Q J Med 2001; 94: 599-607
© 2001 Association of Physicians
Passive transfer of scrub typhus plasma to patients with AIDS: a descriptive clinical study
From the Retrovirology Department, USAMC, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand, 1 Chiangrai Regional Hospital, Chiangrai, Thailand, and 2 Human Plasma Product Services, Lille, France
Received 3 July 2001 and in revised form 24 August 2001
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Summary |
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We examined the HIV-inhibitory effects previously found to be associated with scrub typhus infection. Individual 500 ml units of plasma from donors with mild scrub typhus were safety-tested, subjected to virucidal heat treatment, and administered to 10 HIV-1-infected recipients who were not receiving antiretroviral drugs. HIV-1 copy number fell three-fold or more in two recipients, and virus burden was reduced for 8 weeks in 70% (7/10) of recipients of a single plasma infusion, compared with the mean of three pre-infusion measurements. Scrub typhus donor plasma inhibited HIV-1 in vitro compared with normal human plasma and media controls. In the clearest in vivo response, reduction in viral load was accompanied by clinical improvement, a switchback from the syncytia-inducing to the non-syncytia-inducing phenotype, and decreases in CD8 cells and IL-6 levels. Scrub typhus infections can generate heat-stable, transferable plasma factors that exert prolonged anti-HIV effects. Whether variability in the results is due to different scrub typhus infections, different HIV infections or different individual responses, is unclear.
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Introduction |
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Transient rises in HIV viral load have been reported following immune activation by intercurrent infection,1,2 immunization,3,4 and even tuberculin skin testing.5 Unexpectedly, a drop in viral load was observed in some HIV-1-infected individuals not receiving antiretroviral drugs during acute co-infection with scrub typhus, a chigger-borne zoonosis common in rural Asia. Copy number fell to below detectable limits in some patients.6 Sera from HIV-uninfected scrub typhus patients and sera from mice inoculated with Orientia tsutsugamushi, the causative agent of scrub typhus, inhibited HIV-1 replication in vitro.6 A substantial reduction of copy number was associated with scrub typhus co-infection in only 40% of HIV-infected subjects, suggesting that circulating HIV-inhibitory substances appear during some, but not all, scrub typhus infections. We passively transferred plasma from individual patients infected with O. tsutsugamushi to individual recipients with AIDS to test this hypothesis. We also sought to obtain information about donor and recipient determinants of HIV inhibition by scrub typhus.
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Methods |
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We obtained written informed consent from patients using a protocol approved by the Ethical Review Committee of the Thai Ministry of Public Health and by the Human Subjects Research Review Board, Office of the Surgeon General, Department of the United States Army. Clinical studies were carried out at Chiangrai Regional Hospital in northern Thailand.
Reduction of risk of plasma infectivity
Every attempt was made to reduce the risk of transmitting infectious agents to plasma recipients. All potential donors completed a uniform donor history questionnaire following guidelines of the American Association of Blood Banks. Individuals with tattoos, a history of drug use, exposure to blood or blood products, non-sterile skin penetration, or contact with individuals infected with viral hepatitis or HIV, were not asked to donate plasma. Prior to taking plasma and 1 month later, potential donors were tested for anti-HIV antibody, Treponema pallidum antigen, anti-hepatitis C antibody, dengue infection, hepatitis B surface antigen, and malaria. Only plasma units taken from donors free of detectable transmissible diseases with serum bilirubin levels <1.0 mg/dl, serum alanine transferase concentrations <80 U/l, and serum aspartamine transferase levels <100 U/l were used. Blood was withdrawn into a closed bag and subjected to cell separation by rapid centrifugation (Mistral 6000) to obtain approximately 500 ml plasma. Red blood cells were returned to the donor. Plasma was tested by RT-PCR for HIV RNA, and by PCR for HCV RNA, and cultured for aerobic and anaerobic bacteria. Blood specimens were tested for the presence of O. tsutsugamushi by inoculation of patient blood into mice and passage.7 Units of plasma were only used if all screening test results were negative. Plasma was frozen at -70 °C until the day of infusion. As an additional safety measure, each unit was then thawed, transferred to a sterile blood bag and heated at 50 °C for 3 h to inactivate transmissible viruses.8
