Identification of genotypically diverse Cryptococcus neoformans and Cryptococcus gattii isolates by Luminex xMAP technology

A Luminex suspension array, which had been developed for identification of Cryptococcus neoformans and Cryptococcus gattii isolates, was tested by genotyping a set of 58 mostly clinical isolates. All genotypes of C. neoformans and C. gattii were included. In addition, cerebrospinal fluid (CSF) obtained from patients with cryptococcal meningitis was used to investigate the feasibility of the technique for identification of the infecting strain. The suspension array correctly identified haploid isolates in all cases. Furthermore, hybrid isolates possessing two alleles of the Luminex probe region could be identified as hybrids. In CSF specimens, the genotype of the cryptococcal strains responsible for infection could be identified after optimization of the PCR conditions. However, further optimization of the DNA extraction protocol is needed to enhance the usability of the method in clinical practice.     

Comparison of the Luminex Respiratory Virus Panel Fast Assay with In-House Real-Time PCR for Respiratory Viral Infection Diagnosis

The Luminex xTAG Respiratory Virus Panel (RVP) assay has been shown to offer improved diagnostic sensitivity over traditional viral culture methods and to have a sensitivity comparable to those of individual real-time nucleic acid tests for respiratory viruses. The objective of this retrospective study was to test a new, streamlined version of this assay, the RVP Fast assay, which requires considerably less run time and operator involvement. The study compared the performance of the RVP Fast assay with those of viral culture, a direct fluorescent assay (DFA), and a panel of single and multiplex real-time PCRs in the testing of 286 respiratory specimens submitted to the Edinburgh Specialist Virology Centre for routine diagnosis of viral infection between December 2007 and February 2009. At least one respiratory viral infection was detected in 13.6% of specimens by culture and DFA combined, in 49.7% by real-time PCR, and in 46.2% by the RVP Fast assay. The sensitivity and specificity of the RVP Fast assay compared to the results of real-time PCR as the gold standard were 78.8% and 99.6%, respectively. Real-time PCR-positive specimens missed by the RVP Fast assay generally had low viral loads or were positive for adenovirus. Additionally, a small number of specimens were positive by the RVP Fast assay but were not detected by real-time PCR. For some viral targets, only a small number of positive results were found in our sample set using either method; therefore, the sensitivity of detection of the RVP Fast assay for individual targets could be investigated further with a greater number of virus-positive specimens.Viral infections of the respiratory tract have traditionally been diagnosed in the laboratory by culture of respiratory specimens and direct fluorescent assay (DFA). However, the availability of real-time PCR has allowed us to detect respiratory viruses with greater sensitivity and shorter turnaround times (12). In recent years, a number of new respiratory viruses have been identified, so we must now consider a wider range of viruses in our diagnoses (see, e.g., references 1 and 14). However, the number of fluorophores that can be differentiated in a multiplex real-time PCR assay limits the number of viral targets that can be detected.One solution is to screen each specimen with several different multiplex real-time PCRs to cover a large number of viruses (4). An alternative, the xTAG respiratory virus panel (RVP) assay (Luminex Molecular Diagnostics Inc., Toronto, Canada), is based on suspension microarray technology, which enables the detection of a large number of targets in a single reaction (6, 9). The xTAG RVP assay has been shown to offer results comparable or superior to those of culture/DFA and nucleic acid tests for the diagnosis of respiratory viral infections (7, 10). Recently, the RVP assay has been used successfully for the detection of etiological agents in outbreaks of respiratory illness (3, 15).The latest version of this test, the RVP Fast assay, has a simpler protocol and a shorter turnaround time than the original assay but still detects 19 different viral and subtype targets: influenza A virus (with additional subtyping: H1, H3, and H5), influenza B virus, respiratory syncytial virus A (RSV-A), RSV-B, parainfluenza virus 1 (PIV-1), PIV-2, PIV-3, PIV-4, adenovirus, human metapneumovirus, coronaviruses 229E, NL63, OC43, and HKU1, enterovirus/rhinovirus (EV/RhV), and human bocavirus. Here we compare the performance of the RVP Fast assay with those of culture/DFA and in-house real-time PCR assays, using respiratory specimens collected for routine viral testing.     

