Direct detection and characterization of foot-and-mouth disease virus in East Africa using a field-ready real-time PCR platform.

Effective control and monitoring of foot-and-mouth disease (FMD) relies upon rapid and accurate disease confirmation. Currently, clinical samples are usually tested in reference laboratories using standardized assays recommended by The World Organisation for Animal Health (OIE). However, the requirements for prompt and serotype-specific diagnosis during FMD outbreaks, and the need to establish robust laboratory testing capacity in FMD-endemic countries have motivated the development of simple diagnostic platforms to support local decision-making. Using a portable thermocycler, the T-COR™ 8, this study describes the laboratory and field evaluation of a commercially available, lyophilized pan-serotype-specific real-time RT-PCR (rRT-PCR) assay and a newly available FMD virus (FMDV) typing assay (East Africa-specific for serotypes: O, A, Southern African Territories [SAT] 1 and 2). Analytical sensitivity, diagnostic sensitivity and specificity of the pan-serotype-specific lyophilized assay were comparable to that of an OIE-recommended laboratory-based rRT-PCR (determined using a panel of 57 FMDV-positive samples and six non-FMDV vesicular disease samples for differential diagnosis). The FMDV-typing assay was able to correctly identify the serotype of 33/36 FMDV-positive samples (no cross-reactivity between serotypes was evident). Furthermore, the assays were able to accurately detect and type FMDV RNA in multiple sample types, including epithelial tissue suspensions, serum, oesophageal-pharyngeal (OP) fluid and oral swabs, both with and without the use of nucleic acid extraction. When deployed in laboratory and field settings in Tanzania, Kenya and Ethiopia, both assays reliably detected and serotyped FMDV RNA in samples (n = 144) collected from pre-clinical, clinical and clinically recovered cattle. These data support the use of field-ready rRT-PCR platforms in endemic settings for simple, highly sensitive and rapid detection and/or characterization of FMDV.

Isothermal method for hydrate studies using a transparent variable volume cell.

The measurements of hydrate dissociation points are generally achieved using the well-established isochoric method. This method implies determination of the total pressure of the system under study, as a function of temperature. It is quite time consuming, especially at higher pressures. Working at higher pressures requires equilibrium cells with thicker walls, which compromises on fast heat exchange. The use of a variable volume cell is therefore quite attractive as it allows for the measurements of hydrate dissociation pressure under isothermal conditions. This paper describes a transparent variable volume cell used for efficient and rapid measurements via the isothermal procedure.

Specific detection of Rinderpest virus by real-time reverse transcription-PCR in preclinical and clinical samples from experimentally infected cattle.

A highly sensitive detection test for Rinderpest virus (RPV), based on a real-time reverse transcription-PCR (rRT-PCR)system, was developed. Five different RPV genomic targets were examined, and one was selected and optimized to detect viral RNA in infected tissue culture fluid with a level of detection ranging from 0.59 to 87.5 50% tissue culture infectious doses (TCID(50)) per reaction depending on the viral isolate. The strain sensitivity of the test was validated on 16 RPV strains belonging to all three phylogenetic branches described for RPV. No cross-reactivity was detected with closely related peste des petit ruminants or with symptomatically similar viruses, including all seven serotypes of foot-and-mouth disease virus, two serotypes of vesicular stomatitis virus, bluetongue virus, and bovine herpes virus type 2. In samples from experimentally infected cattle, our real-time RT-PCR test was significantly more sensitive than the gold standard test of virus isolation, allowing the detection of the disease 2 to 4 days prior to the appearance of clinical signs. The comparison of clinical samples with putative diagnostic value from live animals showed that conjunctival swabs and blood buffy coat were the samples of choice for epidemiological surveillance, while lymph nodes performed the best as postmortem specimens. This portable and rapid real-time RT-PCR has the capability of the preclinical detection of RPV and provides differential diagnosis from look-alike diseases of cattle. As RPV is declared globally eradicated, this test provides an important rapid virus detection tool that does not require the use of infectious virus and allows the processing of a large number of samples.

The use of contrast-enhanced ultrasound in the characterisation of focal liver lesions.

