The immunoregulatory landscape of human tuberculosis granulomas.

Tuberculosis (TB) in humans is characterized by formation of immune-rich granulomas in infected tissues, the architecture and composition of which are thought to affect disease outcome. However, our understanding of the spatial relationships that control human granulomas is limited. Here, we used multiplexed ion beam imaging by time of flight (MIBI-TOF) to image 37 proteins in tissues from patients with active TB. We constructed a comprehensive atlas that maps 19 cell subsets across 8 spatial microenvironments. This atlas shows an IFN-γ-depleted microenvironment enriched for TGF-ß, regulatory T cells and IDO1+ PD-L1+ myeloid cells. In a further transcriptomic meta-analysis of peripheral blood from patients with TB, immunoregulatory trends mirror those identified by granuloma imaging. Notably, PD-L1 expression is associated with progression to active TB and treatment response. These data indicate that in TB granulomas, there are local spatially coordinated immunoregulatory programs with systemic manifestations that define active TB.

Human papillomavirus genotype distribution patterns in Zimbabwe; is the bivalent vaccine sufficient?

BACKGROUND: Vaccination against Human papillomavirus (HPV) is the primary preventative strategy that has been shown to reduce the burden of HPV related diseases. Zimbabwe introduced the bivalent vaccine (HPV 16/18) in the vaccination program targeting prepubescent girls in 2018. This review is an analysis of the distribution of HPV genotypes from various studies conducted in Zimbabwe to ascertain the effectiveness of the bivalent vaccine and make recommendations for future HPV vaccine choices. SUMMARY: Zimbabwean studies have mostly reported on cervical HPV in the urban areas. The most frequent HPV genotypes from cervical sites were 16, 18, 33, 35, 45, 56 and 58. These were identified from samples with normal cytology, pre-cancer and invasive cervical cancer. The few studies that have been done in rural areas reported HPV 35 as the most frequent cervicovaginal genotype. From the anal region of individuals reporting for routine screening, HPV 16, 18, 35 52 and 58 were the most frequent. A study on genital warts identified HPV 6, 11, 16, 40, 51and 54. In a study on children with recurrent respiratory papillomatosis (RRP), HPV 6 and 11 were the most common and HPV 35 was also identified in these children. There is no available published data on HPV distribution in head and neck cancers in Zimbabwe. KEY MESSAGES: Given that 83% of cervical cancers in Zimbabwe are caused by HPV 16/18, the bivalent vaccine could cover a significant proportion of HPV related cervical cancer. The current limitation of the bivalent vaccine is its failure to prevent benign lesions such as genital warts and RRP or all cervical cancer cases in Zimbabwe. For the prevention of most HPV related conditions, the nonavalent vaccine would be the most appropriate option for the Zimbabwean population. Currently there is no vaccine that includes HPV 35, yet this genotype was frequently identified in HPV related diseases. Vaccine developers may need to consider HPV 35 when manufacturing the next generation HPV vaccines. Furthermore, boys should also be included in HPV vaccination programs to improve herd immunity, as well as prevent RRP and HPV-related head and neck cancers.

Uses and limitations of small-volume biopsies for the diagnosis of lymphoma.

Although surgical biopsy remains the gold standard for the diagnosis of lymphoma, small-volume biopsies including fine-needle aspiration and core needle biopsy are increasingly being used as a first line diagnostic tool. Small-volume biopsies are safe, rapid and cost effective; however, diagnostic utility varies by lymphoma subtype. It is important for pathologists and clinicians to recognize both the strengths and limitations of such biopsies.

Correlation of p53 immunohistochemistry with TP53 mutational status and overall survival in newly diagnosed acute myeloid leukaemia.

AIMS: TP53-mutated acute myeloid leukaemia (AML) is associated with an adverse prognosis and poor response to traditional chemotherapy regimens. Next-generation sequencing (NGS) is considered the gold standard method to determine TP53-mutational status; however, molecular assays are costly and time-consuming. In contrast, immunohistochemistry (IHC) can be performed within 1 day of biopsy. We sought to determine an optimal threshold of staining with p53 IHC to predict TP53-mutational status. METHODS AND RESULTS: We identified 142 consecutive patients with newly diagnosed AML with concurrent NGS analysis diagnosed between 2019 and 2020. All cases were stained for p53 IHC and images were scored for the percent of strongly stained p53+ cells by a combination of manual counting and image analysis. We then correlated percent positive staining with mutational status and clinical outcomes. We determined that a threshold of ≥7% strongly stained cells by p53 IHC correlated with the presence of a TP53 mutation with a sensitivity of 67%, specificity of 100%, positive predictive value of 100% and negative predictive value of 90%. TP53 mutation and the presence of ≥7% staining by IHC were associated with shorter overall survival by univariate analysis (P < 0.01). CONCLUSION: If the limitations of this study are carefully considered, our findings suggest that p53 protein expression as evaluated by IHC could be used to rapidly predict TP53-mutational status with high specificity and assist in risk stratification in newly diagnosed AML.

