Ambispective epidemiological observational study of varicella-zoster virus infection: An 18 year-single-center Bulgarian experience

BACKGROUND Varicella(chickenpox)and herpes zoster(shingles)are outcomes of varicella-zoster virus(VZV)infection,and understanding their incidence trends is vital for public health planning.AIM To conduct an ambispective epidemiological study by analyzing the main epidemiological characteristics of VZV infection during an 18 year-period(2000-2018).METHODS We used descriptive and epidemiological methods to characterize chickenpox in Bulgaria,the city of Plovdiv and the region for a period of 18 years(2000-2018).RESULTS The average incidence of varicella-zoster infection for the period 2000–2018 in the Plovdiv region was estimated at 449.58‰.The highest relative share of the infection was assessed in the month of January at 13.6%,and the lowest in the months of August and September at 2.9%(both months).The age group most affected by the infection was 1-4 years,followed by 5-9 years.This corresponds to the so-called"pro-epidemic population"-a phenomenon typical for airborne infections,confirming their mass impact on the perpetuation of VZV infection.CONCLUSION Our findings reveal significant insights into VZV epidemiology,including age-specific incidence rates,clinical manifestations,and vaccination impact.This comprehensive analysis contributes to the broader understanding of VZV infec-tion dynamics and may inform evidence-based preventive measures.

Varicella-Zoster Virus Encephalitis with Hyponatremia in an Immunocompetent Elderly Patient:A Case Report

As a common cause of viral encephalitis,varicella-zoster virus(VZV)may invade the central nervous system of immunosuppressed patients during reactivation.Herein,we report a rare case of an immunocompetent patient with VZV encephalitis who developed severe hyponatremia and was considered to have a suspected primary infection.The patient was diagnosed with the support of second-generation sequencing and had persistent hyponatremia after being cured.Although rare,this case suggests that VZV encephalitis may occur in unexpected patients and present with unusual clinical manifestations,requiring advanced detection methods and clinical expertise for resolution.

Varicella-zoster virus-associated meningitis,encephalitis,and myelitis with sporadic skin blisters:A case report

BACKGROUND Varicella-zoster virus(VZV)generally causes chickenpox at first infection in childhood and then establishes latent infection in the dorsal root ganglia of the spinal cord or other nerves.Virus reactivation owing to an impaired immune system causes inflammation along spinal nerves from the affected spinal segment,leading to skin manifestations(herpes zoster).Viremia and subsequent hematogenous transmission and nerve axonal transport of the virus may lead to meningitis,encephalitis,and myelitis.One such case is described in this study.CASE SUMMARY A 64-year-old man presented with dysuria,pyrexia,and progressive disturbance in consciousness.He had signs of meningeal irritation,and cerebrospinal fluid(CSF)analysis revealed marked pleocytosis with mononuclear predominance and a CSF/serum glucose ratio of 0.64.Head magnetic resonance imaging revealed hyperintense areas in the frontal lobes.He had four isolated blisters with papules and halos on his right chest,right lumbar region,and left scapular region.Infected giant cells were detected using the Tzanck test.Degenerated epidermal cells with intranuclear inclusion bodies and ballooning degeneration were present on skin biopsy.Serum VZV antibody titers suggested previous infection,and the CSF tested positive for VZV-DNA.He developed paraplegia,decreased temperature perception in the legs,urinary retention,and fecal incontinence.The patient was diagnosed with meningitis,encephalitis,and myelitis and was treated with acyclovir for 23 days and prednisolone for 14 days.Despite gradual improvement,the urinary retention and gait disturbances persisted as sequelae.CONCLUSION VZV reactivation should be considered in differential diagnoses of patients with sporadic blisters and unexplained central nervous system symptoms.

Varicella-zoster virus meningitis with hypoglycorrhachia: A case report

BACKGROUND Varicella-zoster virus(VZV)is a common viral infection,but meningitis is a rare complication of VZV infection.The cerebrospinal fluid glucose of viral meningitis is usually within the normal range,which is different from bacteria,fungi,and cancerous meningitis.This paper reports a case of VZV meningitis with hypoglycorrhachia and the relevant literature was reviewed.CASE SUMMARY We report a case of an immunocompetent 39-year-old male,presenting with severe headache and fevers,without meningeal signs or exanthem,found to have VZV meningitis by the metagenomic next-generation sequencing of cerebrospinal fluid.The cerebrospinal fluid analysis revealed hypoglycorrhachia(cerebrospinal fluid glucose of 2.16)and he was treated successfully with intravenous acyclovir.Our literature review identified only ten cases diagnosed with VZV meningitis with hypoglycorrhachia previously reported to date in the English literature whose cerebrospinal fluid glucose was from 1.6 to 2.7mmol/L,with a ratio of cerebrospinal fluid to serum glucose from 0.30 to 0.49.CONCLUSION Although rare,the cerebrospinal fluid of patients with VZV meningitis may have hypoglycorrhachia,which broadens the understanding of the disease.

