Evaluation of Detection and Efficacy of Decontamination of Respiratory Pathogens Including SARS-CoV-2 on Basic Military Trainee Gas Masks.

INTRODUCTION: Basic military trainee (BMT) gas mask training poses a potential mechanism of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) transmission. After training, gas masks are decontaminated. Insufficient decontamination can lead to viral transmission in the next training class. To our knowledge, the decontamination process has not been validated for efficacy in removing respiratory pathogens such as SARS-CoV-2. MATERIALS AND METHODS: Inactivated strains of SARS-CoV-2, influenza A and B, and Bordetella pertussis were separately inoculated onto gas masks in the emitter area (n = 5). Pathogen detection in swabs collected from gas masks was performed by real-time polymerase chain reaction (RT-PCR) using the BioFire® RP2.1 panel and Biomeme Franklin system. For decontamination efficacy experiments, pathogens were inoculated onto gas masks, and contaminated areas were swabbed before and after decontamination, with detection using both PCR platforms. Lastly, 65 gas masks were swabbed after gas mask training, and again after the trainees and guardians decontaminated the masks, to identify the presence of any respiratory pathogen exhaled onto the gas masks. RESULTS: All four pathogens were detected by both PCR platforms. The BioFire® FilmArray® was more sensitive than the Biomeme platform. Decontamination resulted in undetectable levels of all three viruses. B. pertussis was detected on one mask after decontamination. Experiments with live B. pertussis validated that decontamination eliminated all viable bacteria from gas masks. For BMT sampling, all masks were negative for SARS-CoV-2. One mask tested positive for coronavirus 229E. Once decontaminated, all masks tested negative. CONCLUSIONS: BMT gas masks can be monitored for the presence of respiratory pathogens using RT-PCR. The decontamination process removed all viable respiratory pathogens tested from the gas masks. This study demonstrates that RT-PCR can be used to conduct pathogen surveillance on BMT gas masks after training and that the current decontamination process is effective to eliminate respiratory viruses including SARS-CoV-2.

Evaluating sensitivity and specificity of the Biomeme Franklin™ three9 real-time PCR device and SARS-CoV-2 go-strips assay using clinical samples.

We evaluated the sensitivity and specificity of the Biomeme Franklin™ three9 Real-Time PCR Thermocycler and Biomeme SARS-CoV-2 Go-Strips in the detection of SARS-CoV-2. The Biomeme Franklin™ three9 platform is a portable, battery-operated system that could be used in remote settings. We assessed performance of the Biomeme SARS-CoV-2 detection system at a wide range of viral concentrations, examined cross-reactivity of the SARS-CoV-2 Go-Strips against several near-neighbor respiratory pathogens, and evaluated agreement against the BioFire® Respiratory Panel 2.1 in four clinical sample types. Our data indicate the Biomeme Go-Strips can reliably detect SARS-CoV-2 at a concentration of 4.2 × 103 copies/mL. No cross reactivity of the Go-Strips targets was detected against any of the tested near-neighbor respiratory pathogens. Cohen's kappa statistics ranged from 0.68 to 0.92 between results from the Biomeme SARS-CoV-2 Go-Strips and the BioFire® Respiratory Panel 2.1 in all the different sample types. Compared to the BioFire® Respiratory Panel 2.1, the Biomeme SARS-CoV-2 Go-Strips demonstrated statistically significantly lower sensitivity in 3 out of 5 sample types. Overall, our study demonstrates the Biomeme Franklin™ three9 used with the SARS-CoV-2 Go-Strips is an effective system for the detection of SARS-CoV-2 that could potentially be used in a remote or austere environment.

Exploration of Epigenetic State Hyperdopaminergia (Surfeit) and Genetic Trait Hypodopaminergia (Deficit) During Adolescent Brain Development.

