Acceptability, feasibility, and likelihood of stakeholders implementing the novel BPaL regimen to treat extensively drug-resistant tuberculosis patients.

BACKGROUND: BPaL, a 6 month oral regimen composed of bedaquiline, pretomanid, and linezolid for treating extensively drug-resistant tuberculosis (XDR-TB) is a potential alternative for at least 20 months of individualized treatment regimens (ITR). The ITR has low tolerability, treatment adherence, and success rates, and hence to limit patient burden, loss to follow-up and the emergence of resistance it is essential to implement new DR-TB regimens. The objective of this study was to assess the acceptability, feasibility, and likelihood of implementing BPaL in Indonesia, Kyrgyzstan, and Nigeria. METHODS: We conducted a concurrent mixed-methods study among a cross-section of health care workers, programmatic and laboratory stakeholders between May 2018 and May 2019. We conducted semi-structured interviews and focus group discussions to assess perceptions on acceptability and feasibility of implementing BPaL. We determined the proportions of a recoded 3-point Likert scale (acceptable; neutral; unacceptable), as well as the overall likelihood of implementing BPaL (likely; neutral; unlikely) that participants graded per regimen, pre-defined aspect and country. We analysed the qualitative results using a deductive framework analysis. RESULTS: In total 188 stakeholders participated in this study: 63 from Kyrgyzstan, 51 from Indonesia, and 74 from Nigeria The majority were health care workers (110). Overall, 88% (146/166) of the stakeholders would likely implement BPaL once available. Overall acceptability for BPaL was high, especially patient friendliness was often rated as acceptable (93%, 124/133). In contrast, patient friendliness of the ITR was rated as acceptable by 45%. Stakeholders appreciated that BPaL would reduce workload and financial burden on the health care system. However, several stakeholders expressed concerns regarding BPaL safety (monitoring), long-term efficacy, and national regulatory requirements regarding introduction of the regimen. Stakeholders stressed the importance of addressing current health systems constraints as well, especially in treatment and safety monitoring systems. CONCLUSIONS: Acceptability and feasibility of the BPaL regimen is high among TB stakeholders in Indonesia, Kyrgyzstan, and Nigeria. The majority is willing to start using BPaL as the standard of care for eligible patients despite country-specific health system constraints.

Effective repair of articular cartilage using human pluripotent stem cell-derived tissue.

In an effort to develop an effective source of clinically relevant cells and tissues for cartilage repair a directed differentiation method was used to generate articular chondrocytes and cartilage tissues from human embryonic stem cells (hESCs). It has previously been demonstrated that chondrocytes derived from hESCs retain a stable cartilage-forming phenotype following subcutaneous implantation in mice. In this report, the potential of hESC-derived articular-like cartilage to repair osteochondral defects created in the rat trochlea was evaluated. Articular cartilage-like tissues were generated from hESCs and implanted into the defects. After 6 and 12 weeks, the defects were evaluated histologically and immunohistochemically, and the quality of repair was assessed using a modified ICRS II scoring system. Following 6 and 12 weeks after implantation, hESC-derived cartilage tissues maintained their proteoglycan and type II collagen-rich matrix and scored significantly higher than control defects, which had been filled with fibrin glue alone. Implants were found to be well integrated with native host tissue at the basal and lateral surfaces, although implanted human cells and host cells remained regionally separated. A subset of implants underwent a process of remodeling similar to endochondral ossification, suggesting the potential for a single cartilaginous implant to promote the generation of new subchondral bone in addition to repair of the articular cartilage. The ability to create cartilage tissues with integrative and reparative properties from an unlimited and robust cell source represents a significant advance for cartilage repair that can be further developed in large animal models before clinical- setting application.

Gold Nanoparticle-Based Detection of Low Molecular Weight AGEs from In Vitro Glycated Haemoglobin A0 Samples.

Protein glycation is a major biochemical event that takes place in the plasma of diabetic patients due to increased sugar levels. Extensive glycation leads to the formation of advanced glycation end products (AGEs) that is well known for having detrimental effects on diabetic patients. In the current work, we have glycated the physiologically important protein Haemoglobin A0 in vitro to study AGE formation and activity by using them as a template for gold nanoparticle (GNPs) synthesis. It was found that the surface plasmon resonance of synthesised GNPs showed high correlation with the extent of glycation. On fractionation, the glycated Haemoglobin A0 segregated into two distinct population of products, one consisting of proteinaceous, cross-linked larger fragments of Haemoglobin A0 and a second population of non-proteinaceous low molecular weight AGEs. Only low molecular weight AGEs contributed to synthesis of GNPs upon using the fractions as a template, substantiating the principle of proposed GNP-based assay. Owing to its physiological importance, AGEs can be used as a diagnostic means for diabetes and its associated complications. In this study, we have employed the high reactivity of AGEs for the development of a GNP-based novel colorimetric sensor to enable their detection. Our proposed GNP-based sensing could have high clinical significance in detecting diabetes and its associated complexities.

