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PubMed2026

Assessment of differentially culturable tubercle bacteria assays for the detection of tuberculosis infection in asymptomatic household contacts and the implications for intra-household transmission: a longitudinal cohort study.

BACKGROUND: Conventional culture methods for tuberculosis diagnosis miss differentially culturable tubercle bacteria (DCTB), which grow only in liquid assays supplemented with growth-enhancing factors. This limitation, combined with inadequate contact tracing and screening, often fails to identify asymptomatic individuals, with live bacilli detectable by enhanced culture methods. This shortfall results in undiagnosed reservoirs of bacteria, potentially fuelling ongoing transmission. In this study, we aimed to investigate whether DCTB assays provide greater sensitivity by detecting more Mycobacterium tuberculosis infections than conventional culture and whether this enhanced detection improves the resolution of intrahousehold transmission mapping. In addition, we sought to evaluate whether DCTB populations can progress to conventional culture positivity, thereby highlighting their clinical and epidemiological relevance. METHODS: In this prospective observational longitudinal cohort study, drug-susceptible or rifampicin-resistant tuberculosis index participants aged 12 years or older, were recruited from primary healthcare clinics from two South African districts. Inclusion criteria were informed consent, Xpert MTB/RIF Ultra-positive results, tuberculosis symptoms (>2 weeks), provision of baseline samples, at least one consenting household contact, and documented HIV status. Household contacts of the index patients and control households were also recruited. Sputum specimens were collected at baseline and 2, 4, 8, 12, and 16 months from the index participants and household contacts. Samples were analysed by conventional mycobacterial growth indicator tube (MGIT) culture, and colony-forming unit assays to identify viable bacteria. Enhanced culture to detect DCTB involved serial dilution of sputum in liquid culture, supplemented with M tuberculosis culture filtrate as a source of growth stimulatory factors. Whole-genome sequencing (WGS) of cultured isolates was performed to trace household transmission. FINDINGS: Between June 1, 2020, and Feb 6, 2024, 293 index participants (183 [62%] male), 701 household contacts (453 [65%] female), and 122 control participants (67 [55%] female) were enrolled. At baseline, 249 (85%) of 293 index participants and 110 (16%) of 701 household contact sputum samples were positive for M tuberculosis by MGIT conventional culture. For baseline MGIT-negative specimens, DCTB assays detected M tuberculosis in an additional 21 (7%) of 293 index participants and 26 (4%) of 701 household contacts. Over 16 months of follow-up, DCTB assays identified 61 (8·7%) of 701 additional tuberculosis-positive household contacts not detected by conventional culture. WGS-guided transmission mapping using conventional culture identified transmission in 16 (15%) of 104 households, whereas DCTB assays detected an additional 19 (18%) of 104 transmission events. No evidence of intrahousehold transmission was found in the remaining 69 (66%) of 104 tuberculosis-positive households. Over the 16-month follow-up period, conventional culture identified 233 positive household contacts, of which 195 (84%) were asymptomatic. DCTB assays detected an additional 94 cases of M tuberculosis positivity in household contacts, of which 79 (84%) were asymptomatic. In control households, tuberculosis prevalence at baseline was two (2%) of 122, with an additional three (3%) of 122 identified during follow-up. INTERPRETATION: DCTB assays provide substantial value by detecting asymptomatic individuals missed by conventional culture, revealing a potentially important reservoir of subclinical infection, which could sustain transmission. In addition, DCTB detection uncovers transmission linkages missed by conventional culture, providing a more comprehensive understanding of M tuberculosis transmission dynamics and highlighting the need to incorporate enhanced culture methods into diagnostic and surveillance strategies, to strengthen early case identification and tuberculosis control efforts. FUNDING: National Institutes of Health.

Humans↗
PubMed1979

Relevance of colony forming assays to bone marrow transplanation with particular reference to grafting for aplastic anaemia.

The value of bone marrow colony-forming assays in monitoring transplanted patients has been assessed by comparing results from two pairs of bone marrow recipients. One pair received marrow from their identical twins for acute lymphoblastic leukaemia; the other pair were grafted with allogeneic marrow from their siblings for aplastic anaemia. One of each pair showed successful engraftment while in the others the grafts failed. The colony-forming assay was then used to investigate marrow function in five grafted aplastic patients. Of these, four rejected their first grafts and required further immunosuppression before engraftment could be accomplished. The remaining patient was immunosuppressed at the outset with antithymocyte globulin (ATG) and her first graft was successful. Sera from all five patients inhibited colony formation by normal human marrow and it is suggested that this activity was related to graft rejection as well as to the pathogenesis of the condition.

