Mixed Chimerism and Secondary Graft Failure in Allogeneic Hematopoietic Stem Cell Transplantation for Aplastic Anemia

Mixed chimerism (MC) and/or secondary graft failure (SGF) with recipient- or donor-type chimerism is a major obstacle in allogeneic transplantation for aplastic anemia (AA). From a registry database in Japan, patients with AA age >15 years who underwent a first allogeneic bone marrow or peripheral blood stem cell transplantation between 2000 and 2014 and achieved engraftment were included in this study. MC that did not require either granulocyte-colony stimulating factor (G-CSF) or transfusion support (group 1), MC (not SGF) that required G-CSF and/or transfusion support (group 2), SGF with MC or complete recipient-type chimerism (group 3), and SGF with complete donor-type chimerism (group 4) developed in 26, 16, 19, and 17 patients, respectively. The overall median duration of follow-up for survivors was 1727 days. The overall survival (OS) was 90.4% at 1 year and 83.5% at 5 years in patients without MC or SGF (n = 340), which was not different from the OS in groups 1 and 2. However, inferior OS was observed in group 3 (1 year, 52.1%; 5 years, 52.1%) and group 4 (1 year, 82.4%; 5 years, 56.3%). In multivariate analyses, the use of fludarabine (Flu) and the absence of irradiation in conditioning were associated with the development of SGF with MC or complete recipient-type chimerism, and the use of Flu in conditioning was associated with SGF with complete donor-type chimerism. In conclusion, the use of Flu may affect the occurrence of SGF with both recipient-type and donor-type chimerism.     

Blood lymphocyte chimerism associated with IVF and monochorionic dizygous twinning: case report

We report on dizygotic (DZ) twins, conceived by IVF and ICSI with assisted hatching, who each had a mixture of 46,XX and 46,XY cells in blood lymphocytes. The female twin had mild genitalia abnormalities but further study revealed anatomically normal reproductive anatomy. Chromosome and fluorescence in situ hybridization studies of buccal, skin and ovarian tissue were normal, as were buccal tissue DNA studies. Fetal ultrasound and fetal membrane pathology were consistent with a monochorionic, diamniotic placenta (MCDAP). These twins thus have blood chimerism but are not chimeric in the other tissues studied. The mechanism for the chimerism could be due to either placental vascular anastamoses (after the development of the haematoblast stem cells) or due to an admixture of trophoblast cells during early blastocyst development. Such trophoblast cell admixtures would be restricted to the extraembryonic tissues so that general physical development in the fetus is normal and without somatic cell chimerism. This case in combination with others previously reported suggests that in IVF conceptions, the prevalence of blood chimerism associated with twinning, and the occurrence of DZ twinning associated with MCDAP, may be higher than previously thought.     

Prognostic Utility of Routine Chimerism Testing at 2 to 6 Months after Allogeneic Hematopoietic Cell Transplantation

The utility of routine chimerism analysis as a prognostic indicator of subsequent outcomes after allogeneic hematopoietic cell transplantation (HCT) with myeloablative conditioning regimens remains controversial. To address this controversy, routine chimerism test results at 2 to 6 months after HCT with myeloablative conditioning regimens were evaluated for association with subsequent risk of chronic graft-versus-host disease (GVHD), nonrelapse mortality (NRM), relapse, and overall mortality. Only 70 of 1304 patients (5%) had < 95% donor-derived cells in the marrow. Low donor chimerism in the marrow occurred more often in patients with low-risk diseases compared with those with higher-risk diseases and was significantly associated with a reduced risk of chronic GVHD. Among 673 patients evaluated, 164 (24%) had < 85% donor-derived T cells in the blood. Low donor T cell chimerism was more frequent in patients with low-risk diseases compared with those with higher-risk diseases, in those who received conditioning with busulfan compared with those who received conditioning with total body irradiation, and in those with lower-grade acute GVHD. Low donor T cell chimerism in the blood was significantly associated with a reduced risk of chronic GVHD but not with a reduced risk of relapse, NRM, or overall mortality. Routine testing of chimerism in the marrow and blood at 2 to 6 months after HCT with myeloablative conditioning regimens may be helpful in documenting engraftment in clinical trials, but provides only limited prognostic information in clinical practice.     