BACKGROUND: The risk for all addictive drug and non-drug behaviors, especially, in the unmyelinated Prefrontal Cortex (PFC) of adolescents, is important and complex. Many animal and human studies show the epigenetic impact on the developing brain in adolescents, compared to adults. Some reveal an underlying hyperdopaminergia that seems to set our youth up for risky behaviors by inducing high quanta pre-synaptic dopamine release at reward site neurons. In addition, altered reward gene expression in adolescents caused epigenetically by social defeat, like bullying, can continue into adulthood. In contrast, there is also evidence that epigenetic events can elicit adolescent hypodopaminergia. This complexity suggests that neuroscience cannot make a definitive claim that all adolescents carry a hyperdopaminergia trait. OBJECTIVE: The primary issue involves the question of whether there exists a mixed hypo or hyper-dopaminergia in this population. METHOD: Genetic Addiction Risk Score (GARS®) testing was carried out of 24 Caucasians of ages 12-19, derived from families with RDS. RESULTS: We have found that adolescents from this cohort, derived from RDS parents, displayed a high risk for any addictive behavior (a hypodopaminergia), especially, drug-seeking (95%) and alcohol-seeking (64%). CONCLUSION: The adolescents in our study, although more work is required, show a hypodopaminergic trait, derived from a family with Reward Deficiency Syndrome (RDS). Certainly, in future studies, we will analyze GARS in non-RDS Caucasians between the ages of 12-19. The suggestion is first to identify risk alleles with the GARS test and, then, use well-researched precision, pro-dopamine neutraceutical regulation. This "two-hit" approach might prevent tragic fatalities among adolescents, in the face of the American opioid/psychostimulant epidemic.

Cannabis-Induced Hypodopaminergic Anhedonia and Cognitive Decline in Humans: Embracing Putative Induction of Dopamine Homeostasis.

Over years, the regular use of cannabis has substantially increased among young adults, as indicated by the rise in cannabis use disorder (CUD), with an estimated prevalence of 8. 3% in the United States. Research shows that exposure to cannabis is associated with hypodopaminergic anhedonia (depression), cognitive decline, poor memory, inattention, impaired learning performance, reduced dopamine brain response-associated emotionality, and increased addiction severity in young adults. The addiction medicine community is increasing concern because of the high content of delta-9-tetrahydrocannabinol (THC) currently found in oral and vaping cannabis products, the cognitive effects of cannabis may become more pronounced in young adults who use these cannabis products. Preliminary research suggests that it is possible to induce 'dopamine homeostasis,' that is, restore dopamine function with dopamine upregulation with the proposed compound and normalize behavior in chronic cannabis users with cannabis-induced hypodopaminergic anhedonia (depression) and cognitive decline. This psychological, neurobiological, anatomical, genetic, and epigenetic research also could provide evidence to use for the development of an appropriate policy regarding the decriminalization of cannabis for recreational use.

High Genetic Addiction Risk Score (GARS) in Chronically Prescribed Severe Chronic Opioid Probands Attending Multi-pain Clinics: an Open Clinical Pilot Trial.

Millions of Americans experience pain daily. In 2017, opioid overdose claimed 64,000 lives increasing to 84,000 lives in 2020, resulting in a decrease in national life expectancy. Chronic opioid use results in dependency, drug tolerance, neuroadaptation, hyperalgesia, potential addictive behaviors, or Reward Deficiency Syndrome (RDS) caused by a hypodopaminergia. Evaluation of pain clinic patients with the Genetic Addiction Risk Score (GARS) test and the Addiction Severity Index (ASI- Media Version V) revealed that GARS scores equal to or greater than 4 and 7 alleles significantly predicted drug and alcohol severity, respectively. We utilized RT-PCR for SNP genotyping and multiplex PCR/capillary electrophoresis for fragment analysis of the role of eleven alleles in a ten-reward gene panel, reflecting the activity of brain reward circuitry in 121 chronic opioid users. The study consisted of 55 males and 66 females averaging ages 54 and 53 years of age, respectively. The patients included Caucasians, African Americans, Hispanics, and Asians. Inclusion criteria mandated that the Morphine Milligram Equivalent (MME) was 30-600 mg/day (males) and 20 to 180 mg/day (females) for treatment of chronic pain over 12 months. Ninety-six percent carried four or more risk alleles, and 73% carried seven or more risk alleles, suggesting a high predictive risk for opioid and alcohol dependence, respectively. These data indicate that chronic, legally prescribed opioid users attending a pain clinic possess high genetic risk for drug and alcohol addiction. Early identification of genetic risk, using the GARS test upon entry to treatment, may prevent iatrogenic induced opioid dependence.