Plasma cell myeloma: role of histopathology, immunophenotyping, and genetic testing.

Myeloma is a malignant neoplasm of plasma cells with complex pathogenesis. Diagnosis and risk stratification require the integration of histology, radiology, serology, and genetic data. Bone marrow biopsies are essential for myeloma diagnosis by providing material for histologic and cytologic assessment as well as immunophenotypic and genetic studies. Flow cytometry and genetic studies are, in particular, becoming increasingly important for diagnosis, risk stratification, and assessment of treatment response. Myeloma has traditionally been characterized by recurrent cytogenetic abnormalities that can be divided into two subtypes: hyperdiploid, characterized by trisomies, and non-hyperdiploid, characterized by translocations involving chromosome 14. These abnormalities are thought to be primary events, initiating a premalignant state, which progresses to myeloma through the acquisition of secondary mutations. The emergence of next-generation sequencing has led to the discovery of numerous mutations and gene fusions that comprise the heterogenous genomic landscape of myeloma. As the underlying pathogenesis of myeloma continues to be delineated, possible therapeutic targets have also emerged. Herein, we describe the importance of histology, immunophenotype, and mutational analysis in the assessment of myeloma.

Clinical sensitivity and interpretation of PCR and serological COVID-19 diagnostics for patients presenting to the hospital.

The diagnosis of COVID-19 requires integration of clinical and laboratory data. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic assays play a central role in diagnosis and have fixed technical performance metrics. Interpretation becomes challenging because the clinical sensitivity changes as the virus clears and the immune response emerges. Our goal was to examine the clinical sensitivity of two most common SARS-CoV-2 diagnostic test modalities, polymerase chain reaction (PCR) and serology, over the disease course to provide insight into their clinical interpretation in patients presenting to the hospital. We conducted a single-center, retrospective study. To derive clinical sensitivity of PCR, we identified 209 PCR-positive SARS-CoV-2 patients with multiple PCR test results (624 total PCR tests) and calculated daily sensitivity from date of symptom onset or first positive test. Clinical sensitivity of PCR decreased with days post symptom onset with >90% clinical sensitivity during the first 5 days after symptom onset, 70%-71% from Days 9 to 11, and 30% at Day 21. To calculate daily clinical sensitivity by serology, we utilized 157 PCR-positive patients with a total of 197 specimens tested by enzyme-linked immunosorbent assay for IgM, IgG, and IgA anti-SARS-CoV-2 antibodies. In contrast to PCR, serological sensitivity increased with days post symptom onset with >50% of patients seropositive by at least one antibody isotype after Day 7, >80% after Day 12, and 100% by Day 21. Taken together, PCR and serology are complimentary modalities that require time-dependent interpretation. Superimposition of sensitivities over time indicate that serology can function as a reliable diagnostic aid indicating recent or prior infection.

Future winters present a complex energetic landscape of decreased costs and reduced risk for a freeze-tolerant amphibian, the Wood Frog (Lithobates sylvaticus).

Winter climate warming is rapidly leading to changes in snow depth and soil temperatures across mid- and high-latitude ecosystems, with important implications for survival and distribution of species that overwinter beneath the snow. Amphibians are a particularly vulnerable group to winter climate change because of the tight coupling between their body temperature and metabolic rate. Here, we used a mechanistic microclimate model coupled to an animal biophysics model to predict the spatially explicit effects of future climate change on the wintering energetics of a freeze-tolerant amphibian, the Wood Frog (Lithobates sylvaticus), across its distributional range in the eastern United States. Our below-the-snow microclimate simulations were driven by dynamically downscaled climate projections from a regional climate model coupled to a one-dimensional model of the Laurentian Great Lakes. We found that warming soil temperatures and decreasing winter length have opposing effects on Wood Frog winter energy requirements, leading to geographically heterogeneous implications for Wood Frogs. While energy expenditures and peak body ice content were predicted to decline in Wood Frogs across most of our study region, we identified an area of heightened energetic risk in the northwestern part of the Great Lakes region where energy requirements were predicted to increase. Because Wood Frogs rely on body stores acquired in fall to fuel winter survival and spring breeding, increased winter energy requirements have the potential to impact local survival and reproduction. Given the geographically variable and intertwined drivers of future under-snow conditions (e.g., declining snow depths, rising air temperatures, shortening winters), spatially explicit assessments of species energetics and risk will be important to understanding the vulnerability of subnivium-adapted species.

