Johnny D. Callahan

Development and Evaluation of Serotype- and Group-Specific Fluorogenic Reverse Transcriptase PCR (TaqMan) Assays for Dengue Virus

ABSTRACT Five fluorogenic probe hydrolysis (TaqMan) reverse transcriptase PCR (RT-PCR) assays were developed for serotypes 1 to 4 and group-specific detection of dengue virus. Serotype- and group-specific oligonucleotide primers and fluorogenic probes were designed against conserved regions of the dengue virus genome. The RT-PCR assay is a rapid single-tube method consisting of a 30-min RT step linked to a 45-cycle PCR at 95 and 60°C that generates a fluorogenic signal in positive samples. Assays were initially evaluated against cell culture-derived dengue stock viruses and then with 67 dengue viremic human sera received from Peru, Indonesia, and Taiwan. The TaqMan assays were compared to virus isolation using C6/36 cells followed by an immunofluorescence assay using serotype-specific monoclonal antibodies. Viral titers in sera were determined by plaque assay in Vero cells. The serotype-specific TaqMan RT-PCR assay detected 62 of 67 confirmed dengue virus-positive samples, for a sensitivity of 92.5%, while the group-specific assay detected 66 of 67 confirmed dengue virus-positive samples, for a sensitivity of 98.5%. The TaqMan RT-PCR assays have a specificity of 100% based on the serotype concordance of all assays compared to cell culture isolation and negative results obtained when 21 normal human sera and plasma samples were tested. Our results demonstrate that the dengue virus TaqMan RT-PCR assays may be utilized as rapid, sensitive, and specific screening and serotyping tools for epidemiological studies of dengue virus infections.

Detection of Foot-and-Mouth Disease Virus: Comparative Diagnostic Sensitivity of Two Independent Real-Time Reverse Transcription-Polymerase Chain Reaction Assays

Rapid and accurate diagnosis is central to the effective control of foot-and-mouth disease (FMD). It is now recognized that reverse-transcription polymerase chain reaction (RT-PCR) assays can play an important role in the routine detection of FMD virus (FMDV) in clinical samples. The aim of this study was to compare the ability of 2 independent real-time RT-PCR (rRT-PCR) assays targeting the 5′ untranslated region (5′UTR) and RNA polymerase (3D) to detect FMDV in clinical samples. There was concordance between the results generated by the 2 assays for 88.1% (347 of 394) of RNA samples extracted from suspensions of epithelial tissue obtained from suspect FMD cases. The comparison between the 2 tests highlighted 19 FMDV isolates (13 for the 5′UTR and 6 for the 3D assay), which failed to produce a signal in 1 assay but gave a positive signal in the other. The sequence of the genomic targets of selected isolates highlighted nucleotide substitutions in the primer or probe regions, thereby providing an explanation for negative results generated in the rRT-PCR assays. These data illustrate the importance of the continuous monitoring of circulating FMDV field strains to ensure the design of the rRT-PCR assay remains fit for purpose and suggest that the use of multiple diagnostic targets could further enhance the sensitivity of molecular methods for the detection of FMDV

A serosurvey for HTLV-I among high-risk populations and normal adults in Egypt.

The prevalence of antibodies to human T-cell lymphotropic virus type I (HTLV-I) was determined in high-risk groups and normal adults in Egypt. Among 647 individuals tested, 6 (0.9%) were confirmed positive by western blot analysis. These included 2 (0.7%) of 279 drug addicts, 1 (3.3%) of 30 patients with sexually transmitted diseases, and 3 (2.2%) of 133 healthy individuals. Antibody was not detected in 47 blood recipients or 158 prostitutes. There was no correlation between sex or geographical location and HTLV-I infection. Fifty-three of the 647 sera (8%) were initially reactive by ELISA, but only 12 sera were repeatedly reactive. Since only 4 of these repeatedly reactive sera were confirmed by the western blot, the frequency of false positives using the DuPont screening ELISA was 1.2% (8/643). Two additional sera, confirmed positive by western blot, had been reactive, but not repeatedly, by ELISA. In comparison to the prevalence of HTLV-I antibody among risk groups in many parts of the world, the prevalence in Egypt was low.

Time for HIV-1/HIV-2 combination tests?

We should like to report the case of an individual in the Middle East infected with HIV-2 whose serum was negative by our routine ELISA tests for HIV-1. We propose that screening for HIV-2, in addition to HIV-1, be performed on individuals who are clinically determined to be at risk for HIV infection. Commercially available HIV-1/HIV-2 combination tests may serve to identify both infectious agents.

Evaluation of five hepatitis C virus screening tests and two supplemental assays: performance when testing sera from sexually transmitted diseases clinic attendees in the USA.