PURPOSE: To determine the potential application of contrast-enhanced ultrasound in the characterisation of focal liver lesions encountered in radiological practice at a district general hospital. MATERIALS & METHODS: Retrospective analysis of 68 sequential patients undergoing contrast-enhanced ultrasound (CEUS) of liver. All patients were referred for CEUS following identification of 1 or more focal liver lesions on conventional ultrasound or CT imaging. After baseline US examination (Acuson), a bolus of 1.0-2.4 ml of SonoVue (Bracco, UK) was administered intravenously. CEUS images were obtained during arterial, portal venous and delayed phases. Patients were followed up for a mean period of 6 months. The CEUS diagnosis was compared to that indicated by other imaging modalities, histopathology, and clinical follow up. RESULTS: CEUS correctly identified malignant liver lesions in 19 patients, with the final diagnosis confirmed by histopathology in 5 cases and clinico-radiological follow up in 14 cases. 47 patients were correctly identified with benign liver lesions on CEUS imaging, with all these cases confirmed on clinico-radiological follow up. In the detection of malignancy, the sensitivity was 95.0% and the specificity was 97.9%. CONCLUSIONS: In our experience to date, contrast-enhanced ultrasound imaging is highly accurate in characterising malignant and benign focal liver lesions. It therefore has significant potential for utilisation in most general radiology departments.

A language programme to increase the verbal production of a child dually diagnosed with Down syndrome and autism.

BACKGROUND: The incidence of children dually diagnosed with Down syndrome and autism is estimated to be as high as 11%. There is a paucity of research investigating linguistic treatment interventions for such children. This single-subject experiment examined a programme designed to increase the language production and verbal behaviour of a 9-year-old dually diagnosed boy who had been receiving a 15-h/week home-based applied behaviour analysis (ABA) programme. METHODS: Training principles were derived from previously empirically validated research in discrete trail learning and natural environment teaching, as well as modified incidental teaching procedures. The crux of the language programme involved withholding reinforcement until a spoken request was made. RESULTS: Language production noticeably increased for each target area after the introduction of the language programme and was maintained at a 9-month follow-up session. CONCLUSIONS: A combined treatment approach incorporating direct instruction, natural environment teaching and incidental teaching can be effective in increasing and maintaining responsive and spontaneous speech in a child with Down syndrome diagnosed with autism. Replication studies are needed with such multiple dually diagnosed children to further evaluate the effectiveness and generalizability of this combined language programme.

Preclinical diagnosis of African swine fever in contact-exposed swine by a real-time PCR assay.

A fluorogenic probe hydrolysis (TaqMan) PCR assay for African swine fever virus (ASFV) was developed and evaluated in experimentally infected swine. This sensitive and specific one-step single-tube assay, which can be performed in 2 h or less, detected viral DNA in tonsil scraping samples 2 to 4 days prior to onset of clinical disease. Thus, the assay would have application for preclinical diagnosis of African swine fever and surveillance and/or emergency management of a disease outbreak.

Diagnostic evaluation of a real-time reverse transcriptase PCR assay for detection of classical swine fever virus.

A fluorogenic-probe hydrolysis (TaqMan)-reverse transcriptase (RT) PCR for classical swine fever virus (CSFV) was evaluated for diagnostic sensitivity and specificity by using clinical samples obtained from the Dominican Republic, where the disease is enzootic. The sensitivity of this test, using nasal swab samples taken from both symptomatic and asymptomatic animals, exceeded the diagnostic sensitivity of virus isolation (100% versus 72.4%, respectively) with little loss of specificity (98.9% versus 100%, respectively). At the herd level, three of four infected farms were identified by virus isolation, while the CSFV real-time RT-PCR assay identified all four infected premises. This simple and accurate test permits rapid detection of CSFV in affected herds.

Detection of U.S., Lelystad, and European-like porcine reproductive and respiratory syndrome viruses and relative quantitation in boar semen and serum samples by real-time PCR.

