A simple and rapid immunochromatographic test kit for rabies diagnosis

In rabies endemic countries, funds and infrastructure are often insufficient to employ the approved gold standard for the definitive diagnosis of rabies: the direct fluorescent test. In the present study, two types (type 1 and 2) of an ICT kit were evaluated for detection of rabies. These were developed using monoclonal antibodies which recognize epitope II and III of the nucleoprotein of rabies virus. Both kits specifically detected all rabies virus strains and there was no cross reactivity with Lyssaviruses (Lagos, Mokola and Duvenhage), Rhabdovirus (VSV and Oita 296/1972) and other common canine-pathogenic viruses. In type 1, a single type of monoclonal antibody was used. It was capable of detecting recombinant nucleoprotein and showed sensitivity of 95.5% (42/44) and specificity of 88.9% (32/36) using brain samples from rabid dogs. In contrast, type 2 which was made of two different monoclonal antibodies had a lower sensitivity of 93.2% (41/44) and higher specificity of 100% (36/36). These ICT kits provide a simple and rapid method for rabies detection. They need neither cold chain for transportation nor complicated training for personnel. This diagnostic test is suitable for rabies screening, particularly in areas with a high prevalence of rabies and where the fluorescent antibody test is not available.     

Assessment of immunochromatographic test for rapid lymphatic filariasis diagnosis

Two rapid immunodiagnostic tests (ICT Filariasis test), developed for the quick diagnosis of Wuchereria bancrofti infection, have been validated in laboratory and field situation. The aim of this study was to assess the performance and usefulness of this antigen capture assay as a diagnostic method in three foci of lymphatic filariasis, located in the South Pacific (Society archipelago, French Polynesia), with different levels of endemicity. A sample of 1,595 patients was tested with this assay in parallel with a reference Og4C3 antigen capture assay and microfilariae detection. A second-generation ICT test, available for whole blood analysis, was also tested in parallel with the first generation test, developed for serum analysis, on a sample of 50 reference cases. The correspondence between the results obtained with the two rapid tests was excellent, without any influence of rheumatoid factors, but the sensitivity was in both cases slightly inferior to the one obtained with the ELISA reference test. This seems particularly true in epidemiological situation where a high proportion of amicrofilaraemic, adult worm carriers are observed.     

16S rRNA gene sequence analysis of Photobacterium damselae and nested PCR method for rapid detection of the causative agent of fish pasteurellosis.

The causative agent of fish pasteurellosis, the organism formerly known as Pasteurella piscicida, has been reclassified as Photobacterium damselae subsp. piscicida on the basis of 16S rRNA gene sequence comparisons and chromosomal DNA-DNA hybridization data; thus, this organism belongs to the same species as Photobacterium damselae subsp. damselae (formerly Vibrio damselae). Since reassignment of P. damselae subsp. piscicida was based on only two strains, one objective of the present work was to confirm the taxonomic position of this fish pathogen by sequencing the 16S rRNA genes of 26 strains having different geographic and host origins. In addition, a nested PCR protocol for detection of P. damselae based on 16S rRNA was developed. This PCR protocol was validated by testing 35 target and 24 nontarget pure cultures, and the detection limits obtained ranged from 1 pg to 10 fg of DNA (200 to 20 cells). A similar level of sensitivity was observed when the PCR protocol was applied to fish tissues spiked with bacteria. The PCR approach described in this paper allows detection of the pathogen in mixed plate cultures obtained from asymptomatic fish suspected to be carriers of P. damselae subsp. piscicida, in which growth of this bacterium cannot be visualized. Our results indicate that the selective primers which we designed represent a powerful tool for sensitive and specific detection of fish pasteurellosis.     

Evaluation of a standardized F1 capsular antigen capture ELISA test kit for the rapid diagnosis of plague

Rapid detection of soluble F1 capsular antigen in serum, bubo fluid or urine of patients proved to be a valuable tool in the presumptive diagnosis of plague. We evaluated a F1 capsular antigen capture ELISA resembling a commercially available test kit. The minimal detectable concentration was 4 ng/ml. The specificity was 100% when investigating 47 sera from healthy Malagasy subjects and 98.4% when 365 sera from German blood donors were studied. Sensitivity was determined on sera (n=11) and buboes (n=18) from bacteriologically confirmed Malagasy plague patients. Sensitivity was 90.1% for serum and 100% for buboes. A standardized F1 capsular antigen capture ELISA test kit might be well suited for the early detection of plague particularly in non-endemic areas where clinical microbiological laboratories have only limited access to alternative techniques for rapid identification of Yersinia pestis.     

