Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses.

Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), and in vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping.

Emergent 2009 influenza A(H1N1) viruses containing HA D222N mutation associated with severe clinical outcomes in the Americas.

BACKGROUND: During the 2010-2011 influenza season, a small sub-group of 2009 influenza A(H1N1) viruses (hereafter referred to as 2009 A(H1N1)) emerged that was associated with more severe clinical outcomes in Ecuador and North America. Genetically, the haemagglutinin (HA) of this sub-clade was distinct from HAs found in viruses associated with severe outbreaks in 2010 from the United Kingdom and from other global specimens isolated earlier in the season. OBJECTIVE: We report the emergence of a novel 2009 A(H1N1) variant possessing a re-emergent HA D222N mutation obtained from patients with severe respiratory illnesses and phylogenetically characterise these D222N mutants with other severe disease-causing variants clustering within a common emerging sub-clade. CASE REPORTS: In early 2011, three cases of 2009 A(H1N1) infection, two from Quito, Ecuador, and one from Washington, DC, USA, were complicated by severe pneumonia requiring mechanical ventilation, resulting in one fatality. These cases were selected due to the reported nature of the acute respiratory distress (ARD) that were captured in Department of Defence (DoD)-sponsored global influenza surveillance nets. RESULTS: Genetically, the 2009 A(H1N1) strains isolated from two of the three severe cases carried a prominent amino acid change at position 222 (D222N) within the primary HA receptor binding site. Furthermore, these cases represent an emerging sub-clade of viruses defined by amino acid changes within HA: N31D, S162N, A186T and V272I. Phylogenetically, these viruses share a high degree of homology with strains associated with recent fatal cases in Chihuahua, Mexico. DISCUSSION: Previously, enhanced virulence associated with the change, D222G, has been clinically linked to severe morbidity and mortality. Initial observations of the prevalence of a novel sub-clade of strains in the Americas suggest that viruses with a re-emergent D222N mutation may too correlate with severe clinical manifestations. These findings warrant heightened vigilance for emerging sub-clades of 2009 A(H1N1) and presumptive clinical implications.

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.

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.

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.

Diagnostic approach to acute diarrheal illness in a military population on training exercises in Thailand, a region of campylobacter hyperendemicity.

High rates of Campylobacter fluoroquinolone resistance highlight the need to evaluate diagnostic strategies that can be used to assist with clinical management. Diagnostic tests were evaluated with U.S. soldiers presenting with acute diarrhea during deployment in Thailand. The results of bedside and field laboratory diagnostic tests were compared to stool microbiology findings for 182 enrolled patients. Campylobacter jejuni was isolated from 62% of the cases. Clinical and laboratory findings at the time of presentation were evaluated to determine their impact on the posttest probability, defined as the likelihood of a diagnosis of Campylobacter infection. Clinical findings, the results of tests for inflammation (stool occult blood testing [Hemoccult], fecal leukocytes, fecal lactoferrin, plasma C-reactive protein), and the numbers of Campylobacter-specific antibody-secreting cells in peripheral blood failed to increase the posttest probability above 90% in this setting of Campylobacter hyperendemicity when these findings were present. Positive results by a Campylobacter-specific commercial enzyme immunoassay (EIA) and, less so, a research PCR were strong positive predictors. The negative predictive value for ruling out Campylobacter infection, defined as a posttest probability of less than 10%, was similarly observed with these Campylobacter-specific stool-based tests as well the fecal leukocyte test. Compared to the other tests evaluated, the Campylobacter EIA is a sensitive and specific rapid diagnostic test that may assist with diagnostic evaluation, with consideration of the epidemiological setting, logistics, and cost.

Inhibition of dengue virus replication by mycophenolic acid and ribavirin.

Dengue viruses (DEN), mosquito-borne members of the family Flaviviridae, are human pathogens of global significance. The effects of mycophenolic acid (MPA) and ribavirin (RBV) on DEN replication in monkey kidney (LLC-MK2) cells were examined. MPA (IC(50)=0.4+/-0.3 microM) and RBV (IC(50)=50.9+/-18 microM) inhibited DEN2 replication. Quantitative real-time RT-PCR of viral RNA and plaque assays of virions from DEN2-infected and MPA (10 microM)- and RBV (> or =200 microM)-treated cells showed a fivefold increase in defective viral RNA production by cells treated with each drug. Moreover, a dramatic reduction of intracellular viral replicase activity was seen by in vitro replicase assays. Guanosine reversed the inhibition of these compounds, suggesting that one mode of antiviral action of MPA and RBV is by inhibition of inosine monophosphate dehydrogenase and thereby depletion of the intracellular GTP pool. In addition, RBV may act by competing with guanine-nucleotide precursors in viral RNA translation, replication and 5' capping.

