Minimal infectious dose and dynamics of Babesia microti parasitemia in a murine model.

BACKGROUND: Babesia microti is a parasite that infects red blood cells (RBCs) in mammals. It is transmitted to humans by tick bites, transfusion, organ transplantation, and congenital acquisition. Although the Babesia natural history and seroprevalence in donors have been well described, gaps in knowledge relevant to transfusion remain. STUDY DESIGN AND METHODS: Mice were infected with dilutions of parasitized blood to address the minimal infectious dose and the kinetics of parasitemia by quantitative polymerase chain reaction (qPCR) and of antibodies by enzyme immunoassay. RESULTS: In immunocompetent DBA/2 mice infected with 100 parasitized RBCs (pRBCs) and in immunodeficient NSG mice infected with 63 pRBCs, parasitemia was detectable in five of five mice each. Peak parasitemia up to 2 × 107 pRBCs/mL at 2 to 3 weeks or 5 × 108 pRBCs/mL at 6 weeks was observed for DBA/2 and NSG mice, respectively. Protracted fluctuating parasitemia was observed for 8 months in DBA/2 mice, whereas NSG mice exhibited a high-plateau parasitemia. Antibody titers continued to increase until 6 to 18 weeks in DBA/2 mice and remained high through 6 months. This study also investigated the analytical performance of Babesia assays that detect parasite DNA or RNA using a blinded panel. A Babesia assay targeting parasite RNA was approximately 10-fold more sensitive compared to qPCR targeting DNA. CONCLUSION: The mice in this study were highly susceptible to Babesia infection using as few as 1 to 2 log pRBCs and maintained chronic parasitemia. If the infectious dose in human transfusion recipients is comparably low, a highly sensitive assay targeting parasite RNA may safeguard the blood supply, particularly before antibody detection.

Assessment of nucleic acid modification induced by amotosalen and ultraviolet A light treatment of platelets and plasma using real-time polymerase chain reaction amplification of variable length fragments of mitochondrial DNA.

BACKGROUND: Pathogen inactivation methods are increasingly used to reduce the risk of infections after transfusion of blood products. Photochemical treatment (PCT) of platelets (PLTs) and plasma with amotosalen and ultraviolet A (UVA) light inactivates pathogens and white blood cells through formation of adducts between amotosalen and nucleic acid that block replication, transcription, and translation. The same adducts block the amplification of nucleic acids using polymerase chain reaction (PCR) in a manner that correlates with the number of adducts formed, providing a direct quality control (QC). Current QC measures for PCT rely on indirect methods that measure the delivered UVA dose or percent residual amotosalen after illumination, rather than directly measuring nucleic acid modification. STUDY DESIGN AND METHODS: Endogenous mitochondrial DNA (mtDNA), which is detectable in PLT and plasma units, was chosen as a target for the quantification of photochemically induced modifications. DNA was extracted from untreated or amotosalen and UVA-treated PLTs or plasma, and mtDNA fragments of variable lengths were quantified using a real-time PCR inhibition assay. RESULTS: PCT induced increasing real-time PCR inhibition of mtDNA amplification for larger amplicon sizes. Amplification was unaffected by treatment with amotosalen or UVA alone, whereas up to 3 log inhibition was observed after PCT. Blinded PCR testing of a panel of 110 samples each, from PLT or plasma components prepared for routine use within a blood center, allowed 100% discrimination between untreated and treated units. CONCLUSION: Our initial findings indicate that an adequately sensitive, quantitative real-time PCR inhibition assay targeting mtDNA could provide a valuable tool to confirm and monitor PCT.

Quantitation of HTLV-I and II proviral load using real-time quantitative PCR with SYBR Green chemistry.

