Evaluation of a PCR-electrospray ionization mass spectrometry platform for detection and identification of fungal pathogens directly from prospectively collected bronchoalveolar lavage specimens.

The incidence of invasive fungal infections is on the rise worldwide due to the growth of the immunocompromised population. We report here the use of a diagnostic assay that utilizes a universal extraction method, broad spectrum PCR amplification and analysis via electrospray ionization mass spectrometry (PCR/ESI-MS) to detect and identify more than 200 pathogenic fungi directly from bronchoalveolar lavage (BAL) specimens in less than 8 hours. In this study, we describe both analytical and clinical performance of the assay, when run with prospectively collected clinical BAL specimens. In 146 patients with probable and possible fungal infections defined by EORTC/MSG (European Organization for Research and Treatment of Cancer/Mycoses Study Group) criteria, the PCR/ESI-MS assay demonstrated a sensitivity of 90.9% (95% CI: 76.4-96.9%) and a specificity of 82.3% (95% CI: 74.2-88.2%). This data demonstrates the utility of a non-culture based broad fungal targets molecular diagnostic tool for rapid and accurate diagnosis of invasive fungal infections in patients at risk of developing fungal diseases.

Molecular Epidemiology of Adenovirus Type 21 Respiratory Strains Isolated From US Military Trainees (1996-2014).

BACKGROUND: The circulation of human adenovirus type 21 (HAdV21) in the United States has been documented since the 1960s in association with outbreaks of febrile respiratory illness (FRI) in military boot camps and civilian cases of respiratory disease. METHODS: To describe the molecular epidemiology of HAdV21 respiratory infections across the country, 150 clinical respiratory isolates obtained from continuous surveillance of military recruit FRI, and 23 respiratory isolates recovered from pediatric and adult civilian cases of acute respiratory infection were characterized to compile molecular typing data spanning 37 years (1978-2014). RESULTS: Restriction enzyme analysis and genomic sequencing identified 2 clusters of closely related genomic variants readily distinguishable from the prototype and designated 21a-like and 21b-like. A-like variants predominated until 1999. A shift to b-like variants was noticeable by 2007 after a 7-year period (2000-2006) of cocirculation of the 2 genome types. US strains are phylogenetically more closely related to European and Asian strains isolated over the last 4 decades than to the Saudi Arabian prototype strain AV-1645 isolated in 1956. CONCLUSIONS: Knowledge of circulating HAdV21 variants and their epidemic behavior will be of significant value to local and global FRI surveillance efforts.

Analytical characterization of an assay designed to detect and identify diverse agents of disseminated viral infection.

BACKGROUND: Diverse viruses often reactivate in or infect cancer patients, patients with immunocompromising infections or genetic conditions, and transplant recipients undergoing immunosuppressive therapy. These infections can disseminate, leading to death, transplant rejection, and other severe outcomes. OBJECTIVES: To develop and characterize an assay capable of inclusive and accurate identification of diverse potentially disseminating viruses directly from plasma specimens. STUDY DESIGN: We developed a PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) assay designed to simultaneously detect and identify adenovirus, enterovirus, polyomaviruses JC and BK, parvovirus B19, HSV-1, HSV-2, VZV, EBV, CMV, and herpesviruses 6-8 in plasma specimens. The assay performance was characterized analytically, and the results from clinical plasma samples were compared to the results obtained from single-analyte real time PCR tests currently used in clinical practice. RESULTS: The assay demonstrated sensitivity and specificity to diverse strains of the targeted viral families and robustness to interfering substances and potentially cross reacting organisms. The assay yielded 94% sensitivity when testing clinical plasma samples previously identified as positive using standard-of-care real-time PCR tests for a single target virus (available samples included positive samples for 11 viruses targeted by the assay). CONCLUSIONS: The assay functioned as designed, providing simultaneous broad-spectrum detection and identification of diverse agents of disseminated viral infection. Among 156 clinical samples tested, 37 detections were made in addition to the detections matching the initial clinical positive results.

Identification of a novel human papillomavirus by metagenomic analysis of samples from patients with febrile respiratory illness.

