South Africa has the largest HIV epidemic globally, with ~7.5 million people living with HIV in 2021. Adolescent girls (AG) and young women (YW), aged 15–19 years and 20–24 years, are twice as likely to be living with HIV as their male counterparts. The national HIV prevalence for young women was 9.1% (2021), with limited data on disease severity.This study assessed very advanced HIV disease (CD4 < 100 cells/μL) in adolescent girls and young women (AGYW) in South Africa.A retrospective descriptive study analysed data collated from the National Health Laboratory Service database for 2017 to 2021 calendar years for AGYW. National and provincial specimen volumes, the percentage of tests with a CD4 < 100 cells/μL and ≥ 100 cells/μL, and the median and interquartile ranges, were calculated. Logistic regression determined the odds ratio for a CD4 < 100 cells/μL, controlling for age category.Data for 1 199 010 CD4 specimens indicated a significant decrease in volumes of 34% from 287 410 (2017) to 189 533 (2021). The percentage of samples with a count < 100 cells/μL ranged from 4.9% to 5.2% for YW versus 5.6% to 6.1% for AG. Provincial data for a CD4 count < 100 cells/μL ranged between 4.5% and 8.3% in AG and 3.6% to 6.3% for YW. Logistic regression indicated a 24% higher likelihood for AG having a CD4 count < 100 cells/μL.The study reported a higher proportion of very advanced HIV disease for AG versus YW nationally, with provincial disparity needing further analysis. What this study adds: This study provides important insights into very advanced HIV disease for adolescent girls (AG) and young women (YW). This is of extreme concern, given the reported vertical transmission rates of 3.51% in 2021. There is, however, a need to assess what other factors could result in a low CD4 count, that is, failing treatment or disengaging care.1 In addition, 650 000 deaths from AIDS-related illnesses were reported.2 Of PLWH, only 28.7 million (74.7%) accessed antiretroviral therapy (ART) in the same year, despite effective HIV treatment and tools to prevent, detect and treat opportunistic infections.3 In 2021, women and girls accounted for 63% of all new HIV infections in sub-Saharan Africa,1 with adolescent girls  and young women  accounting for 25% of new HIV infections, while only representing 10% of the population.4 Six out of seven new HIV infections among adolescents are among girls.2 Girls and young women aged 15 to 24 years are twice as likely to be living with HIV than young men of the same age.2Globally, an estimated 38.4 million people were living with HIV (PLWH) in 2021, of which 1.5 million people became newly infected.6 The ART clinical guidelines for the management of HIV recommend that all PLWH are eligible to start ART, irrespective of CD4 count.7 As a baseline investigation, CD4 testing is required to identify eligibility for cotrimoxazole prophylaxis as well as screening for cryptococcal antigenaemia.7 CD4 count should be monitored for patients with virological failure at six-monthly intervals .7 For women aged 15 to 49 years, a national prevalence of 24.5% is reported compared to 12.1% for men.8 Of the 210 000 new infections in 2021, the majority were in women aged 15–49 years .8 A national prevalence of 9.1% was reported for young women (YW) and 3.0% for young men (defined as ages 15 to 24 years).8 Furthermore, knowledge about HIV prevention among YW in 2021 was only 46.1%.8 Ninety-five percent of people at risk of HIV infection should have access to an appropriate, prioritised person-centred and effective combination of prevention strategies. In addition, 90% of adolescents and young people should receive comprehensive sexuality education. HIV incidence has remained largely unchanged in the highest-burdened communities in South Africa.10 Given that adolescent girls and young women (AGYW) primarily acquire infections from older men who are often unaware of their HIV status and less likely to be on ART, the minimal decrease in HIV incidence in this sub-population in South Africa is not surprising.13 It has been reported that sexual partnering between young women and older men is a key feature of the sexual networks driving HIV transmission.11South Africa has the largest HIV epidemic globally with an estimated 7.5 million PLWH in 2021 and a national prevalence of 18.3%.14 Advanced HIV disease is defined as a CD4 cell count < 200 cells/μL and is associated with higher mortality rates due to opportunistic infections such as tuberculosis and cryptococcal meningitis.15 A local study in three high-burden districts in the Mpumalanga, KwaZulu-Natal and Gauteng provinces, which assessed late presentation for newly diagnosed HIV-positive individuals, reported that men, non-pregnant women and those accessing care in facilities located in townships and inner cities were more likely to present late for HIV care.14 HIV-positive individuals presenting late for care have an increased risk of mortality, as high as 40% of all AIDS-related deaths.14 Using national laboratory data, Carmona et al. reported that between 2005 and 2011, the proportion of patients entering into care with a CD4 count < 200 cells/μL declined from 46.8% to 35.6%.13 In contrast, from 2011 to 2016, the proportion of patients entering ART with advanced HIV disease remained relatively unchanged, ranging from 32.9% to 34.8%.13 The same study reported that for women seeking care, the proportion with CD4 < 200 cells/μL varied from 42.8% in 2005 to 26.5% by 2016.13 The study findings indicated gender disparity in late presentation between the years reported.13The majority of PLWH in South Africa are aware of their status (94% based on 2021 estimates), but are diagnosed in later stages of HIV infection.16 There is thus a real need for better monitoring of very advanced HIV disease, especially in a sub-population such as AGYW, already with a higher risk of infection due, in part, to risky behaviour resulting in high rates of teenage pregnancies.17Local studies reported that 5.4% of specimens from AGYW reported a CD4 count < 100 cells/μL for the 2019 calendar year in South Africa, defined as very advanced HIV disease.There is a lack of data and awareness about AGYW living with HIV. Furthermore, there is limited data for advanced HIV disease for AGYW. This study, using CD4 laboratory data for the period 2017 to 2021, aimed to describe very advanced HIV disease in AGYW.18 It has 268 laboratories across the nine provinces and serves approximately 80% of the South African population.18 CD4 testing is decentralised to 49 out of 268 laboratories within the NHLS and is based on an integrated tiered service delivery model.20Data are reported for testing performed by the National Health Laboratory Service (NHLS), with a mandate to provide laboratory services for public sector health facilities across South Africa.The retrospective descriptive study design was used to analyse secondary laboratory CD4 data for the 2017 to 2021 calendar years. The sample population studied included AG (15–19 years) and YW (20–24 years) presenting for CD4 testing. Data were extracted from the Corporate Data Warehouse (CDW) of the NHLS.23 This algorithm uses the patient’s first name, last name, date of birth, gender, and hospital folder number for matching.24 In the data set, a patient might have had more than one test performed during the study period. The aim was to indirectly determine the extent and timeframe of, specifically, very advanced HIV disease. Therefore, data were categorised as < 100 cells/μL and ≥ 100 cells/μL. Data were prepared and analysed using SAS statistical software . The logistic regression was conducted using STATA SE .The following variables were provided in the extract: (1) unique patient identifier, (2) episode number, (3) age (in years), (4) ethnicity, (5) health facility, (6) province, (7) result reviewed date and (8) CD4 count value. An episode number is a unique identifier used within the laboratory information system to link specimens to a patient visit. For this work, the extract was restricted to women aged 15 to 24 years. The result reviewed date was used to extract the year and month of testing. Age in years was captured on the laboratory request form by the healthcare worker and recorded in the laboratory information system. The following age categories were assigned: (1) 15–17 years, (2) 18–19 years, (3) 20–22 years and (4) 23–24 years. Data were categorised as either AG or YW using the captured age (in years). The CD4 count was categorised as follows: (1) ≤ 10 cells/μL, (2) 11 cells/μL – 29 cells/μL, (3) 30 cells/μL – 49 cells/μL, (4) 50 cells/μL – 99 cells/μL and (5) ≥ 100 cells/μL. The CDW developed a unique patient identifier generated by a probabilistic matching algorithm that includes fuzzy logic matching.P-value are reported. A P-value of < 0.05 indicated significant associations between the categorical variable of interest with CD4 < 100 cells/μL.A decision tree was used to depict the number of specimens with a CD4 count < 100 cells/μL, with the median indicated for AG and YW. Annual test volumes for AG and YW were assessed, with the percentage change year-on-year indicated. CD4 descriptive statistics included the median and interquartile range (IQR). The data were analysed by age category, with the chi-squared test used to assess any significant associations for the CD4 category, assuming an alpha of 0.05. For AG and YW, the percentage of specimens with a CD4 count ≤ 10 cells/μL, 11 cells/μL – 29 cells/μL, 30 cells/μL – 49 cells/μL, 50 cells/μL – 99 cells/μL and ≥ 100 cells/μL for each year was calculated nationally and at the provincial level. Logistic regression was applied to determine the risk factors associated with a CD4 < 100 cells/μL (binary dependent), controlling for age category as an independent variable using STATA. The baseline comparative group was the age category 23–24 years. The odds ratio (OR), 95% confidence interval (CI) and Ethical clearance was obtained from the Human Research Ethics Committee  at the Faculty of Health Sciences, University of the Witwatersrand. The study approval number is M220163. As analyses only include secondary laboratory data, patient consent was not required. No patient identifiers were used for the study.Of all the CD4 testing done between 2017 and 2021, 1 199 010 tests met the inclusion criteria for women aged 15 to 24 years. Following the de-duplication of data, this equates to 778 712 patients tested. Of all patients tested, 64.9% had one CD4 test during the test period, 21.5% had two tests, and 8.4% had three . CD4 and patient age reported a skewness of 0.78 and –0.70. Ethnicity was provided for only 101 680 (8.5%) of the specimens.n = 301 260) for CD4 had a count < 100 cells/μL (median: 37 cells/μL) compared to 5.1% for YW (n = 897 742) (median: 45 cells/μL). For AGYW with a count < 100 cells/μL, a median CD4 of 34 cells/μL was reported for the 15–17 age group, 40 cells/μL for the 18–19-year-olds, 45 cells/μL for the 20–22-year-olds and 46 cells/μL for the 23–24-year-olds.Overall, AGYW had a median CD4 count of 485 cells/μL . Most ofTest volumes for AGYW decreased from 287 410 to 189 533 between 2017 and 2021 . A year-For AG, testing decreased from 69 582 in 2017 to 50 330 by 2021. A year-on-year annual percentage change of between 2.4% (2019) and –14.8% (2020) was reported. Overall, AG reported a decrease of –27.7% between 2017 and 2021. The percentage of specimens with a CD4 count < 100 cells/μL ranged from 