Improving naltrexone compliance and outcomes with putative pro- dopamine regulator KB220, compared to treatment as usual.

A recent analysis from Stanford University suggested that without any changes in currently available treatment, prevention, and public health approaches, we should expect to have 510,000 deaths from prescription opioids and street heroin from 2016 to 2025 in the US. In a recent review, Mayo Clinic Proceedings (October 2019), Gold and colleagues at Mayo Clinic reviewed the available medications used in opioid use disorders and concluded that in private and community practice adherence is more important as a limiting factor to retention, relapse, and repeat overdose. It is agreed that the primary utilization of known opioid agonists like methadone, buprenorphine and naloxone combinations, while useful as a way of reducing societal harm, is limited by 50% of more discontinuing treatment within 6 months, their diversion, and addiction liability. Opioid agonists may have other unintended consequences, like continuing the down regulation of dopamine systems. While naltrexone would be expected to have opposite effects, adherence is also low even after detoxification and long acting naltrexone injections. Recent studies have shown Naltrexone is beneficial by attenuation of craving via "psychological extinction" and reducing relapse. Buprenorphine is the MAT of choice currently but injectable Naltrexone plus an agent to improve dopaminergic function and tone may renew interest amongst addiction physicians and patients. Understanding this dilemma there is increasing movement to opt for the non-addicting narcotic antagonist Naltrexone. Even with extended injectable option there is still poor compliance. As such, we describe an open label investigation in humans showing improvement of naltrexone compliance and outcomes with dopamine augmentation with the pro- dopamine regulator KB220 (262 days) compared to naltrexone alone (37days). This well studied complex consists of amino-acid neurotransmitter precursors and enkephalinase inhibitor therapy compared to treatment as usual. Consideration of this novel paradigm shift may assist in not only addressing the current opioid epidemic but the broader question of reward deficiency in general.

In Search of Reward Deficiency Syndrome (RDS)-free Controls: The "Holy Grail" in Genetic Addiction Risk Testing.

BACKGROUND: The search for an accurate, gene-based test to identify heritable risk factors for Reward Deficiency Syndrome (RDS) was conducted based on hundreds of published studies about the role of dopamine in addictive behaviors, including risk for drug dependence and compulsive/impulsive behavior disorders. The term RDS was first coined by Blum's group in 1995 to identify a group of behaviors with a common neurobiological mechanism associated with a polymorphic allelic propensity for hypodopaminergia. OBJECTIVES: To outline the process used to select risk alleles of reward genes for the Genetic Addiction Risk Score (GARS) test. Consequently, to address the limitations caused by inconsistent results that occur in many case-control behavioral association studies. These limitations are perhaps due to the failure of investigators to adequately screen controls for drug and alcohol use disorder, and any of the many RDS behaviors, including nicotine dependence, obesity, pathological gambling, and internet gaming addiction. METHODS: Review of the literature related to the function of risk alleles of reward genes associated with hypodopaminergia relevant case-control association studies for the selection of alleles to be measured by the Genetic Addiction Risk Score (GARS) test. RESULTS: The prevalence of the DRD2 A1 allele in unscreened controls (33.3%), compared to "Super-Controls" [highly screened RDS controls (3.3%) in proband and family] is used to exemplify a possible solution. CONCLUSION: Unlike one gene-one disease (OGOD), RDS is polygenetic, and very complex. In addition, any RDS-related behaviors must be eliminated from the control group in order to obtain the best possible statistical analysis instead of comparing the phenotype with disease-ridden controls.

Hypodopaminergia and "Precision Behavioral Management" (PBM): It is a Generational Family Affair.

