Storage and Transportation of "HIV" RNA in Plasma Samples up to 45°C in a Lyophilized Stabilizer.

BACKGROUND: Blood or plasma samples from rural areas are often transported under suboptimal conditions to central laboratories. The negative influence of different storage temperatures during transportation as well as long transportation times on the stability of unprotected HIV RNA is well known. Therefore, the correct and reliable quantification of HIV RNA might be very difficult. A stabilization solution for the storage and transportation of plasma samples was developed which stabilizes RNA for seven days up to 45°C without viral load changes. METHODS: Blood samples from HIV positive individuals were collected into EDTA containing tubes. The isolated plasma samples in Germany were pipetted into pre-prepared RNA stabilization tubes and incubated for seven days at 45°C. HIV-1 RNA quantification was performed on a HIV-1 LCx m 2000 system from Abbott and a Qiagen/Artus HI Virus-1 RG RT-PCR Kit on a Rotor-Gene Q PCR machine. In addition, plasma samples were collected and tested using existing SOP for storage and transportation in Nigeria. Plasma samples were treated with and without stabilization solution and the AMPLICOR HIV-1 MONITORTM test was used to determine viral load. RESULTS: Seventy-four stabilized plasma samples were tested in Germany and results were compared to those tested unprotected within two hours. No significant changes of viral load were detected up to seven days and 45°C in case of stabilized samples. In contrast RNA of the same unprotected samples was no longer detectable after one day at 45°C. Additionally, 22 plasma samples were investigated on day zero and under field conditions in Nigeria without changes of the viral load after seven days under given temperature conditions. CONCLUSIONS: No cooling chain is necessary for the storage and/or transportation of plasma samples treated with the new RNA stabilization solution for up to seven days. The use of this solution to preserve plasma RNA will be very helpful in countries where the environmental temperature is higher than 30°C, thus addressing the problem of unreliable viral load results due to suboptimal storage or transportation conditions. Further, the costs of storage and transportation of samples for viral load quantification could be significantly reduced.

A new triplex real time PCR which distinguishes between MRSA, MSSA, and mecA coagulase negative strains by means of melting point analysis using SYTO 9.

BACKGROUND: A new screening method was developed for the detection and identification of methicillin resistant staphylococcus aureus (MRSA) from sterile sites or mixed flora samples (inguinal or nose swabs). METHODS: After rapid treatment of samples, the method consists of two steps, template DNA preparation by a simple and rapid boiling procedure and a multiplex real time PCR. The triplex PCR system simultaneously detects the following targets, (i) the integration site for the open reading frame X (orfX) of the staphylococcal cassette chromosome mec type I - V (SCCmec), (ii) the mecA gene which codes for the penicillin-binding protein PBP-2a, and (iii) an internal control (IC) which can be amplified with the SCCmec primer system. A new buffer system, which contains the fluorescent dye SYTO 9, allows a reproducible real time PCR for the discrimination of the above mentioned PCR products by means of a high resolution melting point analysis (HRM). RESULTS: This new PCR system distinguishes between MRSA, MSSA, and mecA positive but coagulase-negative staphylococci (CoNS) strains. An internal control confirms the integrity of the PCR run. The HRM shows three melting points specific for each amplification product. 78.75 degrees C for the mecA gene, 83.15 degrees C for the SCCmec/orfX fragment and 88.25 degrees C for the internal control. CONCLUSIONS: This new multilocus MRSA PCR system is a fast and inexpensive alternative to commercially available test systems and costs only five to six euros.

An improved PCR method for detection of HIV-1 proviral DNA of a wide range of subtypes and recombinant forms circulating globally.

Proviral DNAs are being measured increasingly as a marker of the efficacy of highly active anti-retroviral therapy (HAART) and is accepted for the early diagnosis of perinatal HIV-1 infections. This requires a standardized test which enables the detection of a wide range of subtypes worldwide including O, N and circulating recombinant forms (CRFs). Based on a previous publication, a PCR - Test for HIV-1 provirus detection in peripheral blood mononuclear cells (PBMCs) was developed. Blood samples from 80 individuals infected with HIV-1 and 20 persons negative for HIV-1&2 from Africa and Germany were tested for the presence of HIV-1 provirus DNA. The primer system used enables the detection of proviral DNA despite the high concentrations of human DNA. The limit of detection was determined to be 5 copies per 10(5) cells. All 20 samples from persons negative for HIV were negative for HIV-1 proviral DNA while provirus DNA was amplified from 76 of the 80 (95%) samples from persons infected with HIV. The amplified products were detected by gel-electrophoresis, flow cytometry and real-time PCR. All three detection systems provided the same results.

A new affordable flow cytometry based method to measure HIV-1 viral load.

Most commercially available assays for diagnosis of HIV infection have shown shortcomings in the detection and quantification of rare genotypes of the virus. Most of the assays do not detect subtype O (outlier) and/or N (nonmajor, nonoutlier) or new circulating recombinant forms (CRFs), which are becoming more important in sub-Saharan Africa. Furthermore, the commonly available tests require costly measuring devices and expensive test kits, which are not easily affordable for developing countries. This study was designed to explore solutions to the problem of viral load assays in developing countries. Two forward primers, digoxygenin (DIG) and dinitrophenol (DNP) labeled, and one biotin (BIO) labeled reverse primer were used to amplify both, the HIV-1-5'LTR (long terminal repeat) region and an internal standard sequence. The two polymerase chain reaction (PCR)-products were captured by anti-DIG and anti-DNP antibody coated microparticles. Flow cytometric analyses were carried out after labeling with streptavidin-R-phycoerythrine. The primer system used recognized all HIV-1 subtypes. A coamplified internal standard warranted the functionality of the PCR and allows reproducible viral load measurements. Two drawbacks of current viral load measurements are overcome by the flow cytometry based test described hereof. First, all known worldwide relevant HIV-1 subtypes including subtypes O, N, and new CRFs are quantifiable with high sensitivity (50 to >1 x 10(6) copies per PCR). Second, the cost per test can be reduced to less than 12 US$ instead of the current 50-100 US$. Additionally, the test described in this report offers the possibility to perform complete monitoring program (CD4 T-cell count, CD4% and viral load) for the first time, with the same device for HIV-infected persons.