Knowledge, attitudes, and practices of cervical Cancer screening among HIV-positive and HIV-negative women participating in human papillomavirus screening in rural Zimbabwe.

BACKGROUND: Women in low- and middle-income countries are at the highest risk of cervical cancer yet have limited access to and participation in cervical cancer screening programs. Integrating self-collected, community-based screening offers a potential primary screening method in areas of limited resources. In this paper, we present a study evaluating knowledge, attitudes, and practices of cervical cancer and Human Papilloma Virus (HPV) in rural Zimbabwe. METHODS: We performed a community-based cross-sectional knowledge, attitudes and practices of HPV and cervical cancer study in rural Zimbabwe from January 2017-May 2017. Women were selected for the study via random number generation from complete lists of inhabitants in the study area if they satisfied the inclusion criteria (≥30-years-old, ≤65-years-old, not pregnant, intact uterus). If selected, they participated in a 19-question structured knowledge, attitudes and practices survey. The questionnaire included questions on demographics, education, knowledge of HPV, cervical cancer, and risk factors. Chi-squared tests were evaluated comparing knowledge, attitudes and practices relating to HPV and cervical cancer screening with actual infection with HPV. Women were also offered a voluntary HIV and self-collected HPV screening. RESULTS: Six hundred seventy-nine women were included in the knowledge, attitudes and practices survey. Most women (81%) had heard of cervical cancer while the majority had not heard of HPV (12%). The number of women that had been screened previously for cervical cancer was low (5%). There were no significant differences between and within groups regarding knowledge of cervical cancer and actual overall infection with HR-HPV, HPV 16, and HPV 18/45 test results. CONCLUSIONS: Most women in rural Zimbabwe have heard of cervical cancer, but the number that had been screened was low. Extending existing outreach services to include cervical cancer screening, potentially including HPV screening, should include cervical cancer/HPV education and screening triage. This approach would serve to bridge the gap between knowledge and screening availability to address some of the barriers to cervical cancer care still affecting women in many regions of the world.

Evaluating for Human Herpesvirus 6 in the Liver Explants of Children With Liver Failure of Unknown Etiology.

BACKGROUND: Liver failure of unknown etiology (LFUE) has a transplant-free survival rate <25%. Human herpesvirus 6 (HHV-6) may be associated with LFUE, but studies are limited by small sample size. METHODS: We identified all children who underwent liver transplant for LFUE at a single quaternary children's hospital; 51/65 cases could be age matched with controls (children who underwent liver transplant for metabolic liver disease). Quantitative polymerase chain reaction for HHV-6 was performed on DNA from formalin-fixed paraffin-embedded liver explant tissue. RESULTS: HHV-6 was detected in 34/51 cases (66.7%) and 19/51 controls (37.3%) (P = .005). Average HHV-6 viral load was 213207 copies/106 cells in positive cases (range: 7293-1102030) and 38115 copies/106 cells in positive controls (range: 1382-122375) (P = .0008). HHV-6 was present significantly more often in cases compared to controls in patients younger than 6 years. In particular, in patients younger than 3 years, HHV-6 was present in 13/27 cases (48.1%) and 2/27 controls (7.4%) (P = .0009). CONCLUSIONS: HHV-6 was detected in liver explants significantly more often and in higher quantities in children transplanted for LFUE compared to controls, suggesting HHV-6 should be evaluated in young children who present with LFUE.

Community-based self-collected human papillomavirus screening in rural Zimbabwe.

BACKGROUND: In low- and middle-income countries (LMIC), women have limited access to and uptake of cervical cancer screening. Delayed diagnosis leads to poorer outcomes and early mortality, and continues to impede cancer control disproportionately in LMIC. Integrating self-collected, community-based screening for High Risk-Human Papilloma Virus (HR-HPV) into existent HIV programs is a potential screening method to identify women at high risk for developing high-risk cervical lesions. METHODS: We implemented community-based cross-sectional study on self-collection HR-HPV screening in conjunction with existing community outreach models for the distribution of antiretroviral therapy (ART) and the World Health Organization Expanded Program on Immunization (EPI) outreach in villages in rural Zimbabwe from January 2017 through May 2017. RESULTS: Overall, there was an 82% response rate: 70% of respondents participated in self-collection and 12% were ineligible for the study (inclusion criteria: age 30-65, not pregnant, with an intact uterus). Women recruited in the first 2-3 months of the study had more opportunities to participate and therefore significantly higher participation: 81% participation (additional 11% ineligible), while those with fewer opportunities also had lower participation: 63% (additional 13% ineligible) (p < 0.001). Some village outreach centers (N = 5/12) had greater than 89% participation. CONCLUSIONS: Integration of HR-HPV screening into existing community outreach models for HIV and immunizations could facilitate population-based screening to scale cancer control and prevention programs in sub-Saharan Africa. Community/village health workers (CHW/VHW) and village outreach programs offer a potential option for cervical cancer screening programs to move towards improving access of sexual and reproductive health resources for women at highest risk.

