Astrocytes are essential to virtually all brain processes, from ion homeostasis to neurovascular coupling to metabolism, and even play an active role in signaling and plasticity. Astrocytic dysfunction can be devastating to neighboring neurons made inherently vulnerable by their polarized, excitable membranes. Therefore, correcting astrocyte dysfunction is an attractive therapeutic target to enhance neuroprotection and recovery following acquired brain injury. However, the translation of such therapeutic strategies is hindered by a knowledge base dependent almost entirely on rodent data. To facilitate additional astrocytic research in the translatable pig model, we present a review of astrocyte findings from pig studies of health and disease. We hope that this review can serve as a road map for intrepid pig researchers interested in studying astrocyte biology. Neurotrauma and neurodegenerative diseases that impact these essential astrocytic functions can be devastating to the brain and the organism as a whole  or to synthesize essential molecules like glutamate, as well as providing antioxidant protection to neighboring neurons and performing the essential task of fixing NHews, see ,6,7,8). ews, see ,10,11. TThe overwhelming majority of our astrocyte knowledge stems from research in rodents due to their low cost and ease-of-use. However, recent genetic brain atlas comparisons between mice, pigs, and humans revealed greater variability between humans and mice relative to humans and pigs , and theBeyond direct brain injury, we will also discuss studies of astrocytes in pigs that have experienced indirect brain injuries through mechanisms such as cardiac arrest, sepsis, and others. While we will have inevitably overlooked some research works, we believe that this neurotrauma-forward review provides a near-comprehensive assessment of studies that have assessed astrocytes in pigs. Such a review would be impossible in rodent literature, further emphasizing the relative lack of, and need for, astrocyte research in pigs.To date, there have been several studies that have investigated the structure and function of astrocytes in the pig brain. Porcine astrocytes have been investigated in vivo, in slice culture, histologically, and using cell culture techniques. Here, we review the details of these studies to highlight what is known about porcine astrocytes. We only discuss elements of these publications that are relevant to astrocytes in the porcine brain. Furthermore, as it can be difficult to find histological antibodies for pig specimens, we summarize all available antibody information from the reviewed papers in The astrocytic sodium-dependent glutamate transporters GLAST (GLutamate ASpartate Transporter) and GLT-1 (GLutamate Transporter-1) are responsible for clearing glutamate from the extracellular space. Glutamate is the primary excitatory neurotransmitter in the mammalian brain and also, paradoxically, a potent neurotoxin. Therefore, one of the most obvious and important tasks astrocytic glutamate transporters perform is the constant clearance of glutamate from the extracellular space to prevent excitotoxic neuronal death. However, astrocytic glutamate transporters and associated calcium signals appear integral to many brain functions. Glutamate uptake into fine astrocytic processes results in local reversal of the sodium/calcium exchanger that positions astrocytic mitochondria near GLT-1 clusters that are servicing active synapses . Aside fThe Pow lab published a detailed article describing the expression patterns of the glutamate transporters GLAST, GLT-1alpha, and GLT-1v in the Large White/Landrace porcine brain . They coThe Pow lab followed up on this with a publication describing the associated expression of the glutamate transporter GLAST and the astrocyte intermediate filament protein GFAP in the 1-day-old piglet brain . In thisThis group also demonstrated that the GLAST1c splice variant is expressed in the piglet brain . RT-PCR Throughout this review, it will become evident that histological staining of GFAP is by far the most common technique used to study astrocytes, primarily via morphological analyses or simply by measuring stain intensity. This section describes studies in pigs that focused on GFAP itself.Blechinberg and colleagues published a report detailing the expression of the GFAP isoforms GFAPα, GFAPε, and GFAPκ in the adult and developing porcine brain . They ut2α is the predominant prostaglandin produced by porcine astrocytes [1α, PGE2, and LTC4/D4. Application of phorbol 12,13-dibutyrate (PDB), a protein kinase C (PKC) activator, onto astrocyte cultures led to increased levels of PGF2α. PDB application did not increase levels of 6-keto-PGF1α, PGE2, or LTC4/D4. This PDB-induced increase in PGF2α was prevented when cultures were treated with the drugs indomethacin, quinacrine (phospholipase A2/PLA2 inhibitor), or isoquinolinylsulfonylmethyl piperazine (PKC inhibitor) at the same time as PDB. Application of 4α-phorbol 12,13-didecanoate (PDD), which does not activate PKC, did not lead to increased levels of PGF2α in porcine astrocyte cultures. These results indicate that the major prostaglandin produced by porcine cerebral astrocytes is PGF2α and that PKC activation increases the production of PGF2α through a mechanism that may involve PLA2. In a separate study, they also demonstrated that the administration of interleukin 1α (IL-1α) to cultured porcine astrocytes rapidly increases their production of PGF2α [2α was increased two-fold when 11 μg/mL of IL-1α was added and four-fold when 22 μg/mL of IL-1α to astrocyte media. To our knowledge, equivalent dosing in humans has not been determined. Levels of 6-keto-PGF1α and PGE2 did not change with IL-1α addition. These results confirm the results of their previous study indicating that PGF2α is the major prostaglandin produced by porcine astrocytes.The Busija lab published two reports detailing prostaglandin synthesis in cultured porcine cerebral astrocytes ,32. The trocytes . These cThe Zimmer lab conducted a study to investigate the cultured porcine astrocyte expression of major histocompatibility complex (MHC) antigens and their ability to induce the proliferation of human T-lymphocytes . They reIonescu and colleagues reported that astrocytes harvested from the adult porcine cortex express the markers GFAP, S100β, CD14, and interferon-γ receptor 2 (IFN-γ-R2) . This grThe Parfenova group published a detailed report investigating the antioxidant and cytoprotective effects of sulforaphane (SFN) in porcine cortical astrocyte cultures . They deBarnes et al. investigated the presence of the cell surface peptidases aminopeptidase N (AP-N) and dipeptidyl peptidase IV (DPP-IV) in porcine striatal astrocytes . They reCa channel activation in myocytes. Additionally, treatment of porcine cortical brain slices with the selective astrocyte toxin L-2-α-aminoadipic acid (L-AAA) blocked glutamate-induced dilation of arterioles. These results provide evidence that glutamate-induced dilation of monocyte arterioles is dependent on astrocyte-derived HO.Leffler and colleagues investigated the role of astrocytes in the dilation of porcine cerebral arteriole myocytes . In porc2+ signaling in arteriolar smooth muscle cells in live brain slices from the porcine cortex [2+ spark activation and reduction in intracellular Ca2+ concentration in arteriolar smooth muscle cells were both prevented. L-AAA treatment also increased glutamate-stimulated Ca2+ wave frequency in smooth muscle cells. This experiment demonstrates that astrocytes modulate glutamate-induced Ca2+ sparks, Ca2+ waves, and global intracellular Ca2+ levels in smooth muscle cells.Authors from this same group then investigated the role of astrocytes in the mediation of Cae cortex . Followi2+ ionophore that held free intracellular calcium concentration constant) to astrocyte cultures each increased astrocyte CO production in a concentration-dependent manner. Administration of glutamate also increased intracellular calcium concentration in astrocyte cultures in a concentration-dependent manner. When astrocyte cultures were treated with the endoplasmic reticulum Ca2+-ATPase blocker thapsigargin, which depleted intracellular Ca2+ stores, glutamate-induced Ca2+ signaling was blocked, and steady-state intracellular calcium levels were elevated, suggesting that glutamate stimulates Ca2+ release from the ER which in turn leads to intracellular calcium concentration. Glutamate did not increase astrocyte CO production when Ca2+ levels were held constant with ionomycin, suggesting that glutamate stimulates astrocyte CO production via Ca2+. HO was inhibited by CrMP, which blocked glutamate-induced CO production in cultured astrocytes. Additionally, treatment of astrocytes with thapsigargin reduced basal CO levels and blocked glutamate-stimulated CO increase in astrocytes. Treatment of cultures with the calmodulin blocker calmidazolium also blocked glutamate-stimulated CO increase in astrocytes. Overall, these results suggest a mechanism by which glutamate causes the ER to release Ca2+, which leads to elevated intracellular Ca2+ levels, in turn leading to Ca2+-calmodulin-dependent HO activation and CO production in porcine cortical astrocytes. Astrocytic CO production then dilates cerebral arterioles.This group then investigated the mechanism of glutamate-induced carbon monoxide (CO) production in cultured porcine astrocytes . They deThis group also utilized Western blotting and immunohistochemistry to demonstrate that newborn porcine cortical astrocytes express the hydrogen sulfide-producing enzyme cystathionine β-synthase, which is important for cysteine synthesis .Several different groups have published protocols outlining the isolation of cerebral astrocytes from the adult porcine brain ,41,42. TThe Bobilya group and others have demonstrated that astrocytes can be cultured together with porcine brain capillary endothelial cells to create an in vitro blood–brain barrier model ,43,44. IThe Osterberg lab published a detailed report about the structure of the adult porcine olfactory bulb (OB) . Using GThe Eppenberger group utilized GFAP immunohistochemistry and transmission electron microscopy to examine the astrocyte morphology in the porcine retina . They deNoda and colleagues described the expression of myocilin, the product of the gene MYOC/TIGR that is responsible for the pathogenesis of primary open-angle glaucoma in astrocytes of the optic nerve head . UtiliziRipodas and colleagues reported that GFAP+ astrocytes in the inner layers of the porcine retina (nerve cell layer and ganglion cell layer) extend processes that make contact with blood vessels . These aLee and colleagues reported the presence of heat shock protein 27 (HSP27) in the porcine retina . In the Carreras and colleagues used immunohistochemistry and transmission electron microscopy to study the astrocyte cell–cell adhesions in the prelaminar region of the