Development of an immunochromatographic kit for rapid diagnosis of H5 avian influenza virus infection

Highly pathogenic avian influenza (HPAI) caused by the H5N1 subtype has given rise to serious damage in poultry industries in Asia. The virus has expanded its geographical range to Europe and Africa, posing a great risk to human health as well. For the control of avian influenza, a rapid diagnosis by detecting the causative virus and identifying its subtype is essential. In the present study, a rapid diagnosis kit combining immunochromatography with enzyme immunoassay which detects the H5 HA antigen of influenza A virus was developed using newly established anti-H5 HA monoclonal antibodies. The present kit specifically detected all of the H5 influenza viruses tested, and did not react with the other HA subtypes. H5 HA antigens were detected from swabs and tissue homogenates of chickens infected with HPAI virus strain A/chicken/Yamaguchi/7/04 (H5N1) from 2 days post inoculation. The kit showed enough sensitivity and specificity for the rapid diagnosis of HPAI.     

New lyophilized kit for rapid radiofluorination of peptides

Radiolabeling compounds with positron-emitting radionuclides often involves a time-consuming, customized process. Herein, we report a simple lyophilized kit formulation for labeling peptides with (18)F, based on the aluminum-fluoride procedure. The prototype kit contains IMP485, a NODA (1,4,7-triazacyclononane-1,4-diacetate)-MPAA (methyl phenylacetic acid)-di-HSG (histamine-succinyl-glycine) hapten-peptide, [NODA-MPAA-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH(2)], used for pretargeting, but we also examined a similar kit formulation for a somatostatin-binding peptide [IMP466, NOTA-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Throl] bearing a NOTA ligand to determine if the benefits of using a kit can be extended to other AlF-binding peptides. The NODA-MPAA ligand forms a single stable complex with (AlF)(2+) in high yields. In order to establish suitable conditions for a facile kit, the formulation was optimized for pH, peptide to Al(3+) ratio, bulking agent, radioprotectant, and the buffer. For optimal labeling, the kit was reconstituted with an aqueous solution of (18)F(-) and ethanol (1:1), heated at 100-110 °C for 15 min, and then simply and rapidly purified using one of two equally effective solid-phase extraction (SPE) methods. Al(18)F-IMP485 was isolated as a single isomer complex, in high yield (45-97%) and high specific activity (up to 223 GBq/μmol), within 20 min. The labeled product was stable in human serum at 37 °C for 4 h and in vivo, urine samples showed the intact product was eliminated. Tumor targeting of the Al(18)F-IMP485 in nude mice bearing human colon cancer xenografts pretargeted with an anti-CEACAM5 bispecific antibody showed very low uptake (0.06% ± 0.02 ID/g) in bone, further illustrating its stability. At 1 h, pretargeted animals had high Al(18)F-IMP485 tumor uptake (28.1% ± 4.5 ID/g), with ratios of 9 ± 4, 123 ± 38, 110 ± 43, and 120 ± 108 for kidney, liver, blood and bone, respectively. Tumor uptake remained high at 3 h postinjection, with increased tumor/nontumor ratios. The NOTA-somatostatin-binding peptide also was fluorinated with good yield and high specific activity in the same kit formulation. However, yields were somewhat lower than those achieved with IMP485 containing the NODA-MPAA ligand, likely reflecting this ligand's superior binding properties over the simple NOTA. These studies indicate that (18)F-labeled peptides can be reproducibly prepared as stable Al-F complexes with good radiochemical yield and high specific activity using a simple, one-step, lyophilized kit followed by a rapid purification by SPE that provides the (18)F-peptide ready for patient injection within 30 min.     

Evaluation of a kit for rapid detection of group A streptococci in a pediatric emergency department.

We evaluated a kit for the rapid detection of group A streptococci from throat swabs (Culturette Brand 10-Minute Group A Strep ID, Marion Scientific, Division of Marion Laboratories, Inc., Kansas City, Missouri) in the laboratory and in a busy pediatric emergency department. The sensitivity of the kit in the laboratory was 80% for all specimens and 94% for specimens with more than 10 colony-forming units of group A streptococci; the specificity was 99%. After initial training, emergency department pediatricians and nurses achieved sensitivities of 72% and 69% respectively. The specificity achieved by the pediatricians was 76% initially but 96% after further training. Untrained residents achieved a sensitivity of 58%. We conclude that this kit is potentially useful in the hands of adequately trained personnel, but without training the accuracy of the results is unacceptable. We recommend that the kit be used by designated staff trained and monitored by laboratory personnel.     

