Volunteer unrelated donor experience after administration of filgrastim and apheresis for the collection of haemopoietic stem cells: the Australian perspective.

BACKGROUND: Voluntary donations of peripheral blood stem cells after administration of filgrastim (granulocyte-colony stimulating factor, G-CSF) are undertaken throughout the world by healthy individuals, but the short-, medium- and long-term adverse events during and after donation are not fully understood. AIMS: We document the experience of donors of peripheral blood stem cells mobilised by G-CSF at Australian Bone Marrow Donor Registry collection centres. METHODS: When the Australian Bone Marrow Donor Registry commenced collecting mobilised peripheral blood stem cells, based on data used for registration of G-CSF, all adverse reactions in donors were documented prospectively to determine the rate and severity of events. A total of 512 consecutive first-time donors assessed between July 2001 and March 2010 were included in this study. RESULTS: The median age at work-up was 40 years and 71% of donors were male. A large proportion of donors (91%) experienced bone pain during administration of G-CSF, and in fewer numbers headache (61%) and fatigue (61%). Bone pain was associated with a body mass index of overweight/obese (P = 0.03). Headache (P = 0.03), muscle pain (P = 0.03) and fatigue (P = 0.001) were all significantly associated with female sex. More than a quarter (28%) of donations involved a range of complications at collection. CONCLUSION: The incidence of short- and medium-term symptoms and events observed provide support for the information provided to unrelated donors at counselling. Follow up of the consequences of unrelated voluntary donation remains important to provide accurate and relevant information to prospective donors.

The largest prison outbreak of TB in Western Europe investigated using whole-genome sequencing.

BACKGROUND: In March 2011, the Department of Public Health East in Ireland were notified of two cases of TB in two prisoners sharing a cell. We define the resulting outbreak and highlight the role of public health and laboratory-based molecular epidemiology in mapping and control of a prison outbreak.METHODS: Cases were identified through clinical presentation, contact tracing, case-finding exercise or enhanced laboratory surveillance. Mycobacterium tuberculosis isolates were genotyped and underwent whole-genome sequencing (WGS).RESULTS: Of the 34 cases of TB linked to the outbreak, 27 were prisoners (79%), 4 prison officers (12%) and 3 community cases (9%). M. tuberculosis was isolated from 31 cases (culture positivity: 91%). A maximum of six single-nucleotide polymorphisms separated the isolates, with 22 being identical, suggestive of a highly infectious 'super-spreader´ within the prison. Isolates belonged to the Beijing sub-lineage, and were susceptible to first-line anti-TB agents. A case-finding exercise incidentally detected a prisoner with multidrug-resistant TB. Of the 143 prison officers screened, 52% had latent TB infection. Litigation costs exceeded five million euros.CONCLUSION: This constitutes the largest prison outbreak of TB in Western Europe investigated using WGS. A robust prison entry TB screening and education programme is required to effect better TB control, and prevent future outbreaks and attendant litigation.

Effect of skin testing and segregation on the prevalence of bovine tuberculosis, and molecular typing of Mycobacterium bovis, in Ethiopia.

In 2002, the prevalence of bovine tuberculosis (tb) among 500 cattle on Holeta Farm, near Addis Ababa, Ethiopia, was 48 per cent, and the farm was divided into positive and negative herds. After three consecutive rounds of skin testing and segregation of skin test-positive and -negative animals, the prevalence of bovine tb was reduced from 14 per cent to 1 per cent in the negative herd in a year. Spoligotyping of 41 isolates from 17 cows gave an identical and unique spoligotype pattern, which can be represented as the binary number 1100000101111110111111100010000000000100000, where 1 indicates the presence of a spacer and 0 represents a loss. This spoligotype pattern had not previously been reported on the Mycobacterium bovis spoligotype database, and it was therefore designated SB1176, Ethiopian M bovis strain 1 (EMbs1). The variable number tandem repeat (VNTR) profile of the strain was 5254(*)33.1, which differed from the VNTR profile of strains reported in Great Britain.

Tuberculosis Caused by Mycobacterium orygis in Dairy Cattle and Captured Monkeys in Bangladesh: a New Scenario of Tuberculosis in South Asia.

