Pooling nasopharyngeal/throat swab specimens to increase testing capacity for influenza viruses by PCR

Real-time PCR methodology can be applied to rapidly and accurately detect influenza viruses. During times of surge testing or enhanced pandemic surveillance, public health laboratories (PHLs) may experience overwhelming demand for testing, even while the prevalence of positive specimens remains low. To improve laboratory capacity and testing efficiency during surges, we evaluated whether nasopharyngeal (NP)/throat swab specimens can be pooled and tested for the presence of the 2009 H1N1 influenza virus without a reduction in sensitivity. Pools of 10 specimens were extracted and concentrated upon elution on the MagNA Pure LC instrument, and real-time PCR was performed on the Applied Biosystems 7500 Fast platform, using the CDC swine influenza virus real-time RT-PCR detection panel (rRT-PCR swine flu panel). Specimens in positive pools were singly re-extracted and retested by PCR to identify individual positive samples. Initial studies showed that spiking a pool of nine negative specimens (100 μl each) or 900 μl of virus transport medium with 100 μl of a positive clinical specimen caused no loss of sensitivity by rRT-PCR testing. Pools containing either multiple positive specimens or specimens positive for other respiratory viruses also showed no negative effect on crossing threshold (C(T)) values. To test the robustness of the pooling protocol, a panel of 50 blinded samples was sent to three PHLs and tested in five pools of 10. All PHLs correctly identified the positive specimens. This study demonstrates the feasibility of using a pooling strategy to increase capacity and conserve resources during surge testing and periods of enhanced influenza surveillance when the prevalence is low.     

Evaluation of Aspergillus PCR protocols for testing serum specimens

A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was requested. The information provided was made anonymous, and meta-regression analysis was performed to determine any procedural factors that significantly altered PCR performance. Ninety-seven percent of protocols were able to detect a threshold of 10 genomes/ml on at least one occasion, with 83% of protocols reproducibly detecting this concentration. Sensitivity and specificity were 86.1% and 93.6%, respectively. Positive associations between sensitivity and the use of larger sample volumes, an internal control PCR, and PCR targeting the internal transcribed spacer (ITS) region were shown. Negative associations between sensitivity and the use of larger elution volumes (≥100 μl) and PCR targeting the mitochondrial genes were demonstrated. Most Aspergillus PCR protocols used to test serum generate satisfactory analytical performance. Testing serum requires less standardization, and the specific recommendations shown in this article will only improve performance.     

Detection of methicillin-resistant Staphylococcus aureus directly from nasal swab specimens by a real-time PCR assay

Screening for colonization with methicillin-resistant Staphylococcus aureus (MRSA) is a key aspect of infection control to limit the nosocomial spread of this organism. Current methods for the detection of MRSA in clinical microbiology laboratories, including molecularly based techniques, require a culture step and the isolation of pure colonies that result in a minimum of 20 to 24 h until a result is known. We describe a qualitative in vitro diagnostic test for the rapid detection of MRSA directly from nasal swab specimens (IDI-MRSA; Infectio Diagnostic, Inc., Sainte-Foy, Quebec, Canada), based upon a real-time PCR and direct detection of MRSA via amplicon hybridization with a fluorogenic target-specific molecular beacon probe. Samples from 288 patients were analyzed for the presence of MRSA with the IDI-MRSA assay, compared to detection by either direct plating or enrichment broth selective culture methods. The diagnostic values for this MRSA screening method were 91.7% sensitivity, 93.5% specificity, 82.5% positive predictive value, and 97.1% negative predictive value when compared to culture-based methods. The time from the start of processing of specimen to result was approximately 1.5 h. In our hands, the IDI-MRSA assay is a sensitive and specific test for detection of nasal colonization with MRSA and providing for same-day results, allowing more efficient and effective use of infection control resources to control MRSA in health care facilities.     

Rapid detection of adenovirus in throat swab specimens by PCR during respiratory disease outbreaks among military recruits

We evaluated the performance of a generic PCR test to detect adenoviruses (AdV) in throat swab specimens collected from asymptomatic and ill military recruits with acute respiratory disease. Samples (n = 210) were collected at entry to basic training and at the time of large outbreaks of AdV-associated acute respiratory disease among military recruits at Fort Jackson, South Carolina, from 1997 to 1998. Compared to cell culture, a sensitivity of 99% and a specificity of 98% were noted for the PCR method to detect AdV in throat swabs. Similar results were obtained with or without DNA extraction, suggesting the absence of significant inhibitors for the PCR method in throat swab samples. No AdV was detected by culture or PCR in throat swabs from healthy recruits, suggesting the absence of latency or asymptomatic shedding. Throat swab specimens proved to be adequate, noninvasive samples to rapidly diagnose respiratory disease in young adults. This generic direct PCR proved to be a useful test for the rapid diagnosis of AdV-associated respiratory disease, detecting all serotypes tested to date and furnishing results within 6 h of specimen arrival. The use of this direct, rapid, sensitive, and specific assay would assist health care providers and public health practitioners in the early diagnosis, management, and control of AdV-associated respiratory disease.     