optic nerve head (ONH) . They reCarreras and colleagues used GFAP immunohistochemistry, transmission electron microscopy, and perfusion with a fluorescent tracer to examine the pathways of fluid exchange between astrocytes within the prelaminar tissue of the porcine ONH . They deCarreras and colleagues also published a report detailing the expression of glucose transporters in the ONH . They deBalaratnasingam and colleagues detailed astrocyte distribution in the lamina cribrosa, pre-laminar region, and post-laminar region of the porcine ONH ,56,57,58Kimball and colleagues reported on the co-expression of GFAP and the astrocyte aquaporin (AQP) channels AQP1, AQP4, and AQP9 in the ONH, retina, and myelinated optic nerve (MON) of the porcine eye . They foEderra and colleagues reported on the expression of the high-affinity nerve growth factor (NGF) receptor tyrosine kinase A (TrkA) in the porcine retina . They peVarious research groups have conducted examinations of astrocyte response to injury in a range of porcine models. While undoubtedly not exhaustive, our literature review is extensive, and we summarize the number of studies in each porcine injury model that investigated astrocytes to provide a glimpse at where the focus has been thus far . These sSeveral research groups have investigated astrocyte response to injury in the porcine brain using models of hypoxia (oxygen availability deficiency) and/or ischemia (blood supply restriction). The Traystman lab utilized a piglet model of asphyxic cardiac arrest to examine striatal astrocyte response at 24-, 48-, and 96 h following hypoxic–ischemic (H-I) injury . They reLee and colleagues examined whether H-I injury, with and without therapeutic hypothermia and rewarming, affected the expression of endoplasmic reticulum to nucleus signaling-1 protein (ERN1) expression in astrocytes . ERN1 isLee and colleagues also examined changes in astrocyte morphology and the correlation of these changes to fractional anisotropy (FA) from diffusion tensor imaging (DTI) following H-I injury and excitotoxic injury caused by striatal quinolinic acid (QA) injection . They exThe Pow lab described the altered expression of GFAP and GLAST following hypoxia in the 1-day-old piglet brain . They reThe Pow lab also examined the expression of the exon-9 skipping form of the glutamate-aspartate transporter EAAT1 (GLAST1b) following hypoxic injury to piglets . They reThis same group then published a detailed article describing astrocyte structural changes following H-I injury in the newborn piglet brain . By injeThis group then published another report detailing changes in the presence of glutamine synthetase (GS), the glutamate detoxification enzyme that converts ammonia and glutamate into glutamine, in a newborn porcine model of H-I injury . They reThis group also investigated the presence of two phosphorylated GFAP proteins, p8GFAP and p13GFAP, following H-I injury in newborn piglets . They reRuzafa and colleagues exposed newborn pigs to hypoxia and then examined astrocyte changes in the retina and superior colliculus at 4 h following injury . They reZheng and Wang investigated changes in lactate and glucose metabolism in the basal ganglia between 2 and 72 h following hypoxic–ischemic injury in piglets . In thisParfenova and colleagues investigated the role of astrocyte-produced carbon monoxide in both in vitro porcine astrocyte cultures and an in vivo model of neonatal asphyxia in the piglet brain . They reSpellicy and colleagues recently published an article detailing their use of high-content image (HCI) analysis to examine changes in astrocyte morphology following middle cerebral artery occlusion (MCAO) in the adult Yucatan porcine brain . Four weBaker and colleagues developed a graded cortical control impact (CCI) brain injury model in 3-week-old piglets . They deLafrenaye and colleagues performed central fluid percussion injury (cFPI) in Yucatan mini pigs and collected blood samples at various time points up to 6 h post-injury . They foCurrently, the only preclinical model that recreates the mechanisms and manifestations of human TBI relies on head rotational acceleration injury in pigs . The corTo our knowledge, just three previous reports on the effects of explosive blast injury in the porcine brain have included an analysis of astrocyte response to this injury. In 2011, explosive blast injury was performed on Yorkshire swine to simulate a blast experienced in an open field (blast tube), tactile vehicle, or a building . SeventyThe Rosenthal group performed water intoxication in female swine as a mechanism to investigate cerebral edema leading to elevated intracranial pressure . They toZhou and colleagues induced intracerebral hemorrhage in adult male pigs by injecting blood into the frontal lobe to study the expression of the integrin-associated protein cluster of differentiation 47 (CD47) following injury . CD47 plVogt and colleagues studied the effects of multiple trauma associated with hemorrhagic shock (MT/HS) in male pigs . They peLeffler and colleagues performed a set of experiments examining the effects of glial toxin L-AAA on pig astrocytes in culture and in vivo . They reThe Parfenova lab has also published two research articles which examine the effects of cerebral astrocyte response to neonatal seizure caused by bicuculline administration ,95. BothLiberski and colleagues experimentally infected porcine with bovine spongiform encephalopathy (BSE) and performed transmission electron microscopy imaging to examine the ultrastructural pathology caused by this neurodegenerative disorder . They reThe Reynolds research group investigated brain injury caused by lung-protective mechanical ventilation in a porcine model . FollowiSharma et al. examined changes in GFAP expression and astrocyte morphology via transmission electron microscopy following 12 min of untreated cardiac arrest (ventricular fibrillation) in piglets . They reIn 1989, Laursen and colleagues examined GFAP expression following total cardiopulmonary bypass (CPB) for two hours in pigs . They diAround 30 years later, Stinnett and colleagues examined changes in fractional anisotropy (FA) obtained from diffusion tensor imaging and the correlation of these changes to changes in GS+GFAP+ astrocyte numbers following mild and severe CPB in piglets . They rePapadopolous and colleagues utilized electron microscopy to explore astrocyte morphology changes in the cortex following sepsis induced by fecal peritonitis in adolescent pigs . They reDiemer and Tonnesen investigated glial changes following portocaval anastomosis (PCA) and either total or partial hepatic artery clamping/devascularization . UtiliziKristiansen and colleagues induced acute liver failure in pigs by placing an end-to-end portocaval shunt and ligating the hepatic arteries and analyzed the frontal lobe, pons, and cerebellum with electron microscopy imaging 8 h later . They reKanai and colleagues induced acute hepatic failure in male mini-pigs by administration of 0.05 mg/kg alpha-amanitin and 1 ug/kg lipopolysaccharide (LPS) in the splenic vein and investigated the effect of this injury on blood serum S100B levels with ELISA several hours after injury . They reSenna occidentalis  seeds [S. occidentalis may precede glial cell death resulting from poisoning.The Cholich group induced hepatic encephalopathy by poisoning pigs with 5–10% s) seeds . Brain hFinnie and colleagues published a case report on the presence of AIIA in the brains of pigs that were subject to salt poisoning resulting from at least 2 days of water deprivation . UtiliziTaenia solium (T. solium) [T. solium. This astrogliosis resulting from infection was more pronounced around cysticeri  found in the cortex compared to the medulla. They also noted that some lesions were surrounded by astrocyte endfeet forming a glial scar, while others were surrounded by blood vessels that lacked astrocyte endfeet (indicative of BBB loss). Overall, astrogliosis is prominent after T. solium neurocysticercosis.The Leifsson group examined changes in the astrocyte phenotype following the natural infection of pigs with the parasite  solium) . They deAeschynomene indica (A. indica) seeds in adult pigs [A. indica ingestion.Riet-Correra et al. examined the effects of ingesting 13% ult pigs . A. indiIntrauterine growth restriction (IUGR), caused by placental insufficiency, leads to fetal development in a chronic hypoxic environment that, in turn, leads to neurological disabilities. Wixey and colleagues published a report detailing the effects of IUGR on astrocyte density and morphology in postnatal-day 1 (p1) and 4 (p4) piglets . They rePrenatal maternal infections can lead to fetal brain abnormalities. Antonson and colleagues investigated astrocyte changes in the Large White/Landrace porcine fetal hippocampus following maternal infection with porcine reproductive and respiratory syndrome virus (PRRSV) . PregnanRecently the Pankratova Lab published a study investigating the exposure of porcine fetuses to intra-amniotic injection of 1 mg liposaccharide (LPS), which can cause intrauterine inflammation at embryonic gestation day 103 (E103) prior to cesarean section delivery at E106 . They anBrunse and colleagues investigated the effect of preterm birth, often associated with impaired neurodevelopment and necrotizing enterocolitis, on perivascular astrocyte coverage in the hippocampus and striatum of the porcine brain at 8 h and 5 days following birth . Followi2 and 5% CO2 for 72 h) caused significantly decreased myocilin expression and no change in optineurin expression. Exposing porcine optic nerve head astrocytes to dexamethasone (500 nM added to culture media for 2 weeks) caused significantly decreased optineurin expression and significantly increased myocilin expression. The differing responses in expression levels of these two genes under different stressors indicate that myocilin and optineurin induce glaucoma via different mechanisms.In an earlier section, we described a publication that reported on the amino acid sequences of the glaucoma-associated genes myocilin and optineurin . In thisBalaratnasingam and colleagues examined astrocyte damage in the porcine optic nerve head following an acute increase in intraocular pressure (IOP) for 3, 6, 9, or 12 h . There wThis group also reported astrocyte changes following argon laser-induced axotomy of the porcine retinal ganglion cell axon . A reducHistorically, studies that assess astrocytes in the context of injury or disease suffer from an overreliance on GFAP and simplistic “reactive” scoring, and many of the above pig studies suffer from this oversimplification. Astrocyte reactivity is actually quite heterogeneous, and a great deal can be learned from a closer examination of expression patterns, morphological changes, and other properties ,119. How


