Kerstin Malm

Prevalence of human T-lymphotropic virus type 1 and 2 infection in Sweden

Background: Prevalence data on human T-lymphotropic virus types 1 and 2 (HTLV-1/2) in Sweden have not been updated since 1995. The seroprevalence among blood donors at that time was 0.2/10,000. A few years earlier, a high prevalence of HTLV-2 was found in intravenous drug users (IDUs) in Stockholm (3.4%). The objective of this study was to update information on the seroprevalence of HTLV in several study groups. Methods: Serum samples from pregnant women, hepatitis C virus (HCV)-positive individuals, and IDUs in Stockholm were investigated for HTLV-1/2 antibodies. Data from the mandatory HTLV-1/2 screening (2003–2006) of in vitro fertilization (IVF) clients were compiled, as well as data from new blood donors. Results: Eight out of 35,000 IVF patients were positive for anti-HTLV-1/2 (seroprevalence 2.3 per 10,000). Of the anti-HCV-positive individuals (n = 355), 1 sample was HTLV-1-positive (28.2 per 10,000). From 1995 to 2007, 18 HTLV-positive new blood donors were identified out of approximately 550,000 individuals tested (0.3 per 10,000). Thirty-five of 1079 tested IDUs were screening reactive. Conclusions: Since the start of screening in 1994, there has been no increased seroprevalence of HTLV-1/2 among blood donors in Sweden. Seroprevalence among Swedish IVF patients is 10 times higher than among blood donors. This finding is comparable to a 2003 European seroprevalence study of pregnant women in 7 countries. However, the possibility that the IVF group includes individuals with a higher risk of acquiring sexually transmitted infections, including HTLV, than the general population cannot be ruled out.

Evaluation of a New Screening Assay for HTLV-1 and -2 Antibodies for Large-Scale Use

Laboratory testing for Human T‐lymphotropic Virus type 1 and 2 (HTLV‐1 and ‐2) infections has become routine in blood transfusion, tissue transplantation and clinical diagnoses in many countries worldwide. Screening is usually based on the detection of antibodies to HTLV‐1 and/ or ‐2. The number of commercially available assays is limited, and among them, ELISA tests based on microtiter format are most commonly used. Recently, the new rHTLV‐I/II assay (Abbott Laboratories, Abbott Park, IL) was released; this assay was developed for an automatic large‐scale screening platform. This assay was evaluated using pre‐characterized serum panels and routine samples from the clinical laboratory. The sensitivity was 100% for HTLV‐1 and ‐2 (99/99 and 42/42, respectively, including one sample that was dually reactive, HTLV‐1 + 2). To test assay specificity, panels of blood donor sera, specimens from patients with autoimmune diseases and some viral infections were used. False‐reactive samples from previous HTLV diagnoses were also included. With these panels, the specificity was 99.4% (619/623). However, the four false‐reactive samples all belonged to the group of samples that were previously considered as false‐reactive for HTLV‐antibodies. All other samples were negative by the rHTLV‐I/II assay, and thus 100% specificity was obtained. The 1,412 samples tested in the clinic by this assay in routine use were all negative (100% specificity). Taken together, the overall specificity was 99.8%. The assay was sensitive, specific and appropriate for the large‐scale screening of samples for HTLV‐1/2 antibodies. J. Med. Virol. 82:1606–1611, 2010.

Analytical evaluation of nine serological assays for diagnosis of syphilis

The diagnosis of syphilis is most frequently dependent on antibody detection with serological assays. Assays for both treponemal and non‐treponemal antibodies are needed to provide a sensitive and specific diagnosis. For decades, a first screening has been done with non‐treponemal assays, followed by treponemal. However, in recent years, following laboratory automation, the reverse sequence screening algorithms have been developed, using a treponemal assay as the initial screening test.

Performance of Liaison XL automated immunoassay platform for blood-borne infection screening on hepatitis B, hepatitis C, HIV 1/2, HTLV 1/2 and Treponema pallidum serological markers.

Aim of the study was to evaluate performance of a new fully automated platform, DiaSorin‐LIAISON® XL (DiaSorin S.p.A, Vercelli, Italy), in blood donor screening, specifically for hepatitis B surface antigen (HBsAg), hepatitis B core antibodies (anti‐HBc), hepatitis C antibodies (anti‐HCV), HIV p24 antigen, HIV antibodies, human T‐lymphotropic virus types 1 and 2 (HTLV‐1/2) and Treponema pallidum antibodies.

Droplet digital PCR for absolute quantification of proviral load of human T-cell lymphotropic virus (HTLV) types 1 and 2

BACKGROUND Human T-lymphotrophic virus (HTLV) types 1 and 2 cause lifelong infection whereby most infected individuals are asymptomatic whilst a minority develop infection-related disease. These latter patients invariably have been found to have high proviral load (PVL). Therefore, infected patients are monitored by determining the proportion of lymphocytes that are infected with HTLV-1/2. An increase in PVL has been shown to represent an increasing risk of developing HTLV-associated diseases. Monitoring of PVL requires a reliable and sensitive method. In this study assays based on droplet digital PCR (ddPCR) were established and evaluated for detection and quantification of HTLV-1/2. OBJECTIVES To develop two parallel assays to detect the tax genes and determine the PVL of HTLV-1 and -2. STUDY DESIGN Sixty-seven clinical samples from patients infected with HTLV-1 or HTLV-2 were analysed. The samples had previously been analysed with a qPCR and a comparison between ddPCR and qPCR was performed. The specificity of the assays were determined by analyzing samples from 20 healthy blood donors. RESULTS The ddPCR was a stable and sensitive method for detection and quantification of HTLV-1 and -2. When comparing the qPCR and ddPCR the correlation was high (Pearsons correlation coefficient 0.96). The variability of the ddPCR was very low with intra-assay coefficient of variation (CV) of 0.97-3.3% (HTLV-1) and 1.7-8.2% (HTLV-2) and inter-assay CV of 1.8-6.1% (HTLV-1) and 1.2-12.9% (HTLV-2). CONCLUSIONS The ddPCR reliably quantified HTLV DNA in clinical samples and could be a useful tool for monitoring of PVLs in HTLV-infected individuals.