Polymerase chain reaction for detection of Mycobacterium leprae in nasal swab specimens.

The polymerase chain reaction based on the selective amplification of a 531-bp fragment of the gene encoding the proline-rich antigen of Mycobacterium leprae was applied to nasal swab specimens from leprosy patients, occupational contacts, and endemic and nonendemic controls. To prevent false-positive amplification, we used dUTP and uracil-DNA-glycosylase in all polymerase chain reactions. False-negative reactions were detected by using a 531-bp modified template as an internal control. Amplification products were found in 55% of untreated patients, in 19% of the occupational contacts, in 12% of endemic controls, and in none of the nonendemic controls. This study strongly suggests that not only leprosy patients but also healthy persons may carry M. leprae. We concluded that polymerase chain reaction is a reliable method to detect M. leprae in nasal specimens. The method holds promise for studying the spread and transmission of M. leprae within a population.     

Evaluation of real-time polymerase chain reaction assays for the detection of herpes simplex virus in swab specimens

An in-house herpes simplex virus (HSV) real-time polymerase chain reaction (PCR) hydrolysis probe assay and three commercial real-time HSV assays were evaluated for the detection of HSV from genital, oral, ocular and dermal clinical samples. Five hundred and sixty samples from patients displaying signs and symptoms of HSV infection were used to evaluate the in-house HSV assay. A representative 151 samples were processed using the artus(R) HSV-1/2 TM PCR Kit, SmartCycler(R) Non-typing and SmartCycler(R) Typing ASR kits. The in-house PCR assay demonstrated an overall positivity rate of 38% (211/560), equating to a 14% increase in HSV detection compared to 24% for culture (135/560). All 76 culture-negative, in-house PCR-positive samples were confirmed using at least one HSV commercial kit. The in-house, SmartCycler(R) Non-typing, artus(R) and SmartCycler(R) Typing assays showed improved sensitivity (100%, 100%, 98% and 99%, respectively) compared to culture (37%), and all real-time assays were highly specific (100%). The in-house and commercial real-time HSV PCR assays performed well and, combined with careful clinical interpretation, should improve the detection, differentiation and quantification of HSV from mucocutaneous swab samples.     

Rapid identification of human adenovirus types 3 and 7 from respiratory specimens via multiplex type-specific PCR

The rapid diagnosis of human adenovirus (Ad) infection is crucial for the timely recognition of epidemics. Moreover, identification of the serotypes known to cause serious disease can be helpful in therapeutic intervention. A multiplex PCR assay was developed for the rapid detection of adenovirus type 3 (Ad3) and Ad7 directly from clinical specimens. For this assay, three primer pairs (primers were based on the conserved and hypervariable regions of the hexon) were designed in order to simultaneously amplify all adenoviral serotypes and discriminate between Ad3 and Ad7. In our preliminary analysis, this multiplex PCR assay generated amplicons of the consensus primers from all 106 adenoviral isolates of diverse serotypes and proved able to correctly identify Ad3 and Ad7. This assay was subsequently applied to the detection of Ad3 and Ad7 in respiratory specimens. Among the 127 nasal aspirates from which an adenovirus was grown, the sensitivity with which any serotype could be detected was 91% (115/127). Two of the 53 nasal aspirates which did not grow Ads yielded adenovirus-specific bands, which were confirmed by sequencing analysis. Among the 115 specimens which produced common adenoviral bands, the sensitivity with which Ad3 could be detected was 93% (26/28), and the sensitivity with which Ad7 could be detected was 100% (35/35). Five out of the 115 specimens were proved to harbor more than one type of Ad via sequencing analysis of the amplicons, suggesting mixed infection with at least two different serotypes. In conclusion, this multiplex PCR system can be utilized in the rapid identification of Ad3 and Ad7 directly from clinical specimens. Furthermore, this method constitutes a diagnostic strategy for the detection of coinfection by different Ad serotypes.     