5.6% (2020) to 6.1% (2017). The median CD4 ranged between 462 cells/μL in 2017 and 480 cells/μL by 2021.For a CD4 < 100 cells/μL, test volumes for YW decreased from 11 137 in 2017 to 7225 by 2021 . Between 4.9% (2020) and 5.2% (2018) reported a count of < 100 cells/μL. The median CD4 increased from 479 cells/μL in 2017 to 503 cells/μL by 2021.P-value of < 0.05 (chi-squared test) was reported for the association between the CD4 and age categories.For AGYW, there were 10.7% tests performed for the 15–17 years age category, 14.4% for the 18–19-year-olds, 20–22 and 23–24 years age categories, 38.2% for the 20–22-year-olds and 36.7% for the 23–24-year-olds during the 2017 to 2021 period . For theFor the period 2017 to 2021, the percentage of AG specimens with a CD4 category of < 10 cells/μL was 1.2%, 1.3% for 11 cells/μL – 29 cells/μL, 1.0% for 30 cells/μL – 49 cells/μL and 2.3% for 50 cells/μL – 99 cells/μL (data not shown). In comparison, for YW, 0.8%, 1.0%, 0.9%, and 2.4% were reported for the CD4 categories as indicated.For AG, a range of 93.9% (2017) to 94.4% (2020) had a CD4 count ≥ 100 cells/μL . In compFor the ≤ 10 cells/μL category, a range of 1.1% – 1.3% was reported for AG compared to 0.8% – 0.9% for YW. For AG, a range of 1.3% – 1.4% was reported for ranges of 11 cells/μL – 29 cells/μL, 0.9% – 1.1% for 30 cells/μL – 49 cells/μL, and 2.2% – 2.4% for 50 cells/μL – 99 cells/μL. Ranges of 1.0% – 1.1%, 0.9% – 0.9% and 2.2% – 2.4% were reported for YW in the same CD4-range categories .The provincial analysis revealed that for AG, between 4.5%  and 8.3% (Limpopo) reported a count < 100 cells/μL. Between 3.6%  and 6.3% (Northern Cape) of YW had a count < 100 cells/μL . NationaP ≤ 0.0001). In comparison, the 20–22 years age group is 10% less likely to have a CD4 count < 100 cells/μL . No significant difference was noted for the 18–19 years age group when compared to the 23–24 years age group (P = 0.693).For the 15–17 years age category, an odds ratio of 1.24 (CI: 1.21–1.27) was reported, 1.00 (CI: 0.98–1.03) for 18–19-year-olds and 1.90 (CI: 0.89–0.92) for 20–22-year-olds . These r7 and the impact of coronavirus disease 2019 (COVID-19).25 Any proportion of AGYW presenting late for care is of extreme concern where vertical transmission rates are as low as 3.51%.8 At the national level, the proportion of specimens (the vast majority adults of 18 years and older) representing very advanced HIV disease was 11.5% (2018), 11.4% (2019), 10.8% (2020) and 10.8% (2021) (data not shown).27 However, lower levels of very advanced HIV in AGYW compared to national data suggest that enhanced focus on this group may lead to early diagnosis and treatment.28The proportion of specimens with very advanced HIV disease was highest for those aged 15 to 17 years, despite reporting a higher median CD4. Overall, for AGYW, levels of very advanced HIV disease remained above 5% between 2017 and 2021, despite significant changes in test volumes. The decrease in test volumes may be due in part to guideline changes,30 It has been reported that AGYW are at the epicentre of the HIV epidemic in southern Africa, contributing a disproportionate ~30% of new infections and seroconverting between 5 and 7 years earlier than their male peers.31 Evidence-based specific healthcare interventions for AGYW, such as the determined, resilient, empowered, AIDS-free, mentored and safe  initiative, aim to reduce new HIV infections by empowering AGYW to reduce their risk, strengthen families, mobilise communities for change and reduce the risk in men who are likely to be their male sex partners.33 The goals of the DREAMS initiative are to ensure that AGYW have access to prevention technologies and strategies, and the opportunity to complete high school and graduate HIV-negative and without pregnancies.34 This approach should hopefully address AGYW at the highest risk of HIV acquisition through political commitment, leadership, financial and human resource investments, advocacy efforts, and a focus on the highest priority settings.10 Based on our findings, these interventions should be focussed on those aged 15–17 years who were more likely to have very advanced HIV disease. However, prevention intervention should be targeted at earlier age groups with very advanced HIV disease.This study reveals high rates of very advanced HIV disease, suggesting HIV seroconversion in much younger girls, especially worrying bearing in mind the trajectory of HIV disease progression.32 Recent systematic reviews cite a history of sexually transmitted infections, alcohol use, multiple partners, early marriage, being out of school, inconsistent condom use and engaging in transactional sex32 where AGYW primarily acquire infections from older HIV-positive men (20s and early 30s).10 It has been reported by Karim et al. that AGYW have the least power in society, and bear an enormous burden of both intimate partner violence (IPV) and HIV.35 A key intervention to reduce gender-based violence (GBV) is primary and secondary education of AGYW.36 This may reduce rates of HIV infection, delay childbearing, lower infant and maternal mortality rates, and improve other development outcomes.37 Bearing the disparity in HIV prevalence between AGYW (9.1%) and young men (3.0%) in mind, it appears that GBV and IPV are important drivers of HIV transmission for AGYW and unintended pregnancies.38 However, there is an interplay of psychosocial gender-power disparities and socioeconomic and other social anomalies which lead to vulnerability in women in Africa.10A myriad of factors are associated with HIV infection vulnerability among AGYW.28 A local study reported that the coverage of prevention of mother-to-child transmission ART  ranged from 63.9 (Eastern Cape) to 87.4% in KwaZulu-Natal.39 Our data showed a correlation between very advanced disease in AG in Limpopo and Mpumalanga provinces with mother-to-child-transmission data.39 For YW, a correlation was found for Eastern Cape, Limpopo and Mpumalanga provinces.39The provincial data revealed similar outcomes to national data. For AG, higher levels of very advanced HIV disease in the Free State, Gauteng and Limpopo provinces (above 7%) is a concern. Similarly, over 6% of testing for YW reported CD4 counts < 100 cells/μL in the Free State, Gauteng, Northern Cape and Western Cape provinces. These findings indicate provincial disparities in levels of very advanced HIV disease in YW. This is similar to findings reported by two separate local studies assessing late presentation.40 This would include integrating clinical and laboratory aggregate findings to clearly understand why and where late presentation for AGYW persists. Unfortunately, without matching clinical data it is not possible to assess access to care and longitudinal follow-up. The integration of clinical and laboratory data would be critical to developing longitudinal cohorts and providing outcomes to laboratory data. Once a clearer understanding is obtained from the data analysis, policymakers can develop and implement focussed programmatic interventions.To understand why AGYW are presenting late for HIV care, further analysis is required at the health district and sub-district levels.7 It is not possible to determine whether CD4 testing was performed for AGYW on ART.The study used laboratory data to assess very late presentation in AGYW. Due to the absence of matching clinical data, it is not possible to determine whether CD4 testing was performed for baseline clinical/laboratory evaluation before ART.41 However, study findings of year-on-year decreases in testing for AGYW do not match the reported decrease in population estimates,41 that is, between 2019 and 2020, a 15.7% testing reduction was reported for AGYW compared to a 0.2% reduction for mid-year population estimates.41 Possible contributing factors to the steady yearly decline could relate to the non-youth-friendly perception of healthcare facilities and healthcare worker biases regarding the provision of sexual health services to AGYW.42 Data are also not available to assess whether AGYW were on treatment and whether any programmatic interventions had taken place. This would require the integration of clinical and laboratory data systems not currently in place in South Africa.45The study findings reveal that testing specifically for AGYW decreased year-on-year since 2017, with reductions of up to 15.7% reported between years. In comparison, for national CD4 data , a percentage reduction of between 5% and 8% annually is noted (data not shown). The change in HIV guidelines, that is, universal test and treat, is one of the contributing factors to the annual decrease in CD4 test volumes. Comparative results spanning across 10 years of CD4 testing (2012 to 2022) indicate that the percentage of 15–24-year-old women’s contribution to total CD4 tests has not changed significantly (< 2%) over time (data in preparation for publication). As a response to limit the spread of severe acute respiratory syndrome coronavirus 2 infections, the implementation of social distancing and lockdown measures in 2020 led to restrictions in healthcare accessibility and may have contributed to the declines seen from 2020 but was not a contributor to prior yearly declines. Mid-year population estimated between 2017 and 2021 indicated a decrease of 6.3% reported for AG compared to 6.7% for YW (data not shown).46 National and provincial representative surveys in South Africa have reported a reduction of the vertical transmission of HIV from > 25% in the absence of vertical transmission prevention interventions to 2.6% by 2012–2013.46 Unfortunately, data for mothers who present for delivery without having accessed antenatal/prenatal care are not available. Although the probabilistic matching algorithm was used, we would require earlier data to conduct a first-ever CD4 analysis, which is out of the scope of this study. Work is underway to develop an extended HIV cohort algorithm that would make it possible to analyse first-ever CD4 trends. This may help to identify vertical transmission (using HIV DNA polymerase chain reaction testing), with follow-up CD4 testing when the AGYW present for pre-ART HIV care.An important limitation is that some of the AGYW presenting with very advanced HIV disease were infected at birth .One of the limitations of this study is that it only reported the immunological status of AGYW presenting for CD4 testing. Correlating their immune status to their clinical staging was out of the scope of this study. Data analysis was further subject to the accuracy of patient age captured on the request form by the healthcare worker and laboratory clerk.32 The integration of clinical and laboratory data would be critical to develop longitudinal cohorts and provide outcomes to laboratory data.This study demonstrates the value of utilising secondary laboratory data (stratified by age and gender) to identify potential weaknesses in the healthcare of individuals with very advanced HIV disease. This data have made it possible to describe unacceptable levels of very advanced HIV disease in sub-populations such as AGYW. The provincial disparity of very advanced HIV disease for AGYW indicates that further analysis is required with clinical data to elucidate where interventions are required. It is important to focus on interventions that ensure that AGYW have access to appropriate healthcare services, prevention technologies and strategies, and complete high school and graduate HIV-negative.


