Magnetic bead flocculation test: Improving the diagnosis of tuberculous meningitis (TBM) in low-resource settings.

BACKGROUND: Despite several recent advances in detection techniques, there is still an unmet need for simple tests for the diagnosis of tuberculous meningitis (TBM). Therefore, in an effort towards developing a simple and rapid diagnostic test for resource-poor settings, we designed an assay in which magnetic bead flocculation test (MBF) was used to detect the amplified DNA. Multi-targeted (using two multicopy gene targets IS6110 and IS1081) loop-mediated isothermal amplification (MLAMP) was used for amplification. METHODS: MLAMP-MBF assay was performed on CSF samples of 600 patients, out of which 120 were definite TBM (culture confirmed), 280 were probable TBM and 200 were non-TB controls, based on Marais's criteria. The performance of assay was evaluated by comparing the result of definite TBM with culture and that of probable TBM with composite reference standard consisting of clinical, microbiological(smear/culture) and radiological parameters. RESULTS: The overall sensitivity of MLAMP-MBF (using any of the two gene targets) was 89.5% and specificity was 100%. The sensitivity was 96.6% (116/120) in diagnosing definite TBM and 86.4% (242/280) in diagnosing probable TBM. The sensitivity of IS1081 was 88% and that of IS6110 was 83% in diagnosing TBM. Specificity of both the gene targets was 100%. There were 20 cases positive only by IS1081 LAMP and 6 cases positive only by IS6110; thus 26 of 400 (6.5%) TBM cases could be additionally detected following multi-targeted approach. CONCLUSION: MLAMP-MBF is a sensitive, robust, cost-effective and promising technique for diagnosis of TBM in low-resource high-endemic settings.

Calibration of replacement international standards of diphtheria and tetanus toxoids for use in flocculation test.

The 1st International Reference Reagents (IRR) of Diphtheria and Tetanus Toxoids for Flocculation Test (DIFT and TEFT) were established by the WHO in 1988. These reagents are essential for the standardization of assays used to calculate Lf units of toxoids. Candidate replacement materials were provided by several European vaccine manufacturers and were formulated and freeze-dried at NIBSC. This paper provides a summary of the results of an international collaborative study including 18 laboratories from 16 countries, which examined the candidate replacement materials in a variety of methods. Materials 02/176 and 04/150 were proposed and adopted by the Expert Committee on Biological Standardization of WHO in October 2007 as 2nd WHO International Standards of Diphtheria and Tetanus Toxoid for use in Flocculation Test. The replacement standards were assigned the value of 1100 and 690Lf/ampoule, respectively, based on results of flocculation tests carried out using provided reagents. Material coded 02/176 fully complied with the WHO specifications for stability, residual moisture content, precision of fill and sterility. Stability of material coded 04/150 was slightly lower than expected but predictions were based only on 2-year data and were to be further monitored, post-adoption.

Toxoid flocculation assay by laser light-scattering.

Internationally accepted designations of antigen content for toxoid vaccines are provided by the WHO in Lf (limes flocculationis) units, based on the formation of antigen-antibody complexes. The current assay method for Lf determination involves observation of the complexes by eye, making the development of a more objective system highly desirable. Here we report a novel detection system using a laser light-scattering platelet aggregometer. The system was highly reproducible and more objective than the current method. Only three sets of duplicate data were sufficient for statistically significant determination of toxoid Lf by parabolic regression.

Seasonal change in the number of Cryptosporidium parvum oocysts in water samples from the rivers in Hokkaido, Japan, detected by the ferric sulfate flocculation method.

An epidemiological study was carried out in natural water supplies of Hokkaido, one of the largest dairy prefectures in Japan. To investigate the prevalence of Cryptosporidium parvum (C. parvum) oocysts water samples were collected from three rivers in the eastern area of Hokkaido from August 1999 to October 2001, and C. parvum oocysts were collected and purified by the ferric sulfate flocculation method. The oocysts were detected using the immunofluorescent assay test (IFAT) and 4', 6-diamidino-2-phenylindole (DAPI) staining. The seasonal change in the number of oocysts detected was observed. Oocysts increased in numbers from the late summer to the early autumn (from August to November), thereafter, they exhibited a trend to decrease until December, when no oocysts could be detected. The maximum number of oocysts detected in the three rivers was 3.50, 5.00 and 3.33 oocysts/l, respectively. The oocyst density in river water changed in relation to the season in 1999, 2000 and 2001. This report first cleared up the seasonal changes in C. parvum oocysts number in river water.

