Comparison of four RNA extraction methods for the detection of porcine reproductive and respiratory syndrome virus by RT-PCR

We investigated the effect of repeated cold stress (RCS) on the capsaicin-evoked release of glutamate from the primary afferent fibers of the rat, and compared this with the effect of inoculation of complete Freund's adjuvant (adjuvant inoculation). The release of glutamate was measured using a fluorometric on-line continuous monitoring system in which the immobilized glutamate dehydrogenase column was connected to an in vitro superfusion system. In the presence of 0.3 microM tetrodotoxin, the application of 1 microM capsaicin to spinal dorsal horn slices evoked glutamate release (18.6 +/- 1.2 pmol mg(-1) protein, n = 11). In rats subjected to RCS (RCS rats), the release of glutamate evoked by 1 microM capsaicin was markedly increased to 272% (n = 6, P < 0.05) of the value for the control group, although the basal release was not significantly altered (n = 6, P > 0.05). Adjuvant inoculation produced a significant increase in the basal and capsaicin (1 microM) evoked release of glutamate to 141 and 344% (n = 6, P < 0.05) of the value for the control group, respectively. The present results suggest that the facilitated release of glutamate from capsaicin-sensitive primary afferent terminals in the spinal dorsal horn is, at least in part, involved in the hyperalgesia of RCS rats as well as the complete Freund's adjuvant-induced hyperalgesia.     

Rapid detection and typing of herpes simplex virus DNA in clinical specimens by the hybrid capture II signal amplification probe test

A second-generation signal amplification, nucleic acid-based test for the rapid detection and typing of herpes simplex virus (HSV) DNA was developed and evaluated with artificial and clinical specimens. The analytical sensitivity of the Hybrid Capture II (HC II) HSV DNA assay was determined by testing either cloned HSV DNA or total genomic HSV DNA titrations and resulted in detection thresholds of between 5 x 10(3) and 1 x 10(4) copies per assay. Specificity was assessed by testing a panel of bacteria and viruses commonly found in the female genital tract. Sensitivity was assessed by testing 112 ulcerative genital lesions by the HC II assay and comparing the results to those obtained by routine cell culture. Discrepant results were resolved by PCR testing. After resolution of the discrepant results, the sensitivity of the HC II assay compared to the consensus result (the results of two of three tests, the HC II assay, culture, and PCR, were in agreement) was 93.2% (41 of 44 specimens), and the specificity was 100% (60 of 60 specimens). Culture gave a sensitivity of 84.1% (37 of 44 specimens) and a specificity of 100% (60 of 60 specimens) compared to the consensus result. The results of HSV typing by the HC II assay and culture agreed in all cases. The HC II assay is a rapid and accurate assay for detecting and typing HSV types 1 and 2, with a sensitivity comparable to that of culture and greater ease of use than culture.     

Proof of hepatitis A virus negative-sense RNA by RNA/DNA-hybrid detection: a method for specific detection of both viral negative- and positive-strand RNA species

The detection of hepatitis A virus (HAV) negative-strand RNA, which is synthesized during replication of the positive-strand RNA genome, proved to be difficult. We developed a method for the specific detection of HAV negative-strand RNA by RNA-DNA hybridization and luminescence detection using an anti-RNA:DNA hybrid antibody. This method, which is also applicable for the specific detection of positive-strand RNA, offers a simple, yet relatively rapid and certain means of detecting low amounts of RNA such as HAV negative-strand RNA. By using appropriate hybridization DNA probes, the method should be applicable for the detection of single-stranded RNA species of different viruses in general.     

Identification of hepatitis E virus in clinical specimens: amplification of hydroxyapatite-purified virus RNA and restriction endonuclease analysis

Mutants null for the cathepsin B-like cysteine proteinase gene (cpc) of Leishmania mexicana have been generated by targeted gene disruption. The gene deletion was confirmed using a polymerase chain reaction (PCR) method with cpc-specific primers and genomic DNA isolated from the mutants. cpc was re-expressed in the null mutants from an episomal vector. Re-expression of the enzyme (CPC) was detected by Western blotting with a specific anti-peptide antiserum. The cpc null mutants grew apparently normally as promastigotes and amastigotes in axenic cultures, but they showed greatly reduced infectivity to macrophages in vitro with only a low percentage of the cells being infected. Re-expression of cpc in the null mutant increased the parasite's infectivity in vitro. The null mutant parasites formed lesions in mice at a similar rate as wild type parasites, although somewhat smaller lesions were produced. The results suggest that although the cysteine proteinase encoded by cpc plays a role in the parasite's interaction with macrophages it alone is not crucial for infectivity or virulence.     