Multiplexed typing of Mycobacterium avium subsp. paratuberculosis types I, II, and III by Luminex xMAP suspension array

Differentiation among types I, II, and III is the primary step in typing Mycobacterium avium subsp. paratuberculosis. We propose an innovative approach based on detection of gyrase B (gyrB) gene polymorphisms by suspension array technology, with high discriminatory power and high-throughput potential.     

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 (10¹ to 10⁸ 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.     

Meta-Analysis of the Diagnostic Efficacy of the Luminex xTAG Respiratory Viral Panel FAST v2 Assay for Respiratory Viral Infections

PurposeAcute respiratory viral infections pose significant morbidity and mortality, making it essential to diagnose respiratory viral infections rapidly. In this study, the diagnostic efficacy of the Luminex xTAG Respiratory Virus Panel (RVP) FAST v2 test was evaluated on respiratory viral infections.Materials and MethodsInformation was retrieved from electronic databases, including Embase, Web of Science, PubMed, and Cochrane Library, for systematic review. Studies that fulfilled predefined inclusion criteria were included. After the extraction of information, statistical software was utilized for quality evaluation, data analysis, and assessment of publication bias.ResultsEighty groups in fourfold tables from nine articles were included to perform statistical analyses. Therein, the mean specificity and mean sensitivity of Luminex xTAG RVP FAST v2 test for the detection of respiratory viral infections were 0.99 (0.98–0.99) and 0.88 (0.87–0.90), respectively. Additionally, the negative and positive likelihood ratios were 0.14 (0.11–0.19) and 87.42 (61.88–123.50), respectively. Moreover, the diagnostic odds ratio and area under the curve of summary receiver operating characteristic were 714.80 and 0.9886, respectively.ConclusionThe Luminex xTAG RVP FAST v2 test could be a reliable and rapid diagnostic method for multiple respiratory viral infections.     

Development of a Fourfold Multiplexed Opsonophagocytosis Assay for Pneumococcal Antibodies against Additional Serotypes and Discovery of Serological Subtypes in Streptococcus pneumoniae Serotype 20

Opsonophagocytic killing assays (OPAs) are important in vitro surrogate markers of protection in vaccine studies of Streptococcus pneumoniae. We have previously reported the development of a 4-fold multiplexed OPA (MOPA) for the 13 serotypes in Prevnar 13. Because new conjugate vaccines with increased valence are being developed, we developed 4-fold MOPAs for an additional 13 serotypes: serotypes 6C and 6D, plus the 11 serotypes contained in Pneumovax but not in Prevnar 13. A high level of nonspecific killing (NSK) was observed for three serotypes (10A, 15B, and 33F) in multiple batches of baby rabbit complement. The NSK could be reduced by preadsorbing the complement with encapsulated, as well as unencapsulated, pneumococcal strains. The MOPA results compared well with the results of single-serotype OPA for all serotypes except for serotype 3. For serotype 3, the results obtained from the MOPA format were ∼40% higher than those of the single-serotype format. Interassay precision of MOPA was determined with 5 serum samples, and the coefficient of variation was generally <30% for all serotypes. MOPA was also specific for all serotypes except for serotype 20; i.e., free homologous polysaccharide (PS), but not unrelated PS, could completely and efficiently inhibit opsonization. However, serotype 20 PS from ATCC could efficiently inhibit opsonization of one serotype 20 target strain but not three other type 20 target strains even at a high (>80 mg/liter) PS concentration. This suggests the presence of serologic heterogeneity among serotype 20 strains.     

Elimination of False-Positive Results in a Luminex Assay for Pneumococcal Antibodies