Plasma donors
Adults aged 21–45 years with mild scrub typhus infection confirmed by a clear positive dot-blot ELISA test (Dip-s-Ticks, Integrated Diagnostics)7 and no other medical problems were asked to donate plasma. Neither patients with severe O. tsutsugamushi infection nor individuals at risk for exposure to blood borne pathogens were recruited. Exclusion criteria were hypotension (systolic pressure of <90 mmHg and/or diastolic pressure <60 mmHg), impaired consciousness, tachypnoea (respiratory rate >30 bpm), or any sign of severe scrub typhus. Demographic information and admission characteristics of the 12 plasma donors are shown in Table 1. Plasma was taken without incident, and O. tsutsugamushi infection responded well to doxycycline treatment. All donors were asymptomatic at the 1-month follow-up visit. We originally intended to transfer scrub typhus plasma to 10 recipients, and recruited 12 donors to provide a margin of error should post-collection test results preclude some plasma units from use. However, no markers of bacterial-, rickettsial- or viral-infection were found in any plasma. A second unit of plasma was therefore given to two patients (coded RE1 and RE3) 8 months after their first scrub typhus plasma infusion.
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Plasma recipients
Only HIV-seropositive individuals who were not taking antiretroviral drugs and had <200 CD4+ cells/µl of blood were enrolled, since this made them ineligible to receive antiretroviral drugs under Thai Ministry of Public Health protocols. Patients with fever, active opportunistic infection or chest radiographic evidence of tuberculosis or Pneumocystis carinii pneumonia were not eligible to receive plasma. Plasma from one Rh- and ABO-matched donor was chosen at random from all remaining matched units using random number tables, and 500 ml was given by slow intravenous infusion over 2 h to each recipient. This volume of plasma had been safely given to patients with advanced AIDS.9 Recipients were hospitalized during transfusion and monitored closely in hospital for possible adverse effects for 24 h after receiving plasma. All HIV-seropositive individuals evaluated for the study received the highest attainable standard of care in Thailand,10 which included co-trimoxazole for the prevention of Pneumocystis carinii pneumonia, but did not include antiretroviral drugs.
HIV virus load and lymphocyte subsets
Three copy number measurements were made during the week prior to plasma infusion, and the mean value was taken as baseline. HIV-1 viral load was then measured 1, 2, 3, 7, 10, 14, 21, 28, 35, 42, 49 and 56 days after plasma infusion (Chiron bDNA 3.0, Chiron Corporation, limit of detection 50 copies/ml, CV 15%) at the Department of Retrovirology, AFRIMS. Specimens for copy number determinations obtained at different time points from the same patient were assayed simultaneously on the same run. The Department of Retrovirology is part of an international consortium of QA/QC for bDNA assays and is an approved laboratory for vaccine clinical trials. Intra-lab variations in viral load are within 0.10–0.15 log; inter-lab variation is within 0.2 log. CD4 and CD8 counts were monitored at monthly intervals using two-colour flow cytometry and a FACScan instrument (Becton Dickinson Immunocytometry Systems).
In vitro HIV inhibition
Complement inactivated plasma from scrub typhus donors was assessed for in vitro inhibition of HIV using cryopreserved peripheral blood mononuclear cells (PBMCs) infected with a non-laboratory-adapted Thai strain of clade E virus (NP1668). HIV replication was assessed at 3, 7, 10 and 14 days after infection by measuring the amount of p24 antigen in the culture supernatant.6 The ten donor plasma were tested during the same experiment.