BACKGROUND/AIMS: This case series presents the novel Genetic Addiction Risk Score (GARS®) coupled with a customized pro-dopamine regulator matched to polymorphic reward genes having a hypodopaminergic risk. METHODS: The proband is a female with a history of drug abuse and alcoholism. She experienced a car accident under the influence and voluntarily entered treatment. Following an assessment, she was genotyped using the GARS, and started a neuronutrient with a KB220 base indicated by the identified polymorphisms. She began taking it in April 2018 and continues. RESULTS: She had success in recovery from Substance Use Disorder (SUD) and improvement in socialization, family, economic status, well-being, and attenuation of Major Depression. She tested negative over the first two months in treatment and a recent screening. After approximately two months, her parents also decided to take the GARS and started taking the recommended variants. The proband's father (a binge drinker) and mother (no SUD) both showed improvement in various behavioral issues. Finally, the proband's biological children were also GARS tested, showing a high risk for SUD. CONCLUSION: This three-generation case series represents an example of the impact of genetic information coupled with an appropriate DNA guided "Pro-Dopamine Regulator" in recovery and enhancement of life.

Death by Opioids: Are there non-addictive scientific solutions?

In the face of the current Opioid crisis in America killing close to 800,000 people since 2004, we are proposing a novel approach to assist in at least attenuating these unwanted premature deaths. While we applaud the wonderful efforts of our governmental institutes and professional societies (NIDA, NIAAA, ASAM, ABAM ) in their extraordinary efforts in combating this continued dilemma, the current approach is failing, and other alternative approaches should at least be tested. These truths present a serious ethical dilemma to scientists, clinicians and counselors in the Reward Deficiency Syndrome (RDS) treatment community. It is important to realize that the current DSM-5 does not actually accurately display the natural brain reward process. The human brain has not been designed to carve out specific drugs like opioids, alcohol, nicotine, cocaine, benzodiazepines or cannabis and process addictions such as gambling as distinct endophenotypes. This is true in spite of natural ligands for cannabinoids, endorphins, or even benzodiazepines. The most accurate endophenotype is indeed reward dysfunction (e.g hypodopaminergic or hyperdopaminergic). With this mind, we are hereby proposing that the current Medication Assisted Treatment (i.e. 'MAT') expands to needed individuals as an initial "Band-Aid" to reduce harm avoidance, with the long-term goal of prophylaxis. So, to be clear, there may be other promising modalities other than MAT such as repetitive transcranial magnetic stimulation (rTMS), exercise and even new medications with positive allosteric modulators of GABA-A receptors, as well as the highly researched Genetic Addiction Risk Score (GARS) coupled with precision KB220Z. This will induce "dopamine homeostasis" to effectively rebalance and restore healthier brain function by promoting the cross talk between various brain regions (e.g. Nucleus accumbens, cingulate gyrus, hippocampus etc.) resulting in dopamine homeostasis. Our laudable goal is to not only save lives, but to redeem joy and improve the quality of life in the recovery community through scientifically sound natural non-addicting alternatives.

Adenovirus-Associated Acute Appendicitis: An Under-Recognized Relationship?

OBJECTIVE: Acute appendicitis (AA) is one of the most common causes of a surgical abdomen worldwide, occurring most frequently in those age 10 to 29 years. Adenovirus (ADV) is a rare but reported cause of AA in children and a well-recognized cause of intussusception in infants and young children. Annually, about 36,000 basic military trainees (BMTs) undergo initial training at Joint Base San Antonio Lackland, Texas. Before reintroduction of the ADV 4/7 vaccine in November 2011, one-third of BMTs developed an adenoviral upper respiratory tract infection (URI) during the 8.5 weeks of training. We hypothesized that ADV may be a common cause of AA in the BMT population given their young age and high incidence of adenoviral URIs. The objective of this study was to determine the frequency with which ADV, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and enterovirus were associated with AA in a population of young adults. MATERIALS AND METHODS: This study was a retrospective review of patient charts and existing pathological tissue specimens of all BMTs who underwent appendectomy at the Wilford Hall Medical Center from January 1, 2003, to August 31, 2011. Pathological tissue samples from 112 BMTs were assayed by quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC) for viral targets. RESULTS: ADV DNA was detected in 16 of 112 samples (14%) via qPCR: ADV 4 in 13 cases, ADV B14 in 1 case, and nontypable ADV in 2 cases. IHC was positive in only the ADV B14 case (0.9%). All cases were negative for CMV, EBV, and enterovirus. CONCLUSION: By using qPCR, this study demonstrated an association between ADV and AA higher than has been previously reported: ADV was detected in 14% of AA cases in this series versus in only 0.23% of AA cases in previous studies (p < 0.01). There was no evidence of CMV, EBV, or enterovirus association with AA in this study. Comparison of qPCR to IHC shows that histologic analysis may overlook evidence of ADV in appendiceal tissue: qPCR is significantly more sensitive than light microscopy and IHC for detecting ADV in this setting. Because ADV 4 was detected in 81% of those with positive qPCR, the recently licensed live oral ADV vaccine might be useful for primary prevention against AA. Prospective studies evaluating young adults presenting with AA for evidence of infection with ADV are needed to determine if a causal relationship exists.