In our 14-valent Luminex assay for pneumococcal antibodies, we identified two groups of sera that caused false-positive results. The first group bound nonspecifically to the Luminex microspheres. The second group reacted specifically with bovine serum albumin (BSA). We describe here methods that eliminated the false-positive reactivity of both groups.We previously described a multiplexed pneumococcal antibody assay based on the Luminex xMAP technology (11). Several other laboratories subsequently also described multiplexed Luminex assays for detecting these antibodies (1, 6, 15). In addition, the Luminex xMAP technology has been used for multiplexed assays to measure antibody responses to vaccines for other infectious diseases, including those caused by Neisseria meningitidis, Haemophilus influenza type b, Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, Bacillus anthracis, and papilloma virus (3, 4, 10, 12, 13, 17). Waterboer et al. (18) documented an intrinsic problem with the use of the Luminex technology for serological assays. They found that some human sera bind directly to the carboxylated MicroPlex (formally MultiAnalyte) microspheres, causing a very high level of nonspecific background. These workers also found that SeroMAP microspheres, introduced by Luminex Corporation specifically for use in serological assays, reduced but did not eliminate the nonspecific-binding problem.Using our original protocol (11) for the 14-valent pneumococcal antibody assay with lot B MicroPlex microspheres, we encountered serum samples with very high-level false-positive results that were near or above the analytical measurement range (AMR) of the assay (Table ​(Table1).1). Approximately 15 of every 1,000 sera tested for pneumococcal antibodies exhibited this behavior. We termed these samples “polyspecific,” although they did not react specifically to pneumococcal polysaccharides (PnPs). We tested a panel of 33 of these polyspecific sera and 1 control serum sample not showing polyspecific reactivity against an unconjugated MicroPlex microsphere and an unconjugated SeroMAP microsphere. The serum samples used in this study were submitted to ARUP Laboratories for pneumococcal antibody testing. All samples were deidentified according to protocols approved by the University of Utah Institutional Review Board (no. 7275). Serum samples were diluted 1:25 in phosphate-buffered saline (PBS), pH 7.2, with 5 μg/ml pneumococcal C-polysaccharide (C-Ps) (Staten Serum Institut, Copenhagen, Denmark), 5 μg/ml pneumococcal polysaccharide 22F (American Type Culture Collection, Manassas, VA), and 0.0015% bromcresol purple (BCP) (Sigma-Aldrich, St. Louis, MO). A MicroPlex (region 7) (Luminex Corporation, Austin, TX) microsphere and a SeroMAP (region 8) (Luminex Corporation, Austin, TX) microsphere were pelleted by centrifugation and resuspended in blocking/storage (B/S) buffer consisting of PBS with 0.1% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO) or in BSA-free StabliGuard immunoassay stabilizer (SG01) (SurModics, Inc., Eden Prairie, MN). Serum dilutions were incubated with the uncoupled microspheres for 20 min at room temperature with shaking, washed once with PBS by filtration, incubated for 20 min at room temperature with shaking with phycoerythrin (PE)-labeled affinity-purified anti-human IgG (γ) (Southern Biotech, Birmingham, AL) in B/S buffer, and washed once with PBS. Microspheres were counted with a Luminex 100 analyzer. The MicroPlex and SeroMAP microspheres were compared in the two diluents in the same assay run, with the same sample dilutions and PE conjugate, to eliminate run-to-run variation. As shown in Fig. ​Fig.1A,1A, all 33 of the polyspecific sera tested reacted strongly to the unconjugated MicroPlex microsphere suspended in B/S buffer, with median fluorescence intensity (MFI) values that ranged from 905 to 18,674. In contrast, the MFI of the control serum sample was 38. Compared to those for the MicroPlex microsphere, the MFI values for the SeroMAP microsphere suspended in B/S buffer were low. All 33 of the polyspecific sera, however, had background MFI values above 110, compared to the control serum sample, which had an MFI of 28. Twenty-four of the 33 sera (72.7%) had MFI values above the cutoff value of 200. A background MFI value of 200 could falsely elevate the pneumococcal antibody assay results by 0.1 μg/ml or more for 5 of the 14 serotypes. If the long-term protective level after pneumococcal vaccine immunization is considered to be 1 μg/ml, a background MFI level of 200 could lead to misinterpretation of protective status. In addition, 10 of the polyspecific sera had background MFI levels above 500 with the SeroMAP microsphere, and 5 of these sera had MFI levels above 1,000. Two of the polyspecific sera, no. 3 and 23, had very high levels of nonspecific reactivity to the SeroMAP microspheres, with MFI values of 4,877 and 2,666, respectively.Open in a separate windowFIG. 1.Nonspecific reactivity of human sera to Luminex microspheres. Shown are median fluorescence intensities for 33 polyspecific sera and a control serum sample reacted against unconjugated MicroPlex (clear bars) and SeroMAP (solid bars) microspheres. (A) Microspheres suspended in B/S buffer. (B) Microspheres suspended in StabliGuard.

TABLE 1.

