Treatment outcome of tuberculosis patients detected using accelerated vs. passive case finding in Myanmar.

SETTING: Several projects involving accelerated or active case finding (ACF) of tuberculosis (TB) cases are being implemented in Myanmar. However, there is a concern that patients detected using ACF have poorer TB treatment outcomes than those detected using passive case finding (PCF). OBJECTIVE: To assess differences in the demographics, clinical profile and treatment outcomes of patients detected using ACF and PCF. DESIGN: Retrospective cohort study of TB patients diagnosed and enrolled for treatment during 2014-2016. RESULTS: Of 16 048 patients enrolled, 2226 (16%) were detected using ACF; the treatment success rate (cured and completed) was 88%. A higher proportion of cases detected using ACF were aged 55 years, human immunodeficiency virus (HIV) negative and sputum smear-positive pulmonary TB. After adjusting for differences in demographic and clinical characteristics, we found that treatment outcomes in patients detected using ACF and PCF were not significantly different (adjusted relative risk [aRR] 0.89, 95%CI 0.78-1.00). Male sex, age  55 years, patients with a previous history of TB and HIV positivity were independently associated with unsuccessful outcomes. CONCLUSION: ACF detected a significant proportion of TB cases in study townships; treatment outcomes in cases detected using ACF and those detected using PCF were similar. More tailored interventions are needed to improve treatment outcomes in patients at a higher risk of unsuccessful treatment outcomes.

Sickle liver disease--an unusual presentation in a compound heterozygote for HbS and a novel beta-thalassemia mutation.

A 38-year-old Ghanaian man presented with a 6-month history of worsening pruritus, jaundice, and ascites. He was previously fit and well and rarely drank alcohol. Screening tests for chronic liver disease including viral, autoimmune, and other metabolic causes including iron overload were unremarkable. A liver biopsy performed at the referring hospital demonstrated intralobular cholestasis and cirrhosis. He was listed for liver transplantation but subsequently developed sepsis with multiple organ failure and died. The sickle solubility test was positive. Blood smear showed cells consistent with liver failure and no sickle cells. Hemoglobin electrophoresis revealed HbA2 2.8%, HbF 0.5%, and HbS greater than HbA (49.6% vs. 41.3%) in the absence of blood transfusion. Sequence analysis of the beta-globin genes showed he was a compound heterozygote for the Hbs mutation at codon 6 (CAG --> GTG) and a novel mutation at position 844 of intron 2 (betaIVS2-844 C --> A). A diagnosis of sickle hepatopathy causing decompensated cirrhosis was made. This case is unusual insomuch as this patient was asymptomatic for over 35 years and represents a novel presentation of sickle cell disease. Sickle cell disease should be considered in appropriate patients when unusual presentations of liver disease arise.

Determinants of iron status and bilirubin levels in congenital dyserythropoietic anaemia type I.

Seven untransfused patients with congenital dyserythropoietic anaemia type I were investigated to assess the determinants of both iron overload and serum bilirubin levels. The serum ferritin concentration was increased in all patients and non-transferrin-bound iron (NTBI) was increased in all but one patient. None of the patients showed the C282Y mutation in the hereditary haemochromatosis gene, HFE. One patient was homozygous for the H63D mutation in this gene. The data indicated that differences in the extent of iron overload were not mediated by co-inheritance of the C282Y mutation in the HFE gene but could largely be explained by differences in the severity of anaemia and ineffective erythropoiesis, and in the age of the patient. In one patient an unusually high plasma bilirubin level was associated with the variant A[TA]7TAA configuration in the TATA box of the uridine diphosphate glucuronosyltransferase (UGT-1A) gene promoter, the mutation found in most patients with mild Gilbert's syndrome.

An in vitro system for expression analysis of mutations of the beta-globin gene: validation and application to two mutations in the 5' UTR.

