HSV-1 DNA Research Articles

A-297 Feasibility of a novel Seegene Multiplex PCR panel for the simultaneous detection of HSV-1, HSV-2, VZV and Mpox: a focus on inclusion, confirmation and LOD assessment

Abstract Background Characterized by atypical presentations of short prodromal symptoms, variations in clinical staging, non-uniform presentation of rash, and varied syndromes of proctitis and pharyngitis, the WHO declared the 2022 Mpox outbreak a global public health emergency in July 2022. The overlap of clinical presentation with other causes of vesicular lesions identified gaps in the diagnostic tests available for MPXV and highlighted the need for a multiplex PCR approach to the diagnosis of HSV, VZV, and MPXV among patients presenting with vesicular lesions. Currently, no FDA cleared multiplex approach offering a single unified approach to the diagnosis of HSV, VZV, and MPXV from a single sample exists. Multiplex assays offer a role in diagnostic support where clinicians are challenged to determine the causative agent because of overlap. Furthermore, no assay capable of providing MPXV clade II reporting, needed for compliance to select agent reporting rules, exist for routine clinical labs. This study outlines the initial feasibility of Seegene’s multiplex HSV, VZV, MPXV PCR assay, highlighting the LOD assessment from the 1st WHO International Standards, ZeptoMetrix® NATtrolTM Controls, and custom plasmids. Methods Gene targets representing conserved, differentiated regions of HSV-1, HSV-2, VZV, MPXV, and MPXV clade II were identified through phylogenetic analysis. A total of 28 oligonucleotide combinations, were applied to Seegene’s proprietary amplification and detection process creating a multiplex assay capable of 8 unique detection signals in 5 channels using the BioRad Opus® 96. 1st WHO International Standards (IS) for HSV-1/2, VZV, ZeptoMetrix® NATtrolTM and custom designed plasmids controls were titrated and spiked into Copan UTM supplemented with 5% HeLa cells and used to define target LOD panels ranging in concentrations tested with multiple replicates. Sample processing, DNA extraction, and PCR amplification were performed on the Seegene Starlet® automated liquid handler and BioRad® OPUS 96 using Seegene software. Results Detection rates for HSV-1 at target concentrations of 1x102 IU/ml (WHO) and 6.17x100 copies/ml (NATtrolTM) were 100%. Detection of HSV-1 with plasmid DNA at target concentrations of 1.25x101 was 95%. Detection rates for HSV-2 were 100% at 5x102 IU/ml (WHO) and 5.56x101 copies/ml (NATtrolTM). Detection of HSV-2 with plasmid DNA at target concentrations 2.5E1 was 100%. For VZV, detection rates of 100% were observed at LOD challenge pools comprising 1x102 IU/ml (WHO) and 1.85x101 copies/ml (NATtrolTM). Detection of VZV with plasmid DNA at target concentrations 2.5x101 was 95%. For MPXV, detection rates of 100% were observed when challenged with LOD pools at 1.67x101 copies/ml respectively. Detection of MPXV with plasmid DNA at target concentrations 6.25x100 was 95%. Confirmation of clade II reporting for MPXV was determined using phylogenetic analysis. Conclusions Standardize of Seegene’s multiplex HSV-1, HSV-2, VZV, MPXV assay using traceable quantitated standard resulted in >95% detection rates of 6.17x100 copies/ml for HSV-1, 5.56x101 copies/ml for HSV-2, 1.85x101 copies/ml for VZV, and 1.67x101 copies/ml for MPXV respectively. A single multiplex PCR assay offers advantages for the simultaneous detection of HSV, VZV, and MPXV DNA in lesions specimens where clinical features present overlap and in patients whose clinical history is difficult to obtain.

A nonhuman primate model for genital herpes simplex virus 2 infection that results in vaginal vesicular lesions, virus shedding, and seroconversion.