Comparison of Clinical Features in a Population of Basic Military Trainees Versus the General Department of Defense Beneficiary Population Presenting With Influenza.

INTRODUCTION: Upper respiratory tract infection (URI) is a well-documented cause of morbidity, extra expense, and lost training time among basic military trainees (BMTs). The goal of this study was to characterize the clinical presentation of influenza in the BMT population and to better understand how this presentation differs from that of the general Department of Defense (DoD) beneficiary population (non-BMTs). MATERIALS AND METHODS: Clinical and demographic data were collected in a prospective study that enrolled DoD beneficiaries presenting to medical treatment facilities in San Antonio, Texas, with URI symptoms between January 2005 and March 2011. Vital signs and symptom duration were collected at the time of enrollment along with basic demographic information. RESULTS: Among 4,448 participants enrolled, 466 (10.5%) tested positive for influenza: 198 of 3,103 BMTs (6.4%) vs. 268 of 1,345 non-BMTs (20%) (p < 0.01); 412 of 466 had complete data for nine symptom-related variables. BMTs were more likely to be Caucasian males and younger than non-BMTs. BMTs had a higher temperature at the time of presentation (101.5°F vs. 100.5°F, p < 0.01). BMTs presented less frequently than non-BMTs with chills (79.7% vs. 94.4%, p < 0.01), malaise (62.1% vs. 87.0%, p < 0.01), nausea (30.2% vs. 43.0%, p < 0.01), and vomiting (12.1% vs. 21.7%, p = 0.01). Multiple logistic regression analysis showed that BMTs were less likely to have the four symptoms compared to non-BMTs even after controlling for gender and age (chills: odds ratio [OR] = 0.3, 95% confidence interval [CI] = 0.1-0.6, p < 0.01; malaise: OR = 0.5, 95% CI = 0.3-0.8, p < 0.01; nausea: OR = 0.5, 95% CI = 0.3-0.8, p < 0.01; vomiting: OR = 0.4, 95% CI = 0.2-0.8, p < 0.01). Although there was no difference in the frequency of subjective fever between the two groups, reported duration of fever was significantly shorter in BMTs than non-BMTs: median of 1 day (range 0-10) vs. 2 days (range 0-8) (p < 0.01). BMTs presented with a composite symptom index mean of 6.2 (standard deviation = 1.4) symptoms, whereas non-BMTs presented with a mean of 6.9 (standard deviation = 1.3) symptoms (p < 0.01). CONCLUSIONS: The pretest probability of a BMT presenting with URI symptoms having influenza is significantly lower than that for the general DoD beneficiary population. BMTs with influenza presented sooner, with higher fever, and with fewer overall symptoms than the general DoD beneficiary population. These differences are likely attributable to early reporting and response bias and less likely attributed to age. Military efforts to identify BMTs with suspected influenza infection early and to refer them for treatment promptly are efficacious.

Changes in Clinical Presentation and Epidemiology of Respiratory Pathogens Associated With Acute Respiratory Illness in Military Trainees After Reintroduction of Adenovirus Vaccine.