HIV-1 subtype and phenotype
The HIV-1 subtype of plasma recipients was determined using PBMCs from infected subjects and nested PCR, using primers that differentiate HIV subtypes B and E. Virus isolation was performed on the day of plasma transfer, and then 28 and 56 days later using the subject's PBMCs co-cultured with phytohaemagglutinin-stimulated PBMCs from a seronegative Thai donor at a 1:1 ratio. The biological phenotype of all isolates was determined using the CD4+ T cell line MT-2 and the technique described by the Division of AIDS, NIAID.11 Cultures were scored as either syncytium-inducing (SI) or non-syncytium-inducing (NSI).
Assays for donor plasma substances possibly relevant to HIV inhibition by scrub typhus
Vasculitis with endothelial damage is thought to be the key pathogenic mechanism in scrub typhus, and damaged endothelial cells release endothelin-1. O. tsutsugamushi induces IL-8 in an endothelial cell line.12 Plasma concentrations of endothelin-1 and IL-8 were determined by commercially available ELISA kits (R&D Systems). Secretory leukocyte protease inhibitor (SLPI) is a heat-stable plasma substance with HIV-inhibitory activity.13 SLPI was measured by ELISA with commercially available kits (R&D Systems).
Markers of immune activation in plasma recipients
High concentrations of IL-6 and IgE are associated with advanced HIV infection and immune activation.14,15 Plasma IL-6 concentration was determined by ELISA with commercially available kits (R&D Systems, limit of detection 0.7 pg/ml) and IgE was assayed by solid-phase fluorescent assay in two recipients.
Protein database search for potential cross-reactive components
The intracellular pathogen Orientia (formerly Rickettsia) tsutsugamushi that causes scrub typhus has a growth cycle similar to that of HIV-1, and spreads by budding from the surface of infected cells. Prominent budding is unusual among rickettsial organisms, and was one reason cited for the removal of O. tsutsugamushi from the genus Rickettsia.16 We looked for further similarities between HIV-1 and O. tsutsugamushi by examining protein databases to identify potential cross-reactive components, and we compared the sequences of such components with protein domain databases.17
Statistical analysis
The total viral load burden during the two months following plasma transfer was estimated by calculating the area under the copy number curve (AUC) after converting raw copy number data to percentages of the baseline value.18 AUC was calculated using the trapezoidal rule, according to a compartmental model-independent formula:
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Paired tests were used to compare clinical and laboratory indices in recipients before and after plasma transfer. Body weights were distributed normally, and values were analysed parametrically. AUC, CD4+ cells, CD8+ cells, CD4/CD8 ratio, hematocrit, white blood cell count, and platelet counts were not normally distributed, and values were analysed using nonparametric tests. Two-sided significance testing was performed in all cases. The Wilcoxon Rank Sum Test determined distribution-free confidence limits for the median copy number.
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Results |
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Patients
Eighty patients with mild scrub typhus were evaluated as donors, but two individuals (1%) were unwilling to give plasma. Fifty-four of the remaining 78 patients were excluded because of abnormal laboratory test results, most frequently raised serum bilirubin or transaminase levels and hepatitis B surface antigenemia. Twelve patients were excluded because fever could not be documented, or there were signs and symptoms atypical of O. tsutsugamushi infection.
All 18 HIV-infected subjects identified as eligible volunteered to receive plasma. However, eight subjects were excluded during the week prior to infusion because of fever. Five men and five women, mean age 29 (SD±5) years, received a mean of 9.3 (SD±1.4) ml/kg of plasma without significant side-effects. One subject (RE2) was lost to follow-up after 1 month. The mean body weight of the 10 recipients was 53.7 (SD±8.0) kg before plasma transfer, 54.5 (SD±8.0) kg 1 month later and 55.6 (SD±8.3) kg 2 months later. The mean (95%CI) increase in body weight at one month was 0.8 kg (0–1.6, p=0.05, paired t test) and at 2 months was 1.4 kg (0.5–2.3 kg, p=0.006, paired t test). One month after infusion, CD4+ cell counts and hematocrit were significantly higher than pre-infusion values (p<0.05, Wilcoxon signed rank test), while white blood cell count was significantly lower (Table 2). Two months after infusion, hematocrit and platelet counts were significantly higher than pre-infusion values, and white count remained significantly lower (p<0.05, Wilcoxon signed rank test).
