Improvement of an ultrasensitive human immunodeficiency virus type 1 real-time reverse transcriptase-polymerase chain reaction targeting the long terminal repeat region.

BACKGROUND: Human immunodeficiency virus Type 1 (HIV-1) assays applying nucleic acid testing (NAT) rely on HIV-1 sequence-specific primers and probes. Their hybridization can be limited or abolished by genetic polymorphisms occurring in the target sequence. STUDY DESIGN AND METHODS: Blood donations are routinely tested for HIV-1/2 antibodies and for HIV-1 RNA in our blood transfusion unit. Recently, HIV-1 RNA was undetectable with an established in-house real-time long terminal repeat (LTR) reverse transcriptase-polymerase chain reaction (RT-PCR) in two cases, whereas serologic assays were positive. The reason for this discrepancy was elucidated by sequencing of the NAT target region in the respective single donations. An improved primer was designed and tested on HIV-1 reference panels and blood donations to ensure reliable detection of HIV-1 RNA. RESULTS: Direct sequencing of the target region, isolated from samples of two unrelated HIV-positive blood donors, revealed one and four mismatches in the hybridization domain of the forward primer, respectively. Both viruses belong to HIV-1 Subtype B. LTR RT-PCR with an additional forward primer was suitable for all strains of HIV-1 tested with high sensitivity. CONCLUSIONS: Surveillance of HIV-1 genetic diversity is essentially required to continually evaluate its impact on performance of diagnostic and patient monitoring assays.

Fatal outcome of hepatitis A virus (HAV) infection in a traveler with incomplete HAV vaccination and evidence of Rift Valley Fever virus infection.

Hepatitis A virus (HAV) infection is rarely fatal except in patients with chronic liver disease. In the case reported here, an elderly women died of HAV infection 12 years after incomplete HAV vaccination. The possible role of a concordant Rift Valley fever virus infection acquired in Kenya is discussed.

Hantavirus infections by Puumala or Dobrava-Belgrade virus in pregnant women.

BACKGROUND: Hantavirus infection in humans usually occurs via inhalation of infectious aerosolized excreta of rodents. Horizontal human-to-human transmission was reported only for the highly virulent Andes virus. The likelihood of vertical transmission and the clinical outcome of hantavirus infections in pregnancy is still unpredictable. OBJECTIVES: Very few data were published about the impact of hantaviruses in pregnancy. Here we present four cases of pregnant women infected by European hantaviruses. The risk of vertical virus transmission was investigated. STUDY DESIGN: Four pregnant women with clinical signs of acute hantavirus disease were investigated for hantavirus IgM and IgG after onset of clinical symptoms. Furthermore, the newborns were tested for presence of viral RNA and antibodies in cord blood and, if any parameter was found positive, 8-12 months after delivery. RESULTS: Four women suffered from a hantavirus infection, two of them due to infection by Puumala virus and two by Dobrava-Belgrade virus. Three women delivered healthy babies by vaginal route and one woman by Caesarean section (week 28). In no case hantavirus RNA was detected in cord blood after delivery or in the 8-12 month old babies. Hantavirus IgG was detectable in the cord blood of 3 babies (but not in the preterm child); these antibodies disappeared after 8-12 months indicating a passive transfer of immunoglobulins. No child had any clinical sign of hantavirus infection. CONCLUSIONS: In this study, the absence of vertical hantavirus transmission was demonstrated for pregnant women with onset of hantavirus disease between gestation weeks 14 and 28.

Standardized peptidome profiling of human urine by magnetic bead separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

BACKGROUND: Peptidome profiling of human urine is a promising tool to identify novel disease-associated biomarkers; however, a wide range of preanalytical variables influence the results of peptidome analysis. Our aim was to develop a standardized protocol for reproducible urine peptidome profiling by means of magnetic bead (MB) separation followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). METHODS: MBs with defined surface functionalities (hydrophobic interaction, cation exchange, and metal ion affinity) were used for peptide fractionation of urine. Mass accuracy and imprecision were calculated for 9 characteristic mass signals (M(r), 1000-10,000). Exogenous variables (instrument performance, urine sampling/storage conditions, freezing conditions, and freeze-thaw cycles) and endogenous variables (pH, urine salt and protein concentrations, and blood and bacteria interferences) were investigated with urine samples from 10 male and 10 female volunteers. RESULTS: We detected 427 different mass signals in the urine of healthy donors. Within- and between-day imprecision in relative signal intensities ranged from 1% to 14% and from 4% to 16%, respectively. Weak cation-exchange and metal ion affinity MB preparations required adjustment of the urinary pH to 7. Storage time, storage temperature, the number of freeze-thaw cycles, and bacterial and blood contamination significantly influenced urine peptide patterns. Individual urine peptide patterns differed significantly within and between days. This imprecision was diminished by normalization to a urinary protein content of 3.5 microg. CONCLUSION: This reliable pretreatment protocol allows standardization of preanalytical modalities and facilitates reproducible peptidome profiling of human urine by means of MB separation in combination with MALDI-TOF MS.