Background. Adenovirus (Ad) has long been the predominant cause of acute respiratory illness (ARI) in military trainees. In 2011, live oral Ad vaccines for serotypes 4 and 7 were reintroduced into US basic military training populations. This study evaluated the impact on clinical presentations and other respiratory pathogens. Methods. The Center for Advanced Molecular Detection at Joint Base San Antonio-Lackland prospectively collects demographic, clinical, and polymerase chain reaction data from respiratory specimens (throat swab and nasal wash) among Air Force trainees presenting for care of ARI. Results. From June 2008 to August 2013, 2660 trainees enrolled and were tested for selected respiratory pathogens. Post-vaccine introduction (VI), reported systemic symptoms were less frequent, including fever (38% vs 94%) and myalgia (37% vs 67%; P < .01). Median temperature and heart rate decreased (98.4 vs 101.3°F, 81 vs 96 beats per minute; P < .01). Ad detection decreased for all Ad (3% vs 68%), Ad4 (1% vs 70%), 7 (0% vs 8%), 14 (0% vs 5%), and 3 (0.1% vs 2%); P < .01). Rhinovirus and cases with no pathogen identified increased in frequency (35% vs 18%, 51% vs 14%; P < .01). Conclusions. Acute respiratory illness in military trainees post-VI is associated with decreased severity of systemic symptoms and reduced fever and heart rate. Marked reductions in frequency of Ad serotypes are seen, including those in the vaccine, with no serotype shift. However, detection of several other respiratory pathogens, most notably rhinovirus, is observed in increasing proportions, and a majority are now undiagnosed clinical syndromes.

Comparison of diagnostic test performance in a population of high risk young adults versus a general population presenting with influenza.

BACKGROUND: Upper respiratory tract infection (URI) is a well-documented cause of morbidity, extra expense and lost training time among basic military trainees (BMTs). OBJECTIVES: The goal of this study is to better understand how influenza diagnostic tests perform in the BMT population, and how this performance differs from the general population. STUDY DESIGN: Laboratory test data was collected in a prospective study that enrolled Department of Defense beneficiaries presenting to medical facilities in San Antonio, TX with URI symptoms between January 2005 and March 2011. Three laboratory tests for influenza were performed during the study period: polymerase chain reaction (PCR), enzyme immunoassay (EIA), and viral culture. Patients were grouped into BMT and non-BMT populations and the tests from each of these populations were compared for statistical differences. Similar comparisons were made with various sub-groups to include: influenza A versus influenza B, and influenza A subtypes: (H1N1) versus (H3N2) versus (H1N1)pdm09. RESULTS: Among 4448 participants enrolled, 466 (10.5%) tested positive for influenza. Sensitivity of viral culture differed between BMTs and non-BMTs: 63% versus 41% (p<0.01). There was no difference in the sensitivity of PCR or EIA between the two populations. The sensitivities of viral culture, EIA and PCR were higher in those infected with influenza A than in those infected with influenza B. The sensitivity of viral culture was significantly higher in (H1N1)pdm09 subtype cases. CONCLUSIONS: Viral culture performed better in BMTs than in non-BMTs. These differences are likely attributable to the younger age of the BMTs.

Pandemic influenza virus 2009 H1N1 and adenovirus in a high risk population of young adults: epidemiology, comparison of clinical presentations, and coinfection.

BACKGROUND: In 2009, pandemic H1N1 influenza virus (2009 H1N1) emerged worldwide, causing morbidity and mortality that disproportionately affected young adults. Upper respiratory infection (URI), largely due to adenovirus, is an endemic cause of morbidity in military training. Whether clinical presentations differ or excess morbidity results from coinfection is unclear. METHODS: The Center for Advanced Molecular Detection evaluates epidemiology and rapid diagnostics of respiratory pathogens in trainees with URI. From May 1, 2009, to November 30, 2009, demographic, clinical, and PCR data from throat and nasal specimens for adenovirus and 2009 H1N1 were prospectively collected. RESULTS: 375 trainees with URI enrolled and were tested for both adenovirus and 2009 H1N1 by PCR (median age 20; 89% male). Adenovirus PCR was positive in 72% (96% serotype E-4) and 2009 H1N1 in 20%. Males were more likely to have adenovirus and females more likely to have 2009 H1N1 (p  =  0.047). Subjects with 2009 H1N1 presented an average of 1 week earlier in training, had shorter illness duration before enrollment, less sore throat, diarrhea, and fewer abnormal findings on throat exam. Coryza and cough were more common with 2009 H1N1 compared to adenovirus. Subjects with 2009 H1N1 were less likely to have adenovirus than those without, despite persistently high frequencies of adenovirus detections during peak 2009 H1N1 weeks (15% vs. 83%, p < 0.01). Coinfection with adenovirus and 2009 H1N1 was rare (4%). Rates of hospitalization and pneumonia did not differ between the adenovirus, 2009 H1N1, or coinfected groups. CONCLUSION: Military trainees with 2009 H1N1 vs. adenovirus have differing clinical presentations, and males are more likely to have adenovirus. Despite high frequencies of adenovirus infection, coinfection with adenovirus and 2009 H1N1 is rare and apparently does not result in increased morbidity.