IgG concentrations in serum before (protocol 1) and after (protocol 2) removal of nonspecific binding to microspheres

近来我国马群中呼吸道疾病流行的病因学研究

通过病因学，血清学和流行病学研究，弄清了近期我国马群中类流感疾病流行的病因为马２亚型流感病毒。采用多克隆和单克隆抗体对分离物进行抗原性分析，结果提示，从我国马群中分离到的流感病毒株是马２（Ｈ３Ｎ８）亚型流感病毒，它绝然不同于过去我们发现的禽的Ｈ３Ｎ８流感病毒。估计它是近期内从国外马群传入我国。     

Evaluation of Luminex xTAG Gastrointestinal Pathogen Panel Assay for Detection of Multiple Diarrheal Pathogens in Fecal Samples in Vietnam

Diarrheal disease is a complex syndrome that remains a leading cause of global childhood morbidity and mortality. The diagnosis of enteric pathogens in a timely and precise manner is important for making treatment decisions and informing public health policy, but accurate diagnosis is a major challenge in industrializing countries. Multiplex molecular diagnostic techniques may represent a significant improvement over classical approaches. We evaluated the Luminex xTAG gastrointestinal pathogen panel (GPP) assay for the detection of common enteric bacterial and viral pathogens in Vietnam. Microbiological culture and real-time PCR were used as gold standards. The tests were performed on 479 stool samples collected from people admitted to the hospital for diarrheal disease throughout Vietnam. Sensitivity and specificity were calculated for the xTAG GPP for the seven principal diarrheal etiologies. The sensitivity and specificity for the xTAG GPP were >88% for Shigella spp., Campylobacter spp., rotavirus, norovirus genotype 1/2 (GI/GII), and adenovirus compared to those of microbiological culture and/or real-time PCR. However, the specificity was low (∼60%) for Salmonella species. Additionally, a number of important pathogens that are not identified in routine hospital procedures in this setting, such as Cryptosporidium spp. and Clostridium difficile, were detected with the GPP. The use of the Luminex xTAG GPP for the detection of enteric pathogens in settings, like Vietnam, would dramatically improve the diagnostic accuracy and capacity of hospital laboratories, allowing for timely and appropriate therapy decisions and a wider understanding of the epidemiology of pathogens associated with severe diarrheal disease in low-resource settings.     

Development of a Multiplex Primer Extension Assay for Rapid Detection of Salmonella Isolates of Diverse Serotypes