The performances of five screening tests (recombinant peptide-based first and second generation tests from Abbott and Ortho, and a synthetic peptide-based test from Biochem Immunosystems) and two supplemental tests: recombinant peptide- based, Abbott neutralization test and Chiron second generation recombinant immunoblot assay (RIBA 2), were evaluated for their ability to detect hepatitis C virus (HCV) antibodies in a population of 276 individuals attending a sexually transmitted diseases (STD) clinic in the USA. Although the five screening tests produced a variable number (35-62) of repeatedly reactive samples, only 13% (36/276) were classified as true positives by the supplemental tests. Thirty-four of the 36 were reactive by all screening tests and 32 of the true positives were reactive by both supplemental tests, while 2 did not neutralize but were reactive in the RIBA 2 test. Of the remaining 2 of the true positives which were discordant by several of the screening assays, 1 was confirmed by both supplemental assays but the other required a chemiluminescent enhancement technique to show positivity in RIBA 2. The sensitivities of the first and second generation Abbott and Ortho tests ranged from 97% to 100% and that of the Biochem test was 94%. The specificities of these tests ranged from 89.2% to 99.6%. The second generation Ortho test presented 9.4% (26/276) false positives. The use of second generation Ortho as a screening test would lead to an excessive number of confirmatory false positives. the positive predictive values of the screening tests ranged from 58.1% to 97.1%. Although the synthetic peptide based Biochem test exhibited the best overall indices, the presence of 2 false negative results would prevent its use as a singular screening test. Nevertheless its high specificity may lend itself to be used as a second screening test before confirmatory testing with RIBA 2.

Multiple blood-borne and sexually transmitted infections in sexually transmitted disease clinic and hospital emergency room patient populations.

The degree of coinfections with blood-borne or sexually transmitted pathogens (HIV-1, HTLV-I/II, HBV, HCV, HDV, and Treponema pallidum) were assessed in individuals attending sexually transmitted diseases (STD) clinic and patients admitted to a hospital through the emergency room in Baltimore. Enzyme-linked immunosorbent assays (ELISA), immunoblots, and card tests were used to screen the sera. Nearly one third of the individuals in both populations were infected with one or more pathogens. With some minor exceptions, all individuals with dual or multiple infections had antibodies reactive with the HBV core antigen. There was a strong overall association between the presence of antibodies to HIV-1 and the presence of antibodies to HBV core and HCV in both populations. Additionally, the presence of HIV-1 antibodies was significantly associated with the presence of HTLV-I/II antibodies and HBV surface antigen in the STD population and with a positive RPR test result in the H/ER population. We suggest that HIV-1 and/or HTLV-I/II infected individuals in STD clinic and emergency rooms are highly likely to have had past infections with HBV or HCV.

More support for the use of HIV combination assays

We have recently reported in the Journal a case of an HIV-2 infected person who was not detected using routine HIV-1 screening or confirmatory assays. This prompted us to determine if other HIV-2 infected persons may have been similarly misdiagnosed. Since the initial report we retested 600 individuals from 32 African countries or the Gulf Region using the HIV-1/2 combination assay (Abbott recombinant HIV-1/2 combination). Sera from these 600 individuals were originally tested by the Abbott HIV-1 recombinant ELISA and were negative. Upon retesting with the combination assay 1 serum was found to be repeatedly reactive (mean OD=1.467 cutoff=0.236). This serum had an OD=0.109 (cutoff=0.207) by the HIV-1 ELISA. Both ELISAs were repeated and results were reproducible. A recombinant HIV-1/2 combination dot-blot test (Abbott Test Pack) was also positive. Western blots got HIV-1 and HIV-2 were performed on this serum and results indicated unequivocally that antibodies to HIV-2 were present (reactivity to all major antigens). The HIV-1 Western blot produced indeterminate results showing strong reactions to only p24 and p31. Other HIV-1 screening assays (HIVCHEK and Behring competitive ELISA) were performed and were negative. In contrast to the HIV-2 positive serum previously reported this serum was reactive by the confirmatory IFA for HIV-1. The patient was from the Ivory Coast making the diagnosis credible. He appeared well and was originally tested as part of a national surveillance program focused on foreign students planning to remain in Egypt for longer than 30 days. Identification of another HIV-2 infected individual negative for antibodies to HIV-1 during initial testing substantiates our initial view that the time for combination assays has arrived. 2 potentials missed HIV-2 infections in a geographic location (Egypt) where the prevalence of HIV infection in general is low and where HIV-2 infection is not endemic clearly document the need for tests capable of detecting this similar infection. (full text)

Field evaluation of two commercial RT-rtPCR assays for porcine reproductive and respiratory syndrome virus detection using sera from ill and healthy pigs, China

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious respiratory virus causing severe morbidity in pigs worldwide. Control strategies for PRRSV often rely on detecting PRRSV, culling or isolating sick pigs, disinfecting pig barns, vaccination, and monitoring for virus spread. Given the high economic impact of PRRSV on pig farms, there is a great need for rapid and reliable PRRSV detection assays. We compared the performance of 2 commercial reverse-transcription real-time PCR (RT-rtPCR) assays, the VetMAX PRRSV NA and EU reagents (ABI assay) and the PRRSV general RT-rtPCR kit (Anheal assay), for the molecular detection of PRRSV in sera collected from pigs in China. Between June and September 2015, sera were collected from 219 healthy and 104 suspected PRRSV-infected pigs on 4 farms in China. Employing blinding, the 2 assays were run by 2 laboratories (Guangzhou Animal Health Inspection Institute [GAHII] and Sun Yat-sen University [SYSU] laboratories) and compared. Although both assays detected PRRSV with 100% specificity at both laboratories, the sensitivity (95% vs. 78% at GAHII; 94% vs. 72% at SYSU Laboratory) and the reproducibility (kappa value 0.933 vs. 0.931) were slightly better for the ABI assay compared to the Anheal assay.