The most commonly used animal models for evaluating the efficacy of HSV-2 candidate vaccines are mice and guinea pigs. While numerous HSV-2 vaccine candidates have been tested in these animals and were effective in reducing disease and mortality, these results did not predict the effectiveness of the vaccines in human trials. Infection of rhesus macaques rarely results in lesions or HSV-2 specific antibody responses. In seeking an animal model that better recapitulates human disease and that might be more predictive of the efficacy of prophylactic vaccines than mice and guinea pigs, we evaluated Cebus apella (C. apella), a New World primate, in an HSV-2 genital infection model. Infectious HSV-2 was cultured from vaginal swabs from all 4 animals for 9-14 days after intravaginal inoculation of HSV-2 seronegative monkeys. Two of 4 monkeys had vesicular lesions in the vagina or vulva. No neurological symptoms were noted. Recurrent lesions and HSV-2 DNA shedding after acute disease resolved was infrequent. UV irradiation of the genital area did not induce recurrent genital lesions or virus shedding. All 4 monkeys developed HSV-2 neutralizing antibodies as well as virus-specific CD4 and CD8 T cell responses. Reinfection of animals 15 to 19 months after primary infection did not result in lesions; animals had reduced virus shedding and a shorter duration of shedding compared with that during primary infection, suggesting that primary infection induced protective immunity. Primary fibroblasts from C. apella monkeys supported the growth of HSV-2 in vitro; in contrast, HSV-2 did not replicate above the titer of the input inoculum in fibroblasts from rhesus macaques. These observations suggest that the C. apella monkey has potential to serve as a model for evaluating the efficacy of prophylactic vaccines, antivirals, or monoclonal antibodies to HSV-2.

Comparison of the Accuracy of HSV1 and HSV2 Antibody Tests with PCR in the Diagnosis of Recurrent Genital Herpes.

To assess the accuracy of HSV1and HSV2 antibody testing in identifying genital herpes infection. A cohort of 299 patients previously diagnosed with recurrent genital herpes, confirmed via PCR, were tested using ELISA for HSV1 and HSV2 IgM and IgG antibodies. The study compared the accuracy of HSV1 and HSV2 antibody tests in diagnosing genital herpes. Among 299 patients, 14 tested positives for HSV1 DNA. Of these, 9 had HSV1 IgG antibodies, but none had HSV2 IgG antibody. Among 278 patients with HSV2 DNA, 149 had HSV1 IgG, 9 had HSV2 IgG, and 97 had both. Seven patients had both HSV1 and HSV2 DNA; 3 had HSV1 IgG, 1 had HSV2 IgG, and 3 had both. The accuracy of HSV1 IgG for HSV1 infection was 64.2%, and for HSV1 and HSV2 co-infection, 85.7%. The accuracy of HSV2 IgG for HSV2 infection was 38.1%, and for HSV1 and HSV2 co-infection, 57.1%. The combined antibody positivity accuracy was 34.9%. Genital herpes is primarily caused by HSV2 (92.98%). A smaller percentage is HSV1 (4.67%) or co-infection (2.34%). Despite relatively low diagnostic accuracy (34.9-85.7%) for antibody detection, combined antibody testing is necessary. Herpes DNA testing is recommended for accurate diagnosis. Absence of antibodies does not rule out genital herpes and clinical assessment is essential.

Gene editing for latent herpes simplex virus infection reduces viral load and shedding in vivo

Anti-HSV therapies are only suppressive because they do not eliminate latent HSV present in ganglionic neurons, the source of recurrent disease. We have developed a potentially curative approach against HSV infection, based on gene editing using HSV-specific meganucleases delivered by adeno-associated virus (AAV) vectors. Gene editing performed with two anti-HSV-1 meganucleases delivered by a combination of AAV9, AAV-Dj/8, and AAV-Rh10 can eliminate 90% or more of latent HSV DNA in mouse models of orofacial infection, and up to 97% of latent HSV DNA in mouse models of genital infection. Using a pharmacological approach to reactivate latent HSV-1, we demonstrate that ganglionic viral load reduction leads to a significant decrease of viral shedding in treated female mice. While therapy is well tolerated, in some instances, we observe hepatotoxicity at high doses and subtle histological evidence of neuronal injury without observable neurological signs or deficits. Simplification of the regimen through use of a single serotype (AAV9) delivering single meganuclease targeting a duplicated region of the HSV genome, dose reduction, and use of a neuron-specific promoter each results in improved tolerability while retaining efficacy. These results reinforce the curative potential of gene editing for HSV disease.