Mycobacterium orygis, commonly known as the oryx bacillus and a newly proposed Mycobacterium tuberculosis complex subspecies, was isolated from 18 cattle in a dairy farm and two captured rhesus monkeys in a zoo in Bangladesh. All the infected animals had tuberculosis lesions in their lungs, suggesting transmission and infection with M. orygis by an airborne route. The 20 isolates were analysed using a range of conventional and molecular typing methods, and RD-deletion typing and sequencing of selected genes confirmed the isolates as M. orygis. Multiple-locus variable-number tandem repeat analysis (MLVA) allowed the isolates to be divided into three clusters based on the relatedness of their MLVA profiles. The two monkey isolates shared the same MLVA pattern with 15 of the cattle isolates, whereas the remaining three cattle isolates had different patterns, even though the latter animals had been kept in the same dairy farm. The diversity observed among isolates may suggest the bacteria have been established in this area for a long period. This study along with other recent findings that report the detection of M. orygis from animals as well as humans originating from South Asia potentially indicate endemic distribution of M. orygis in South Asia.

The evolution of mycobacterial pathogenicity: clues from comparative genomics.

Comparative genomics, and related technologies, are helping to unravel the molecular basis of the pathogenesis, host range, evolution and phenotypic differences of the slow-growing mycobacteria. In the highly conserved Mycobacterium tuberculosis complex, where single-nucleotide polymorphisms are rare, insertion and deletion events (InDels) are the principal source of genome plasticity. InDels result from recombinational or insertion sequence (IS)-mediated events, expansion of repetitive DNA sequences, or replication errors based on repetitive motifs that remove blocks of genes or contract coding sequences. Comparative genomic analyses also suggest that loss of genes is part of the ongoing evolution of the slow-growing mycobacterial pathogens and might also explain how the vaccine strain BCG became attenuated.

Genomics of Mycobacterium bovis.

The imminent completion of the genome sequence of Mycobacterium bovis will reveal the genetic blueprint for this most successful pathogen. Comparative analysis with the genome sequences of M. tuberculosis and M. bovis BCG promises to expose the genetic basis for the phenotypic differences between the tubercle bacilli, offering unparalleled insight into the virulence factors of the M. tuberculosis complex. Initial analysis of the sequence data has already revealed a novel deletion from M. bovis, as well as identifying variation in members of the PPE family of proteins. As the study of bacterial pathogenicity enters the postgenomic phase, the genome sequence of M. bovis promises to serve as a cornerstone of mycobacterial genetics.

Comparative genomics of the leprosy and tubercle bacilli.

To achieve the quantum leap in understanding required to overcome two major human diseases, leprosy and tuberculosis, systematic and comparative genome analysis has been undertaken. New insight into the biology of their causative agents has been obtained and the principle findings are reported here.

Response of Mycobacterium smegmatis to acid stress.

Evidence was sought for the existence of an inducible acid tolerance response in Mycobacterium smegmatis. Exposure of M. smegmatis to a sub-lethal, adaptive acidic pH was found to confer a significant level of protection against subsequent exposure to a lethal pH, compared to unadapted cells. Adaptation was dependent on de novo protein synthesis.

Symmetrical peripheral gangrene: a new presentation of an old disease.

This report concerns two cases and a review of the literature on the subject of symmetrical peripheral gangrene. Symmetrical peripheral gangrene is defined as symmetrical distal ischemic damage in two or more sites in the absence of major vascular occlusive disease. It occurs in patients who are septic and have disseminated intravascular coagulation and in nonseptic patients who have cardiogenic or hypovolemic shock. The syndrome is devastating and rare, and controlled studies of its etiology and management are lacking. Recommendations are presented for its prevention and treatment. Cooperative multicenter studies may be necessary to obtain valid data about its prevention and management.

The phylogeny and population structure of Mycobacterium bovis in the British Isles.