Food-borne salmonellosis is a major manifestation of gastrointestinal disease in humans across the globe. Accurate and rapid identification methods could positively impact the identification of isolates, enhance outbreak investigation, and aid infection control. The SNaPshot multiplex system is a primer extension-based method that enables multiplexing of single nucleotide polymorphisms (SNPs). Here the method has been developed for the identification of five Salmonella serotypes, commonly detected in the United Kingdom, based on serotype-specific SNPs identified in the multilocus sequence typing (MLST) database of Salmonella enterica. The SNPs, in genes hemD, thrA, purE, and sucA, acted as surrogate markers for S. enterica serovars Typhimurium, Enteritidis, Virchow, Infantis, and Braenderup. The multiplex primer extension assay (MPEA) was conducted in two separate panels and evaluated using 152 Salmonella enterica isolates that were characterized by MLST. The MPEA was shown to be 100% specific and sensitive, within this collection of isolates. The MPEA is a sensitive and specific method for the identification and detection of Salmonella serotypes based upon SNPs seen in MLST data. The method can be applied in less than 6 h and has the potential to improve patient care and source tracing. The utility of the assay for identification of Salmonella serotypes directly from clinical specimens and food samples warrants further investigation.Food-borne salmonellosis is an important public health problem, causing substantial morbidity and mortality worldwide. Transmission of salmonella to humans has been linked to multiple sources, including contaminated or undercooked food. Therefore, development of rapid and sensitive methods for detection of Salmonella serovars directly from clinical specimens and environmental samples may have a significant impact on the disease burden caused by this pathogen. Tools developed for such purposes could help both in preventing the spread of outbreaks and in clinical diagnosis. Traditional methods for diagnosis of Salmonella strains, including culture on selective media and biochemical and serological identification methods, have been successful in clinical diagnostic laboratories and in epidemiological surveillance (1, 10, 13, 26). However, these methods may require up to 3 days to provide results and are not as amenable to high-throughput analyses as some more-recently proposed molecular typing methods. Many molecular methods have been described for detection of Salmonella serovars (4, 6, 24, 25, 31, 33); however, these methods are not always rapid assays and generally do not permit serovar identification. In addition, these approaches cannot be applied directly to clinical and environmental samples and require a bacterial culture, which increases analysis time and can lead to culture-based biases.Multilocus sequence typing (MLST) is a high-resolution genotyping technique that has emerged as a powerful tool for determining the global epidemiology and population structure of many bacterial pathogens, including Salmonella enterica (18, 32). MLST detects sequence variability within particular genes to determine the genetic relatedness of organisms (12). The method is reproducible, and the data generated are easy to interpret and directly comparable among laboratories (9). However, MLST remains expensive and impractical for routine examination of bacterial genotype. Therefore, the use of informative single nucleotide polymorphisms (SNPs) has been recently suggested as a cost-effective alternative to full MLST characterization (2, 3). SNP typing, using various genetic targets, has been reported to be a robust method for analysis of relationships of S. enterica serovar Typhi (20, 22, 23). An advantage of the SNPs observed across MLST loci is that they represent neutral genetic variation in the bacterial genome (12) and thus could be used for developing accurate, rapid, economical, and phylogenetically meaningful typing methods that may lead to significant improvements in diagnosis and surveillance for Salmonella serotypes.The SNaPshot multiplex system (Applied Biosystems) can be used to genotype multiple known SNPs simultaneously using a multiplex primer extension assay (MPEA) approach. These assays monitor the single-base extension of unlabeled oligonucleotide primers, which bind to a cDNA template adjacent to target SNPs. DNA polymerase extends the primer at the polymorphic site, incorporating a fluorescently labeled dideoxynucleoside triphosphate (ddNTP), and the fluorescently labeled extension products are separated and visualized using capillary electrophoresis. The SNaPshot approach has been demonstrated to be efficient in various fields of research, such as forensic investigation (5, 21) and clinical diagnosis (11). The approach has been applied in microbiological investigations, though until recently, this has been for analysis of mammalian systems when interacting with bacteria (8, 14-16, 19, 28).In the present study, we have developed an MPEA for rapid “molecular serotyping” of Salmonella isolates by targeting 15 SNPs identified by interrogation of the MLST database for salmonella.(Some of this work was presented as a poster at the Health Protection Agency Conference, Warwick, United Kingdom, September 2009.)     

Evaluation and Implementation of FilmArray Version 1.7 for Improved Detection of Adenovirus Respiratory Tract Infection

The BioFire FilmArray respiratory panel is a multiplex PCR technology capable of detecting a number of bacteria and viruses that cause respiratory tract infection. The assay is technically simple to perform and provides rapid results, making it an appealing option for physicians and laboratorians. The initial product released by BioFire (version 1.6) was reported to have poor sensitivity for adenovirus detection and was therefore of concern when testing immunocompromised patients. This study evaluates the redesigned FilmArray assay (version 1.7) for detection of adenovirus. In this evaluation, we performed both retrospective and prospective verification studies, as well as a detailed serotype analysis. We found that version 1.7 demonstrated improved adenovirus sensitivity. In retrospective studies, sensitivity improved from 66.6% to 90.5%, and in prospective studies, it improved from 42.7% to 83.3%. In addition, when 39 clinically relevant serotypes were tested, 8 were not detected by version 1.6 and only 1 was not detected by version 1.7. The limit of detection remained the same when tested against serotype 4 but improved by 2 log units for serotype 7. Lastly, turnaround time analyses showed that the FilmArray assay was completed 3 h and 9 min after collection, which was more than a 37-h improvement over the previous multiplex PCR assay performed in our laboratory.     