The stigma of mental illness is a complex socialphenomenon that is widespread throughout the world, even amonghealth care professionals.Assessing attitudes towards mental illness among familymedicine residents in TunisiaThis is a descriptive cross-sectional study among of familymedicine residents enrolled at the Faculty of Medicine in Monastir(Tunisia), conducted over a period of 3 months (July 2022 to October2022). The CAMI scale was used to assess the attitude towards mental illness. Sociodemographic data were collected through a pre-established questionnaire. The data were analyzed using SPSS software 26 thversion.Our population was made up of 95 family medicineresidents, divided into 28 males and 67 females. Sex ratio was 2.39. The average of age was 28 years with extremes 25 and 35 years.46.3% (n=44) of the residents were enrolled in the first year, 22.1%(n=21) enrolled in the second year and 31.6% (n=30) enrolled in thethird year. 88.4% (n=84) of the residents did a psychiatric rotationduring their training.We found that 47.4% of residents (n=45) had a positive attitudetowards mental illness.Improving the attitudes of primary care physicianstowards people with mental illness is necessary to provide goodquality care to these patientsNone Declared


Advancements in single-cell-related technologies have opened new possibilities for analyzing rare cells, such as circulating tumor cells (CTCs) and rare immune cells. Among these techniques, single-cell proteomics, particularly single-cell mass spectrometric analysis (scMS), has gained significant attention due to its ability to directly measure transcripts without the need for specific reagents. However, the success of single-cell proteomics relies heavily on efficient sample preparation, as protein loss in low-concentration samples can profoundly impact the analysis. To address this challenge, an effective handling system for rare cells is essential for single-cell proteomic analysis. Herein, we propose a microfluidics-based method that offers highly efficient isolation, detection, and collection of rare cells . The detailed fabrication process of the micropillar array-based microfluidic device is presented, along with its application for CTC isolation, identification, and collection for subsequent proteomic analysis. Over the past decade, the detection and analysis of rare cells have emerged as a crucial and transformative field, holding significant implications for disease diagnosis, treatment, and our understanding of fundamental biological processes ,2,3,4. RMicrofluidics has emerged as a highly effective tool for detecting rare cells, demonstrating remarkable detection efficiency ,34,35,36The proposed micropillar based microfluidic device relies on the specific capture of rare cells such as circulating tumor cells (CTCs) based on their unique surface marker . As illuAZ 400k developer Chromium etchant 5-inch chromium coated glass plate covered with positive photoresist film 4-inch silicon wafers Tweezers for 4-inch silicon wafer Acetone Isopropanol (IPA) 6-inch wide-open round beakers N2 dryerHexamethyldisiloxane (HMDS) SU8 3035 photoresist SU8 developer Polydimethylsiloxane (PDMS) Sylgard 184 kit: silicone elastomer base and curing agent Trichlorosilane (TPOS) Weighing dish Petri dish (15 cm) Weighing scale Razor blade or scalpel PDMS punches Microscope slides 99% ethanol Deionized (DI) water1 mL pipet tips Stainless steel dispensing needle with luer lock connection Cleaned and bagged high-purity white silicone rubber tubing PTFE light wall tubing 3D printed supporting structureSterile Luer-Lock syringes (3 mL and 5 mL) 2+/Mg2+ ions Phosphate-buffered saline (PBS) without CaAvidin Bovine serum albumin (BSA) Biotinylated anti-EpCAM Cell line Cell culture medium Fetal bovine serum (FBS) Trypsin-EDTA (0.25% Trypsin with EDTA 4Na) Vybrant dye DiD Sterile 1.7-mL centrifuge tubes Maskless Aligner Spin coater Digital Programmable Stirring Hot Plates Contact Mask Aligner (Suss MA6 Mask Aligner)Desiccator with a vacuum connection Programmable Oven Plasma treater Programmable, dual-channel infusion and withdrawal modular syringe pump .Pipet  (Eppendorf) Serological pipet 2 incubator for cell culture COBiosafety hood Centrifuge (Eppendorf) Mini centrifuge This section includes three main parts: (1) the microfabrication of the micropillar based device; (2) the functionalization of the device using antibodies; (3) the process of samples using the microfluidic device and the release of captured CTCs.CRITICAL STEP The fabrication of the micropillar based device is critical as its performance largely depends on the fabrication outcome. As shown in Draw the layout of the micropillar-based microfluidic device using CAD software and convert the file into DXF form.Load the chromium and positive photoresist coated glass plate to the maskless aligner.Pattern writing on the photoresist layer of the glass plate using the maskless aligner.Dilute the AZ 400K developer 5 times .Develop the glass plate with the diluted AZ 400K developer for 20 s.Rinse the glass plate with running DI water for 10 s.Soak the glass plate in the chrome etchant for 1 min.Rinse the glass plate with running DI water for 10 s.Strip off the undeveloped photoresist in undiluted AZ 400K developer for 30 s.Rinse the chrome mask with running DI water for 10 s.2 gun.Dry the chrome mask with the N12.Bath a 4-inch silicon wafer in acetone for 10 min.13.Spray wash the silicon wafer with IPA to remove acetone residues.14.Bake the wafer at 120 °C for 10 min until fully dry and cool it down to room temperature.15.Vaporize HMDS onto the silicon wafer to create hydrophobic surfaces.16.Spin-coat a layer of 50 µm-thick SU-8 3035 photoresist (2000 rpm for 30 s).17.Soft bake the wafer on a hotplate at 60 °C for 1 min.18.Ramp up the hotplate temperature to 95° C at 450 °C/h.19.Incubate the wafer at 95 °C for 10 min.20.2).Expose the wafer using the contact mask aligner  in a weighing boat.30.Place the PDMS mixture in a vacuum for 15–30 min to remove bubbles.31.Dispense PDMS mixture on the silicon mold.32.Incubate PDMS in the oven at 75 °C for 2–4 h.33.Cut off the polymerized PDMS substrate with fluidic feature from the silicon mold using a scalpel or razor blade.34.Punch holes at the inlet and outlet of the PDMS substrate using a punch with inner diameter of 1.2 mm.35.Treat an unused silicon wafer with TPOS as described from Step 24–27.36.Apply 10 g of degassed liquid PDMS mixture on the silicon wafer.37.Spin the wafer at 1000 rpm for 1 min.38.Incubate the PDMS coated silicon wafer at 100 °C for 5 min.39.Treat the PDMS substrate with an oxygen plasma treater for 1 min.40.Treat the PDMS film on the silicon wafer with the oxygen plasma treater for 1 min.41.Bind the PDMS substrate with the PDMS film.42.Incubate the sandwiched layers at 100 °C for 1 min in the oven.43.Cool the bonded device down to room temperature.44.Gently cut off the excessive PDMS thin film using the scalpel.45.Peel the microfluidic device off the silicon wafer.46.Physically attach the microfluidic device on a microscope slide for sample processing.47.Prepare 2 sections of silicone rubber tubes using a scalpel or razor blade. One with a length of 5 cm. The other with a length of 10 cm.48.Prepare 2 sections of PTFE light wall tubes with a length of 8 mm using a scalpel or razor blade.49.Couple the silicone rubber tubes with the PTFE light wall tubes by inserting the PTFE light wall tube to one end of the silicone rubber with a depth of 4 mm.50.Connect the short-length silicone rubber tube to the inlet of the chip by inserting the coupled PTFE light wall tube.51.Connect the long-length silicone rubber to the outlet of the chip by inserting the coupled PTFE light wall tube.52.Connect the short-length silicone rubber tube to a 1 mL pipet tip used as a sample reservoir. Keep the pipet tube vertical to the chip using a 3D printed hanger or fixture.53.Fix a 5 mL Luer-locked syringe on the syringe pump.54.Cap the syringe with a blunt-tip mixer nozzle.55.Connect the long-length silicone rubber tube to the blunt-tip mixer nozzle.56.Introduce 300 µL of 99% ethanol to the sample reservoir.57.Withdraw 250 µL the ethanol at a flow rate of 2 µL/s using the syringe pump.58.Introduce 200 µL of DI water to the sample reservoir and withdraw 200 µL at a flow rate of 2 µL/s.59.Introduce 200 µL of PBS to the sample reservoir and withdraw 200 µL at a flow rate of 2 µL/s.60.Introduce 100 µL of avidin to the sample reservoir and withdraw 100 µL at a flow rate of 2 µL/s.61.Incubate for 15 min at room temperature.62.Introduce 300 µL of PBS to the sample reservoir and withdraw 300 µL at a flow rate of 2 µL/s.63.Dilute anti-EpCAM 25 times (4 µL of antibodies in 100 µL of PBS at a concentration of 20 µg/mL)64.Introduce 100 µL of anti-EpCAM to the sample reservoir and withdraw 100 µL at a flow rate of 2 µL/s.65.Incubate for 20 min at room temperature.66.Introduce 300 µL of 1% BSA to the sample reservoir and withdraw 300 µL at a flow rate of 2 µL/s.67.Trypsinize PC9 cancer cells by adding 5 mL of 0.25% trypsin-EDTA followed by incubating for 5 min at 37 °C.68.Neutralize the trypsin-EDTA by adding 10 mL of whole culture medium (RPMI 1640 supplemented with 10% FBS and 1% penicillin streptomycin).69.g for 4 min.Spin down the cells at 1200× 70.g for 4 min.Discard the supernatant and wash the cells with 10 mL of PBS at 1200× 71.Discard the supernatant and resuspend the cells in 1 mL of PBS.72.Determine the concentration of cells using a hemocytometer.73.6 cells/mL.Aliquot 1 mL of PC9 cells in PBS at a concentration of 1 × 1074.Add 5 µL of Vybrant DID dye in the cell sample and mix cell by pipetting.75.Incubate the mixture at 37 °C for 15 min.76.g for 4 min.Spin down the cells at 1200× 77.Wash the cells 3 times with 1 mL of PBS.78.Resuspend the cells in 1 mL of PBS.79.Determine the concentration of cells using a hemocytometer.80.3 cells/mL through serial dilution.Aliquot 1 mL of cell sample with a concentration of 1081.Add 50 µL of cells into 1 mL of whole blood sample supplemented with 1.8 mg/mL EDTA and mix well.82.Load the spiked sample to the sample reservoir of the microfluidic device.83.Withdraw 1.05 mL of the sample at a flow rate of 1 µL/s.84.Wash the microfluidic device by withdrawing 300 µL of PBS at a flow rate of 2 µL/s.85.Repeat Step 83 two times.86.Clamp the silicone tubes of both inlet and outlet.87.Dislodge the microfluidic device from the sample reservoir and the syringe.88.Inspect the microfluidic device under a fluorescence microscope. Fluorescently labeled PC9 cell can be detected under the APC channel.89.Mark the positions with captured PC9 cells using a marker.90.Detach the microfluidic device from the microscope slide.91.Punch though the microfluidic device of the captured PC9 position using a PDMS punch with an inner diameter of 5 mm.92.Collect the dislodged piece of the PDMS with captured PC9 cells in a 1.7 mL centrifuge tube.93.Fill the centrifuge tube with 1 mL of cell culture medium to keep the viability of the captured PC9 cellsThis work is to develop a microfluidic system for the isolation of rare cells such as CTCs for single cell proteomic analysis. The success of the system relies on three aspects: the fabrication of the microfluidic device, the functionalization of the microfluidic device and the detection of rare cells. For the fabrication of microfluidic device, the essential component is the silicon mold. To ensure the quality of the silicon mold, the surface of the silicon wafer should be cleaned thoroughly. The acetone can efficiently remove organic contamination from the silicon wafer. In some occasions , the silicon wafer can be cleaned with piranha solution. However, piranha solution is highly corrosive, users should be properly trained and handle the solution with extra caution. To strengthen the adhesion of SU8, HMDS vapor is used to create a hydrophobic surface on the silicon wafer. Alternatively, the silicon wafer can be treated with buffered oxide etch (BOE) to remove silicon oxide and a superior hydrophobic surface can be made. The channel height of the microfluidic device is 50 µm which requires a relatively thick SU8 layer. Therefore, the SU8 coated silicon wafer should be handled with patience. To prevent bubbles, the SU8 3050 bottle can be heated in water bath at 60 °C for 10 min. To achieve optimal uniformity, the SU8 coated silicon wafer can be baked at 60 °C for 10 min and ramp the baking temperature up to 95 °C at a ramping rate of 120 °C/h. For exposure, following the manufacturer’s protocol is optimal. It is advised not to over exposure SU8 which will make the photoresist more fragile and less adhesive to the silicon wafer. The development of unexposed SU8 is critical. Over development can cause feature damage and detachment, while not sufficient development can leave significant residue on the silicon surface which will affect the later process. Hard bake after development is