A novel multiplex real-time PCR assay for the detection of cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1/2 and strategies for application to blood screening

A multiplex real-time PCR has been developed to simultaneously detect transfusion-transmissible pathogens cytomegalovirus, Epstein-Barr virus and herpes simplex virus, as well as to provide sample quality testing, for the conserved regions of the cytomegalovirus UL123 gene, the Epstein-Barr virus BKRF1 gene, and the herpes simplex virus 1/2 UL30 gene, tested on 500 blood donors and 320 transfusion recipients. The laboratory sensitivities for all 3 pathogens were 100 copies/μL. Compared to the commercial real-time PCR reference kit, the multiplex real-time PCR assay for the detection of CMV, EBV and HSV presented 100% consistency. In 820 whole blood samples, the multiplex real-time PCR assay identified 34 (4.15%) positive for CMV DNA, 15 (1.83%) positive for EBV DNA, and 6 (0.73%) positive for HSV DNA. For blood transfusions in high-risk groups, whole blood herpes virus test should be included in the spectrum of pathogen testing for blood donors and recipients.

Instantaneous Inactivation of Herpes Simplex Virus by Silicon Nitride Bioceramics.

Hydrolytic reactions taking place at the surface of a silicon nitride (Si3N4) bioceramic were found to induce instantaneous inactivation of Human herpesvirus 1 (HHV-1, also known as Herpes simplex virus 1 or HSV-1). Si3N4 is a non-oxide ceramic compound with strong antibacterial and antiviral properties that has been proven safe for human cells. HSV-1 is a double-stranded DNA virus that infects a variety of host tissues through a lytic and latent cycle. Real-time reverse transcription (RT)-polymerase chain reaction (PCR) tests of HSV-1 DNA after instantaneous contact with Si3N4 showed that ammonia and its nitrogen radical byproducts, produced upon Si3N4 hydrolysis, directly reacted with viral proteins and fragmented the virus DNA, irreversibly damaging its structure. A comparison carried out upon testing HSV-1 against ZrO2 particles under identical experimental conditions showed a significantly weaker (but not null) antiviral effect, which was attributed to oxygen radical influence. The results of this study extend the effectiveness of Si3N4's antiviral properties beyond their previously proven efficacy against a large variety of single-stranded enveloped and non-enveloped RNA viruses. Possible applications include the development of antiviral creams or gels and oral rinses to exploit an extremely efficient, localized, and instantaneous viral reduction by means of a safe and more effective alternative to conventional antiviral creams. Upon incorporating a minor fraction of micrometric Si3N4 particles into polymeric matrices, antiherpetic devices could be fabricated, which would effectively impede viral reactivation and enable high local effectiveness for extended periods of time.

Metagenomic search of viral coinfections in herpes simplex encephalitis patients

Little is known about concomitant central nervous system (CNS) infections by more than one virus. Current diagnostics are based on molecular tests for particular pathogens making it difficult to identify multi-viral infections. In the present study, we applied DNA- and RNA-based next-generation sequencing metagenomics (mNGS) to detect viruses in cerebrospinal fluids from 20 patients with herpes simplex encephalitis. Coinfection was detected in one patient: sequences in cerebrospinal fluids matched enterovirus A (2.660 reads; 4% of recovered genome) and enterovirus B (1.571 reads; 13% of recovered genome). Subsequent PCR combined with serotyping allowed to identify human echovirus 6, a representative of enterovirus B. Several other mNGS hits (human pegivirus, Merkel cell polyomavirus, human papillomavirus type 5) were not considered to represent a genuine signal as they could not be confirmed by specific RT-PCR/PCR. HSV DNA, while being detectable by PCR in every patient, was detected by mNGS in only one. In conclusion, contaminations and false signals may complicate mNGS interpretation; however, the method can be useful in diagnostics of viral coinfections in CNS, particularly in the case of rare pathogens.

