Enteric viruses are commonly found obligate parasites in the gastrointestinal (GI) tract. These viruses usually follow a fecal-oral route of transmission and are characterized by their extraordinary stability as well as resistance in high-stress environments. Most of them cause similar symptoms including vomiting, diarrhea, and abdominal pain. In order to come in contract with mucosal surfaces, these viruses need to pass the three main lines of defense: mucus layer, innate immune defenses, and adaptive immune defenses. The following atypical gastrointestinal infections are discussed: SARS-CoV2, hantavirus, herpes simplex virus I, cytomegalovirus, and calicivirus. Dysbiosis represents any modification to the makeup of resident commensal communities from those found in healthy individuals and can cause a patient to become more susceptible to bacterial and viral infections. The interaction between bacteria, viruses, and host physiology is still not completely understood. However, with growing research on viral infections, dysbiosis, and new methods of detection, we are getting closer to understanding the nature of these viruses, their typical and atypical characteristics, long-term effects, and mechanisms of action in different organ systems. In recent years, the morbidity rates for waterborne diseases have increased drastically and they have now become a public concern around the world ,2. Based5 and 1011 viral particles per gram of stool. These samples are composed of many genera, including noroviruses, adenoviruses, parvoviruses, astroviruses, rotaviruses, Hepatitis E viruses, Hepatitis A viruses, and as enteroviruses that include polioviruses, Coxsackie viruses, and echoviruses [These symptoms are caused by enteric viruses, representing commonly found obligate parasites in the gastrointestinal (GI) tract. When it comes to gastroenteritis or viral hepatitis, patients can extract between 10oviruses ,5. Apartoviruses .The infection with an enteric virus follows a fecal-oral route of transmission. The virus then infects the GI tract and directly interacts with the host microbiome . One of Humans can come in contact with enteric viruses in different ways. In many instances, enteric virus infections come from contaminated water and food, usually when sewage effluent water is involved. This is especially seen in developed countries, where food crops are sometimes grown on land irrigated with sewage-polluted water ,10. If aIn recent years, scientists have deeply analyzed the interplay between enteric viruses, the host immune system, and the host microbiome . In studLactobacillus crispatus-dominant microbiota, as opposed to the rapid distribution seen with a Lactobacillus iners-dominant microbiota or when Gardnerella vaginalis is abundant [We still have a lot to understand about how bacteria, viruses, and host physiology interact. Despite this, a growing corpus of research is starting to illuminate the exciting role that commensal and probiotic microbes play in the host’s ability to defend itself against viral infections . A cruciabundant .Numerous disease models have been used to conduct substantial research on the effect of the gut microbiota on GI barrier permeability, particularly its relationship with hyperpermeability, sometimes known as the “leaky gut” . AlthougStreptomycetes, blocks the co-receptor of the Zika virus, TIM1 [Antimicrobial peptides are protective substances found in almost all living things, from bacteria to people . All impus, TIM1 .Lactobacillus delbrueckii 5 kDa bacteriocin has no impact on the early stages of influenza virus infection, but it does decrease the generation of viral proteins in infected cells [Bacillus genus. In particular, viral glycoprotein gD’s intracellular location is changed yet virus multiplication up to the protein synthesis step is unaffected. As opposed to subtilosin, enterocin CRL35 from Enterococcus faecium was discovered to reduce glycoprotein gD synthesis. This suggests that enterocin CRL35 disrupts the infectious cycle of HSV at a significantly earlier stage [On the other hand, some bacteriocins interfere with the late stages of the viral cycle to diminish cytopathic effects and viral release yield even though they do not impede viral entrance. For instance, a ed cells . Similarer stage . The aimAny modification to the makeup of resident commensal communities from those found in healthy individuals has been generically referred to as dysbiosis . The symThere is a desperate need to detect viruses not only in humans but in water environments as well. Novel methods of detection that are reliable, cost-effective, and time-efficient could improve the overall quality of life of humans and decrease the mortality rates caused by enteric viruses worldwide. One of the significant challenges in water control represents determining the number of enteric viruses in water that can cause infections. Firstly, the concentration of enteric viruses in these water environments is deficient . SecondlAll methods for analyzing water samples can be divided into two main parts, including sample concentration determination and virus detection. In the past, the most common methods used for purifying water from viruses included ultracentrifugation, entrapment ultrafiltration, electropositive membrane, adsorption/elution protocols, and others . NowadayToday, the COVID-19 outbreak represents one of the most significant worldwide threats to public health. It is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first identified in a patient in Wuhan City, Hubei Province, China . This paThe high-risk groups of this infection were noticed to be older male patients who have cardiovascular diseases, hypertension, or diabetes . The infSince the apical sides of lung epithelial cells in the alveolar space have a high expression of the ACE2 receptor, SARS-CoV-2 can quickly enter and destroy these cells. This is in concordance with the fact that many early cases of lung damage caused by these infections can be seen in the distal airways. When it comes to the innate immune response in the airway, the primary cells involved include epithelial cells, dendritic cells, and alveolar macrophages, that fight viruses before adaptive immunity .Interestingly, young children and infants are usually at high risk for hospital admissions because of potential respiratory infections. However, in COVID-19 patients, recent data shows that pediatric patients have generally milder symptoms when compared to older patients. There is a strong correlation between the severity of COVID-19 and the amount of viral loads. Since children can have less viral load in these infections, this could explain the difference in severity . Many hyMoreover, the ACE2 expression could explain differences in disease severity among different gender groups. ACE2 gene can be found on the X-chromosome. Men have higher circulating ACE2 levels than women . This coMore specifically, immunofluorescent techniques show that the ACE2 receptor is abundantly expressed in glandular cells of the rectal, duodenal, and gastric epithelia. On the other hand, the esophageal epithelium composed of squamous cells has fewer expression levels of the ACE2 receptor when compared to glandular cells . AdditioThe symptoms of COVID-19 can range from mild to severe illness and even, in some cases, death . Even thA meta-analysis involving 47 studies with 10,890 patients in total suggested that the most common gastrointestinal manifestations of this virus include vomiting and nausea (7.8% of patients), diarrhea (7.7% of patients), and abdominal pain (2.7% of patients) . In a stBacteroidetes as well as Firmicutes as their main bacteria. In the lungs, Proteobacteria can also be one of the most prevalent microorganisms [Human lungs and gut possess distinct niches of microorganisms. Both the lungs and the gut contain rganisms .The gut microbiota can affect pulmonary health in many different ways. The relationship between these two environments is seen through the so-called “gut–lung axis”, which enables them to communicate with each other . This coBacterial infections often occur as a complication due to viral pneumonia. This can heavily affect the course of the disease, which is especially dangerous for critically ill patients . HoweverFirmicutes. More specifically, seven of these were negatively correlated with the development of the disease. In the Firmicutes phylum, C. hathewayi, Coprobacillus, and Clostridium ramosum were the main bacteria that positively correlated with the severity of the illness [In recent studies, the association between microbiome and the severity of SARS-CoV-2 has been mentioned. In these studies, the fecal microbiome among 7 antibiotic-free patients had experienced other rigors of infection, including mild, moderate, and severe. Association with COVID-19 severity was observed with 23 bacteria taxa. Out of these, 15 belonged to the phylum  illness .Eubacterium rectale, Faecalibacterium Prausnitzii, Clostridium leptum, and Clostridium butyricum was observed. However, when compared to their negative controls, these patients also possessed an increased amount of common opportunistic pathogens such as Enterococcus and Enterobacteriaceae [Gut microbiome dysbiosis in SARS-CoV-2 was also analyzed in a study from 2020. In COVID-19 patients, the abundance of bacteria that produce butyrates such as eriaceae .Veillonella, Streptococcus, Rothia, and Actinomyces, when compared to their negative controls. In the feces of negative controls, an abundance of genera Fusicatenibacter, Faecalibacterium, and Romboutsia were also noticed [Similar studies focused on the specific gut microbiota fluctuations in COVID-19 patients were also conducted. It was found that the feces of COVID-19 patients were enriched with opportunistic pathogenic genera, including  noticed .Even though COVID-19 can be airborne in most cases, some believe the virus could also be transmitted following an oral-fecal route. There is concern that the gastrointestinal tract could be a repository for reinfections. This is because the virus was found in the surface cells of the colon, small intestine, and colon. The number of atypical cases and clinical symptoms involving the gastrointestinal tract is increasing. Additionally, infected individuals were shown to have a prolonged shedding of viral