Evaluation of HIV serial and parallel serologic testing algorithms in Abidjan, Côte d'Ivoire.

OBJECTIVE: To evaluate HIV serologic testing algorithms based on a combination of three enzyme linked immunosorbent assays (ELISA) for the confirmation of HIV infection in Abidjan, Côte d'Ivoire, where HIV-2 and HIV-1 non-B subtypes are prevalent. METHODS: A total of 1069 human sera with known serologic status, in addition to a seroconversion and low titer antibody panel were initially tested by six ELISA to determine the sensitivity, specificity and delta values of the assays. On the basis of the performance of the assays, three ELISA (Enzygnost, ICE 1.0.2, and Vironostika) were selected for use in a parallel and serial testing algorithm in analyzing 8283 consecutively collected sera. In the parallel testing algorithm, sera concordantly reactive or non-reactive by Enzygnost and ICE 1.0.2 were considered as true positive or true negative, respectively. In the serial algorithm, sera reactive by Enzygnost were retested by ICE 1.0.2. Sera with discordant results were tested by Vironostika, and the results was considered definitive. All reactive sera, plus a random sample of negative sera were tested for confirmation by Peptilav. In addition, a random sample of reactive sera was tested by Western blot. RESULTS: All ELISA had 100% sensitivity; specificities ranged from 96.8 to 100%. Positive and negative delta values of the ELISA were high (range, 6.89 to 46.07 and -2.05 to -5.75, respectively). Of the 8283 sera, 2054 were considered true positives and were correctly classified by the parallel testing algorithm (sensitivity, 100%). Of the 6229 true negative sera, 6226 were negative by the parallel testing algorithm (specificity, 99.95%). The sensitivity of the serial algorithm was 99.96%, and specificity was 99.95%. None of the 250 concordant ELISA-negative sera in the algorithm that were randomly tested in Peptilav was positive; similarly, all of the 103 concordant ELISA-positive sera were confirmed by Western blot. The three-ELISA algorithm resulted in reagent cost-savings of at least 50% compared with the Peptilav-based algorithm. CONCLUSION: These results suggest that a combination of ELISA using different principles or antigens in a serial or parallel algorithm is an efficient and cost-effective alternative to the standard algorithm in areas where HIV-1 and HIV-2 are prevalent.

Comparison of NucliSens and Amplicor monitor assays for quantification of human immunodeficiency virus type 1 (HIV-1) RNA in plasma of persons with HIV-1 subtype A infection in Abidjan, Côte d'Ivoire.

We compared the sensitivity and accuracy of the NucliSens assay and those of both the standard and modified (addition of a new primer set, primer mix 1, supplied by Roche) Amplicor HIV Monitor assays to quantify human immunodeficiency virus type 1 (HIV-1) RNA in persons infected with HIV-1 subtype A in Abidjan, Côte d'Ivoire. Seventy-one plasma samples from HIV-1-seropositive persons at different stages of HIV infection and 15 samples from HIV antibody-negative persons were analyzed. The HIV-1 genetic subtype was determined either by DNA sequencing or by a restriction fragment length polymorphism assay. Of the 71 samples, 70 (98%) were subtype A and 1 was subtype G. Of the 70 subtype A samples, the proportion of RNA-positive plasma samples and mean HIV-1 RNA levels were significantly higher by the modified HIV Monitor assay (n = 67 [96%]; mean RNA levels, 5.2 log10 HIV-1 RNA copies/ml) than the NucliSens assay (n = 56 [80%]; 4.3 log10 HIV-1 RNA copies/ml) or the standard HIV Monitor assay (n = 44 [63%]; mean RNA levels, 3.8 log10 HIV-1 RNA copies/ml) (all P values were <0.05). The HIV-1 RNA levels by the modified HIV Monitor assay correlated significantly with those by the NucliSens assay (r = 0.76; P < 0.001) and the standard HIV Monitor assay (r = 0.57; P < 0.001), as did the RNA levels by the NucliSens and the standard HIV Monitor assays (r = 0.60; P < 0. 001). Lower CD4 cell counts were significantly correlated with higher HIV-1 RNA levels by all three assays (r = -0.47 for the NucliSens assay, -0.45 for the standard HIV Monitor assay, and -0.62 for the modified HIV Monitor assay). These results indicate that the modified HIV Monitor assay has the highest sensitivity and efficiency at quantifying the levels of RNA in persons infected with HIV-1 subtype A and thus constitutes a valuable tool for the monitoring of RNA levels in areas of Africa were HIV-1 subtype A is predominant.