Impacts of extreme climatic events on the energetics of long-lived vertebrates: the case of the greater flamingo facing cold spells in the Camargue.

Most studies analyzing the effects of global warming on wild populations focus on gradual temperature changes, yet it is also important to understand the impact of extreme climatic events. Here we studied the effect of two cold spells (January 1985 and February 2012) on the energetics of greater flamingos (Phoenicopterus roseus) in the Camargue (southern France). To understand the cause of observed flamingo mass mortalities, we first assessed the energy stores of flamingos found dead in February 2012, and compared them with those found in other bird species exposed to cold spells and/or fasting. Second, we evaluated the monthly energy requirements of flamingos across 1980-2012 using the mechanistic model Niche Mapper. Our results show that the body lipids of flamingos found dead in 2012 corresponded to 2.6±0.3% of total body mass, which is close to results found in woodcocks (Scolopax rusticola) that died from starvation during a cold spell (1.7±0.1%), and much lower than in woodcocks which were fed throughout this same cold spell (13.0±2%). Further, Niche Mapper predicted that flamingo energy requirements were highest (+6-7%) during the 1985 and 2012 cold spells compared with 'normal' winters. This increase was primarily driven by cold air temperatures. Overall, our findings strongly suggest that flamingos starved to death during both cold spells. This study demonstrates the relevance of using mechanistic energetics modelling and body condition analyses to understand and predict the impact of extreme climatic events on animal energy balance and winter survival probabilities.

Glass reinforced plastic (GRP) boats and the impact on coastal environment - Evidence of fibreglass ingestion by marine bivalves from natural populations.

Classified as marine debris, man made materials are polluting the world's oceans. Recently, glass reinforced plastic (GRP) has been shown to degrade and contaminate the coasts. In this pioneering study, fibreglass particles have been detected in the soft parts of oysters and mussels collected from natural populations, in front of an active boatyard. The presence of particulate glass, with concentrations up to 11,220 particles/kg ww in Ostrea edulis and 2740 particles/kg ww in Mytilus edulis, was confirmed by micro Raman spectroscopy. The results showed higher accumulation during the winter months, when boat maintenance activities are peaking and, through repair work, the release of glass fibres in the environment is more likely. Bivalves are considered high risk species due to their sessile nature and extensive filter feeding behaviour. The microparticle inclusion may contribute to adverse impacts on physiological processes and eventually to a decline in the overall health and subsequent death of the animal. The high costs involved in the proper GRP disposal and the lack of recycling facilities worldwide lead to boat abandonement and further contamination of the coasts. For the first time this study presents the extensive fibreglass contamination of natural bivalve populations, in a popular South England sailing harbour, designated a biological and geological site of specific scientific interest (SSRI).

Molecular Diagnostics of Plasma Cell Neoplasms.

Genetic characterization of myeloma at diagnosis by interphase fluorescence in situ hybridization and next-generation sequencing (NGS) can assist with risk stratification and treatment planning. Measurable residual disease (MRD) status after treatment, as evaluated by next-generation flow cytometry or NGS on bone marrow aspirate material, is one of the most important predictors of prognosis. Less-invasive tools for MRD assessment such as liquid biopsy approaches have also recently emerged as potential alternatives.

Co-creation of human papillomavirus self-sampling delivery strategies for cervical cancer screening in rural Zimbabwe: nominal group technique.