Adolescent↗
PubMed1979

Radiation sensitivity change of hemopoietic cells induced by hydroxyurea.

Hemopoietic tissues of mice contain cells capable of proliferation and differentiation to form endogenous spleen colonies (Endo-CFU-S) in irradiated animals. Hydroxyurea (HU) given before irradiation of C57BL mice for endogenous CFU assay increased the number of surviving CFU-S twofold. Either HU or HSATT given after the initial stimulus abolished this rise. In vivo and in vitro 'suicide' assays of spleen cells indicated that cells were stimulated by the HU or the initial stimulus to enter into DNA synthesis shortly after stimulation. The resting CFU cell population appeared to be positioned close to DNA-S phase and responded to the stimulus by entry into S phase. Whole body irradiation at intervals after HU revealed a changing radiation sensitivity of the endogenous CFU-S consistent with drug-induced progression with decreased radiation sensitivity of the S phase cohort of cells.

Animals↗
PubMed1978

Collection, cryopreservation and subsequent viability of haemopoietic stem cells intended for treatment of chronic granulocytic leukaemia in blast-cell transformation.

We have stored at -196 degrees C peripheral blood buffy coat (BC) and bone marrow (BM) cells collected from 47 patients with chronic granulocytic leukaemia in the chronic phase. Dimethyl sulphoxide (DMSO) 10% was used as cryoprotective agent. As these cells include CFUc and probably pluripotential stem cells they may be transfused as part of the management of patients who enter blast cell transformation. The mean numbers of nucleated cells collected and stored per procedure was about 9 times greater for BC collections than for BM harvests (106 +/- 49 (SD) X 10(9) versus 11.9 +/- 6.6 X 10(9) respectively). Agar CFUc assay showed that stored cells may remain viable for up to 5 years. Since in vitro studies showed that CFUc proliferation is not inhibited by low concentrations of DMSO the removal of all DMSO during cell reconstitution before transfusion may not be necessary. If autologous BC cells are capable of repopulating the BM of patients treated for CGL in blast cell transformation the routine collection and storage of BC rather than BM cells may be desirable for all newly diagnosed patients.

Blood Preservation↗
PubMed1979

Discontinuous density gradient separation of human mononuclear leucocytes using Percoll as gradient medium.

The use of a new commerically available medium (Percoll) for fractionation of human mononuclear leucocytes is described. Cells can be fractionated on the basis of their densities with high reproducibility. The separated cells were characterized by morphological and functional criteria. Monocytes can be obtained in the low density fractions with a purity of 70%--90%. Lymphocytes were found in high density fractions with a purity up to 99%. No separation between E-rosette forming (E-RFC) and surface immunoglobulin-bearing lymphocytes was obtained. However, a reduced number of high avidity E-rosette forming lymphocytes (HAE-RFC) was found within low density lymphocytes. Best spontaneous DNA synthesis and reaction in mixed leucocyte culture (MLC) were obtained with cells isolated from the 1.066/1.068 density interface, whereas the stimulation with phytohemagglutinin (PHA) or pokeweed mitogen (PWM) had a peak response with cells from the 1.064/1.066 density interface. Colony forming myelopoietic stem cells and colony forming T-lymphocytes were detected in fractions of low density.

Cell Count↗
PubMed1979

Proliferative capacity of erythropoietic stem cell lines and aging: an overview.

The earliest bone marrow precursor cell types, often called stem CELLS, have a very large capacity for self renewal. This makes them a useful model system in which to test the hypothesis that normal somatic cells have a limited proliferative capacity. Marrow precursor cells differentiate and multiply to replenish the supply of various blood cell types that constantly turn over. Especially with erythrocyte production, this function is well difined and can be tested rigorously to determine whether a significant amount of the stem cell proliferative capacity is exhausted. Functional tests generally show that marrow stem cell lines are exhausted after three to six serial transplantations into successive recipients; the few exceptions are cases in which functioning by cells from the irradiated recipients has not been ruled out. Genetic markers unambiguously identifying marrow stem cell lines from the original donor are necessary for clear cut interpretations of transplantation experiments. No significant differences are found when comparing erythrocyte production by marrow stem cell lines from old and young adult donors. This suggests that little or none of the erythropoietic stem cell's proliferative capacity is exhausted by a lifespan of normal functioning.