Microbiology laboratory and the management of motherchild varicella-zoster virus infection

Varicella-zoster virus, which is responsible for varicella(chickenpox) and herpes zoster(shingles), is ubiquitous and causes an acute infection among children, especially those aged less than six years. As 90% of adults have had varicella in childhood, it is unusual to encounter an infected pregnant woman but, if the disease does appear, it can lead to complications for both the mother and fetus or newborn. The major maternal complications include pneumonia, which can lead to death if not treated. If the virus passes to the fetus, congenital varicella syndrome, neonatal varicella(particularly serious if maternal rash appears in the days immediately before or after childbirth) or herpes zoster in the early years of life may occur depending on the time of infection. A Microbiology laboratory can help in the diagnosis and management of mother-child infection at four main times:(1) when a pregnant woman has been exposed to varicella or herpes zoster, a prompt search for specific antibodies can determine whether she is susceptible to, or protected against infection;(2) when a pregnant woman develops clinical symptoms consistent with varicella, the diagnosis is usually clinical, but a laboratory can be crucial if the symptoms are doubtful or otherwise unclear(atypical patterns in immunocompromised subjects, patients with post-vaccination varicella, or subjects who have received immunoglobulins), or if there is a need for a differential diagnosis between varicella and other types of dermatoses with vesicle formation;(3) when a prenatal diagnosis of uterine infection is required in order to detect cases of congenital varicella syndrome after the onset of varicella in the mother; and(4) when the baby is born and it is necessary to confirm a diagnosis of varicella(and its complications), make a differential diagnosis between varicella and other diseases with similar symptoms, or confirm a causal relationship between maternal varicella and malformations in a newborn.

Review of varicella-zoster virus infections in pregnant women and neonates

Even though varicella is rare in pregnancy, the disease can lead occasionally to disastrous illnesses for both the mother and her neonate. By contrast, normal zoster is not associated with special problems during pregnancy and peri- natal period. Pregnant women, who contract varicella, are at risk of varicella pneumonia which must be regarded as medical emergency. At any stage during pregnancy, chickenpox may cause intrauterine infection. The consequences for the fetus depend on the time of maternal disease. During the first two trimesters, maternal varicella may result in congenital varicella syndrome which may occur in nearly 2%. Typical symptoms are skin lesions in dermatomal distribution, neurological defects, eye diseases, and skeletal anomalies. Maternal infection near term is associated with a substantial risk of intrauterine acquired neonatal chickenpox in the neonate. If the mother develops varicella rash between day 4 (5) ante partum and day 2 post partum, generalized neonatal varicella leading to death in about 20% of the cases has to be expected. The present paper reviews the clinical consequences and the currently available concepts of prevention, diagnosis, and therapy of varicella-zoster virus infections during pregnancy.

Varicella-zoster virus meningitis after spinal anesthesia:A case report

BACKGROUND Headache is a common complication of regional anesthesia.The treatment of post spinal anesthesia headache varies depending on the cause.Although meningitis is rare,it can cause significant harm to the patient.Post dural puncture headache and septic meningitis are the most commonly suspected causes of post spinal anesthesia headache;however,other causes should also be considered.CASE SUMMARY A 69-year-old woman was scheduled for varicose vein stripping surgery under spinal anesthesia.The procedure was performed aseptically,and surgery was completed without any complications.After 4 d,the patient visited the emergency room with complaints of headache,nausea,and anorexia.Clinical examination revealed that the patient was afebrile.Considering the history of spinal anesthesia,post dural puncture headache and septic meningitis was initially suspected,and the patient was treated with empirical antibiotics.Subsequently,varicella-zoster virus PCR test result was positive,and all other test results were negative.The patient was diagnosed with meningitis caused by varicella-zoster virus and was treated with acyclovir for 5 d.The headache improved,and the patient was discharged without any problems.CONCLUSION Viral meningitis due to virus reactivation may cause headache after regional anesthesia.Therefore,clinicians should consider multiple etiologies of headache.