Evaluation of the Veris MDx™ system for quantification of Hepatitis B DNA and Hepatitis C and HIV-1 RNA in a medium sized University Hospital

Introduction: In the diagnosis and treatment of Hepatitis B (HBV), Hepatitis C (HCV) and HIV, it is crucial to detect and quantify viral nucleic acid. Patients on therapy are monitored continuously ...

Droplet Digital PCR for absolute quantification and determination of proviral load of HTLV-1 and 2

HTLV proviral load in PBMCs is one of the few laboratory parameters used for clinical follow up and assessment of development of symptomatic disease associated with HTLV infection. High levels of proviral load are associated with symptomatic disease (e.g. ATLL, HAM/TSP) and occurance of higher levels among asymptomatic individuals has been suggested prognostic for development of symptomatic HTLV disease. Commonly, standard PCR methods including standard curves have been used. Droplet Digital PCR (ddPCR) is a new method that enables absolute quantification of DNA and RNA without the use of a standard curve. In ddPCR, the PCR sample is partitioned into 20 0 nanoliter-sized droplets prior to PCR and based on the number of positive droplets and Poisson algorithms the concentration of the sample can be determined. We have established and evaluated an assay for identification, quantification and determination of PVL of HTLV-1 and 2 in PBMC using ddPCR. For determination of PVL, the human gene RPP30 was quantified and used as reference [1]. The samples were extracted using the QIAamp DNA Blood Mini kit on the QIAcube (Qiagen) prior to ddPCR. Sixty HTLV-1 – positive (PVL 0.01%-59.8%) and 12 HTLV-2 – positive samples (PVL 0.02%-11.3%) previously analyzed with real-time PCR at Imperial College, St Marys Hospital, London (Prof Graham Taylor) as well as 20 negative samples and dilution series of both HTLV-1 and 2 were examined. The results of ddPCR were in concordance with real-time-PCR, however a few weak positive samples were only detected by one of the methods, see Figure 1. The detection levels of the ddPCR were determined to 1-2 copies per PCR reaction for both HTLV-1 and 2. Both the intra- and inter-assay variability of ddPCR was low, 0.97-3.34% and 1.84-6.13% respectively. In conclusion, ddPCR allows for reliable and sensitive determination of PVL for both HTLV-1 and 2.

P5.087 Evaluation of the Multiplex AmpliSens HCV/HBV/HIV-FRT Real-Time PCR For Simultaneous Qualitative Detection of Hepatitis C RNA, Hepatitis B DNA and HIV RNA

Background Human donors of tissues and organs are obliged to undergo analysis for several blood transmitted infections. Serological assays are used, but for ideal sensitivity particularly for early infections these assays are beneficially supplemented with a nucleic acid amplification test (NAAT). For this as well as other diagnostic purposes, we have evaluated the multiplex AmpliSens HCV/HBV/HIV-FRT real-time PCR for simultaneous qualitative detection of HCV RNA, HBV DNA and HIV RNA in clinical plasma samples. Methods Clinical plasma samples with known concentrations (according to viral load assays from Roche Diagnostics) of HCV (n = 34; range: 25 – 4.9×106 IU/mL), HBV (n = 30; 20 – 7.6×104 IU/mL) and HIV (n = 32; 34 – 4.7×105 c/mL); and samples from virus-negative blood donors (n = 100) were tested. Nucleic acid was isolated from 1 mL plasma on the MagNA Pure Compact using its Total Nucleic Acid Isolation kit I-Large Volume (Roche Diagnostics). The multiplex AmpliSens HCV/HBV/HIV-FRT real-time PCR (Central Research Institute of Epidemiology, Moscow, Russia) was run on a Rotor-Gene Q PCR instrument (Qiagen). Results To date, 96 samples with various viral loads of HCV (n = 34), HBV (n = 30) and HIV (n = 32), have been analysed. Only three samples with very low concentrations of HCV (< 25–59 IU/mL) were false negative, and no false positive samples have been found. Complete data of the study will be presented at the meeting. Conclusion The multiplex AmpliSens HCV/HBV/HIV-FRT real-time PCR proved to be highly sensitive and specific. Accordingly, this rapid, technically simple and low cost assay might be effectively used for screening of human donors as well as for other diagnostic purposes

HTLV-2 infection still prevalent among older injecting drug users in Stockholm, Sweden – indications of limited spread to the younger generation

Results Among 1079 investigated subjects, 35 were found to be positive for antibodies to HTLV-1 and/or HTLV-2, giving an overall HTLV-prevalence of 3.2%. Of these, 2 (0.2%) had antibodies to HTLV-1, 28 to HTLV-2 (2.6%) and 5 (0.5%) had non-typeable antibodies to HTLV. The overall study group had similar age and sex distribution as in 1995. However, the HTLV-positive individuals were 10 years older than the HTLV-negative group (mean age) and compared to the cases studied in 1995.