Is Agent Orange (AO) exposure during the Vietnam War (1966 to 1975) associated with incident hepatocellular carcinoma (HCC)?This cohort study of 296 505 eligible veterans, no association between AO and incident HCC was observed. However, among veterans, viral hepatitis, nonalcoholic fatty liver disease, and alcohol and tobacco use were the most important clinical risk factors for HCC, which were further modified by cirrhosis status.These findings suggest that AO exposure is not associated with incident HCC. This cohort study assessed the association of exposure to Agent Orange and hepatocellular carcinoma among Vietnam veterans. Hepatocellular carcinoma (HCC) and its mortality are on the rise. Viral hepatitis and alcohol are leading risk factors; however, other risk factors among veterans are less defined, including Agent Orange (AO), an herbicide linked to several cancers.To assess the association of AO exposure and HCC in a national cohort of Vietnam veterans.International Classification of Diseases, Ninth Revision and International Statistical Classification of Diseases and Related Health Problems, Tenth Revision scores or calculated Fibrosis-4 scores. Data were collected from January 1, 2019, to December 31, 2020, and analyzed from December 2020 to October 2023.This retrospective cohort study included Vietnam veterans who served between 1966 and 1975, were male, were older than 18 years at the time of deployment, and had established follow-up in the Veterans Affairs (VA) between 2000 and 2019. Veterans with AO exposure were identified in the disability data via validated clinical surveys. Relevant clinical risk factors for cirrhosis and HCC were collected. Patients were stratified based on cirrhosis status, as defined by consecutive diagnosis found by documented Incident HCC was the primary outcome. AO and HCC association was estimated using a multivariable Cox regression analysis, with death and liver transplant as competing events.P <.001) or Black individuals , and having a diagnosis of viral hepatitis , alcohol-associated liver disease , and nonalcoholic fatty liver disease (NAFLD)  were associated with HCC. Among veterans without cirrhosis, hypertension  and diabetes  were also associated with HCC. Early smoking and alcohol use were significant risk factors for HCC.Of the 296 505 eligible veterans , 170 090 (57%) had AO exposure  and 35 877 (12.1%) had cirrhosis. Veterans who were not exposed to AO were more likely to smoke (109 689 of 126 413 [86.8%] vs 146 061 of 170 090 [85.9%]); use alcohol (54 147 of 126 413 [42.8%] vs 71 951 of 170 090 [42.3%]) and have viral hepatitis (47 722 of 126 413 [37.8%] vs 58 942 of 170 090 [34.7%]). In a multivariable competing risk model, AO exposure was not associated with HCC. Among veterans with cirrhosis, self-identification as Hispanic individuals (aHR, 1.51; 95% CI, 1.30-1.75; In this large nationwide cohort study of Vietnam veterans, AO exposure was not associated with HCC. Smoking, alcohol, viral hepatitis, and NAFLD were the most important clinical risk factors for HCC. For instance, as data emerged demonstrating the efficacy of mitigating the risk of liver complications with direct acting antiviral therapies for hepatitis C virus (HCV),3 stakeholders including the US Centers for Disease Control and Prevention and the US Preventative Services Task Force recommended universal HCV screening for adults.4 Similarly, as the metabolic syndrome and its hepatic manifestation of nonalcoholic fatty liver disease (NAFLD) were recognized an independent risk factors for HCC, society guidelines including recommended screening for NAFLD in patients with diabetes.5Hepatocellular carcinoma (HCC) incidence is increasing globally with a relative 5-year survival of only 18%, with cirrhosis being the leading risk factor.6 More recently, the national Hepatic Innovation Team Collaborative shifted their focus on improving the care of veterans living with cirrhosis by using automated and integrated clinical systems.8 Similarly, demonstrating how environmental exposures during combat affect health has also been emphasized within the VHA and is linked to a veterans’ medical service connection and ability to access health care.9 This is reflected in current government policies, including the 2022 Sergeant First Class Health Robinson Honoring Our Promise to Address Comprehensive Toxics (PACT) Act,10 signed into law by the Biden administration. The PACT Act is the largest health care benefit expansion for veteran exposed to toxins. This includes recognizing Agent Orange (AO)’s association with many malignant neoplasms.12The Veterans Healthcare Administration (VHA) is the largest integrated health care system in the United States serving 9 million veterans yearly. The VHA has placed a large emphasis on population health and implementing quality improvement measures to expedite the delivery of care to veterans. For instance, HCV screening and treatment were implemented in the VHA in a more comprehensive manner than any other health care system in the US.13 Most herbicides contained 2,4,5-trichlorophenoxyacetic acid , which was contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), including in AO.13 It was recognized as early as 1970 that 2,4,5-T was teratogenic and was banned from US domestic use.14 An estimated 366 kg of TCDD was sprayed in South Vietnam,15 with large and persistent clinical ramifications to this day. Over the years, AO has been studied extensively given its association with several cancers, including bladder, Hodgkin and non-Hodgkin lymphoma, prostate, and lung and soft tissue sarcomas, leading to its classification in 1997 as a carcinogen by the International Agency for Research on Cancer.16 Its effects on HCC remain controversial. While animal models link TCDD to HCC,17 epidemiological studies in veterans have not been conclusive. These studies have been hindered by small or restricted sample sizes, short-term follow up, and a lack of consideration of other key clinical confounders for HCC, such as HCV and alcohol use, which are prevalent in veterans.22 Type 2 diabetes has also been linked to AO exposure and is recognized by the VHA as an AO-associated condition, which is also an independent risk factor for HCC.23 Given the new government incentives to expand eligibility of veterans and the lack of strong data linking AO to HCC, large, statistically rigorous studies addressing the limitations of previous work are needed to better evaluate if AO as a clinically relevant risk for HCC. In the current study, we investigate the association between AO and incident HCC using the largest cohort to-date of Vietnam veterans with a long-follow up in the VHA.During the Vietnam war, the US military sprayed herbicides to defoliate forests to clear land of military occupation and control enemy food supplies.STROBE) reporting guideline.24 This study was approved by the institutional review board of the Veterans Affairs Greater Los Angeles Healthcare System (GLAHCS) with a waiver of informed consent because of the retrospective design.This cohort study was conducted according to the Strengthening the Reporting of Observational Studies in Epidemiology (The US VHA is comprised of 168 Veteran Affairs (VA) Medical Centers across the US and its territories and is the largest integrated, nationwide health care system in the US. The VHA contains an integrated network of electronic health data that can be accessed by approved researchers. For this study, we used the VA Informatics and Computer Infrastructure (VINCI) platform to extract demographics, clinical laboratory tests, medical comorbidities, alcohol use questionnaires and clinical outcomes data from the Corporate Data Warehouse (CDW), which is the national data repository for VA electronic health records.To capture veterans who primarily obtained their care within the VHA, we selected veterans who had at least 2 outpatient visits that were separated by at least 6 months within a window of 3 years. From this group, we identified Vietnam veterans using their combat service location with the combat start date between the years January 1, 1966, and December 31, 1975. We excluded women  and those who were younger than age 18 years at the time of deployment. Veterans were then followed up with from 2000 to 2019. The year 2000 was selected as the starting window because October 1999 was the earliest time when CDW data became available  having a health condition associated with AO per VHA (updated over time) and (2) having served in a location with exposure to AO. If a veteran believes they have a condition associated with AO but is not currently recognized by the VHA, the veteran must show evidence that the medical condition started during military service or scientific evidence  that show the condition is associated with AO. Additionally, we confirmed these methods to identify veterans who were AO exposed with VINCI Services. We also conducted a manual record review of a random set of 19 patients categorized as AO exposed who were seen at the local VA and found that 95% were correctly identified to have been exposed to AO during the Vietnam War.Veterans who were flagged as having AO exposure during the Vietnam War were categorized as AO exposed, while veterans deployed to Vietnam without exposure to AO were categorized as AO nonexposed. AO exposure was defined as any veteran who was flagged in the disability data under 27 All medical comorbidities were identified the first time they occurred in the veteran’s electronic health record. Diagnoses were identified using previously validated International Classification of Diseases, Ninth Revision (ICD-9) and International Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10) codes.26 HIV and hepatitis B virus (HBV)28 or HCV29 were identified using laboratory data   or smoker .Relevant clinical covariates and baseline characteristics were selected a priori based on their association with HCC. Age at deployment, sex, race and ethnicity  and medical comorbidities were extracted  validated ICD-9 and ICD-10 for decompensated cirrhosis 32; or (3) having a Fibrosis-4 (FIB-4) score of more than 3.25 for at least 2 consecutive years  level and alanine aminotransferase (ALT) value:A diagnosis of cirrhosis was defined using: (1) validated 34 We next evaluated the number of patients with the validated ICD-9 and ICD-10 for cirrhosis and decompensated cirrhosis and an elevated AST to Platelet Ratio Index (APRI) score35 (excludes age):All laboratory data in the FIB-4 calculations were within the last 3 months for each year. For each diagnosis, the earliest date was used to define the occurrence. While FIB-4 is a commonly used noninvasive tests clinically to assess the risk of cirrhosis, it can overestimate it given the large component of age, which can be a confounder given the aging population of Vietnam veterans.36 Second, we used ICD-9 and ICD-1032 codes if they occurred twice in the patient’s outpatient medical records, which has been well validated with positive estimated values of 84% to 94%.33 To assess the accuracy of