particles in their stools ,62,63,64Even though the oral-fecal transmission explanation has recently gained much attraction, the origin of COVID-19 in the gastrointestinal tract and its clinical shedding in patients’ stools is still controversial. The reverse transcription-polymerase chain reaction could detect only the viral fragments as opposed to the whole virus. Additionally, stool cultures for this virus cannot be used to confirm its viral presence accurately. More studies are needed to consider the incubation period, transmission mechanism, and duration of infectivity .More severe gastrointestinal manifestations of COVID-19 include bloody diarrhea. In rare cases, patients can also experience hemorrhagic colitis and constipation ,66. In aViral load can be measured using real time-PCR. An increasing viral load implies that there is an increase in the patient’s viral particles which leads to more severe symptoms. On the other hand, a lower or down-trending viral load could point to milder symptoms or recovery stages. For respiratory tract samples, the viral load usually reaches a peak around the onset of symptoms. It then gradually decreases in the next 1–3 weeks. In most cases, RNA generally becomes undetectable after two weeks of symptom onset .For stool samples, viral shedding can be present even after the respiratory clearance. However, it is usually erratic. In some cases, it can even continue for approximately two months after the symptom onset. The viral load needs to be examined in different organs to form an accurate association between viral load, disease progression, and symptom severity .Bunyaviridae [Hantavirus belongs to the family aviridae . They poaviridae .This is a rare virus that can attack humans and causes human pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS). Information about the symptoms that possibly occurred due to this virus date back to the first millennium and the Middle Age in England and China ,74. The This virus can be spread through rodents. A person can get infected if they inhale aerosols contaminated with the virus in the form of rodent excreta . BecauseHCPS can lead to cardiovascular irregularities and pneumonia. HFRS is characterized by hemorrhagic manifestations and renal failure. The hemorrhagic manifestations can range from petechiae to intense internal bleeding . The incSimplexvirus subfamily Alphaherpesvirinae in the family Herpesviridae [Herpes simplex virus belongs to the genus sviridae . There asviridae ,87,88,89This virus can be transmitted if a person comes in contact with contaminated bodily fluids such as saliva. The early beginnings of the infection are characterized by its replication at the site of the infection. The virus then travels to the dorsal root ganglia down an axon. This is the location where the virus achieves its latency. In this period, the virus is in a non-infectious state. At different periods, HSV-1 can reactivate itself and attack the host ,91,92,93Although herpetic infection can affect any part of the GI tract, it most frequently affects the esophagus and anorectum . AlthougNo matter the location, localized ulceration, neutrophils in the lamina propria, and an inflammatory exudate that frequently incorporates shed epithelial cells are typical histologic findings. Additionally, crypt abscesses and perivascular lymphocytic cuffing may be detected in the anorectum. Only a small percentage of biopsy specimens include multinucleate large cells and distinctive viral inclusions. The squamous epithelium near ulcer margins and sloughed cells in the exudate are the ideal places to look for viral inclusions. Among all diagnostic tools, viral culture is the most useful. Specific techniques include in situ hybridization and immunohistochemistry. Other viral infections that might affect the GI tract, such as CMV and varicella-zoster, are primarily included in the differential diagnosis. When herpetic infection is present, mixed infections are frequently seen. Herpetic infections frequently self-limit in immunocompetent patients, but they can spread and cause life-threatening sickness in immunocompromised people .Immunocompetent and immunocompromised individuals can contract cytomegalovirus (CMV) infection anywhere in the GI tract. In individuals with a compromised immune system, such as those with AIDS, and following solid organ or bone marrow transplantation, CMV is best known as an opportunistic virus. In healthy individuals, first infections typically resolve on their own. The patient’s immune system and the infection site influence the symptoms. The most typical clinical signs are weight loss, fever, stomach pain, and diarrhea (either bloody or watery). Hypertrophic gastropathy and protein-losing enteropathy, similar to Ménétrier’s disease, are rare but significant conditions connected to pediatric CMV infection .Additionally, chronic GI conditions such as Crohn’s disease and ulcerative colitis may have secondary CMV added. In these circumstances, CMV superinfection is linked to steroid-refractory sickness, toxic megacolon, and a greater mortality rate in addition to exacerbations of the underlying illness. Immunohistochemistry testing for CMV is advised by certain experts as part of the routine analysis of biopsies in patients with steroid-refractory ulcerative colitis .A stunning array of gross lesions are brought on by CMV. The most typical type is ulceration. There may be one or more ulcers, which may be shallow or deep. Linear ulcers and segmental ulcerative lesions might resemble Crohn’s disease. Mucosal bleeding, pseudomembranes, and obstructive inflammatory masses are some other gross abnormalities. The CMV infection has a broad histologic spectrum, ranging from hardly noticeable inflammation to profound ulcers with apparent granulation tissue and necrosis. Routine H&E preparations may reveal distinctive “owl’s eye” viral inclusions, which can either be intracytoplasmic or intranuclear . It is mIt is possible to notice multiple apoptotic enterocytes and crypt abscesses, crypt atrophy, and crypt loss. Immunocompromised patients may develop distinctive inclusions with essentially no concomitant inflammatory reaction. When only a few unique inclusions are observed in biopsy specimens, the diagnosis could be easily overlooked. Immunohistochemistry and multilevel examinations may be extremely helpful in locating the seldom cells that have inclusions. Viral culture, PCR assays, in situ hybridization, serologic analyses, and antigen tests are other diagnostic tools. However, the presence of CMV in culture does not necessarily indicate a current infection since the virus might be excreted for months to years following primary infection .Other viral infections, particularly adenoviruses, are primarily included in the differential diagnosis of CMV. Adenovirus inclusions are often intranuclear, crescent-shaped, and typically seen in the surface epithelium. CMV inclusions naturally occur in stromal or endothelial cells, have an “owl’s eye” shape, and can be seen in the nucleus or cytoplasm. The most striking similarity between CMV and adenovirus infection occurs during the ballooning degeneration stage, right before cell lysis .Given the similarity of the clinical and histologic characteristics, distinguishing between CMV infection and graft-versus-host disease in recipients of bone marrow transplants may be particularly challenging. In this situation, it is best to rule out CMV infection using immunohistochemistry or in situ hybridization investigations because delaying antiviral medication if CMV infection is not detected. Additionally, various ailments might coexist. When there is a lot of apoptosis, crypt necrosis, and dropout, and there is not much inflammation, graft-versus-host disease is more likely to occur. Graft-versus-host disease is made more likely by the presence of active nests of endocrine cells .Caliciviridae represents a family of positive-sense single-stranded RNA viruses that infect both humans and animals. Phylogenetically speaking, these viruses can be divided into the following genera: Lagoviruses, Vesiviruses, Sapoviruses, and Noroviruses . SapovirIn recent decades, an increased prevalence of gastroenteritis has been noticed worldwide. A higher mortality rate has also been seen in developing countries. Apart from this, gastroenteritis outbreaks can occur in institutions such as hospitals ,108,109.Noroviruses follow an oral-fecal transmission route, but a few airborne transmission cases have also been reported. The infection dose of these viruses is very low, and the incubation period is from 10 to 51 h. One-third of infected individuals shed the virus before the symptoms occur .Human norovirus is sometimes referred to as “stomach flu” or “winter vomiting disease”. From 2006 to 2010, CDC reported that more than half of outbreaks caused by foodborne diseases were associated with this virus. During this period, the European Union also reported a large number of infections caused by this virus .The most common symptom in adults is vomiting . A largeSapovirus infection outbreaks have also been more frequent in recent years . When coEnteric viruses are stable and unusually resistant obligate parasites in the gastrointestinal tract that cause symptoms such as vomiting, abdominal pain, and diarrhea. The mechanisms of action and long-term effects of viral infections on host physiology are still not completely understood. This data gap is especially true for atypical viral infections, so more studies on this topic need to be conducted. The data obtained from these studies could be used to find the most optimal method of detection and treatment option. The viruses mentioned in this paper include SARS-CoV2, hantavirus, herpes simplex virus, cytomegalovirus, and calicivirus. These viruses share some commonly exhibited symptoms. However, their atypical gastrointestinal manifestations differ drastically. Many believe that the association between dysbiosis, any modification to the makeup of resident commensal communities from those found in healthy individuals, and viral infections are crucial in understanding susceptibility to these viruses and the treatment outcome. By understanding the underlining mechanisms of dysbiosis, we are getting closer to understanding atypical viral infections that cause gastrointestinal symptoms.