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.Due to unique risk factors that include crowding and increased physical and psychological stress, military recruits in training are highly susceptible to outbreaks of acute respiratory disease (ARD), which are most often caused by viruses (26, 29). Human adenovirus (HAdV) infections have been recognized for decades as being important causes of ARD among military trainees in North America, Europe, and Asia (5, 8, 10, 15, 25, 27, 30, 32). The HAdV serotypes most frequently associated with ARD in both military and civilian communities include subspecies B1 HAdV-3, HAdV-7, and HAdV-21 and species E HAdV-4. The association of subspecies B2 HAdV infection with ARD has historically been rarely reported and restricted mostly to closed community outbreaks, some of which were documented among military trainees (1, 2, 8, 24, 36).From 11 January 2005 to 14 October 2005, a total of 226 male participants aged 18 to 24 years were enrolled in a study designed to detect and characterize viral agents responsible for ARD in the Singapore military recruit population. Analysis of laboratory data collected during this period identified influenza virus as the primary causative viral agent of respiratory illness among Singapore recruits in training (51 influenza virus-positive isolates out of 226 tested cases [22.5%]). HAdV-associated ARD cases were detected sporadically between January and October 2005. Thirty symptomatic trainees (13.3%) tested positive for HAdV during this period by a PCR assay described previously by Echavarria and colleagues (6). The temporal distribution of confirmed HAdV-associated cases of ARD is shown in Fig. ​Fig.1.1. Cases of adenovirus-associated ARD were detected between February and June 2005 and in October 2005.Open in a separate windowFIG. 1.Temporal distribution of HAdV-associated cases of ARD diagnosed among Singapore military recruits during 2005.One case of coinfection with influenza A virus was detected among these patients. Recruits testing positive for HAdV were between 19 and 21 years old. The examination of clinical characteristics of the HAdV-positive cases of ARD showed that 23.3% of the recruits reported shortness of breath, 50% reported nasal congestion, 80% reported a headache, 80% reported body ache, and 13.3% reported signs of nausea or vomiting. The identified influenza A virus coinfection did not increase the severity of the respiratory symptoms. One patient presented with additional symptoms of conjunctivitis, but no eye swab samples were collected. Adenovirus isolation was accomplished for 27 of the 30 positive clinical specimens. All virus isolates were initially typed as species B HAdVs by PCR as described previously by Metzgar and colleagues (22). Isolates were further characterized by restriction enzyme analysis and sequencing of the hexon and fiber genes as described previously by Kajon and Erdman (13). Digestion with BamHI identified 26 of 27 isolates as belonging to subspecies B2 HAdVs and one isolate as being HAdV-3. Digestion of viral DNAs with BclI, BglII, BstEII, DraI, HindIII, PstI, SmaI, and XbaI determined that the 26 subspecies B2 isolates were identical and identified them as corresponding to genome type 11a (Fig. ​(Fig.2A)2A) (17). The HAdV-3 isolate was identified as belonging to genome type 3a2 (Fig. ​(Fig.2B)2B) (16).Open in a separate windowFIG. 2.(A) Restriction enzyme analysis of a representative HAdV-11a isolate (SNG1222). (B) Restriction enzyme analysis of HAdV-3a2 isolate SNG1206. M, 1-kbp and 100-bp molecular markers.By using the primer sets described in Table ​Table1,1, identical hexon and fiber sequences were obtained for three randomly selected HAdV-11a isolates (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ607010","term_id":"241992696","term_text":"FJ607010"}}FJ607010 and {"type":"entrez-nucleotide","attrs":{"text":"FJ603103","term_id":"284808702","term_text":"FJ603103"}}FJ603103 for isolate SNG1218, accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ607011","term_id":"241992698","term_text":"FJ607011"}}FJ607011 and {"type":"entrez-nucleotide","attrs":{"text":"FJ603104","term_id":"284808704","term_text":"FJ603104"}}FJ603104 for isolate SNG1222, and accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ607012","term_id":"241992700","term_text":"FJ607012"}}FJ607012 and {"type":"entrez-nucleotide","attrs":{"text":"FJ603105","term_id":"284808706","term_text":"FJ603105"}}FJ603105 for isolate SNG1223). Alignment of the sequences of the hexon gene corresponding to hypervariable regions 1 to 7 using the Basic Local Alignment Search Tool (BLAST) optimized for highly similar sequences (megablast) against the NCBI GenBank database (http://www.ncbi.nlm.nih.gov) showed the three examined viruses to correspond to serotype 11 (19, 28). By using ClustalW implemented in Lasergene (DNASTAR, Inc., Madison, WI), the sequences for both the hexon and the fiber genes of the Singapore HAdV-11a strain were found to be highly similar to those reported for Asian and Middle Eastern HAdV-11 strains circulating over the last 2 decades in association with respiratory disease (Table ​(Table2).2). In addition, the complete genomic sequence obtained at the Walter Reed Army Institute of Research for isolate SNG1222 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ597732","term_id":"257122613","term_text":"FJ597732"}}FJ597732) was found to be 99.9% identical to that reported previously for strain QS-DLL, isolated in China in 2006 during an outbreak of ARD (33) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ643676","term_id":"256028986","term_text":"FJ643676"}}FJ643676).The identified differences between these two genomes are listed in Table ​Table2.2. The results of this study suggest that, in contrast to other geographic locations where HAdV-3, -4, -7, and -21 rank among the most prevalent serotypes, HAdV-11 may be a relatively more important causative agent of ARD in military training facilities in Singapore. The circulation of HAdV-11 in South East Asia has been documented since the early 1960s in association with conjunctivitis, pharyngoconjuctival fever, ARD, and hemorrhagic cystitis among immunocompromised individuals (7, 9, 14, 17, 24, 31, 34-36). The work of Wadell and colleagues demonstrated the existence of two main clusters of relative homology for HAdV-11 genome types: the cluster of prototype-like genomic variants with a tropism for the renal epithelium, and the a-like cluster, with a distinct tropism for the respiratory tract. In addition to their unique restriction site maps, the p-like and a-like genomes differ in the sequences of the fiber gene involving the receptor binding domains (17, 20, 21). As noted by others previously (33), the fiber of the 11a-like genomes is more closely related to the fiber of the prototype strain of HAdV-14, de Wit (99.5% identity), than to the fiber of the prototype strain of HAdV-11, Slobitski (94.4% identity), suggesting that this genomic variant of HAdV-11 is an intertypic recombinant 11-14.