Quantitative real-time PCR assay panel for detection and type-specific identification of epidemic respiratory human adenoviruses.

Outbreaks of human adenovirus (HAdV) acute respiratory illness (ARI) have been well documented among civilians and unvaccinated military recruits. Among the 7 recognized HAdV species (A to G), species B (particularly serotypes 3, 7, 11, 14, and 21) and E (serotype 4) have more often been associated with epidemic ARI. Rapid detection and type-specific identification of these viruses would enhance outbreak response and help guide prevention and control measures. To this end, we developed type-specific real-time quantitative PCR (qPCR) assays for HAdV types 3, 4, 7, 11, 14, 16, and 21 targeting the HAdV hexon gene. All type-specific qPCR assays reproducibly detected as few as 10 copies/reaction of quantified hexon recombinant plasmids with a linear dynamic range of 8 log units (10(1) to 10(8) copies); in contrast, a generic qPCR assay that detects all HAdV types run concurrently detected between 10 and 100 copies/reaction, depending on the virus type. No nonspecific amplifications were observed with concentrated nucleic acid from 51 HAdV prototype strains or other common respiratory pathogens. All members of a panel of 137 previously typed HAdV field isolates and positive clinical specimens were correctly characterized by the type-specific qPCR assays; two different HAdV types were detected in three of the clinical specimens and confirmed by amplicon sequencing. The qPCR assays permit sensitive, specific, and quantitative detection and identification of seven clinically important respiratory HAdVs and should provide a convenient adjunct to classical typing methods for a rapid response to HAdV outbreaks.

Clinical validation of multiplex real-time PCR assays for detection of bacterial meningitis pathogens.

Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae are important causes of meningitis and other infections, and rapid, sensitive, and specific laboratory assays are critical for effective public health interventions. Singleplex real-time PCR assays have been developed to detect N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA and serogroup-specific genes in the cap locus for N. meningitidis serogroups A, B, C, W135, X, and Y. However, the assay sensitivity for serogroups B, W135, and Y is low. We aimed to improve assay sensitivity and develop multiplex assays to reduce time and cost. New singleplex real-time PCR assays for serogroup B synD, W135 synG, and Y synF showed 100% specificity for detecting N. meningitidis species, with high sensitivity (serogroup B synD, 99% [75/76]; W135 synG, 97% [38/39]; and Y synF, 100% [66/66]). The lower limits of detection (LLD) were 9, 43, and 10 copies/reaction for serogroup B synD, W135 synG, and Y synF assays, respectively, a significant improvement compared to results for the previous singleplex assays. We developed three multiplex real-time PCR assays for detection of (i) N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA (NHS assay); (ii) N. meningitidis serogroups A, W135, and X (AWX assay); and (iii) N. meningitidis serogroups B, C, and Y (BCY assay). Each multiplex assay was 100% specific for detecting its target organisms or serogroups, and the LLD was similar to that for the singleplex assay. Pairwise comparison of real-time PCR between multiplex and singleplex assays showed that cycle threshold values of the multiplex assay were similar to those for the singleplex assay. There were no substantial differences in sensitivity and specificity between these multiplex and singleplex real-time PCR assays.

Genome sequences of human adenovirus 14 isolates from mild respiratory cases and a fatal pneumonia, isolated during 2006-2007 epidemics in North America.