Value-based healthcare (VBHC) provides a framework to improve care by improving patient outcomes and reducing healthcare costs. To support value-based decision making in clinical practice we evaluated healthcare costs and cost drivers in perioperative care for pituitary tumour patients.We retrospectively assessed financial and clinical data for surgical treatment up to the first year after surgery of pituitary tumour patients treated between 2015 and 2018 in a Dutch tertiary referral centre. Multivariable regression analyses were performed to identify determinants of higher costs.271 patients who underwent surgery were included. Mean total costs (SD) were €16339 (13573) per patient, with the following cost determinants: surgery time , length of stay , admission to higher care unit , emergency surgery  and postoperative cerebrospinal fluid leak . Intradural  and combined transsphenoidal/transcranial surgery  were associated with higher costs than standard. Further, higher costs were found in these baseline conditions: Rathke’s cleft cyst , giant adenoma , third ventricle invasion  and dependent functional status . In patients with uncomplicated course, costs were €8879 (3210) and with complications €17551 (14250).Length of hospital stay, and complications are the main drivers of costs in perioperative pituitary tumour healthcare as were some baseline features, e.g. larger tumors, cysts and dependent functional status. Costs analysis may correspond with healthcare resource utilization and guide further individualized care path development and capacity planning. Clinically apparent pituitary adenomas are rare with an incidence of 3.9-7.4 persons per 100.000 per year and a prevalence of 1 per 1000 persons –4, with There are disease-specific treatment guidelines , 3, 5–7,per se. Therefore, this study aims to evaluate 1) total in-hospital costs during pituitary tumour surgery, and up to the first year after; 2) how total costs are attributable to different cost domains ; and 3) which determinants are drivers of total costs. Based on our results and prior knowledge we will propose strategies for improving VBHC care pathways.A comprehensive cost evaluation of perioperative care with extensive analyses of cost drivers for pituitary tumour patients is currently lacking. Previous cost evaluation studies in pituitary healthcare were limited to the surgical procedure , index hThis retrospective study was performed using data of consecutive surgically treated pituitary tumour patients between January 2015 and December 2018 at the Leiden University Medical Centre (LUMC), a tertiary referral centre for pituitary surgery in the Netherlands. Patients were eligible if diagnosed with a pituitary adenoma, Rathke’s cleft cyst (RCC) or craniopharyngioma. Patients with other tumours in the (para)sellar region  or uncertain diagnosis were excluded. Clinical data were collected from electronic patient charts and in-hospital costs data were extracted from a financial database constructed for VBHC research. Data of patients were obtained after approval of the scientific committee of the department, after which a waiver was obtained from the institution medical ethics committee, local study number (G19·011).Preoperative evaluation of all patients includes imaging (MRI and CT-scan), comprehensive endocrine work-up of all pituitary axes, and a neuro-ophthalmological assessment when indicated. During a combined consultation with an endocrinologist and neurosurgeon, the main indication for surgery , aim of surgery , chiasm decompression) and intended effect  are established, depending on the patient’s symptoms and surgical feasibility of pituitary tumour resection. Based on last preoperative imaging all pituitary tumours, including RCC’s and craniopharyngioma’s, were classified as micro- (<10mm), macro- (10-40mm) or giant (>40mm) adenoma for comparability. Third ventricle invasion was established and cavernous sinus invasion was defined as a KNOSP-score of ≥3 . PatientEndoscopic transsphenoidal surgery is generally performed by two neurosurgeons and open microscopic transcranial surgery by one surgeon. Patients with complex nasal anatomy or extensive skull base destruction are operated together with an ENT-surgeon. Most surgical resections were performed endoscopically transsphenoidal. When necessary, an extended or intradural approach was performed. In some cases of giant adenomas or post-operative apoplexy in a suprasellar adenoma remnant, a craniotomy or combined endoscopic transsphenoidal and microscopic transcranial surgery was performed. We described our surgical approach in detail in a recent review .Following surgery, patients were discharged at postoperative day 2 or 3 when eligible for a short-stay protocol, instead of at postoperative day 5 . Length Our costs analyses adhere to the Consolidated Health Economic Evaluation Report Standards . Direct Total costs during follow-up and the distribution of total costs into cost domains  were established for the total cohort. Surgical costs were based on costs for resources during the surgical procedure, including surgery materials, surgeons, nurses and monitoring devices. Hospitalization costs comprised expenditures for the general ward, ICU, physician visits and nurses, including readmissions. Diagnostic investigations or consultations costs consisted of laboratory tests, radiology, pathology and (para)medic consultations. Prescribed medication at our hospital and expenditures at other facilities than the LUMC, such as the general practitioner, were not included.Data analyses were performed using the SPSS Statistics Version 25. For categorical variables, the number of patients and corresponding percentage of the total population were calculated. For continuous variables, the mean with range and standard deviation are reported. Missing data were excluded from analyses using pairwise deletion.First of all we report the mean total costs and how these costs are distributed amongst different cost domains  of the total cohort. For each cost domain, both absolute cost data and percentage of total costs are reported. Subsequently, univariable and multivariable linear regression analyses were performed to identify factors associated with higher costs. For each assessed determinant, a separate multivariable analysis was performed, estimating the association between the determinant of interest and total costs, corrected for the relevant confounders. Confounders were defined as variables associated with both the determinant and the outcomes, but not being caused by the determinant, i.e., not laying within the causal path between the determinant and outcomes . ConfounFurthermore, we analyse how total costs of determinants were distributed amongst different cost domains, expecting that some determinants would increase costs among a specific cost domain. For example, larger tumour size may require prolonged, complex surgery and therefore increased surgery costs may the main cost driver. Complications may prolong length of stay and mainly increase hospitalization costs. By analysing how each cost domain contributes to total costs we aim to investigate whether increased total costs are explained by specific parts of the healthcare pathways, which may serve as target for future value-based healthcare initiatives.A total of 271 patients with a pituitary tumour met the inclusion criteria , VFD , vision loss , apoplexy , prevention of disease progression , growth of tumour residual , hydrocephalus , headache , CSF leak , cranial nerve deficit , nose congestion , behavioural alterations  or to perform a biopsy . The surgical aim was GTR , decompression , emergency , exploration , debulking  or biopsy . The intended effect was curative or remission , recover VFD , recover vision loss  prevent vision loss , reduce mediation dosage , recover cranial nerve function , recover CSF leak , protect pituitary function , obtain histopathological diagnosis , improve nose congestion  or reduce headache.Patients underwent surgery in 2018 , 2017 , 2016  or 2015  on an elective basis  or as an emergency  with a mean surgery duration of 189 (130) minutes. Endoscopic trans-sphenoidal surgery was performed in 260 (96%) patients, of which 22 patients (8%) underwent extended endoscopic surgery and in 16 cases (6%) intradural trans-sphenoidal surgery was performed. Other surgical procedures included endoscopic transventricular , transcranial  and combined endoscopic transsphenoidal/microscopic transcranial surgery . Most procedures were performed by two neurosurgeons, in 43 (16%) surgery was performed together with an ENT-surgeon. Intra-operative CSF leak occurred in 94 (35%) and in 43 (16%) skull base reconstruction with nasoseptal flap was performed.Mean length of stay (LOS) was 6 (7) days. 18 patients (7%) were admitted to the medium or intensive care (IC) unit during index hospitalization with a mean LOS of 2 (2) days. Postoperative complications were hyponatremia or SIADH , CSF leak , epistaxis , intracerebral haemorrhage or hematoma , cortisol deficiency , panhypopituitarism , thromboembolic event , vision deterioration , hypothalamic injury  or other  hypopituitarism . Diabetes insipidus (DI) occurred in 90 (33%) and resolved within 48 hours , 2 weeks  or 6 months . In 18 (7%) DI persisted for over 6 months (DI 4). CSF leak was treated with surgery , ELD  or both ELD and surgery .Mean total costs were €16339 (13573) per patient for surgery and the first-year post-operative care. Total costs comprised mean costs for surgery , hospitalization , irradiation  and diagnostic investigations or consultations . For the total cohort, costs for surgical care account for 55% of the total costs, hospitalizations for 28%, diagnostic investigations or consultations for 17%. Costs for irradiation comprise less than 1% of total costs with only few (n=9) subjected to radiotherapy in this study period (data not shown). Costs stratified for different pituitary tumour diagnoses and tumour size are presented in Results are displayed in Surgical factors associated