Microchimerism in twins

Introduction

The aim of this paper was to report the occurrence of peripheral blood chimerism in newborns from bigeminal pregnancies.

Material and methods

Cord blood collected from 50 pairs of twins constituted the biological material studied. Analyses included: DNA isolation, quantitative and qualitative assessment of DNA preparations, hybridization analysis of SLS type as well as of MLS type, and analysis of microsatellite sequences with regard to polymorphisms using polymerase chain reaction.

Results

The presence of additional fragments of DNA in peripheral blood lymphocytes was found in four out of fifty pairs of monozygotic twins (8%) at locus D7S21 (7p22, n = 3) and locus D12S11 (12q24.3, n = 1). In these cases, the presence of additional DNA fragments was also proved by analysis of microsatellite sequence polymorphisms at loci HUMPLA2A1 (pancreatic phospholipase A-2, 12q23), HUMCYARO (cytochrome P450, 15q21.1) and HUMvWF (von Willebrand factor, 12p13).

Conclusions

The results of our study confirm the occurrence of chimerism in twins and constitutes the starting point for further studies aimed at determining the clinical significance of chimerism in twins both for women and fetuses.     

Fetal immune suppression as adjunctive therapy for in utero hematopoietic stem cell transplantation in nonhuman primates

In utero hematopoietic stem cell transplantation could potentially be used to treat many genetic diseases but rarely has been successful except in severe immunodeficiency syndromes. We explored two ways to potentially increase chimerism in a nonhuman primate model: (a) fetal immune suppression at the time of transplantation and (b) postnatal donor stem cell infusion. Fetal Macaca nemestrina treated with a combination of the corticosteroid betamethasone (0.9 mg/kg) and rabbit thymoglobulin (ATG; 50 mg/kg) were given haploidentical, marrow-derived, CD34+ -enriched donor cells. Animals treated postnatally received either donor-derived T cell-depleted or CD34+ -enriched marrow cells. Chimerism was determined by traditional and real-time polymerase chain reaction from marrow, marrow progenitors, peripheral blood, and mature peripheral blood progeny. After birth, the level of chimerism in the progenitor population was higher in the immune-suppressed animals relative to controls (11.3% +/- 2.7% and 5.1% +/- 1.5%, respectively; p = .057). Chimerism remained significantly elevated in both marrow (p = .02) and fluorescence-activated cell sorted and purified CD34+ cells (p = .01) relative to control animals at > or = 14 months of age. Peripheral blood chimerism, both at birth and long term, was similar in immune-suppressed and control animals. In the animals receiving postnatal donor cell infusions, there was an initial increase in progenitor chimerism; however, at 6-month follow-up, the level of chimerism was unchanged from the preinfusion values. Although fetal immune suppression was associated with an increase in the level of progenitor and marrow chimerism, the total contribution to marrow and the levels of mature donor progeny in the peripheral blood remained low. The level of long-term chimerism also was not improved with postnatal donor cell infusion.     

Stable mixed hematopoietic chimerism after bone marrow transplantation for sickle cell anemia.

A multicenter investigation of allogeneic bone marrow transplantation for children with sickle cell disease was conducted that included 27 European and North American transplant centers. Fifty-nine patients who ranged in age from 3.3 to 15.9 years (median, 10.1 years) received HLA-identical sibling marrow allografts between September 1991 and April 2000. Fifty-five patients survive, and 50 survive free from sickle cell disease, with a median follow-up of 42.2 months (range, 11.8 to 115 months) after transplantation. Of the 50 patients with successful allografts, 13 developed stable mixed donor-host hematopoietic chimerism. The level of donor chimerism, measured > or =6 months after transplantation in peripheral blood, varied between 90% and 99% in 8 patients. Five additional patients had a lower proportion of donor cells (range, 11% to 74%). Among these 5 patients, hemoglobin levels varied between 11.2 and 14.2 g/dL (median, 11.3 g/dL; mean, 12.0 g/dL). In patients who had donors with a normal hemoglobin genotype (Hb), the Hb S fractions were 0%, 0%, and 7%, corresponding to donor chimerism levels of 67%, 74%, and 11%, respectively. Among patients who had donors with sickle trait, the Hb S fractions were 36% and 37%, corresponding to donor chimerism levels of 25% and 60%, respectively. Thus, allograft recipients with stable mixed chimerism had Rb S levels similar to donor levels, and only 1 patient required a red blood cell transfusion beyond 90 days posttransplantation. None of the patients have experienced painful events or other clinical complications related to sickle cell disease after transplantation. These observations strongly suggest that patients with sickle cell disease who develop persistent mixed hematopoietic chimerism after transplantation experience a significant ameliorative effect.     