the VACCR, we identified 40 cases and confirmed the diagnosis by conducting a manual record abstraction at GLAHCS. Of the 40 patients selected in the VACCR at GLAHCS, 90% were correctly identified, as confirmed by manual record abstraction of hepatology, gastroenterology, or oncology clinical notes. Using the ICD-9 and ICD-10 codes, we identified 3469 veterans with HCC. Of those, we explored 31 cases, and HCC was correctly identified in 74% of the cases. We found that most incorrectly identified cases were secondary malignant neoplasms to the liver from other primary cancers, mostly of gastrointestinal origin (67% of the cases). To enrich cases with HCC using the ICD-9 and ICD-10 algorithm, we identified all gastrointestinal cancer malignant neoplasms using ICD-9 and ICD-10 , which constitutes a national repository of all cancer cases within the VHA since 1995. In the VACCR, each case is validated by a manual record abstraction using the North American Association for Central Cancer Registries standards with more than 90% accuracy.37 which provides reliable and quality-controlled data for death in veterans and a sensitivity of more than 90%.38 Next, we cross-referenced the death date from the VACCR, CDW, and MDR. We defined death outcomes as in MDR death file, in CDW death , or in VACCR death data. All death dates were between January 1, 2000, and December 31, 2019, with the latter date used as a censor date. Liver transplantation (LT) was identified using validated ICD-9 and ICD-10 codes ,Patient baseline characteristics are presented as the number and percentage of patients or mean (SD). Covariates were selected based on known clinical associations with HCC. HBV and HCV were combined into 1 covariate . NASH and NAFLD diagnoses were also combined into 1 covariate. Diabetes, hypertension, and HBV and HCV were used as time-dependent covariates. Given the link between diabetes and obesity, we assessed the interaction between the 2 diagnoses. Interactions were also assessed between AO and viral hepatitis, age, alcohol, NAFLD or NASH, smoking, and diabetes.We conducted a competing risk analysis to assess the risk of incident HCC as a primary outcome with death and LT as competing events. Type-specific adjusted hazard ratios (aHRs) are reported for both univariate and multivariate models. Covariate selection was determined a priori and included age (at deployment); race and ethnicity; smoking; portal hypertension; and etiologies of cirrhosis, including NAFLD or NASH, viral hepatitis, alcohol associated liver disease, hemochromatosis, alpha-1-antitrypsin, secondary biliary or unspecified biliary cirrhosis, and autoimmune hepatitis. Given that cirrhosis is the most important risk factor for HCC and that cirrhosis could not be ascertained until after CDW were available, which was approximately in the year 2000, the results were stratified into cirrhosis and noncirrhosis status for each veteran prior to any statistical analyses. Furthermore, to adjust for the impact of cirrhosis on HCC, we conducted inverse probability weighting (IPW) model.P <.05, and tests were 2-sided. Data were collected from January 1, 2019, to December 31, 2020, and analyzed from December 2020 to October 2023.All statistical analyses were conducted using SAS version 9.4 (SAS Institute). Statistical significance was set at P <.001) nor alcohol use  among veterans based on AO exposure (P <.001). Most veterans who were exposed to AO had been deployed for 1 year or less , 91 014 veterans  had been deployed for 1 to 2 years, 13 510 veterans  had been deployed 2 to 3 years, 5031 veterans  had been deployed 3 to 4 years, and 3428 veterans  had been deployed 4 or more years. Mean (SD) deployment time was 1.11 (0.66) years.We identified 296 505 male veterans during the Vietnam War who were 18 years or older at the time of deployment . Of these veterans, 170 090 (57%) had AO exposure  . ALD wasexposure . CirrhosP = .98) and without cirrhosis . The lack of association between AO and incident HCC based on cirrhosis status was also substantiated using the APRI score, which is another surrogate marker for advanced fibrosis and cirrhosis (eTable 4 in At a mean (SD) of follow-up time is 51.1 (1.9) years , we found 2654 veterans who developed HCC in our final cohort (1463 [0.9%] in the AO exposed group and 1191 [0.9%] in the AO nonexposed group). HCC incidence rate was 0.00018 per year (0.00018 per year and 0.00017 per year). In our multivariable model adjusting for other confounders of HCC, we found that AO was not associated with HCC both in veterans with cirrhosis  HCC as an event with death as the only competing risk, and (2) HCC as an event and death was the censored event if occurred before HCC (instead of censored at last follow up in 2019) and transplantation as the competing risk. After adjusting for all the HCC risk factors, neither analysis found an association between AO and HCC risk. Given the inverse association between age and incident HCC, we explored the association between AO and age further. There were no significant interactions between age and AO exposure which was further confirmed by assessing their association after stratification of age at deployment (eMethods in There was no difference in LT occurrence based on AO exposure status (366 of 170 090 [0.22%] vs 296 of 126 413 [0.23%]). Overall mortality was lower in the AO exposed (12 589 of 23 706 [7.4%]) vs nonexposed group (11 117 of 23 706 [8.8%]).P <.001) or Black individuals  had a higher risk for HCC compared with non-Hispanic White individuals.We found that self-reported race and ethnicity were associated with incident HCC . VeteranP <.001), NAFLD or NASH , and ALD . Dyslipidemia was inversely associated with the risk of HCC  in this cohort. Diabetes  and hypertension  were only significant risk factors in the noncirrhosis group, even after adjusting for obesity and assessing the interactions between obesity and type 2 diabetes.In the subgroup of patients with cirrhosis (35 877 [12.1%]), the most important risk factors for HCC included viral hepatitis . To explore this further, we assessed the association between smoking and alcohol with age for veterans with and without cirrhosis and stratified age by decades. For both alcohol and smoking, we found that early exposure during ages 20 to 30 years were significantly associated with incident HCC, independent of cirrhosis status (eTables 6 to 8 in P = .047), this was not reproduced using the age-independent APRI score  should be prioritized and modified to mitigate the rise in HCC among veterans.HCC is a complex disease that results from many clinical and environmental risk factors that can change over time.44 The HSE has been attributed to early physical training and health care access and has been shown to occur as far out as 40 years from war.45To appropriately assess the risk of AO exposure and HCC development, we defined our control group as veterans who were deployed to Vietnam but were not flagged in the VHA as having been exposed to AO. This study design allows to control for other nonmeasurable factors, such as other potential geographical, diet, and environmental-related exposures that are not captured by the electronic health records. Using a non-Vietnam War group as a control may not have appropriately isolated these differences and could have potentially introduced bias. This is especially relevant given the consideration of the healthy soldier effect (HSE) that has previously been described, where veterans who are deployed are less likely to develop chronic diseases, including during the Vietnam War.51 However, few studies have addressed this in the VHA. Our findings support that even within the VHA, where patients have access to health care, racial and ethnic disparities persist. Small studies have proposed that mental health and substance use may drive these disparities; however, larger studies are needed to understand these observations.We found that self-reported race and ethnicity were significantly associated with incident HCC. Specifically, patients who self-identified as Hispanic or non-Hispanic Black were more likely to develop HCC, compared with White individuals, even after adjusting for key clinical confounders. These findings suggest that other nonmeasured variables could contribute to these disparities. Several studies have described that Black individuals are at higher risk for HCC-related death, independently of tumor stage.52 it remains limited resource due to the scarcity of organs and a complex evaluation process that affects candidates every level of the evaluation, including referral, eligibility and listing, especially in veterans who have been shown to disproportionally receive fewer LTs.53 Therefore, HCC risk modification of clinical factors is even more imperative in veterans to prevent HCC.We found that a minority of veterans underwent LT. While LT is a life-saving measure for patients with advanced liver disease,54 to occur and thus may not be captured in shorter studies. However, given the limitation of data availability within the VHA only starting in the years 1999 and 2000, events prior these dates would not have been available for our review. Therefore, our cohort may be biased to a potentially healthier veteran population. Although sex differences have been described in HCC, this could not be addressed in our cohort given that less than 1% of all Vietnam veterans were female. Rare etiologies of liver disease, such as Wilson disease (84 veterans), could not be robustly evaluated in our study. Finally, since our interest was to capture veterans who primarily received their care within the VHA, we could not capture events that occurred outside of the VHA, including for those associated with liver transplantation.While we present the largest cohort of US Vietnam veterans with AO exposure with long-term follow, our study has notable limitations. Due to the retrospective nature of the study, residual confounders between exposure and outcomes may be present. While we used robust methods to identify AO exposure, veterans who may not claim an AO-associated disability may not have been captured accordingly. Others have used similar methods to ours to identify AO exposed cases; however, we recognize that since AO cannot be directly measured, there is a potential for misclassification of cases. Using the age at deployment allowed us to determine if there was a potential dose-response given the length of exposure; however, this was not identified. The long follow-up period of our cohort allowed us to identify HCC cases, which can take 20 to 40 yearsOur results suggest that AO was not a significant risk factor for HCC development and does not appear to modify the association between other known risk factors, such as diabetes, on HCC risk. We show that HCC risk is dynamic based on whether or not there is a background of cirrhosis and that risk modification should be tailored to the veteran’s individual risk.


