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ABSTRACTWe have developed a novel multiplex reverse transcription-PCR ligase detection reaction (RT-PCR/LDR) assay for the detection of West Nile virus (WNV) in both clinical and mosquito pool samples. The method relies on the amplification of three different genomic regions, one in the coding sequence of nonstructural protein NS2a and two in nonstructural protein NS5, to minimize the risk of detection failure due to genetic variation. The sensitivity of the PCR is complemented by the high specificity of the LDR step, and the detection of the LDR products can be achieved with capillary electrophoresis (CE) or a universal DNA microarray. We evaluated the limit of detection by both one-step and two-step multiplex RT-PCR/LDR/CE approaches, which reached, respectively, 0.005 and 0.017 PFU. The assay demonstrated 99% sensitivity when mosquito pool samples were tested and 100% sensitivity with clinical samples when the one-step approach was used. The broad strain coverage was confirmed by testing 34 WNV isolates belonging to lineages 1 and 2, and the high specificity of the assay was determined by testing other flaviviruses, as well as negative mosquito pool and clinical samples. In summary, the multiplex RT-PCR/LDR assay could represent a valuable complement to WNV serological diagnosis, especially in early symptomatic patients. In addition, the multiplexing capacity of the technique, which can be coupled to universal DNA microarray detection, makes it an amenable tool to develop a more comprehensive assay for viral pathogens.

The accuracy and acceptability of saliva human immunodeficiency virus type 1 (HIV-1) antibody testing were compared with serum testing in a study of paired specimens from HIV-1-seropositive and HIV-1-seronegative Ugandan adults attending a clinic for sexually transmitted diseases. Saliva collection was performed with the Omni-sal device (Saliva Diagnostic Systems, Vancouver, Wash.), and antibody testing was performed by a rapid filter paper assay (Test-Pack; Abbott Laboratories, Abbott Park, Ill.). Relative to serum testing, the sensitivity of saliva testing was 95% (195 of 205) and the specificity was 99% (295 of 297). The sensitivity of saliva testing was higher for patients with elevated levels of beta-2 microglobulin in sera and greater numbers of HIV-1-related symptoms. Pre- and poststudy interviews indicated that saliva testing did not foster inordinate fears of saliva exposure. The development of saliva tests that are inexpensive and do not require electricity is needed.

Sequential plasma samples obtained from 16 individuals who seroconverted were tested for the presence of antibody to human immunodeficiency virus type 1 (HIV-1) by an antigen conjugate enzyme immunoassay (EIA) and a conventional antibody conjugate assay. In 11 of these individuals, the antigen conjugate assay detected antibody to HIV-1 2 to 11 days (mean, 5.5 days) earlier than the antibody conjugate assay. In 11 individuals, HIV-1 p24 antigen was detected a median of 6.5 days (range, 3 to 14 days) prior to positivity by the antigen conjugate EIA. Using class-specific probes, we determined the profiles of immunoglobulin M (IgM), IgG, and IgA antibodies for each individual and correlated these profiles with the EIA signals from both assays. In general, the appearance of IgM exhibited a peak at about 1 week postseroconversion, which was followed by gradually declining levels. Absorbance levels for IgG antibody, however, rose steadily and reached a plateau after 3 to 5 weeks. The levels of IgA were generally low and variable. In contrast to the progressive increase in EIA absorbance observed by the antibody conjugate assay, the antigen conjugate assay displayed a rapid early rise in absorbance which generally coincided with the transient expression of IgM antibody. The subsequent gradual increase coincided with rising levels of IgG. Because the configuration of the antigen conjugate EIA allows for an increased sensitivity for IgM compared with that for other classes of immunoglobulins, these results suggest that earlier detection of antibody to HIV-1 is due to the detection of IgM antibody during the early phase of seroconversion.