Dengue viremia in blood donors from Honduras, Brazil, and Australia.

BACKGROUND: Dengue fever and hemorrhagic disease are caused by four dengue virus (DENV) serotypes (DENV-1 to -4), mosquito-borne flaviviruses with increasing incidence, and expanding global distributions. Documented transfusion transmission of West Nile virus raised concern regarding transfusion-transmitted DENV. METHODS: A DENV RNA assay was developed based on transcription-mediated amplification (TMA) blood screening assays routinely used by blood centers worldwide. Sensitivity was established by endpoint dilution analyses of DENV-1 RNA transcript and pedigreed tissue culture standards for all four DENV-serotypes. Frozen plasma samples were tested from 2994 donations from Honduras (September 2004-January 2005), 4858 donations from Brazil (February-April 2003), and 5879 donations from Australia (March-September 2003). Type-specific polymerase chain reaction (PCR) assays were used to quantify and genotype TMA repeat-reactive samples; viral cultures, type-specific antibody, and antigen assays were also performed. RESULTS: The TMA assay detected 14.9 copies per mL DENV-1 transcript (95% detection limit), with comparable sensitivity for all four serotypes. Honduran donors yielded 9 TMA repeat-reactive samples (0.30%); 8 were confirmed by PCR, with 3 DENV serotypes detected and viral loads from fewer than 3 x 10(4) to 4.2 x 10(4) copies per mL; and 4 samples yielded infectious virus. Three (0.06%) Brazilian samples tested repeat-reactive; 2 (0.04%) were PCR-positive (serotypes DENV-1 and -3; 12 and 294 copies/mL). No Australian donor samples tested repeat-reactive. CONCLUSION: Dengue viremia rates among asymptomatic blood donors ranged from 0.30 percent in Honduras to 0.04 percent in Brazil. Future studies are needed to establish rates of transfusion transmission by viremic donations and clinical consequences in recipients.

Dengue virus in blood donations, Puerto Rico, 2005.

BACKGROUND: A single instance of transfusion-transmitted dengue infection has been reported. The high incidence of dengue in endemic countries, the high proportion of asymptomatic infection, and the median 5-day viremia, however, suggest that transfusion-associated dengue transmission may be more widespread than documented. STUDY DESIGN AND METHODS: The prevalence of dengue virus (DENV) RNA was determined in all blood donations to the American Red Cross in Puerto Rico from September 20 to December 4, 2005, using a specific type of nucleic acid amplification test called transcription-mediated amplification (TMA). TMA-positive donations were defined as those having two repeatedly reactive TMA results. TMA-positive donations were tested by enzyme-linked immunosorbent assay for immunoglobulin M (IgM) antibodies, by reverse transcription-polymerase chain reaction (RT-PCR), and by viral culture. RESULTS: Twelve (0.07%) of 16,521 blood donations tested were TMA-positive. Four were positive by RT-PCR (DENV serotypes 2 and 3). Virus was cultured from 3 of 4 RT-PCR-positive donations. One of the 12 TMA-positive donations was IgM-positive. Only 5 donations remained TMA-positive when diluted 1:16, as is done for routine minipool screening for other transfusion-transmissible viral infections (hepatitis C, human immunodeficiency, West Nile viruses [WNVs]). CONCLUSION: Nearly 1 in 1000 blood donations contained DENV RNA, and virus could be cultured from TMA-positive donations, suggesting a transfusion transmission risk similar to that which existed in the United States for WNV before universal donation screening. Similar to WNV, IgM antibody screening is likely to be ineffective, and some potentially infectious donations will be missed by minipool screening. Transfusion transmission should be considered in patients with dengue after blood transfusion.

Performance evaluation of the PROCLEIX West Nile virus assay on semi-automated and automated systems.