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There were no significant changes in CD8+ cell counts for the 10 plasma recipients as a group. However, there were changes in CD8+ cell count at the individual level. CD8+ cell counts for RE1 were 1065 cells/ml, 977 cells/ml and 808 cells/ml at 0, 1, and 2 months, respectively, after plasma transfer. CD8+ cell counts for RE9 were 470 cells/ml, 602 cells/ml, and 660 cells/ml, at 0, 1, and 2 months, respectively, after plasma transfer.
HIV virus load
A three-fold or more reduction in copy number occurred in 2/10 recipients of a single unit of scrub typhus plasma, compared with the mean of three pre-infusion values (Figure 1). RE1 copy number at day 28 was 34772 copies/ml, compared with a preinfusion average of 173412 copies/ml. RE6 copy number at day 35 was 12693 copies, compared with a preinfusion average of 44833.
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The value of 5600 is the AUC if the baseline copy number were unchanged (Table 3). AUC% is generally <100 if copy count declines and >100 if copy count increases. For example, RE1 had an AUC of 1884, or 34% of baseline. The AUC during the 2 months after plasma transfer was <5600 in 6 of 9 recipients followed for 2 months, and <2800 in the individual only followed for 1 month (Table 3). The median viral burden was therefore reduced in 7/10 recipients, and was 88% of pre-infusion levels (range 34–181%) for the group as a whole.
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Two patients (RE1 and RE3) received a second unit of plasma 8 months after the first under identical conditions as followed for the initial plasma transfer. Again, the baseline was the average of three measurements made during the week preceding the second infusion. There was no significant response in patient RE3, but a further drop in viral load was measured in subject RE1 (Figure 2). The viral burden for RE1 was reduced by 32% after the second infusion (AUC 68% of steady state copy number). Overall, there was a three-fold fall or greater in copy number after 3/12 infusions (25%) and a decrease in AUC after 8/12 infusions (67%).
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HIV-1 typing and in vitro inhibition
HIV-1 virus was isolated from 32/35 attempts (91%). At least two isolates were made from each plasma recipient. All HIV-1 viruses isolated were subtype E. Five of the nine (56%) isolates made before plasma infusion and 14 of the 23 (61%) isolates made after plasma transfer were of the SI phenotype. A switchback from the more pathogenic SI phenotype to the NSI phenotype occurred in two patients, RE1 and RE8. All donor plasma exhibited in vitro HIV-inhibitory activity in the PBMC assay (Figure 3). Supernatant p24 antigen concentrations were lower in cells cultured with scrub typhus plasma than with either media or normal plasma controls after 7, 10, and 14 days of culture. However, plasma batches which appeared to produce a reduction in viral load upon in vivo infusion did not show any particular ability to inhibit replication in vitro.
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Donor endothelin-1, IL-8 and SLPI
Median donor plasma endothelin-1 concentration was 4.5 pg/ml (range 2.3–8.5 pg/ml) on the day of plasma donation (day 0) and 3.4 pg/ml (range 2.2–5.5 pg/ml) 2 months later. Median donor plasma SLPI concentration on day 0 was 47341 ng/ml (range, 37845–71436 ng/ml) and 45029 ng/ml on day 56 (range 31358–64073 ng/ml). Median day 0 concentration of IL-8 was 4.0 pg/ml (range 0–17.7 pg/ml) and day 56 median concentration was 1.1 pg/ml (range 0–6.7 pg/ml).