with higher costs were emergency surgery, e.g. for hydrocephalus and reduced visual acuity,  compared to elective surgery. One additional minute in OR was associated with a €62 increase in costs . Surgery indications VFD , reduced visual acuity  or hydrocephalus  were associated with increased costs, compared to a more elective indication or preventive surgery (e.g. growing mass close to the optic chiasm without VFD yet). In reference to GTR as surgical aim, emergency surgery  and debulking  were also related to higher costs. When the intended effect of surgery was to lower medication dosage or alleviate hydrocephalus, costs were €7407  and €49111  higher than achieving remission, respectively. Compared to endoscopic trans-sphenoidal surgery, combined transsphenoidal/transcranial  and intradural surgery  were associated with higher costs, whereas endoscopic transventricular surgery was related to lower costs . Furthermore, surgery performed by two neurosurgeons and ENT surgeon together  compared to two neurosurgeons, intra-operative CSF leak  and skull base reconstruction with nasoseptal flap  compared to reconstruction methods without nasoseptal flap  were associated with higher costs.As expected, LOS in days , high care unit admission  and LOS in days at the high care unit  were associated with higher cost. Complications including intracerebral haemorrhage or hematoma , postoperative CSF leak , hypothalamic injury , thromboembolic event , new panhypopituitarism  and diabetes insipidus class 3  or 4  compared to no diabetes insipidus were also related to higher costs. Of patients with postoperative CSF leak, surgical treatment  was associated with higher costs compared to external lumbar drain (ELD) placement. Surprisingly, LOS was shorter in patients with ELD compared to surgically treated patients (data not shown). Visiting the emergency department or being readmitted within 30 days after surgery were not significantly related to increased costs. Patients with persistent hypersecretion , reduced visual acuity  or a visible tumour residual on MRI  were also associated with higher costs compared to remission of hormonal hypersecretion, normalisation of visual acuity and complete resection of tumour mass.We performed additional multivariable analyses to compare the effect of cost outliers on the association between clinical diagnosis and total costs. We excluded cases with >€80.000 (n=3) and this changed the association of clinical diagnoses and total costs for patients with RCC , craniopharyngioma  and Cushing’s disease  (data not shown).Based on cost determinants, we analysed total costs, OR duration and LOS of patients with uneventful or complex course. Uneventful cases were defined as patients with micro- or macroadenoma undergoing elective TSA performed by two neurosurgeons, without (post)operative complications and a mean LOS of ≤ 3 days. Likewise, patients were complex if at least one cost determinant was present   for the year after surgery, with higher mean costs in patients with RCCs (€23263) and craniopharyngiomas (€36129) compared to pituitary adenomas €14462). In line with previous studies, total costs were mostly attributable to direct costs of the surgical procedure 55%) and to a lesser extent to hospitalization 28%), irradiation, and diagnostic investigations or consultations (17%) 462. In l, 43. Add5% and to380 for t%, irradiInterestingly, we found that patients with apoplexy had lower costs. We attempted to address this finding by exploring differences in patients’ characteristics. In patients with apoplexy, we found that mean OR time was 30 min shorter compared to the total cohort, but other cost-driving factors  did not explain lower costs. Thus, the explanation for lower costs in patients with apoplexy remains speculative, e.g. shared follow-up with regional centre, incomplete diagnostic workup preoperatively. It is also important to note that higher costs in patients undergoing emergency surgery are not attributable to apoplexy, but to patients having CSF leak or hydrocephalus.Costs evaluations are necessary to improve care paths in line with the VBHC approach. Structural changes can be made within healthcare trajectories to limit the use of expensive or unnecessary interventions that do not improve outcome or add value for patients . Moreover, cost determinants can be used as proxy for healthcare utilization. Therefore, insights in costs enable physicians to consider costs and outcomes simultaneously in clinical decision making and make more individualized paths. For the first time, this study provides a costs baseline, since reference data for European patients with pituitary tumours was lacking, and enables us to identify cost-drivers, relate costs to clinical outcomes and detect changes in cost over time. By doing so, we can evaluate the effectiveness of future value-improving initiatives during different parts of the care trajectory.It is difficult to compare our outcomes to those of earlier studies, because of different healthcare systems and cost assessment methods. Economic evaluations for patients with pituitary tumours undergoing surgery have been mainly performed in the USA –45, 48, In contrast to previous studies, we did not find significant associations between costs and age , smokingThe previously mentioned cost determinants can be used for value-improving initiatives. However, not all determinants may reduce costs effectively, because they are not subject to preventive measures or extremely rare. For example, preoperative panhypopituitarism may be associated with higher costs, but preventing panhypopituitarism is already an important treatment objective. Regardless, these unmodifiable determinants may be used as a sign of more complex disease course and may be relevant in-patient counselling. Likewise, though hypothalamic injury was the strongest cost-driver, this complication was present in only one case and therefore care adjustments likely have a low impact on overall healthcare costs.LOS is a clear cost driver of in-hospital costs , 45 withHigher costs in functionally dependent patients are likely explained by longer LOS and more complications after surgery –62. DespSome unmodifiable determinants of cost are tumours related to the third ventricle, RCCs and craniopharyngiomas, as they are associated with more complications. These patients showed higher, more varying costs, OR time and LOS , 73. ThoIdentifying patients at risk for postoperative CSF leak and implementing preventive measures likely improves outcomes and reduce costs, thereby improving value for patients and allocating resources  more efficiently. Postoperative CSF leak was related to over €14000 additional costs per case and occurred in 9,6% and 7,5% in the total cohort or in patients with pituitary adenoma, respectively. Compared to previous studies reporting a prevalence between 0,9 and 5,2% , 78, 79 Despite previous cost evaluations, this study is to our knowledge the first European study providing a comprehensive overview of the cost of perioperative healthcare for patients with pituitary tumours in a tertiary referral centre. Though, this study has several limitations. First, this study is retrospective, with all inherent limitations. Another limitation is the single centre-nature, with our centre being a tertiary referral centre and both nationally and internationally endorsed pituitary expertise centre, receiving referrals from throughout the country, including more complex cases . Hence, accurate assessment of healthcare usage is subject to hospital registration data, which differs in different hospitals. Consequently, differences in costs between Dutch tertiary referral centres may be partially accountable to the quality of in-hospital costs registration.Limitations in our costs assessment were that we did not correct for inflation, which may have confounded our cost results across the years. Also, we only included direct costs incurred at our institution, thereby neglecting indirect  costs and costs incurred at other institutions. Finally, the costs for medication were not included in this analysis since these are not included in hospital costs. This likely results in underestimating the costs of patients receiving healthcare at other facilities or pharmacological treatment . We encourage future studies to adhere to cost evaluation guidelines to promote interpretability of results . Cost-evIt is also important to point out that this study focussed on perioperative healthcare, thereby disregarding the costs of the preoperative trajectory and follow-up care after one year. During of the preoperative care different factors might be profound contributors to higher costs. For example, functional imaging for Cushing’s disease or frequent consultations in prolactinoma patients might be associated with higher costs in preoperative healthcare. Finally, we did not exclude patients with inordinately high costs due to a complicated course which possibly confounded the effect size of some determinants. The two most expensive patients in our cohort costed over €100.000 each. They both presented with hydrocephalus and had a complicated course with surgically treated postoperative CSF leak. One of these patients had a NFA and also experienced intracerebral haemorrhage, multiple organ failure, several infections and hypothalamic damage and was admitted for 69 days. The other patient was functionally dependent, and surgery was complicated by phlebitis and hematemesis secondary to stage IV oesophagitis, being admitted for 44 days. Though we attempted to alleviate confounding effects in the multivariable analyses, we cannot preclude these patients confounded the effect sizes of some cost determinants , and as shown in analyses with or without cost outliers, cost outliers influence the significance of some cost determinants  (data not shown).Insight in healthcare costs and their determinants is necessary to curtail costs and facilitate VBHC initiatives. The present study provides a concise overview of costs and its determinants, which will serve as reference point for future value-improving initiatives and as a proxy for resource utilization. With this information, future studies can investigate costs and outcomes more specifically , so that healthcare pathways can be adjusted strategically according to the VBHC framework. For example, differentiated healthcare pathways (The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by LUMC medical ethics committee, local study number G19·011. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.AD, FV, and EH collected the data, and AD and FV conducted the data analysis. The study was performed under supervision of NB. AD and FV wrote the primary version of the manuscript. AZ, MV, AP, WF, and NB supervised the data analysis, contributed to the interpretation of the results, and reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Biomarker testing is indispensable for the implementation of precision medicine (PM) in oncology. The aim of this study was to assess the value of biomarker testing from a holistic perspective based on the example of advanced non-small cell lung cancer (aNSCLC).A partitioned survival model was populated with data from pivotal clinical trials of first-line treatments in aNSCLC. Three testing scenarios were considered; “no biomarker testing” encompassing chemotherapy treatment, “sequential testing” for EGFR and ALK encompassing treatment with targeted- or chemotherapy, and “multigene testing” covering EGFR, ALK, ROS1, BRAF, NTRK, MET, RET and encompassing treatment with targeted- or immuno(chemo)therapy. Analyses of health outcomes and costs were run for nine countries . A 1-year and 5-year time horizon was applied. Information on test accuracy was combined with country-specific information on epidemiology and unit costs.Compared to the no-testing scenario, survival improved and treatment-related adverse events decreased with increased testing. Five-year survival increased from 2% to 5–7% and to 13–19% with sequential testing and multigene testing, respectively. The highest survival gains were observed in East Asia due to a higher local prevalence of targetable mutations. Overall costs increased with increased testing in all countries. Although costs for testing and medicines increased, costs for treatment of adverse events and end-of-life care decreased throughout all years. Non-health care costs (sick leave and disability pension payments) decreased during the first year but increased over a 5-year horizon.The broad use of biomarker testing and PM in aNSCLC leads to more efficient treatment assignment and improves health outcomes for patients globally, in particular prolonged progression-free disease phase and overall survival. These health gains require investment in biomarker testing and medicines. While costs for testing and medicines would initially increase, cost decreases for other medical services and non-health care costs may partly offset the cost increases. Biomarker testing has become indispensable for the implementation of precision medicine (PM) in oncology , 2. TradOn the cost side, PM affects health care costs in various ways . The admLung cancer is the second most diagnosed cancer type and the most common cause of cancer-related death globally, with an estimated 2.2 million new cases and 1.8 million deaths in 2020 . The oveThe poor outcomes of lung cancer patients indicate a large need for improved diagnostic and treatment approaches. Over the past two decades, the development of molecular profiling and targeted therapeutic agents has heralded the introduction of PM, particularly in aNSCLC –14. New The aim of this study was to assess the value of biomarker testing–in terms of health improvements and cost changes–for the implementation of PM in oncology based on the example of aNSCLC. A holistic perspective, including the impact on patient health, the health system, and the treasury , was applied. High-income and middle-income countries representative of different regions of the world were analyzed and different scenarios reflecting past and future biomarker testing were used.A health economic model was developed for the treatment of aNSCLC and a health care and treasury perspective applied. Systemic therapy is the standard treatment in aNSCLC, according to guidelines from the American National Comprehensive Cancer Network (NCCN) and the European Society for Medical Oncology (ESMO) , 16. ThrAnalyses were run for nine high-income and middle-income countries across all continents, Australia, Brazil, China, Germany, Japan, Poland, South Africa, Turkey, and the United States. The choice of countries was informed by data availability, population size, and representativeness for similar countries in their respective region. No low-income countries were included because it seemed unrealistic to assume the wide adoption of PM within the next 10 years in these countries.Three testing scenarios were defined in the model to illustrate the gradual introduction of biomarker testing followed by the administration of matching systemic therapies in the first-line treatment of aNSCLC; see The second scenario was “sequential testing,” which included upfront sequential and single biomarker testing for EGFR and anaplastic lymphoma kinase (ALK) followed by treatment with targeted therapy for mutation-positive patients and with chemotherapy for all other patients. This scenario modeled the situation in high-income countries just before the introduction of immunotherapy in 2015 and the current situation in many middle-income countries.The third scenario was “multigene testing,” which included upfront next-generation sequencing (NGS) testing and treatment with targeted therapy if tested positive for the presence of EGFR, ALK, ROS proto-oncogene 1 (ROS1), B-Raf proto-oncogene (BRAF), neurotrophic tyrosine receptor kinase (NTRK), mesenchymal epithelial transition (MET), or ret proto-oncogene (RET), and testing for PD-L1 status and treatment with immuno(chemo)therapy for all other patients. This scenario modeled the situation in 2022 or the near future in most high-income countries.The model included four main outcome measures per patient. The first measure was survival, measured as both the number of life years after treatment start (separated in the model into years spent progression-free and in progression after first-line therapy) and the absolute survival rate as the proportion of patients alive after a certain number of years. The second measure was the number of treatment-related adverse events (AEs), which served as a proxy measure for quality of life. The third measure was health care costs, calculated as the sum of costs for tests, medicines, administration of medicines, treatment of treatment-related AEs, other medical services, and end-of-life care. The fourth measure was non-health care costs from a treasury perspective, calculated as the sum of public social security payments for temporary sick leave and disability pension due to early retirement. Productivity losses were not considered.A 1-year and 5-year time horizon was applied in the measurement of all outcomes. The first year after diagnosis was supposed to capture the period when patients receive first-line therapy, while the 5-year cutoff is the standard length in analyses of long-term survival .via progression or death, were included for each first-line systemic therapy in aNSCLC. Kaplan-Meier curves for the therapies were sourced from publications of pivotal clinical trials and then digitalized. Survival functions for the OS and PFS curves were estimated using a Weibull distribution for all therapies. Health effects and costs were attached to each state and therapy.A partitioned survival model with three health states–progression-free, progressed, and dead–and a monthly cycle length was developed . InformaAt the start of the analysis, a biopsy was assumed to be performed before the administration of first-line therapies. In the first scenario, no additional biomarker testing was performed. In the second scenario, all patients were first tested for EGFR mutations using a singleplex polymerase chain reaction (PCR) test, and if negative for ALK mutations using an immunohistochemistry assay. In the third scenario, all patients were tested for the presence of all included mutations simultaneously using an NGS test as well as a separate PD-L1 test. Country-specific prevalences of mutations and gene expressions in NSCLC and information on the accuracy of the tests (sensitivity and specificity) were obtained from public sources; see A total of 25 first-line systemic therapies in aNSCLC were included in the scenarios. This comprised first-line therapies with an approval by the United States Food and Drug Administration (US FDA) until December 31, 2021; see The occurrence of treatment-related AEs for all included therapies was sourced from publications of pivotal clinical trials based on the reported frequency irrespective of grade; see The model also included other monthly medical service costs reflecting outpatient visits, interventions, and unplanned hospitalizations unrelated to AEs from a previous publication for the United States . These cThe dead state in the model included a one-time cost for end-of-life care . Unit costs for 1 month of end-of-life care were obtained from public sources for each country; see Non-health care costs included public payments for temporary sick leave during the progression-free state and for disability pension in the progressed state. Only patients of working age [defined to range from 18 years to the official retirement age for men and women in each country ] who couAs the model horizon covered up to 5 years, future health outcomes and costs were discounted with a 3% annual discount rate. All costs were expressed in US-dollars ($) per patient in 2021 prices and exchange rates using information from the World Bank  during the first year of treatment; see Although the total costs increased with sequential and multigene testing in all countries over a 5-year horizon, the different types of health care and