To further understand the epidemic of bovine tuberculosis in Great Britain, Northern Ireland and the Republic of Ireland, we identified 16 mutations that are phylogenetically informative for Mycobacterium bovis strains from these regions. We determined the status of these mutations among a collection of 501 strains representing the molecular diversity found in these three regions of the British Isles. The resulting linear phylogenies from each region were concordant, showing that the same lineage of M. bovis was present. The dominance of this lineage is unique within Europe, and suggests that in the past the populations were homogenous. Comparison of approximately 500 strains isolated in 2005 from each region by spoligotype and 5 locus VNTR profiling, revealed distinct differences in the genotype frequencies and sub-lineage makeup between each region. We concluded that whilst each region shared the same major phylogenetic lineage of M. bovis, more recent evolution had resulted in the development of region-specific populations. Regional differences in the M. bovis populations suggest that it may be possible to identify the movement of strains from one region to another.

Exploring the use of molecular epidemiology to track bovine tuberculosis in Nigeria: an overview from 2002 to 2004.

Tuberculosis remains a major public health problem in Nigeria. While human to human transmission of Mycobacterium tuberculosis is clearly of major importance in driving the tuberculosis epidemic in Nigeria, the impact of Mycobacterium bovis transmission from infected cattle is largely unknown. Molecular epidemiology of M. bovis in Nigeria will increase our understanding of this endemic disease and provide tools to assess cattle-to-human transmission. Between 2002 and 2004, molecular techniques including spoligotyping, variable number of tandem repeats (VNTR) typing and deletion typing were used to track and analyze a sample of strains of the M. tuberculosis complex circulating in the cattle population in Ibadan, Southwestern Nigeria. In all, 180 isolates were typed with a view to elucidating epidemiological information on circulating strains, occurrence of transborder transmission and molecular diversity of the M. bovis strains. Results obtained showed that 99% (178/180) of the isolates were M. bovis, while the remaining were M. tuberculosis and M. africanum. In all, strains of M. bovis had 34 different spoligotypes: strains with spoligotype pattern SB0944 (as designated by www.mbovis.org) were the most common (46% of strains). This molecular type is also common in countries neighbouring Nigeria. Strains with this spoligotype pattern could be further divided into 40 different VNTR types. This analysis shows the value of simple molecular epidemiological techniques applied to strains of M. bovis and suggests that further epidemiological studies will shed more light on the transmission dynamics of bovine tuberculosis locally and across neighbouring African countries.

Antigen mining to define Mycobacterium bovis antigens for the differential diagnosis of vaccinated and infected animals: A VLA perspective.

The urgency for new and improved cattle vaccines and diagnostic reagents has been acknowledged by the UK Government, and development of cattle vaccine is a research priority. Significant progress has been made to develop specific antigens that allow the differentiation of bacille Calmette-Guerin (BCG) vaccinated and M. bovis-infected cattle [diagnosis of infected from vaccinated individuals (DIVA) test]. This progress has been greatly facilitated by the completion of the genome sequences of Mycobacterium tuberculosis, M. bovis and BCG Pasteur. In this study, we describe how we applied this knowledge, through comparative genome and transcriptome analysis, to define DIVA antigens that complemented the prototype DIVA antigens ESAT-6 and CFP-10 by increasing their test sensitivity. In addition, we draw general conclusions from our experience, and discuss potential future approaches in this area.

Field evaluation of a novel differential diagnostic reagent for detection of Mycobacterium bovis in cattle.

In the search for improved tools with which to control bovine tuberculosis, the development of enhanced immunodiagnostic reagents is a high priority. Such reagents are required to improve the performance of tuberculin-based reagents and allow the discrimination of vaccinated cattle from those infected with Mycobacterium bovis. In this study, we identified the immunodominant, frequently recognized peptides from Rv3873, Rv3879c, Rv0288, and Rv3019c, which, together with peptides comprising the current lead diagnostic antigens, ESAT-6 and CFP-10, were formulated into a peptide cocktail. In a test of naturally infected cattle, this cocktail was significantly better than tuberculin was for identifying skin test-negative animals with confirmed bovine tuberculosis. In addition, the specificity of this cocktail was not compromised by Mycobacterium bovis BCG vaccination. In summary, our results prioritize this peptide-based, fully synthetic reagent for assessment in larger trials.

Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays.