We describe the setting up of an in vitro expression system for the analysis of mutations of the beta-globin gene. The system is based on the stable transfection of a normal or mutated beta-globin gene into mouse erythroleukaemia (MEL) cells. The expression construct contains an Agamma gene as an internal control and both globin genes are under the control of the HS2 element of the beta LCR. The system enables analysis of transcription, RNA processing and transport, as well as mRNA stability. With non-mutant genes, high-level expression of both beta and Agamma genes is seen and both mRNAs are stable. The system was validated by comparing the expression of the beta654 thalassaemia splicing mutation in MEL cells with its well-characterized expression in vivo. The level of the initial transcript, the proportion of abnormally spliced mRNA and its instability during erythroid cell maturation were all faithfully reproduced. The system was used to examine the mechanism by which two mutations in the beta-globin 5' untranslated region (5' UTR) result in beta thalassaemia. Surprisingly, the mechanism appeared to differ in the two cases, with the C-G substitution at position +33 affecting transcription, whereas the -T deletion at position +10 resulted in a translational defect. The stably transfected MEL cells, with an internal control and an endogenous enhancer, appear to be a valid and realistic experimental model, superior to transient expression studies. This system should find wide application in the analysis of the effects and mechanisms of gene inactivation in mutations affecting the beta-globin as well as other genes.

Typing of urinary JC virus DNA offers a novel means of tracing human migrations.

Although polyomavirus JC (JCV) is the proven pathogen of progressive multifocal leukoencephalopathy, the fatal demyelinating disease, this virus is ubiquitous as a usually harmless symbiote among human beings. JCV propagates in the adult kidney and excretes its progeny in urine, from which JCV DNA can readily be recovered. The main mode of transmission of JCV is from parents to children through long cohabitation. In this study, we collected a substantial number of urine samples from native inhabitants of 34 countries in Europe, Africa, and Asia. A 610-bp segment of JCV DNA was amplified from each urine sample, and its DNA sequence was determined. A worldwide phylogenetic tree subsequently constructed revealed the presence of nine subtypes including minor ones. Five subtypes (EU, Af2, B1, SC, and CY) occupied rather large territories that overlapped with each other at their boundaries. The entire Europe, northern Africa, and western Asia were the domain of EU, whereas the domain of Af2 included nearly all of Africa and southwestern Asia all the way to the northeastern edge of India. Partially overlapping domains in Asia were occupied by subtypes B1, SC, and CY. Of particular interest was the recovery of JCV subtypes in a pocket or pockets that were separated by great geographic distances from the main domains of those subtypes. Certain of these pockets can readily be explained by recent migrations of human populations carrying these subtypes. Overall, it appears that JCV genotyping promises to reveal previously unknown human migration routes: ancient as well as recent.

Erythroblastic inclusions in dominantly inherited beta thalassemias.

While the precipitation of unstable variant beta-globin chains has been implicated as a major pathogenic mechanism in dominantly inherited beta thalassemia, their instability and presence in intra-erythroblastic inclusions have not been conclusively shown. We report the investigation of two cases of dominantly inherited beta thalassemia due to heterozygosity for the beta-codon 121 G-T mutation. In one case, we were able to demonstrate the presence of an abnormal beta-globin chain in both peripheral blood reticulocytes and bone marrow erythroblasts, and to assess its stability in relation to the substantial amounts of mutant beta mRNA transcript. The serum transferrin receptor (TfR) level was markedly increased, an indication of increased erythropoietic activity. In both cases, we could show by immunoelectron microscopy that the intra-erythroblastic inclusion bodies, a prominent feature of diseases in this category, contained not only precipitated alpha-globin chains, but also beta chains. The data confirm previous suggestions that the cellular pathology underlying this group of beta thalassemias is related to the synthesis of highly unstable beta-globin chain variants, which fail to form functional tetramers and precipitate intracellularly with the concomitant excess alpha chains, leading to increased ineffective erythropoiesis.

Myelodysplastic syndrome with karyotype abnormality is associated with elevated F-cell production.