As part of a virus discovery investigation using a metagenomic approach, a highly divergent novel Human papillomavirus type was identified in pooled convenience nasal/oropharyngeal swab samples collected from patients with febrile respiratory illness. Phylogenetic analysis of the whole genome and the L1 gene reveals that the new HPV identified in this study clusters with previously described gamma papillomaviruses, sharing only 61.1% (whole genome) and 63.1% (L1) sequence identity with its closest relative in the Papillomavirus episteme (PAVE) database. This new virus was named HPV_SD2 pending official classification. The complete genome of HPV-SD2 is 7,299 bp long (36.3% G/C) and contains 7 open reading frames (L2, L1, E6, E7, E1, E2 and E4) and a non-coding long control region (LCR) between L1 and E6. The metagenomic procedures, coupled with the bioinformatic methods described herein are well suited to detect small circular genomes such as those of human papillomaviruses.

Five genome sequences of subspecies B1 human adenoviruses associated with acute respiratory disease.

Five genomes of human subspecies B1 adenoviruses isolated from cases of acute respiratory disease have been sequenced and archived for reference. These include representatives of two prevalent genomic variants of HAdV-7, i.e., HAdV-7h and HAdV-7d2. The other three are HAdV-3/16, HAdV-16 strain E26, and HAdV-3+7 strain Takeuchi. All are recombinant genomes. Genomics and bioinformatics provide detailed views into the genetic makeup of these pathogens and insight into their molecular evolution. Retrospective characterization of particularly problematic older pathogens such as HAdV-7h (1987) and intriguing isolates such as HAdV-3+7 strain Takeuchi (1958) may provide clues to their phenotypes and serology and may suggest protocols for prevention and treatment.

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.

Computational analysis of adenovirus serotype 5 (HAdV-C5) from an HAdV coinfection shows genome stability after 45 years of circulation.

Adenovirus coinfections present opportunities for genome recombination. Computational analysis of an HAdV-C5 field strain genome, recovered from a patient with acute respiratory disease and coinfected with HAdV-B21, shows that there was no exchange of genomic material into HAdV-C5. Comparison of this genome to the sparsely amplified prototype demonstrates a high level of sequence conservation and stability of this genome across 45 years. Further, comparison to a version of the prototype that had been passaged in laboratory settings shows stability as well. HAdV genome stability and evolution are considerations for applications as vaccines and as vectors for gene delivery. In the annotation analysis, a single sequencing error in the HAdV-C5_ARM (Adenovirus Reference Material) genome is noted and may lead to erroneous annotation and biological interpretations.

Molecular epidemiology and brief history of emerging adenovirus 14-associated respiratory disease in the United States.

BACKGROUND: First isolated in the Netherlands in 1955 during an outbreak of acute respiratory disease (ARD) among military recruits, human adenovirus 14 (HAdV-14) has historically been considered rare. With no precedent of circulation in North America, HAdV-14 has been isolated from military and civilian cases of ARD of variable severity since 2003 in the United States. METHODS: Ninety-nine isolates from military and civilian cases from different geographic locations and circulation periods were characterized by restriction enzyme analysis of viral DNA and select gene sequencing. RESULTS: All examined viruses were found to be identical and to belong to a new genome type designated "HAdV-14p1" (formerly known as "14a"). Comparative alignments of E1A, hexon, and fiber gene sequences with other subspecies B2 HAdVs suggest that HAdV-14p1, like the closely related HAdV-11a, arose from recombination among similar HAdV-11 and HAdV-14 ancestral strains. A deletion of 2 amino acids in the knob region of the fiber protein is the only identified unique characteristic of HAdV-14p1. CONCLUSION: The current geographic distribution of HAdV-14p1 involves at least 15 states in the Unites States. The role of the fiber mutations in the recent emergence of HAdV-14p1 ARD in North America warrants further study.

Multiplexed Luminex xMAP assay for detection and identification of five adenovirus serotypes associated with epidemics of respiratory disease in adults.

Several serotypes of human adenovirus (HAdV) cause acute respiratory disease (ARD) among healthy adults, sometimes generating broad outbreaks with high attack rates and occasional fatalities. Timely serotype identification provides valuable epidemiological information and significantly contributes to prevention (vaccination) strategies. The prevalence of specific serotypes causing ARD varies geographically. HAdV-3, HAdV-4, HAdV-7, HAdV-14, and HAdV-21 are the serotypes most commonly found in adult populations in the Western Hemisphere. Unfortunately, conventional serotype identification is a tedious process which can take a week or longer. For this reason, new molecular methods for serotype identification are needed. Commercially available rapid antigen and PCR assays for the detection of HAdV are universal but do not distinguish between the different serotypes. We describe the development of a sensitive and specific multiplex assay capable of identifying serotypes 3, 4, 7, 14, and 21. Two sets of primers were used for nonspecific (universal) PCR amplification, and serotype-specific probes coupled to Luminex tags were used for target-specific extension (TSE). PCR and TSE primers were designed using known hexon gene sequences of HAdV. The TSE products of HAdV-3, HAdV-4, HAdV-7, HAdV-14, and HAdV-21 were correctly identified using the Luminex xMAP fluid microsphere-based array system. No cross-reactivity with other respiratory pathogens or other HAdV serotypes was observed. This multiplexed assay can be expanded to include more serotypes and will allow broad and rapid detection and identification of adenoviral serotypes in a high-throughput environment.