Transmission of porcine reproductive and respiratory syndrome virus (PRRSV) via boar semen has been documented. Since semen is widely disseminated for artificial insemination and the virus can cause significant health and economic consequences, it is essential to have well-validated, rapid diagnostic techniques to detect and quantitate the virus for diagnostic and research purposes. Previously, boar semen was tested by a nested PCR (nPCR) assay which was compared to the "gold standard" swine bioassay. A correlation of 94% was observed, indicating that, most of the time, PCR detected infectious virus. Subsequently, a real-time PCR targeting the 3' untranslated region of the PRRSV genome was compared with nPCR by testing 413 serum and semen samples from PRRSV-inoculated and control boars. There was 95% agreement between the results of the two tests, with the majority of samples with discordant results containing virus at the lower range of detection by the assays. The virus in all samples was quantitated by using a standard curve obtained by serial dilution of an in vitro transcript. By using the in vitro transcript, the lower limit of sensitivity was observed to be approximately 33 copies/ml. Reactivity with a panel of more than 100 PRRSV isolates from various geographical regions in the United States was also documented. No reactivity with nine nonrelated swine viruses was noted. A real-time PCR was also developed for the detection of the European Lelystad virus and the European-like PRRSV now found in the United States. In six of six PRRSV-inoculated boars, peak levels of viremia occurred at 5 days postinoculation (DPI) and were most consistently detectable throughout 22 DPI. In five of six boars, PRRSV was shed in semen for 0 to 2 days during the first 10 DPI; however, one of six boars shed the virus in semen through 32 DPI. Therefore, in general, the concentration and duration of PRRSV shedding in semen did not correlate with the quantity or duration of virus in serum. These differences warrant further studies into the factors that prevent viral replication in the reproductive tract.

Technical aspects and complications of laparoscopic banding for morbid obesity--a radiological perspective.

Morbid obesity is a significant clinical problem in the western world. Various surgical restrictive procedures have been described as an aid to weight reduction when conservative treatments fail. Adjustable laparoscopic gastric banding (LAPBAND) has been popularized as an effective, safe, minimally invasive, yet reversible technique for the treatment of morbid obesity. Radiological input is necessary in the follow-up of these patients and the diagnosis of complications peculiar to this type of surgery. In this review we will highlight the technical aspects of radiological follow-up and the lessons learnt over the last 5 years.

Rapid detection of classical swine fever virus by a portable real-time reverse transcriptase PCR assay.

A fluorogenic-probe hydrolysis (TaqMan)-reverse transcriptase PCR assay for classical swine fever virus (CSFV) was developed and evaluated in experimentally infected swine. The assay detected CSFV, representing different phylogenetic groupings, but did not amplify viral RNA from related pestiviruses. The assay met or exceeded the sensitivity (1 to 100 50% tissue culture infective doses per ml) of viral cultures of samples from experimentally infected animals. Viral RNA was detected in nasal and tonsil scraping samples 2 to 4 days prior to the onset of clinical disease. The assay can be performed in 2 h or less, thus providing a rapid method for the diagnosis of classical swine fever.

Testicular microlithiasis: prevalence and tumor risk in a population referred for scrotal sonography.

OBJECTIVE: Considerable accrued evidence points to an association between testicular microlithiasis, intratubular germ cell neoplasia, and testicular tumor. This study assesses both the prevalence of testicular microlithiasis revealed on sonography in a referred population and the concurrent tumor risk. MATERIALS AND METHODS: Over a 32-month period (April 1996 through November 1998), 4892 scrotal sonographic examinations were performed in 4819 patients at four referral centers. All patients underwent high-resolution (7- to 10-MHz) imaging. Using a computerized word search (n = 4102; testicular microlithiasis, calcification, microliths, calcific foci, tumor, neoplasm, cancer, hyperecho, hypoecho, heterogen, and carcinoma) and manual retrieval (n = 790), cases of tumor, testicular microlithiasis (>5 microliths per sonogram), and testicular microlithiasis plus tumor were pulled and retrospectively reviewed. The presence and type of tumor were confirmed at histology after orchidectomy. RESULTS: Fifty-four tumors were found among 4892 scrotal sonograms (28 seminomas, 14 teratomas, 8 mixed germ cell tumors, 2 Leydig cell tumors, and 2 non-Hodgkin's lymphomas). Testicular microlithiasis was present in 33 patients, giving a prevalence of 0.68%. Concurrent tumor and testicular microlithiasis were detected in seven patients, a relative risk of tumor in testicular microlithiasis was 21.6-fold (95% confidence limits: 10. 6-fold, 44.2-fold). In one patient with testicular microlithiasis, a previous orchidectomy for mixed germ cell tumor had been performed (not included in the relative risk calculation). CONCLUSION: In a referred population of 4819 patients the prevalence of testicular microlithiasis was 0.68% and the relative risk of concurrent tumor was 21.6-fold. Sonographic surveillance of testicular microlithiasis cases for tumor is mandatory.

Validity of the Stanford-Binet Intelligence Scale-IV: its use in young adults with mental retardation.