A Trivalent HSV-2 gC2, gD2, gE2 Nucleoside-Modified mRNA-LNP Vaccine Provides Outstanding Protection in Mice against Genital and Non-Genital HSV-1 Infection, Comparable to the Same Antigens Derived from HSV-1.

HSV-1 disease is a significant public health burden causing orofacial, genital, cornea, and brain infection. We previously reported that a trivalent HSV-2 gC2, gD2, gE2 nucleoside-modified mRNA-lipid nanoparticle (LNP) vaccine provides excellent protection against vaginal HSV-1 infection in mice. Here, we evaluated whether this HSV-2 gC2, gD2, gE2 vaccine is as effective as a similar HSV-1 mRNA LNP vaccine containing gC1, gD1, and gE1 in the murine lip and genital infection models. Mice were immunized twice with a total mRNA dose of 1 or 10 µg. The two vaccines produced comparable HSV-1 neutralizing antibody titers, and surprisingly, the HSV-2 vaccine stimulated more potent CD8+ T-cell responses to gE1 peptides than the HSV-1 vaccine. Both vaccines provided complete protection from clinical disease in the lip model, while in the genital model, both vaccines prevented death and genital disease, but the HSV-1 vaccine reduced day two vaginal titers slightly better at the 1 µg dose. Both vaccines prevented HSV-1 DNA from reaching the trigeminal or dorsal root ganglia to a similar extent. We conclude that the trivalent HSV-2 mRNA vaccine provides outstanding protection against HSV-1 challenge at two sites and may serve as a universal prophylactic vaccine for HSV-1 and HSV-2.

Association of Herpesvirus and Periodontitis: A Clinical and Laboratorial Case-Control Study.

A significant influence of the Herpesviridae family in the progression of periodontal disease has been suggested. The aim of this study was to investigate the potential association of four Herpesviruses (HSV-1, HSV-2, cytomegalovirus [CMV], and Epstein-Barr virus [EBV]) with periodontal disease using a qualitative test for evaluating the presence or absence of viral DNA in crevicular fluid samples of both healthy periodontal patients and periodontal compromised patients. A case-control study was conducted in 100 participants at a university clinic. A qualitative test was used for evaluating the presence/absence of viral DNA in crevicular fluid samples of both healthy periodontal patients and periodontal compromised patients, and considering the periodontitis staging (stage II, stage III, and stage IV) and grading (grade A, grade B, and grade C). The distribution of the same exposure variables to the periodontitis staging and grading was compared using Chi-square, Fisher's exact, and Gamma tests depending on the variable characteristics. The significance level was set at 5%. The association of the variables: age, sex, diabetes, smoking, alcohol, and oral hygiene was also considered. The prevalence of Herpesviridae family virus DNA was 6% for the periodontal healthy group and 60% for the periodontitis group (roughly 60% on periodontitis stages II, III, and IV, p <0.001; and twofold increase in moderate and rapid progression grades compared with the slow progression grade, p <0.001). HSV1 DNA was prevalent in all periodontitis stages and grades. HSV 2, EBV, and CMV DNA had increasing prevalence rates in more severe stages (stages III and IV, p <0.001); while considering periodontitis grade, HSV2 (p = 0.001), CMV (p = 0.019) and EBV (p <0.001) DNA were prevalent only in grades B and C, with EBV DNA registering a marked prevalence in grade C. A significant different distribution of Herpesviridae virus DNA per each stage of disease was registered.

Single therapeutic dose of an antiviral UL29 siRNA swarm diminishes symptoms and viral load of mice infected intranasally with HSV-1.