Evaluation of three flocculation methods for the purification of Cryptosporidium parvum oocysts from water samples.

AIMS: Evaluation of three flocculation methods for the purification of Cryptosporidium parvum oocysts from tap water. METHODS AND RESULTS: Ferric sulphate, aluminium sulphate and calcium carbonate were compared for their recovery efficiency of C. parvum oocysts from tap water. Lower mean recovery was achieved by calcium carbonate (38.8%) compared with ferric sulphate (61.5%) and aluminium sulphate (58.1%) for the recovery of 2.5 x 10(5) oocysts l(-1); 2.5 oocysts l(-1) and 1 oocyst l(-1) were adequately purified using ferric sulphate flocculation. In vitro excystation experiments showed that ferric sulphate flocculation does not markedly reduce the viability of oocysts. CONCLUSIONS: Ferric sulphate flocculation is a simple and effective tool for the purification of C. parvum oocysts from tap water. SIGNIFICANCE AND IMPACT OF THE STUDY: The high recovery rates and low impact on oocyst viability provided by ferric sulphate flocculation might be useful for the detection of Cryptosporidium oocysts in environmental water samples.

[Diagnostic valve of ELISA-IgG for trichinosis using Melcher's antigen]. / Valor diagnóstico de la ELISA-IgG para triquinosis, usando antígeno de Melcher.

An enzyme-linked immunosorbent assay (ELISA) for trichinosis using a Melcher's antigen was developed for the detection of IgG antibodies in 41 serum samples from patients confirmed or suspected to have trichinosis by strong clinical and epidemiological evidences. ELISA- IgG was compared with a precipitin test (PT), a bentonite floculation test (BFT) and an indirect hemagglutination test (IHAT). The cut-off value was determined using serum samples from 67 apparently healthy persons employing two serum dilutions (1:100 and 1:500) with three standard deviations (SD). The sensitivity of ELISA- IgG was 97.6% and 95.2% using serum dilutions of 1:100 and 1:500 respectively, whereas the values for the other tests were: PT (92.7%), BFT (63.4%) and IHAT(85.4%). According to MacNemar test, ELISA-IgG did not present statistical significance (p > 0.05) compared with PT and IHAT, whereas all of them were different compared with BHT (p > 0.01). In order to find out the specificity of ELISA-IgG, additional 124 serum samples from individuals with other parasitoses, such as cysticercosis (31), fascioliasis (17), hydatidosis (51) and toxocariasis (25) were also tested. ELISA-IgG presented a specificity of 99.5% with both serum dilutions. The positive predictive values were 97.6 and 97.5%, whereas the negative one were 99.5 and 99.0% for 1:100 and 1:500 serum dilutions respectively. The use of ELISA-IgG and Melcher's antigen in the diagnosis of human trichinosis is discussed.

Comparison of maternal sera, cord blood, and neonatal sera for detecting presumptive congenital syphilis: relationship with maternal treatment.

The incidence of congenital syphilis has increased rapidly over the past few years. Most infected mothers and their newborns are asymptomatic at birth and diagnosis depends on serologic testing during pregnancy and at delivery. This study was initiated to compare maternal sera, cord blood, and neonatal sera for detecting presumptive congenital syphilis and to assess the role of maternal treatment (administration of penicillin to the mother at least 1 month before delivery) on the serologic results at the time of delivery. The serologic results from all live deliveries complicated by a positive maternal and/or neonatal test for syphilis during a 12-month period were compared using chi 2 analysis and multiple comparisons for proportions. Of 3306 livebirths, 73 (2.2%) were complicated by a positive maternal or neonatal serology. At delivery, the serologic test was positive in 68 (94%) of 72 maternal sera, 30 (50%) of 60 cord sera, and 43 (63%) of 68 neonatal sera. In the absence of maternal treatment, 95% of the maternal sera, 66% of the cord blood samples, and 86% of the neonatal sera were positive. If the mother had been treated, 94% of maternal sera, 36% of cord sera, and 39% of neonatal sera were positive. Cord blood and neonatal sera appear to be inferior to maternal sera for detecting prenatal exposure to syphilis. Cord serology is also inferior to neonatal serology at 2 to 3 days of age. The most effective way to identify newborns at risk for congenital syphilis is to obtain a maternal serologic diagnosis during pregnancy and to test maternal and neonatal sera at delivery.