Dynamical study of varicella-zoster virus model in sense of Mittag-Leffler kernel

The primary varicella-zoster virus(VzV)infection that causes chickenpox(also known as varicella),spreads quickly among people and,in severe circumstances,can cause to fever and encephalitis.In this paper,the Mittag-Leffler fractional operator is used to examine the mathematical representation of the vzV.Five fractional-order differential equations are created in terms of the disease's dynamical analysis such as S:Susceptible,V:Vaccinated,E:Exposed,I:Infectious and R:Recovered.We derive the existence criterion,positive solution,Hyers-Ulam stability,and boundedness of results in order to examine the suggested fractional-order model's wellposedness.Finally,some numerical examples for the VzV model of various fractional orders are shown with the aid of the generalized Adams-Bashforth-Moulton approach to show the viability of the obtained results.

Insights into varicella-zoster virus assembly from the B-and C-capsid at near-atomic resolution structures

Varicella-zoster is a highly communicable virus that can be transmitted through the airborne route.About one quarter of people are infected with this virus.Previous studies have described the structure of A-capsid and a blurred reconstruction of the C-capsid with icosahedral symmetry.In this study,we have determined the more precise detailed structures of the varicella-zoster virus(VZV)B-and C-capsid in icosahedral symmetry using a combination of block-based reconstruction and symmetry relaxation strategies.In addition,we are reporting structural details of the portal vertex reconstructions in five-fold symmetry and portal reconstructions in twelve-fold symmetry.The structures unveil the basis for the high thermal stability of the VZV capsid.The conformational flexibility of structural elements of the capsid plays a role in the assembly of the capsid and drives processes critical for the viral life cycle.The results of the study open up new avenues for the development of drugs against a highly prevalent and contagious pathogen.

Immune Responses to Varicella-Zoster Virus Glycoprotein E Formulated with Poly(Lactic-co-Glycolic Acid) Nanoparticles and Nucleic Acid Adjuvants in Mice

The subunit herpes zoster vaccine Shingrix is superior to attenuated vaccine Zostavax in both safety and efficacy,yet its unlyophilizable liposome delivery system and the limited supply of naturally sourced immunological adjuvant QS-21 still need to be improved.Based on poly(lactic-co-glycolic acid)(PLGA)delivery systems that are stable during the lyophilization and rehydration process and using a double-emulsion(w/o/w)solvent evaporation method,we designed a series of nanoparticles with varicella-zoster virus antigen glycoprotein E(VZV-g E)as an antigen and nucleic acids including polyinosinic-polycytidylic acid(Poly I:C)and phosphodiester Cp G oligodeoxynucleotide(Cp G ODN),encapsulated as immune stimulators.While cationic lipids(DOTAP)have more potential than neutral lipids(DOPC)for activating g E-specific cell-mediated immunity(CMI)in immunized mice,especially when g E is encapsulated in and presented on the surface of nanoparticles,PLGA particles without lipids have the greatest potential to induce not only the highest g Especific Ig G titers but also the strongest g E-specific CMI responses,including the highest proportions of interferon-c(IFNc)-and interleukin-2(IL-2)-producing CD4?/CD8?T cells according to a flow cytometry assay and the greatest numbers of IFN-c-and IL-2-producing splenocytes according to an enzyme-linked immunospot(ELISPOT)assay.These results showed that immune-stimulating nucleic acids together with the PLGA delivery system showed promise as a safe and economical varicella and zoster vaccine candidate.

IFN-a production by human mononuclear cells infected with varicella-zoster virus through TLR9-dependent and-independent pathways

Understanding the defense mechanisms of the host of an organism is important for infection control.In previous studies,we demonstrated that interferon-a(IFN-a),but not IL-12,was produced by human peripheral blood mononuclear cells infected with varicella-zoster virus(VZV).Here,we investigated what kind of cell(s)and which signal molecule(s)are involved in IFN-a production.Using cell isolation and ELISA,we found that plasmacytoid dendritic cells(pDCs)were responsible for IFN-a production during VZV infection.We also found that Toll-like receptor 9(TLR9)was involved in VZV-induced IFN-a production because inhibitory CpG oligodeoxynucleotide inhibited IFN-a production.UV-inactivated VZV-induced IFN-a production was lower than that of active VZV,indicating another TLR9-independent pathway.Further studies demonstrated that double-stranded RNA-dependent protein kinase,but not DNA-dependent protein kinase was involved in VZV-induced IFN-a production.Together,these results suggest that pDCs play an important role in IFN-a production during VZV infection through TLR9-dependent and-independent pathways.