Immunohistochemical expression of Bcl-2 in oral epithelial dysplasia and oral squamous cell carcinoma.

BACKGROUND: The B cell lymphoma-2 gene is a proto-oncogene whose protein product inhibits apoptosis. Its role is associated with keeping cells alive, but not by stimulating them to proliferation, as other proto-oncogenes do. Increased expression of protein product of Bcl-2 gene appears in the early phase of carcinogenesis leading to apoptosis impairment and in consequence to the progression of neoplastic changes. OBJECTIVE: To evaluate and compare the expression of Bcl-2 protein in oral epithelial dysplasia and oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: Sixty cases of formalin-fixed paraffin-embedded archival specimens comprising of 30 cases of leukoplakia with oral epithelial dysplasia and 30 cases of OSCC were taken for immunohistochemical analysis using monoclonal antibody against anti-human Bcl-2 oncoprotein. RESULTS: Immunostaining for Bcl-2 protein was identified in basal and parabasal layers as granular cytoplasmic staining in oral epithelial dysplasia. In OSCC, Bcl-2 immunoreactivity was most prominent in the peripheral cells of the infiltrating tumor islands which diminished toward the center in well-differentiated and moderately differentiated OSCC, whereas stronger and more diffuse expression of Bcl-2 oncoprotein was seen in poorly differentiated OSCC. Overall positivity of 26.7% (8/30) was observed in oral epithelial dysplasia and 30% (9/30) in OSCC in this study. INTERPRETATION AND CONCLUSION: Altered expression of Bcl-2 oncoprotein may be an early molecular event which leads to prolonged cell survival, increased chances of accumulation of genetic alterations, and subsequent increase in malignant transformation potential.

Analysis of abnormal clones by the fluorescent aerolysin method in paroxysmal nocturnal haemoglobinuria and other marrow disorders.

INTRODUCTION: Flow cytometry is the most sensitive and specific diagnostic modality for the assessment of clone size in paroxysmal nocturnal haemoglobinuria (PNH) and other bone marrow failure states. In this study, we attempt to distinguish PNH from aplastic anaemia (AA) and myelodysplastic syndromes (MDS) associated with PNH clones at diagnosis by clone size, clinical and laboratory features. METHODS: A total of 29 samples included 19 PNH cases and 10 AA/MDS cases with PNH clones. Flow cytometry was performed using fluorescent aerolysin (FLAER)-based assay and comparison of clinical features, laboratory parameters and PNH clone size was carried out at diagnosis. RESULTS: The PNH clone size on granulocytes varied from 0.4% to 99.2% and correlated with the clone size on monocytes (r = 0.966; P < 0.001). Paroxysmal nocturnal haemoglobinuria clone size on granulocytes (median = 34.6%) and monocytes (median = 49.9%) was always larger than erythrocytes (median = 10.9%). The median clone size in PNH (median granulocytes = 74.9%, monocytes = 71.8%) was significantly greater than in AA/MDS associated with PNH clone (median granulocytes = 2.9%, monocytes = 6%). In PNH patients, a significant negative correlation was seen between PNH clone on monocytes and the haemoglobin concentration. CONCLUSION: In our small study using the FLAER method, the clone size was >70% in majority of PNH cases. In other marrow disorders like AA/MDS, the clone size was usually <10%.

First isolations in India of Candida nivariensis, a globally emerging opportunistic pathogen.

We randomly screened 363 yeast isolates during 2008 for their ability to form white colonies on CHROM agar Candida medium. Two of these isolates (0.5%) were identified as Candida nivariensis based on detailed phenotypic characterization and DNA sequencing. One was recovered from the sputum of an HIV-positive patient with a pneumonic lesion and the second from the blood of a diabetic with oropharyngeal lesions. Direct DNA sequencing of the D1/D2 region of 28S rRNA gene and/or the internal transcribed spacer (ITS) regions of rDNA confirmed that both of the isolates were C. nivariensis. The carbohydrate assimilation profiles with the ID 32 C and VITEK 2 yeast identification systems revealed only glucose assimilation. In vitro antifungal susceptibility profiles by broth microdilution and Etest methods revealed susceptibility of both isolates to fluconazole, itraconazole, voriconazole, amphotericin B and 5-flucytosine, with low MICs for posaconazole and caspofungin. These results document the occurrence of Candida nivariensis for the first time in India and focus on its potential as an opportunistic human pathogen.

Immunohistochemistry is a more sensitive marker for the detection of myeloperoxidase in acute myeloid leukemia compared with flow cytometry and cytochemistry.