[Cytotoxicity of ferric chloride used as an adjuvant of organic acid precipitation]. / La cytotoxicité du chlorure ferrique utilisé comme adjuvant de la précipitation organique acide.

The objective of this study was the amelioration of the organic acid precipitation technique currently used as a secondary stage for viruses concentration using ferric chloride (FeCl3) as an adjuvant. To carry out this work successfully, we have studied the cytotoxicity effect of FeCl3 towards cell lines usually used for the investigation of enteric viruses. Two kinds of african green monkey kidney cells, MA104 and BGM, were used as a cellular model. The results showed that optimum concentration of FeCl3 for either BGM or MA104 cell survival and organic acid precipitation varies from 0.5 to 1 mM. The cytotoxicity test used in this work was simple, easy to realize and gives an appreciable information about the cytotoxicity dose of a given biological and chemical product.

Comparison of maternal and newborn serologic tests for syphilis.

OBJECTIVE: To compare the cord blood, newborn serum, and maternal serum for the diagnosis of congenital syphilis. DESIGN: Retrospective chart review. SETTING: Kings County Hospital Center, Brooklyn, NY. PATIENTS: Three hundred forty-eight mother-newborn pairs with positive syphilis serology. MEASUREMENTS AND RESULTS: One hundred fifteen newborns (33%) had rapid plasma reagin tests of cord blood that were nonreactive. Their mothers had positive serologic findings. There were 10% false-positive cord blood samples (cord blood rapid plasma reagin tests reactive, newborn serum rapid plasma reagin tests nonreactive) and 5% false-negative cord blood samples (cord rapid plasma reagin tests nonreactive, newborn serum rapid plasma reagin tests reactive). Thirty-three newborns had congenital syphilis. Seven newborns had cord titers fourfold higher than their mothers'; only four of these newborns had congenital syphilis. Maternal serology is superior to cord blood analysis for identifying newborns at risk of congenital syphilis.

Pretest temperature effects on CAP syphilis serology survey samples.

The College of American Pathologists syphilis serology survey participants periodically question survey-sample stability when the samples have possibly been exposed to temperature extremes during shipment. In this study it was demonstrated that short-term pretest incubations at temperatures above 58 degrees C (136.4 degrees F) usually cause a reduction in both rapid plasma reagin and FTA-ABS reactivity. No reactivity changes occurred in either test with short-term exposures to temperatures between -80 degrees C and 58 degrees C.

Interpreting serologic tests for syphilis.

Since syphilis is most often diagnosed by serologic studies, the correct interpretation of these tests is critical. Serologic tests are classified as nontreponemal of treponemal, according to the antigen employed. Flocculation tests are generally used for routine screening because of their simplicity. The treponemal procedures are more specific, but false-positive results may still occur. Results vary with the procedure, stage of disease and treatment. Significant difficulties remain with the serologic diagnosis of neurosyphilis and congenital syphilis.

[Use of the bacterial flocculation test for assessing the mutagenic properties of carcinogenic nitrosamines and nitramines]. / Ispol'zovanie bakterial'nogo floktuatsionnogo testa dlia otsenki mutagennykh svoistv kantserogennykh nitrozaminov i nitraminov.

Fluctuation test developed by Green allows to detect the mutagenicity of trace and non-toxic chemical concentrations. Mutagenic activity of both carcinogenic nitramines--nitrodimethylamine, nitrodiethylamine and nitromorpholine (0.1--10 mM) and their nitrosamine analogues-nitrosodimethylamine, nitrosodiethylamine and nitrosomorpholine (0.01--0.1 mM) was detected in fluctuation test with Salmonella typhimurium TA 98 and TA 1530 as indicator organism in the presence of postmitochondrial supernatant from liver of rats treated with Aroclor 1254. Nearly all compounds appeared to be mutagenic even in the absence of co-factors of microsomal oxygenation.