Herpes simplex virus type 1 (HSV-1) is a human pathogen that causes recurrent infections. Acyclovir-resistant strains exist and can cause severe complications, which are potentially untreatable with current therapies. We have developed siRNA swarms that target a 653 base pair long region of the essential HSV gene UL29. As per our previous results, the anti-UL29 siRNA swarm effectively inhibits the replication of circulating HSV strains and acyclovir-resistant HSV strains in vitro, while displaying a good safety profile. We investigated a single intranasal therapeutic dose of a siRNA swarm in mice, which were first inoculated intranasally with HSV-1 and given treatment 4h later. We utilized a luciferase-expressing HSV-1 strain, which enabled daily follow-up of infection with in vivo imaging. Our results show that a single dose of a UL29-targeted siRNA swarm can inhibit the replication of HSV-1 in orofacial tissue, which was reflected in ex vivo HSV titers and HSV DNA copy numbers as well as by a decrease in a luciferase-derived signal. Furthermore, the treatment had a tendency to protect mice from severe clinical symptoms and delay the onset of the symptoms. These results support the development of antiviral siRNA swarms as a novel treatment for HSV-1 infections.

Molecular and Serological Epidemiology of Herpes Simplex Virus Type 1 and 2 in Pregnant Women of Gorgan City, North East of Iran.

As one of the most widespread sexually transmitted infections, Herpes Simplex Virus (HSVs) globally account for 60-95% of persistent infections in adults. This infection is prevalent in women of gestational age and is likely to be transmitted from the infected mother to her neonate. Additionally, it gives rise to devastating complications in neonates. This study was designed to estimate the molecular and serological prevalence of HSV-1 and 2 in pregnant women of Gorgan city, North East of Iran. Vaginal secretions and blood specimens of 315 pregnant women referred to an educational hospital in the North east of Iran were tested for HSV-1 and HSV-2 using multiplex PCR and ELISA assays. Chi-Square test was utilized to evaluate the association of qualitative variables and the level of significance was set at p≤0.05. Moreover, statistical analysis was performed using SPSS V.19.0. HSV-1 and HSV-2 DNA was detected in 5.7% and 8.3% of participants, respectively. Given the serological analyses of total HSV-1 and HSV-2 antibodies, 92.7% (239/315) of patients were IgG positive and 5.4% (17/315) were IgM positive. The rate of HSV-1 and 2 in the present study was lower than that reported by World Health Organization (WHO). This study emphasizes the conduction of further investigations on HSVs since these viruses are probably playing significant role in sexually transmitted infections.

Investigating the Role of Adenovirus, Herpes Simplex 1, 2, and Varicella Zoster Virus in Incidence of Conjunctivitis in Tehran, Iran

"Conjunctivitis is one of the most common ocular diseases across the world. In this study, the prevalence of viral Conjunctivitis in patients who were referred to the Eye specialized Hospitals was evaluated. Some of the most important viruses that cause conjunctivitis are Human Adenoviruses (HAdV), Herpes Simplex 1, 2 (HSV-1, HSV-2), and Varicella Zoster Virus (VZV). The clinical diagnosis of eye viral infections is difficult and often controversial. Thus, research to experiment with these causative factors is of pivotal importance. In this cross-sectional study, 200 conjunctival swab samples were collected from symptomatic patients with signs of conjunctivitis who had been referred to two hospitals. After DNA extraction, Multiplex Real-Time PCR was carried out in two separate reactions for each sample. Among 200 collected samples, 34 (17%) and 8 (4%) were positive for Adenovirus and HSV-1 DNA, respectively. There were no HSV-2 and VZV conjunctivitis. No significant correlation was identified between gender and conjunctivitis (P-value: 0.845); however, there was a close correlation between age and conjunctivitis (Pvalue: 0.05). In this study, 66.3% of Adenoviral conjunctivitis occurs in the summer compared to 33.7% in spring. No significant seasonal variation was observed for HSV-1. Our results showed that adenovirus has a key role in causing viral conjunctivitis. "

A Rare Case of Anti-N-Methyl-D-Aspartate Receptor Encephalitis in an Infant Presenting with Regression and Movement Disorder.

A Rare Case of Anti-N-Methyl-D-Aspartate Receptor Encephalitis in an Infant Presenting with Regression and Movement Disorder.