TABLE 1.

Primers used for amplification and sequencing of hexon and fiber genes of Singapore HAdV-11 isolates^a

Human Adenovirus Type 7 Infection Associated with Severe and Fatal Acute Lower Respiratory Illness and Nosocomial Transmission

A 23-year-old male died of severe pneumonia and respiratory failure in a tertiary hospital in Beijing, and 4 out of 55 close contacts developed fever. Molecular analysis confirmed human adenovirus type 7 (HAdV7) as the causative agent. We highlight the importance of early diagnosis and treatment and proper transmission control of HAdV7.     

Comparison of Established Diagnostic Methodologies and a Novel Bacterial smpB Real-Time PCR Assay for Specific Detection of Haemophilus influenzae Isolates Associated with Respiratory Tract Infections

Haemophilus influenzae is a significant causative agent of respiratory tract infections (RTI) worldwide. The development of a rapid H. influenzae diagnostic assay that would allow for the implementation of infection control measures and also improve antimicrobial stewardship for patients is required. A number of nucleic acid diagnostics approaches that detect H. influenzae in RTIs have been described in the literature; however, there are reported specificity and sensitivity limitations for these assays. In this study, a novel real-time PCR diagnostic assay targeting the smpB gene was designed to detect all serogroups of H. influenzae. The assay was validated using a panel of well-characterized Haemophilus spp. Subsequently, 44 Haemophilus clinical isolates were collected, and 36 isolates were identified as H. influenzae using a gold standard methodology that combined the results of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and a fucK diagnostic assay. Using the novel smpB diagnostic assay, 100% concordance was observed with the gold standard, demonstrating a sensitivity of 100% (95% confidence interval [CI], 90.26% to 100.00%) and a specificity of 100% (95% CI, 63.06% to 100.00%) when used on clinical isolates. To demonstrate the clinical utility of the diagnostic assay presented, a panel of lower RTI samples (n = 98) were blindly tested with the gold standard and smpB diagnostic assays. The results generated were concordant for 94/98 samples tested, demonstrating a sensitivity of 90.91% (95% CI, 78.33% to 97.47%) and a specificity of 100% (95% CI, 93.40% to 100.00%) for the novel smpB assay when used directly on respiratory specimens.     