non-health care costs included in the model exhibited different trajectories; see Medicine costs increased around 2–10-fold in most countries with sequential testing compared to no testing over a 5-year horizon. With multigene testing, medicine costs increased mostly by a factor of 14–58 compared to no testing. These large relative increases were primarily driven by older and low-cost off-patent chemotherapies being replaced with newer on-patent targeted therapies and immunotherapies. The longer administration time of newer medicines (often until disease progression or unacceptable toxicity) compared to chemotherapy (often a fixed number of treatment cycles) also contributed to the cost increase.Medicine administration costs decreased by up to 50% with sequential testing. This was because patients with targetable mutations switched from receiving intravenously administered medicines to oral medicines for which no administration costs arose. Administration costs with multigene testing over a 5-year horizon were higher than with no testing and sequential testing (except in China and Japan) despite a higher proportion of patients receiving oral targeted therapies. This was caused by patients without targetable mutations receiving intravenous immuno(chemo)therapy for a longer time than only chemotherapy.Costs of treatment-related AEs decreased with sequential and multigene testing. The decrease was particularly strong (64–72% decrease) in the first year with multigene testing. This mirrors the development seen in Other medical service costs for outpatient visits, interventions, and unplanned hospitalizations unrelated to AEs decreased with sequential testing (13–25%) and multigene testing (27–40%) in the first year but increased over the 5-year horizon  in all countries. The initial decrease was driven by lower monthly costs for these services in patients receiving targeted therapy and immuno(chemo)therapy. The subsequent increase was driven by the prolonged survival of patients with sequential and multigene testing.End-of-life care costs decreased in all countries by up to 33% with sequential testing and 58% with multigene testing in the first year, mirroring the increase in the absolute survival rate shown in The two types of non-health care costs were affected in different ways by increased biomarker testing. With sequential and multigene testing, sick leave payments increased already during the first year and continued to increase over the 5-year horizon  in all countries (except in the United States with no such payments), driven by prolonged survival. Disability pension payments decreased by 31–45% during the first year in all countries with multigene testing due to the prolonged progression-free disease phase. Yet these payments increased over the 5-year horizon with multigene testing (36–62%) as more patients maintained a survival advantage compared to the no-testing scenario.To confirm the robustness of the main results, a sensitivity analysis of the accuracy  of the biomarker tests in all testing scenarios was carried out; see The treatment possibilities of aNSCLC have changed radically with the introduction of numerous targeted therapies and immuno(chemo)therapy. The administration of these treatments requires a change from single biomarker testing to multigene testing. Multigene testing avoids overly long delays in treatment start and enables the administration of PM as initial therapy.The results in this study showed that multigene testing followed by the administration of matching PM improved various health outcomes compared to no testing or sequential testing in all countries. This included an improved survival rate, an increased number of life years, a prolonged progression-free disease phase, and a reduced number of treatment-related AEs. The largest improvements in life years were observed in East Asian countries due to a higher prevalence of targetable mutations in the local patient population. Multigene testing also improved treatment assignment (for patients with EGFR and ALK mutations) because the sensitivity and specificity of NGS testing was higher than the sensitivity/specificity of the single biomarker tests in the sequential testing scenario.For all countries, total costs increased with sequential testing and multigene testing compared to no testing, yet this development was not shared by all individual cost items. The testing costs for multigene testing were higher than for sequential testing, but the increase in testing costs was dwarfed by the increase in medicine costs. The latter was partly the result of on-patent medicines replacing off-patent chemotherapy medicines in all countries. The replacement of intravenously administered chemotherapy with oral agents reduced medicine administration costs in some countries. Health care costs for the treatment of AEs and end-of-life care also decreased with multigene testing. Non-health care costs decreased initially with multigene testing in some countries thanks to a prolonged progression-free disease phase but increased over time in line with overall survival.Clinical guidelines from the NCCN, ESMO, the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology recommend molecular testing in aNSCLC when feasible and to be performed with NGS , 25, 26.The value of NGS testing in aNSCLC is increasing with the identification of additional oncogenic drivers and the development of matching targeted therapies. The third scenario in the model included seven targets besides PD-L1, based on all US FDA approved first-line medicines. Additional targets that may become relevant in the coming years are KRAS, HER2, and PIK3CA –31. For Multigene testing is also set to become increasingly important in earlier stages of NSCLC for the adjuvant treatment after surgery. In 2020, the US FDA approved the first EGFR-targeted medicine in this setting , and resAs a partitioned survival model with three states was used, the assignment of patients in these states at different points in time by treatment is critical. The Kaplan-Meier curves of treatments used for this purpose typically had fewer than 5 years of follow-up. Weibull distributions were used to estimate all survival functions for the full 5-year horizon of the model, but these survival functions go to zero with increasing time. The functions do not capture survival plateaus that are indicative of cure well, leading to an underestimation of the true size of the survival outcomes. In addition, the model feeds on survival data from different clinical trials with somewhat heterogenous patient populations due to varying inclusion criteria across clinical trials.The analysis assumed that all patients are tested and would receive appropriate treatment corresponding to their test results. In clinical practice, not every patient with aNSCLC gets tested and not every tested patient will receive the appropriate medicine. A recent analysis in European countries indicated that the use of targeted therapy and immuno(chemo)therapy was below the level recommended in clinical guidelines .Medicine costs were the second largest cost item in the scenario with multigene testing in most countries. It is important to reiterate that the analysis drew on country-specific list prices of all medicines in the absence of publicly available net prices. List prices may deviate from net prices due to confidential rebates granted by pharmaceutical companies to the payer. For example, an analysis of branded prescription medicines in the United States showed that the average size of rebates increased markedly over the last decade, reaching over 50% of the list prices in 2019 . The medThe estimation of medicine costs for subsequent treatment lines in the progression state was approximated by assigning costs to all additional patients in the progressed health state compared to the previous cycle. This may have underestimated the medicine costs in the progressed state, as the difference did not take into account patients who had died in the previous cycle.Unit costs for biomarker tests, treatment of AEs, and other medical services were only obtained for the United States. The conversion of these costs to other countries took into account differences in PPP, but this should still be considered a crude conversion. The true costs of these three cost items in other countries could thus be either overestimated or underestimated. The unit costs of NGS testing were sourced from a recent publication for the year 2020 . These cThe use of PM in oncology is set to increase in the coming years thanks to advances in the understanding of molecular mechanisms and expressions involved in the growth of cancer tumors. A prime example is aNSCLC, where the number targetable mutations have already multiplied during the last decade. This development has created a need to improve biomarker testing, moving away from upfront single biomarker testing to multigene testing. Other cancer types might face similar circumstances in the near future.The example in this study illustrates that the gradual introduction of PM in aNSCLC improves health outcomes in countries around the world, with the largest improvements in survival observed in East Asia due to a higher prevalence of targetable mutations in the local patient population. Although the use of PM in aNSCLC leads to increased costs for biomarker testing and medicines in all countries, other health care costs are decreased, notably for the treatment of AEs and end-of-life care. Testing costs are also minimal in relation to medicine costs. Non-health care costs decrease in the first year after treatment start in some countries but increase subsequently. The overall results demonstrate the importance to apply a wider perspective in the assessment of the value of PM in oncology.The original contributions presented in this study are included in the article/TH and PL: conceptualization. TH and CM: data analysis and writing—original draft. All authors contributed to the data interpretation, writing—review and editing, and approved the submitted version.


