York, San Francisco, and Los Angeles) in 2013 and from 2017 to 2020. The quantitative data indicate that language ability is negatively associated with stigma. Major themes emerged related to communication, including the impact of language barriers on HIV care and the positive impact of language facilitators-family members/friends, case managers, or interpreters-who can communicate with healthcare providers in the AALWH's native language. Language barriers negatively impact access to HIV-related services and thus result in decreased adherence to antiretroviral therapy, increased unmet healthcare needs, and increased HIV-related stigma. Language facilitators enhanced the connection between AALWH and the healthcare system by facilitating their engagement with health care providers. Language barriers experienced by AALWH not only impact their healthcare decisions and treatment choices but also increase levels of external stigma which may influence the process of acculturation to the host country. Language facilitators and barriers to health services for AALWH represent a target for future interventions in this population.

Across sub-Saharan Africa, men are less likely to know their HIV status than women, leading to later treatment initiation. Little is known about how experiences with general health services affect men's use of HIV testing. We used data from a 2019 community-representative survey of men in Malawi to understand frequency and cause of men's negative health service experiences (defined as men reporting they "would not recommend" a facility) and their association with future HIV testing. We conducted univariable and multivariable logistic regressions to determine which aspects of health facility visits were associated with would-not-recommend experiences and to determine if would-not-recommend experiences 12-24 months prior to the survey were associated with HIV testing in the 12 months prior to the survey. Among 1,098 men eligible for HIV testing in the 12 months prior to the survey, median age was 34 years; 9% of men reported at least one would-not-recommend experience, which did not differ by sociodemographics, gender norm beliefs, or HIV stigma beliefs. The factors most strongly associated with would-not-recommend experiences were cost (aOR 5.8, 95%CI 2.9-11.4), cleanliness (aOR 4.2, 95%CI 1.8-9.9), medicine availability (aOR 3.3, 95%CI 1.7-6.4), and wait times (aOR 2.7, 95%CI 1.5-5.0). Reporting a would-not-recommend experience 12-24 months ago was associated with a 59% decrease in likelihood of testing for HIV in the last 12 months (aOR 0.41; 95% CI:0.17-0.96). Dissatisfaction with general health services was strongly associated with reduced HIV testing. Coverage of high-priority screening services like HIV testing may benefit from improving overall health system quality.

The prevalence of "long COVID" is just one of the conundrums highlighting how little we know about the lung's response to viral infection, particularly to syndromecoronavirus-2 (SARS-CoV-2), for which the lung is the point of entry. We used an in vitro human lung system to enable a prospective, unbiased, sequential single-cell level analysis of pulmonary cell responses to infection by multiple SARS-CoV-2 strains. Starting with human induced pluripotent stem cells and emulating lung organogenesis, we generated and infected three-dimensional, multi-cell-type-containing lung organoids (LOs) and gained several unexpected insights. First, SARS-CoV-2 tropism is much broader than previously believed: Many lung cell types are infectable, if not through a canonical receptor-mediated route (e.g., via Angiotensin-converting encyme 2(ACE2)) then via a noncanonical "backdoor" route (via macropinocytosis, a form of endocytosis). Food and Drug Administration (FDA)-approved endocytosis blockers can abrogate such entry, suggesting adjunctive therapies. Regardless of the route of entry, the virus triggers a lung-autonomous, pulmonary epithelial cell-intrinsic, innate immune response involving interferons and cytokine/chemokine production in the absence of hematopoietic derivatives. The virus can spread rapidly throughout human LOs resulting in mitochondrial apoptosis mediated by the prosurvival protein Bcl-xL. This host cytopathic response to the virus may help explain persistent inflammatory signatures in a dysfunctional pulmonary environment of long COVID. The host response to the virus is, in significant part, dependent on pulmonary Surfactant Protein-B, which plays an unanticipated role in signal transduction, viral resistance, dampening of systemic inflammatory cytokine production, and minimizing apoptosis. Exogenous surfactant, in fact, can be broadly therapeutic.

Bacteroides fragilis is a Gram-negative commensal bacterium commonly found in the human colon, which differentiates into two genomospecies termed divisions I and II. Through a comprehensive collection of 694 B. fragilis whole genome sequences, we identify novel features distinguishing these divisions. Our study reveals a distinct geographic distribution with division I strains predominantly found in North America and division II strains in Asia. Additionally, division II strains are more frequently associated with bloodstream infections, suggesting a distinct pathogenic potential. We report differences between the two divisions in gene abundance related to metabolism, virulence, stress response, and colonization strategies. Notably, division II strains harbor more antimicrobial resistance (AMR) genes than division I strains. These findings offer new insights into the functional roles of division I and II strains, indicating specialized niches within the intestine and potential pathogenic roles in extraintestinal sites.ImportanceUnderstanding the distinct functions of microbial species in the gut microbiome is crucial for deciphering their impact on human health. Classifying division II strains as Bacteroides fragilis can lead to erroneous associations, as researchers may mistakenly attribute characteristics observed in division II strains to the more extensively studied division I B. fragilis. Our findings underscore the necessity of recognizing these divisions as separate species with distinct functions. We unveil new findings of differential gene prevalence between division I and II strains in genes associated with intestinal colonization and survival strategies, potentially influencing their role as gut commensals and their pathogenicity in extraintestinal sites. Despite the significant niche overlap and colonization patterns between these groups, our study highlights the complex dynamics that govern strain distribution and behavior, emphasizing the need for a nuanced understanding of these microorganisms.

ObjectivesSome drugs that augment cell-intrinsic defenses or modulate cell death/survival pathways have been reported to selectively kill cells infected with HIV or Simian Immunodeficiency Virus (SIV), but comparative studies are lacking. We hypothesized that these drugs may differ in their ability to kill cells infected with intact and defective proviruses.DesignTo investigate this hypothesis, drugs were tested ex vivo on peripheral blood mononuclear cells (PBMC) from nine antiretroviral therapy (ART)-suppressed individuals.MethodsWe tested drugs currently in clinical use or human trials, including auranofin (p53 modulator), interferon alpha2A, interferon gamma, acitretin (RIG-I inducer), GS-9620/vesatolimod (TLR7 agonist), nivolumab (PD-1 blocker), obatoclax (Bcl-2 inhibitor), birinapant [inhibitor of apoptosis proteins (IAP) inhibitor], bortezomib (proteasome inhibitor), and INK128/sapanisertib [mammalian target of rapamycin mTOR] [c]1/2 inhibitor). After 6 days of treatment, we measured cell counts/viabilities and quantified levels of total, intact, and defective HIV DNA by droplet digital PCR (Intact Proviral DNA Assay).ResultsObatoclax reduced intact HIV DNA [median = 27-30% of dimethyl sulfoxide control (DMSO)] but not defective or total HIV DNA. Other drugs showed no statistically significant effects.ConclusionObatoclax and other Bcl-2 inhibitors deserve further study in combination therapies aimed at reducing the intact HIV reservoir in order to achieve a functional cure and/or reduce HIV-associated immune activation.