Myeloperoxidase (MPO) is an unequivocal marker of myeloid differentiation which is routinely detected using cytochemistry (CC), flow cytometry (FC) and immunohistochemistry (IH). Several studies have compared the use of two of these methods, but to our knowledge none has compared all three techniques. We compared the performance of these three modalities in the detection of MPO in 158 cases of acute myeloid leukaemia (AML). Discrepancies were noted in a total of 28 cases. Of 110 cases in which all three modalities were performed, 23 cases showed discrepancies. CC was the least sensitive marker, being negative in 11 of 23 cases in the presence of positive IH and/or FC. IH was the most sensitive marker with only one case being negative in the presence of a positive result by FC and/or CC. The results highlight the necessity of employing more than one method in determining the presence of MPO and confirm the important role of IH in the diagnosis of AML particularly in cases where MPO is not detected by CC and FC.

Molecular characterization of field isolates and vaccine strains of infectious bursal disease virus.

The present investigation was conducted to study the genetic heterogenicity and molecular polymorphism among the field isolates and vaccine strains of infectious bursal disease virus (IBDV). Samples of bursa of Fabricius from 15 suspected outbreaks of infectious bursal disease (IBD) were subjected to agar gel precipitation test (AGPT), virus isolation and reverse transcription-polymerase chain reaction (RT-PCR) combined with restriction fragment length polymorphism (RFLP). Nine out of 15 samples were found positive in AGPT while 14 were found positive both by virus isolation and RT-PCR. PCR amplified 474bp fragment from the variable region of VP2. Sac I, Stu I, Alu I, Ssp I and Mbo I restriction enzymes were used for characterization of all the 14 IBDV isolates and four reference vaccine strains. Sac I, Stu I, Alu I and Ssp I could differentiate classical virulent IBD (cvIBD) vaccine virus strains from very virulent IBD (vvIBD) field isolates by their varying restriction patterns. Based on above results two field isolates (VPL and VMK) were placed in cvIBD virus group and 12 field isolates were placed in vvIBD virus group. Virus neutralisation test (VNT) using rabbit raised Georgia strain anti-serum, however, could not differentiate between cvIBD virus and vvIBD virus. It was concluded that RT-PCR combined with RFLP assay using restriction enzymes Sac I, Stu I, Alu I and Ssp I can be used for rapid differentiation and classification of field isolates of IBDV.

Prognostic features for response and survival in elderly patients with de novo acute myeloid leukemia treated with mitoxantrone and intermediate dose cytarabine.

Forty-three fit elderly patients with de novo acute myeloid leukemia (AML) received chemotherapy with mitoxantrone and intermediate dose cytarabine (MIDAC) in a phase II clinical trial conducted by the Australasian Leukaemia and Lymphoma Group. The main aim of the study was to evaluate the tolerability and efficacy of MIDAC in inducing durable remissions. While the chemotherapy was generally well tolerated, less than half the patients achieved complete remission (CR) after induction and many of those in CR could not receive planned consolidation cycles. The median overall survival for all patients was 6.5 months and the median disease-free survival for those achieving CR was 8.3 months. Only 2 patients survived beyond 4 years. Factors significantly associated with shorter survival were adverse cytogenetics, marrow dysplasia and increasing age. These results suggest that only selected elderly patients with AML are likely to benefit from aggressive chemotherapy and that novel therapies are required to improve the poor prognosis of this group.

Pure red cell aplasia with the onset of graft versus host disease.

Pure red cell aplasia (PRCA) occurred in the fourth month after an ABO-compatible nonmyeloablative allograft coincident with the cessation of immunosuppression and the onset of limited chronic GVHD. No secondary causes could be identified. Erythropoiesis was restored promptly and durably with the resumption of immunosuppression. A clonal T cell receptor gamma rearrangement was detected in peripheral blood lymphocytes prior to the onset of PRCA. PRCA should be added to the list of immunohaematological complications of GVHD.

Spontaneous rupture of the spleen as the presenting feature of the blastoid variant of mantle cell lymphoma.

Spontaneous splenic rupture is rare, and particularly so as the initial presentation of a lymphoproliferative disorder. Although rare cases of splenic rupture have been reported in mantle cell lymphoma there has not been a report of the blastoid variant presenting in this manner. We report such a case in a 64-year-old man.

Bone marrow immunohistology of plasma cell neoplasms.

The application of immunohistology to the spectrum of plasma cell disorders has yet to be incorporated widely into routine haematology practice. This technique enables the direct visualisation of specific surface and cytoplasmic antigens in the context of the individual cell and the surrounding anatomical neighbourhood. This review outlines the role of bone marrow immunohistology in the laboratory evaluation of patients with suspected and established plasma cell neoplasms and its emerging role in understanding myeloma biology for possible future therapeutic application.