373. Development of multiplex loop-mediated isothermal amplification (LAMP) based on a microfluidic chip for detection of HSV-1 and 2 and VZV.

Abstract Background In the era of universal varicella vaccination, point-of-care testing for herpes simplex viruses 1 and 2 (HSV-1, HSV-2) and varicella-zoster virus (VZV) is becoming more important because of the increase in the number of breakthrough varicella cases. The Loop-mediated isothermal amplification (LAMP) method is a reliable method for rapid diagnostic tests for infectious diseases. Meanwhile, microfluidics technology is revolutionary in total system miniaturization, including facile parallelization through multiplexing and process-reduced reagent volumes. It is easy to create miniaturized chips containing multiple chambers according to the needs. In this study, we sought to develop multiplex LAMP for the detection of HSV-1, HSV-2, and VZV DNA using in-house microfluidic chips. Methods The polydimethylsiloxane-based chip consists of microfluidic channels and 5 reaction chambers. The specific primers were pre-spotted and dried in each chamber. The condition of LAMPs and each specific primer were described previously. The sensitivity of microfluidic LAMPs was confirmed using plasmids that contain the target sequences. In order to assess the reliability of clinical use, 35 swab samples (without DNA extraction) collected from patients with vesicular skin lesions were used. The result of microfluidic multiplex LAMP was compared with conventional LAMPs and real-time PCRs. Results Detection limits of HSV-1 and 2 and VZV LAMP-based on a microfluidic chip were 500 copies per reaction. Among 35 swab samples, HSV-1, HSV-2, and VZV DNA were detected in 10 (28.6%), 4 (11.4%), and 12 (34.3%) samples, respectively. The results of microfluidic LAMPs were completely consistent with the results of conventional LAMPs. HSV-1 DNA was detected in one LAMP negative sample by real-time PCR. One HSV-1 LAMP positive sample was negative in real-time PCR. If real-time PCR was used as a standard, sensitivity of HSV-1, HSV-2, and VZV LAMPs based on microfluidic chip were 90%, 100%, and 100%, and the specificity of three methods were 96%, 100% and 100%, respectively. Conclusion Multiplex LAMP, based on a microfluidic chip that detects three alpha herpesviruses, may be useful in laboratory diagnosis of patients with vesicular skin lesions. Disclosures All Authors: No reported disclosures.

2168. Viral DNAemia and Herpesvirus Seropositivity are Associated with Mortality in Pediatric Patients with Severe Sepsis

