Combination of reverse transcriptase PCR analysis and immunoglobulin M detection on filter paper blood samples allows diagnostic and epidemiological studies of measles.

As measles control and elimination campaigns progress, laboratory confirmation of clinically diagnosed measles cases becomes increasingly important. However, in many tropical countries collection and storage of clinical specimens for this purpose are logistically complicated. In this study it is shown that blood samples spotted on filter paper are suitable for the laboratory diagnosis of measles using a combination of reverse transcriptase PCR (RT-PCR) analysis and immunoglobulin M (IgM) detection. First, it was shown that in vitro measles virus (MV)-infected cells diluted in human blood and spotted on filter paper can be detected by RT-PCR. Small amounts of infected cells remained detectable after 25 weeks of storage of the filter paper at room temperature, 4 weeks at 37 degrees C, or 2 weeks at 45 degrees C. Subsequently, this RT-PCR was applied to filter paper blood samples collected from 117 clinically diagnosed measles patients in Sudan in 1997 and 1998. Prior laboratory diagnosis had confirmed 90 cases as acute MV infections, while 27 proved to be nonmeasles rash disease cases. Positive RT-PCR signals were detected in filter paper blood samples of 43 of the 90 confirmed cases (48%) but in none of the 27 nonmeasles cases. In addition, MV-specific IgM levels measured in reconstituted filter paper samples correlated well with those measured in plasma samples. Measles diagnosis based on the combination of filter paper RT-PCR and IgM detection had a sensitivity and specificity of 99 and 96%, respectively. An advantage of this diagnostic approach is that sequencing of RT-PCR products allows phylogenetic analysis of the MV strain involved.

Quantitative assessment of hepatitis B virus DNA in chronic hepatitis B: comparison of two solution hybridization assays.

We compared two solution hybridization assays, the AffiProbe assay (Orion Corporation) and the Abbott HBV-DNA assay (Abbott), for quantitative measurement of hepatitis B virus (HBV) DNA in serum samples obtained from chronic hepatitis B surface antigen (HBsAg) carriers. Forty transversally collected (group 1) and 83 serially collected (group 2) serum samples from chronic hepatitis B patients were tested with both assays. The serial serum samples were obtained from 6 patients who underwent alpha-interferon therapy with different outcomes (nonresponse, hepatitis B e antigen [HBeAg] seroconversion, HBeAg and HBsAg seroconversion). In group 1 a good correlation (r = 0.91; P < 0.001) was found between the HBV-DNA results of the two assays. Two samples (5%) were HBV-DNA positive according to the Abbott but negative according to the AffiProbe assay; for all other samples the HBV-DNA status corresponded. In group 2 the assays gave colinear HBV-DNA results during follow-up of 5 of the 6 patients (correlation for the total group: r = 0.90; P < 0.001). Nevertheless, in both groups the AffiProbe assay yielded about 5-10 times higher HBV-DNA levels than the Abbott HBV-DNA assay (P < 0.001). These discordant results were most probably due to standardization differences of the positive control samples of the two test systems. This observation underlines the need for international standardization of HBV-DNA and uniform reference panels.

HBV-DNA detection by gene amplification in acute hepatitis B.

Serum samples from 62 women, inadvertently infected with hepatitis B virus in an in vitro fertilization program, were tested for the presence of hepatitis B virus-DNA using the polymerase chain reaction. Under conditions of a strict spatial separation of DNA extraction, amplification and product analysis, we succeeded in detection of as few as 360 hepatitis B virus particles per milliliter. Hepatitis B virus-DNA was detected with a high frequency during HBsAg and HBeAg antigenemia (98.5%) but also in the convalescent phase after appearance of antibody to HBsAg (18.2%). However, all patients with hepatitis B virus-DNA in convalescent sera were hepatitis B virus-DNA negative 3 to 6 mo later. All patients with HBeAg-positive samples showed hepatitis B virus-DNA positivity by polymerase chain reaction. For acute hepatitis, gene amplification restores the relationship between HBeAg and hepatitis B virus-DNA observed in serum from chronic hepatitis B patients and calls attention to the prolonged presence of hepatitis B virus-DNA in serum after generally accepted criteria for resolution of the infection have been reached.