A multiplex RT-PCR assay for analysis of relative transcript levels of different members of multigene families: application to Arabidopsis calmodulin gene family

In systemic amyloidosis, widespread amyloid deposition interferes with organ function, frequently with fatal consequences. Diagnosis rests on demonstrating amyloid deposits in the tissues, traditionally with histology although scintigraphic imaging with radiolabeled serum amyloid P component (SAP) has lately been developed as a specific noninvasive alternative. We report a detailed analysis of the abnormal turnover of SAP in patients with systemic amyloidosis and an assessment of its clinical value. METHODS: Iodine-123-labeled human SAP (200 MBq) SAP was injected intravenously into 49 patients with histologically proven systemic AA- or AL- amyloidosis and in 7 control subjects. Plasma clearance and whole-body retention of labeled SAP were analyzed over 48 hr using plasma sampling, whole-body gamma camera imaging and measurement of radioactivity in the urine. The rate of SAP synthesis and interstitial exchange were determined, and the size of the amyloid compartment was compared with clinical estimates of whole-body amyloid load and patient survival. RESULTS: All plasma time-activity curves were biphasic. In comparison with control subjects, patients with amyloidosis showed significantly faster plasma disappearance [4-hr value: AA 48% +/- 18%, AL 45% +/- 15% versus 65% +/- 8% (p < 0.05)], higher total-body retention 48 hr p.i. [AA 74% +/- 14%, AL 73% +/- 17% versus 46% +/- 15% (p < 0.01)] and especially higher extravascular retention 48 hr p.i. [AA 59% +/- 16%, AL 58% +/- 19% versus 30% +/- 14% (p < 0.01)]. Extravascular retention correlated with clinical estimation of the amyloid load. If extravascular retention values in patients with AL amyloidosis were over 60%, survival was decreased (median 4 versus 23 mo, p < 0.001). Markedly increased interstitial exchange rates were present in amyloidosis (AA 64 +/- 61, AL 50 +/- 37 versus 18 +/- 8 mg/hr), whereas the SAP synthesis rate did not differ from the control values (AA 5.0 +/- 3.0, AL 5.5 +/- 3.2 versus 4.5 +/- 1.4 mg/hr). CONCLUSION: The presence of systemic amyloidosis is characterized by accelerated initial clearance of 123I-SAP from the plasma and increased interstitial exchange rate and extravascular retention. These findings reflect reversible binding of radiolabeled SAP to amyloid deposits and provide clinically useful information for diagnosis, monitoring of therapy and prognosis in patients with systemic amyloidosis.     

Rapid enzymatic analysis for human immunodeficiency virus type 1 DNA in clinical specimens

A clinical procedure for rapid detection of human immunodeficiency virus type 1 (HIV-1) by DNA amplification is demonstrated. The rapid procedure reduces handling requirements and amplification time and eliminates use of radioactivity for the detection of the amplification product. Total leukocyte lysates are the amplification substrates. Two conserved regions in the HIV-1 genome are amplified by 45 cycles of a two-temperature thermal cycle and the amplification products are detected by ultraviolet light after electrophoresis on agarose gels. Twenty-four specimens clinically diagnosed by detection of antibody (IgG) to HIV-1 were confirmed by the rapid DNA amplification procedure. In a blind study, 56 samples positive for HIV-1 DNA were detected in 503 individuals by the current classical polymerase chain reaction method; the same 56 positive samples were also detected by the rapid amplification protocol. No false-positive or false-negative results were obtained. The turnaround time for analysis has been reduced to < 24 h without compromising test results.     

Rapid detection of Vibrio cholerae O139 Bengal from stool specimens by PCR

In a previous study using pure bacterial cultures in a PCR assay, a primer pair corresponding to a unique chromosomal region of Vibrio cholerae O139 Bengal generated an amplicon from only V. cholerae O139 Bengal. PCR with the same primer pair was used to screen 180 diarrheal stool specimens. All the 67 V. cholerae O139 culture-positive stool specimens were positive by PCR, and the remaining specimens, which contained either other recognized enteric pathogens or no pathogens, were all negative by PCR.     

Rapid detection of species of the opportunistic yeast Trichosporon by PCR

Trichosporon species are opportunistic pathogens, associated with a high mortality rate in immunocompromised patients. Oligonucleotide primers were used to amplify a 170-bp fragment of small-subunit ribosomal DNA of all species in the genus Trichosporon by PCR. The primers amplify DNAs of all species in the genus Trichosporon, including six causative agents of trichosporonosis. DNAs of other medically important yeasts, such as Candida albicans and Cryptococcus neoformans, are not amplified by this detection system.     

Genotyping by multiplex polymerase chain reaction for detection of endemic hepatitis B virus transmission

A nested polymerase chain reaction (PCR) protocol was developed for rapid genotyping of hepatitis B virus (HBV). During the first PCR round, a universal HBV primer pair was used to amplify the entire pre-S region of the HBV genome. Within the pre-S region, many nucleotide exchanges are observed. These are partly correlated to the serological hepatitis B surface antigen subtypes. Five additional subtype-specific primers were selected from that region which, together with two universal non-group-specific primers, generated specific combinations of two to four DNA fragments of defined sizes. By this approach, 55 hepatitis B surface antigen-positive patients from a pediatric oncology unit in Germany were analyzed. Fifty-four patients who had been infected within 2 years had an identical pattern in the multiplex PCR, suggesting a common source of infection and person-to-person transmission within the unit. One child who was infected 5 years later had a different PCR pattern and, therefore, must have been infected from a different source. Furthermore, 109 serum samples taken from pregnant Cameroonian women and 25 serum samples from their babies taken 6 months after birth were analyzed. In one case, mother-to-infant transmission of the virus was demonstrated. Apart from its role in epidemiological studies on HBV, multiplex PCR may also be a useful tool for rapid genetic analysis in other fields if there is a moderate degree of sequence variation which enables the design of specific primers.     