Varicella-zoster virus ORF7 interacts with ORF53 and plays a role in its trans-Golgi network localization

Varicella-zoster virus(VZV) is a neurotropic alphaherpesvirus that causes chickenpox and shingles. ORF7 is an important virulence determinant of VZV in both human skin and nerve tissues,however, its specific function and involved molecular mechanism in VZV pathogenesis remain largely elusive. Previous yeast two-hybrid studies on intraviral protein-protein interaction network in herpesviruses have revealed that VZV ORF7 may interact with ORF53, which is a virtually unstudied but essential viral protein. The aim of this study is to identify and characterize VZV ORF53, and to investigate its relationship with ORF7. For this purpose, we prepared monoclonal antibodies against ORF53 and, for the first time, characterized it as a ~40 k Da viral protein predominantly localizing to the trans-Golgi network of the infected host cell. Next, we further confirmed the interaction between ORF7 and ORF53 by co-immunoprecipitation and co-localization studies in both plasmid-transfected and VZV-infected cells. Moreover, interestingly, we found that ORF53 lost its trans-Golgi network localization and became dispersed in the cytoplasm of host cells infected with an ORF7-deleted recombinant VZV, and thus ORF7 seems to play a role in normal subcellular localization of ORF53. Collectively, these results suggested that ORF7 and ORF53 may function as a complex during infection, which may be implicated in VZV pathogenesis.

Truncated glycoprotein E of varicella-zoster virus is an ideal immunogen for Escherichia coli-based vaccine design

Varicella-zoster virus(VZV)is a highly infectious agent responsible for both varicella and herpes zoster disease.Despite high efficacy,there remain safety and accessibility concerns with the licensed vaccines.Here,we sought to produce a VZV g E immunogen using an E.coli expression system.We found that the soluble expression and yield of g E protein could be enhanced via C-terminal truncations to the protein,thereby facilitating a robust and scalable purification process for the purpose of vaccine manufacturing.The lead truncated g E(aa 31–358),hereafter referred to as tg E,was a homogenous monomer in solution and showed excellent antigenicity.Finally,we assessed and compared the immunogenicity of tg E with commercial v Oka LAV and Shingrix vaccine.We found that aluminum-adjuvanted tg E was immunogenic as compared with v Oka LAV.When adjuvanted with AS01B,a two-dose immunization of tg E showed comparable or better potency in antibody responses and cell-mediated immunity with those of the Shingrix vaccine at the same dosage,especially in terms of the proportion of IFN-γ-expressing CD4^(+)T cells.In conclusion,this method of E.coli-mediate tg E expression offers a cost-effective and scalable strategy to generate an ideal VZV g E immunogen for the development of both varicella and zoster vaccines.

Complicated multiple organ infection of Purpureocillium lilacinum and varicella-zoster virus infection in a patient with Evans’ syndrome

Purpureocillium lilacinum(P lilacinum)is a rare pathogenic fungus,which mainly involves immunocompromised individuals.Here,we report a case of complicated multiple-organ infections involving skin,lungs,and spleen in a 63-year-old female with Evans’syndrome after 9 months of glucocorticoid treatment.Microbial examinations of skin biopsy and blood samples revealed Plilacinum infections.Posaconazole was effectivein this patient.During anti-fungitreatment,she developedvaricella-zoster virus infection and was diagnosed through next-generation sequencing examination.In conclusion,Plilacinum may affect different organ systems and is susceptible to posaconazole treatment.The molecular-based methods like microbial cell-free DNA sequencing could provide accurate and timely identification of a wide range of infections.

Interleukin 1βreceptor and synaptic dysfunction in recurrent brain infection with Herpes simplex virus type-1

Several experimental evidence suggests a link between brain Herpes simplex virus type-1 infection and the occurrence of Alzheimer’s disease.However,the molecular mechanisms underlying this association are not completely understood.Among the molecular mediators of synaptic and cognitive dysfunction occurring after Herpes simplex virus type-1 infection and reactivation in the brain neuroinflammatory cytokines seem to occupy a central role.Here,we specifically reviewed literature reports dealing with the impact of neuroinflammation on synaptic dysfunction observed after recurrent Herpes simplex virus type-1 reactivation in the brain,highlighting the role of interleukins and,in particular,interleukin 1βas a possible target against Herpes simplex virus type-1-induced neuronal dysfunctions.