preferred. The thermos cracks during SU8 development will disappear after the hard bake. The success of the silicon mold can be determined by inspection using an optical profilometer. The measurement of the channel height can be performed. The good mold should have a relatively uniform channel height in different positions of the silicon mold and the variation should be smaller than 5 µm.For the functionalization of the microfluidic device, preventing bubbles is important. When withdrawing ethanol, tapping the microfluidic device to drive the bubbles out can be a good trick for bubble removal. Ethanol is used for degassing the microfluidic device, it should be washed out thoroughly with DI water. Redundant ethanol can cause the precipitation of salt in PBS. The functionalization of the microfluidic device with antibodies allows the microfluidic device to capture rare cells by targeting their specific markers. The antibody immobilization can be inspected by introducing excessive amount of fluorescently labeled secondary antibodies. A fluorescence signal calibration curve can be generated to determine the saturated concentration of primary antibodies.When processing blood samples, it is advisable to gently agitate the sample in the inlet reservoir with a pipette to regularly to keep the homogeneity of the sample and prevent clogging near the inlet of the device. The detection of rare cells is through the identification of specific biomarkers on the rare cells. The protocol here shows the process of a spiked sample which simulate actual clinical samples, and the target cells are pre-labeled. However, for actual clinical sample processing, labeling of rare cells are usually performed after sample processing. For example, some CTCs can be labeled with fluorescently tagged anti-cytokeratin (CK) and DAPI, while normal white blood cells can be labeled with fluorescently tagged anti-CD45 and DAPI. In this case, CTCs can be detected as CK+/DAPI+/CD45−. When a target cell is detected and located, its position can be roughly marked. If the device works properly, the capture efficiency should be more than 90% and the purity should be higher than 70% ,56,57. IAfter the whole chip scan, the microfluidic device can be detached from the microscope slide, and the PDMS punch can be used to punch through the whole chip and dislodge the small PDMS piece containing the target cell. For this step, it is important to the punch straight to ensure the target cell is within punch zone. Following the target cell collection, the cell can either by cultured in cell culture medium for a short period of time or be lysed for proteomic analysis. As a sample preparation tool, the micropillar-based device is applicable for a variety of scMS technologies, such as label-free and multiplexed scMS ,60, tranOverall, this work describe a microfluidic device that can be used for easily capture, detection and release of rare cells. The fabrication of the device is straightforward and reproducible. Due to the moderate shear rate inside the microfluidic device, the captured rare cells remain high viability. Therefore, the captured cell is amenable for culture and further analysis. Also, since the silicon molds are reusable, the micropillar-based device acquire the advantage of other PDMS-based devices—highly reproducible with relatively low cost per chip. PDMS materials will be the main consumables for replicating the micropillar-based devices. This microfluidic device will empower the field of single cell proteomics.


Since 2008, the Improving Access to Psychological Therapies (IAPT) programme has offered adults in England evidence-based psychological treatments for common mental disorders (CMDs) such as depression and anxiety disorders. However, inequalities in access have not been explored at the national level.Using a unique individual patient dataset that linked 2011 Census information of English residents to national IAPT data collected between April 2017 and March 2018, we estimated the rate of access by a wide range of socio-demographic characteristics that are not routinely available. A large household survey was used to estimate the prevalence of probable CMDs by these socio-demographic characteristics. We estimated the probability of access to IAPT amongst people with CMDs by comparing the rates of access from IAPT data and the estimates of prevalence of CMDs from the household survey. Both unadjusted and adjusted (for important patient characteristics) access rates were estimated in logistic regression models.As a proportion of those with a probable CMD, access to IAPT varied markedly by socio-demographic characteristics. Older adults, males, people born outside of the UK, people with religious beliefs, people from Asian ethnic backgrounds, people reporting a disability and those without any academic or professional qualifications were underrepresented in IAPT services nationally, in adjusted models.The identification of patients who may be underrepresented in IAPT provides an opportunity for services to target outreach and engagement with these groups. Further understanding of barriers to access should help increase equity in access. In England, one in six adults are estimated to have a common mental disorder (CMD)  . The cohort was also linked to death registration data using NHS number, to remove participants who died during the reporting periods. Study participants are the adult population in England aged 18 (at the start of the reporting period) to 100 years, who were usual residents in England when they were enumerated in the 2011 Census. Participants living in Wales at the time of 2011 Census were excluded from the study.Out of the 1 009 035 IAPT referrals received within the 2017/18 reporting period, we identified 941 008 unique patients. Out of the 941 008 individuals, 766 829 (81.5%) were successfully linked to the 2011 Census .We used data from UKHLS waves 8 to 10, to measure probable CMDs within the population at an equivalent time to the IAPT data. The UKHLS data was accessed via the UK Data Archive with addition of lower super output area information requested through the special licence. Interviews for which sample months were between April 2017 and March 2018 were pooled into a financial year 2017/18 dataset equivalent to the IAPT reporting period. Survey weights were scaled to adjust for reduced likelihood to respond over time following UKLHS guidance , qualifications, region of England, religious affiliation, and sex (See online Supplementary Table S1). To estimate the prevalence in the general population, the socio-demographic characteristics came from the UKHLS interviews that were available closest to the 2011 Census date (±26 months) or the first adult interview for someone aged 16 years.For the analysis of access to IAPT, the main outcome was having received at least one treatment session in IAPT between April 2017 and March 2018.For the analysis of the prevalence of CMDs using the UKHLS, the main outcome was having a General Health Questionnaire (GHQ)-12 score of 4+ using the standard scoring method to proxy probable CMD.GHQ-12 is a validated measure that is commonly used to estimate non-psychotic psychiatric morbidity and CMDs  and of having a CMD . We fitted unadjusted models and models adjusted for all other exposures using our 18+ population. We restricted the population to individuals aged 25+ when estimating the effects of qualifications and National Statistics Socio-Economic Classification (NS-SEC) to minimise the influence of measurement error between the Census and IAPT time points.In the UKHLS sample, we used analysis weights for the self-completion questionnaire to adjust for differences in selection probabilities and non-response. We deleted observations with missing data and used inverse probability weighting based on a logistic regression model to account for non-randomness of the missing data.We used an indirect method to estimate the access to IAPT amongst people with probable CMDs. We estimated the proportion of individuals with CMD who received treatment in IAPT, by dividing the rates of people receiving treatment through IAPT services by the self-reported CMD rates. If for a particular group, the rate of access to IAPT was 3%, and the self-reported CMD rate was 15%, then the rate of access to IAPT among people with probable CMDs was 20% (0.03/0.15). To obtain 95% confidence intervals, we ran a Monte Carlo simulation with one million iterations, drawing each rate from a normal distribution with a mean equal to the estimated rate and a standard deviation equal to the standard error and calculating the ratio. The 2.5th and 97.5th percentiles of the resulting distributions were used to estimate the 95% confidence intervals  received at least one treatment session through IAPT services (around 1 in 50) in 2017/18. The UKHLS cohort comprised 15 614 participants of which 18.1% had a probable CMD.Our study population  were preThe proportion of people with a CMD in UKHLS; the proportion of people accessing IAPT services; and those accessing IAPT as a proportion of those with a probable CMD broken down by socio-demographic characteristic can be found in Adults aged 25- to 34-years were most likely to have a probable CMD  and adults 65 and over least likely to access IAPT [4.7% (95% CI 4.3–5.1%)]. Adjusting for other factors had little effect on the estimated differences in access to IAPT by age and sex.Males were less likely to have a probable CMD [15.9% (95% CI 14.7–17.1%)] and to receive treatment through IAPT [1.4% (95% CI 1.3–1.4%)]. There is a statistically significant underrepresentation of males in IAPT with only 8.5% (95% CI 7.9–9.2%) of males with CMD entering this service compared to 10.8% (95% CI 10.3–11.4%) of females. When adjusting for other factors the difference reduced slightly but remained significant.The Mixed ethnic group had the highest proportion of probable CMDs [30.4% (95% CI 21.9–38.8%)] when comparing against the other ethnic groups (range 14.1 to 21.8%). They were also most likely to be receiving treatment in IAPT [2.9% (95% CI 2.8–3.1%)]. When estimating the proportion of individuals with CMD who received treatment in IAPT, 9.7% (95% CI 7.6–13.5%) of the Mixed ethnic group received treatment compared to 10.2% (95% CI 9.7–10.7%) of the White ethnic group. The Asian ethnic group [7.2% (95% CI 6.2–8.7%)] were significantly underrepresented in IAPT when compared to the White ethnic group. Adjusting for other factors reduced the differences, but the Asian ethnic group remained significantly underrepresented in IAPT compared to the White ethnic group.Those born in the UK were (non-significantly) more likely to have a probable CMD over those born outside of the UK [19.6% (95% CI 18.7–20.6%) compared to 17.0% (95% CI 14.6–19.4%)]. However, those born outside the UK were significantly less likely to receive treatment in IAPT [1.2% (95% CI 1.2–1.3%)] compared to 2.0% (95% CI 2.0–2.0%) and consequently only 7.2% (95% CI 6.3–8.4%) of those with probable CMD received treatment in IAPT compared with 10.4% (95%CI 9.9–10.9%) of UK born patients. This difference reduced but remained significant when adjusted for other factors.CMDs affected 19.6% (95% CI 18.7–20.5%) of people whose first language was English compared to 15.4% (95% CI 12.7–18.1%) of people for who English was not their first language. Also, there was a higher proportion of patients receiving treatment in IAPT where English was their first language [2.0% (95% CI 2.0–2.0%) compared to 1.1% (95% CI 1.0–1.1%)] where English was not their first language. When comparing there was a significant difference in representation with those where English is not the first language being underrepresented [6.9% (95% CI 5.9–8.4%)] compared to those where English was the first language [10.1% (95% CI 9.7–10.6%)]. Adjusting for other factors made this difference non-significant.There was no difference in the proportion of people with a probable CMD by religious affiliation . People with no religion were significantly more likely to be treated in IAPT [2.4% (95% CI 2.4–2.5%) compared to 1.7% (95% CI 1.7–1.8%)]. Those with a religious affiliation and probable CMD were significantly underrepresented in IAPT [9.1% (95% CI 8.6–9.7%) compared with 12.4% (95% CI 11.6–13.3%)]. Adjusting for other factors reduced the difference, but having a religious affiliation remained significantly associated with being underrepresented in IAPT.Disabled people had higher levels of probable CMD [31.6% (95% CI 29.5–33.8%)] compared to those who were not disabled [16.4% (95%CI 15.5–17.3%)] and there was a higher proportion of disabled people receiving treatment in IAPT [2.2% (95% CI 2.1–2.2%) compared to 1.9% (95% CI 1.9–1.9%)]. However, only 6.8% (95% CI 6.4–7.3%) of disabled people with a probable CMD are being treated in IAPT compared with 11.5% (95%CI 10.8–12.1%) of non-disabled people, indicating disabled people are