A sensitive F-cell assay has been used to examine the production of fetal haemoglobin (Hb F) in a group of 77 adult patients with myelodysplastic syndrome (MDS), and a control group composed of 100 normal blood donors. Although the mean F-cell percentage in the MDS group (6.0%) is not statistically different from that in the normal blood donors (3.1%), a higher proportion of myelodysplastic patients have elevated F-cell values and the magnitude of the increases is greater than that observed in blood donors. In order to investigate the association further, the karyotypes of the MDS patients have been examined. 13/21 (61.9%) of the MDS patients with karyotypic abnormalities have F-cell values > 5%, compared to only 6/56 (10.9%) of the MDS patients with a normal karyotype and 11/100 (11%) of the blood donors. The observed difference in the distributions of F cells between the two subgroups of patients with MDS is highly significant (P < 0.0001).

Somatic mutations detected by mini- and microsatellite DNA markers reveal clonal intratumor heterogeneity in gastrointestinal cancers.

We investigated clonal intratumor heterogeneity by comparing different areas of each tumor in 20 gastrointestinal cancers from female patients (1 esophageal cancer, 5 stomach cancers, and 14 colorectal cancers). In all 19 cases informative for X-inactivation analysis with the M27 beta and/or the phosphoglycerate kinase probes, the tumors were clonal. Separate areas from a given tumor showed identical X-inactivation patterns, providing evidence for its single-cell origin. Of 20 cancers, 11 showed p53 gene mutations (base pair insertions, point mutations, and one case of a base pair deletion) in exons 5-8. A particular p53 gene mutation was identical in all tumor areas investigated per case. The minisatellite probes detected loss of heterozygosity or new mutant alleles at 1p33, 1q21, 5q35, 17p13, or 18q21. In seven cases mutations at particular loci were restricted to one or two areas per tumor, while in another seven cases they were common to all tumor areas. Loss of heterozygosity or new alleles detected at the microsatellite loci D2S123, D3S1611, D5S107, D17S261, or D18S34 [(CA)n repeats] were common to all tumor areas in 7 of 19 cases. In another seven cases, however, microsatellite mutations at these loci were restricted to one to three areas per tumor. Tracing clonal intratumor heterogeneity would permit one to study the hierarchy of mutational events in cancers where no premalignant lesions can be harvested. Most important, our study indicates that clonal intratumor heterogeneity might lead to sampling errors in the molecular diagnosis of cancer biopsy specimens when using mini- or microsatellite markers.

Beta-thalassemia unlinked to the beta-globin gene in an English family.

An inherited hypochromic microcytic anemia transmitted in an autosomal manner has been observed in three generations of an English family. Affected members had the hallmarks of heterozygous beta-thalassemia, ie, elevated levels of hemoglobin A2 and imbalanced globin chain synthesis. However, despite extensive sequence analysis, no mutations could be found in or around the beta-globin genes of either the propositus or two other affected members from two different generations. Linkage analysis using restriction fragment length polymorphisms in the beta-globin gene cluster clearly showed that the gene responsible for the beta-thalassemia phenotype segregates independently of the beta-gene complex. Therefore, this condition represents a novel form of the disease.

Clonal allele loss in gastrointestinal cancers.

Using a panel of DNA probes for hypervariable DNA regions we screened 52 gastrointestinal carcinomas for clonal allele losses on chromosomes 1, 5, 7, 12, 16 and 17. A total of 24/35 informative cases of colorectal cancers showed loss of constitutional heterozygosity at a locus on chromosome 17p, while 9/31 cases informative for a locus on 5q showed allele loss. Loss of sequences at 5q was linked to allele loss at 17p with a single exception. In gastric cancers loss of heterozygosity most frequently occurred at 1q (5/10 tumours) and at 12q (6/11 tumours). Gastrointestinal tumours show consistent chromosomal losses and the loci involved are different in gastric and colorectal cancers.

Analysis of somatic mutations at human minisatellite loci in tumors and cell lines.