BACKGROUND: Human T-lymphotropic virus type I (HTLV-I) is linked etiologically with adult T cell leukemia/lymphoma and HTLV-I-associated myelopathy/tropical spastic paraparsis (HAM/TSP). Human T-lymphotropic virus type II (HTLV-II) is associated with HAM/TSP and, in HIV coinfected patients only, rare cases of cutaneous T cell lymphoma. Proviral load may be important in the pathogenesis of HTLV-associated disease. MATERIALS AND METHODS: A real time quantitative PCR assay using SYBR Green intercalation was established. Primers targeting the tax region were standardized against MT2 and MOT cell line DNA for HTLV-I and HTLV-II, respectively. HTLV-I/II copy number was normalized to the amount of cellular DNA by quantitation of the HLA-DQ alpha gene. We measured proviral load in peripheral blood mononuclear cells (PBMCs) in a large cohort of 120 HTLV-I and 335 HTLV-II seropositive former blood donors. We also assessed the intra- and inter-assay reproducibility of the assay. RESULTS: Proviral load for HTLV-I infected patients ranged from 3.1 x 10(0) to 1.8 x 10(5)copies/10(6) PBMCs with a mean of 1.6 x 10(4) and a median of 3.0 x 10(3). HTLV-I was undetectable in 7 of 120 cases (5.8%). Proviral load for HTLV-II infected patients ranged from 1.1 x 10(0) to 1.0 x 10(6)copies/10(6) PBMCs with a mean of 2.8 x 10(4) and a median of 5.0 x 10(2). HTLV-II was undetectable in 31 out of 335 cases (9.3%). CONCLUSION: The assay has excellent dynamic range from 10(6) to 10(0)copies/reaction, good intra- and inter-assay reproducibility, and a lower limit of detection of a single copy per reaction. The sensitivity and high dynamic range allow determination of a broad range of HTLV-I/II proviral load in clinical subjects. This assay will facilitate the study of the relationship between proviral load and pathogenesis.

The role of transplacental microtransfusions of maternal lymphocytes in in utero HIV transmission.

BACKGROUND: The mechanisms of HIV transmission from mothers to infants are poorly understood. A possible mechanism of in utero transmission is transplacental transfer of HIV-infected maternal leukocytes into the fetal circulation during pregnancy. OBJECTIVE: To determine if the frequency of in utero HIV infection correlates with presence or levels of maternal cells (MCs) in placenta-derived cord blood. METHODS: DNA was extracted from dried cord blood spots (DBS) from newborns born to HIV+ mothers and corresponding maternal DBS specimens. Paired mother-infant samples were probed to identify unique maternal sequences targeted by 24 allele-specific real-time polymerase chain reaction assays. Infant DBS-derived DNA was then probed in replicate analyses for noninherited maternal allelic sequences. Rates of detection and levels of MCs in DBS samples of HIV(+) and HIV(-) newborns were compared. RESULTS: Of 114 mother-infant pairs with informative alleles, 38 newborns were HIV(+) and 76 HIV(-), based on detection of HIV DNA/RNA at birth. MC were detected in 23 of 38 HIV(+) newborns (60.5%) and in 47 of 76 HIV(-) newborns (61.8%). The mean and median concentrations of nucleated MCs in DBS for the HIV(+)/MC(+) newborns (n = 23) were 0.33% and 0.27%, respectively, compared with 0.09% and 0.10% for the HIV(-)/MC(+) newborns (n = 47) (2-sample T test for means: P = 0.78). CONCLUSIONS: There was no significant difference in rates of detection or concentrations of MC in DBS between HIV(+) and HIV(-) newborns. Therefore, we could not demonstrate a correlation between MC in DBS, assumed to reflect levels of in utero maternal-fetal cell trafficking, and the risk of in utero HIV transmission.

Microchimerism decades after transfusion among combat-injured US veterans from the Vietnam, Korean, and World War II conflicts.

BACKGROUND: Blood transfusion after traumatic injury can result in microchimerism (MC) of donor white cells (WBCs) in the recipient as late as 2 to 3 years postinjury, the longest prospective follow-up to date. The purpose of this study was to determine how long transfusion-associated MC lasts after traumatic injury. STUDY DESIGN AND METHODS: A group of US combat veterans who received transfusions who responded to a recruitment notice was retrospectively evaluated. Their blood was sampled, and MC was assessed by quantitative allele-specific polymerase chain reaction detection of differences at the HLA-DR locus or a panel of insertion-deletion polymorphism loci. Results of veterans were compared to those from an age- and gender-matched blood donor control group, from whom WBCs were retrieved from leukoreduction filters. RESULTS: Among 163 combat veterans who received transfusion and 150 control subjects who did not receive transfusions, 16 (9.8%) of the veterans and 1 (0.7%) control subject had evidence of MC (relative risk, 14.7; 95% confidence interval, 2.0-110). The veterans with MC included 3 who served in WWII (7% of subjects from that conflict), 5 in Korea (18%), and 6 in Vietnam (7%). CONCLUSIONS: Transfusion for combat-related injury can result in MC that lasts for 60 years, suggesting that it may involve permanent engraftment. MC is rare among male blood donors who did not receive transfusions, who are probably representative of individuals who have not had postnatal allogeneic exposures.

Long-term variations in human T lymphotropic virus (HTLV)-I and HTLV-II proviral loads and association with clinical data.