significantly underrepresented in IAPT. After adjustment, these differences were attenuated but remained significant, suggesting having a disability is negatively associated with access to IAPT.Having no academic or professional qualifications was associated with a higher level of probable CMDs [20.1% (95% CI 17.0–23.3%)] compared to below a degree qualification [19.1% (95% CI 17.8–20.4%)] and degree level and above [16.9% (95%CI 15.5–18.2%)] . DespiteFurther analyses on local area deprivation, region and NS-SEC can be found in This study found that older adults, males, people born outside of the UK, people with a religious affiliation, people from Asian ethnic backgrounds, people with a disability and those without any academic or professional qualifications appear underrepresented in IAPT as a proportion of those with a probable CMD.Research acknowledges that older adults are underrepresented in mental health treatment (Department of Health, Men are less likely to seek support than women for their mental health (Bogdanova, Cooper, Ahmad, McManus, & Shoham, Research reports that marginalised groups are at higher risk of poorer mental health (Nyashanu, Ganga, & Chenneville, There was a significant underrepresentation of those born outside of the UK compared to those born in the UK in IAPT, confirming previous research suggesting that migrants are likely to be disadvantaged regarding access to support for mental health issues (Bhavsar et al., Religion can be an important factor affecting attitudes to accessing support for mental health (Loewenthal et al., Previous research has identified complex relationships between school experiences, educational qualifications and mental health problems (Esch et al., Lastly, although the self-referral model has been designed to improve the overall access to IAPT, studies have found that the self-referral process can be overwhelming and act as a barrier in access for certain socio-demographics (Thomas et al., This study benefitted from a unique dataset linking the 2011 Census to national IAPT data. Unlike studies solely based on sample frames, we generated high-quality population-based information with large sample size and high data coverage. Data collected by UKHLS provided large representative sample of the English population with a probable CMD which increased validity of the findings. This study used a validated and reliable measure to identify probable CMDs.The 2017/18 IAPT dataset was used for the analysis, and therefore findings may not reflect the current representation in IAPT which showed relative increases in referrals that started treatment across all socio-demographic groups (NHS Digital, Despite GHQ-12 being a reliable and commonly used measure, previous studies have questioned validity of GHQ-12 for identifying CMDs Donath, . We usedLastly, we only included individuals seen at least once in IAPT, meaning we evaluated the initial access to mental health services as an unmet need. This does not consider the quality and efficacy of the care they receive. Retention of patients, treatment length and outcomes should be evaluated to fully understand socio-demographic differences in access to mental health services. Due to data availability, we could not include or control for clinical severity scores, so future iterations of this study should incorporate these to provide a more accurate representation of people with probable CMD in IAPT.This study aimed to identify the representativeness of the IAPT population amongst people with probable CMDs. The study found underrepresentation of several key groups including older adults, males, ethnic minority groups, those born outside of the UK and those with no academic or professional qualifications. Whilst these results are not necessarily unique to IAPT in the context of mental healthcare, they should inform future expansion of IAPT services targeting these groups to ensure they get equal treatment of their CMD. Future research should consider repeating the study with later years of IAPT and 2021 Census data, and consider to further explore the measure for probable CMD prevalence by comparing GHQ-12 to the more detailed CIS-R based on the next APMS survey. To address knowledge gaps, future research should also incorporate analysis looking at differences in treatment length, dropout rates and outcomes of patients who completed minimal treatment in IAPT. This will provide more accurate representation of people with CMDs in IAPT and greater understanding of the unmet need. Research should also incorporate patients' perspective of barriers to access to mental health services.Sharland et al. supplementary materialSharland et al. supplementary materialSharland et al. supplementary material


The pandemic of Covid-19 has overwhelmed healthcare systems including healthcare workers(HCWs) imposing additional physical and mental workloads.We aimed to evaluate the impact of the pandemic on the professional quality of life of HCWs.We conducted a cross-sectional study among HCWs working in the Covid-19 unit in Sfax, Tunisia in December 2021. We used a self-administrated questionnaire including sociodemographic and professional information. Professional issues were assessed by The Professional Quality of Life scale.Our population consisted of 69 participants. The mean age was 31± 6 years. The sex ratio (M/F) was 1.1. Sixty-eight per cent had a university education. On a scale from 0 to 10,76.8% rated their Health Status greater than or equal to 8 and the mean score was 8.89±1. Medical history of Covid-19 infection was found in 37.7% of paricipants and 94,1% were vaccinated against SARS Cov 2.Most of the workers showed moderate to high levels of compassion (65.2% and 31.9 % respectively). Fifty-five per cent showed moderate burnout levels. Only 2.9% of the population had a high level of secondary traumatic stress.Secondary traumatic stress was associated with age . The males were more affected with burnout symptoms. Vaccination Status was associated with compassion satisfaction (p=0.042). Health Status Evaluation was not correlated with compassion satisfaction, burnout or secondary traumatic stress.The current pandemic has affected the HCWs system professional and social lives. A long follow-up should be maintained to support HCWs dealing with the pandemic.None Declared


Heterodera glycines) is the most yield-limiting pathogen of soybean in the US. This study was carried out in order to provide updated information on SCN virulence phenotypes in Indiana. A total of 124 soil samples were collected from soybean fields in 2020 and all of them tested positive for SCN. The virulence phenotypes of 42 representative SCN populations were determined with seven soybean indicator lines using the standard HG type test. The most predominant HG types were 2.5.7 and 1.2.5.7, which accounted for 64% and 14% of the SCN populations tested, respectively. None of the SCN populations tested were rated as HG type 0, compared with 28% of the populations in a previous survey in Indiana during 2006–2008. Nearly 88% of the SCN populations evaluated in this study overcame the resistance provided by PI 88788, which is the most common source of resistance in soybean, up from 56% in the 2006–2008 survey. Approximately 14% of SCN populations tested were virulent to PI 548402 (Peking), in contrast to 0% in the 2006–2008 survey. This study reveals a trend of increasing virulence of SCN populations to resistant sources of soybean in Indiana. The results highlighted the importance of rotating soybean varieties with different types of resistance and identifying new sources of resistance for sustainable management of SCN.The soybean cyst nematode (SCN,  Glycine max (L.) Merr.) is one of the most important leguminous crops globally, providing protein and oil for humans and livestock. In Indiana, over 5 million acres of soybeans were planted and harvested in 2020  with a modified method of sieving and decanting procedure , PI 88788 (#2), PI 90763 (#3), PI 437654 (#4), PI 209332 (#5), PI 89772 (#6), PI 548316 (#7), PI 567516C, and Williams 82 (the susceptible check) were used to determine SCN population HG type . The soyData analysis: The average number of SCN females from 5 replicates per indicator line was used to determine the FI and the HG type of each SCN population. The HG type was determined by assaying which indicator lines had an FI ≥ 10%. Microsoft Excel  was used for the descriptive statistics of the FI datasets. R  and RStudio  were used to conduct Pearson's product moment correlation analysis for the seven indicator lines and PI 567516C.3 of soil. The soil sample with the highest SCN population density was collected from a field in Noble County, and the soil sample with the lowest population density was from Tippecanoe County  had a population density greater than 500 eggs and juveniles per 100 cm3 of soil. Forty-two SCN populations from 26 counties in Indiana were selected for the SCN HG type test using Williams 82 as the susceptible check  were tested positive for SCN. The SCN population densities of the samples ranged from 165 to 19,840 eggs and juveniles per 100 cme County . The avele check   that originated from Carroll, Grant, Huntington, Pulaski, White, Whitley counties were capable of reproducing on PI 548402  with FI ≥ 10%  and 2. TOn PI 88788 (HG type indicator line #2), 37 out of 42 HG-typed populations (88.1%) reproduced with an FI ≥ 10%  and 2. TFor the soybean line PI 209332 (HG type indicator line #5), 90.5% of the SCN populations (38 out of 42) tested had an FI ≥ 10% . The SCNThe soybean line PI 548316 (HG type indicator line #7) had the highest number of SCN populations with an FI ≥ 10%. All but one of the 42 (97.6%) SCN populations tested had an FI ≥ 10% on PI 548316  and 2. TOnly two of the SCN populations tested had an FI ≥ 10% on PI 90763 (HG type indicator line #3) with FI = 15.6% and 20.3%, and they were from Benton County and Kosciusko County, respectively . None ofP < 0.01) with each other. PI 548402 (#1) was positively correlated with PI 88788 (#2), PI 209332 (#5) (P < 0.05) and PI 89772 (#6), PI 548316 (#7) (P < 0.01)  . No sign < 0.01) .Monitoring SCN virulence phenotypes is critical for establishing management recommendations for soybean growers and guiding soybean breeding efforts . In thisDifferent approaches had been employed by previous studies to collect soil samples for surveying the SCN virulence phenotypes, depending on the available resources. For example, the last similar study conducted in Indiana during 2006–2008 used soil samples submitted by growers to the Purdue Nematode Diagnostic Lab . A recenIn this study, a total of seven different HG types were detected in 42 SCN field populations collected in 2020 in Indiana. The 42 SCN samples were from 26 counties in Indiana . When moThe HG types of SCN populations from the 26 counties found in this study provided insight into the overall trends of SCN virulence in Indiana in 2020. The most predominant SCN virulence phenotype was HG type 2.5.7. Out of the 42 SCN populations tested, 64.3% were HG type 2.5.7, followed by HG type 1.2.5.7 (14.3%), HG type 5.7 (7.1%), HG type 2.3.5.7 (4.8%), HG type 7 (4.8%), HG type 2.7 (2.4%) and HG type 2 (2.4%). Similar patterns of SCN virulence phenotypes were observed in 52 SCN populations in Illinois and 48 in Missouri, where the HG type 2.5.7 was prevalent .In contrast, HG type 7 was the predominant SCN virulence phenotype in North Dakota, South Dakota and Minnesota . The difOur study of SCN populations collected in 2020 revealed a significant shift in SCN virulence phenotypes in Indiana compared to the last survey conducted during 2006–2008 . The soiRhg1-b resistance allele to confer resistance against SCN . Furthermore, more than 88% of SCN populations tested in our study had an FI ≥ 10% on PI 88788 (indicator line #2), compared to 56% in 2006–2008 . The siginst SCN . TherefoNone of the SCN populations in the 2006–2008 survey of Indiana had an FI ≥ 10% on PI 548402 . In this study, 14.3% of the SCN field populations tested showed significant reproduction on PI 548402 (FI ranging from 12.6% to 38.1%) and were all determined to be HG type 1.2.5.7. This is the first time that SCN populations virulent against PI 548402 (Peking) have been reported in Indiana. Although PI 88788-type and Peking-type resistances are different, a positive correlation between the FI values of the indicator line #1 (Peking) and #2 (PI 88788) was detected in this study. Similar correlations between #1 and #2 were also reported in previous SCN surveys conducted in North Dakota and Missouri . Our datNone of the SCN populations studied in the 2006–2008 survey had an FI ≥ 10% on PI 90763 (#3) . In thisRhg1-b or Rhg4 (PI 567516C has a unique, though not well elucidated, mechanism of resistance, and does not rely on  or Rhg4 . The cur or Rhg4 . PI 5675Supplementary Material DetailsClick here for additional data file.