Emergence of a new human adenovirus type 4 (Ad4) genotype: identification of a novel inverted terminal repeated (ITR) sequence from majority of Ad4 isolates from US military recruits.

BACKGROUND: Ad4 is the principal etiological agent of acute respiratory disease (ARD) in the US military. Discovery of the novel 208bp inverted terminal repeated (ITR) sequence from a recent Ad4 Jax78 field isolate was totally distinct from the analogous 116bp ITR of Ad4 prototype. OBJECTIVES: To investigate the origin and distribution of the novel Ad4 ITR sequence from ARD infections. STUDY DESIGN: Direct sequencing of ligated Ad ITR termini. RESULTS: The new Ad4 ITR was highly homologous with the ITRs of human Ad subgroup B. The left post-ITR region of Ad4 Jax78 was found to be highly homologous to the corresponding region of subgroup B Ads: 81% for Ad11 and 98% for Ad3 and Ad7. The right post-ITR region of Ad4 Jax78 contained a truncated classic ITR of the Ad4 prototype. CONCLUSIONS: The Ad4 Jax78 ITR most likely evolved from Ad4 prototype by substituting the Ad4 prototype ITR with the subgroup B Ads ITR. The ITR-based PCR assays developed from this study can be used to distinguish the new Ad4 genotype from the classical Ad4 prototype. The new Ad4 genotype was first detected in 1976 from Georgia, USA, and is the main causative agent of ARD infections in US military population.

Development and evaluation of an efficient 3'-noncoding region based SARS coronavirus (SARS-CoV) RT-PCR assay for detection of SARS-CoV infections.

The severe acute respiratory syndrome (SARS) epidemic originating from China in 2002 was caused by a previously uncharacterized coronavirus that could be identified by specific RT-PCR amplification. Efforts to control future SARS outbreaks depend on the accurate and early identification of SARS-CoV infected patients. A real-time fluorogenic RT-PCR assay based on the 3'-noncoding region (3'-NCR) of SARS-CoV genome was developed as a quantitative SARS diagnostic tool. The ideal amplification efficiency of a sensitive SARS-CoV RT-PCR assay should yield an E value (PCR product concentration increase per amplification cycle) equal to 2.0. It was demonstrated that the 3'-NCR SARS-CoV based RT-PCR reactions could be formulated to reach excellent E values of 1.81, or 91% amplification efficacy. The SARS-CoV cDNA preparations derived from viral RNA extract and the cloned recombinant plasmid both exhibit the identical amplification characteristics, i.e. amplification efficacy using the same PCR formulation developed in this study. The viral genomic copy (or genomic equivalences, GE) per infectious unit (GE/pfu) of SARS-CoV used in this study was also established to be approximate 1200-1600:1. The assay's detection sensitivity could reach 0.005 pfu or 6-8 GE per assay. It was preliminarily demonstrated that the assay could efficiently detect SARS-CoV from clinical specimens of SARS probable and suspected patients identified in Taiwan. The 3'-NCR based SARS-CoV assay demonstrated 100% diagnostic specificity testing samples of patients with acute respiratory disease from a non-SARS epidemic region.

Detection of Shigella by a PCR assay targeting the ipaH gene suggests increased prevalence of shigellosis in Nha Trang, Vietnam.

Shigella spp. are exquisitely fastidious gram-negative organisms which frequently escape detection by traditional culture methods. To get a more complete understanding of the disease burden caused by Shigella in Nha Trang, Vietnam, real-time PCR was used to detect Shigella DNA. Randomly selected rectal swab specimens from 60 Shigella culture-positive patients and 500 Shigella culture-negative patients detected by population-based surveillance of patients seeking care for diarrhea were processed by real-time PCR. The target of the primer pair is the invasion plasmid antigen H gene sequence (ipaH), carried by all four Shigella species and enteroinvasive Escherichia coli. Shigella spp. could be isolated from the rectal swabs of 547 of 19,206 (3%) patients with diarrhea. IpaH was detected in 55 of 60 (93%) Shigella culture-positive specimens, whereas it was detected in 87 of 245 (36%) culture-negative patients free of dysentery (P < 0.001). The number of PCR cycles required to detect a PCR product was highest for culture-negative, nonbloody diarrheal specimens (mean number of cycles to detection, 36.6) and was lowest for children with culture-positive, bloody diarrheal specimens (mean number of cycles, 25.3) (P < 0.001). The data from real-time PCR amplification indicate that the culture-proven prevalence of Shigella among patients with diarrhea may underestimate the prevalence of Shigella infections. The clinical presentation of shigellosis may be directly related to the bacterial load.

Evaluation of a rapid quantitative diagnostic test for adenovirus type 4.