Do monochorionic dizygotic twins increase after pregnancy by assisted reproductive technology?

Although monochorionic (MC) dizygotic twins (DZT) are extremely rare in natural pregnancy, six pairs of such twins have successively been reported in a recent short period. All six cases of MC DZT were the products of pregnancy by assisted reproductive technology (ART). In this overview, we summarize these six cases and discuss possible mechanisms of this twinning and clinical implications of confined blood cell chimerism (CBC). The placental MC membrane was diagnosed ultrasonographically in all cases and pathologically in four. The presence of CBC was confirmed in four cases by haplotyping at polymorphic marker loci in peripheral blood leukocytes, karyotyping of lymphocytes and skin fibroblasts, and/or ABO blood group typing. As CBC is attributable to placental vessel anastomosis between DZT, it may become a risk factor for twin–twin transfusion syndrome (TTTS), mortality, and for other complications in twins. MC DZT may produce psychological trauma, especially in a girl/woman when she grows up and is known to be chimeric for a male karyotype and vice versa, although genital organs are generally normal—unlike freemartin in cattle. In addition, CBC in twins may mislead physicians when genotyping for a disease-susceptibility test is performed in medical practice in the near future. Blood group chimera may also cause confusion if a blood transfusion is necessary. Therefore, sufficient informed consent prior to ART and genetic counseling before/after birth are absolutely necessary for improved quality of life. It is most likely that all six cases are the consequence of fusion between two outer cell masses from two zygotes. The ART used in the six MC DZT included in vitro fertilization-embryonic transfer (IVF-ET) into the uterus, FSH-induced superovulation followed by intrauterine insemination, and/or intracytoplasmic sperm injection (ICSI). The use of an ovulation-inducing agent and implantation of several fertilized eggs at close sites are probably the events common among these cases. Assisted hatching, simultaneous ET, the use of eggs that have developed to the blastcyst stage, and cell culture procedures that lead to changes of the nature of cell surface, all may increase the chance of a cell fusion. This chance hypothesis can simply explain why MC DZT are very rare in natural pregnancy. Large-scale research on the prevalence of ART-associated MC DZT and long-term follow-up of the twins are essential.     

Hematopoietic Cell Transplantation with Reduced Intensity Conditioning Using Fludarabine/Busulfan or Fludarabine/Melphalan for Primary Immunodeficiency Diseases

Purpose

The purpose of our study was to compare the safety and efficacy of hematopoietic cell transplantation (HCT) using fludarabine (Flu)-based reduced intensity conditioning (RIC) with busulfan (BU) or melphalan (Mel) for primary immunodeficiency diseases (PID).

Methods

We retrospectively analyzed transplant outcome, including engraftment, chimerism, immune reconstitution, and complications in 15 patients with severe combined immunodeficiency (SCID) and 27 patients with non-SCID PID. The patients underwent Flu-based RIC-HCT with BU (FluBU: 7 SCID, 16 non-SCID) or Mel (FluMel: 8 SCID, 11 non-SCID). The targeted low-dose BU with therapeutic drug monitoring was set to 30 mg hour/L for SCID.

Results

The 2-year overall survival of all patients was 79.6% and that of patients with SCID in the FluBU and FluMel groups was 100% and 62.5%, respectively. In the FluBU group, all seven patients achieved engraftment, good immune reconstitution, and long-term survival. All five patients receiving umbilical cord blood transplantation achieved complete or high-level mixed chimerism and sufficient specific IgG production. In the FluMel group, six of eight patients achieved complete or high-level mixed chimerism. Viral reactivation or new viral infection occurred in one FluBU group patient and four FluMel group patients. In the non-SCID group, 10 of 11 patients (91%) who received FluMel achieved complete or high-level mixed chimerism but had variable outcomes. Patients with WAS (2/2 patients), NEMO deficiency (2/2 patients), and X-linked hyper IgM syndrome (2/3 patients) who received FluBU achieved complete or high-level mixed chimerism and long-term survival.