Abstract Background Sepsis is a leading cause of pediatric mortality. Little attention has been paid to latent and reactivated viral infection in critically ill children with sepsis. We report a large multi-center study of viral DNAemia and herpesvirus seropositivity in pediatric patients with severe sepsis. Methods We enrolled 401 pediatric patients from 9 Pediatric Intensive Care Units (PICUs). Patient samples were tested via qPCR for EBV, CMV, HSV, adenovirus, HHV6, BK and parvovirus B19 DNA. CMV, EBV, HSV and HHV6 IgG were also measured to classify patients as having no infection (IgG-negative without DNAemia), acute infection (IgG-negative with DNAemia), reactivated infection (IgG-positive with DNAemia), or latent infection without reactivation (IgG-positive without DNAemia). Results 55% of enrolled patients were male, 39% previously healthy, and 27% immunocompromised. 56% had documented infection(s) on enrollment (63% bacterial, 50% viral, and 2% fungal). 11% died in the PICU. Viral DNAemia was detected in 61% of immunocompromised patients and 46% of non-immunocompromised patients. DNAemia with 2 or more viruses on study, detected in 21% of patients, was independently associated with increased mortality in both immunocompromised (OR 5.5 [1.6, 22.6] p=0.023) and non-immunocompromised patients (OR 3.9 [1.4, 11.5] p=0.015). Viral detection was due to reactivated infection in 91% of patients with EBV DNAemia, 63% with CMV, and 100% with HSV and HHV-6, making acute infection rare. Viral seropositivity (HSV 33%, CMV 42%, EBV 61%, HHV6 98%) and latent infection (HSV 30%, CMV 35%, EBV 45%, HHV6 75%) were both common. Adjusted mortality was higher in patients seropositive for EBV (OR 9.1 [2.8, 172.8], p=0.002) compared to those seronegative for EBV. For HHV-6, mortality was higher in patients with viral reactivation compared to latent infection (18% vs. 7%, p=0.024). Mortality in patients with no detected viral infection (IgG-negative without DNAemia) was low (≤ 5% for each virus). Pediatric Sepsis Mortality and Viral DNAemia ICU mortality was significantly associated with the number of viruses detected during ICU stay in both non-immunocompromised patients (light grey bars) and immunocompromised patients (dark grey bars). In patients with 0 viruses detected, morality was less than 10% in both groups. If 1 virus was detected, mortality was 6% in non-immunocompromised patients and 14% in immunocompromised patients. In the 47 non-immunocompromised patients in which 2 or more viruses were detected, mortality was almost 25% and in the 31 immunocompromised patients mortality neared 40%. Among all patients, additional viral detection of 3, 4 or more viruses by PCR continued to increase risk of death, with mortality nearing 45% in patients who had 4 or more viruses detected during their ICU admission. Conclusion Viral DNAemia was common and associated with mortality in pediatric patients with severe sepsis. DNAemia with 2 or more viruses increased mortality risk, even in previously healthy patients. EBV seropositivity was strongly associated with PICU mortality, independent of concurrent viral DNAemia. Disclosures Andrew H. Walton, MS, RevImmune: Grant/Research Support|Thermo Fisher: Spouse employed by Thermo Fisher.

430: VIRAL DNAEMIA IS ASSOCIATED WITH MORTALITY IN PEDIATRIC PATIENTS WITH SEVERE SEPSIS

Introduction: Sepsis is a leading cause of pediatric mortality. Little attention has been paid to the contribution of latent and reactivated viral infection to critical illness in children with sepsis. We report a large multicenter study of viral DNAemia and herpesvirus seropositivity in pediatric patients with severe sepsis. Methods: We enrolled 401 pediatric patients from 9 Pediatric Intensive Care Units (PICUs). Patient samples were tested via qPCR for EBV, CMV, HSV, adenovirus, HHV6, BK and parvovirus B19 DNA. CMV, EBV, HSV and HHV6 IgG were also measured to classify patients as having no infection (IgG-negative without DNAemia), acute infection (IgG-negative with DNAemia), reactivated infection (IgG-positive with DNAemia), or latent infection without reactivation (IgG-positive without DNAemia). Results: 55% of enrolled patients were male, 39% previously healthy, and 27% immunocompromised. 56% had documented infection(s) on enrollment (63% bacterial, 50% viral, and 2% fungal). 10% died in the PICU. Viral DNAemia was detected in 61% of immunocompromised patients and 46% of non-immunocompromised patients. DNAemia with 2 or more viruses on study, detected in 21% of patients, was independently associated with increased mortality in both immunocompromised (OR 5.51 [1.6, 22.6] p=0.023) and non-immunocompromised patients (OR 3.93 [1.4, 11.5] p=0.015). Viral detection was due to reactivated infection in 91% of patients with EBV DNAemia, 63% with CMV, and 100% with HSV and HHV-6, making acute infection rare. Viral seropositivity (HSV 33%, CMV 42%, EBV 61%, HHV6 98%) and latent infection (HSV 30%, CMV 35%, EBV 45%, HHV6 75%) were both common. Adjusted mortality was higher in patients seropositive for EBV (OR 9.1 [2.78, 172.8], p=0.002) compared to those seronegative for EBV. For HHV-6, mortality was higher in patients with viral reactivation compared to latent infection (18% vs. 7%, p=0.024). Mortality in patients with no detected viral infection (IgG-negative without DNAemia) was low (≤ 5% for each virus). Conclusions: Viral DNAemia was common and associated with mortality in pediatric patients with severe sepsis. DNAemia with 2 or more viruses increased mortality, even in previously healthy patients. EBV seropositivity was strongly associated with PICU mortality, independent of concurrent viral DNAemia.