Broad spectrum respiratory pathogen analysis of throat swabs from military recruits reveals interference between rhinoviruses and adenoviruses.

Military recruits experience a high incidence of febrile respiratory illness (FRI), leading to significant morbidity and lost training time. Adenoviruses, group A Streptococcus pyogenes, and influenza virus are implicated in over half of the FRI cases reported at recruit training center clinics, while the etiology of the remaining cases is unclear. In this study, we explore the carriage rates and disease associations of adenovirus, enterovirus, rhinovirus, Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis in military recruits using high-density resequencing microarrays. The results showed that rhinoviruses, adenoviruses, S. pneumoniae, H. influenzae, and N. meningitidis were widely distributed in recruits. Of these five agents, only adenovirus showed significant correlation with illness. Among the samples tested, only pathogens associated with FRI, such as adenovirus 4 and enterovirus 68, revealed strong temporal and spatial clustering of specific strains, indicating that they are transmitted primarily within sites. The results showed a strong negative association between adenoviral FRI and the presence of rhinoviruses in recruits, suggesting some form of viral interference.

Outbreak of febrile respiratory illness associated with adenovirus 11a infection in a Singapore military training cAMP.

Outbreak cases of acute respiratory disease (ARD) associated with subspecies B2 human adenovirus 11a (HAdV-11a) infection were detected during 2005 in a military basic training camp in Singapore. The Singapore HAdV-11a strain is highly similar to other Asian strains of HAdV-11, including strain QS-DLL, which is responsible for the recently described 2006 outbreak of ARD in China.

Evaluation of multiplex type-specific real-time PCR assays using the LightCycler and joint biological agent identification and diagnostic system platforms for detection and quantitation of adult human respiratory adenoviruses.

Every year, thousands of basic military trainees in each service of the U.S. Armed Forces experience acute respiratory disease. The majority of this disease burden results from infection with human adenoviruses. We designed single- and multiplex assays that detect and discriminate adenovirus types B3, E4, B7, B11, B14, and B21. A total of 116 oropharyngeal swab specimens obtained from patients at the Naval Health Research Center were used to validate the new assays. Type-specific singleplex assays were designed and used independently to successfully identify 94 representative patient specimens. The lower limits of detection for our singleplex real-time PCR assays were calculated to be 50, 500, 500, 50, 50, and 50 genomic copies per reaction for human adenovirus type B3 (HAdV-B3), HAdV-E4, HAdV-B7, HAdV-B11, HAdV-B14, and HAdV-B21, respectively. These were then multiplexed to increase efficiency and tested against singleplex assays using titrated controls. The HAdV-B3/B11 and HAdV-E4/B7 multiplex assays were as sensitive and specific as they were individually. The HAdV-B14/B21 multiplex assay was not as efficient at detecting HAdV-B14 as the singleplex assay. Interestingly, a statistically significant difference was found between the viral loads of HAdV-B14 and those of HAdV-B3, -E4, -B7, and -B21 (P < 0.001). The assays did not cross-react with other adenoviruses, influenza virus, respiratory syncytial virus, or respiratory disease-causing bacteria. These assays have the potential to be useful as clinical diagnostic tools for the detection of HAdV infection in adult populations.

Genomic characterization of human adenovirus 36, a putative obesity agent.