Recipient IL-6 and IgE
Median plasma IL-6 concentration in the recipients of plasma transfer was 1.7 pg/ml (range 0.0–2.7pg/ml) on day 0, 1.3 pg/ml (range 1.0–2.5 pg/ml) on day 28, and 1.5 pg/ml (range 0.7–7.1 pg/ml) on day 56. Serum IgE levels in recipient RE1 were 7500 kU/l prior to plasma transfer, 3582 kU/l at 28 days and 3413 kU/l at 56 days. The pre-infusion IgE level in patient RE9 was 2585 kU/l, and IgE levels were 2580 kU/l at day 28 and 2493 kU/l at day 56.
Protein database search
A search of the NCBI protein database for short, nearly exact matches revealed a 10-amino-acid match (8 identical and 2 conserved) between the O. tsutsugamushi Karp strain 47 kDa antigen (accession number gi|1220501) and HIV envelope protein (accession number gi|2250974).
47 kDa : 433 TLREIVTNIK 442
HIVgp120 : 214 TLRQIVTNLK 223
Pattern : TLR + IVTN + K
The sequence match within the envelope protein is at the C-terminal portion of the V1-V3 loop and might be accessible to antibody. Shorter matches between the scrub typhus 47kDa antigen, other O. tsutsugamushi antigens, and HIV-1 gp120 and Pol protein sequences were retrieved. Nine of 13 domains of the O. tsutsugamushi 47 kDa antigen were homologous with serine protease domains.
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Discussion |
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The data from this clinical study support and extend earlier findings6 that showed HIV-inhibitory effects associated with scrub typhus infection. The HIV-1 viral load response to acute co-infection with O. tsutsugamushi was variable, and included a transitory fall to below the limit of detection.6 Changes in HIV-1 viral load after passive transfer of scrub typhus plasma varied (Figure 1). HIV RNA levels did not fall below the limit of detection, but copy number fell by three-fold or greater in two patients after a single infusion of plasma. The total viral burden during the 2 months after each plasma transfer was approximated by the AUC, which reflects both transient and longer term changes in copy number.17 The AUC was less than that predicted had viral load remained at the pre-infusion level in 7/10 patients who received scrub typhus plasma (Table 3). These findings suggest that some scrub typhus plasma contained HIV-inhibitory factors. It is also possible that all plasma contained HIV-suppressor factors but that, for whatever reason, not all subjects responded to them.
We attempted to single out characteristics of HIV suppression by further analysing the clearest in vivo response, that of patient RE1 (Figure 1). During the week prior to plasma transfer, this patient's median HIV-1 RNA virus load was 166404 (95%CI 101310–245525) copies/ml. The median value of all virus load measurements during the 2 months following plasma infusion was 58666 (95%CI 44901–70931) copies/ml. It might be argued that an infection prior to plasma transfer could have raised the baseline copy number and, therefore, that the reduction in viral load observed after the infusion of plasma was simply a return to baseline. However, this explanation is unlikely, since a three-fold fall in viral load also followed the second infusion of plasma (Figure 2). There was no evidence of infection during the two weeks preceding the second plasma transfer, nor were there major fluctuations in virus load (Figure 2). A switchback to the less pathogenic non-syncytia-inducing (NSI) phenotype was detected prior to the second plasma infusion to RE1. The next two isolates were also NSI, whereas the previous virus isolates from this patient had yielded the more pathogenic SI variant. Interestingly, HIV-1 phenotype also appeared to be affected during naturally occurring scrub typhus co-infection; HIV isolates from such co-infected patients were invariably NSI variants.6 One month after receiving the first plasma infusion, RE1 was able to return to her job in a fruit orchard. A subjective increase in appetite was accompanied by a gain in body weight from 57.0 kg pre-infusion to 58.0 kg at day 28 and 59.5 kg at day 56. IL-6 concentrations fell after plasma was transferred to RE1 (Table 4), as occurs with effective antiretroviral therapy.19 A significant correlation between weight loss and IL-6 has been reported, and heightened IL-6 synthesis has been proposed as a source of IgE overproduction in HIV infection.20 The IgE concentration in patient RE1 fell from 7500 kU/l (normal limit <100 kU/l) before infusion, to 3582 kU/l on day 28 and 3413 kU/l on day 56. This was not a controlled study, so it is possible that plasma infusion coincided with some other change that prompted a remission.