Early trials of long-term lenalidomide use reported an increased incidence of second primary malignancy (SPM), including acute myeloid leukaemia and myelodysplastic syndrome. Later, meta-analysis suggested the link to be secondary to lenalidomide in combination with melphalan.NCT01554852). Here we present an analysis of SPM incidence and profile the SPM type to determine the impact of autologous stem cell transplantation (ASCT) and lenalidomide exposure in 4358 patients treated on study. Data collection took place from the start of the trial in May 2010, to May 2019, as per the protocol timeline. The Median follow-up following maintenance randomisation was 54.5 and 46.1 months for TE and TNE patients, respectively.Myeloma XI is a large, phase III randomised trial in-which lenalidomide was used at induction and maintenance, in transplant eligible (TE) and non-eligible (TNE) newly diagnosed patients . Although the TNE lenalidomide maintenance patients had the greatest SPM incidence (15.4%), this was not statistically significant when compared to the observed patients .The SPM incidence was higher in patients who received lenalidomide at induction and maintenance (double exposure), when compared to those treated with lenalidomide at one time point (single exposure). Again, this was most marked in TNE patients where the overall SPM incidence was 16.9% in double exposed patients, compared to 11.7% in single exposed patients, and 11.2% in patients who did not receive lenalidomide (p = 0.04). This is likely an effect of treatment duration, with the median number of cycles being 27 in the TNE double exposed patients, vs 6 in the single exposure patients.Haematological SPMs were uncommon, diagnosed in 50 patients (incidence 1.1%). The majority of cases were diagnosed in TE patients treated with lenalidomide maintenance , suggesting a possible link with melphalan. Non-melanoma skin cancer incidence was highest in patients receiving lenalidomide maintenance, particularly in TNE patients, where squamous cell carcinoma and basal cell carcinoma were diagnosed in 5.5% and 2.6% of patients, respectively. The incidence of most solid tumour types was higher in lenalidomide maintenance patients.Mortality due to progressive myeloma was reduced in patients receiving lenalidomide maintenance, noted to be 16.6% compared 22.6% in those observed in TE patients and 32.7% compared to 41.5% in TNE patients. SPM related mortality was low, 1.8% and 6.1% in TE and TNE lenalidomide maintenance patients, respectively, compared to 0.4% and 2.8% in those being observed.This provides reassurance that long-term lenalidomide treatment is safe and associated with improved outcomes in TE and TNE populations, although monitoring for SPM development should be incorporated into clinic review processes.10.13039/501100000289Cancer Research UK [C1298/A10410].Primary financial support was from  Initial studies assessing lenalidomide as both a continuous and maintenance strategy reported an increased risk of second primary malignancy (SPM), including myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). Following this, in 2014, a meta-analysis inclusive of seven phase 3 studies and 3218 patients found that the risk of haematological SPM (hSPM) appeared to be linked to lenalidomide when used in combination with oral melphalan. Recently, in 2022, further meta-analysis of SPM incidence in patients receiving lenalidomide treatment for all haematological cancer types, suggests that the SPM risk is unique to patients with myeloma.Long-term data on SPM incidence in large series of uniformly treated patients is lacking. In addition, no analysis has reported on the profile of SPM developed, or whether lenalidomide treatment at both induction and maintenance poses an additional risk. Lenalidomide is undoubtably effective in myeloma, but a greater understanding of the true SPM risk may further influence its application. Here, we address these unanswered questions, reporting SPM profiles and incidence in 4358 patients treated in the NCRI Myeloma XI trial.Lenalidomide is safe and effective as post ASCT maintenance and as continuous therapy in the TNE setting. SPM risk is increased, but myeloma related death is reduced, and SPM mortality is low, providing reassurance for its use. There is no current recommendation regarding the duration of lenalidomide therapy in long-term responders, but this may be taken into consideration for future trial design. We recommend that clinic review should include discussion around SPM, including skin changes and symptoms/signs of solid tumours, particularly in TNE patients, who have an innately greater risk of secondary carcinogenesis.Outcomes in myeloma continue to improve.Early studies assessing lenalidomide maintenance reported an increased incidence of SPM, including MDS and AML.Interim safety data from the Myeloma XI Trial, inclusive of 2732 patients, with a median follow-up of 34.3 months from trial entry and 24.2 months from maintenance randomisation did not reveal an increased incidence of haematological hSPM in the maintenance lenalidomide patients, irrespective of ASCT status. There was an increased incidence of all cancers, but this was only statistically significant in older, TNE patients, defined as ≥74 years of age. The SPM mortality was 1%, suggesting that the survival benefit associated with lenalidomide use may outweigh the risks.Here we report the long-term incidence of SPM in Myeloma XI, inclusive of all 4358 treated patients. We determine SPM incidence and profile the types of malignancies seen in TE and TNE patients, including whether there is an impact on SPM development in patients treated with lenalidomide at both induction and maintenance. This forms the largest assessment of SPM profiling in a uniformly treated, newly diagnosed TE and TNE patients.NCT01554852). As per the statistical analysis plan, the SPM analysis includes the safety population, not the intention to treat. This analysis reports SPM incidence in all patients who received at least one cycle of trial prescribed treatment.Myeloma XI is a phase III, multicentre, randomised, open-label and adaptive design trial conducted in 110 National Heath Centre Hospitals , solid tumours and haematological malignancies.All patients recruited were assessed for transplant eligibility and then randomised between thalidomide, lenalidomide or carfilzomib (TE only) containing induction regimes. Patients not achieving at least a very good partial remission, may have also received bortezomib intensification prior to ASCT or maintenance randomisation, as per protocol, All statistical analyses were undertaken in SAS  according to the Trial Statistical Analysis Plan. Cumulative incidence function curves were estimated by non-parametric maximum likelihood estimationAll patients included provided written informed consent. The study is now closed for accrual, but follow-up continues for long-term analysis. The study was approved by the national ethics review board , institutional review boards of the participating centres, and the competent regulatory authority , and was undertaken according to the Declaration of Helsinki and the principles of Good Clinical Practice as espoused in the Medicines for Human Use  Regulations.JJ, DC and TM had access to the data set. All study authors agreed on the decision to submit this manuscript for publication. The funders of the study have not been involved in data collection, data analysis, data interpretation or writing of the paper.Since May 2010, 4358 patients entered and received induction therapy. Median follow up at the data cut-off was 60 months (interquartile range 47–76). Of the 4358 patients that initiated treatment, 2532 entered the TE pathway and 1826 entered the TNE pathway. Within the TE pathway 1008 patients received CTD, 1014 received CRD and 510 received KCRD induction. In the TNE pathway 910 patients received CTDa and 916 received CRDa, The median follow-up since maintenance randomisation is 46 and 55 months, for TE and TNE patients, respectively. A breakdown of follow-up times according to treatments received in the TE and TNE pathway is detailed in Three hundred and seventy-six SPM in 318 patients were identified. Of the 318 patients, 277 had 1 SPM and 41 patients had 2 or more. The median age at SPM diagnosis was 73 years (range 50–90) and the median time to first diagnosis from induction randomisation was 35.7 months (range 1.4–96.6).Overall trial-related SPM incidence was 7.3%, with an incidence rate of 2.4 per 100 person years. The cumulative incidence of SPM in the whole trial population was 4.1%  3.5%–4.7%), 7.4% (95% CI 6.6%–8.3%) and 10.9% (95% CI 9.6–12.2%) at 3, 5 and 7 years, respectively.One hundred and thirty-eight TE (incidence 5.5%) patients developed an SPM compared to 180 TNE patients (incidence 9.9%), Within the TE pathway 37 patients (incidence 1.5%) developed an SPM during induction. The cumulative incidence in patients who received CTD was 2.0%, 4.7%, and 8.3% at 3, 5 and 7 years, respectively. This was comparable to the incidence in patients who received CRD, being 3.1%, 5.8% and 9.3% (Pepe–Mori p = 0.08). Patients receiving KCRD had an SPM incidence of 2.7% and 8.1% at 3 and 5 years, Within the TNE pathway 61 patients (incidence 3.3%) developed an SPM during induction. Those treated with CTDa had a cumulative SPM incidence of 6.5%, 9.8% and 12.5% at 3, 5 and 7 years, respectively. Patients treated with CRDa had a cumulative SPM incidence of 6.0%, 10.4% and 14.3% at the same time points (Pepe–Mori p = 0.47), This data shows that SPM incidence does not appear to be significantly affected by the treatment given at induction, although SPM incidence is higher in older patients.Eighty-five (incidence 5.9%) TE patients developed an SPM during maintenance. Patients who received lenalidomide ± vorinostat (n = 875) maintenance had a cumulative SPM incidence of 4.3%, 10.5% and 12.4% at 3, 5 and 7 years. In observed patients (n = 566) the cumulative SPM incidence was 2.2%, 4.1% and 5.9% at 3, 5 and 7 years (Pepe-Mori p = 0.006) One hundred and ten (incidence 13.2%) TNE patients developed an SPM during maintenance. In those who received lenalidomide ± vorinostat maintenance (n = 493) the cumulative SPM incidence was 9.8% and 17.2% at 3 and 5 years, respectively. In observed patients (n = 340) the cumulative SPM incidence was 6.4% and 10.2% for 3 and 5 years (Pepe-Mori p = 0.10), respectively, In the TE pathway, the median time to first SPM development in lenalidomide maintenance patients (from maintenance randomisation) was 35 months (range 1–69) and 31 months in those being observed (range 1–75). In the TNE pathway, the median time to first SPM development was 28 months (range 2–71) in those receiving lenalidomide, compared to 29 months (range 4–90) in those being observed.These data reveal that SPM incidence is higher in patients who received lenalidomide maintenance, compared to those being observed.The median age of the TNE patients was 74 years and, therefore, this was used as a cut-off for defining advanced age. The cumulative SPM incidence in TNE patients ≤74 who were randomised to lenalidomide ± vorinostat was 8.7% (95% CI 5.2–12.2) at 3 years and 16.6% (95% CI 11.6–21.7) at 5 years. In observed patients, the cumulative incidence was 7.7% (95% CI 3.7–11.7) and 10.8% (95% CI 5.9–15.8) at 3 and 5 years, respectively (Pepe Mori p = 0.30), In the TNE patients >74 the cumulative SPM incidence in patients who received lenalidomide ± vorinostat was 11.1% (95% CI 7.0–15.2) and 17.7% (95% CI 12.2–23.2) at 3 and 5 years, respectively. In those randomised to observation the cumulative incidence was 4.9% (95% CI 1.6–8.3) and 9.5% (95% CI 4.5–14.6) at 3 and 5 years, respectively (Pepe Mori p = 0.09), These data illustrate the impact of age on SPM development, with the greatest incidence noted in patients >74 receiving lenalidomide.Fifty patients (incidence 1.1%) developed a hSPM, 36 within the TE pathway (incidence 1.4%) and 14 (incidence 0.8%) in the TNE pathway. Of the 50 patients, 32 received lenalidomide maintenance, 25 in the TE pathway (incidence 2.8%) and 7 in the TNE pathway (incidence 1.4%). The median time to hSPM development in the TE and TNE patients was 51.8 months (range 12.4–82.9) and 42.3 months (range 22.9–78.9). The median age at time of hSPM diagnosis was 79 (range 72–83) in TNE patients and 68 (range 50–76) in TE patients. The most diagnosed hSPM were MDS (N = 21), AML (n = 13), DLBCL (n = 6) and B-ALL (n = 4) One hundred and eighty-three solid malignancies (including melanoma and fibroxanthoma but excluding BCC and SCC) in 177 patients were confirmed (incidence 4.1%). Seventy-six (incidence 3%) TE patients developed an SPM compared to 101 (incidence 5.5%) TNE patients. The median age of SPM diagnosis was 67 (range 53–76) years and 77 (range 65–90) years for the TE and TNE pathways, respectively. The median time to SPM diagnosis from induction randomisation was 38.8 (range 2.4–84.7) months in the TE pathway and 32.0 (range 1.4–97.1) months in the TNE pathway. The most diagnosed were prostate (22 cases), breast (20), colon (19), lung (16) and melanoma (16), One hundred and forty-three NMSC were diagnosed in 106 patients (incidence 2.4%), In summary, these data illustrate that hSPM were uncommon and mainly diagnosed in TE patients. Solid tumours and NMSC formed the majority of SPM, with the greatest incidence in TNE patients.Thirty-eight different cancer types were diagnosed in patients who had been through maintenance randomisation. Of the 38 different types, 17 were noted in both observation and lenalidomide maintenance groups, 16 were confined to only lenalidomide maintenance patients and five solely in those being observed. The incidence of each cancer type was low. In all lenalidomide maintenance patients, only squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) were diagnosed in >1% of patients. No individual SPM type was diagnosed in >1% of patients being observed, In TE patients, 28 different cancer types were diagnosed. Most cases were noted in the lenalidomide maintenance patients, although the overall incidence of all cancer types was low. Only BCC (1.6%), MDS (1.3%) and SCC (1.1%) were diagnosed in >1% of lenalidomide maintenance patients. In the observed patients only prostate cancer (1.2%) was diagnosed in >1%. Haematological SPMs were almost confined to the lenalidomide maintenance patients, particularly MDS (1.3%) and AML (0.6%), diagnosed in a total of 16 patients, compared to 1  patient being observed, In TNE patients, 28 different cancer types were diagnosed. Few hSPMs were diagnosed, with only 8 cases, 7 in patients receiving maintenance lenalidomide. The incidence of NMSC was marked, particularly in the lenalidomide patients, with an SCC incidence of 5.5% and BCC incidence of 2.6%, These data show that the incidence of each SPM type was low, apart from NMSC which were common, particularly in the TNE lenalidomide maintenance patients.Patients were grouped according to no lenalidomide treatment, single exposure, i.e., at induction or maintenance, or double exposure i.e., at both induction and maintenance. In TE patients the SPM incidence was greater in the double exposed patients, although this was not statistically significant , single exposed vs double exposed 7.6% (p = 0.20)). Despite this, the trial related incidence of individual SPM types was low, being <1%, except for BCC (1.4%), SCC (1.2%) and MDS (1.1%) noted in >1% of patients treated with lenalidomide and both induction and maintenance. Haematological SPMs were almost confined to the lenalidomide-treated patients, with most in the double exposed group, In the TNE patients, there was also a greater SPM incidence in the double exposed patients , single exposed vs double exposed 16.9% (p = 0.04)). Non-melanoma skin cancers were common, particularly in the double exposed patients, with an SCC incidence of 4.6% and BCC incidence of 3.1%. No SPM type was diagnosed in >1% of non-exposed patients. In the double exposed patients, prostate cancer (1.2%) and melanoma (1.2%) were also noted in >1% of patients, These data show that the incidence of SPM is highest in patients who received lenalidomide at both induction and maintenance. This is likely to be as a consequence of treatment duration.At the median follow-up point of 54.5 months and 46.1 months for TE and TNE patients, respectively, we determined the outcome for all patients, including the proportion of patients who died because of SPM.In TE patients treated with lenalidomide maintenance, 13 (incidence 1.8%) deaths were secondary to an SPM, compared to 121 (incidence 16.6%) myeloma-related and 18 (incidence 2.5%) non-myeloma related deaths. This compared to 2 (incidence 0.4%) SPM deaths, 117 (incidence 22.6%) myeloma-related and 19 (incidence 3.7%) non-myeloma related deaths in the observed patients.In TNE lenalidomide maintenance patients, 25 (incidence 6.1%) died due to a SPM, 133 (incidence 32.7%) due to progressive myeloma and 44 (incidence 10.8%) due to other causes. In the observed TNE patients, 9 (incidence 2.8%) died due to an SPM, 131 (incidence 41.5%) due to progressive myeloma and 28 (incidence 8.9%) secondary to non-myeloma related issues, This data shows that lenalidomide maintenance is associated with reduced myeloma related mortality and a low risk of SPM related death.This is the largest single trial analysis of SPM incidence associated with lenalidomide as both an induction and maintenance treatment option, in newly diagnosed TE and TNE eligible, uniformly treated patients.Significantly, in TE patients, the overall SPM incidence rate was 7.7% in those randomised to lenalidomide maintenance, compared to 3.2% in those being observed (p = 0.006). Importantly however, there was a low risk of SPM related death, noted in 1.8% of lenalidomide maintenance treated TE patients. In addition, lenalidomide maintenance reduced the myeloma mortality rate, with 16.6% of patients dying because of progressive disease, compared to 22.6% of the TE patients being observed. Overall, the data in the TE patients is positive, with improved survival and a low risk of death due to secondary carcinogenesis. With increasing number of patients receiving lenalidomide maintenance routinely post ASCT, these data provide reassurance.TNE patients have the highest SPM incidence, particularly those receiving lenalidomide maintenance, where the overall incidence was 15.4%, compared to 10% in those being observed (p = 0.10). The highest incidence was noted in the older TNE patients, defined as those >74 years, where the overall incidence was 22.5% in those receiving lenalidomide maintenance and 13.9% in those being observed (p = 0.09). In the general UK population, cancer incidence per 100 person years is noted to be between 0.66 and 2.02 for patients aged between 70 and 84, far lower than the overall rate of between 3.1 and 6.1 for the TNE patients, Haematological SPM incidence was low, with an overall incidence of 1.1% for all trial participants. Furthermore, the incidence remained low in lenalidomide maintenance patients, with an incidence of 2.8% in TE patients and 1.4% in TNE patients. The higher incidence rate in TE patients may be linked to the use of melphalan ASCT conditioning, consistent with previous meta-analysis.Solid SPMs developed in TNE patients were consistent with the general population, with colon, prostate, breast, and lung frequently diagnosed, Almost a third of SPM patients developed a NMSC. This was particularly marked in the TNE patients, where 73 of the 180 (41%) SPM patients were diagnosed with a NMSC, compared to 33/138 (24%) TE patients. In both pathways, most cases were diagnosed in patients receiving lenalidomide maintenance. The data reveals the predisposition to these malignancies in immunocompromised patients.The overall incidence of SPM was highest in patients who received lenalidomide at both induction and maintenance. This was however only significant in TNE patients treated at both time points, who had received fewer cycles than the TE group (median 34 cycles TE vs 27 TNE). This suggests that the risk of SPM is not conclusively linked to accumulative dose in all patient groups. The benefit of continuous lenalidomide in patients with long-term remissions is a consideration for future trial design.,It is important to note some potential confounding factors when interpreting these data. Patients being observed may be reviewed less frequently and as a result, there could be the potential for the under-reporting of SPMs. The trial data also indicates an overall survival benefit in lenalidomide maintenance treated TE patients and therefore this group of patients will have more time to develop an SPM, when compared to those being observed. The study is also open label and therefore patients receiving lenalidomide maintenance would have been counselled regarding the small SPM risk. This could result in increased vigilance and reporting of signs and symptoms of potential SPM in the lenalidomide maintenance patients. Of note, we also pooled the lenalidomide and lenalidomide + vorinostat data, due to low numbers of combination maintenance patients, poor tolerance of vorinostat in the combination maintenance setting and a lack of outcome improvement, meaning a separation of the analysis was not indicated.The data provide reassurance that lenalidomide maintenance is associated with a low incidence of hSPM, low risk of SPM death and improved survival, particularly post ASCT. There is an increased SPM incidence in lenalidomide maintenance patients, but a third of cases are low risk, NMSC. The increased SPM incidence is not statistically significant in TNE patients, illustrating the predisposition to carcinogenesis in older patients with myeloma, irrespective of whether lenalidomide maintenance is used or not. Patients treated with long-term lenalidomide need to be counselled regarding the signs and symptoms of possible SPM, particularly skin cancers. Clinic review should include a history and examination, focused on identifying possible SPM, so intervention can be made appropriately and in a timely fashion.The Myeloma XI study design and protocol was developed by GJM, GHJ and FED. DAC, TM and JRJ analysed and interpreted the data presented in the manuscript. Statistical analysis was undertaken by DAC and TM. JRJ and DAC created the figures and tables. JRJ, GHJ, KDB and RGO reviewed SPM cases. JRJ drafted the first version of the manuscript. JRJ, DAC, TM, CP, FED, RS, AB, MWJ, MFK, CO, MR, MTD, RGO, KDB, GC, GJM and GHJ contributed to amendments and approval of the final version for submission.All requests for data not provided in the manuscript and supplementary material must be requested from the corresponding author in the first instance. Requests will be discussed with the trial management group and approval sought from the independent trial steering committee. Only requests that have a methodologically sound proposal will be considered. All shared data will be de-identified.DAC is a DSMB statistician for academically led investigator-initiated study in multiple myeloma; CP has received honoraria, undertaken consultancy and received research funding from BMS/Celgene; AB has received honoraria from BMS/Celgene, GSK, Sanofi, Amgen, Takeda and Janssen, received travel support from Amgen, Sanofi and BMS/Celgene, and undertaken consultancy for BMS/Celgene, Janssen, Takeda and Sanofi. MWJ has received honoraria from Pfizer, Janssen, BMS and Sanofi, and received meeting support from Janssen and Merinari Stemline; MFK has received research funding from BMS/Celgene and Janssen, honoraria from BMS/Celgene, Takeda and Abbvie and undertaken consultancy for Adaptive, Abbvie, BMS/Celgene, Pfizer, GSK, Karyopharm and Seattle Genetics; RGO has undertaken consultancy from Janssen and Beigene, received honoraria from Astra Zeneca, Janssen and Beigene, and received meeting support from Beigene; GHJ has received honoraria and research funding from BMS/Celgene. Celgene corporation, Merck Sharpe and Dohme and Amgen have provided unrestricted educational grants relating to this study, paid to the Clinical Trials Research Unit, University of Leeds.