Evaluation of clinical specimens for influenza A virus positivity using various diagnostic methods

The diagnostic method for Influenza A virus, utilizing the SERION ELISA Antigen kit (SERION EIA), if results were evaluated according to the manufacturer's instructions, has repeatedly failed to detect a great number of clinical samples positive by virus isolation and RT-PCR. Therefore we compared the SERION EIA with the one-step 44/107L-Px immunocapture enzyme immunoassay (44/107L-Px EIA), developed in our laboratory (Tkácová and Varecková, J. Virol. Methods 60, 65-71, 1996). Seventy-three clinical specimens, of which 65 were positive by virus isolation (used as reference method), were tested by both EIAs. By the SERION EIA, out of the 65 reference-positive samples only 8 (12%) were positive, 5 (8%) were ambiguous, and 52 (80%) were negative, which corresponded to the sensitivity of 12%. On the contrary, the sensitivity of the 44/107L-Px EIA was 74%. However, the calculation of cut-off values for the evaluation of positivity of clinical specimens in these two assays were not the same. If the evaluation procedure used for the 44/107L-Px EIA was applied to the SERION EIA, the sensitivity and the specificity of both EIAs became comparable, namely 71% and 100% for the SERION EIA and 74% and 100% for the 44/107L-Px EIA, respectively. From these results it follows that not the detection ability of the SERION EIA, but the evaluation procedure recommended by its manufacturer led to a loss of large number of positive specimens.     

Comparative study of nasopharyngeal aspirate and nasal swab specimens for diagnosis of acute viral respiratory infection

Paired nasopharyngeal aspirate (NPA) and nasal swab (NS) samples from 475 children hospitalized for acute respiratory infection were studied for the detection of influenza virus, parainfluenza virus, respiratory syncytial virus, and adenovirus by immunofluorescence test, viral culture, and multiplex PCR assay. The overall sensitivity of viral detection with NPA specimens was higher than that obtained with NS specimens.     

Rapid antigen testing for the surveillance of influenza epidemics

Objective   To assess the use of a 'near patient' test for rapid antigen detection to obtain the more timely acquisition of data for the surveillance of influenza epidemics.
Methods   To the classical cell culture system used for the surveillance of influenza, a 'near patient' test was added. The cell culture method was applied for the detection of influenza virus in specimens sent to our laboratory. In contrast, the 'near patient' test was used directly by practitioners in their practices to screen patients for the presence of influenza virus antigen.
Results   The results for two seasons are presented. The 'near patient' test was able to detect a developing influenza epidemic with the same reliability as clinical consultation reports for influenza-like illness or the conventional culture method. However, the results obtained were available 9 days earlier on average, compared with cell culture. Because of this, results concerning the epidemics could be announced via the internet more rapidly. Although the 'near patient' test demonstrated a lower sensitivity than detection by conventional cell culture, the sensitivity was still sufficiently high to reveal the characteristics of the epidemics in the community.
Conclusions   Rapid influenza testing is a reliable tool for influenza surveillance and, compared with traditional methods (virus detection on cell culture and monitoring of influenza-like illness), provides faster results. Although the 'near patient' test has limited sensitivity compared with cell culture, results were consistent over two seasons, and suggest that rapid testing should be part of a surveillance program.     

Evaluation of nasal and nasopharyngeal swab collection for the detection of Epstein‐Barr virus in nasopharyngeal carcinoma

Epstein‐Barr virus detection using nasopharyngeal swabs has been suggested as a potential screening test that could improve the specificity of current EBV‐based serological assays. However, application requires insertion of the swab deep into the nasopharynx, a procedure not amenable to non‐clinic screening. We reasoned that swabbing the more easily accessible nasal cavity might provide an appealing alternative for NPC detection. Patients > 18 years of age diagnosed with histologically confirmed NPC were recruited from the Otolaryngology Department at the National Taiwan University Hospital. ENT clinicians collected both nasal and nasopharyngeal swabs. EBV DNA and cellular beta‐globulin DNA were quantified using quantitative PCR targeting a highly‐conserved region of the BKRF1 gene. EBV DNA was detectable (non‐zero) in all 34 nasopharyngeal swabs and above the positivity threshold of 1666 EBV copies in 30 (88.2%) patients. EBV DNA was detectable in 50% of 34 nasal swabs and above the positivity threshold in four (11.8%) patients. Average EBV DNA levels were >3‐fold higher (P < 0.001) in nasopharyngeal compared to nasal swabs. Among the 17 NPC patients with detectable EBV DNA in both swab types, we observed correlation (P < 0.01) between EBV DNA measurements. Our data represent the first evaluation of EBV DNA collected from nasal swabs. Given current EBV DNA amplification techniques, nasopharyngeal swabs remain more sensitive than nasal swabs for NPC detection.     

Evaluation of the Abbott TESTPACK RSV enzyme immunoassay for detection of respiratory syncytial virus in nasopharyngeal swab specimens.