Central nervous system (CNS) infections carry a substantial burden of morbidity and mortality worldwide, and accurate and timely diagnosis is required to optimize management. Metagenomic next-generation sequencing (mNGS) has proven to be a valuable tool in detecting pathogens in patients with suspected CNS infection. By sequencing microbial nucleic acids present in a patient's cerebrospinal fluid, brain tissue, or samples collected outside of the CNS, such as plasma, mNGS can detect a wide range of pathogens, including rare, unexpected, and/or fastidious organisms. Furthermore, its target-agnostic approach allows for the identification of both known and novel pathogens. This is particularly useful in cases where conventional diagnostic methods fail to provide an answer. In addition, mNGS can detect multiple microorganisms simultaneously, which is crucial in cases of mixed infections without a clear predominant pathogen. Overall, clinical mNGS testing can help expedite the diagnostic process for CNS infections, guide appropriate management decisions, and ultimately improve clinical outcomes. However, there are key challenges surrounding its use that need to be considered to fully leverage its clinical impact. For example, only a few specialized laboratories offer clinical mNGS due to the complexity of both the laboratory methods and analysis pipelines. Clinicians interpreting mNGS results must be aware of both false negatives-as mNGS is a direct detection modality and requires a sufficient amount of microbial nucleic acid to be present in the sample tested-and false positives-as mNGS detects environmental microbes and their nucleic acids, despite best practices to minimize contamination. Additionally, current costs and turnaround times limit broader implementation of clinical mNGS. Finally, there is uncertainty regarding the best practices for clinical utilization of mNGS, and further work is needed to define the optimal patient population(s), syndrome(s), and time of testing to implement clinical mNGS.

Virulent infectious agents such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and methicillin-resistant Staphylococcus aureus (MRSA) induce tissue damage that recruits neutrophils, monocyte, and macrophages, leading to T cell exhaustion, fibrosis, vascular leak, epithelial cell depletion, and fatal organ damage. Neutrophils, monocytes, and macrophages recruited to pathogen-infected lungs, including SARS-CoV-2-infected lungs, express phosphatidylinositol 3-kinase gamma (PI3Kγ), a signaling protein that coordinates both granulocyte and monocyte trafficking to diseased tissues and immune-suppressive, profibrotic transcription in myeloid cells. PI3Kγ deletion and inhibition with the clinical PI3Kγ inhibitor eganelisib promoted survival in models of infectious diseases, including SARS-CoV-2 and MRSA, by suppressing inflammation, vascular leak, organ damage, and cytokine storm. These results demonstrate essential roles for PI3Kγ in inflammatory lung disease and support the potential use of PI3Kγ inhibitors to suppress inflammation in severe infectious diseases.

Controlling the principal African malaria vector, the mosquito Anopheles gambiae, is considered essential to curtail malaria transmission. However, existing vector control technologies rely on insecticides, which are becoming increasingly ineffective. Sterile insect technique (SIT) is a powerful suppression approach that has successfully eradicated a number of insect pests, yet the A. gambiae toolkit lacks the requisite technologies for its implementation. SIT relies on iterative mass releases of nonbiting, nondriving, sterile males which seek out and mate with monandrous wild females. Once mated, females are permanently sterilized due to mating-induced refractoriness, which results in population suppression of the subsequent generation. However, sterilization by traditional methods renders males unfit, making the creation of precise genetic sterilization methods imperative. Here, we introduce a vector control technology termed precision-guided sterile insect technique (pgSIT), in A. gambiae for inducible, programmed male sterilization and female elimination for wide-scale use in SIT campaigns. Using a binary CRISPR strategy, we cross separate engineered Cas9 and gRNA strains to disrupt male-fertility and female-essential genes, yielding >99.5% male sterility and >99.9% female lethality in hybrid progeny. We demonstrate that these genetically sterilized males have good longevity, are able to induce sustained population suppression in cage trials, and are predicted to eliminate wild A. gambiae populations using mathematical models, making them ideal candidates for release. This work provides a valuable addition to the malaria genetic biocontrol toolkit, enabling scalable SIT-like confinable, species-specific, and safe suppression in the species.

Cluster randomized trials (CRTs) often enroll large numbers of participants; yet due to resource constraints, only a subset of participants may be selected for outcome assessment, and those sampled may not be representative of all cluster members. Missing data also present a challenge: if sampled individuals with measured outcomes are dissimilar from those with missing outcomes, unadjusted estimates of arm-specific endpoints and the intervention effect may be biased. Further, CRTs often enroll and randomize few clusters, limiting statistical power and raising concerns about finite sample performance. Motivated by SEARCH-TB, a CRT aimed at reducing incident tuberculosis infection, we demonstrate interlocking methods to handle these challenges. First, we extend Two-Stage targeted minimum loss-based estimation to account for three sources of missingness: (i) subsampling; (ii) measurement of baseline status among those sampled; and (iii) measurement of final status among those in the incidence cohort (persons known to be at risk at baseline). Second, we critically evaluate the assumptions under which subunits of the cluster can be considered the conditionally independent unit, improving precision and statistical power but also causing the CRT to behave like an observational study. Our application to SEARCH-TB highlights the real-world impact of different assumptions on measurement and dependence; estimates relying on unrealistic assumptions suggested the intervention increased the incidence of TB infection by 18% (risk ratio [RR]=1.18, 95% confidence interval [CI]: 0.85-1.63), while estimates accounting for the sampling scheme, missingness, and within community dependence found the intervention decreased the incident TB by 27% (RR=0.73, 95% CI: 0.57-0.92).

We evaluated the HIV-1 DNA reservoir in peripheral blood mononuclear cells (PBMC) and cerebrospinal fluid (CSF) in people with HIV (PWH) and associations to cognitive dysfunction. Using the intact proviral DNA assay (IPDA), an emerging technique to identify provirus that may be the source of viral rebound, we assessed HIV DNA in CSF and PBMC in PWH regardless of antiretroviral therapy (ART). CSF was used as a sampling surrogate for the central nervous system (CNS) as opposed to tissue. IDPA results (3' defective, 5' defective, and intact HIV DNA) were analyzed by compartment (Wilcoxon signed rank; matched and unmatched pairs). Cognitive performance, measured via a battery of nine neuropsychological (NP) tests, were analyzed for correlation to HIV DNA (Spearman's rho). 11 CSF and 8 PBMC samples from PWH were evaluated both unmatched and matched. Total CSF HIV DNA was detectable in all participants and was significantly higher than in matched PBMCs (p ​= ​0.0039). Intact CSF HIV DNA was detected in 7/11 participants and correlated closely with those in PBMCs but tended to be higher in CSF than in PBMC. CSF HIV DNA did not correlate with global NP performance, but higher values did correlate with worse executive function (p ​= ​0.0440). Intact HIV DNA is frequently present in the CSF of PWH regardless of ART. This further supports the presence of an HIV CNS reservoir and provides a method to study CNS reservoirs during HIV cure studies. Larger studies are needed to evaluate relationships with CNS clinical outcomes.

IntroductionIdentifying the optimal approaches to offering HIV prevention to meet the needs of those at risk is a high priority, particularly given the expanding toolkit of biomedical HIV prevention options. An ongoing study in rural East African communities evaluated the uptake of choices in product, testing mode and location of care delivery through a structured patient-centred HIV prevention delivery model. In this qualitative study, we sought to understand clients' experiences of this "dynamic choice prevention model" (DCP) and highlight pathways of action to inform HIV prevention delivery models.MethodsIn-depth semi-structured interviews were conducted from November 2021 through March 2022 with a purposively selected sample of n = 56 participants in DCP trials (across outpatient departments, antenatal clinics and community settings), and n = 21 healthcare providers (total n = 77). A seven-person multi-regional team translated and inductively coded transcript data. We used a framework analysis approach to identify emergent themes.ResultsIndividuals taking up HIV pre-exposure prophylaxis (PrEP) reported feelings of relief, liberation from fears of acquiring HIV and satisfaction with being able to take action despite partners' behaviours. Couples used a range of approaches afforded by the study to persuade partners to get tested and opt for PrEP. Post-exposure prophylaxis (PEP) use was less common, although women welcomed it in the event of sexual coercion or assault. Participants discussed switching from PEP to PrEP after familiarizing themselves with usage and ascertaining ongoing risk. Participants felt respected by providers, trusted them and appreciated being able to contact them directly for telephone support. Prevention uptake was hindered by stigma, limited experience with and knowledge of prevention methods, gendered and generational power dynamics within intimate partnerships and families, and negative perceptions of methods due to the products themselves. Participants anticipated long-acting injectable PrEP could solve their challenges regarding pill size, daily pill burden and the likelihood of unwanted disclosure.ConclusionsDiverse preferences and barriers to uptake of prevention require a choice of HIV prevention options, locations and delivery modalities-but in addition, flexible, competent and friendly care provision is crucial to promote uptake. Helping clients feel valued, and addressing their unique needs and challenges, enables their agency to prioritize their health.