A novel microarray-based PCR assay for the detection of HSV-1, HSV-2, and VZV skin infections: A retrospective analysis

Prevalence of HSV-1, HSV-2, and VZV infection ranges from 20% to 90%. Viral reactivation is common and results in a significant individual and socioeconomic burden. Pathognomonic skin manifestations are not always present, impairing definitive clinical diagnosis. We evaluated the performance of a novel microarray-based multiplex PCR system (Euroarray, Euroimmun Medizinische Labordiagnostika) for the molecular detection of these pathogens. In this retrospective study, 50 consecutive specimens positive for HSV-1, HSV-2, or VZV (pre-characterized by qPCR) were analyzed. Two hundred-and-five negative test results were applied as a control group. The microarray successfully detected the respective pathogens in all samples that yielded a qPCR quantifiable amount of DNA. Two and one specimens containing VZV and HSV-1 DNA beneath the limit of quantification tested microarray negative. Microarray specificity was 100%. The microarray is a useful tool for diagnosing viral infections of skin and mucous membranes, allowing rapid differentiation between three pathogens in a single assay.

Meganuclease targeting HSV-1 protects against herpetic keratitis: Application to corneal transplants

Herpes simplex virus (HSV) infection is a leading cause of corneal blindness. However, keratoplasty is only rarely proposed due to the high frequency of graft failure and associated recurrences. Gene therapy of the corneal graft might provide sustained protection against HSV infection. To test that hypothesis, we designed a meganuclease specific to an HSV-1 DNA sequence coding for major capsid protein (UL19) and selected an adeno-associated virus type-2 as the vector. Meganuclease was transduced into corneas and its effect was challenged invitro, exvivo, and then invivo in a rabbit HSV-1-infection model of stromal keratitis and endotheliitis. Invivo, meganuclease exposure resulted in fewer infected stromal and endothelial cells, and protected against corneal opacification and edema. Exvivo, HSV-1 infection rates of meganuclease-treated human corneas were drastically reduced. Furthermore, genetically engineered corneas transplanted invivo into rabbit eyes protected against HSV-1 infection. This genome-editing technology targeting HSV-1 opens new opportunities to manage severe post-herpetic corneal blindness by providing infected patients with genetically protected corneal transplants.

A case report of severe systemic herpes simplex virus-1 (HSV-1) infection with multi-organ involvement after a course of oral corticosteroid treatment

BackgroundHerpes simplex virus (HSV) rarely causes organ-invasive infection. Diagnosis and treatment for such infections are often delayed, and mortality is high. We present the first reported case of disseminated HSV-1 infection in an adult causing liver failure, myocarditis, and encephalitis in a patient who recovered after receiving parenteral acyclovir treatment.Case presentationA 46-year-old female presented with fever, chills, and malaise after 2 weeks of oral corticosteroid treatment for uveitis. She was diagnosed with disseminated HSV-1 infection with multi-organ involvement causing hepatitis, encephalitis, and myocarditis. Diagnosis was made timely using serum polymerase chain reaction (PCR) for HSV DNA and the patient was given intravenous acyclovir treatment promptly, which led to her survival without significant morbidity.ConclusionsClinicians should have a low threshold for suspecting HSV infection and ordering HSV PCR to decrease morbidity and mortality when there is a high clinical suspicion of systemic HSV infection with multi-organ involvement. Serum PCR for HSV DNA is an excellent modality for an initial diagnostic approach. Further research is warranted to elucidate causality between a course of corticosteroid therapy and systemic HSV-1 infection without major immunosuppressive comorbidities or treatments.

SECONDARY HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS IN A PATIENT WITH COVID-19 AND HERPES SIMPLEX VIRUS (HSV): A CASE REPORT

SECONDARY HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS IN A PATIENT WITH COVID-19 AND HERPES SIMPLEX VIRUS (HSV): A CASE REPORT