The Abbott TESTPACK RSV assay (Abbott Laboratories, North Chicago, Ill.), a rapid (20-min) enzyme immunoassay, was compared with culture and direct immunofluorescence (DFA) of nasopharyngeal cells for the detection of respiratory syncytial virus (RSV) in nasopharyngeal swab specimens. Nasopharyngeal swab specimens, collected from 234 infants, were placed in viral transport medium. Portions of specimen in transport medium were used for each test. Of 234 specimens, 70 (30%) were culture positive, 103 (44%) were DFA positive, 107 (46%) were culture or DFA positive, and 112 (48%) were TESTPACK RSV positive. Of 19 specimens positive by TESTPACK RSV but negative by culture or DFA, 15 were positive by the blocking assay. A total of 122 specimens were culture, DFA, or blocking assay positive; TESTPACK RSV detected 108 specimens (sensitivity, 89%). The specificity, positive predictive value, and negative predictive value of TESTPACK RSV as compared with those of culture, DFA, and the blocking assay were 96, 96, and 89%, respectively. By comparison, the sensitivity, specificity, positive predictive value, and negative predictive value of combined culture and DFA were 88, 100, 100, and 88%, respectively. TESTPACK RSV is a rapid and reliable enzyme immunoassay for the direct detection of RSV antigen in nasopharyngeal swab specimens.     

Rectal swab for detection of norovirus by real-time PCR: similar sensitivity compared to faecal specimens

Rapid diagnosis and immediate infection control precautions are cornerstones in the prevention of norovirus outbreaks. However, faecal sampling for norovirus PCR—the standard of care—is time consuming. From 2009 to 2011, parallel faecal and rectal swab samples were consecutively obtained from patients with acute gastroenteritis presenting at our emergency department. In total, 109 complete sample pairs of 108 patients were analysed by specific norovirus real-time PCR. The sensitivity of real-time PCR was 97.3% (36/37) for both sampling methods. A rectal swab is a reasonable option for detection of norovirus by real-time PCR, if a stool specimen is not readily available.     

PCR and restriction endonuclease analysis for rapid identification of human adenovirus subgenera

Subgenus identification of adenoviruses is of clinical importance and is as informative as identification by serotype in most clinical situations. A PCR-based identification of adenovirus subgenera A, B, C, D, E, and F and sometimes serotypes is described. The PCR uses nonnested primer pair ADRJC1-ADRJC2, which targets a highly conserved region of the adenovirus hexon gene, has a sensitivity of 10 to 40 copies of adenovirus type 2 (Ad2) DNA, and generates 140-bp PCR products from adenovirus serotypes representative of all the subgroups. The PCR products of all subgroups can be differentiated on the basis of the restriction fragment patterns produced by a total of five restriction endonucleases. In addition, serotypes Ad40 and Ad41 (subgroup F) and important serotypes of subgroup D (Ad8, Ad10, Ad19, and Ad37) can easily be differentiated, but serotypes within subgroups B and C cannot. The method was assessed by blind subgenus identification of 56 miscellaneous clinical isolates of adenoviruses. The identities of these isolates at the subgenus level by the PCR correlated 91% (51 of 56) with the results of serotyping by the neutralization test, and 9% (5 of 56) of clinical isolates produced discordant results.     

Digital dashboard design using multiple data streams for disease surveillance with influenza surveillance as an example

Background

Great strides have been made exploring and exploiting new and different sources of disease surveillance data and developing robust statistical methods for analyzing the collected data. However, there has been less research in the area of dissemination. Proper dissemination of surveillance data can facilitate the end user''s taking of appropriate actions, thus maximizing the utility of effort taken from upstream of the surveillance-to-action loop.

Objective

The aims of the study were to develop a generic framework for a digital dashboard incorporating features of efficient dashboard design and to demonstrate this framework by specific application to influenza surveillance in Hong Kong.

Methods

Based on the merits of the national websites and principles of efficient dashboard design, we designed an automated influenza surveillance digital dashboard as a demonstration of efficient dissemination of surveillance data. We developed the system to synthesize and display multiple sources of influenza surveillance data streams in the dashboard. Different algorithms can be implemented in the dashboard for incorporating all surveillance data streams to describe the overall influenza activity.

Results

We designed and implemented an influenza surveillance dashboard that utilized self-explanatory figures to display multiple surveillance data streams in panels. Indicators for individual data streams as well as for overall influenza activity were summarized in the main page, which can be read at a glance. Data retrieval function was also incorporated to allow data sharing in standard format.