Construction of a kit of reference strains for rapid genetic mapping in Bacillus subtilis 168.

A set of nine reference strains bringing convenient markers in the genetic background of Bacillus subtilis Marburg 168 has been prepared to allow rapid mapping of new markers.     

细菌生理特性快速检测试剂盒研制

选取各代谢途径中利用某些特定碳源,如蔗糖、葡萄糖、甘露醇等95种碳源作为测试碳源,分别加入96孔酶标板中.选用指示剂噻唑兰(MTT)来鉴定细菌利用特征碳源情况.同时选取不同种类和不同浓度的抗生素,不同起始pH、不同NaCl浓度加入96孔酶标板中,测定细胞是否生长,可以快速地获得该菌的抗生素耐受性谱.试验还从指示剂浓度、培养基的固化、接菌浓度、观察时间等方面对试剂盒进行优化,得到一种最佳的试剂盒鉴定系统BiobiqA(碳源利用谱)和BiobiqB(生理抗性谱).用模式菌株对试剂盒进行测试和验证,结果表明,试剂盒具有操作简便、结果准确、节省成本、节约时间等优点,可以进行细菌生理特性的快速检测.     

Assessment of the Accuprobe Listeria monocytogenes culture identification reagent kit for rapid colony confirmation and its application in various enrichment broths.

The Accuprobe Listeria monocytogenes Culture Identification Reagent Kit, a nonradioactive probe, was evaluated as a colony confirmation test and in different selective or nonselective enrichment broths. The probe was 100% sensitive and 100% specific when applied to isolated colonies. The minimal detection limit in physiological saline was established to be about 10(5) CFU of L. monocytogenes. Hybridization done directly in broths seeded with L. monocytogenes showed variable results. Three nonselective broths (Todd-Hewitt broth, brain heart infusion broth, and tryptic soy broth) and one selective broth (FDA) gave positive reactions at an inoculum of 5 x 10(6) CFU, whereas two other selective broths (UVM, and PALCAMY) gave negative reactions with up to 10(8) and 10(9) CFU. In FDA broth, the level of detection of L. monocytogenes was not modified by the presence of other organisms in mixed cultures.     

Assessment of the Accuprobe Listeria monocytogenes culture identification reagent kit for rapid colony confirmation and its application in various enrichment broths.

The Accuprobe Listeria monocytogenes Culture Identification Reagent Kit, a nonradioactive probe, was evaluated as a colony confirmation test and in different selective or nonselective enrichment broths. The probe was 100% sensitive and 100% specific when applied to isolated colonies. The minimal detection limit in physiological saline was established to be about 10(5) CFU of L. monocytogenes. Hybridization done directly in broths seeded with L. monocytogenes showed variable results. Three nonselective broths (Todd-Hewitt broth, brain heart infusion broth, and tryptic soy broth) and one selective broth (FDA) gave positive reactions at an inoculum of 5 x 10(6) CFU, whereas two other selective broths (UVM, and PALCAMY) gave negative reactions with up to 10(8) and 10(9) CFU. In FDA broth, the level of detection of L. monocytogenes was not modified by the presence of other organisms in mixed cultures.     

Evaluation of rapid immunochromatographic assay kit for HBsAg-screening using whole blood

A rapid immunochromatographic assay kit using whole blood to screen hepatitis B surface antigen was developed and evaluated by using sera from 240 patients. The reference diagnosis was based on the results obtained with GENEDIA Anti-HBs Rapid kit which is very similar to the above kit except for the use of serum. The test demonstrated a good correlation with the reference immunochromatographic assay kit, that is, the sensitivity and the specificity of the kit was 100%, respectively. The rapid test kit using whole blood should be more convenient and useful for the diagnosis of hepatitis B virus because the kit does not need machines and time to prepare serum. In addition, this kit is safe from inadvertent infection during sample treatment because the blood is sterilized with hydrogen peroxide, eliminates the procedure required to prepare serum and reduces the possibility of exposure to infectious agents.     

The india-ink immunoreaction: a method for the rapid diagnosis of encephalitozoonosis.

Sera from 37 rabbits were assayed for antibodies against Encephalitozoon cuniculi (Nosema cuniculi) by the india-ink immunoreaction and the indirect fluorescent antibody tests: all animals seropositive to the former were also positive to the latter test. 27 of the rabbits were also tested for skin hypersensitivity and then autopsied. Animals positive to the skin test were also positive to the serological tests. At autopsy 18 of 22 rabbits positive in the immunological tests showed lesions typical of encephalitozoonosis. Sera from 200 rabbits originating from 6 institutes were assayed by the india-ink test: seropositive rabbits were found from all institutes (9.1 to 81.9% incidence), with serum titres ranging from 1:125 to 1:5000. The india-ink test appears to be a rapid and convenient method for diagnosis of encephalitozoonosis in rabbits.     