Varicella-zoster virus as a causative agent of acute retinal necrosis in younger patients

Background: Herpes virus is considered to be the pathogen of acute retinal necrosis (ARN) infection. Previous studies have that patients with ARN caused by the varicella-zoster virus (VZV) are often older, and patients with herpes simplex virus (HSV) induced ARN are considerably younger. However, in our clinical work, we find that VZV is also a pathogen in younger ARN patients. We, therefore, aimed to analyze the common etiology of younger ARN patients. Methods: A retrospective analysis was made of 20 eyes (18 patients) diagnosed as having ARN in the Department of Ophthalmology of Peking Union Medical College Hospital from 2014 to 2016. All patients were reviewed for demographic data, clinical course, clinical manifestations, time from onset to initial physician visit, duration of follow-up, visual acuity at both presentation and final visit, and treatment strategies. A paired t test was used to compare visual acuity between the presenting vision and those of final follow-up. Vitreous or aqueous specimens from 18 eyes of 18 patients were analyzed with multiplex polymerase chain reaction (mPCR)/quantitative PCR (qPCR) and xTAG-liquid chip technology (xTAG-LCT) to determine the causative virus of ARN. Results: Final best visual acuity (BCVA) improved significantly from 1.36±0.95 (median 20/400) to 0.95±0.82 (median 20/100)￠=2.714, P = 0.015) after systemic and intravitreal antiviral treatment combined with or without pars plana vitrectomy. PCR and xTAG-LCT results showed four of the five samples in the younger group (32.2±5.2 years) and 12 of the 13 samples in the senior group (53.6±4.9 years) were positive for VZV, and two of the five samples in the younger group were positive for HSV-1. Conclusions: This study demonstrates that VZV is also a common causative virus for ARN in younger patients. Considering this finding, a systemic antiviral treatment protocol should be immediately changed to intravenous ganciclovir when the patient does not respond to acyclovir before determining the causative virus, especially in younger patients.

LNP-CpG ODN-adjuvanted varicella-zoster virus glycoprotein E induced comparable levels of immunity with Shingrix^(TM) in VZV-primed mice

Latent varicella-zoster virus(VZV)may be reactivated to cause herpes zoster,which affects one in three people during their lifetime.The currently available subunit vaccine Shingrix^(TM) is superior to the attenuated vaccine Zostavax®in terms of both safety and efficacy,but the supply of its key adjuvant component QS21 is limited.With ionizable lipid nanoparticles(LNPs)that were recently approved by the FDA for COVID-19 mRNA vaccines as carriers,and oligodeoxynucleotides containing CpG motifs(CpG ODNs)approved by the FDA for a subunit hepatitis B vaccine as immunostimulators,we developed a LNP vaccine encapsulating VZV-glycoprotein E(gE)and CpG ODN,and compared its immunogenicity with Shingrix^(TM) in C57BL/6J mice.The results showed that the LNP vaccine induced comparable levels of gE-specific IgG antibodies to Shingrix^(TM) as determined by enzymelinked immunosorbent assay(ELISA).Most importantly,the LNP vaccine induced comparable levels of cellmediated immunity(CMI)that plays decisive roles in the efficacy of zoster vaccines to Shingrix^(TM) in a VZVprimed mouse model that was adopted for preclinical studies of Shingrix^(TM) .Number of IL-2 and IFN-γsecreting splenocytes and proportion of T helper 1(Th1)cytokine-expressing CD4^(+)T cells in LNP-CpG-adjuvanted VZV-gE vaccinated mice were similar to that of Shingrix^(TM) boosted mice.All of the components in this LNP vaccine can be artificially and economically synthesized in large quantities,indicating the potential of LNP-CpGadjuvanted VZV-gE as a more cost-effective zoster vaccine.