The PROCLEIX West Nile virus assay (WNV assay) is a qualitative nucleic acid test based on transcription-mediated amplification (TMA). The assay was used under an investigational protocol in the United States to screen blood donations for West Nile virus (WNV) RNA starting in the summer of 2003, and was licensed by the FDA in December 2005 for use on the PROCLEIX System, also known as the enhanced semi-automated system (eSAS). Performance characteristics for the assay were determined on both eSAS and the fully automated PROCLEIX TIGRIS (TIGRIS) System. Detection of both lineage 1 and lineage 2 WNV was demonstrated on both systems. For lineage 1, the 95% detection limit was 8.2 copies/ml for eSAS and 9.8 copies/ml for the TIGRIS system. For lineage 2, > or =95% detection was seen at > or =30 copies/ml on both systems. The overall specificity of the assay was >99.9% in fresh and frozen plasma specimens. Reproducibility studies on the TIGRIS system yielded > or =99.1% agreement with expected results for the 3-member panel tested (0, 30, and 100 copies/ml). The WNV assay exhibited robust performance in cadaveric specimens and specimens representing various donor and donation conditions, including specimens from different plasma collection tubes that were subjected to multiple freeze/thaw cycles; specimens with elevated levels of endogenous substances; specimens containing other viruses and microorganisms; and specimens from patients with autoimmune and other diseases. Overall, these studies demonstrate high sensitivity, specificity, and reproducibility of the WNV assay on both the semi-automated and automated systems.

Detection of West Nile virus in the Mexican blood supply.

BACKGROUND: West Nile virus (WNV) is the etiologic agent of an emerging disease in the Western Hemisphere that can be transmitted to humans by blood transfusion. WNV first appeared in the United States in 1999, in Canada in 2001, and in Mexico in 2002. The aim of this nationwide study was to determine the prevalence of WNV in blood donors in Mexico as a first step in preventing its transfusion-associated transmission. STUDY DESIGN AND METHODS: In July and August 2004, a total of 3856 fresh plasma specimens collected from each state's center for blood transfusion in 29 of 31 Mexican states were screened with an investigational WNV assay (Procleix,(R) Gen-Probe Inc. and Chiron Corp.), a nucleic acid test based on transcription-mediated amplification (TMA). Reactive specimens were confirmed with a second TMA-based test, the alternative WNV assay (Gen-Probe), and with WNV capture enzyme-linked immunosorbent assays (ELISAs) for detection of immunoglobulin M (IgM) and IgG antibodies. In addition, 3714 frozen plasma samples collected in 2002 and 2003 were similarly tested. RESULTS: One of 3856 fresh samples from an asymptomatic donor from Chihuahua was reactive by both TMA-based tests and IgM ELISA, suggesting a recently acquired infection. The observed percentage of viremic donors blood donors was 0.03 percent. Results from frozen samples were not included in the prevalence calculation and none were TMA-reactive for WNV. CONCLUSIONS: WNV is present in the Mexican blood supply and measures should be taken to reduce the risk of transfusion transmission.

Sensitive detection of genetic variants of HIV-1 and HCV with an HIV-1/HCV assay based on transcription-mediated amplification.

This paper describes a comprehensive study of hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) genotype sensitivity of the transcription-mediated amplification (TMA)-based HIV-1/HCV assay, developed and manufactured by Gen-Probe Incorporated (San Diego, CA) for screening human plasma specimens in blood bank settings. The TMA HIV-1/HCV assay is a qualitative, in vitro nucleic acid testing system used for initial screening. HIV-1 and HCV discriminatory assays are used to distinguish between HIV-1 and HCV infection or co-infection. In this study, multiple unique specimens representing HCV genotypes 1-6 were tested at various dilutions. The results show that the TMA HIV-1/HCV assay and the TMA HCV discriminatory assay have similar HCV genotype sensitivity, as both assays detected all six genotypes at 100 copies/ml and nearly all replicates tested at 30 copies/ml. Similarly, numerous unique specimens representing HIV-1 group M subtypes (A-G), HIV-1 group N, and group O specimens were tested at various dilutions. The TMA HIV-1/HCV assay and the TMA HIV-1 discriminatory assay were found to have similar HIV-1 subtype sensitivity; all variants at 100 copies/ml and nearly all at 30 copies/ml were detected. These results indicate that the TMA assays meet the sensitivity requirements for blood screening in blood banks worldwide.