Increased levels of serum antibody titers against human adenovirus 36 (HAdV-D36) are associated with human obesity and experimental obesity in laboratory animals. While HAdV-D36 has been studied as an infectious agent implicated in obesity for over a decade, the complete genome sequence and its analysis have yet to be reported. A detailed analysis of the genome sequence of HAdV-D36 may be important to understand its role in obesity. Genomic and bioinformatic comparisons with other HAdVs identified differences that suggested unique functions. Global pairwise genome alignment with all sequenced human adenovirus D (HAdV-D) genomes revealed areas of nonconserved sequences in the hexon, E3 CR1 beta, E3 CR1 gamma, and fiber genes. Phylogenetic analysis of all HAdV-D36 proteins confirmed that this virus belongs to species Human adenovirus D. This genomic analysis of HAdV-D36 provides an important tool for comprehending the role that this unique adenovirus may play in human obesity. Low amino acid sequence identity in the E3 CR1 beta and CR1 gamma genes may suggest distinctive roles for these proteins. Furthermore, the predicted molecular models of the HAdV-D36 fiber protein seem to implicate a unique tissue tropism for HAdV-D36.

Viral agents responsible for febrile respiratory illnesses among military recruits training in tropical Singapore.

BACKGROUND: Military personnel are highly susceptible to febrile respiratory illnesses (FRI), likely due to crowding, stress and other risk factors present in the military environment. OBJECTIVE: Our objective was to investigate the viral etiological agents responsible for FRI among military recruits training in a tropical climate in Singapore. STUDY DESIGN: From March 2006 through April 2007, a total of 1354 oropharyngeal (throat) swabs were collected from military recruits who reported sick with an oral temperature of > or =38 degrees C and a cough and/or sore throat. Real-time polymerase chain reaction (PCR) was used to assay for the presence of influenza A and B viruses and adenoviruses (H-AdV), and conventional PCR used for the remaining respiratory viruses in all specimens. RESULTS: Influenza A virus was the dominant infection with a laboratory-confirmed incidence of 24% (326/1354) and a predominance of the H3N2 subtype. The temporal pattern for influenza A virus infections coincided with the nation-wide pattern in the civilian community. Detection rates of 12% (159/1354) and 2.7% (5/1354) were obtained for influenza B virus and other respiratory viruses, respectively. CONCLUSIONS: The laboratory findings identified influenza A virus as the primary causative viral agent for FRI in the Singapore military, in strong contrast to findings from temperate countries and countries where recruits are often vaccinated for influenza. Our results suggest that influenza vaccination should be considered as a requirement to reduce the incidence of influenza infections. This is the first report describing respiratory infections in a tropical military setting, in a developed country in Asia.

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.

Adenovirus microsatellite reveals dynamics of transmission during a recent epidemic of human adenovirus serotype 14 infection.

This study reveals diverse-length polymorphisms in long mononucleotide repeats (microsatellites) in several serotypes of epidemic human respiratory adenovirus. The length of one of these microsatellites, a homopolymeric thymidine [poly(T)] repeat, is measured in 68 isolates of adenovirus serotype 14. These isolates were collected during a series of sudden and sometimes fatal outbreaks among both military recruits and civilians as the virus emerged for the first time in the United States in 2006 and 2007. The results demonstrate the usefulness of adenoviral microsatellites as high-resolution molecular strain markers. The described homopolymer is hypervariable in length, varying from 12 to 17 bp in the analyzed sample set. All intermediate lengths were identified in at least one isolate. Furthermore, the specific length of the marker is stable for significant periods of time (up to 7 months) at individual sites where the virus is in consistent circulation. The microsatellite also can maintain specific length identity through site-to-site transmission events, as determined by the analysis of isolates from three advanced training sites that appeared to be subject to pathogen transfer from one of the affected recruit training installations. Public database searches revealed that the polymorphic nature of the microsatellite extends to other species B serotypes, and that other polymorphic microsatellites can be identified readily in a variety of epidemic respiratory adenovirus clades. This study shows that microsatellites are a ubiquitous source of polymorphic markers for human adenoviruses and demonstrates their use through an epidemiological analysis of isolates from a recent North American epidemic.

Outbreak of severe respiratory disease associated with emergent human adenovirus serotype 14 at a US air force training facility in 2007.