Conclusion: MWA is effective and safe as a local treatment for recurrent HCC after initial surgical resection.Purpose: To evaluate the efficacy and safety of microwave ablation (MWA) as a treatment for recurrent hepatocellular carcinoma (HCC) after initial successful surgical resection. Methods: This retrospective study included 40 patients  with 48 recurrent lesions of HCC after initial surgical resection that were treated by percutaneous MWA. Several parameters including complications, technical success, local tumor progression (LTP), intrahepatic distant recurrence (IDR), overall survival (OS), and progression-free survival (PFS) were evaluated in order to investigate the safety and efficacy of MWA for these recurrent HCC lesions after surgical treatment. Results: All MWA treatments were performed without complications or procedure-related deaths. Technical success was achieved in all cases. Two cases developed LTP at a rate of 5%, and IDR occurred in 23 cases at a rate of 57.5% (23/40). The 1-, 2-, 3-, 4-, and 6-year OS rates were 97%, 89.2%, 80.3%, 70.2%, and 60.2%, respectively. The 1- and  Primary liver cancer, of which hepatocellular carcinoma (HCC) is the most common type, belongs to the most common newly diagnosed cancers and cancer-related deaths worldwide . There aAs we know, there was only one study in the literature by Zhang et al. , which iTherefore, this retrospective study focuses on evaluating the treatment of recurrent HCC after curative surgical resection by MWA, mainly according to complications, oncological outcome, and survival rates.This retrospective cohort study was approved by our institutional review board.In this single-center study, a total of 40 patients  with 48 recurrent HCC tumors were treated by percutaneous CT-guided MWA at our department for diagnostic and interventional radiology between 1 July 2013 and 1 April 2022, were enrolled. The inclusion criteria were (1) patients with recurrent HCC after curative R0 resection (2) patients with solitary recurrent tumor with a maximum axial diameter of <5 cm, (3) patients with a maximum number of 3 tumors each ≤3 cm, and (4) sufficient coagulation status. The exclusion criteria were (1) patients with R1 or R2 resection (2) patients who were treated by resection and ablation for the primary HCC before recurrence (3) patients with extrahepatic metastases, (4) patients with vascular invasion (5) decompensated liver function (Child Pugh class C) (6) patients with recurrent HCC, which were treated by TACE as a neoadjuvant treatment prior to MWA.Percutaneous MWA was performed under CT guidance  by a radiologist with many years of experience in local ablation. All MWA sessions were carried out under conscious sedation and in concordance with device protocol settings. The latest abdominal contrast-enhanced cross-sectional imaging of the patients was evaluated, and recent hematological and coagulation parameters were verified prior to ablation. Each patient underwent an unenhanced abdominal CT scan before MWA to determine the best access way for the MWA antenna. Local anesthesia was carried out afterwards. When the local anesthesia had taken effect, the microwave antenna was inserted into the recurrent tumor under sterile conditions. After verification of the correct position of the antenna, subsequently, thermal ablation was carefully started. Fluoroscopic scans were used to continuously check the MWA process. After finishing the ablation session, the microwave antenna was pulled out, while sealing the needle track.For assessment of tumor response, contrast-enhanced MRI  was performed by administration of intravenous contrast medium. We performed the first contrast-enhanced MRI 24 h post-ablation for evaluation of the ablation area. Patients underwent surveillance imaging every 3 months for the first year and every 6 months afterward to evaluate for tumor recurrence.Sex, age, etiology of liver disease, liver cirrhosis, Child Pugh class, surgical approach, time to recurrence after surgical resection, number and location of tumors, maximum axial diameters of tumor and ablation area, number of used antennas, ablation time, applied power, complications, technical success, LTP, IDR, follow-up time, overall survival (OS), and progression-free survival (PFS) were investigated in all patients’ cases. Complications were evaluated using the classification recommended by the Cardiovascular and Interventional Radiological Society of Europe (CIRSE) . Time top-value of <0.05 was considered statistically significant. Quantitative variables are shown as average ± standard deviation. Categorical variables are presented using frequencies and percentages.SPSS  was used for the statistical analysis. Kaplan–Meier method was used to calculate and analyze the survival and the log–rank test was used for the comparison. A The most common cause of liver disease was chronic viral hepatitis, followed by alcohol abuse at rates of 40% (16/40) and 22.5% (9/40), respectively. Liver cirrhosis was reported in 27 patients at a rate of 67.5%. Of these patients, 26 had a Child Pugh class A at a rate of 96.3%, and one had Child Pugh class B at a rate of 3.7%. Most surgical resections were performed by laparotomy at a rate of 77.5% (31/40). Laparoscopic surgical resection was carried out in 9 cases at a rate of 22%. The mean time to recurrence after surgical resection was 1.5 years. After curative surgical resection, a total of seven patients developed tumor recurrence within ≤6 months at a rate of 17.5%; 32.5% (13/40) of the patients had their tumor recurrence within 6 to ≤12 months. In 25% (10/40) of patients occurred, the recurrence within 12 to ≤24 months, and in another 25% (10/40) of the patients, after more than 24 months. Most of the included patients had solitary and small-sized (≤2 cm) recurrent tumors.The rates of patients who had 1, 2, or 3 recurrent tumors were 82.5% (33/40), 15% (6/40), and 2.5% (1/40), respectively. The distribution of tumor size was as follows; 75% (36/48) of the tumors were smaller than 2 cm, 4.2% (2/48) were 2 cm, and 20.8% (10/48) were larger than 2 cm. The most recurrent tumors were located in the right liver lobe at the rate of 56.25% (27/48). The remaining 21 of 48 tumors were located in the left liver lobe at a rate of 43.75%. The average diameter of the recurrent tumor was 1.8 ± 0.8 cm. The patients’ and tumors’ characteristics are summarized in A total of 48 MWA treatments were performed. Technical success was achieved in all MWA treatments 100% (48/48). Each recurrent tumor was only treated by one MWA antenna. The average ablation time was 9 ± 2.5 min. The average applied power was 86 ± 20.7 watts. The average diameter of the ablation area was 4.5 ± 0.9 cm. There were no treatment-related complications or treatment-related deaths reported. A total of 15 patients at a rate of 37.5% showed complete hepatic response after MWA without developing IDR or LTP. Hepatic recurrence, including IDR and LTP, was observed in 25 patients at a rate of 62.5%. The rates of LTP and IDR were 5% (2/40), and 57.5% (23/40), respectively. The patients who developed recurrence after MWA were mainly treated by TACE and local ablation after thoroughly discussing the case at the multidisciplinary tumor board. Results are summarized in The average follow-up time was 3.2 ± 2.5 years. The 1-, 2-, 3-, 4-, and 6-year OS rates were 97%, 89.2%, 80.3%, 70.2%, and 60.2%, respectively. The estimated median PFS time was 13.6 months. The 1-and 3-year PFS rates were 50.2% and 34.6%, respectively. Survival rates for all patients are summarized in p-value: 0.1) or PFS  between patients with or without liver cirrhosis. There was no statistically significant difference in OS  or PFS  between patients with solitary recurrent HCC and patients with multiple recurrent HCC lesions.There was no statistically significant difference in OS (Liver transplantation is an optimal curative treatment for HCC since it not only treats the tumor but also chronic liver disease ,21. LiveUnfortunately, there are no established guidelines or standards for the management of recurrent HCC. Surgical treatment can be conducted in only 20% of patients with HCC recurrence . PatientTACE, which has a wide range of indications for patients with HCC including neoadjuvant, bridging and palliative among others , is the When it comes to local thermal ablation, MWA has several advantages over RFA. Using MWA, higher temperatures can be achieved in tumor tissue, and it generates a larger ablation . This stLee et al.  analyzedAn advantage of local ablation over surgical therapy is the higher organ preservation and, thus, the better-preserved liver function posttheraputical . Feng etPeng et al.  showed tThis study was limited by some points and factors. As a single-center retrospective study, the patient population was not large, and selection bias could occur. In addition, this study did not include a control group and did not directly compare MWA with any other possible and available treatments for recurrent HCC, such as RFA, TACE, or repetitive surgical resection, for instance. Another limitation was that this study did not focus on the prognostic factor for the outcome and survival of HCC patients with recurrent HCC that were treated by MWA. A comparative prospective randomized study with a larger number of patients and the presence of a control group should be conducted in the future for better analysis and investigation of different treatments for recurrent HCC after initial surgical resection.In conclusion, we show that MWA is an effective, safe, and suitable treatment for recurrent HCC after curative surgical resection. It also represents a parenchyma-saving therapy for HCC, especially in previously surgically treated patients who already have reduced liver volume. MWA provided long OS and PFS without complications or long hospitalization stays and should be considered among the treatments for managing recurrent HCC after successful initial surgical resection.