China has low human papillomavirus(HPV) vaccination rate due to lack of public funding and mistrust in domestic vaccines. This pilot study evaluated the feasibility and preliminary effectiveness of an innovative pay-it-forward strategy, which has a participant receive a subsidized vaccine and offers her an opportunity to donate to support other girls, in improving HPV vaccine uptake among 15–18-year-old adolescent girls.A two-arm randomized controlled pilot trial was performed in one vaccination clinic in Western China. Adolescent girls (via caregivers) were invited to participate the study via online dissemination of the pilot study. Eligible ones were then randomly allocated using a sealed envelope method into standard-of-care or pay-it-forward arm in a 1:1 ratio. Pay-it-forward participants received hand-written postcard messages, a subsidized vaccine, and an opportunity to donate and (or) write postcards for future recipient girls. Standard-of-care participants self-paid for vaccines. The primary outcome was the first-dose HPV vaccine uptake, which was calculated using multivariable logistic regression, presented as crude/adjusted odds ratios (cORs/aORs) and 95% con dence intervals (CIs). Standard scales were used to assess the feasibility of the program.cOR = 10.76, 95% CI: 1.31–88.47, P = 0.027; aOR = 12.12, 95% CI: 1.37–107.29, P = 0.025). The completion rate of full schedule of HPV vaccination in the two arms was 100% (49/49) and 95.1% (39/41), respectively. Of all 49 vaccinated girls in the pay-it-forward arm, 38 (77.6%) donated to support future participants, and the total donation accounted for 33.3% of prepaid subsidization. Among caregivers in the pay-it-forward arm, 97.6% (41/42) believed that this strategy was feasible.A total of 100 participants (50 in each arm) were recruited from Jan 4, 2022 to Feb 18, 2022. The HPV vaccine uptake rate was 98% (49/50) in the pay-it-forward arm and 82% (41/50) in the standard-of-care arm , ChiCTR2200055542. Retrospectively registered on 11 January 2022,  Cervical cancer is the most common cancer affecting women’s health worldwide, and is the fourth leading cause of cancer death among women . Over 34®, Gardasil® and Gardasil 9® and two domestic vaccines Cecolin® and Walrinvax®) have been approved in China. But the HPV vaccination rate is low in the country. In 2019, among females aged 15–24 years, only 11% of them were vaccinated against HPV, and Western China had the lowest vaccination rate at 8.6% in comparison to Central (10.6%) and Eastern China (13.7%)[HPV vaccine is the most cost-effective preventive measure . CurrentThe World health Organization (WHO) recommends to include HPV vaccine in national immunization program and suggested priority populations for vaccination . Up to dThe pay-it-forward innovation may be such a solution. Pay-it-forward has a girl receive community supported and subsidized HPV vaccination and then offers an opportunity for her to donate/write postcard messages to encourage other girls to vaccinate. Our previous studies have shown that pay-it-forward effectively increased service uptake, including two trials focused on gonorrhoea and chlamydia testing and one trial on in influenza vaccination , 18. HowThe two-arm RCT compared the effectiveness of a pay-it-forward strategy against the standard-of-care approach in increasing HPV vaccination among 15–18-year-old adolescent girls. The pilot was conducted in Yulin Community Health Center (CHC) in the downtown area of Chengdu, Western China. CHCs in China are the urban primary care system that provides essential medical care, preventive services, and routine health promotion and management for local residents . Given tParticipants were adolescent girls living in the communities served by Yulin CHC. The eligibility criteria were: 1) aged between 15–18 years, 2) no HPV vaccination history, 3) no prior allergies to vaccination; and 4) have a legal guardian consenting to participate the study. Exclusion criteria included: 1) age below 15 or above 18 years, 2) received HPV vaccination, 3) risk of allergy to HPV vaccines by clinical assessment, or 4) refused to consent to participate. Participant eligibility was screened by a research assistant (RA).Eligibility of participants who came to the clinic after the online promotion was screened and verified by RAs. They introduced the program and obtained written informed consent from caregivers and girls who agreed to participate. Eligible participants were randomly assigned to either the standard-of-care arm or the pay-it-forward arm at a ratio of 1:1 using a sealed envelope method. Individual randomization involved two steps. First, 100 random digits were generated by SPSS (version 21.0) and sorted from smallest to largest, the smaller 50 digits were assigned as the control arm and the larger 50 digits were assigned as the pay-it-forward arm. These digits along with assigned intervention materials were assembled into envelopes. Second, each enrolled participant drew an envelope in a sequential order on a temporal basis and was assigned to either of the arms. Different RAs assembled the envelopes and conducted assignment independently. All staff were unaware of which treatment one individual participant was receiving until the participant opened the envelope. The recruiting RA then guided participants to a private room according to the assigned arm for intervention delivery which was conducted by other RAs. Assignment and interventions delivered to participants were blinded to vaccination personnel in the clinic and data analysts. However, it was impossible to completely blind the treatment one participant received to the RAs who delivered the intervention due to the nature of a behavioral intervention. But since the assignment and delivery procedures were independently done by different people, it was impossible for the two assistants who delivered interventions to determine or alter the possibility of an individual to receive a treatment.An educational pamphlet that provided a brief introduction to cervical cancer, HPV vaccination, types and costs of HPV vaccine products was given to all participants in both two arms. This educational pamphlet is provided in routine practices by CHCs and details can be found in Additional le 1. All participants completed a paper questionnaire survey  immediately after receiving the intervention.Participants in the pay-it-forward arm were told the market prices of various HPV vaccines and that previous participants donated to support their vaccination (a subsidy of US$ 149.8 per person). If they decided to receive the subsidy and a vaccination, they were then also given an opportunity to donate to support more future participants. The donation was voluntary and anonymous, and donation decisions did not affect their receiving a subsidy nor any other services at the clinic. Besides, participants were shown handwritten postcard messages and invited to create their own messages .Participants in the standard-of-care arm needed to pay out of pocket at the standard market price if they decided to receive HPV vaccination. However, they didn’t receive community-engaged messages or information relevant to pay-it-forward.Girls (via caregivers) who decided to vaccinate made an appointment to visit the clinic again within two weeks for administration of vaccination. Clinical staff assessed clinical eligibility again for receiving an HPV vaccine. We followed up with all participants who booked an appointment and collected data on the first-shot, second-shot and full schedule of vaccination 6 months after intervention delivery. After vaccination and while they waited, girls and/or their caregivers from the pay-it-forward arm were provided with an opportunity to contribute, monetarily and/or non-monetarily by sending a warm message in a designed postcard  to help future girls to receive HPV vaccination.The primary outcome was the first-dose HPV vaccine uptake in two arms. The secondary outcomes were the completion of the full schedule (3-doses) of HPV vaccination in both arms and the donation amount from the pay-it-forward arm. The number of participants who received at least one shot and their choices of vaccines were also analyzed. Paper-based questionnaires were used to collect data on a): sociodemographic characteristics; b): willingness to be vaccinated and its related reasons; c) their donation behavior after the receipt of the first-dose (pay-it-forward arm only). Electronic questionnaires were also implemented to investigate parental attitudes towards their preference to the types of HPV vaccines. The acceptability, appropriateness and feasibility were evaluated by using the standard Acceptability of Intervention Measure (AIM), Intervention Appropriateness Measure (IAM), and Feasibility of Intervention Measure (FIM) Scales  that have been well evaluated before. Each scTwo trained master students were recruited to double-enter data from the paper to computer-based database (EpiData) independently. All finished double-entry databases were validated by running EpiData (version 3.1). Any inconsistency found between the two databases was adjusted and corrected until the databases agreed. As final check, one of databases was chosen and underwent a final consistency check and then was finalized for analysis.2 tests) and Student t-tests were used to compare the characteristics and outcomes between two arms (the standard of care vs. pay-it-forward). Multivariable logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (95% CIs) adjusted for relevant parameters  as identified in preliminary data analysis. SPSS Statistics (version 21.0) and Microsoft Excel 2010 were used to analyze the data. Statistical significance was assessed by two-tailed tests with level of 0.05.No power analysis was performed to estimate the sample size as for a pilot study to evaluate the feasibility and potential effectiveness of a pay-it-forward strategy . QuantitA total of 100 participants were included for final analysis . The meacOR = 10.76, 95%CI: 1.31–88.47, P = 0.027, Additional file 5). Adjusted for parents’ gender, education, and family income, we found that the health behavior of receiving first-dose HPV vaccination in the pay-it-forward arm was 12.12 times of that in the standard-of-care arm . The completion of full schedule HPV vaccination in the pay-it-forward arm was 100%, however, it was 95.1% in the control arm  in the pay-it-forward arm vs. 82% (41/50) in the standard-of-care arm . Comparetrol arm .Except for 5 girls who chose 2v-HPV vaccine because they were ineligible for the 9v-HPV vaccine due to the indicated age from the instruction, 98% (48/49) in the pay-it-forward arm and 90% (37/41) in the control arm chose 9v-HPV vaccines. The choice of vaccine types was generally consistent in both arms . When asThe primary reason for caregivers to vaccinate their daughters was that HPV vaccine could protect them from developing cervical cancer  actually contributed some amount through the pay-it-forward program, and 26 (68.4%) donated $49.9 and above. The mean donation per person was $69.4 (SD = 49.9). The largest donation was $149.8 and the median donation was about $49.9 (IQR 69.6). The total amount of donations ($2433.6) accounted for 33% of the prepaid fees ($7488.7) and could cover 16 individuals in the future for HPV vaccination .Out of 50 participants in the pay-it-forward arm, we obtained responses from 42 individuals. Thirty-eight (90.5%), 41(97.6%) and 41(97.6%) of 42 participants believed that the pay-it-forward strategy was acceptable, appropriate, and feasible, respectively. The scale scores for acceptability, appropriateness and feasibility were 18.57(SD = 3.04), 18.81(SD = 1.84), and 18.71(SD = 1.92), respectively. More than 95% of the participants had consent or full consent attitudes .For the first time, our study contributes to the literature by assessing the feasibility, acceptability, appropriateness and preliminary effectiveness of a social innovation intervention using a two-arm RCT design, developing new methods for HPV vaccination engagement among adolescent girls, and enhancing vaccine uptake. Our findings suggest that the pay-it-forward strategy is feasible, acceptable and appropriate to implement among the target population. This strategy might increase HPV vaccine uptake among the 15–18 adolescent girls compared with a standard-of-care strategy for vaccination in China. This strategy increased vaccine uptake, elicited financial contribution, improved the public engagement and was correlated with vaccine confidence.The finding that those who took part in the pay-it-forward intervention had higher HPV vaccine uptake than those in the standard-of-care arm is consistent with previous intervention studies using pay-it-forward interventions to improve health services uptake and influenza vaccine uptake , 18, 23.We also observed that, among those enrolled in the pay-it-forward arm, the majority of them (77.6%) voluntarily donated to support another girl after receiving an HPV vaccine. The average donation was $69.4 and the total donation could cover 33% of prepaid fees which was similarly the previous studies , 23. ThePay-it-forward interventions have additional social benefits, fostering community engagement , 26. ComThe findings observed suggested that parents in both arms preferred to the 9v-HPV vaccines even when the 9v-HPV vaccines were unavailable or their daughters were ineligible to get the 9v-HPV vaccines in mainland China. This phenomenon is representative of the general public especially among the metropolitan areas that the choose of HPV vaccines is based on the misleading principle that “the more valent, the better” rather than “the earlier, the better”. As evidence has shown that 87% of cervical cancer and 97% of cervical intraepithelial neoplasia grade 3 (CIN3) could be prevented when administrated at an early age (12–13 years) even with the bivalent HPV (2v-HPV) vaccines –31. ThisThe study has several limitations that should be considered and revised for the formal study. First, an easy sampling through online recruitment was conducted because it was implemented during the COVID-19 pandemic period. People were inconvenient to go outside because of the lock-downs. Although the online recruitment was an easy way to recruit a sufficient number of eligible participants within a short time, it led to selection bias. As those who paid attention on the posts and advertisements from the local CHC were well educated and had higher annul income as well as higher health concerns on themselves and their family members and that’s why we see HPV vaccine uptake was high in both arms in this pilot study. Thus, randomization based on the residency list to recruit is highly recommended. The protocol was revised accordingly as well. Second, the high uptake might due to the availability and accessibility of the 9v-HPV vaccines as well as the waive of its waiting time/appointment, as even up to date, the 9v-HPV vaccines are in huge demand and the average waiting time was from several months to 2 years. This might be an important confounding factor that affected our study result. To avoid this confounding in our formal RCT, we will need to revise the protocol from to reserve all kinds of HPV vaccines on purpose for our study to only provide the participants with any kind of HPV vaccine that was in full stock during their routine clinical practice.In conclusion, the pilot RCT showed that pay-it-forward strategy was feasible, acceptable and appropriate to implement among adolescent girls aged 15–18 years of age. It had potential to improve HPV vaccination rate among girls of appropriate age, although conclusive closure cannot be made due to the limited sample size, locations and important selection bias and confounders found. The pilot data helped inform the refinement of the intervention package and implementation process in vaccination clinics for the formal two-arm RCT with multi-center and expanded sample size.