Antibiotics are vital in combating infectious diseases, yet their increasing use fosters resistance. Antimicrobial resistance (AMR) is rising in Nepal due to factors such as indiscriminate, inappropriate, and inadequate antibiotic usage. This study aims to explore the association between demographic factors and the prevalence of specific bacterial strains within the surveyed population. Additionally, it seeks to determine the antibiotic resistance patterns of these bacteria. Antibiotic susceptibility or resistance data were retrieved from the Medical Records Department (MRD) of the Manmohan Memorial Medical College and Teaching Hospital (MMMCTH) in Kathmandu. Samples from patients with certain types of bacterial infections were included, with 56 from sputum reports, 46 from urine, and 8 from blood samples out of 110 retrieved. Analysis revealed that sputum samples were mostly from older males, while urine samples were mostly from females. Yet, gender did not significantly influence bacterial presence across sample types. Overall, Escherichia coli was the most prevalent bacterium (74%), followed by Salmonella typhi (25%), Staphylococcus aureus (25%), and Klebsiella pneumoniae (23%) isolated from different type of clinical samples. Altogether, 6-15 antibiotics were assessed for sensitivity, with 2–6 antibiotics showing sensitivity to blood bacteria, 1-6 antibiotics demonstrating sensitivity to sputum bacteria, and 3–8 antibiotics exhibiting sensitivity to urine bacteria. Many investigated antibiotics were resistant, only gentamicin exhibited sensitivity for all types of bacteria found in blood, sputum and urine. These findings underscore the importance of discerning bacterial resistance patterns for effective antimicrobial treatment selection.

Antigenically sequence variable M proteins of the major bacterial pathogen Streptococcus pyogenes (Strep A) are responsible for recruiting human C4b-binding protein (C4BP) to the bacterial surface, which enables Strep A to evade destruction by the immune system. The most sequence divergent portion of M proteins, the hypervariable region (HVR), is responsible for binding C4BP. Structural evidence points to the conservation of two C4BP-binding sequence patterns (M2 and M22) in the HVR of numerous M proteins, with this conservation applicable to vaccine immunogen design. These two patterns, however, only partially explain C4BP binding by Strep A. Here, we identified several M proteins that lack these patterns but still bind C4BP and determined the structures of two, M68 and M87 HVRs, in complex with a C4BP fragment. Mutagenesis of these M proteins led to the identification of amino acids that are crucial for C4BP binding, enabling formulation of new C4BP-binding patterns. Mutagenesis was also carried out on M2 and M22 proteins to refine or generate experimentally grounded C4BP-binding patterns. The M22 pattern was the most prevalent among M proteins, followed by the M87 and M2 patterns, while the M68 pattern was rare. These patterns, except for M68, were also evident in numerous M-like Enn proteins. Binding of C4BP via these patterns to Enn proteins was verified. We conclude that C4BP-binding patterns occur frequently in Strep A strains of differing M types, being present in their M or Enn proteins, or frequently both, providing further impetus for their use as vaccine immunogens.

Historically neglected by microbial ecologists, soil viruses are now thought to be critical to global biogeochemical cycles. However, our understanding of their global distribution, activities and interactions with the soil microbiome remains limited. Here we present the Global Soil Virus Atlas, a comprehensive dataset compiled from 2,953 previously sequenced soil metagenomes and composed of 616,935 uncultivated viral genomes and 38,508 unique viral operational taxonomic units. Rarefaction curves from the Global Soil Virus Atlas indicate that most soil viral diversity remains unexplored, further underscored by high spatial turnover and low rates of shared viral operational taxonomic units across samples. By examining genes associated with biogeochemical functions, we also demonstrate the viral potential to impact soil carbon and nutrient cycling. This study represents an extensive characterization of soil viral diversity and provides a foundation for developing testable hypotheses regarding the role of the virosphere in the soil microbiome and global biogeochemistry.

IntroductionThere is little HIV counselling that directly meets the needs of men in Eastern and Southern Africa, limiting men's knowledge about the benefits of HIV treatment and how to overcome barriers to engagement, contributing to poorer HIV-related outcomes than women. Male-specific approaches are needed to improve men's outcomes but may be difficult for healthcare workers (HCWs) to implement with fidelity and quality in low-resource settings. We developed a male-specific counselling curriculum which was implemented by male HCWs and then conducted a mixed-methods quality assessment.MethodsWe audio-recorded counselling sessions to assess the quality of implementation (n = 50) by male HCWs from two cadres (nurse, n = 10 and lay cadre, n = 10) and conducted focus group discussions (FGDs) with HCWs at 6 and 9 months after rollout to understand barriers and facilitators to implementation. Counselling sessions and FGDs were translated, transcribed and analysed using thematic analysis adapted from WHO Quality Counselling Guidelines. We assessed if sessions were respectful, informative, interactive, motivating and included tailored action plans for overcoming barriers to care. All data were collected September 2021-June 2022.ResultsAll sessions used respectful, non-judgemental language. Sessions were highly interactive with most HCWs frequently asking open-ended questions (n = 46, 92%) and often incorporating motivational explanations of how antiretroviral therapy contributes to life goals (n = 42, 84%). Few sessions included individually tailored action plans for clients to overcome barriers to care (n = 9, 18%). New counselling themes were well covered; however, occasionally themes of self-compassion and safe sex were not covered during sessions (n = 16 and n = 11). HCWs believed that having male HCWs conduct counselling, ongoing professional development and keeping detailed counselling notes facilitated quality implementation. Perceived barriers included curriculum length and client hesitancy to participate in action plan development. Findings were similar across cadres.ConclusionsImplementing high-quality male-specific counselling using male nurses and/or lay cadre is feasible. Efforts to utilize lay cadres should be prioritized, particularly in low-resource settings. Programmes should provide comprehensive job aids to support HCWs. Ongoing training and professional development are needed to (1) improve HCWs' skills in tailored action plans, and (2) sensitize HCWs to the need for self-compassion within male clients to promote holistic sexual health.