Background: Human papillomavirus (HPV) self-sampling is recommended for cervical cancer screening, particularly among women who do not participate in or have access to current screening methods offered in Zimbabwe. Key stakeholder involvement is critical in co-creating acceptable delivery strategies for implementing HPV self-sampling to ensure demand and facilitate uptake by the target population. The main objective of this study was to engage key stakeholders in co-creating acceptable HPV self-sampling delivery strategies for cervical cancer screening in rural Zimbabwe. Methods: We invited key stakeholders and employed a nominal group technique (NGT) for data collection. We employed the NGT to (1) identify barriers to access and utilisation of available cervical cancer screening services and (2) co-create delivery strategies for HPV self-sampling. The workshop included 8 participants (women n = 4, health workers n = 2 and policymakers n = 2). Quantitative data was gathered by ranking ideas and qualitative data were collected from participant group discussions and analysed thematically. The results of the ranking exercise were fed back to the participants for comments. Results: The most significant barriers to accessing and utilising current cervical cancer screening services by women were: Inadequate information and education on cervical cancer, lack of resources and funding for cervical cancer programmes, long distances to nearest health facilities, and low perceived personal risk of cervical cancer. Key stakeholders recommended enhanced education and awareness, results notification, linkage to care, community-based self-sampling, and the choice of sampling devices as potential HPV self-sampling delivery strategies. Conclusion: Our study demonstrated the utility of the NGT for reaching a consensus. Using the NGT, we established priority delivery strategies for HPV self-sampling cervical cancer screening. Adequate education and awareness, early results notification, choice of sampling device and community-based self-sampling were crucial to HPV self-sampling screening in rural Zimbabwe. The proposed delivery strategies can guide the development of guidelines for designing and implementing an HPV self-sampling intervention. We recommend a study to determine women's most preferred HPV self-sampling delivery strategies before implementing the intervention.

Correction: Self-collected and clinician-collected anal swabs show modest agreement for HPV genotyping.

[This corrects the article DOI: 10.1371/journal.pone.0250426.].

Betapapillomaviruses in p16-Negative Vulvar Intraepithelial Lesions Associated with Squamous Cell Carcinoma.

Approximately 40% of vulvar squamous cell carcinoma (vSCC) cases are etiologically associated with high-risk human papillomaviruses (HPVs) of the alpha genera (α-HPV) that cause other anogenital cancers; however, the etiology of α-HPV-negative vSCC is poorly understood. HPVs of the beta genera (ß-HPV) are risk factors for cutaneous squamous cell carcinoma (cSCC) and may be related to carcinomas originating in other cutaneous sites such as the vulva. In this study, we investigate the presence of ß-HPVs, with an emphasis on p16-negative squamous lesions adjacent to vSCC. We subjected 28 vulvar squamous intraepithelial lesions adjacent to vSCC for comprehensive HPV genotyping, p16 and p53 immunohistochemistry, and consensus morphology review. Selected cases were subjected to qPCR and RNA in situ hybridization. Clinical data were obtained from medical records. ß-HPV DNA was detected in eight of ten p16-negative lesions and three of fourteen p16-positive high-grade squamous intraepithelial lesions. The HPV DNA loads in vulvar squamous intraepithelial lesions ranged between less than 1 HPV DNA copy per cell to more than 100 HPV DNA copies per cell. This is, to the best of our knowledge, the first report of the association of p16-negative vulvar intraepithelial squamous lesions with detection of ß-HPVs. These findings expand possible etiologic mechanisms that may contribute to p16-negative lesions of the vulva.

Computer vision quantitation of erythrocyte shape abnormalities provides diagnostic, prognostic, and mechanistic insight.

Examination of red blood cell (RBC) morphology in peripheral blood smears can help diagnose hematologic diseases, even in resource-limited settings, but this analysis remains subjective and semiquantitative with low throughput. Prior attempts to develop automated tools have been hampered by their poor reproducibility and limited clinical validation. Here, we present a novel, open-source machine-learning approach (denoted as RBC-diff) to quantify abnormal RBCs in peripheral smear images and generate an RBC morphology differential. RBC-diff cell counts showed high accuracy for single-cell classification (mean AUC, 0.93) and quantitation across smears (mean R2, 0.76 compared with experts, interexperts R2, 0.75). RBC-diff counts were concordant with the clinical morphology grading for 300 000+ images and recovered the expected pathophysiologic signals in diverse clinical cohorts. Criteria using RBC-diff counts distinguished thrombotic thrombocytopenic purpura and hemolytic uremic syndrome from other thrombotic microangiopathies, providing greater specificity than clinical morphology grading (72% vs 41%; P < .001) while maintaining high sensitivity (94% to 100%). Elevated RBC-diff schistocyte counts were associated with increased 6-month all-cause mortality in a cohort of 58 950 inpatients (9.5% mortality for schist. >1%, vs 4.7% for schist; <0.5%; P < .001) after controlling for comorbidities, demographics, clinical morphology grading, and blood count indices. RBC-diff also enabled the estimation of single-cell volume-morphology distributions, providing insight into the influence of morphology on routine blood count measures. Our codebase and expert-annotated images are included here to spur further advancement. These results illustrate that computer vision can enable rapid and accurate quantitation of RBC morphology, which may provide value in both clinical and research contexts.