Conclusions

RIC-HCT with FluBU is a safe and effective strategy for obtaining high-level donor chimerism, immune reconstitution including B cell function, and long-term survival in patients with SCID. In patients with non-SCID PID, the results varied according to the subtype of the disease. Further prospective studies are required to optimize the conditioning regimen for non-SCID PID.

     

Monitoring of Hematopoietic Chimerism after Transplantation for Pediatric Myelodysplastic Syndrome: Real-Time or Conventional Short Tandem Repeat PCR in Peripheral Blood or Bone Marrow?

Quantitative real-time PCR (qPCR) has been proposed as a highly sensitive method for monitoring hematopoietic chimerism and may serve as a surrogate marker for the detection of minimal residual disease minimal residual disease in myelodysplastic syndrome (MDS), until specific methods of detection become available. Because a systematic comparison of the clinical utility of qPCR with the gold standard short tandem repeat (STR)-PCR has not been reported, we retrospectively measured chimerism by qPCR in 54 children transplanted for MDS in a previous study. Results obtained by STR-PCR in the initial study served as comparison. Because the detection limit of qPCR was sufficiently low to detect an autologous background, we defined the sample as mixed chimera if the proportion of recipient-derived cells exceeded .5%. The true positive rates were 100% versus 80% (qPCR versus STR-PCR, not significant), and mixed chimerism in most cases was detected earlier by qPCR than by STR-PCR (median, 31 days) when chimerism was quantified concurrently in peripheral blood and bone marrow. Both methods revealed a substantial rate of false positives (22.7% versus 13.6%, not significant), indicating the importance of serial testing of chimerism to monitor its progression. Finally, we propose criteria for monitoring chimerism in pediatric MDS with regard to the subtypes, specimens, PCR method, and timing of sampling.     

Tubular and endothelial chimerism in renal allografts using fluorescence and chromogenic in situ hybridization (FISH, CISH) technology

The role of endothelial and tubular chimerism in renal allograft adaptation and rejection varies in different studies. We addressed the correlation between different clinico-pathological settings and sex-chromosomal endothelial and/or tubular chimerism in renal allografts. We examined the presence or absence of the X and Y chromosomes by fluorescence and chromogenic in situ hybridization (FISH, CISH) methodology on paraffin embedded kidney biopsies in 16 gender mismatched renal transplants (1 to 12 years post-transplantation). Twelve patients were male, four female. Four groups were selected: (i) Vascular calcineurin inhibitor toxicity without rejection; (ii) T-cell mediated vascular rejection; (iii) antibody mediated rejection; and (iv) C4d-positivity in AB0-incompatible transplants with or without rejection. Twelve non-transplant kidney biopsies (8 female, 4 male) were used as controls. Tubular chimerism was detected more frequently (69%) than endothelial chimerism (12%) in renal transplants. One of 12 control patients had tubular and endothelial chimeric cells (8%). The Y chromosome occurred in 8/12 male recipients (67%) in tubular epithelial cells and in 5/12 male recipients (42%) in endothelial cells. Double X chromosomes were detected in 3/4 female recipients in tubular epithelium. Tubular chimerism occurred more often with endothelial chimerism and capillaritis without correlation with other parameters, such as rejection. Combined Y chromosomal tubular and lymphatic endothelial chimerism correlated with T-cell mediated vascular rejection in two out of three patients (66%). Combined Y chromosomal tubular and peritubular capillary chimerism correlated with antibody mediated C4d+ rejection in one out of two patients (50%). Tubular and/or endothelial chimerism occur frequently in gender mismatched renal allografts and, when combined, this is associated with T-cell mediated rejection.     

Restricted chimerism of peripheral blood cells after transplantation of allogeneic hemopoietic tissue in rabbits.

Allogeneic bone marrow or spleen cells were transplanted in rabbits treated with a single dose of 110 kmg/g of cyclophosphamide or four doses of 50 mg/kg. Chimerism of peripheral blood cells occurred in about 50% of the recipients, lasting from about the 14th day until 180 days after transplantation. The chimerism was limited to some kinds of cells since dissociation of proliferation of hemopoietic cells and split chimerism of lymphocytes were observed. Dissociation of proliferation of hemopoietic cells consisted in the appearance of only one cell line, namely lymphocytes, whereas the erythrocyte cells did not proliferate. Split chimerism occurred within lymphocytes, manifested by the presence of the donor's T lymphocytes but absence of proliferation of the donor's B lymphocytes.     

