Performance of six commercial enzyme immunoassays and two alternative HIV-testing algorithms for the diagnosis of HIV-1 infection in Kisumu, Western Kenya.

Performances of serological parallel and serial testing algorithms were analyzed using a combination of three ELISA and three rapid tests for the confirmation of HIV infection. Each was assessed individually for their sensitivity and specificity on a blinded panel of 769 retrospective sera of known HIV status. Western blot was used as a confirmatory assay for discordant results. Subsequently, one parallel and one serial testing algorithm were assessed on a new panel of 912 HIV-positive and negative samples. Individual evaluation of the ELISAs and rapid tests indicated a sensitivity of 100% for all assays except Uni-Gold with 99.7%. The specificities ranged from 99.1% to 99.4% for rapid assays and from 97.5% to 99.1% for ELISAs. A parallel and serial testing algorithms using Enzygnost and Vironostika, and Determine followed by Uni-Gold respectively, showed 100% sensitivity and specificity. The cost for testing 912 samples was US$4.74 and US$ 1.9 per sample in parallel and serial testing respectively. Parallel or serial testing algorithm yielded a sensitivity and specificity of 100%. This alternative algorithm is reliable and reduces the occurrence of both false negatives and positives. The serial testing algorithm was more cost effective for diagnosing HIV infections in this population.

13C-labeled D-ribose: chemi-enzymic synthesis of various isotopomers.

Current interest in the use of heteronuclear multidimensional NMR methods to assess the structures, conformations and/or dynamics of oligonucleotides in solution has created an immediate need for nucleosides and their derivatives labeled in various ways with stable isotopes (13C, 2H, 15N and/or 17,18O). This short review focuses exclusively on chemienzymic methods to introduce one or more 13C labels into D-ribose, a precursor to ribo- and 2'-deoxyribonucleosides. It will be demonstrated that five convenient reactions, applied in specific sequences, provide access to 26 of the 32 13C-labeled isotopomers of D-ribose in acceptable yields. While not explicitly discussed herein, these same reactions, appropriately modified, can also be used to insert one or more 2H and/or 17,18O isotopes into this aldopentose.

Multiply 13C-substituted monosaccharides: synthesis of D-(1,5,6-13C3)glucose and D-(2,5,6-13C3)glucose.

D-(1,5,6-13C3)Glucose (7) has been synthesized by a six-step chemical method. D-(1,2-13C2)Mannose (1) was converted to methyl D-(1,2-13C2)mannopyranosides (2), and 2 was oxidized with Pt-C and O2 to give methyl D-(1,2-13C2)mannopyranuronides (3). After purification by anion-exchange chromatography, 3 was hydrolyzed to give D-(1,2-13C2)mannuronic acid (4), and 4 was converted to D-(5,6-13C2)mannonic acid (5) with NaBH4. Ruff degradation of 5 gave D-(4,5-13C2)arabinose (6), and 6 was converted to D-(1,5,6-13C3)glucose (7) and D-(1,5,6-13C3)mannose (8) by cyanohydrin reduction. D-(2,5,6-13C3)Glucose (9) was prepared from 8 by molybdate-catalyzed epimerization.

Stable, isotopically substituted carbohydrates: an improved synthesis of (6-13C)aldohexoses.

1,2-O-Isopropylidene-alpha-D-xylo-pentodialdo-1,4-furanose (1) has been used as the parent aldose in the preparation of D-(6-13C)glucose and L-6-13C)idose via cyanohydrin reduction. The addition of K13CN (pH 6.8, 5 min) to 1 yields D-gluco and L-ido cyanohydrins that are readily reduced with H2 and Pd-BaSO4, to give 1,2-O-isopropylidene-alpha-D-gluco-hexodialdo-1,4-furanose (2; approximately 65%) and 1,2-O-isopropylidene-beta-L-ido-hexodialdo-1,4-furanose (3; 35%). Aldehydes 2 and 3 are reduced in situ with NaBH4, the resulting alcohols are deprotected with aqueous acid, and the aldoses are chromatographed on Dowex 50 X-8 (Ca2+) ion-exchange resin (200-400 mesh), to yield D-(6-13C)glucose (6) and L-(6-13C)idose (7). Molybdate epimerization of 6 and 7 yields D-(6-13C)mannose and L-(6-13C)gulose, respectively. A similar reaction scheme may be applied to methyl 2,3-O-isopropylidene-beta-D-ribo-pentodialdo-1,4-furanoside to generate the remaining four (6-13C)aldohexoses. This route is considerably simpler than the traditional Kiliani-Fischer route, and higher yields are obtained.

Tricyclic antidepressants in serum by a Clin-ElutTM column extraction and high pressure liquid chromatographic analysis.

We sought a method for routine therapeutic monitoring of serum tricyclic antidepressants (TCAs) which offered good reproducibility, detection limits, linearity, and specificity, and which was simple, rapid, and inexpensive to perform. The method described utilizes Clin-Elut columns (Analytichem International, Inc., Lawndale, CA 90206) to facilitate the extraction. The analysis is by high pressure liquid chromatography (HPLC) with a CN bonded phase column, a mobile phase of acetonitrile/pH 7.0 phosphate/methanol and detection at 210 nm. This chromatographic system gives short equilibration times, stable calibration curves, high sensitivity and resolution, short retention times, and long column life. The method is useful for determination of amitriptyline, doxepin, imipramine, nortriptyline, nordoxepin, desipramine, and protriptyline. Trimipramine is used as an internal standard for the tertiary amines and protriptyline for the secondary amines. Recovery is linear from 25 ato 1,000 ng/ml. Rubber stoppers of a new formulation in Vacutainer blood collection tubes (Becton-Dickinson, Rutherford, NJ 07070) do not affect serum TCA levels. Sera from 53 psychiatric patients suffering from endogenous depression were analyzed using the procedure presented. The mean serum level of four patients on amitriptyline therapy having complete remission was 201 ng/ml (range, 123-259). The mean serum level of four patients on imipramine therapy rated as having complete remission was 200 ng/ml (range, 145-258). These values compare well with recently published therapeutic ranges.