Phenotypic and genotypic characterization of Vibrio cholerae isolates from a recent cholera outbreak in Senegal: comparison with isolates from Guinea-Bissau.

A total of 127 strains of Vibrio cholerae (117 V. cholerae O1 and 10 nonagglutinating strains) isolated from a recent cholera outbreak in Senegal and four strains isolated in Guinea-Bissau (during the survey of a cholera epidemic that occurred 10 months before the Senegalese one) were analyzed. Strains were characterized by conventional methods (biochemical and serologic identification, susceptibility to antimicrobial agents), polymerase chain reaction for genes encoding cholera toxin (CtxA), zonula occludens toxin (Zot), and accessory cholera enterotoxin (Ace), and by ribotyping. Conventional methods showed that all strains of V. cholerae O1 belonged to serotype Ogawa, biotype El Tor and were resistant to the vibriostatic agent O129 (2,4-diamino 6,7-diisopropylpteridine phosphate), cotrimoxazole, and chloramphenicol; all strains were sensitive to tetracycline, a drug that has been extensively used in cholera therapy. Most of these V. cholerae O1 (112 strains from Senegal and four strains from Guinea-Bissau) had an intact core region (virulence cassette) and amplified a 564-basepair (bp) fragment of ctxA, a 1083-bp fragment of zot, and a 314-bp fragment of ace. Ribotyping of V. cholerae O1 strains after Bgl I restriction of total DNA revealed that ribotype B5a, which is the predominant ribotype of this seventh pandemic of cholera, was not isolated. Instead, a new ribotype was identified and designated B27 in our data bank. Since O1 isolates from Guinea-Bissau and Senegal have the same biotype, serotype, and ribotype and as the Guinea-Bissau outbreak that preceded the one in Senegal, this emerging ribotype probably came from Guinea-Bissau. Nonagglutinating strains exhibited no resistance to the O129 agent and to the tested antibiotics, they were all negative for virulence cassette, except for one strain with the ctxA and zot genes isolated from a patient with diarrhea, and there was a great variability of ribotypes among these strains. There was no difference between environmental O1 strains isolated from water and strains isolated from patients with cholera, suggesting that fecally contaminated water is an important reservoir for infection.

Field evaluation of a combination of monospecific enzyme-linked immunosorbent assays for type-specific diagnosis of human immunodeficiency virus type 1 (HIV-1) and HIV-2 infections in HIV-seropositive persons in Abidjan, Ivory Coast.

Serologic distinction between human immunodeficiency virus type 1 (HIV-1) and HIV-2 infection is made difficult because of the cross-reactivity and high cost of existing differentiation assays. An evaluation of a strategy based on a combination of monospecific enzyme-linked immunosorbent assays (ELISAs) (CME), was carried out in Abidjan, Ivory Coast, where both HIV-1 and HIV-2 are present, to determine its accuracy and cost-effectiveness. A total of 1,608 (428 HIV-1-positive, 361 HIV-2-positive, 371 dually HIV-1 and HIV-2 [HIV-D] reactive, and 448 HIV-negative) sera that had been serotyped by a line immunoassay (Peptilav) were tested retrospectively by an HIV-1-monospecific (Wellcozyme HIV Recombinant ELISA) and an HIV-2-monospecific (ICE*-HIV-2) assay. The CME strategy gave concordant results for all of the 428 sera scored as HIV-1 by Peptilav. Of the 361 sera scored as HIV-2 by Peptilav, 316 (87.5%) were scored as HIV-2 by CME; the remaining 45 sera were positive by both monospecific ELISAs (mean optical density ratios, 1.36 for Wellcozyme and 11.30 for ICE*-HIV-2) and were classified as HIV-D by CME. Of the 371 sera classified as HIV-D by Peptilav, 344 (92.7%), 21, and 6 were scored as HIV-D, HIV-1, and HIV-2, respectively, by CME. Additional testing of the discrepant samples by two HIV differentiation assays (RIBA and INNO-LIA) gave results that agreed with those by CME for most of the sera. In addition, 267 other sera were tested prospectively by both CME and Peptilav. In the prospective evaluation, CME results agreed with those by Peptilav for all 106 HIV-1 sera and 40 of the 41 HIV-2 sera. However, of the 120 sera scored as HIV-D by Peptilav, 69 (57.5%), 47 (39.2%), and 4 (3.3%) were scored as HIV-D, HIV-1 only, and HIV-2 only, respectively, by CME. All 47 samples scored as HIV-1 by CME and two of four HIV-2 sera gave concordant results by RIBA, whereas 29 of 47 sera scored as HIV-1 by CME and all four HIV-2 sera gave concordant results by INNO-LIA. The reagent cost for the CME strategy was 59% lower than the cost of the Peptilav strategy. These results suggest that a combination of highly sensitive and specific commercially available monospecific ELISAs is a reliable and cost-effective strategy for type-specific serodiagnosis of HIV-1 and HIV-2 infections in HIV-seropositive persons and therefore represents a recommended strategy in areas where both HIV-1 and HIV-2 are endemic.