Aedes spp and one pool of Anopheles spp collected from nine states were sequenced and metagenomic analysis was carried out. We identified a wide diversity of viruses belonging to various families in this sample set. Seven different viruses detected included: Fako virus, Phasi Charoen-like virus, Verdadero virus, Chaq like-virus, Aedes aegypti totivirus, cell fusing agent virus and Tesano Aedes virus. Although there are no reports of these viruses being pathogenic, they are an understudied group in the same families and closely related to known pathogenic arboviruses. Our study highlights the power of next generation sequencing in identifying Insect specific viruses (ISVs), and provide insight into mosquito vectors virome in Nigeria.Mosquito vectors are a tremendous public health threat. One in six diseases worldwide is vector-borne transmitted mainly by mosquitoes. In the last couple of years, there have been active Yellow fever virus (YFV) outbreaks in many settings in Nigeria, and nationwide, entomological surveillance has been a significant effort geared towards understanding these outbreaks. In this study, we used a metagenomic sequencing approach to characterize viruses present in vector samples collected during various outbreaks of Yellow fever (YF) in Nigeria between 2017 and 2020. Mosquito samples were grouped into pools of 1 to 50 mosquitoes, each based on species, sex and location. Twenty-five pools of  It is estimated that half of the world's population is in danger of viral illnesses spread by mosquitoes and causing death in millions of people annually. Rift Valley fever virus (RVF), Dengue virus (DENV), Zika virus (ZIKV), Chikungunya virus (CHIKV), Yellow fever virus (YFV), Japanese encephalitis virus (JEV), and Ross River virus (RRV) are just a few of the mosquito-borne viruses that have caused disease epidemics both in humans and animals. Additionally, an increasing number of viruses specific to arthropods, classified as insect-specific viruses (ISVs), have been identified in the last two decades in diverse mosquito populations around the world8. ISVs and pathogenic arboviruses evolutionary relationship remains uncertain9. However, evolution from arthropod-specific viruses has been assumed for the genus Flavivirus. Pathogenic viruses are thought to have evolved from insect-specific to dual host viruses12.One in six diseases worldwide is transmitted by mosquito vectors11. Recently, in Nigeria, metagenomic sequencing was used to identify an ongoing yellow fever outbreak and its aetiology and inform real-time public health actions, resulting in accurate and timely disease management and control13. A greater understanding of the virome in mosquito species in Nigeria could allow for a more accurate assessment of mosquito-borne disease risk, vector competence and mosquito management.Metagenomic sequencing has increased exponentially the number of mosquito-borne viruses isolated in the last couple of years and further provided fresh insight into the enormous complexity and variety of invertebrate RNA virusesAedes spp) in sites where there were active YFV cases. Next-generation sequencing (NGS) was carried out on 26 pools of 1300 mosquitoes (50 mosquitoes per pool) across nine (9) states in Nigeria using a metagenomic protocol as previously described14. In this paper we present our findings and discuss the implications.In the course of various YFV outbreaks in Nigeria between 2017 and 2020, we collected vector samples , Iflaviridae (Tesano Aedes virus), Partitiviridae (Chaq-like virus and Verdadero virus), Flaviviridae (Cell fusing agent virus) and Totiviridae (Aedes aegypti totivirus (AaTV) were detected.Metagenomics analysis carried out on sequenced samples revealed the presence of a wide range of viruses Fig. . The folAedes aegypti (n = 12 pools), Aedes albopictus (n = 10 pools), Aedes simpsoni complex (n = 2) Aedes luteocephalus and Anopheles coustani (n = 1 pool each) with Aedes aegypti and Aedes albopictus accounting for more than 80% of the mosquito pools analyzed. Of the 26 pools, irrespective of the mosquito species, the number of pools positive for Aedes aegypti totivirus (AaTV), cell fusing agent virus (CFAV), Chaq like-virus (CLV), Phasi Charoen like phasivirus (PCLV) (L segment), PCLV (M segment), PCLV (S segment), Verdadero virus (VdV), Fako virus (FKV) and Tesano Aedes virus (TeAV), were 7, 1, 3, 13, 13, 12, 2, 1 and 7 respectively . Table . Table 115 was attempted to characterize the diversity of these viruses and their evolutionary relationship with previously reported viruses. A heatmap showing the distribution of viruses is shown in Supplementary Fig. Genome assembly of viruses detected by our metagenomics pipelineAedes aegypti totivirus (AaTV) out of the seven pools with reads for this virus. Phylogenetic analysis revealed that the Aedes aegypti totivirus sequences from this study clustered closely together and fell in the same major clade. Within the major clade, the sequence from Kwara state (BIS 100c) branched out on its own while the Ebonyi sequences  clustered together, which could imply a localized/within-state spread of the virus. The short branch lengths of the sequences also show the limited diversity of the virus in Nigeria  genomes for Phylogenetic analysis revealed similar clustering patterns across the three segments of the virus in Nigeria. According to states, there is no observed clustering pattern showing the virus's limited diversity in the country Figs. , 4, 5.Fi, Chaq-like virus, Fako virus and Verdadero virus) were assembled from our sequenced data. We did not construct phylogenetic trees due to the lack of substantial genomes on NCBI for proper comparison. In which cases, only one to three genomes are available in the database.Other viruses  and a partial first segment out of the nine segments of the Fako virus; reovirus of the genus Dinovernavirus. The first report of the virus is in mosquito pools from Cameroun17. The virus has shown evidence of vertical transmission17. It increases the growth of Dengue virus 1 (DENV-1) in a concentration-dependent manner under laboratory conditions17. Members of this family are known to infect insects with no reports of human infection. On average, our sequences were 96% similar to the only sequence for the TeAV available on NCBI  for Tesano Aedes virus (TeAV) in mosquito pools. TeAV is a member of the Iflaviridae family and was first isolated from Mosquito samples in GhanaBI Table .Totiviridae family and a group of dsRNA viruses that infect fungi, protozoa, or invertebrates21. AaTV was highly distributed and present in mosquito pools from Kwara and Ebonyi States. Phylogenetic analysis of the virus genomes revealed that the Nigerian Strains clustered together in the same major clade, independent of the American/Asian/European lineages, implying continuous evolution and diversity of the virus  and medically important flaviviruses. This information could be valuable in understanding how ISFs enable/inhibit transmission of arboviruses in nature and their possible use as agents of biological control of vectors2. A study by Baidaliuk et al., 2019 evaluated how CFAV affects ZIKV and DENV-1 in vitro and vivo29. Their findings showed a negative correlation both in-vitro and in-vivo, indicating a decrease in transmission in both viruses due to the presence of CFAV.In addition, we had one partial genome assembly of a flavivirus; Cell fusing agent virus. Many medically important arboviruses belong to the Aedes aegypti larvae30. The phylogenetic tree based on the S segment (Nucleocapsid) and M segment (glycoprotein) displayed these segments as being in a separate cluster independent of previously detected lineages 32 during an arbovirus surveillance program. The relationship between PCLV and CHIKV transmission is still unknown and needs further investigation.Furthermore, we assembled 13 genomes for the L and M segments, as well as 12 for the S segment of Phasi Charoen-like-phasivirus (PCLV)- a bunyavirus first isolated from the Phasi Charoen district of Thailand from wild-caught es Figs. . However012 Fig. . We have34. In this study, three (3) Chaq-like viruses were found from Aedes spp pools. The BLASTn search results of the three sequences from our study showed high nucleotide sequence identity (98.7%) to strain CLv.PozaRica20 (MT742176.1) was isolated from Aedes aegypti in Mexico in 2020. Showing the virus may be widely disseminated in the mosquito vector. Only three sequences are available on NCBI for this virus as of 23rd of October 2021, with limited information.Partitiviruses are known to infect a vast host, including plants, fungi with some members of this genus recently discovered in arthropodsAedes aegypti colony from Poza Rica, Mexico. The name of the virus was derived from the Spanish word for “true” Verdadero7. There is no report on human infection by this virus. Generally, we did not observe any unique differences in viruses detected between male and female mosquito pools from the same collection site.Another Partivirus genome assembled in this study is the Verdadero virus Table ; Fig. 6./in vitro conditions.Generally, reports on the effects of ISVs on pathogenic arbovirus transmissions are controversial and conflicting from various studies and in vivo9. Furthermore, the discovery of ISVs within the families of pathogenic viruses provided insights into the evolution and adaptation of these groups of viruses35. For example, ISVs belonging to Flaviviridae and Bunyaviridae families are thought to be ancient viruses with distinct lineages that have evolved at the same time and diversified with their vector hosts37. Studies establishing vertical transmission38 and evidence of ISVs genomic sequence integration in the genome of insect vectors39 have supported the hypothesis. Against this background, many pathogenic arboviruses probably gained their dual-host range by an adaptive evolution process that conferred the ability to infect vertebrates to ISVs. There is limited information on the pathogenicity of ISVs to their insect host. Furthermore, ISVs were considered possible biological control agents for vectors and arboviruses of public health importance due to their characteristic lack