BACKGROUND: Human adenovirus 14 (HAdV-14) is a recognized causative agent of epidemic febrile respiratory illness (FRI). Last reported in Eurasia in 1963, this virus has since been conspicuously absent in broad surveys, and was never isolated in North America despite inclusion of specific tests for this serotype in surveillance methods. In 2006 and 2007, this virus suddenly emerged in North America, causing high attack rate epidemics of FRI and, in some cases, severe pneumonias and occasional fatalities. Some outbreaks have been relatively mild, with low rates of progression beyond uncomplicated FRI, while other outbreaks have involved high rates of more serious outcomes. METHODOLOGY AND FINDINGS: In this paper we present the complete genomic sequence of this emerging pathogen, and compare genomic sequences of isolates from both mild and severe outbreaks. We also compare the genome sequences of the recent isolates with those of the prototype HAdV-14 that circulated in Eurasia 30 years ago and the closely related sequence of HAdV-11a, which has been circulating in southeast Asia. CONCLUSIONS: The data suggest that the currently circulating strain of HAdV-14 is closely related to the historically recognized prototype throughout its genome, though it does display a couple of potentially functional mutations in the fiber knob and E1A genes. There are no polymorphisms that suggest an obvious explanation for the divergence in severity between outbreak events, suggesting that differences in outcome are more likely environmental or host determined rather than viral genetics.

Health care transmission of a newly emergent adenovirus serotype in health care personnel at a military hospital in Texas, 2007.

BACKGROUND: Adenoviruses can cause outbreaks of febrile respiratory illness in military trainees, but until 2007, adenovirus serotype 14 (Ad14) was never associated with such outbreaks. From April through June 2007, 15 trainees at one base were hospitalized for pneumonia due to Ad14. Subsequent reports of febrile respiratory illness among health care personnel suggested nosocomial transmission. METHODS: Health care personnel participants completed a questionnaire and provided blood and nasal wash specimens for Ad14 diagnostic testing. We defined a confirmed case of Ad14 infection as one with titers > or = 1:80 or nasal wash specimens positive for Ad14 by polymerase chain reaction, whereas a possible case was defined by titers of 1:20 or 1:40. We also collected environmental samples. RESULTS: Among 218 tested health care personnel, 35 (16%) had titers > or = 1:20; of these, 7 had possible cases and 28 had confirmed cases of infection. Confirmed case patients were more likely to report febrile respiratory illness (57% vs 11%; P< .001) and to have had direct contact with patients with Ad14 infection (82% vs 62%; P.04 ). Of the 23 confirmed case patients with direct contact with Ad14-infected patients, 52% reported that patients were not in contact and droplet precautions at the time of exposure. Ad14 was recovered from several hospital surfaces. CONCLUSION: Our findings of possible nosocomial transmission of Ad14 highlight the need to reinforce infection control guidelines.

Evaluation and validation of a real-time PCR assay for detection and quantitation of human adenovirus 14 from clinical samples.

In 2007, the Centers for Disease Control and Prevention (CDC) reported that Human adenovirus type 14 (HAdV-14) infected 106 military personnel and was responsible for the death of one U.S. soldier at Lackland Air Force Base in Texas. Identification of the responsible adenovirus, which had not previously been seen in North America and for which rapid diagnostic tools were unavailable, required retrospective analysis at reference laboratories. Initial quarantine measures were also reliant on relatively slow traditional PCR analysis at other locations. To address this problem, we developed a real-time PCR assay that detects a 225 base pair sequence in the HAdV-14a hexon gene. Fifty-one oropharyngeal swab specimens from the Naval Health Research Center, San Diego, CA and Advanced Diagnostic Laboratory, Lackland AFB, TX were used to validate the new assay. The described assay detected eight of eight and 19 of 19 confirmed HAdV-14a clinical isolates in two separate cohorts from respiratory disease outbreaks. The real-time PCR assay had a wide dynamic range, detecting from 10(2) to 10(7) copies of genomic DNA per reaction. The assay did not cross-react with other adenoviruses, influenza, respiratory syncytial virus, or common respiratory tract bacteria. The described assay is easy to use, sensitive and specific for HAdV-14a in clinical throat swab specimens, and very rapid since turnaround time is less than four hours to obtain an answer.