The objective of this study was to compare the performance of the AmpFire HPV assay to the Xpert® HPV assay in detection of both HPV and clinically significant cervical disease.Low- and middle-income countries (LMICs) account for nearly 85% of the global cervical cancer burden, yet have the least access to high-performance screening. International guidelines recommend human papillomavirus testing (HPV) as primary screening, yet implementation is inhibited by the cost of HPV testing.Atila AmpFireWe utilized stored cervical specimens from a prospective cohortstudy of women living with human immunodeficiency virus (HIV) in Botswana conducted from May to July 2018. Positive and negative percent agreement was calculated for the AmpFire and Xpert assays, as was detection of high-grade cervical dysplasia.63 stored cervical specimens haddetectable DNA after thawing and were included in the analysis. The positive percent agreement was 91.2% (95%CI: 76.3–98.1) and negative percent agreement was 79.3% (95% CI: 60.3–92.0). Six cases positive by AmpFire but negative by Xpert were HPV genotypes 35, 52 (n=2), 58, 68, and co-infection with HPV 45 and 68. Both Xpert and AmpFire assays detected HPV in all 10 samples of women who had high-grade cervical dysplasia.The AmpFire HPV assay demonstrated excellent analytic performance in both detection of HPV and clinically significant cervical disease. AmpFire HPV is a promising option to increase access to affordable, type-specific HPV screening for cervical cancer in LMICs. The objective of this study was therefore to compare the AmpFire HPV assay to Xpert HPV assay using stored cervical samples, and to compare the performance of both tests for detecting clinically significant cervical disease.Atila AmpFire15We utilized 67 stored cervical swab specimens from a prospective cohort study of women living with HIV in Botswana conducted from May to July of 2018. The study investigated the performance of primary high-risk HPV testing followed by triage evaluation with cytology, visual inspection with acetic acid and colposcopy to diagnose pre-invasive cervical disease in women with living with HIV in Botswana. Histopathology outcomes were available for all HPV specimens which were positive by original Xpert testing.®. HPV specimens were then placed in a PreservCyt® transport medium and taken to the Botswana Harvard AIDS Institute Laboratory for HPV testing as previously described.15 All participants who tested positive for high-risk HPV returned for colposcopy  and directed biopsy. Biopsy specimens were sent in formalin to the Botswana National Health Laboratory for processing and results were reported as benign, cervical intraepithelial neoplasia (CIN) 1, 2, 3 or invasive cervical cancer. Results were categorized according to clinical significance, where clinically significant pre-cancer or cancer included CIN 2, 3 or invasive cancer (CIN2+) and benign clinical disease included histopathological reports of benign and CIN1.In the study, provider-collected samples were taken from the cervix for HPV testing and cervical cytology using a Cervex-brushHPV genotyping was performed in the original study using the Xpert assay according to the instruction’s manual. The Xpert assay was used to qualitatively genotype the 14 high-risk HPV genotypes, including 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. The results were reported as HPV 16, HPV 18/45, and other high-risk HPV genotypes. The input volume of cervical cell suspension for this assay was 1mL per sample.Stored specimens were thawed and prepared for assay processing in March 2021. One milliliter of each specimen was briefiy vortexed and transferred to a sterile 1.5mL eppendorf tube. The tubes were then centrifuged for 30 minutes at a maximum speed followed by a complete supernatant removal and subsequently 30μL of the lysis buffer was added and then thoroughly vortexed to resuspend the cell pellet. The whole content from each sample tube was transferred into a 0.2mL polymerase chain reaction (PCR) tube. Incubation of the PCR tubes was then performed at 95 °C for 10 minutes in a thermocycler.https://atilabiosystems.com/multiplex-high-risk-hpv-by-fluorescent-detection/). This assay detects 15 individual high-risk HPV genotypes in a single tube, including HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, and 68 (the same types as the Xpert HPV assay plus HPV 53). AmpFire uses the cyanine5 (CY5) fluorophore for HPV 16, the carboxyrhodamine (ROX) fluorophore for HPV18 and other high-risk HPV genotypes using the fluoresce in amidites (FAM) fluorophore, all combined with an internal control using the HEX fluorophore. The Real-Time PCR was programmed using Bioer Real-Time PCR system for an isothermal reaction setting of 60°C while taking fluorescence readings at the FAM/HEX/CY5/ROX channels once every minute for a total of 60 minutes. The thermocycling software system automatically reported the results of the cycle threshold values for each amplification curve in all fluorescence channels.Samples were then genotyped using the AmpFire by fluorescent detection according to the instruction manual , negative percent agreement (both results were negative), and discordant if the results of each assay were different. The 95% CIs of the positive and negative percent agreements were calculated. P-values were also calculated, and P < 0.05 was considered statistically significant. Comparisons to histopathology were descriptive.67 stored cervical samples were available for validation. When Amp re was run on thawed specimens, 4 (6%) samples had invalid controls, leaving 63 samples with detectable DNA that was genotyped and included in the analysis. In the original study, Xpert detected HPV in 34 samples while 29 samples tested negative. AmpFire detected HPV in 37 of the 63 samples, classified into 13 of the 15 high-risk HPV genotypes detected by AmpFire . Using XOf the samples with discordant Xpert and AmpFire results, the three cases that were negative by AmpFire had been classified as “other high-risk HPV” by Xpert. The six cases that were positive by AmpFire but negative by Xpert were HPV genotypes 35, 52 (n=2), 58, 68, and co-infection with HPV 45 and 68. Additional notes on type-specific discordance are found in The results of the HPV assays were compared to the histopathologic diagnosis in the original study . Among t24 Finally, our study supports the performance of AmpFire in cervical screening in women with HIV, which is essential for increasing access to HPV-based screening in LMICs, which bear the greatest burden of HIV-attributable cervical cancer.25The AmpFire HPV assay demonstrated excellent analytic performance in detecting high-risk HPV genotypes, demonstrating 85.7% overall agreement with the Xpert assay. The AmpFire assay also performed equally well to Xpert HPV in identifying women with clinically significant cervical disease (CIN2+), thus meeting an essential requirement of a high-performance screening test. Our study also demonstrated the benefit of AmpFire in detecting individual HPV genotypes, as 13 of 15 high-risk HPV individual genotypes were detected in this small sample. Individual genotyping offers the possibility of HPV genotype restriction triage for positive HPV results, which is of increasing interest in cervical screening research.26 Our data support expediting the approval process for AmpFire in order to rapidly increase global access to affordable HPV screening.These results show that an affordable and accurate HPV assay with individual HPV genotyping is within reach of the global market. Currently WHO recommends primary cervical screening with HPV assays with particular regulatory approvals.27 Vaginal self-swabs had >95% agreement between AmpFire, both Roche Cobas® 4800 HPV assay, and SeqHPV assay.20 Rates of concordant results between AmpFire and Roche linear array HPV on anal swabs was similarly high (90%).17 In a population of women living with HIV, vaginal self-swabs were found to have 89% positive agreement between AmpFire and Xpert, with AmpFire being slightly more likely to diagnose HPV than Xpert.28 Beyond agreement in HPV results, our findings demonstrate the accuracy of AmpFire in identifying women with CIN2+, data which few other studies have con rmed.25The high rate of agreement between AmpFire and Xpert was most likely due to the similar real time fluorescence detection methodology and the same high-risk HPV genotypes detected by the two assays (aside from HPV 53 which is not included in the Xpert assay). Our finding is in keeping with prior studies evaluating AmpFire compared to WHO prequalified HPV tests in various specimen types. AmpFire successfully detected 15 high-risk HPV genotypes from cervical, vulvar and oropharynx tissue in paraffin embedded blocks with >95% agreement with Linear Array HPV DNA genotyping tests.It is not clear what accounts for the differences in detection of HPV between assays. The use of different materials for the detection process could be a factor to consider. AmpFire produces cell lysates using the kit extraction lysis buffer, whereas Xpert performs cell lysis and purification within the cartridge, ensuring high quality HPV DNA before PCR amplification. Another possible factor is the open reading frames targeted by the Xpert assay which are E6/E7 whereas the AmpFire assay targets E1 and/or L1. Process monitoring to further optimize performance is warranted.30 Other attractive features of the AmpFire HPV assay include ease of use in the laboratory setting, particularly with minor sample preparation for lysis with no need for extensive extraction. There is a short hands-on time to process specimens and it requires only a small sample volume. It uses isothermal amplification, which can be run on any Real-Time PCR or isothermal platform and has a rapid turnaround time to results. AmpFire requires a laboratory infrastructure at present; however, it approximates the requirements for speed and ease of use for point-of-care applications, including a rapid preparation and run-time, and has been used in a same day test-and-treat project.31 With further optimizations and modifications, AmpFire has the potential to integrate clinic-based HPV testing into same-day test-and-treat programs in LMICs.The AmpFire assay is well-suited for HPV detection in most resource limited settings aiming to implement HPV testing. The ability to detect HPV DNA in samples that had been frozen for three years in a practical setting is reassuring; samples can be collected, stored, and tested at a later time when stored in the correct conditions for prolonged periods. Data from other studies support the stability of HPV DNA on dry swabs across unregulated temperatures for at least a month which increases the feasibility of conducting population-based screening with decentralized self-collected vaginal HPV samples that could be stored in variable conditions until transport and testing are possible in a referral laboratory.32 Given the need to expand HPV testing in LMICs, and the affordability of the AmpFire assay relative to other tests on the market, we took this practical approach to our validation in order to expedite its utilization. The high level of agreement observed between the assays is reassuring as agreement should theoretically decrease when comparing against an imperfect reference standard.33This study has several limitations. Our study used a small sample of stored specimens due to feasibility constraints. Additionally, we only have histopathology data on women with positive HPV results, limiting our ability to evaluate the sensitivity and specificity of both assays in detecting CIN2+ in the entire sampled population. Another limitation is the lack of gold standard used as a comparator. However, the Xpert assay is WHO pre-qualified, and in validation of other infectious disease tests it has generally been acceptable to evaluate the interchangeability of a test with a clinically utilized comparator without altering diagnostic accuracy in cases where a gold standard is not available.A larger study with a true gold standard  is necessary to evaluate the performance of AmpFire in the detection of high-grade cervical dysplasia. This study is currently underway by our research group in Botswana and will allow validation of the accuracy of AmpFire in diagnosing CIN2+ in 3000 women. The study will also include histopathologic assessment of a proportion of the women who test negative with AmpFire, such that test performance parameters  can be evaluated.The AmpFire HPV assay demonstrated excellent performance compared to a clinically utilized alternative and is a promising option to increase access to affordable, type-specific HPV screening for cervical cancer in LMICs. Because of its simplicity, low cost, and ease of use, AmpFire can be easily implemented in facilities that currently have no HPV testing. Expedited approval is essential to rapidly scale-up affordable HPV primary screening for cervical cancer.