ObjectiveHIV treatment regimen during pregnancy was associated with preterm delivery (PTD) in the PROMISE 1077 BF trial. Systemic inflammation among pregnant women with HIV could help explain differences in PTD by treatment regimen. We assessed associations between inflammation, treatment regimen, and PTD.Design/methodsA nested 1 : 1 case-control study ( N  = 362) was conducted within a multicountry randomized trial comparing three HIV regimens in pregnant women: zidovudine alone, or combination antiretroviral therapy (ART) with lopinavir/ritonavir and either zidovudine or tenofovir. Cases were women with PTD (

Candida parapsilosis is known to cause severe and persistent outbreaks in clinical settings. Patients infected with multidrug-resistant C. parapsilosis (MDR Cp) isolates were identified in a large Turkish hospital from 2017-2020. We subsequently identified three additional patients infected with MDR Cp isolates in 2022 from the same hospital and two echinocandin-resistant (ECR) isolates from a single patient in another hospital. The increasing number of MDR and ECR isolates contradicts the general principle that the severe fitness cost associated with these phenotypes could prevent their dominance in clinical settings. Here, we employed a multidimensional approach to systematically assess the fitness costs of MDR and ECR C. parapsilosis isolates. Whole-genome sequencing revealed a novel MDR genotype infecting two patients in 2022. Despite severe in vitro defects, the levels and tolerances of the biofilms of our ECR and MDR isolates were generally comparable to those of susceptible wild-type isolates. Surprisingly, the MDR and ECR isolates showed major alterations in their cell wall components, and some of the MDR isolates consistently displayed increased tolerance to the fungicidal activities of primary human neutrophils and were more immunoevasive during exposure to primary human macrophages. Our systemic infection mouse model showed that MDR and ECR C. parapsilosis isolates had comparable fungal burden in most organs relative to susceptible isolates. Overall, we observed a notable increase in the genotypic diversity and frequency of MDR isolates and identified MDR and ECR isolates potentially capable of causing persistent outbreaks in the future.

The presence of HIV in sequestered reservoirs is a central impediment to a functional cure, allowing HIV to persist despite life-long antiretroviral therapy (ART), and driving a variety of comorbid conditions. Our understanding of the latent HIV reservoir in the central nervous system is incomplete, because of difficulties in accessing human central nervous system tissues. Microglia contribute to HIV reservoirs, but the molecular phenotype of HIV-infected microglia is poorly understood. We leveraged the unique "Last Gift" rapid autopsy program, in which people with HIV are closely followed until days or even hours before death. Microglial populations were heterogeneous regarding their gene expression profiles but showed similar chromatin accessibility landscapes. Despite ART, we detected occasional microglia containing cell-associated HIV RNA and HIV DNA integrated into open regions of the host's genome (∼0.005%). Microglia with detectable HIV RNA showed an inflammatory phenotype. These results demonstrate a distinct myeloid cell reservoir in the brains of people with HIV despite suppressive ART. Strategies for curing HIV and neurocognitive impairment will need to consider the myeloid compartment to be successful.

ImportanceUrinary tract infection (UTI) is the second most common infection leading to hospitalization and is often associated with gram-negative multidrug-resistant organisms (MDROs). Clinicians overuse extended-spectrum antibiotics although most patients are at low risk for MDRO infection. Safe strategies to limit overuse of empiric antibiotics are needed.ObjectiveTo evaluate whether computerized provider order entry (CPOE) prompts providing patient- and pathogen-specific MDRO risk estimates could reduce use of empiric extended-spectrum antibiotics for treatment of UTI.Design, setting, and participantsCluster-randomized trial in 59 US community hospitals comparing the effect of a CPOE stewardship bundle (education, feedback, and real-time and risk-based CPOE prompts; 29 hospitals) vs routine stewardship (n = 30 hospitals) on antibiotic selection during the first 3 hospital days (empiric period) in noncritically ill adults (≥18 years) hospitalized with UTI with an 18-month baseline (April 1, 2017-September 30, 2018) and 15-month intervention period (April 1, 2019-June 30, 2020).InterventionsCPOE prompts recommending empiric standard-spectrum antibiotics in patients ordered to receive extended-spectrum antibiotics who have low estimated absolute risk (

ImportancePneumonia is the most common infection requiring hospitalization and is a major reason for overuse of extended-spectrum antibiotics. Despite low risk of multidrug-resistant organism (MDRO) infection, clinical uncertainty often drives initial antibiotic selection. Strategies to limit empiric antibiotic overuse for patients with pneumonia are needed.ObjectiveTo evaluate whether computerized provider order entry (CPOE) prompts providing patient- and pathogen-specific MDRO infection risk estimates could reduce empiric extended-spectrum antibiotics for non-critically ill patients admitted with pneumonia.Design, setting, and participantsCluster-randomized trial in 59 US community hospitals comparing the effect of a CPOE stewardship bundle (education, feedback, and real-time MDRO risk-based CPOE prompts; n = 29 hospitals) vs routine stewardship (n = 30 hospitals) on antibiotic selection during the first 3 hospital days (empiric period) in non-critically ill adults (≥18 years) hospitalized with pneumonia. There was an 18-month baseline period from April 1, 2017, to September 30, 2018, and a 15-month intervention period from April 1, 2019, to June 30, 2020.InterventionCPOE prompts recommending standard-spectrum antibiotics in patients ordered to receive extended-spectrum antibiotics during the empiric period who have low estimated absolute risk (

Enteric pathogens such as Salmonella enterica serovar Typhimurium experience spatial and temporal changes to the metabolic landscape throughout infection. Host reactive oxygen and nitrogen species non-enzymatically convert monosaccharides to alpha hydroxy acids, including L-tartrate. Salmonella utilizes L-tartrate early during infection to support fumarate respiration, while L-tartrate utilization ceases at later time points due to the increased availability of exogenous electron acceptors such as tetrathionate, nitrate, and oxygen. It remains unknown how Salmonella regulates its gene expression to metabolically adapt to changing nutritional environments. Here, we investigated how the transcriptional regulation for L-tartrate metabolism in Salmonella is influenced by infection-relevant cues. L-tartrate induces the transcription of ttdBAU, genes involved in L-tartrate utilization. L-tartrate metabolism is negatively regulated by two previously uncharacterized transcriptional regulators TtdV (STM3357) and TtdW (STM3358), and both TtdV and TtdW are required for the sensing of L-tartrate. The electron acceptors nitrate, tetrathionate, and oxygen repress ttdBAU transcription via the two-component system ArcAB. Furthermore, the regulation of L-tartrate metabolism is required for optimal fitness in a mouse model of Salmonella-induced colitis. TtdV, TtdW, and ArcAB allow for the integration of two cues, i.e., substrate availability and availability of exogenous electron acceptors, to control L-tartrate metabolism. Our findings provide novel insights into how Salmonella prioritizes the utilization of different electron acceptors for respiration as it experiences transitional nutrient availability throughout infection.ImportanceBacterial pathogens must adapt their gene expression profiles to cope with diverse environments encountered during infection. This coordinated process is carried out by the integration of cues that the pathogen senses to fine-tune gene expression in a spatiotemporal manner. Many studies have elucidated the regulatory mechanisms of how Salmonella sense metabolites in the gut to activate or repress its virulence program; however, our understanding of how Salmonella coordinates its gene expression to maximize the utilization of carbon and energy sources found in transitional nutrient niches is not well understood. In this study, we discovered how Salmonella integrates two infection-relevant cues, substrate availability and exogenous electron acceptors, to control L-tartrate metabolism. From our experiments, we propose a model for how L-tartrate metabolism is regulated in response to different metabolic cues in addition to characterizing two previously unknown transcriptional regulators. This study expands our understanding of how microbes combine metabolic cues to enhance fitness during infection.