Rapid shell vial culture technique for detection of enteroviruses and adenoviruses in fecal specimens: comparison with conventional virus isolation method

Detection of enteroviruses and adenoviruses mainly in fecal specimens by rapid culture with inoculation onto cell monolayers in flat-bottom tubes by centrifugation and immunofluorescence staining with genus-specific monoclonal antibodies was compared with that by the conventional virus isolation procedure. For both conventional culture and shell vial culture human lung fibroblast cells and tertiary monkey kidney cells were used. For enterovirus detection, 979 clinical specimens (916 stool specimens, 56 cerebrospinal fluid specimens, and 7 nasopharyngeal swabs) were used. Conventional culture detected 74 enterovirus isolates. A cytopathic effect compatible with the presence of an enterovirus after 3 days of incubation occurred in 25 of the 74 (34%) specimens that eventually became positive. The detection rate for enteroviruses by rapid cell culture after 2 to 3 days of incubation was 42 of 74 (57%). The genus-specific enterovirus monoclonal antibody did not react with strains of echovirus types 22 and 23 or enterovirus type 71. Rapid cell culture for the detection of adenoviruses was performed with 567 clinical specimens (536 stool specimens, 25 cerebrospinal fluid specimens, and 6 miscellaneous specimens), in which 42 adenoviruses were found by conventional culture. Nine of the 42 (21%) adenovirus isolates were detected by conventional culture within 3 days after inoculation, whereas 21 (50%) were found by rapid cell culture within 2 to 3 days. Only two of the nine specimens found to be positive for the enteric adenovirus type 41 by conventional culture as well by a type-specific enzyme-linked immunosorbent assay (ELISA) tested positive by rapid cell culture. In conclusion, the rapid shell vial assay allows the early detection and identification of enteroviruses and adenoviruses in clinical specimens but is markedly less sensitive than the conventional isolation procedure according to the eventual results of the conventional isolation procedure. Conventional cell culture remains a prerequisite for serotyping of enteroviral isolates. On the basis of the results for adenovirus type 41, the rapid detection of adenoviruses was not considered to be useful for the detection of clinically relevant adenoviruses in fecal samples.     

Detection of herpes simplex virus in clinical specimens by cytospin-enhanced direct immunofluorescence

For 275 samples tested for herpes simplex virus (HSV), cytospin-enhanced direct immunofluorescence using Chemicon HSV monoclonal antibodies identified 80 (95%) and culture identified 77 (92%) of 84 confirmed positive specimens. Cytospin-prepared slides contained a greater number of total cells than standard cell spots, resulting in fewer inadequate cell smears and a higher HSV detection rate.     

Antiserum-agar plate method for simultaneous detection and direct isolation of Legionella species in clinical and environmental specimens

Colonies of Legionella pneumophila serotypes 1 through 6, L. micdadei, L. bozemanii, L. dumoffii, and L. gormanii, which were developed on filtered yeast extract agar containing polyvalent antiserum, were surrounded by distinct, specific precipitin rings.     

Novel method for processing respiratory specimens for detection of mycobacteria by using C18-carboxypropylbetaine: blinded study

A novel method for processing respiratory specimens to improve culture and acid-fast staining of mycobacteria is introduced. This new method utilized N,N-dimethyl-N-(n-octadecyl)-N-(3-carboxypropyl)ammonium inner salt (Chemical Abstract Service no. 78195-27-4), also known as C18-carboxypropylbetaine (CB-18). In a blinded, five-center study, CB-18-based processing was compared to the standard method combining NALC and NaOH (NALC/NaOH). A total of 573 respiratory specimens were tested. Individual specimens were split approximately equally; the host institutions processed half of each specimen by the NALC/NaOH method, while the other half was processed with CB-18 at Quest Diagnostics--Baltimore. A total of 106 specimens were culture positive for acid-fast bacilli (AFB). Replacement of the primary decontamination agent with CB-18 caused changes in all diagnostic parameters. Aggregate culture sensitivity improved by approximately 43% (P < 0.01), and smear sensitivity improved by approximately 58% (P < 0.01). The sensitivity of smear relative to that of M. tuberculosis isolates exceeded 93% (P < 0.01) when specimens were processed with CB-18. The average times to a positive result were reduced by 7.3 days in liquid culture (P < 0.01) and 5.3 days on solid media (P < 0.05); however, the CB-18 method had a 20.8% contamination rate in liquid culture versus a rate of approximately 7.5% with NALC/NaOH processing. There were also unusual reductions in liquid culture sensitivity and smear specificity among CB-18-processed specimens. The characteristics of the latter parameters suggested that refinement of the CB-18 processing method should allow further improvements in culture sensitivity. This study showed that the CB-18 method has the potential to improve both smear and culture detection for these important human pathogens.