Optimal practice is constrained when there is a lack of experience, professionalism, and communication among staff, patients, and external agencies. COVID-19 also has had an impact on pharmacists both personally and in their work environments. Exploring how the pandemic has affected pharmacists and their work environment warrants further research. (4) Conclusions: Pharmacists across New Zealand agreed that optimal practices were occurring and considered other factors that were perceived to not affect optimal practice. A human factor S.H.E.L.L framework has been used to analyze themes to understand the optimal practice. The rising body of international literature on the effect of the pandemic on pharmacy practice serves as a foundation for many of these themes. Longitudinal data would be useful in exploring some factors, such as pharmacist well-being over time.(1) Background: The safe performance of pharmacists is an important issue for patients and regulators. It is recognized that pharmacists interact with a variety of healthcare professionals and act as a bridge between other healthcare providers and systems and patients in the health setting. There has been growing activity in exploring factors that impact optimal performance and determinants that are linked with medication errors and practice incidents. The aviation and military industries have used S.H.E.L.L modeling to identify how personnel interact with factors that affect outcomes. A human factors approach is a useful angle to take when trying to improve optimal practice. Little is known about the experiences of New Zealand pharmacists and S.H.E.L.L factors that affect day-to-day practices in their work environment. (2) Methods: We investigated environment, team, and organizational considerations as the determining factors of optimal work practices using an anonymous online questionnaire. The questionnaire was built from a modified version of the software, hardware, environment, and liveware (S.H.E.L.L) model. This identified components of a work system that were vulnerable and that provided risks to optimal practice. Participants were New Zealand pharmacists approached through a subscriber list provided by the regulatory authority of the profession. (3) Results: We received responses from 260 participants (8.56%). The majority of participants indicated that optimal practice was occurring. More than 95% of respondents agreed that knowledge, fatigue interruptions, complacency, and stress affected optimal practice. Equipment and tools, medication arrangement on the shelf, lighting, physical layout, and communication with staff and patients were important factors for optimal practice. A smaller cohort of participants, 13 percent ( The safe performance of pharmacists is an important issue for patients and regulators in New Zealand. It is recognized that pharmacists interact with a variety of healthcare professionals and act as a bridge between other healthcare providers, systems, and patients. Human factor researchers have noted there is growing interest in ascertaining what influences optimal performance and which determinants contribute to medication errors or incidents ,2. Processes investigating the occurrence of a medication error usually recognize that there are factors outside a pharmacist’s direct control. While pharmacists worldwide have continued to contend with greater workloads, the COVID-19 pandemic has had an impact on pressures in the environment in which pharmacists have previously reported working ,4,5,6. GA divergence from optimal performance frequently arises from a series of incidents, with contributions from human and environmental factors. Previous literature in this area is limited and refers to variations in pharmacists or the environments in which they work rather than how these factors interact and what pharmacists perceive as important for the optimal practice to occur. A systematic review of the prevalence and nature of medication errors and medication-related harm following discharge from hospital to community pharmacy found that pharmacist interventions alone, such as medicine reconciliation and electronic communication, did not demonstrate a consistent reduction in adverse effects and errors post-discharge . The rolThe aviation and military industries utilize a framework of human factors to investigate incidents, and this may be applied to healthcare to investigate the environment, team, and organizational considerations as potential factors of optimal work practices. The framework looks beyond cause and effect and examines the interaction between factors . MedicalUnlike other models utilized in assessing risk and errors, the S.H.E.L.L model, as described in The S.H.E.L.L model classifies and helps understand the interplay of human factors in errors that occur in the pharmacy practice setting. It relies on the experience that people make errors, and even in optimal environments, errors can happen and are to be expected ,15,16. TThere is limited knowledge of New Zealand pharmacists’ perceptions of optimal practice and what affects it in their everyday working environments. This research examines New Zealand pharmacists’ ideology about the major factors or determinants contributing to optimal factors and where the evidence for this is by utilizing the S.H.E.L.L framework model.This study aims to investigate the following questions: What do New Zealand pharmacists believe are the major contributing factors to optimal pharmacy practice? What are the S.H.E.L.L factors that affect New Zealand pharmacy practice?TM XM survey. Registered pharmacists working in the New Zealand pharmacy sector who have consented to receiving questionnaires from the New Zealand Schools of Pharmacy in relation to their annual practicing certificate renewal were invited to participate by email with a Qualtrics URL survey link. The survey was available from 1 October 2021 until 30 November 2021. No further reminders of the survey were emailed, and no follow-up non-responder bias was examined [The study was undertaken using a mixed methods approach in a similar manner to the previous publication to answer a query about suboptimal practice in the New Zealand setting . The stuexamined .The questionnaire was adapted from an aviation industry human factor framework utilized to investigate the potential environment, team, and organizational factors or determinants of optimal work practices . The adaThe questionnaire comprised three sections. The demographic data and the perceived factors influencing optimal practice were gathered in the first two sections, while the third investigated the pharmacist’s working environments and their perspective of what was important to them in their workplace practices. A Likert scale to investigate determinants of potential risk and the degree to which they concurred or contradicted with what occurred in their workplace assisted this inquiry. The third section investigated the interactions between software, hardware, environment, and liveware (S.H.E.L.L) . Eight pharmacists were invited to participate in the survey pilot. Modifications to the gender identity queries to include “non-binary” and “prefer to self-describe” options were added. Wider ethnicity options and workplace title demographics to accurately represent current practicing environments were altered. To address further concerns about the framework and its ability to be applied to different workplace settings resulted in additional modifications being made.The analytical software, the IBM Statistical Package for the Social Sciences (IBM SPSS version 19), was utilized for the quantitative data analysis. Frequency data according to participant characteristics were utilized for the descriptive statistics . Inductive methods were used to analyze qualitatively open-text comments submitted by questionnaire participants before the topics were classified . The theThe study was approved by the University of Auckland Human Participants Ethics Committee on 2 July 2021 for three years—Reference Number UAHPEC22669.The overall response rate of 8.56% was derived from the 260 eligible pharmacists in New Zealand that engaged with the survey. The survey was sent out to 3039 eligible pharmacists in New Zealand.n = 85, 32.7%), community pharmacy owners , and community pharmacy managers . The next most common respondents were hospital pharmacists . n = 172, 66.2%) responded than males . Respondents in the 31–35 years and 51–55 years age range  made up the larger categories. The majority of participants were in practice 30 years or more , and 12.7% (n = 33) were in practice 6-10 years, with 11.9% having practiced 26–30 years and 10 % had practiced for two years or less (n = 28).The majority of respondents comprised staff pharmacists in community pharmacies (p < 0.02), workplace norms (p < 0.01), lack of consistency in barcode scanning and not using barcode scanners as intended every time a medication is dispensed (p < 0.05), and communication between pharmacists and pharmacy technicians/pharmacy assistants/senior supervisor/manager/other health professionals in the same workplace as being significant (p < 0.01).Factors that affect optimal pharmacy practice of statistical significance were divided according to the demographic data of the pharmacist participants. Gender differences identified factors relating to inadequate dispensing processes, dissemination, and enforcement of standard operating procedures (SOPs) or procedural guidance/policy (p < 0.02), substance exposure (p < 0.02), familiarity with a given task, previous actions (p < 0.02), lack of assertiveness of members within the team (p < 0.001), lack of teamwork (p < 0.01), and cultural differences (p < 0.03). Factors of significance to years of practice included factors relating solely to inadequate training or experience (p < 0.05). In the work setting, factors such as the arrangement of computers, printers, barcode scanners, and stationery (p < 0.007), fatigue, lack of awareness and inattention, distractions, or interruptions (p < 0.00001), complacency (p < 0.006), stress (p < 0.00001), and communication between staff in the same workplace and between consumers of medicines (p < 0.001) were considered statistically significant factors. Factors that were of significance to the age of participants included workplace norms  agreed that fatigue, lack of awareness, inattention, and stress affected optimal pharmacy practice. p < 0.05), suggesting that for New Zealand pharmacists, these were the main factors that affected optimal performance. These included fatigue and lack of awareness and inattention, stress, distractions or interruptions, communication among pharmacists and pharmacy technicians/pharmacy assistants/senior supervisor/manager/other health professionals in the same workplace, and complacency. Almost all respondents (n = 35) indicated that professional and ethical requirements did not affect optimal practice. A third of respondents did not indicate that cultural differences affected optimal pharmacy practice, while 21 percent (n = 21), thought that dispensing processes, dissemination, and enforcement of standard operating procedures and procedural guidance did not affect pharmacy practice, 21.3% (n = 35) responded that professional and ethical requirements did not affect practice, 26.9% (n = 36) did not think that having a staffroom for rest affected practice, 20.3% (n = 25) did not think substance use affected practice, and 29.5% (n = 41) stated that cultural differences did not affect practice.A smaller cohort of participants, 13.5 percent (p < 0.02) and workplace norms (p < 0.006) as significant factors that affected optimal practice. A factor that was of significance to the age of participants included workplace norms (p < 0.02). An important factor when considering years of practice is inadequate training and experience (p < 0.05). Gender differences identified factors relating to inadequate dispensing processes, dissemination, and enforcement of standard operating procedures (SOPs) or procedural guidance/policy  thought there were inadequate dispensing processes, dissemination, and enforcement of standard operating procedures (SOPs) or procedural guidance/policy. This is possibly due to their vast experiences as practicing pharmacists. Minor themes consistent in responses included practice within the law and legislation, a desire for quality improvement, and best practice