The impacts of microsporidia on host individuals are frequently subtle and can be context dependent. A key example of the latter comes from a recently discovered microsporidian symbiont of Daphnia, the net impact of which was found to shift from negative to positive based on environmental context. Given this, we hypothesized low baseline virulence of the microsporidian; here, we investigated the impact of infection on hosts in controlled conditions and the absence of other stressors. We also investigated its phylogenetic position, ecology, and host range. The genetic data indicate that the symbiont is Ordospora pajunii, a newly described microsporidian parasite of Daphnia. We show that O. pajunii infection damages the gut, causing infected epithelial cells to lose microvilli and then rupture. The prevalence of this microsporidian could be high (up to 100% in the lab and 77% of adults in the field). Its overall virulence was low in most cases, but some genotypes suffered reduced survival and/or reproduction. Susceptibility and virulence were strongly host-genotype dependent. We found that North American O. pajunii were able to infect multiple Daphnia species, including the European species Daphnia longispina, as well as Ceriodaphnia spp. Given the low, often undetectable virulence of this microsporidian and potentially far-reaching consequences of infections for the host when interacting with other pathogens or food, this Daphnia-O. pajunii symbiosis emerges as a valuable system for studying the mechanisms of context-dependent shifts between mutualism and parasitism, as well as for understanding how symbionts might alter host interactions with resources.ImportanceThe net outcome of symbiosis depends on the costs and benefits to each partner. Those can be context dependent, driving the potential for an interaction to change between parasitism and mutualism. Understanding the baseline fitness impact in an interaction can help us understand those shifts; for an organism that is generally parasitic, it should be easier for it to become a mutualist if its baseline virulence is relatively low. Recently, a microsporidian was found to become beneficial to its Daphnia hosts in certain ecological contexts, but little was known about the symbiont (including its species identity). Here, we identify it as the microsporidium Ordospora pajunii. Despite the parasitic nature of microsporidia, we found O. pajunii to be, at most, mildly virulent; this helps explain why it can shift toward mutualism in certain ecological contexts and helps establish O. pajunii is a valuable model for investigating shifts along the mutualism-parasitism continuum.

Endofungal Mycetohabitans (formerly Burkholderia) spp. rely on a type III secretion system to deliver mostly unidentified effector proteins when colonizing their host fungus, Rhizopus microsporus. The one known secreted effector family from Mycetohabitans consists of homologues of transcription activator-like (TAL) effectors, which are used by plant pathogenic Xanthomonas and Ralstonia spp. to activate host genes that promote disease. These 'Burkholderia TAL-like (Btl)' proteins bind corresponding specific DNA sequences in a predictable manner, but their genomic target(s) and impact on transcription in the fungus are unknown. Recent phenotyping of Btl mutants of two Mycetohabitans strains revealed that the single Btl in one Mycetohabitans endofungorum strain enhances fungal membrane stress tolerance, while others in a Mycetohabitans rhizoxinica strain promote bacterial colonization of the fungus. The phenotypic diversity underscores the need to assess the sequence diversity and, given that sequence diversity translates to DNA targeting specificity, the functional diversity of Btl proteins. Using a dual approach to maximize capture of Btl protein sequences for our analysis, we sequenced and assembled nine Mycetohabitans spp. genomes using long-read PacBio technology and also mined available short-read Illumina fungal-bacterial metagenomes. We show that btl genes are present across diverse Mycetohabitans strains from Mucoromycota fungal hosts yet vary in sequences and predicted DNA binding specificity. Phylogenetic analysis revealed distinct clades of Btl proteins and suggested that Mycetohabitans might contain more species than previously recognized. Within our data set, Btl proteins were more conserved across M. rhizoxinica strains than across M. endofungorum, but there was also evidence of greater overall strain diversity within the latter clade. Overall, the results suggest that Btl proteins contribute to bacterial-fungal symbioses in myriad ways.

BackgroundHousehold contact investigations are effective for finding tuberculosis (TB) cases but are hindered by low referral uptake for clinic-based evaluation and testing. We assessed the acceptability and feasibility of in-home testing of household contacts (HHC) using the GeneXpert Edge platform.MethodsWe conducted a 2-arm, randomized study in Eastern Cape, South Africa. HHCs were verbally assessed using the World Health Organization-recommended 4-symptom screen. Households with ≥1 eligible symptomatic contact were randomized. Intervention households received in-home GeneXpert MTB/RIF molecular testing. GeneXpert-positive HHCs were referred for clinic-based treatment. Standard-of-care households were referred for clinic-based sputum collection and testing. We defined acceptability as agreeing to in-home testing and feasibility as generation of valid Xpert MTB/RIF results. The proportion and timeliness of test results received was compared between groups.ResultsEighty-four households were randomized (n = 42 per arm). Of 100 eligible HHCs identified, 98/100 (98%) provided consent. Of 51 HHCs allocated to the intervention arm, all accepted in-home testing; of those, 24/51 (47%) were sputum productive and 23/24 (96%) received their test results. Of 47 HCCs allocated to standard-of-care, 7 (15%) presented for clinic-based TB evaluation, 6/47 (13%) were tested, and 4/6 (67%) returned for their results. The median (interquartile range) number of days from screening to receiving test results was 0 (0) and 16.5 (11-15) in the intervention and standard-of-care arms, respectively.ConclusionsIn-home testing for TB was acceptable, feasible, and increased HHCs with a molecular test result. In-home testing mitigates a major limitation of household contact investigations (dependency on clinic-based referral), revealing new strategies for enhancing early case detection.

BACKGROUNDEarly antiretroviral therapy initiation (ARTi) in HIV-1 restricts reservoir size and diversity while preserving immune function, potentially improving opportunities for immunotherapeutic cure strategies. For antibody-based cure approaches, the development of autologous neutralizing antibodies (anAbs) after acute/early ARTi is relevant but is poorly understood.METHODSWe characterized antibody responses in a cohort of 23 participants following ARTi in acute HIV (

Early stages of deadly respiratory diseases including COVID-19 are challenging to elucidate in humans. Here, we define cellular tropism and transcriptomic effects of SARS-CoV-2 virus by productively infecting healthy human lung tissue and using scRNA-seq to reconstruct the transcriptional program in "infection pseudotime" for individual lung cell types. SARS-CoV-2 predominantly infected activated interstitial macrophages (IMs), which can accumulate thousands of viral RNA molecules, taking over 60% of the cell transcriptome and forming dense viral RNA bodies while inducing host profibrotic (TGFB1, SPP1) and inflammatory (early interferon response, CCL2/7/8/13, CXCL10, and IL6/10) programs and destroying host cell architecture. Infected alveolar macrophages (AMs) showed none of these extreme responses. Spike-dependent viral entry into AMs used ACE2 and Sialoadhesin/CD169, whereas IM entry used DC-SIGN/CD209. These results identify activated IMs as a prominent site of viral takeover, the focus of inflammation and fibrosis, and suggest targeting CD209 to prevent early pathology in COVID-19 pneumonia. This approach can be generalized to any human lung infection and to evaluate therapeutics.