BACKGROUND: The human T lymphotropic virus (HTLV)-I or -II proviral load (VL) may be linked to viral pathogenesis, but prospective data on VL and disease outcomes are lacking. METHODS: Using data from a prospective cohort study of HTLV disease outcomes, we examined baseline VLs with real-time quantitative polymerase chain reaction in 122 HTLV-I- and 319 HTLV-II-infected subjects and serial VLs over the course of 6 visits in a subset of 30 HTLV-I- and 30 HTLV-II-infected subjects. Cox and logistic-regression models were used to test baseline associations, and repeated-measures analysis was used to study variations in VL over time. RESULTS: Over the course of a median of 10.4 years, HTLV-I VLs decreased slightly (slope, -0.017 log(10) copies/10(6) peripheral blood mononuclear cells [PBMCs]/year; P=.042) and HTLV-II VLs did not change (slope, -0.019 log(10) copies/10(6) PBMCs/year; P=.165). Changes in VL over time were associated positively with alcohol use (P=.07) and negatively with black race (P=.03) for HTLV-I and positively with smoking (P=.08) for HTLV-II. In the larger group, there was no association between baseline VL and disease outcomes. In the smaller group with serial VL data, there was an association between increasing VL and bladder or kidney infections for both HTLV-I (P=.005) and HTLV-II (P=.022). CONCLUSIONS: HTLV VLs are stable over time, but alcohol and tobacco intake may affect the progression of VLs. The association between increasing VLs and bladder/kidney infection may be explained by early HTLV-related neuropathologic progression.

Enhanced ascertainment of microchimerism with real-time quantitative polymerase chain reaction amplification of insertion-deletion polymorphisms.

BACKGROUND: The characterization of microchimerism (MC) by gene amplification has been limited by few allogeneic markers, ascertainment bias, and assay analytic performance. To address this, a panel of 12 MC assays based on insertion-deletion (InDel) polymorphisms had been optimized. STUDY DESIGN AND METHODS: The InDel assays were validated with comprehensive in vitro spiking studies at the stochastic limit of detection. Their ability was also determined to ascertain MC of unknown source genotype with both theoretical and actual donor-recipient pairs, and the assays were applied to a clinical population of 73 trauma patients who received transfusions where MC was previously characterized by HLA-based assays alone. RESULTS: In the stochastic spiking experiments, all assays were sensitive to a single copy of target DNA, and no false-positive amplification occurred among 1128 samples studied. Among 219 theoretical donor-recipient pairs, informative alleles existed for 99.5 percent with both InDel and HLA compared to 91.3 percent with HLA alone. In the clinical population, 33 cases of MC were detected (9 more cases than by HLA-DR alone) in the nonleukoreduced (non-LR) group and 8 cases (1 more case than by HLA-DR) in the LR group for the short-term follow-up. Among 27 long-term follow-up samples, 8 cases were detected overall (3 more cases than by HLA-DR alone). CONCLUSION: It is concluded that an InDel-based assay panel has excellent technical performance characteristics while also allowing for ascertainment of some MC cases not detectable with HLA alone. The tandem use of both the InDel and the HLA provides a powerful tool for the enhanced ascertainment of MC.

A prospective study of sexual transmission of human T lymphotropic virus (HTLV)-I and HTLV-II.

BACKGROUND: Cross-sectional studies support sexual transmission of human T lymphotropic virus (HTLV)-I/II; however, prospective incidence data, particularly for HTLV-II, are limited. METHODS: A cohort of 85 HTLV-positive (30 with HTLV-I and 55 with HTLV-II) blood donors and their stable (>or=6 months) heterosexual sex partners were followed biannually over the course of a 10-year period. RESULTS: Four of 85 initially seronegative sex partners of HTLV-I and -II carriers seroconverted, for an incidence rate (IR) of 0.6 transmissions/100 person-years (py) (95% confidence interval [CI], 0.2-1.6). This includes 2 HTLV-I transmissions/219 py (IR, 0.9 transmissions/100 py [95% CI, 0.1-3.3]) and 2 HTLV-II transmissions/411 py (IR, 0.5 transmissions/100 py [95% CI, 0.06-1.8]), with no significant difference by HTLV type. There were 2 male-to-female (IR, 1.2 transmissions/100 py [95% CI, 0.1-4.3]) and 2 female-to-male (IR, 0.4 transmissions/100 py [95% CI, 0.05-1.6) transmissions. HTLV-I or -II proviral load was 2 log10 lower in newly infected partners than in index positive partners who transmitted HTLV (P=.007). CONCLUSIONS: The incidence of sexual transmission of HTLV-II may be similar to that of HTLV-I, and female-to-male transmission may play a more important role than previously thought. HTLV-I and -II proviral load may be lower in sexually acquired infection, because of a small infectious dose.