Human T-lymphotropic virus type III (LAV, HTLV-III) is aetiologically linked to acquired immune deficiency syndrome (AIDS) and persistent general lymphadenopathy (PGL). Specific radioimmunoassays (RIA), enzyme-linked assays, immunofluorescence assays (IFA) and immunoblotting techniques are being used widely to detect serum antibodies to HTLV-III in infected patients and in those at risk of infection. However, these assays do not functionally identify those antibodies that neutralize the infectivity of the virus. We have used three methods of titrating serum neutralizing factors: inhibition of syncytium induction, neutralization of envelope pseudotypes of vesicular stomatitis virus (VSV) and reduction of infectivity of HTLV-III for a cell line permissive to virus replication. We report here that sera from subjects in various disease categories possess only low-level neutralizing activity, even when antibodies to viral membrane antigens are present in high titre. Envelope pseudotypes prepared from four HTLV-III isolates made in three different countries are equally sensitive to neutralization by positive sera, including sera from patients yielding two of the virus isolates.

Urethral and endocervical swabs and self-collected vaginal swabs (SCVSs) and urine specimens are all used as samples for diagnosis of urogenital infection with Chlamydia trachomatis. We have now determined chlamydial organism load in matched specimens from different anatomic sites and examined its relation to clinical signs and symptoms in men and women. Organism load was measured with assays based on the ligase chain reaction or real-time PCR analysis. The mean organism loads in 58 infected men were 1,200 and 821 elementary bodies (EBs) per 100 l of sample for first-void urine (FVU) and urethral swabs, respectively (P > 0.05). Organism load in FVU samples or urethral swabs was positively associated with symptoms (P < 0.01) and clinical signs (P < 0.01) in men. The mean organism loads in 73 infected women were 2,231, 773, 162, and 47 EBs/100 l for endocervical swabs, SCVSs, urethral swabs, and FVU samples, respectively (P < 0.001 for each comparison). Only the presence of multiple symptoms or clinical signs was associated with organism load in women. These results show that FVU is a suitable noninvasive sample type for men, given the fact that its chlamydial load did not differ significantly from that of urethral swabs. Given their higher organism load compared with FVU, SCVSs are the preferred noninvasive sample type for women.Urogenital infection with Chlamydia trachomatis is the most commonly reported bacterial sexually transmitted infection (STI) and continues to be a major public health problem worldwide (4, 35). Given that most chlamydial infections are asymptomatic in both men and women, they often remain undiagnosed and untreated and therefore provide a reservoir for the disease (33). Infection of the upper genital tract may lead to complications, such as epididymitis in men and pelvic inflammatory disease in women. The inflammation and subsequent tissue scarring associated with the latter can lead to more serious sequelae (4).Effective control of chlamydial infection within a population requires early diagnosis and prompt treatment of asymptomatic individuals (28). Targeted and regular screening is also recommended for people in high-risk groups or with a past history of genital chlamydial infection (14). The most common sites of infection in women are the cervix and urethra. Infected cells are shed from the endocervix into the vagina and are present in vaginal secretions. Infected epithelial cells from the urethra and the associated C. trachomatis elementary bodies (EBs) can also be detected in first-void urine (FVU) (3, 16). Potentially suitable clinical specimens for detection of chlamydial infection in women thus include urethral, vaginal, and endocervical swabs, self-inserted tampons, and FVU samples (3, 12). For screening programs, noninvasive specimens, such as vaginal swabs, tampons, and FVU, are preferable to invasive urethral and endocervical swabs because they overcome several barriers associated with the traditional diagnostic pathway (5, 11). Sensitivity of C. trachomatis detection with vag...

BackgroundThe real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories.Principal FindingsHere we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs) enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART) continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi) produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi) produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required.ConclusionsThe combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a simple light detector, the iNAAT-BART combination is ideal for molecular diagnostic assays in both laboratory and low resource settings.

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