IntroductionAdverse sexual and reproductive health (SRH) outcomes, such as unplanned pregnancies and HIV infection, disproportionately affect adolescent girls and young women (AGYW; aged 15-24 years) in east Africa. Increasing uptake of preventive SRH services via innovative, youth-centred interventions is imperative to addressing disparities in SRH outcomes.Methods and analysisFrom 2018 to 2019, we used human-centred design to co-develop a theoretically driven HIV and pregnancy prevention intervention for AGYW at private drug shops called Accredited Drug Dispensing Outlets (ADDOs) in Tanzania. The result, Malkia Klabu (Queen Club), was a customer loyalty programme designed to strengthen ADDOs' role as SRH providers while encouraging uptake of critical SRH prevention products among AGYW. Malkia Klabu members had access to free contraceptives and oral HIV self-test (HIVST) kits and earned punches on a loyalty card for other shop purchases; punches were redeemable for small prizes. Our pilot among 40 shops showed that intervention ADDOs had higher AGYW patronage and distributed more HIVST kits and contraceptives to AGYW relative to business-as-usual (ie, client purchasing) comparison shops. We will conduct a cluster-randomised controlled trial (c-RCT) among 120-140 ADDOs in 40 health catchment areas in Shinyanga and Mwanza Regions (Lake Zone), Tanzania. ADDO shop recruitment includes a 1-month run-in with a tablet-based electronic inventory management system for tracking shop transactions, followed by enrolment, randomisation and a 24-month trial period. Our c-RCT evaluating the human-centred design-derived intervention will assess population impact on the primary outcomes of HIV diagnoses and antenatal care registrations, measured with routine health facility data. We will also assess secondary outcomes focusing on mechanisms of action, evaluate programme exposure and AGYW behaviour change in interviews with AGYW, and assess shop-level implementation strategies and fidelity.Ethics and disseminationEthical approval was granted from both the University of California, San Francisco and the Tanzanian National Institute for Medical Research. Study progress and final outcomes will be posted annually to the National Clinical Trials website; study dissemination will occur at conferences, peer-reviewed manuscripts and local convenings of stakeholders.Trial registration numberNCT05357144.

BackgroundYoung adult (18-30 years) people who inject drugs (PWID) face high hepatitis C virus (HCV) prevalence. In San Francisco, where >60% of PWID lack stable housing, barriers hinder HCV treatment access. We assessed progress towards the World Health Organization's (WHO) HCV elimination goal of an 80% reduction in incidence over 2015-2030, focusing on young (YPWID) and unstably housed PWID in San Francisco.MethodsWe developed a dynamic HCV transmission model among PWID, parameterized and calibrated using bio-behavioural survey datasets from San Francisco. This included 2018 estimates for the antibody-prevalence among PWID (77%) and care cascade estimates for HCV for YPWID (72% aware of their status and 33% ever initiating treatment). Based on programmatic data, we assumed a 53.8% reduction in testing and 40.7% decrease in treatment from 2020 due to the COVID-19 pandemic, which partially rebounded from April 2021 with testing rates then being 31.1% lower than pre-pandemic rates and treatment numbers being 19.5% lower. We simulated different scenarios of how services changed after the pandemic to project whether elimination goals would be met.ResultsContinuing post-pandemic rates of testing and treatment, the model projects an 83.3% (95% credibility interval [95% CrI]:60.6-96.9%) decrease in incidence among PWID over 2015-2030 to 1.5/100pyrs (95% CrI:0.3-4.4) in 2030. The probability of achieving the elimination goal by 2030 is 62.0%. Among YPWID and unstably housed PWID, the probability of achieving the elimination goal by 2030 is 54.8 and 67.6%, respectively. Importantly, further increasing testing and treatment rates to pre-pandemic levels by 2025 only results in a small increase in the probability (67.5%) of the elimination goal being achieved among all PWID by 2030, while increased coverage of medication for opioid use disorder among YPWID and/or housing interventions results in the probability of achieving elimination increasing to over 75%.ConclusionThe COVID-19 pandemic impeded progress toward achieving HCV elimination. Our findings indicate that existing partial rebounds in HCV testing and treatment may achieve the elimination goal by 2030, with an additional scale-up of interventions aimed at YPWID or unstably housed PWID ensuring San Francisco is likely to achieve elimination by 2030.

All-cause COVID-19 hospitalization ≤ 30 days of infection is a common outcome for severe illness in observational/surveillance studies. Milder COVID-19 disease and COVID-19-specific measurements calls for an evaluation of this endpoint. This was a descriptive, retrospective cohort study of adults ≥ 18 who were established in primary care at Veteran Health Administration (VHA) facilities. The outcome was hospitalization within 30 days of a laboratory-confirmed, symptomatic SARS-CoV-2 infection. Between December 15, 2021 and May 1, 2022, a simple random sample of all VA facilities, excluding Puerto Rico or Philippines, was drawn to identify these hospitalized cases and determine whether hospitalization was due to COVID-19-specific causes. A chart review was conducted to record the inpatient clinical team's diagnosis and whether the inpatient team classified the diagnosis as COVID-19 related or not. These data were used to classify hospitalizations as either due to COVID-19-specific causes (direct manifestations of SARS-CoV-2 infection) or non-COVID-19-specific hospitalizations (incidental SARS-CoV-2 infection), A simple random sample of 9966 (12.3%) all-cause hospitalizations (95% CI: 12.1%, 12.5%) was used to select 300 representative patients. Of these, 226/300 (75.3%) were determined to be COVID-19-specific. COVID-19 pneumonia was most common (147/226, 65.0%). The highest proportion of COVID-19-specific hospitalizations occurred among unvaccinated (85.0%), followed by vaccinated but not boosted (73.7%) and boosted (59.4%) (p

Novel mosquito genetic control tools, such as CRISPR-based gene drives, hold great promise in reducing the global burden of vector-borne diseases. As these technologies advance through the research and development pipeline, there is a growing need for modeling frameworks incorporating increasing levels of entomological and epidemiological detail in order to address questions regarding logistics and biosafety. Epidemiological predictions are becoming increasingly relevant to the development of target product profiles and the design of field trials and interventions, while entomological surveillance is becoming increasingly important to regulation and biosafety. We present MGDrivE 3 (Mosquito Gene Drive Explorer 3), a new version of a previously-developed framework, MGDrivE 2, that investigates the spatial population dynamics of mosquito genetic control systems and their epidemiological implications. The new framework incorporates three major developments: i) a decoupled sampling algorithm allowing the vector portion of the MGDrivE framework to be paired with a more detailed epidemiological framework, ii) a version of the Imperial College London malaria transmission model, which incorporates age structure, various forms of immunity, and human and vector interventions, and iii) a surveillance module that tracks mosquitoes captured by traps throughout the simulation. Example MGDrivE 3 simulations are presented demonstrating the application of the framework to a CRISPR-based homing gene drive linked to dual disease-refractory genes and their potential to interrupt local malaria transmission. Simulations are also presented demonstrating surveillance of such a system by a network of mosquito traps. MGDrivE 3 is freely available as an open-source R package on CRAN (https://cran.r-project.org/package=MGDrivE2) (version 2.1.0), and extensive examples and vignettes are provided. We intend the software to aid in understanding of human health impacts and biosafety of mosquito genetic control tools, and continue to iterate per feedback from the genetic control community.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a broad group of compounds mediating microbial competition in nature. Azole/azoline heterocycle formation in the peptide backbone is a key step in the biosynthesis of many RiPPs. Heterocycle formation in RiPP precursors is often carried out by a scaffold protein, an ATP-dependent cyclodehydratase, and an FMN-dependent dehydrogenase. It has generally been assumed that the orchestration of these modifications is carried out by a stable complex including the scaffold, cyclodehydratase, and dehydrogenase. The antimicrobial RiPP micrococcin begins as a precursor peptide (TclE) with a 35-amino acid N-terminal leader and a 14-amino acid C-terminal core containing six Cys residues that are converted to thiazoles. The putative scaffold protein (TclI) presumably presents the TclE substrate to a cyclodehydratase (TclJ) and a dehydrogenase (TclN) to accomplish the two-step installation of the six thiazoles. In this study, we identify a minimal TclE leader region required for thiazole formation, demonstrate complex formation between TclI, TclJ, and TclN, and further define regions of these proteins required for complex formation. Our results point to a mechanism of thiazole installation in which TclI associates with the two enzymes in a mutually exclusive fashion, such that each enzyme competes for access to the peptide substrate in a dynamic equilibrium, thus ensuring complete modification of each Cys residue in the TclE core.ImportanceThiopeptides are a family of antimicrobial peptides characterized for having sulfur-containing heterocycles and for being highly post-translationally modified. Numerous thiopeptides have been identified; almost all of which inhibit protein synthesis in gram-positive bacteria. These intrinsic antimicrobial properties make thiopeptides promising candidates for the development of new antibiotics. The thiopeptide micrococcin is synthesized by the ribosome and undergoes several post-translational modifications to acquire its bioactivity. In this study, we identify key interactions within the enzymatic complex that carries out cysteine to thiazole conversion in the biosynthesis of micrococcin.