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In contrast to RE1, there was no lowering of viral load after plasma transfer to RE9. Every post-infusion copy number was higher than the pre-infusion baseline value (Figure 1). Serum IgE concentration in RE9 was 2585 kU/l before infusion, 2580 kU/l at day 28 and 2493 kU/l at day 56. CD8+ T lymphocyte expansion has been implicated as a component of AIDS pathogenesis, and CD8 cell counts fall in conjunction with successful antiretroviral therapy.21,22 CD8+ cell counts in RE1 fell from a pre-infusion count of 1065 cells/ml to 977 cells/ml at 1 month and 808 cells/ml at 2 months after plasma transfer. CD8 cell counts rose in RE9 from a pre-infusion value of 470 cells/ml to 602 cells/ml 1 month later and 660 cells/ml 2 months later. Plasma infusion to RE1 was not associated with a significant change in CD4 cells. The pre-infusion count was 65 cells/ml, compared with 59 cells/ml and 64 cells/ml one and two months later.
The first plasma unit transferred to RE1 contained higher levels of endothelin-1 (8.5 pg/ml) and IL-8 (17.7 pg/ml) than did other donor plasma, and these substances could be markers of processes occurring during O. tsutsugamushi infection that generate antiretroviral effects. The highest SLPI level was measured in the plasma given to RE1. SLPI has known HIV-inhibitory activity,13 but is produced in a variety of infections. However, reduction of HIV copy number by a co-infection is highly unusual, suggesting that scrub-typhus-specific factors are involved. These factors are as yet undefined, but sequence similarities between O. tsutsugamushi and HIV proteins raise the possibility that scrub typhus infection generates HIV cross-reacting antibody. The 47 kDa scrub typhus surface antigen with sequences similar to HIV envelope protein is known to have T-cell epitopes,23 and its possible relevance to HIV inhibition by O. tsutsugamushi is currently under study.
Plasma recipients had a median pre-infusion CD4+ T lymphocyte count of 63 (range, 15–130) cells/µl. Patients such as these, with advanced AIDS, are less susceptible to antiretroviral interventions than are patients in earlier stages of HIV infection.24 Our data suggest that some scrub typhus infections generate heat stable plasma factors capable of exerting anti-HIV effects even in late-stage HIV infection. Hepatitis G virus is the only other known pathogen that appears to have a beneficial influence on AIDS.25
O. tsutsugamushi infection is common. An estimated one billion people live in endemic areas, and seroprevalence rates of up to 69% have been reported in Thailand.26 There is therefore a large potential pool of research material with which to further explore possible anti-HIV effects associated with scrub typhus. All scrub typhus plasma were HIV-inhibitory in vitro but, as in natural infection, only some inhibited in vivo. The modest increases in weight and hematocrit after the first infusion (Table 2) suggest that the recipients were not harmed and might have benefited from receiving scrub typhus plasma. Plasma with clearly defined in vivo HIV suppressive activity can be used to try to optimize in vitro HIV inhibitory tests and identify an appropriate marker of HIV inhibitory effect. Elucidation of the inhibitory mechanism could generate new tools for the prevention and treatment of HIV infection.
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Acknowledgments |
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The investigation was carried with support arranged by Dr Debbi Birx. Professor WG van Aken kindly reviewed the manuscript and suggested improvements. The opinions or assertions contained in this report are the private views of the authors and are not to be construed as official or as reflecting the views of the US Army.
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Notes |
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Address correspondence to Dr G. Watt, DTM&H, Retrovirology Department, AFRIMS, APO AP 96546. e-mail: wattgh{at}thai.amedd.army.mil
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References |
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