compliance for audit reviews. Regular error reporting and a hands-on approach from owners and managers were featured in addition to positive sector representation, and innovation within the practice was cited as contributing to optimal practice. Those with 30-plus years of practice (p < 0.007).The arrangement of computers, printers, barcode scanners, and stationery to work settings were noted to be statistically significant . Gender, years of practice, and work setting did not statistically affect environmental factors significantly. Age differences identified that substance exposure was a factor that was statistically significant to optimal practice  and adequately protected them from potential exposure to medicines . There was divergence in relation to cluttered layout impacting workload  and noise also negatively impacting performance .With regard to the environment, a consistent topic was the operating environment. This is addressed in greater detail in Subthemes that linked into the operational theme included prioritizing the safety of staff, having robust Standard Operating Procedures (SOPs), and good workflow and timing with adequate teamwork and support. Neatness and tidiness were important to pharmacy design and layout, in addition to adequate lighting and ventilation. A lack of interruptions in task completion, evidence-based practice approaches, and having designated rest periods or breaks were also contributors to optimal practice. Substance exposure to the likes of viruses was identified as a statistically significant factor in optimal practice as a COVID-19 infection was likely to affect a pharmacist’s practice if they were infected.p < 0.00001), lack of awareness and inattention, distractions, or interruptions (p < 0.00006), complacency (p < 0.007), and stress (p < 0.0001). The age of pharmacists highlighted familiarity with a given task as contributing to optimal practice (p < 0.02).Factors that were of significance to the work setting included factors such as fatigue , psychological stress , and emotional stress  affected their performance. The importance of personnel was a repetitive topic that was raised in the survey commentary in reference to liveware. Further information is summarized in p < 0.05) with multiple distractions and interruptions that are not conducive to optimal practice.Minor themes consistent from looking at personnel included staff that had experience, initiative, passion, high levels of professionalism, and integrity. Personnel who were flexible in their approach, were resilient, and practiced patient-centered care were also linked to optimal practicing environments. In contrast to these optimal factors, pharmacists are working under statistically significant stress (p < 0.007). Relationship building is important to any practitioner, and this is supported in the subthemes from p < 0.001), the ability of teams to work together (p < 0.009), and cultural differences (p < 0.028), which highlights the importance of communication as a factor influencing optimal performance. This is reinforced within work settings as communication within established teams and between health consumers are highlighted as important to optimal practice. Gender differences identified communication between colleagues and other health professionals to be statistically significant  and cited challenges with teamwork and/or collaboration and communication .Communication and relationships continued to be important subjects in relation to liveware–liveware interactions. This is elaborated on in greater detail in Subthemes relating to this theme included the maintenance of positive relationships and open communication with district health boards, management, professional bodies, other health care practitioners, and staff. Honesty and trust in communication were also optimal requirements for effective practice. The central role of communication also aligns with the statistical significance of communication between colleagues in the workplace and between health consumers and pharmacists. This study aimed to understand what New Zealand pharmacists associated with optimal practice and to explore how this applied to their current work settings. Registered pharmacists across all sectors were recruited to explore what they associated with optimal practices in their workplaces and, using a human factor framework, assess which areas of the S.H.E.L.L model affected optimal practice and which did not. To understand pharmacists’ perceptions of the diverse influences encountered that affect their work, a mixed methods questionnaire was used . n = 85) made up the majority of respondents, followed by community pharmacy owners , hospital pharmacists , and community pharmacy managers  defined Good Pharmacy Practice (GGP) as a responsive practice to the needs of people who use pharmacists’ provision of optimal and evidence-based care services . Core asThere is no current accepted definition in New Zealand of what optimal pharmacy practice is; however, an analysis based on the S.H.E.L.L framework demonstrates that with respect to Software, good leadership and sector representation, innovations in practice, quality improvement initiatives, and error reporting with compliance and auditing contributes to optimal practice. Workplace norms were an important factor as a proxy measure of whether workplaces completed activities to best practice standards or if there was a culture of deviation from standard practices. Age and years of experience were not statistically important when considered in relation to optimal practice.Respondents also reported from a Hardware perspective that adequate resourcing should include not only enough and correct equipment but also having enough pharmacists and support staff with continued training and remuneration to contribute to optimal practice. When comparing the arrangement of computers, printers, barcode scanners, and stationery to work settings, this was noted to be statistically significant, too. This would not be out of the ordinary from a work setting point of view as this is likely to affect efficiency, which, in turn, affects optimality.An environment where there was adequate lighting and ventilation and standard operating procedures that were followed and reflected best practices all contributed to optimal practice. An environment that put safety first, where there was teamwork, support, and break periods, in addition to a lack of interruptions in tasks, also was thought to contribute to optimal practice. From a liveware point of view, communication between management, staff, District Health Boards, and professional bodies were all offered as being important contributors to optimal practice. Experience, passion for the profession, honesty, and initiative were also highlighted as contributing to an optimal practicing environment. The survey data was collected over the COVID-19 pandemic period, where COVID-19 exposure would have an impact on a pharmacy’s ability to practice optimally.Although our findings are generally positive around optimal practice and what contributes to it in New Zealand pharmacies, there are drivers to suboptimal practice that warrant further research.The study covers the whole New Zealand pharmacy sector. It is critical to highlight that community pharmacy is currently audited within two frameworks: a full audit with notice against 67 criteria or a standard inspection audit without notice against 10 risk-based criteria. The standard inspection audit, which occurs more frequently, looks at inadequate training and education for medicine supply as an important factor conducive to optimal practice but fails to acknowledge other criteria influencing optimal practice. Some of the optimal factors are subjective to measure; however, the lack of a uniform measurement instrument means that risk-based behavior could be increasing, as reported in Quarter 1 and 2 pharmacy quarter audits from 2021/2022 ,31. More organizational culture research in pharmacy needs to be undertaken. The literature around culture in pharmacies provides less evidence of the influence of organizational culture on security and caliber in addition to outcomes of service provision. This needs to be quantitated, and effectiveness criteria for pharmacies need to be determined to map culture against them. A human factors approach could be an acceptable tool to capture the dynamic working environment of pharmacists and give more detailed insight into specific interventions that could identify areas to improve community practices ,33.Additional research is also required to comprehend the various models of pharmacy and the impact of corporatization on the pharmacy sector in New Zealand. As corporatization has increased in pharmacies, there is little evidence available about the role of corporate structures on safety measures and corporate pharmacies’ efficacy in delivering safe patient-centered outcomes . There iThis investigation has several limitations. The survey distribution occurred during the alert Level 4 lockdown in New Zealand, where issues were rife, and the data being collected occurred under challenging conditions. This likely impacted the survey response rate of 8.56%. The sample, in terms of numbers, corresponds with projections of other survey research undertaken in the New Zealand Pharmacy Sector. We recognize there is a likelihood of relatively low generalizability. We understand that there is a risk of ascertainment bias owing to the way in which survey distribution occurred. Pharmacists that declined to provide contact information for research when renewing their annual practicing certificate were not contacted with a survey link. Pharmacists were able to avoid answering survey questions without a return-back function, potentially impacting the accuracy of the response data collected.In relation to pharmacist well-being, the questionnaire was not cultivated with concerns about pre-existing mental health conditions and their perceptions of how this impacted their day-to-day working time. Investigating matters of prior and current health could have considered benefits when assessing the effect of psychological pressure on pharmacists and if it correlates with optimal practice.The questionnaire did not discriminate responses received from community pharmacists or owners in reference to whether they were independent, franchise-based, or corporate-owned pharmacies. Additional research concerning the difference in responses between sectors about optimal factors for practice could inform the direction of the business model practice and direct legislative writing and discussion around pharmacy ownership.This research aimed to explore the extent to which optimal practice is transpiring across pharmacy practice in New Zealand. The study aimed to understand what factors were associated with optimal practice and whether a human factor approach was a useful tool to help understand factors that affect pharmacy as a high-performance humanistic workplace. The S.H.E.L.L model proved to be an effective structure upon which to map the factors that might impact optimal practice. In terms of Software, good leadership and sector representation, innovation in practice, quality improvement initiatives, and error reporting with compliance and auditing contributes to optimal practice. From a Hardware perspective, adequate resourcing, including sufficient and correct equipment, enough pharmacists, and support staff with adequate continued training and appropriate remuneration, contributes to optimal practice. An Environment where there is adequate lighting and ventilation, standard operating procedures that are followed, and reflected best practices all contribute to optimal practice. An environment that puts safety first, where there is teamwork, support, and break periods, in addition to a lack of interruptions in tasks, was also found to contribute to optimal practice. Liveware requirements include communication between management, staff, District health boards, and professional bodies. Experience, passion for the profession, honesty, and initiative were also highlighted as contributing to an optimal practicing environment.Future research should center on the pharmacy organization and how organizational culture impacts outcomes related to optimal practice. A much more defined research agenda needs to be developed to explore the impact of corporatization globally on the pharmacy sector.