Optimization and Validation of a Multiplexed Luminex Assay To Quantify Antibodies to Neutralizing Epitopes on Human Papillomaviruses 6, 11, 16, and 18

A human papillomavirus (HPV) multiplexed competitive Luminex immunoassay first described by Opalka et al. (D. Opalka, C. E. Lachman, S. A. MacMullen, K. U. Jansen, J. F. Smith, N. Chirmule, and M. T. Esser, Clin. Diagn. Lab. Immunol. 10:108-115, 2003) was optimized and validated for use in epidemiology studies and vaccine clinical trials. Optimization increased both the analytical sensitivity and the clinical specificity of the assay to more effectively discriminate the low-titer antibody response of HPV-infected persons from noninfected individuals. The characteristics of the assay that were optimized included monoclonal antibody (MAb) specificity, scaling up the conjugation of virus-like particles (VLPs) to microspheres, VLP concentration, MAb concentration, sample matrix, sample dilution, incubation time, heat inactivation of sample sera, and detergent effects on assay buffer. The assay was automated by use of a TECAN Genesis Workstation, thus improving assay throughput, reproducibility, and operator safety. Following optimization, the assay was validated using several distinct serum panels from individuals determined to be at low and high risk for HPV infection. The validated assay was then used to determine the clinical serostatus cutoff. This high-throughput assay has proven useful for performing epidemiology studies and evaluating the efficacy of prophylactic HPV vaccines.     

Adenovirus Expressing a Bioluminescence Reporter Gene and cMAGI cell Assay for the Detection of HIV-1

We report a fast, highly sensitive method for detecting and testing drug resistance of M-tropic and T-tropic laboratory and primary HIV-1 isolates. cMAGI cells are infected with an adenovirus vector harboring the luciferase reporter gene controlled by HIV-1 Tat-responsive element, TAR. HIV-1 Tat production by HIV-1 chronically infected cells, or by cMAGI cells as early as two days after being acutely infected with HIV-1, is readily monitored in the presence or absence of antiviral drugs. This method is more sensitive than HIV-1 Tat dependant production of beta-galactosidase in the cMAGI cells. The fast answer, ease and sensitivity as well as the possibility of using this method in high throughput screening, makes it an very attractive tool for phenotypic detection of HIV-1 in clinical samples as well as a sensitive assay for monitoring drug resistant HIV-1 variants. This method can also be used for discovery of novel anti HIV-1 drugs.     

Detection of Canine Pneumovirus in Dogs with Canine Infectious Respiratory Disease

Canine pneumovirus (CnPnV) was recently identified during a retrospective survey of kenneled dogs in the United States. In this study, archived samples from pet and kenneled dogs in the United Kingdom were screened for CnPnV to explore the relationship between exposure to CnPnV and the development of canine infectious respiratory disease (CIRD). Within the pet dog population, CnPnV-seropositive dogs were detected throughout the United Kingdom and Republic of Ireland, with an overall estimated seroprevalence of 50% (n = 314/625 dogs). In the kennel population, there was a significant increase in seroprevalence, from 26% (n = 56/215 dogs) on the day of entry to 93.5% (n = 201/215 dogs) after 21 days (P <0001). Dogs that were seronegative on entry but seroconverted while in the kennel were 4 times more likely to develop severe respiratory disease than those that did not seroconvert (P < 0.001), and dogs with preexisting antibodies to CnPnV on the day of entry were significantly less likely to develop respiratory disease than immunologically naive dogs (P < 0.001). CnPnV was detected in the tracheal tissues of 29/205 kenneled dogs. Detection was most frequent in dogs with mild to moderate respiratory signs and histopathological changes and in dogs housed for 8 to 14 days, which coincided with a significant increase in the risk of developing respiratory disease compared to the risk of those housed 1 to 7 days (P < 0.001). These findings demonstrate that CnPnV is present in the United Kingdom dog population; there is a strong association between exposure to CnPnV and CIRD in the kennel studied and a potential benefit in vaccinating against CnPnV as part of a wider disease prevention strategy.     

Molecular Assay for Detection of Genetic Markers Associated with Decreased Susceptibility to Cephalosporins in Neisseria gonorrhoeae

The incidence of antimicrobial-resistant Neisseria gonorrhoeae continues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) in ponA, mtrR, penA, porB, and one N. gonorrhoeae-specific marker (porA). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24 N. gonorrhoeae-negative NAAT specimens, and 34 N. gonorrhoeae-positive NAAT specimens. Twenty-four of the N. gonorrhoeae-positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252 N. gonorrhoeae strains, the agreement between the DNA sequence and real-time PCR was 100% for porA, ponA, and penA, 99.6% for mtrR, and 95.2% for porB. The presence of ≥2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% for porB, 95.8% for ponA and mtrR, and 91.7% for penA. We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins in N. gonorrhoeae. Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results.