Conclusions

The influenza surveillance dashboard serves as a template to illustrate the efficient synthesization and dissemination of multiple-source surveillance data, which may also be applied to other diseases. Surveillance data from multiple sources can be disseminated efficiently using a dashboard design that facilitates the translation of surveillance information to public health actions.     

PCR for specific detection of haemorrhagic enteritis virus of turkeys, an avian adenovirus.

A hexon gene based PCR was developed for specific amplification of DNA sequences from the haemorrhagic enteritis virus (HEV) of turkeys. The hexon genes of different avian adenoviruses were compared for primer construction. Two regions with low sequence homology between HEV and fowl adenovirus (FAV) hexon genes were selected for primer localisation. In correlation with the known sequence data a fragment of 1647 bp was amplified from a live vaccine and spleens of turkeys suffering from haemorrhagic enteritis (HE). All other avian adenoviruses which are able to infect turkeys, i.e. FAV and turkey adenoviruses (TAV), were negative. This is the first PCR for specific detection of HEV DNA which should be useful for rapid diagnosis and epidemiological investigations of HEV infections in turkeys.     

Perirectal swab surveillance for Clostridium difficile by use of selective broth preamplification and real-time PCR detection of tcdB

Active surveillance testing to identify and isolate asymptomatic carriers of toxigenic Clostridium difficile has been limited by the lack of a test that is sensitive, specific, and timely enough to serve as an infection control tool. We tested DNA preamplified from perirectal surveillance specimens in a liquid medium selective for C. difficile by using a modified commercial real-time PCR assay. All fermenting specimens were subcultured, and isolates were tested for toxigenicity. Culture-positive toxigenic isolates served as the gold standard for comparison with the broth preamplification/PCR assay. The limit of detection for the assay was 1 CFU. Relative to toxigenic anaerobic culture, the sensitivity, specificity, and positive and negative predictive values of this assay were 70/70 (100.0%), 422/426 (99.1%), 70/74 (94.6%), and 422/422 (100.0%), respectively. These data demonstrate that selective broth preamplification and real-time PCR of perirectal swab specimens constitute a practical approach to the detection of asymptomatic C. difficile carriage.     

Rapid detection of equine herpesvirus type-1 antigens in nasal swab specimens using an antigen capture enzyme-linked immunosorbent assay.

An antigen capture enzyme-linked immunosorbent assay (ELISA) was developed for the detection of equine herpesvirus type-1 (EHV-1) antigens in nasal swab specimens. The test was designed as a solid phase, amplified sandwich assay in which an EHV-1 specific monoclonal antibody was used to capture virus antigen and polyclonal antisera used to detect antigen bound to the test plates. Eight monoclonal antibodies were tested for their ability to capture virus antigen and one was selected for routine use. The sensitivity and specificity of the ELISA was compared with that of virus isolation using swabs from ponies which were experimentally infected with EHV-1. Of 72 nasal swabs collected, 32 were found to be positive by both virus isolation (VI) and ELISA, a further 15 samples were positive by VI alone, but none of the samples were positive by ELISA and negative by VI. This yielded an overall assay sensitivity of 68% and specificity of 100%. The assay proved useful for diagnosis since virus antigen was detected during the first four days post-infection which corresponded to the acute phase of disease when some clinical symptoms were apparent. In addition, the assay could be completed within one day when antibody coated plates were available.     

Rapid detection of bovine herpesvirus type 1 antigens in nasal swab specimens with an antigen capture enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of bovine herpesvirus type 1 (BHV-1) antigens in nasal swab specimens collected from infected animals. Development of the ELISA involved screening and selection of BHV-1-specific monoclonal antibodies for their ability to capture BHV-1 antigens and for their stability and activity after conjugation to horseradish peroxidase. Forty combinations of capture-conjugate monoclonal antibody pairs were screened for detection of nanogram amounts of purified BHV-1 by using a double-antibody-sandwich ELISA in which antigen and conjugated antibody were simultaneously added to antibody-coated wells. Of the 40 monoclonal antibody pairs, 4 were analyzed further and 1 was selected for routine application to clinical specimens. Of 129 nasal swab specimens collected during the first 10 days after experimental infection with BHV-1, 66 were found to be positive by both virus isolation and ELISA and 34 were positive for infectious virus but negative by ELISA. One specimen was positive by ELISA but negative by virus isolation, and the remaining 28 specimens were negative by both tests. Quantitation of the virus-containing specimens showed that the ELISA had a lower detection limit of 10(3.5) median tissue culture infective doses. The ELISA was judged to be highly useful for diagnosis of BHV-1 infections, since all of the nasal swab specimens that were collected from 12 animals during the first 5 days of the infection, when the clinical signs were the most apparent, were positive.