Development of multiplex short tandem repeat (STR)-PCR for chimerism analysis in patients with hematological malignancies and comparison of chimerism in different sample sources

Polymerase chain reaction analysis of short-tandem repeat (STR) markers (STR-PCR) has been used for chimerism testing to assess engraftment following hematopoietic stem cell transplantation (HSCT). We investigated the informativity of 7 STR loci (FGA, D5S818, SE33, TH01, VWF, PentaE, and D18S51) in 82 pre-HSCT DNA samples from 41 donor/recipient pairs and developed 2 multiplex STR-PCRs using VWF, SE33, and D18S51, D5S818 and FGA, respectively. The multiplex STR-PCRs could distinguish the recipients and donors in 92.7% of the cases. Dilution experiments using mixed DNA showed that the sensitivity of the multiplex STR-PCRs for detecting the minor population was 1-5%. To compare chimerism in different samples such as peripheral blood, mononuclear cells (MNC), and CD3-positive cells (CD3+), we investigated the relationship between the chimerisms at approximately day 30 post-HSCT and the interval from the day of HSCT to achievement of complete chimerism (CC) in 70 patients undergoing HSCT. CC was found in all samples of 54 patients at day 30 post-HSCT, and these samples showed CC thereafter. Eleven patients with mixed chimerism (MC) in all samples or in MNC and CD3+ showed CC at day 60-270 post-HSCT or persistent MC. The remaining 5 patients with MC in only CD3+ showed CC at day 30-60 post-HSCT. Taken together, MNC which can be separated easily may be a useful source for detecting patients who require longer time to achieve CC and those with high risk of graft failure.     

Ultra-Sensitive Droplet Digital PCR for the Assessment of Microchimerism in Cellular Therapies

Current techniques to assess chimerism after hematopoietic stem cell transplantation (HSCT) are limited in both sensitivity and precision. These drawbacks are problematic in the context of cellular therapies that frequently result in microchimerism (donor chimerism <1%). We have developed a highly sensitive droplet digital PCR (ddPCR) assay using commercially available regents with good performance throughout the range of clinically relevant chimerism measurements, including microchimerism. We tested the assay using spiked samples of known donor-recipient ratios and in clinical samples from HSCT recipients and patients enrolled on clinical trials of microtransplantation and third-party virus-specific T cells (VSTs). The levels of detection and quantification of the assay were .008% and .023%, with high levels of precision with samples of DNA content ranging from 1 to 300?ng DNA. From the panel of 29 insertion-deletion probes multiple informative markers were found for each of 43 HSCT donor-recipient pairs. In the case of third-party cellular therapies in which there were 3 DNA contributors (recipient, HSCT donor, and T-cell donor), a marker to detect the cellular product in a background of recipient and donor cells was available for 11 of 12 cases (92%). Chimerism by ddPCR was able to quantify chimerism in HSCT recipients and comparison against standard STR analysis in 8 HSCT patients demonstrated similar results, with the advantage of fast turnaround time. Persistence of donor microchimerism in patients undergoing microtransplantation for acute myeloid leukemia was detectable for up to 57 days in peripheral blood and bone marrow. The presence of microtransplant product DNA in bone marrow T cells after cell sorting was seen in the 1 patient tested. In patients receiving third-party VSTs for treatment of refractory viral infections, VST donor DNA was detected at low levels in 7 of 9 cases. ddPCR offers advantages over currently available methods for assessment of chimerism in standard HSCT and cellular therapies.     