The validity of the Stanford Binet-IV (SB-IV) was assessed. This test and the WAIS-R and WRAT-R were administered to 42 adults previously classified with mild to moderate mental retardation. Validity coefficients between scores on the SB-IV and the other two measures were significant. The mean IQ on the SB-IV (mean Test Composite = 43.26) was significantly lower than that on the Wechsler Adult Intelligence Scale-Revised--WAIS-R (mean Full-Scale IQ = 57.91). With regard to the internal validity of the SB-IV, the intersubtest relationships of each of the four Area scores correlated significantly with the Test Composite (range = .66 to .91). Verbal Reasoning earned the highest correlation (.91). Results support the SB-IV's concurrent, criterion-related, and internal validity for use with young adults who have mental retardation.

Reliability, criterion-related validity and qualitative comments of the Fourth Edition of the Stanford-Binet Intelligence Scale with a young adult population with intellectual disability.

The test-retest reliability and concurrent, criterion-related validity of the Fourth Edition of the Stanford-Binet Intelligence Scale (SB-IV) were examined in a young adult population with intellectual disability. Forty adults with mild to moderate intellectual disability (mean age = 20.8 years; SD = 1.8 years) were administered the SB-IV and retested approximately 5 weeks later (mean = 33.4 days, SD = 1.2). The Vineland Adaptive Behavior Scale: Interview Edition (VABS) was completed by a reliable informant within one week of the SB-IV testing. The test-retest reliability coefficients for the four SB-IV area and composite scores were all significant (P < 0.00). Individual subtest correlations tended to be lower but consistent across the two administrations. Moderate correlations were observed between the VABS composite and SB-IV composite scores. The present results provide support for the temporal reliability of the SB-IV and its concurrent, criterion-related validity in an exceptional sample.

Evaluation of the protective efficacy of a recombinant dengue envelope B domain fusion protein against dengue 2 virus infection in mice.

A recombinant protein containing part of the dengue (DEN) 2 envelope protein was evaluated as a subunit immunogen for vaccination against DEN virus infection. A gene fragment encoding amino acids 298-400 (B domain) of the DEN-2 virus envelope was expressed as a fusion protein with the maltose binding protein (MBP) of Escherichia coli. This recombinant, DEN-2(B)/MBP, was purified and analyzed for its antigenicity, immunogenicity, and ability to protect mice against lethal challenge. The recombinant antigen reacted with a DEN-2 type-specific neutralizing monoclonal antibody (3H5), DEN-2 hyperimmune mouse ascitic fluid, and DEN-2 immune human sera. When administered to mice, DEN-2(B)/MBP elicited a DEN-2 virus neutralizing antibody response that conferred partial protection against challenge infection with a lethal dose of DEN-2 virus administered by intracranial inoculation. In addition, no replication of DEN-2 virus was detectable in the brains of the immunized mice as compared with control mice that were killed six days after challenge. Sera from immunized mice revealed no cross-neutralizing antibody to any of the other DEN serotypes in the plaque-reduction neutralization test. These findings warrant further studies with the DEN-2(B)/MBP antigen as a potential human vaccine candidate. An effective vaccine could prevent thousands of cases of illness and many deaths each year resulting from DEN virus infections.

Molecular analysis of dengue virus attenuation after serial passage in primary dog kidney cells.

The complete nucleotide sequences of the genomes of dengue-1 virus virulent 45AZ5 PDK-O and attenuated vaccine candidate strain 45AZ5 PDK-27 have been determined and compared with the dengue-1 virus Western Pacific (West Pac) 74 parent strain from which 45AZ5 PDK-O was derived. Twenty-five (0.23%) nucleotide and 10 (0.29%) amino acid substitutions occurred between parent strain dengue-1 virus West Pac 74 and virulent strain 45AZ5 PDK-O, which was derived from the parent by serial passage in diploid foetal rhesus lung (FRhL-2) and mutagenized with 5-azacytidine. These substitutions were preserved in the 45AZ5 PDK-27 vaccine. 45AZ5 PDK-O and PDK-27 strains, which differ by 27 passages in primary dog kidney (PDK) cells, show 25 (0.23%) nucleotide and 11 (0.32%) amino acid divergences. These comparative studies suggest that the changes which occurred between the West Pac 74 and 45AZ5 PDK-O strains may alter the biological properties of the virus but may not be important for attenuation. Important nucleotide base changes responsible for attenuation accumulated between 45AZ5 PDK-O and 27.