Molecular characterization of Vibrio cholerae O1 strains isolated in Romania.

A collection of 89 Vibrio cholerae O1 strains, isolated in Romania between 1977 and 1994, and 6 strains from the Republic of Moldavia, was characterized by ribotyping, toxin gene restriction pattern (toxinogenotype) and distribution of cholera toxin gene (ctx), accessory toxin gene (ace) and zonula occludens toxin gene (zot). After Bg/I endonuclease restriction of chromosomal DNA, a total of 18 ribotypes and 21 toxinogenotypes were distinguished. Deletions in the core region of the toxin gene cassette were found in 20% of strains; however, with the exception of one strain, all the isolates contained the ctx gene. Used in association, the three methods of molecular typing provided an accurate characterization of V. cholerae O1 isolates.

Molecular epidemiology of Vibrio cholerae O1 isolates from Colombia.

A total of 173 Vibrio cholerae O1 isolates from the recent cholera epidemic in Colombia was analysed by the polymerase chain reaction (PCR) for the genes encoding the A subunit of cholera toxin (ctxA) and the zonula occludens toxin (zot), and by ribotyping. All isolates were positive for ctx A and zot, which was confirmed by hybridisation. Ribotyping with restriction endonuclease Bg/I digestion of total DNA revealed three ribotypes: B5a comprising 165 (96.4%) isolates, and two new designated ribotypes B20 and B21a in six (3.5%) isolates and two (1.1%) isolates, respectively. These findings have significant public health implications.

Isolation of a new variant of Vibrio cholerae O1: V. cholerae O1 ribotype B27 toxinogenotype TB31 during the last cholera epidemic in Senegal.

A total of 205 Vibrio cholerae O1 isolates from recent cholera epidemic in Senegal were analyzed by conventional methods, polymerase chain reaction (PCR) for genes encoding cholera toxin (ctx A), zonula occludens toxin (zot) and accessory cholera enterotoxin (ace), ribotyping and toxinogenotyping. Ribotyping after Bg1 I digestion of total DNA revealed that ribotype B5a, the predominant ribotype of the seventh pandemic in Africa and Asia, was not isolated. A new ribotype designated B27 in our database is predominant and was associated with a new toxinogenotype designated TB31.

Restriction fragment length polymorphism of the pMJ101-like plasmid and ribotyping in the fish pathogen Vibrio ordalii.

A total of 32 Vibrio ordalii strains were studied for their plasmid content and shown to carry a plasmid of approximately 32 kb. This plasmid was subsequently subjected to restriction fragment length polymorphism (RFLP) studies. Using Hind III, three different restriction patterns were identified while BamH I cleaved the plasmid into a single linear fragment. The results suggest that the 32 kb plasmid is highly conserved but that some variation in restriction pattern occurs. The same set of strains was subjected to ribotyping. Using Mlu I, six different restriction patterns were demonstrated. Strains from the USA and Canada shared profiles with strains from Australia and Japan. Strains from Australia generated a single pattern whereas strains from North America were subdivided into three patterns, and the Japanese strains fell into five patterns. The results suggest that ribotyping in combination with RFLP studies of the pMJ101-like plasmid may be useful in epidemiological studies of V. ordalii.

Clonal diversity of Vibrio cholerae O1 evidenced by rRNA gene restriction patterns.

The rRNA gene restriction patterns of 89 Vibrio cholerae O1 isolates from different geographic origins were studied. The probe was Escherichia coli 16 + 23S rRNA labelled with "ECL Gene detection system". A total of 17 rRNA gene restriction patterns were observed after BglI cleavage. Four patterns (B1 to B4) were only given by biotype cholerae (14 strains studied). Thirteen patterns (B5 to B17) were only given by biotype El Tor (75 strains studied). There was no correlation between serotypes and rRNA gene restriction patterns. This study provides arguments that (1) strains of biotypes cholerae and El Tor are different clones, (2) a cholera pandemic is not a single world-wide epidemic (due to a single clone) but rather a simultaneous occurrence of several epidemics (several clones involved), and (3) epidemic waves of biotype El Tor could be due to the emergence of new clones.