Morphological effects of imatinib mesylate (STI571) on the bone marrow and blood of patients with Philadelphia chromosome (Ph) positive chronic myeloid leukaemia.

There have been few reports on the morphological findings in patients with chronic myeloid leukaemia (CML) undergoing treatment with imatinib mesylate. We examined morphological changes in the marrow and peripheral blood of 27 patients with chronic phase (CP), accelerated phase (AP) and blastic phase (BP) CML, 3 and 6 months after treatment with imatinib. At 3 months there was a significant clearance of leukaemic cells as evidenced by a complete haematological response (CHR) in the peripheral blood in 25 patients, together with reduced marrow cellularity in 25 (median reduction CP 42%; AP/BP 68%) and a reduction in the number of megakaryocytes (13 of 18 CP: five of six AP/BP; three patients did not have an assessable marrow) with an increase in the amount of normal megakaryopoiesis. After 6 months, there was continued morphological improvement in eight of 17 CP patients (one patient died after 3 months) with continued cytogenetic response (7 out of 15 patients with assessable metaphases had no abnormal Ph+ cells and three had <35% Ph+ cells) and maintenance of haematologic response in all patients. After an initial response to treatment at 3 months in the AP/BP group, with CHR and a reduction in cellularity in all patients, we found morphological evidence of a loss of response to treatment, with an increase in leukaemic cells, as evidenced by loss of CHR in three of nine and an increase in median cellularity in five patients. No patient in this group achieved a complete cytogenetic response. In summary, in CML-CP patients treated with 6 months of imatinib, there was a significant reduction in leukaemic cells as evidenced by a haematolological response in the peripheral blood, together with reduced marrow cellularity, restoration of morphologically normal haemopoiesis and a meaningful cytogenetic response. Maintaining a response to treatment appeared less likely to occur in the AP/BP group patients, especially those who did not achieve any cytogenetic response to treatment.

Optimum trephine length in the assessment of bone marrow involvement in patients with diffuse large cell lymphoma.

BACKGROUND: The National Cancer Institute has recommended a bone marrow biopsy length of >/=20 mm for the staging and surveillance of patients with non-Hodgkin's lymphoma. However, there are few published data to support this recommendation, particularly the role of examining multiple levels. PATIENTS AND METHODS: Bone marrow biopsies from 172 patients with newly diagnosed diffuse large cell lymphoma (DLCL) entered in two consecutive trials of the Australasian Leukaemia and Lymphoma Group were analysed. The original haematoxylin and eosin-stained trephine biopsy and two or more deeper sections cut at 0.1-0.2 mm intervals were assessed with respect to the morphology, extent and pattern of lymphomatous involvement. The rate of positive diagnosis was correlated with the length of the biopsy specimen and the number of sections examined. RESULTS: Forty-seven biopsies (27%) demonstrated marrow involvement on examination of a mean of four trephine biopsy sections. The rate of positivity increased with the examination of multiple levels and correlated with increasing trephine length but was not dependent on the number of sites sampled. Twenty per cent of biopsies <20 mm in length were positive for lymphoma; this increased to 35% for biopsies >/=20 mm (P = 0.023). CONCLUSIONS: Morphological bone marrow involvement in DLCL is optimally demonstrated by a 20-mm long trephine biopsy from a single site which is examined at multiple levels (four or more). This obviates the need for bilateral sampling, thereby reducing patient morbidity from the procedure. This study provides evidence to support the National Cancer Institute recommendations regarding trephine biopsy in the staging of DLCL, providing multiple levels are examined.

Portal or hepatic vein thrombosis as the first presentation of a myeloproliferative disorder in patients with normal peripheral blood counts.

Myeloproliferative disorders (MPD) are associated with an increased risk of thrombotic complications. We describe three patients with portal or hepatic vein thrombosis and normal peripheral blood counts who had MPD on bone marrow morphology and growth factor-independent megakaryocyte or erythroid colony growth in vitro. The peripheral blood counts have become abnormal subsequently in two patients. Patients presenting with unexplained portal or hepatic vein thrombosis should be investigated systematically for the presence of a MPD, which may not be apparent using conventional diagnostic criteria.

Sequential transformation of t(8;13)-related disease: a case report.

We report a case of an atypical myeloproliferative disorder with t(8;13) that presented as B-lineage acute lymphoblastic leukaemia (B-ALL). Following induction chemotherapy, the disease manifested as chronic myeloproliferative state, which responded to hydroxyurea. Terminally, the disease transformed into acute myeloid leukaemia (AML) with additional chromosomal abnormalities including monosomy 7. To our knowledge, this is the first case of this rare atypical myeloproliferative disorder with t(8;13) that presented as B-ALL and terminally transformed to AML with additional chromosomal abnormalities.