Venezuelan equine encephalitis and Oropouche virus infections among Peruvian army troops in the Amazon region of Peru.

An outbreak of a febrile illness characterized by headache, ocular pain, myalgia, and arthralgia occurred during June 1994 among Peruvian army troops in Northern Peru. On June 14-16, 1994, clinical data and blood samples were obtained from eight soldiers with a febrile illness, and from 26 others who had a history of febrile illness during the past three months. A follow-up blood sample was obtained 107 days later from four of the febrile and seven of the afebrile soldiers. Serum samples were tested for dengue (DEN), Oropouche (ORO), and Venezuelan equine encephalitis (VEE) IgM and IgG antibodies by an enzyme-linked immunosorbent assay (ELISA). Virus isolation was performed by inoculation of newborn mice and Vero cell cultures. Viral isolates were identified by immunofluorescence, ELISA, and nucleotide sequencing. A VEE virus infection was confirmed in three of the eight febrile soldiers, two by virus isolation, and one by serology. Antigenic analysis indicated that one of the virus isolates was similar to VEE subtype I, variety ID, viruses previously isolated in Colombia and Venezuela. Nucleotide sequence data showed that both viral isolates were identical to one another and closely related to VEE ID viruses previously isolated in Peru, Colombia, and Venezuela. Serologic results showed that two of 26 afebrile soldiers had IgM antibody to VEE and four had IgG antibody to VEE; two febrile soldiers had IgG antibody in their first serum samples. Oropouche-specific IgM antibody was detected in one of the eight febrile and five of the afebrile soldiers, and 18 of the 34 soldiers had low titers of ORO IgG antibody titers, which did not meet the diagnostic criteria for confirmed cases. All soldiers were negative for DEN IgM antibody, and 10 had flavivirus IgG antibody that reacted with DEN antigens. These data indicated that VEE ID virus was one of the causes of illness among Peruvians soldiers and that this was the first association of this VEE subtype with human disease in Peru.

Mechanistic studies of partial-filling micellar electrokinetic chromatography.

The need for coupling micellar electrokinetic chromatography (MEKC) with electrospray mass spectrometry initiates the development of partial-filling MEKC. In comparison with conventional MEKC, only a small portion of the capillary is filled with a micellar solution for performing the separation in partial-filling MEKC. Analytes first migrate into the micellar plug, where the separation occurs, and then into the leading electrophoresis buffer, which is free of surfactants. A theoretical model is proposed for predicting the separation behavior of triazine herbicides in partial-filling MEKC. The comparisons between conventional and partial-filling MEKC in terms of separation efficiency and resolution of triazine herbicides are presented and discussed. The optimization techniques, possible applications, and advantages of partial-filling MEKC are similarly addressed.

Sensitivity of clinically hospitalized adolescents' self-report measures to change over time.

Twenty-five psychiatrically hospitalized adolescents were assessed on three separate occasions (approximately 2 weeks apart) using the Revised Children's Manifest Anxiety Scale (R-CMAS), Beck Depression Inventory (BDI), and Children's Attributional Styles Questionnaire Revised (KASTAN) within 1 week of hospitalization. Attending clinicians also rated each subject concurrently on the Anxiety and Depression factors of the Brief Psychiatric Rating Scale for Children (BPRS-C). Results indicated only modest agreement between self-report measures and clinician ratings over time. Clinician ratings on both BPRS-C factors changed significantly over time, while, of the self-report measures, only the R-CMAS evidenced significant change. Results were discussed in terms of the construct of "negative affectivity," method variance in assessment, and clinical implications.

Criterion-related validity and stability: equivalence of the MMPI and the MMPI-2.

The differential criterion-related validity of the MMPI and MMPI-2 and their stability over a 4-month period of time were examined in a university population by correlating the clinical scales with their counterpart SCL-90-R factors. Fair to moderate correlations were found on all eight paired MMPI scales and SCL-90-R factors, while only two of eight MMPI-2/SCL-90-R pairings were found to be correlated significantly. Further analyses, however, found no significant differences between these MMPI/SCL-90-R and MMPI-2/SCL-90-R correlations. Adequate stability was found between MMPI-2 and SCL-90-R pairs over 4 months, except for the MMPI-2 D scale with the SCL-90-R Depression factor. Several issues related to the equivalency between the MMPI and the MMPI-2 were discussed.