Aging↗
PubMed2026

Glycaemic burden disrupts innate immunity in TB by modulating CD206 expression and macrophage antimicrobial responses.

Tuberculosis (TB) and diabetes mellitus (DM) represent a growing dual global health burden, with chronic hyperglycaemia recognized as a major modifier of host immunity against Mycobacterium tuberculosis (Mtb). Macrophages, central to pathogen recognition, phagocytosis, antigen presentation, and intracellular killing, may be particularly vulnerable to diabetic metabolic dysregulation. This study evaluated phenotypic and functional macrophage alterations in individuals with pulmonary TB, type 2 DM, TB-DM comorbidity, and healthy controls. Surface receptor expression was analysed by multicolour flow cytometry, while phagocytosis and intracellular bacterial clearance were assessed using FITC-labelled Mtb assays and colony-forming unit enumeration. Hyperglycaemia was associated with reduced CD11b, MARCO, and TLR2 expression alongside upregulation of the mannose receptor CD206, which correlated positively with HbA1c levels, indicating a shift toward a permissive M2-like phenotype. Phagocytic uptake of Mtb was significantly impaired and inversely correlated with HbA1c. Antigen-presenting capacity was selectively compromised, with reduced CD80 and CD86 expression in DM and TB-DM groups, while HLA-DR remained unchanged. Intracellular Mtb killing was markedly diminished in diabetic macrophages. These findings demonstrate that chronic hyperglycaemia profoundly disrupts macrophage innate immunity, contributing to increased TB susceptibility and poor infection control in diabetic populations.

Humans↗
PubMed1979

Therapeutic efficiency of spleen or bone marrow CFU in X-irradiated 89Sr marrow-ablated mice.

Experiments were carried out to compare the therapeutic efficiency (TE: number of CFU required to reduce the mortality from 100 to 50 per cent) of spleen or marrow (BM) stem cells (CFU) grafted into lethally irradiated mice (807 rad) which had been previously treated with 89Sr or splenectomized. It was found that during the reconstitution of the haemopoietic organs, the spleen does not provide more than 10 per cent of the functional cells necessary for survival. Besides, the BM-derived CFU growing in 89Sr marrow-ablated mice remain twice as efficient as the spleen-derived ones. Similarly, spleen-derived CFU transplanted into splenectomized mice are half as efficient as BM-derived ones. It may therefore be assumed that haemopoietic stem cells grafted into a foreign microenvironment retain their original kinetics of growth and differentiation during 7 to 10 days after their transplantation.

Animals↗
PubMed1979

Organization of haemopoietic stem cells: the generation-age hypothesis.

This paper proposes that the previous division history of each stem cell is one determinant of the functional organization of the haemopoietic stem cell population. Stem cells from a lineage of stem cells which have generated many stem cells (older stem cells) are used in the animal to form blood before stem cells which have generated few stem cells (younger stem cells). The stem cell generating capacity of a lineage of stem cells is finite. After a given number of generations a stem cell is lost to the stem cell compartment by forming two committed precursors of the cell lines. Its part in blood formation is taken by the next oldest stem cell. We have called this proposal the generation-age hypothesis. Experimental evidence in support of the proposal is presented. We stripped away older stem cells from normal bone marrow and 13 day foetal liver with phase-specific drugs and revealed a younger population of stem cells whose capacity for stem cell generation was three- to four-fold greater than that of the average normal, untreated population. We aged normal stem cells by continuous irradiation and serial retransplantation and found that their stem cell generative capacity had declined eight-fold. We measured the stem cell generative capacity of stem cells in the bloodstream. It was a half to a quarter that of normal bone marrow stem cells and we found a subpopulation of circulating stem cells whose capacity for stem cell generation was an eighth to a fortieth that of normal femoral stem cells. This subpopulation was identified by its failure to express the brain-associated antigen which was present on 75% of normal femoral stem cells but was not found on their progeny, the committed precursors of granulocytes.

Animals↗
PubMed1978

Bone marrow colony-forming cells in acute drug-induced agranulocytosis.

The capacity to differentiate and form colonies in vitro by bone marrow granulocytic precursor cells and its evolution were studied in eight cases of drug-induced acute agranulocytosis. In three cases colonies and clusters count were normal or high. These cases were probably immunological agranulocytosis. In the five other cases this number was very low and returned to normal from three to thirteen days. These were probably cases of marrow suppression by the drug.

Acute Disease↗
PubMed1978

Experimental hypoplastic marrow failure in the mouse.