BackgroundSputum-based testing is a barrier to increasing access to molecular diagnostics for tuberculosis (TB). Many people with TB are unable to produce sputum, and sputum processing increases assay complexity and cost. Tongue swabs are emerging as an alternative to sputum, but performance limits are uncertain.MethodsFrom June 2022 to July 2023, we enrolled 397 consecutive adults with cough >2 weeks at 2 health centers in Kampala, Uganda. We collected demographic and clinical information, sputum for TB testing (Xpert MTB/RIF Ultra and 2 liquid cultures), and tongue swabs for same-day quantitative polymerase chain reaction (qPCR) testing. We evaluated tongue swab qPCR diagnostic accuracy versus sputum TB test results, quantified TB targets per swab, assessed the impact of serial swabbing, and compared 2 swab types (Copan FLOQSWAB and Steripack spun polyester).ResultsAmong 397 participants, 43.1% were female, median age was 33 years, 23.5% were diagnosed with human immunodeficiency virus, and 32.0% had confirmed TB. Sputum Xpert Ultra and tongue swab qPCR results were concordant for 98.2% (95% confidence interval [CI]: 96.2-99.1) of participants. Tongue swab qPCR sensitivity was 92.6% (95% CI: 86.5 to 96.0) and specificity was 99.1% (95% CI: 96.9 to 99.8) versus microbiological reference standard. A single tongue swab recovered a 7-log range of TB copies, with a decreasing recovery trend among 4 serial swabs. Swab types performed equivalently.ConclusionsTongue swabs are a promising alternative to sputum for molecular diagnosis of TB, with sensitivity approaching sputum-based molecular tests. Our results provide valuable insights for developing successful tongue swab-based TB diagnostics.

BackgroundInfection prevention (IP) measures are designed to mitigate the transmission of pathogens in healthcare. Using large-scale viral genomic and social network analyses, we determined if IP measures used during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic were adequate in protecting healthcare workers (HCWs) and patients from acquiring SARS-CoV-2.MethodsWe performed retrospective cross-sectional analyses of viral genomics from all available SARS-CoV-2 viral samples collected at UC San Diego Health and social network analysis using the electronic medical record to derive temporospatial overlap of infections among related viromes and supplemented with contact tracing data. The outcome measure was any instance of healthcare transmission, defined as cases with closely related viral genomes and epidemiological connection within the healthcare setting during the infection window. Between November 2020 through January 2022, 12 933 viral genomes were obtained from 35 666 patients and HCWs.ResultsAmong 5112 SARS-CoV-2 viral samples sequenced from the second and third waves of SARS-CoV-2 (pre-Omicron), 291 pairs were derived from persons with a plausible healthcare overlap. Of these, 34 pairs (12%) were phylogenetically linked: 19 attributable to household and 14 to healthcare transmission. During the Omicron wave, 2106 contact pairs among 7821 sequences resulted in 120 (6%) related pairs among 32 clusters, of which 10 were consistent with healthcare transmission. Transmission was more likely to occur in shared spaces in the older hospital compared with the newer hospital (2.54 vs 0.63 transmission events per 1000 admissions, P < .001).ConclusionsIP strategies were effective at identifying and preventing healthcare SARS-CoV-2 transmission.

Ticks are increasingly important vectors of human and agricultural diseases. While many studies have focused on tick-borne bacteria, far less is known about tick-associated viruses and their roles in public health or tick physiology. To address this, we investigated patterns of bacterial and viral communities across two field populations of western black-legged ticks (Ixodes pacificus). Through metatranscriptomic analysis of 100 individual ticks, we quantified taxon prevalence, abundance, and co-occurrence with other members of the tick microbiome. In addition to commonly found tick-associated microbes, we assembled 11 novel RNA virus genomes from Rhabdoviridae, Chuviridae, Picornaviridae, Phenuiviridae, Reoviridae, Solemovidiae, Narnaviridae and two highly divergent RNA virus genomes lacking sequence similarity to any known viral families. We experimentally verified the presence of these in I. pacificus ticks across several life stages. We also unexpectedly identified numerous virus-like transcripts that are likely encoded by tick genomic DNA, and which are distinct from known endogenous viral element-mediated immunity pathways in invertebrates. Taken together, our work reveals that I. pacificus ticks carry a greater diversity of viruses than previously appreciated, in some cases resulting in evolutionarily acquired virus-like transcripts. Our findings highlight how pervasive and intimate tick-virus interactions are, with major implications for both the fundamental biology and vectorial capacity of I. pacificus ticks.ImportanceTicks are increasingly important vectors of disease, particularly in the United States where expanding tick ranges and intrusion into previously wild areas has resulted in increasing human exposure to ticks. Emerging human pathogens have been identified in ticks at an increasing rate, and yet little is known about the full community of microbes circulating in various tick species, a crucial first step to understanding how they interact with each and their tick host, as well as their ability to cause disease in humans. We investigated the bacterial and viral communities of the Western blacklegged tick in California and found 11 previously uncharacterized viruses circulating in this population.

Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d results in accumulation of phage-encoded mRNAs in the phage nucleus, reduces phage protein production, and compromises virion assembly. Taken together, our data show that the conserved ChmC protein plays crucial roles in the viral life cycle, potentially by facilitating phage mRNA translocation through the nuclear shell to promote protein production and virion development.

The mouse pathogen Citrobacter rodentium is utilized as a model organism for studying infections caused by the human pathogens enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) and to elucidate mechanisms of mucosal immunity. In response to C. rodentium infection, innate lymphoid cells and T cells secrete interleukin (IL)-22, a cytokine that promotes mucosal barrier function. IL-22 plays a pivotal role in enabling mice to survive and recover from C. rodentium infection, although the exact mechanisms involved remain incompletely understood. Here, we investigated whether particular components of the host response downstream of IL-22 contribute to the cytokine's protective effects during C. rodentium infection. In line with previous research, mice lacking the IL-22 gene (Il22-/- mice) were highly susceptible to C. rodentium infection. To elucidate the role of specific antimicrobial proteins modulated by IL-22, we infected the following knockout mice: S100A9-/- (calprotectin), Lcn2-/- (lipocalin-2), Reg3b-/- (Reg3β), Reg3g-/- (Reg3γ), and C3-/- (C3). All knockout mice tested displayed a considerable level of resistance to C. rodentium infection, and none phenocopied the lethality observed in Il22-/- mice. By investigating another arm of the IL-22 response, we observed that C. rodentium-infected Il22-/- mice exhibited an overall decrease in gene expression related to intestinal barrier integrity as well as significantly elevated colonic inflammation, gut permeability, and pathogen levels in the spleen. Taken together, these results indicate that host resistance to lethal C. rodentium infection may depend on multiple antimicrobial responses acting in concert, or that other IL-22-regulated processes, such as tissue repair and maintenance of epithelial integrity, play crucial roles in host defense to attaching and effacing pathogens.