BACKGROUND: In 2007, a US Air Force training facility reported a cluster of severe respiratory illnesses associated with a rare human adenovirus (Ad) serotype, Ad14. We investigated this outbreak to better understand its epidemiology, clinical spectrum, and associated risk factors. METHODS: Data were collected from ongoing febrile respiratory illness (FRI) surveillance and from a retrospective cohort investigation. Because an Ad7 vaccine is in development, Ad7 antibody titers in pretraining serum samples from trainees with mild and those with severe Ad14 illness were compared. RESULTS: During 2007, an estimated 551 (48%) of 1147 trainees with FRI were infected with Ad14; 23 were hospitalized with pneumonia, 4 required admission to an intensive care unit, and 1 died. Among cohort members (n = 173), the Ad14 infection rate was high (50%). Of those infected, 40% experienced FRI. No cohort members were hospitalized. Male sex (risk ratio [RR], 4.7 [95% confidence interval {CI}, 2.2-10.1]) and an ill close contact (RR, 1.6 [95% CI, 1.2-2.2]) were associated with infection. Preexisting Ad7 neutralizing antibodies were found in 7 (37%) of 19 Ad14-positive trainees with mild illness but in 0 of 16 trainees with Ad14 pneumonia (P = .007). CONCLUSIONS: Emergence of Ad14, a rare Ad serotype, caused a protracted outbreak of respiratory illness among military recruits. Most infected recruits experienced FRI or milder illnesses. Some required hospitalization, and 1 died. Natural Ad7 infection may protect against severe Ad14 illness.

A double-blind, placebo-controlled study of the safety and immunogenicity of live, oral type 4 and type 7 adenovirus vaccines in adults.

Adenovirus serotypes 4 (ADV-4) and 7 (ADV-7) are important causes of febrile acute respiratory disease (ARD) in US military recruits. Previously licensed vaccines, which effectively controlled adenovirus-associated ARD, are no longer available. In the Fall of 2004 we conducted this Phase 1 randomized, double-blind, placebo-controlled trial of the live, oral ADV-4 and ADV-7 vaccines made by a new manufacturer to assess their safety and immunogenicity. The adenovirus vaccines were administered orally together in a single dose to thirty subjects. Twenty eight additional subjects received placebo. Subjects were then observed for 8 weeks. The most commonly reported adverse events were nasal congestion (33%), cough (33%), sore throat (27%), headache (20%), abdominal pain (17%), arthralgia (13%), nausea (13%) and diarrhea (13%). None of these rates differed significantly from placebo. The duration of vaccine virus fecal shedding was 7-21 days. Seventy three percent of vaccine recipients seroconverted to ADV-4 (GMT 23.3) while 63% seroconverted to ADV-7 (GMT 51.1) by Day 28. The new ADV-4 and ADV-7 vaccines were safe and induced a good immune response in the study population. Expanded trials for safety and efficacy are in progress.

Rapid detection and molecular serotyping of adenovirus by use of PCR followed by electrospray ionization mass spectrometry.

We have developed a PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) assay for the rapid detection, identification, and serotyping of human adenoviruses. The assay employs a high-performance mass spectrometer to "weigh" the amplicons obtained from PCR using primers designed to amplify known human adenoviruses. Masses are converted to base compositions and, by comparison against a database of the genetic sequences, the serotype present in a sample is determined. The performance of the assay was demonstrated with quantified viral standards and environmental and human clinical samples collected from a military training facility. Over 500 samples per day can be analyzed with sensitivities greater than 100 genomes per reaction. This approach can be applied to many other families of infectious agents for rapid and sensitive analysis.

Abrupt emergence of diverse species B adenoviruses at US military recruit training centers.

BACKGROUND: Adenoviruses (Ads) cause continuous outbreaks of acute respiratory disease (ARD) in US military training facilities. In 1996, the loss of vaccines targeting the dominant recruit-associated serotypes precipitated the reemergence of Ads in these populations. Between 1999 and 2002, serotype 4 accounted for >95% of Ads isolated from recruits and for >50% of ARD cases in training facilities (15,000 cases/year). METHODS: Ads (n=1867) collected between 2002 and 2006 from recruits with ARD at 8 military training facilities in the United States were serotyped by serum neutralization and polymerase chain reaction. RESULTS: The dominance of Ad4 continued through 2005, followed by a simultaneous emergence of diverse species B serotypes at the majority of sites. This included the subspecies B1 serotypes 3, 7, and 21 and the subspecies B2 serotype 14. Ad14 was the most prevalent species B serotype, appearing in high numbers at 3 sites and becoming dominant at 1. CONCLUSIONS: Subspecies B2 Ads have rarely been associated with ARD, and only in Eurasia. This survey represents the first report of AdB2-associated ARD in the Western Hemisphere. The simultaneous emergence of several species B Ads suggests a common external source (the civilian population) and a decrease in preexisting immunity to species B Ads.