In order to study the pathogenesis of aplastic anemia in man, hemopoietic stem cells were investigated in 'aplastic mice' the aplasia being induced by the immunological method. C3H/He (H-2k, Mlsc) received 600 rad whole body x-irradiation followed by the transplantation of 10(7) lymph node cells prepared from B10.BR mic e (H-2k, Mlsb). The C3H/He mice developed pancytopenia and marrow hypoplasia 21 days after these treatments. The total number of nucleated cells, CFU-S and CFU-C in the marrow and the wet weight and CFU-C of the spleen were markedly reduced. These findings are consistent with those of aplastic anemia in man and the model may provide a useful tool for the investigation of the pathogenesis of this anemia. Control mice that received irradiation only recovered from the damage 21 days later, while control mice that receive lymph node cells only showed no hematological changes.

Anemia, Aplastic↗
PubMed1979

In vitro proliferation of haemopoietic cells in the presence of adherent cell layers. I. Culture conditions and strain dependence.

The culture system, in which a marrow-derived adherent cell population, established in vitro, exerts a long-term promoting influence on proliferation of haemopoietic cells, is reproduced. Essential parameters of the system are investigated; it is confirmed that the system is critically dependent on horse serum, and on the in vitro age of the adherent cell layer. The growth-promoting effect on haemopoietic cells seems to be independent of the number of marrow cells per culture flask initially inoculated into the cultures to establish the adherent cell layer. In vitro established marrow-derived adherent cell layers from RFM (H-2f) and BALB/c (H-2d) mice can promote the long-term proliferation of syngeneic and allogenic haemopoietic cells; haemopoietic marrow cells from C3H (H-2k) cannot be maintained on syngeneic or allogeneic (BALB/c, H-2d) adherent cell layers; adherent cell layers of C3H (H-2k) can maintain haemopoietic cells of the H-2d (BALB/c) genotype. This culture system does not reproduce the in vivo phenomenon of allogeneic resistance. The relevance of these findings to the suggestion that the growth-promoting activity of adherent marrow cells on haemopoietic stem cells in vitro duplicates aspects of the in vivo haemopoietic microenvironment is discussed.

Animals↗
PubMed1979

[Self-maintenance of migrating hematopoietic stem cells].

Decreased self-maintenance ability of the migrating stem cells (CFU) from the peripheral blood or ectopic focus of hemopoiesis in comparison to the settled bone marrow CFU, as measured by the spleen colony method or by means of chromosomal markers, has been studied. The competence for myeloid and lymphoid differentiation was essentially the same for migrating and settled stem cells.

Animals↗
PubMed1979

Albumin density gradient purification of canine hemopoietic blood stem cells (HBSC): long-term allogeneic engraftment without GVH-reaction.

Long-term repopulation of the blood-forming organs of dogs, conditioned by wholebody X-irradiation (1200 R midplane dose), was achieved by transfusion of cryopreserved allogeneic blood mononuclear cells (MNC) without causing graft-versus-host-reaction (GVH-R). Donor and recipient dogs were DL-A identical, MLC-negative, no siblings, non-related. The blood stem cells (CFUc) were procured by a 3- to 4-hour continuous-flow leukapheresis. To increase the CFUc concentration in the peripheral blood, dextran sulfate (DS) was administered intravenously beforehand. About 1 x 10(10) MNC, among them about 1 x 10(7) CFUc, were collected and further segregated using a discontinuous albumin density gradient. Less dense cells were to be found in the upper part of the gradient (fraction 2). These cells included most of the CFUc, enriched by a factor of between 275 and 1730 compared to their concentration in the peripheral blood beforehand. After cryopreservation, these cells, when transfused into lethally irradiated dogs, completely repopulated the marrow and lymph nodes, caused no GVH-R and allowed long-term survival. These dogs received no immunosuppressive therapy, either before or after transfusion. More dense MNC were to be found in fraction 3; their transfusion caused a severe GVH-R, followed quickly by death. Fraction 4 was rich in lymphocytes and poor in CFUc. The transfusion of these cells produced a selective plasma-cell hyperplasia of the lymph nodes but failed to repopulate permanently the marrow. The reappearance of the different cell lineages in the marrow and in the peripheral blood after conditioning and transfusion of these cells produced a selective plasma-cell hyperplasia of the lymph nodes but failed to repopulate permanently the marrow. The reappearance of the different cell lineages in the marrow and in the peripheral blood after conditioning and transfusion of the segregated MNC is described in detail.

Animals↗