Low Exposure Busulfan Conditioning to Achieve Sufficient Multilineage Chimerism in Patients with Severe Combined Immunodeficiency

After allogeneic hematopoietic cell transplantation (HCT), the minimal myeloid chimerism required for full T and B cell reconstitution in patients with severe combined immunodeficiency (SCID) is unknown. We retrospectively reviewed our experience with low-exposure busulfan (cumulative area under the curve, 30 mg·hr/L) in 10 SCID patients undergoing either first or repeat HCT from unrelated or haploidentical donors. The median busulfan dose required to achieve this exposure was 5.9 mg/kg (range, 4.8 to 9.1). With a median follow-up of 4.5 years all patients survived, with 1 requiring an additional HCT. Donor myeloid chimerism was generally >90% at 1 month post-HCT, but in most patients it fell during the next 3 months, such that 1-year median myeloid chimerism was 14% (range, 2% to 100%). Six of 10 patients had full T and B cell reconstitution, despite myeloid chimerism as low as 3%. Three patients have not recovered B cell function at over 2 years post-HCT, 2 of them in the setting of treatment with rituximab for post-HCT autoimmunity. Low-exposure busulfan was well tolerated and achieved sufficient myeloid chimerism for full immune reconstitution in over 50% of patients. However, other factors beyond busulfan exposure may also play critical roles in determining long-term myeloid chimerism and full T and B cell reconstitution.     

Immunogenicity and safety of an accelerated hepatitis E vaccination schedule in healthy adults: a randomized,controlled, open-label,phase IV trial

ObjectivesThis study aimed to evaluate the immunogenicity and safety of a hepatitis E (HE) vaccine using an accelerated vaccination schedule (vaccine doses at 0, 7 and 21 days).MethodsA total of 126 participants aged ≥18 years were randomly assigned to receive the hepatitis E virus vaccine in either the accelerated group (0, 7 and 21 days) or the routine group (0, 1 and 6 months). Serology samples were obtained at 0, 21, 28 and 51 days, and 7 months in the accelerated group, or 0, 1, 2 and 7 months in the routine group after the first vaccine injection. Adverse events (AEs) reported during the whole study were analysed.ResultsA total of 126 participants were randomized, 63 for each group. Sixty-two participants in the accelerated group and 63 in the routine group received at least one dose of vaccine; 57 and 63 participants received all three doses and were included in per-protocol set, respectively. In the per-protocol population, at 1 month after the last dose (accelerated group at 51 days versus routine group at 7 months), the seropositive rates were both 100% (57/57 and 63/63, respectively), and the geometric mean concentrations (GMCs) were 8.51 WHO units/mL (95% CI 6.73–10.76) in the accelerated group and 9.67 WHO units/mL (95% CI 7.67–12.20) in the routine group. The ratio of the accelerated group GMC to the routine group GMC was 0.88 (95% CI 0.61–2.17, lower limit of 95% CI > 0.5), indicating that the accelerated vaccination schedule was non-inferior to the routine one. The overall incidence rates of solicited AEs in the accelerated and routine groups were 32.26% (20/62) and 30.16% (19/63), respectively (p 0.800). Most AEs were moderate.ConclusionsAn accelerated schedule is safe and provides protective antibodies in a shorter time compared with the routine schedule. The accelerated schedule should be recommended to adults who are travelling on short notice to an HE-endemic area or during an HE outbreak (Clinical Trial Registration. NCT03168412)     

Use of polymerase chain reaction to provide prognostic information on human cytomegalovirus disease after liver transplantation

Sixty-four consecutive liver transplant patients receiving 76 organs have been monitored for human cytomegalovirus (HCMV) in blood and urine posttransplantation using a polymerase chain reaction (PCR) assay that amplifies a 149 base pair fragment of the glycoprotein B gene. Six hundred and twenty-six blood and 310 urine samples were analysed during surveillance. Thirty-two patients had CMV infection (50%), 12 of whim progressed to HCMV disease. Detection of HCMV in either blood or urine was significantly associated with the presence or development of HCMV disease (blood, P < 0.00001; urine, P = 0.0033). All cases of HCMV disease were detected as PCR-positive in blood, although due to sampling only 50% of these patients were PCR-positive prior to disease onset. HCMV infection and disease were more likely in patients who suffered rejection (P < 0.001). In addition, the median amounts of augmented prednisolone were higher in patients with HCMV infection and disease. In all cases, augmented prednisolone preceded HCMV infection/disease. There was no statistical association between CMV infection and death. Overall, the results show that routine use of PCR for HCMV in surveillance samples of blood and urine of liver transplant recipients can provide diagnostic and prognostic information. However, its ability to provide prognostic information is directly related to the availability of appropriate surveillance samples, emphasising the importance of the routine acquisition of such samples in patient management to allow preemptive anti-HCMV therapy. J. Med. Virol. 51:152–158, 1997. © 1997 Wiley-Liss, Inc.     