Acute respiratory disease (ARD) due to adenoviruses is a reemerging disease in military recruits. It is a challenge for clinicians to accurately diagnose this disease and to appropriately treat affected individuals. This study investigated the utility of a quantitative, rapid-cycle, real-time fluorogenic polymerase chain reaction (PCR) technique for detecting adenovirus type 4 (Ad4) in a clinical setting. Throat swab specimens and clinical data were collected from US Army basic trainees hospitalized with ARD at Fort Jackson, South Carolina. A total of 140 throat swab specimens were collected from 83 subjects. Rapid PCR results (obtained in <2 h) had a sensitivity of 100% and a specificity of 100%, compared with viral culture. There was no difference, qualitative or quantitative, between frozen and fresh samples for PCR detection of Ad4. Individuals with test results positive for Ad4 were hospitalized longer than were individuals with negative test results. Higher virus loads at hospital admission corresponded to longer lengths of stay for Ad4-positive subjects.

Vaccination of human volunteers with monovalent and tetravalent live-attenuated dengue vaccine candidates.

Four serotypes of monovalent live attenuated dengue virus vaccine candidates were tested for reactogenicity and immunogenicity in 49 flavivirus non-immune adult human volunteers. The four monovalent candidates were then combined into a tetravalent formulation and given to another 10 volunteers. Neutralizing antibody seroconversion rates after a single-dose monovalent vaccination ranged from 53% to 100%. Solicited reactogenicity was scored by each volunteer. A composite index, the Reactogenicity Index, was derived by these self-reported scores. Reactogenicity differed among the four serotype candidates with serotype-1 associated with the most vaccine related side effects. A second dose of monovalent vaccines at either 30 days or 90 days was much less reactogenic but did not significantly increase seroconversion rates. Seroconversion rates in the 10 volunteers who received a single dose of tetravalent vaccine ranged from 30% to 70% among the four serotypes. Similar to the monovalent vaccines, a second dose of the tetravalent vaccine at one month was less reactogenic and did not increase seroconversion. A third dose of the tetravalent vaccine at four months resulted in three of four volunteers with trivalent or tetravalent high-titer neutralizing antibody responses.

Differing influences of virus burden and immune activation on disease severity in secondary dengue-3 virus infections.

Dengue hemorrhagic fever (DHF), the most severe form of illness following infection with a dengue virus, is characterized by plasma leakage, thrombocytopenia, and hepatic inflammation. The interrelationships among virus burden, immune activation, and development of DHF were examined in 54 children with secondary dengue-3 virus infections participating in a prospective, hospital-based study. DHF was associated with higher mean plasma viremia early in illness and earlier peak plasma interferon-gamma levels. Maximum plasma viremia levels correlated with the degree of plasma leakage and thrombocytopenia. Maximum plasma levels of interleukin (IL)-10 and soluble tumor necrosis factor receptor-II correlated with the degree of thrombocytopenia, independently of viremia levels. Hepatic transaminase elevation correlated with plasma soluble IL-2 receptor levels and not with viremia levels. Quantitative differences in virus burden and host immune responses, and the timing of type 1 cytokine responses, have differing influences on the severity of disease manifestations during secondary dengue-3 virus infections.

Rapid type-specific diagnosis of adenovirus type 4 infection using a hexon-based quantitative fluorogenic PCR.

A hexon-based fluorogenic polymerase chain reaction (PCR) assay utilizing the 5'-nuclease activity of DNA Taqpolymerase was developed as a rapid and type-specific diagnostic system for adenovirus type 4 (Ad4) detection and quantification. The assay consists of a pair of flanking primers and an internal fluorescence labeled probe that allows real time amplification to quantify the Ad4 virus. One out of 12 flanking primer pairs evaluated (combinations of three forward primers and four reverse primers) was found to be optimal for Ad4 virus detection that yielded background-free operation, i.e., no fluorescent signal generated by non-template controls. The assay was employed to detect Ad4 reference virus strain RI-67, Wyeth Ad4 vaccine strain and 71 different clinical Ad4 isolates from US military recruits used in this study with consistent sensitivity (lower detection limit) of 2-4 pfu per PCR reaction. The assay showed linear Ad4 detection with a dynamic range of greater than five logs (from 2-4 pfu/assay to greater than 10(5) pfu/assay). This Ad4-specific assay did not crossreact with representative members of Ad subgroups A, B, C, D and F at viral concentrations greater than 10(8) pfu/ml. It was also demonstrated that Ad4 viruses could be efficiently detected from throat swabs (71/72 specimens or 98.6% detection sensitivity) of infected patients by the Ad4-specific PCR. In general, there was a good correlation between PCR determined viral titers in throat swabs and time required to detect viral cytopathic effects (CPE) in cell culture. Evaluation of the simple Ad4 specific assay developed in this study could be used to provide a rapid clinically relevant diagnosis of Ad4 infections in patients with acute respiratory disease (ARD).