of replication in mammalian cell lines. Given all these possible applications of ISVs, they are an exciting group of viruses for further investigations.Although ISVs cannot grow in mammalian cell lines, a study in Brazil isolated a novel insect-specific virus, Guapiaçu virus (GUAPV), from the plasma sample of a febrile person13 successfully. An approach targeted at enriching the Yellow fever virus in the mosquito vectors could have resulted in potential YFV assembly.We could not detect YFV from this study, possibly because of our small amount of virus in the vectors that could not be properly amplified in the vectors. This could also be due to limitations in the protocol we used to construct the genomic libraries. Although we have used the same protocol to sequence and assemble YFV in human samplesIdentifying these diverse groups of viruses (ISVs) from our study is a first step in applying local genomics capacity within the country for a holistic approach to disease outbreaks. Further investigation of the pathogenic potential of these viruses, how they enhance/inhibit transmission of circulating arboviruses in Nigeria needs to be carried out.40. Live adult mosquitoes collected in the field were immobilized using Ethyl Acetate. A total of 15 pools were mosquitoes collected at the immature stages and reared to the adult stage, while 11 pools were from mosquitoes collected directly as adults. These adult mosquitoes were unfed and hungry, as they were collected foraging for blood meal. All adult mosquitoes collected were morphologically identified to species level using keys of43 and then pooled based on species and sex into 50 mosquitoes per pool. They were then introduced into well-labelled Eppendorf tubes containing RNAlater. The tubes were stored in freezers for the duration of the surveillance to keep the samples genetically intact. Immature stages (pupae and larvae) are reared to adults before pooling.During active yellow fever virus outbreaks, mosquito samples were trapped across nine (9) states in Nigeria between 2017 and 2020 for metagenomic sequencing. Generally, three trapping methods were used in this study; egg, larval and adult collections. At each sampling point, the coordinates were taken using a global positioning system (GPS) gadget as described byAedes spp  and one pool of Anopheles coustani  pools were 14. Briefly, Vector pools were initially homogenized in 1 ml of cooled Dulbecco's Modified Eagle Medium (DMEM) (composition-500 ml DMEM High Glucose (4.5 g/I) with l-Glutamine), 1 ml Penicillin–Streptomycin, 15 ml Fetal Calf Serum (FCS) 3% and 5 ml Amphotericin B) and 500 ml of Zirconia beads . The contents were macerated for 10 min on the Qiagen Tissuelyser LT followed by centrifuging at 4500×g for 15 min. According to the manufacturer's instructions, the supernatant was further used for RNA extraction using the QIAamp Viral RNA extraction kit . Extracted RNA was turbo Dnased to remove contaminating DNA and cDNA synthesis was carried out according to the published protocol14. Sequencing libraries were made using the Illumina Nextera XT kit. Next generation sequencing using Illumina Miseq V2-500 cycle kit at 251 read length paired-end sequencing was carried out on Illumina Miseq at the African Centre of Excellence for Genomics of Infectious Diseases (ACEGID), Redeemer's University, Ede, Nigeria.A total of 1300 mosquitoes made into twenty-six (26) mosquito pools were sequenced based on the established unbiased protocol.We added nuclease-free water (NTC) as a negative control for each sample preparation step. Before subsequent downstream analysis, we checked the water control for possible contaminations. All sequencing reads taxonomically classified as a given virus were first checked against possible reads from the NTC. We did a reference mapping of our reads against reference genomes on NCBI to rule out false positives. For this study, we only report viruses for which genomes are assembledhttps://innovation.microsoft.com/en-us/premonition). We also carried out genome assembly of individual viruses detected using our publicly available viral-ngs44 and VGEA pipelines45.Bioinformatics analysis was carried out on the sequence data generated to identify viruses present in the samples. First, we demultiplexed individual libraries and removed reads mapping to the human genome or other known technical contaminants . Raw reads from the sequencing machine were passed through Microsoft’s Premonition metagenomics pipeline (https://www.ncbi.nlm.nih.gov/orffinder/) with a 300-nt minimum length and the genome structure was annotated using NCBI Conserved Domain Database version 3.19  with the NCBI viral genome database as references.Assembled genomes were annotated using ORFfinder  were obtained from GenBank. The evolutionary history was inferred using the Maximum Likelihood method and Tamura-Nei model47 with 1000 bootstrap replicates. Bootstrap support is indicated by values on branches. Evolutionary analysis was conducted in MEGA X version 10.1.848.Multiple alignments of nucleotide sequences and deduced amino acids with those of reference strains from GenBank were analyzed using the MAFFT online versionSupplementary Information.


Flavivirus, transmitted by Ixodes species, with groundhogs being the usual mammalian host. The virus is endemic to North America, with peak transmission during the summer and fall. The incubation period is 7–34 days, followed by a prodrome of flu-like symptoms. Although most infected individuals are asymptomatic, the virus can penetrate the CNS to produce a viral encephalitis. The key to the diagnosis is a positive serology.  Powassan is a positive-sense, single-stranded, enveloped RNA virus that is a tick-borne  The patient is a 62-year-old male with a past history of a right putamen infarct, hepatitis C, hypertension, and substance abuse who presented due to acute onset altered mental status, dysarthria, and left-sided facial droop. He had several tick bites around the time of presentation in December. He was empirically treated for possible meningitis, as CSF revealed WBC 370 (80% mononuclear cells); RBC 10, protein 152 mg/dL, and glucose 59 mg/dL. An MRI scan of the brain showed a subacute left putamen stroke. MRAs of the head and neck were unremarkable. A Mayo Clinic Encephalopathy Panel was unremarkable; however, a New York State Arbovirus panel revealed Powassan IgM ELISA as well as Powassan Polyvalent microsphere immunofluorescence assay reactivity. His hospital course was complicated by critical illness myopathy and respiratory failure requiring tracheostomy. Powassan virus is a known etiology for encephalitis in North America. Although the peak incidence of transmission is in the summer and fall, this does not exclude transmission during other seasons. Due to the increasing prevalence of Powassan virus in Lyme-endemic areas particularly in the Midwest and Northeast, United States, patients with an unexplained altered mental status in these regions should be screened for Powassan virus, regardless of the time of year.  The  Flavivirus, transmitted by Ixodes species, with groundhogs and other small- to medium-sized forest rodents being the usual mammalian host [Ixodes tick is most active [Powassan virus, they will present with encephalitis and altered sensorium [Powassan is a positive-sense , single-ian host –4. This t active . Howevert active . The inct active . Despitet active . When paensorium , 4.Routine laboratory studies are typically unremarkable; the cerebrospinal fluid will show a pleocytosis, which can be lymphocytic or polymononuclear predominant , 8.The gold standard for diagnosis is serological testing, particularly immunoglobulin M (IgM) antibody testing utilizing enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay . AlthougThere is insufficient data for standardized treatment, as the treatment is largely supportive. However, case reports suggest the usage of intravenous immunoglobulin and steroids , 3.Powassan virus, such as recurrent headaches, cognitive disruption, and focal neurological deficits [Roughly, 50% of patients will have long term neurological sequelae of deficits . It is edeficits .The patient is a 62-year-old male with a past medical history of a right putamen infarct, hepatitis C, hypertension, and substance abuse who initially presented due to altered mental status, dysarthria, and a left facial droop. He had several tick bites around the time of presentation in December.Magnetic resonance imaging (MRI) of the brain showed subtle acute to subacute left putamen infarct . MagnetiArbovirus Panel, given the patient's known tick exposure. This revealed a positive CSF Powassan IgM ELISA as well as Powassan Polyvalent microsphere immunofluorescence assay reactivity, which confirmed the presence of active Powassan virus in CSF, a surrogate to diagnose Powassan encephalitis.His hospitalization course was complicated by a recurrent stroke; his repeat MRI brain 2 weeks after presentation revealed a small foci of acute infarct involving cerebellum, left basal ganglia, and splenium of corpus callosum . A repeaUnfortunately, the patient required artificial airway and feeding access due to respiratory failure. At the time of discharge, notable neurological symptoms included global aphasia.Powassan virus is a known etiology for encephalitis in North America. Although the peak incidence of transmission of the virus is in the summer and fall when the Ixodes species is most active, this does not exclude transmission in other seasons [Powassan virus. seasons . As wint seasons . This suPowassan virus in Lyme-endemic areas , some experts have suggested screening for Powassan virus in patients with unclear CNS phenomena in Lyme-endemic areas, as the disease is underrecognized [Our patient's diagnosis demonstrates the importance of obtaining a thorough tick exposure history. Due to the increasing prevalence of cognized , 10. Thicognized .This case highlights the importance of obtaining Powassan serology in a patient with an unexplained altered mental status. It also demonstrates the importance of testing for the virus in the appropriate clinical scenario in Lyme-endemic areas, even outside of the normal tick season.