Mycobacterium neoaurum and Mycobacterium bacteremicum sp. nov. as causes of mycobacteremia

Reference isolates of Mycobacterium neoaurum, Mycobacterium aurum, and the nonvalidated species "Mycobacterium lacticola" were the focus of two recent molecular taxonomic studies. On the basis of this grouping, we identified 46 clinical pigmented, rapidly growing mycobacterial isolates. By 16S rRNA gene sequencing, only two major taxa were identified: M. neoaurum and a previously uncharacterized "M. neoaurum-like" group. The M. neoaurum-like group exhibited only 99.7% identity to M. neoaurum by 16S rRNA gene sequencing and 96.5% identity to M. neoaurum by rpoB sequencing and was named M. bacteremicum. No clinical isolates of M. aurum or M. lacticola were identified. Of isolates with known sources, 4/8 (50%) of M. bacteremicum isolates and 22/34 (65%) of M. neoaurum isolates were recovered from blood, and 35% of these were known to be from patients with catheter-related sepsis. MIC and clinical data on these 46 isolates of M. neoaurum and M. bacteremicum along with a review of 16 previously reported cases of infection with the M. neoaurum-M. lacticola group demonstrated that the isolates were highly susceptible to all drugs tested except clarithromycin, and most clinical cases were successfully treated. The clarithromycin resistance suggested the presence of an inducible erm gene reported in other species of rapidly growing mycobacteria. Sequencing studies are currently required to identify these two species. Strain ATCC 25791 (originally submitted as an example of Mycobacterium aurum) is proposed to be the type strain of M. bacteremicum.     

Results of minimally toxic nonmyeloablative transplantation in patients with sickle cell anemia and beta-thalassemia.

We describe previously transfused patients with sickle cell disease (n = 6) and thalassemia (n = 1) who received nonmyeloablative hematopoietic stem cell transplantation (HCT) to induce stable (full or partial) donor engraftment. Patients were 3 to 20 years (median, 9 years) old. All 7 received pretransplantation fludarabine and 200 cGy of total body irradiation; 2 patients also received horse antithymocyte globulin. Patients received bone marrow (n = 6) or peripheral blood stem cells (n = 1) from HLA-identical siblings, followed by a combination of mycophenolate mofetil and cyclosporine or tacrolimus for postgrafting immunosuppression. After nonmyeloablative HCT, absolute neutrophil counts were <0.5 x 10(9)/L and <0.2 x 10(9)/L for a median of 5 days (range, 0-13 days) and 0 days (range 0-13 days), respectively. A median of 0 (range, 0-9) platelet transfusions were administered. No grade IV nonhematologic toxicities were observed. One patient experienced grade II acute graft-versus-host disease. Two months after transplantation, 6 of 7 patients had evidence of donor chimerism (range, 25%-85%). Independent of red blood cell transfusions, these 6 patients initially had increased total hemoglobin and hemoglobin A concentrations and a reduction of reticulocytosis and transfusion requirements. There were no complications attributable to sickle cell disease during the interval of transient mixed chimerism. However, after posttransplantation immunosuppression was tapered, there was loss of the donor graft, and all patients experienced autologous hematopoietic recovery and disease recurrence. One patient did not engraft. The duration of transient mixed chimerism ranged from 97 to 441 days after transplantation in patients 4 and 6, respectively, and persisted until immunosuppressive drugs were discontinued after transplantation. In summary, the nonmyeloablative HCT regimens described here produced minimal toxicity and resulted in transient donor engraftment in 6 of 7 patients with hemoglobinopathies. Although complications from the underlying hemoglobinopathies did not occur during the period of mixed chimerism, these results suggest that stable (full or partial) donor engraftment after nonmyeloablative HCT is more difficult to achieve among immunocompetent pediatric patients with hemoglobinopathies than among adults with hematologic malignancies, perhaps in part because recipients may have been sensitized to minor histocompatibility antigens of their donor by preceding blood transfusions.     