Hypervariable human minisatellite loci show a substantial level of germline instability, and spontaneous mutation rates to new length alleles have been measured directly by pedigree analysis. We now show that mutation events altering the number of minisatellite repeat units are not restricted to the germline, but also arise in other tissues. Mutant alleles can be detected at a very low frequency in lymphoblastoid cell lines and at much higher frequencies in clonal tumor cell populations, most particularly in gastrointestinal adenocarcinomas. Mutant alleles in these tumors are usually present at a dosage equal to or greater than that of the progenitor allele, indicating that most or all of the tumor cells carry the same clonally derived mutant allele. As with germline mutation, the incidence of somatic mutations in tumors varies from locus to locus, with the same locus showing the highest level of germline and somatic instability. Most length changes, as those in the germline, are of only a few repeat units; however, very large changes are also observed, implying that such mutations can occur in the absence of meiosis.

Assessment of clonality in gastrointestinal cancer by DNA fingerprinting.

DNA fingerprinting with three different probes (33.15, 33.6, and alpha-globin 3'HVR) was investigated as a method for the determination of clonality in gastrointestinal tumors. In 29/44 carcinomas the tumor DNA showed clonal somatic mutations that were not seen in the corresponding peripheral blood and normal mucosa samples. The changes consisted of either novel fingerprint bands, losses of bands, or both. The probe 33.15 yielded the highest rate of abnormal DNA fingerprints (21/44 carcinomas). Sequential use of the probes increased the number of cases where clonal fingerprint markers could be detected. One out of five colorectal adenomas also showed a clonal loss of a fingerprint band. In two cases of gastric cancer, DNA from the metastatic tumor had a different DNA fingerprint from that found in the primary carcinoma. DNA fingerprinting offers a novel approach to determining clonality in tumors and may prove useful for the study of tumor progression.

Detection of chromosomal 7 loss in myelodysplasia using an extremely polymorphic DNA probe.

Chromosomal loss is a characteristic feature of the myelodysplastic syndromes (MDS). A method is described which detects chromosomal 7 loss in MDS by DNA analysis using a specific hypervariable region gene probe which has been cloned from a human DNA fingerprint. Loss of one of the chromosomal 7 homologues was demonstrated in 10/118 MDS patients; the ten patients include all the five patients which had previously been shown to have monosomy 7 by cytogenetic analysis. This technique makes it feasible to study serial samples from large numbers of patients for loss of chromosomal material and could be readily applied to the study of other human malignancies.

Identical twin marrow transplantation for 5 patients with chronic myeloid leukaemia: role of DNA finger-printing to confirm monozygosity in 3 cases.

5 patients with chronic myeloid leukaemia (CML) have been treated by BMT from identical twin donors. Syngeneity of the twins was established by conventional methods in the first 2 patients. These included the similarity of phenotype, dermatoglyphics and analysis of red cell isoenzymes and blood groups. 2 of the other patients had received multiple blood transfusions prior to referral for BMT thereby invalidating red cell analysis. However genetic identity was confirmed in these patients by the method of DNA 'finger-printing' which demonstrated identical restriction-fragment length polymorphisms. Conclusive proof of syngeneity in twins prior to BMT is important since it obviates the need for T-cell depletion and/or post-graft immunosuppression to prevent graft-versus-host disease (GVHD). All 5 patients were conditioned with high-dose chemoradiotherapy prior to BMT and all patients are alive and disease-free at a median follow-up of 12 months. In conclusion, we report a new, reliable method for determining syngeneity of twins which has bearing on the technical approach to BMT.

Detection of somatic changes in human cancer DNA by DNA fingerprint analysis.

Minisatellite DNA probes which can detect a large number of autosomal loci dispersed throughout the human genome were used to examine the constitutional and tumour DNA of 35 patients with a variety of cancers of which eight were of gastrointestinal origin. Somatic changes were seen in the tumour DNA in ten of the 35 cases. The changes included alterations in the relative intensities of hybridising DNA fragments, and, in three cases of cancers of gastrointestinal origin, the appearance of novel minisatellite fragments not seen in the corresponding constitutional DNA. The results of this preliminary study suggests that DNA fingerprint analysis provides a useful technique for identifying somatic changes in cancers.