Role of viruses in periodontitis:An extensive review of herpesviruses,human immunodeficiency virus,coronavirus-19,papillomavirus and hepatitis viruses

Periodontitis is the inflammation of the supporting structures around the dentition.Several microbial agents,mostly bacteria,have been identified as causative factors for periodontal disease.On the other hand,oral cavity is a rich reservoir for viruses since it contains a wide variety of cell types that can be targeted by viruses.Traditionally,the focus of research about the oral flora has been on bacteria because the most widespread oral diseases,like periodontitis and dental caries,are outcomes of bacterial infection.However,recently and especially after the emergence of coronavirus disease 2019,there is a growing tendency toward including viruses also into the scope of oral microbiome investigations.The global high prevalence of periodontitis and viral infections may point out to a concomitant or synergistic effect between the two.Although the exact nature of the mechanism still is not clearly understood,this could be speculated through the manipulation of the immune system by viruses;hence facilitating the furthermore colonization of the oral tissues by bacteria.This review provides an extensive and detailed update on the role of the most common viruses including herpes family(herpes simplex,varicella-zoster,Epstein-Barr,cytomegalovirus),Human papillomaviruses,Human immunodeficiency virus and severe acute respiratory syndrome coronavirus 2 in the initiation,progression and prognosis of periodontitis.

Alterations in the gut microbiome after transjugular intrahepatic portosystemic shunt in patients with hepatitis B virus-related portal hypertension

BACKGROUND Gut microbiota(GM)affects the progression and response to treatment in liver diseases.The GM composition is diverse and associated with different etiologies of liver diseases.Notably,alterations in GM alterations are observed in patients with portal hypertension(PH)secondary to cirrhosis,with hepatitis B virus(HBV)infection being a major cause of cirrhosis in China.Thus,understanding the role of GM alterations in patients with HBV infection-related PH is essential.AIM To evaluate GM alterations in patients with HBV-related PH after transjugular intrahepatic portosystemic shunt(TIPS)placement.METHODS This was a prospective,observational clinical study.There were 30 patients(with a 100%technical success rate)recruited in the present study.Patients with esophagogastric variceal bleeding due to HBV infection-associated PH who underwent TIPS were enrolled.Stool samples were obtained before and one month after TIPS treatment,and GM was analyzed using 16S ribosomal RNA amplicon sequencing.RESULTS One month after TIPS placement,8 patients developed hepatic encephalopathy(HE)and were assigned to the HE group;the other 22 patients were assigned to the non-HE group.There was no substantial disparity in the abundance of GM at the phylum level between the two groups,regardless of TIPS treatment(all,P>0.05).However,following TIPS placement,the following results were observed:(1)The abundance of Haemophilus and Eggerthella increased,whereas that of Anaerostipes,Dialister,Butyricicoccus,and Oscillospira declined in the HE group;(2)The richness of Eggerthella,Streptococcus,and Bilophila increased,whereas that of Roseburia and Ruminococcus decreased in the non-HE group;and(3)Members from the pathogenic genus Morganella appeared in the HE group but not in the non-HE group.CONCLUSION Intestinal microbiota-related synergism may predict the risk of HE following TIPS placement in patients with HBVrelated PH.Prophylactic microbiome therapies may be useful for preventing and treating HE after TIPS placement.

Emerging role of liquid biopsy in rat sarcoma virus mutated metastatic colorectal cancer:A case report

BACKGROUND In patients with metastatic colorectal cancer(mCRC),the treatment options are limited and have been proved to be affected by rat sarcoma virus(RAS)mutational status.In RAS wild-type(wt)patients,the combination of antiepidermal growth factor receptor(EGFR)monoclonal antibodies with chemotherapy(CT)is more effective than CT alone.On the other hand,RAS-mutated patients are not eligible for treatment with anti-EGFR antibodies.CASE SUMMARY Eleven patients with initially RAS-mutated mCRC were followed from diagnosis to May 2022.At the time of cell-free DNA determination,five patients had undergone one CT line,five patients had undergone two CT lines,and one patient had undergone three CT lines(all in combination with bevacizumab).At the second and third treatment lines[second line(2L),third line(3L)],patients with neo-RAS wt received a combination of CT and cetuximab.In neo-RAS wt patients treated with anti-EGFR,our findings indicated an increase in progression-free survival for both 2L and 3L(14.5 mo,P=0.119 and 3.9 mo,P=0.882,respectively).Regarding 2L overall survival,we registered a slight increase in neo-RAS wt patients treated with anti-EGFR(33.6 mo vs 32.4 mo,P=0.385).At data cut-off,two patients were still alive:A RAS-mutated patient undergoing 3L treatment and a neo-RAS wt patient who received 2L treatment with anti-EGFR(ongoing).CONCLUSION Our case series demonstrated that monitoring RAS mutations in mCRC by liquid biopsy may provide an additional treatment line for neo-RAS wt patients.