Towards a more rapid diagnosis of rapidly progressive glomerulonephritis.

An attempt was made to provide simple practical guidelines to alert general practitioners to the diagnosis of rapidly progressive glomerulonephritis and lead to early referral to hospital. The duration of illness before referral to this hospital and its effect on outcome in patients with crescentic nephritis were assessed retrospectively from the case notes of 24 patients referred over two years. Four patients had Goodpasture''s syndrome, 11 Wegener''s granulomatosis, seven microscopic polyarteritis, and two idiopathic progressive glomerulonephritis. The duration of symptoms before referral to the local hospital was similar in the four groups of patients and varied from one week to 28 months (mean 10 months). The duration of stay in the local hospital was two, nine, 11, and 180 days in the patients with Goodpasture''s syndrome and a mean of four days (range one to eight) in those with Wegener''s granulomatosis and 10 days (one to 18 days) in those with microscopic polyarteritis. In the local hospital the diagnosis was based on the results of renal biopsy and detection of antibodies to glomerular basement membrane in two patients with Goodpasture''s syndrome and on the results of renal biopsy in seven of the other patients aided by the detection of antibodies to the cytoplasm of neutrophils (ANCA) in 10. Three of the 24 patients died and four required maintenance haemodialysis. Patients who present to their general practitioners with persistent non-specific symptoms should have a urine dipstick test and then blood tests and emergency referral to hospital if necessary. Hospital physicians should be aware of the speed and accuracy with which current assays can confirm a diagnosis of rapidly progressive glomerulonephritis.     

Virus morphology as an aid for rapid diagnosis.

Standard methods of virus diagnosis may take many days to complete. As antiviral drugs are being used with more effectiveness, it becomes more important to develop rapid diagnostic methods. It takes only a few minutes to prepare and examine a specimen for electron microscopy (EM), using the negative staining technique. Viruses in the specimen can readily be identified by their morphology. In order to be detected by EM there must be at least 10(7) virus particles per milliliter of sample. This concentration is frequently found in certain types of specimens. The sensitivity of EM is increased 100-fold if homologous antibody is used to aggregate the virus. Visualization of virus-antibody aggregates forms the basis for serotyping by immunoelectron microscopy (IEM).     

Quantitative trait loci for resistance to fish pasteurellosis in gilthead sea bream (Sparus aurata)

Fish pasteurellosis is a bacterial disease causing important losses in farmed fish, including gilthead sea bream, a teleost fish of great relevance in marine aquaculture. We report in this study a QTL analysis for resistance to fish pasteurellosis in this species. An experimental population of 500 offspring originating from eight sires and six dams in a single mass‐spawning event was subjected to a disease challenge with Photobacterium damselae subsp. piscicida (Phdp), the causative agent of fish pasteurellosis. A total of 151 microsatellite loci were genotyped in the experimental population, and half‐sib regression QTL analysis was carried out on two continuous traits, body length at time of death and survival, and for two binary traits, survival at day 7 and survival at day 15, when the highest peaks of mortality were observed. Two significant QTLs were detected for disease resistance. The first one was located on linkage group LG3 affecting late survival (survival at day 15). The second one, for overall survival, was located on LG21, which allowed us to highlight a potential marker (Id13) linked to disease resistance. A significant QTL was also found for body length at death on LG6 explaining 5–8% of the phenotypic variation.     

The melanophore aggregating response of isolated fish scales: a very rapid and sensitive diagnosis of whooping cough

Pertussis toxin (PT) has been found to block noradrenaline-induced pigment aggregation in fish melanophores, and, based on this, a rapid and highly sensitive assay for PT was developed. Some preliminary results have also indicated that it may be possible to detect PT-like activity in saliva samples from patients with clinically suspected pertussis. In the present study the diagnostic value of the fish melanophore method was evaluated in 70 patients suspected of having pertussis; culture, serology and physician diagnosis were used as reference methods. In 60 of the patients, pertussis was verified by at least one of the reference methods. The melanophore test showed PT-like activity in saliva samples from 58 of the patients. Three patients with reference-verified pertussis showed no PT-like activity in the test; among these, one patient had been immunized and had also been treated with erythromycin during 3 days immediately prior to visiting the hospital. The melanophore test has three major advantages: it allows detection of pertussis in the early and curable stage of the disease; it takes only 2 h to perform; and it requires no sophisticated equipment.