Mycobacterium tuberculosis (Mtb) infects lung myeloid cells, but the specific Mtb-permissive cells and host mechanisms supporting Mtb persistence during chronic infection are incompletely characterized. We report that after the development of T cell responses, CD11clo monocyte-derived cells harbor more live Mtb than alveolar macrophages (AM), neutrophils, and CD11chi monocyte-derived cells. Transcriptomic and functional studies revealed that the lysosome pathway is underexpressed in this highly permissive subset, characterized by less lysosome content, acidification, and proteolytic activity than AM, along with less nuclear TFEB, a regulator of lysosome biogenesis. Mtb infection does not drive lysosome deficiency in CD11clo monocyte-derived cells but promotes recruitment of monocytes that develop into permissive lung cells, mediated by the Mtb ESX-1 secretion system. The c-Abl tyrosine kinase inhibitor nilotinib activates TFEB and enhances lysosome functions of macrophages in vitro and in vivo, improving control of Mtb infection. Our results suggest that Mtb exploits lysosome-poor lung cells for persistence and targeting lysosome biogenesis is a potential host-directed therapy for tuberculosis.

BackgroundWe examined HIV prevalence and transmission dynamics among people who inject drugs in the U.S./Mexico border region during the COVID-19 pandemic.MethodsPeople who inject drugs aged ≥18 years from 3 groups were recruited: people who inject drugs who live in San Diego (SD) and engaged in cross-border drug use in Tijuana, Mexico (SD CBDUs), and people who inject drugs in SD and Tijuana (TJ) who did not engage in cross-border drug use (NCBDUs). We computed HIV prevalence at baseline and bivariate incidence-density rates (IR) at 18-month follow-up. Bayesian phylogenetic analysis was used to identify local transmission clusters, estimate their age, and effective reproductive number (Re) over time within the clusters.FindingsAt baseline (n = 612), 26% of participants were female, 9% engaged in sex work, and HIV prevalence was 8% (4% SD CBDU, 4% SD NCBDU, 16% TJ NCBDU). Nine HIV seroconversions occurred over 18 months, IR: 1.357 per 100 person-years (95% CI: 0.470, 2.243); 7 in TJ NCBDU and 2 in SD CBDU. Out of 16 identified phylogenetic clusters, 9 (56%) had sequences from both the U.S. and Mexico (mixed-country). The age of three youngest mixed-country dyads (2018-2021) overlapped with the COVID-related US-Mexico border closure in 2020. One large mixed-country cluster (N = 15) continued to grow during the border closure (Re = 4.8, 95% Highest Posterior Density (HPD) 1.5-9.1) with 47% engaging in sex work.InterpretationAmidst the COVID-19 pandemic and the border closure, cross-border HIV clusters grew. Efforts to end the HIV epidemic in the U.S. should take into account cross-border HIV-1 transmission from Tijuana. Mobile harm reduction services and coordination with municipal HIV programs to initiate anti-retroviral therapy and pre-exposure prophylaxisis are needed to reduce transmission.FundingThis research was supported by the James B. Pendleton Charitable Trust and the San Diego Center for AIDS Research.

Macrophages (MΦ) infected with human immunodeficiency virus (HIV)-1 or activated by its envelope protein gp120 exert neurotoxicity. We found previously that signaling via p38 mitogen-activated protein kinase (p38 MAPK) is essential to the neurotoxicity of HIVgp120-stimulated MΦ. However, the associated downstream pathways remained elusive. Here we show that cysteinyl-leukotrienes (CysLT) released by HIV-infected or HIVgp120 stimulated MΦ downstream of p38 MAPK critically contribute to neurotoxicity. SiRNA-mediated or pharmacological inhibition of p38 MAPK deprives MΦ of CysLT synthase (LTC4S) and, pharmacological inhibition of the cysteinyl-leukotriene receptor 1 (CYSLTR1) protects cerebrocortical neurons against toxicity of both gp120-stimulated and HIV-infected MΦ. Components of the CysLT pathway are differentially regulated in brains of HIV-infected individuals and a transgenic mouse model of NeuroHIV (HIVgp120tg). Moreover, genetic ablation of LTC4S or CysLTR1 prevents neuronal damage and impairment of spatial memory in HIVgp120tg mice. Altogether, our findings suggest a novel critical role for cysteinyl-leukotrienes in HIV-associated brain injury.

BackgroundLong-acting (LA) antiretrovirals may provide meaningful benefit to people who use drugs and people experiencing homelessness (PEH) who face disproportionate structural and psychosocial barriers in adhering to daily oral HIV antiretroviral therapy or pre-exposure prophylaxis (PrEP), but their use in these populations has not been studied.SettingThe Maria X. Martinez Health Resource Center is a low-barrier (eg, no appointment) community-based clinic serving San Francisco PEH.MethodsA multidisciplinary care model with robust monitoring and outreach support was developed to provide LA antiretroviral therapy (ART) and LA-PrEP to eligible patients experiencing difficulties adhering to oral antiretrovirals. Feasibility was assessed by evaluating the rates of HIV viremia and on-time injections among patients receiving LA antiretrovirals over the first 24 months of program implementation.ResultsBetween November 2021 and November 2023, 33 patients initiated LA-ART or LA-PrEP (median age, 37 years; 27% transgender/nonbinary; 73% non-White; 27% street homeless; 52% sheltered homeless; 30% with opioid use disorder; 82% with methamphetamine use disorder). Among 18 patients with HIV, 14 initiated LA-ART injections with detectable viremia (median CD4 count, 340 cells/mm 3 ; mean log 10 viral load, 3.53; SD, 1.62), 8 had never previously been virally suppressed, and all but 1 achieved or maintained virologic suppression (mean, 9.67 months; SD, 8.30). Among 15 LA-PrEP patients, all remained HIV negative (mean, 4.73 months; SD, 2.89). Of 224 total injections administered, 8% were delayed >7 days.DiscussionThe implementation of LA antiretrovirals is feasible in low-barrier, highly supportive clinical settings serving vulnerable PEH. Expansion of such programs will be critical in ending the HIV epidemic.

Plant viruses are ubiquitous throughout plant communities, but research on viral impacts largely focuses on crops. Little is known about how viruses influence wild plants in their native habitats. To address this gap, we examined virus interactions with wild drought-tolerant perennials in California desert natural areas encroached upon by agriculture. We used metagenomics, targeted diagnostics, and phylogenetics to assess virus diversity and clade relationships, and experiments to investigate viral influence on hosts. We focused on three herbaceous perennials ( Cucurbita foetidissima, C. palmata, and Datura wrightii) and tested the hypothesis that these wild species accumulate virus infections typically found in crops and transmitted by polyphagous insects. We predicted that such infections might be retained across seasons and potentially impair plant performance. Virome profiling revealed a rich community of previously characterized virus species (12 total), with virus community structure varying by site and host species. The dominant viruses in the wild hosts were non-native crop pathogens, including cucurbit aphid-borne yellows virus (CABYV) and cucurbit yellow stunting disorder virus (CYSDV). Targeted testing revealed that CABYV infected as many as 88% of sampled wild Cucurbita individuals, with dual CABYV–CYSDV infections common in natural areas adjacent to desert agriculture. CABYV infections reduced shoot and root production in greenhouse experiments with the two wild Cucurbita species. Phylogenetic analyses suggest that CABYV was introduced to California multiple times from other continents. Our findings provide concerning evidence of ways in which human activities can alter virus pressure on wild plants and potentially contribute to plant decline.