An Intermediate Alemtuzumab Schedule Reduces the Incidence of Mixed Chimerism Following Reduced-Intensity Conditioning Hematopoietic Cell Transplantation for Hemophagocytic Lymphohistiocytosis

Reduced-intensity conditioning (RIC) improves the outcomes of hematopoietic cell transplantation (HCT) in patients with hemophagocytic lymphohistiocytosis (HLH). Proximal (ie, close to graft infusion) dosing of alemtuzumab is associated with a high incidence of mixed chimerism, whereas distal (ie, distant from graft infusion) dosing is associated with less mixed chimerism but more acute graft-versus-host disease (GVHD). The alemtuzumab dose per kilogram of body weight also influences these outcomes. We hypothesized that an intermediate alemtuzumab dosing schedule would reduce mixed chimerism and maintain a low incidence of acute GVHD. In this study, 24 consecutive HCTs were performed in patients with HLH or a related disorder using a novel intermediate alemtuzumab schedule of 1 mg/kg starting on day -14. The cumulative incidences (CIs) of mixed chimerism, upfront acute GVHD grades II-IV, and receipt of additional hematopoietic cell products after HCT were compared in patients treated with a distal alemtuzumab schedule (n = 15) and those treated with a proximal alemtuzumab schedule (n = 33). All patients received fludarabine and melphalan. The CI of mixed chimerism was 31% in the intermediate group, 72% in the proximal group (P < .01), and 75% in the distal group patients who received ≥2 mg/kg alemtuzumab (P = .03). The CI of acute GVHD grades II-IV before the development of mixed chimerism was 4% in the intermediate group, 0% in the proximal group, and 13% in the distal group (P = .04, proximal versus distal). The 1-year CI of administration of additional hematopoietic cell products for mixed chimerism (donor lymphocyte infusion ± hematopoietic stem cell boost ± repeat HCT) was 14% in the intermediate group, 53% in the proximal group (P = .01), and 38% in the distal ≥2 mg/kg alemtuzumab group (P = .02). Our findings indicate that intermediate RIC reduces the incidence of mixed chimerism, is associated with a low incidence of upfront acute GVHD, and decreases the need for additional hematopoietic cell products after HCT.     

Post-Transplant CD34+ Selected Stem Cell “Boost” for Mixed Chimerism after Reduced-Intensity Conditioning Hematopoietic Stem Cell Transplantation in Children and Young Adults with Primary Immune Deficiencies

Mixed chimerism and eventual graft loss occurs in a proportion of children with primary immune deficiencies receiving alemtuzumab, fludarabine, and melphalan reduced-intensity conditioning (RIC) regimens before allogeneic hematopoietic stem cell transplantation (HSCT). We investigated the usefulness of a CD34⁺ selected stem cell “boost” without conditioning to treat mixed chimerism in children and young adults who received predominantly an alemtuzumab, fludarabine, and melphalan RIC regimen for primary immune deficiencies and reported the outcomes. Patients with a primary immune deficiency disorder who were either enrolled on a prospective CD34⁺ boost study for treatment of mixed chimerism from 2011 to 2014 (n = 9) or treated with a CD34⁺ boost on a clinical basis from 2014 to 2016 (n = 3) were included in this analysis. Response to a CD34⁺ boost was defined as a rise in donor chimerism by ≥15% with donor chimerism of at least 20%, stabilization was defined as a rise in chimerism by <15% with donor chimerism ≥ 20%, and no response was defined as any decline in donor chimerism or need for a second HSCT after a CD34⁺ boost. Twelve patients received alemtuzumab, fludarabine, and melphalan. Median age was 4.5 years (range, .9 to 20.6), and median whole blood donor chimerism before the boost was 25% (range, 3% to 61%). Three patients (25%) met criteria for response, 1 patient (8%) was considered to have stabilization, and 8 patients (67%) had no response 12 months after the boost. None of the patients developed any complications from a CD34⁺ boost, including no acute graft-versus-host disease (GVHD). All patients are alive with a median follow-up of 32 months (range, 8 to 79). We conclude that a CD34⁺ selected stem cell boost can be considered for treatment of mixed chimerism after alemtuzumab, fludarabine, and melphalan RIC HSCT in children and young adults with primary immune deficiencies. Approximately one-third of patients can be expected to benefit from a CD34⁺ selected stem cell boost and may avoid the need for a second HSCT. Lack of any GVHD or toxicity makes a stem cell boost an attractive option compared with donor lymphocyte infusions for treatment of mixed chimerism.