Whole-genome comparisons of the tubercle bacilli were undertaken using ordered bacterial artificial chromosome (BAC) libraries of Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG-Pasteur, together with the complete genome sequence of M. tuberculosis H37Rv. Restriction-digested BAC arrays of M. tuberculosis H37Rv were used in hybridization experiments with radiolabelled M. bovis BCG genomic DNA to reveal the presence of 10 deletions (RD1-RD10) relative to M. tuberculosis. Seven of these regions, RD4-RD10, were also found to be deleted from M. bovis, with the three M. bovis BCG-specific deletions being identical to the RD1-RD3 loci described previously. The distribution of RD4-RD10 in Mycobacterium africanum resembles that of M. tuberculosis more closely than that of M. bovis, whereas an intermediate arrangement was found in Mycobacterium microti, suggesting that the corresponding genes may affect host range and virulence of the various tubercle bacilli. Among the known products encoded by these loci are a copy of the proposed mycobacterial invasin Mce, three phospholipases, several PE, PPE and ESAT-6 proteins, epoxide hydrolase and an insertion sequence. In a complementary approach, direct comparison of BACs uncovered a third class of deletions consisting of two M. tuberculosis H37Rv loci, RvD1 and RvD2, deleted from the genome relative to M. bovis BCG and M. bovis. These deletions affect a further seven genes, including a fourth phospholipase, plcD. In summary, the insertions and deletions described here have important implications for our understanding of the evolution of the tubercle complex.

Comparative genomics of the mycobacteria.

The genus mycobacteria includes two important human pathogens Mycobacterium tuberculosis and Mycobacterium lepra. The former is reputed to have the highest annual global mortality of all pathogens. Their slow growth, virulence for humans and particular physiology makes these organisms extremely difficult to work with. However the rapid development of mycobacterial genomics following the completion of the Mycobacterium tuberculosis genome sequence provides the basis for a powerful new approach for the understanding of these organisms. Five further genome sequencing projects of closely related mycobacterial species with differing host range, virulence for humans and physiology are underway. A comparative genomic analysis of these species has the potential to define the genetic basis of these phenotypes which will be invaluable for the development of urgently needed new vaccines and drugs. This minireview summarises the different techniques that have been employed to compare these genomes and gives an overview of the wealth of data that has already been generated by mycobacterial comparative genomics.

Identification of novel Mycobacterium tuberculosis antigens with potential as diagnostic reagents or subunit vaccine candidates by comparative genomics.

An independent review for the British government has concluded that the development of a cattle vaccine against Mycobacterium bovis holds the best long-term prospects for tuberculosis control in British herds. The development of complementary diagnostic tests to differentiate between vaccinated and infected animals is necessary to allow the continuation of test-and-slaughter-based control policies alongside vaccination. Vaccination with M. bovis bacillus Calmette-Guérin (BCG), the only available vaccine, results in tuberculin purified protein derivative sensitivity and has shown varying vaccine efficacies in cattle. Thus, identification of more-specific reagents to distinguish between vaccination and infection, as well as the identification of subunit vaccine candidates for improved tuberculosis vaccines, is a research priority. In the present study, we applied comparative genomics to identify M. bovis-Mycobacterium tuberculosis antigens whose genes had been deleted in BCG Pasteur. In total, 13 open reading frames (ORFs) from the RD1, RD2, and RD14 regions of the M. tuberculosis genome were selected. Pools of overlapping peptides spanning these ORFs were tested in M. bovis-infected (n = 22), BCG-vaccinated (n = 6), and unvaccinated (n = 10) control cattle. All were recognized in infected cattle, with responder frequencies varying between 16 and 86%. In particular, eight highly immunogenic antigens were identified whose potentials as diagnostic reagents or as subunit vaccines warrant further study (Rv1983, Rv1986, Rv3872, Rv3873, Rv3878, Rv3879c, Rv1979c, and Rv1769).

Analysis of the proteome of Mycobacterium tuberculosis in silico.

Novel bioinformatics routines have been used to provide a more detailed definition of the proteome of Mycobacterium tuberculosis H37Rv. Over half of the current proteins result from gene duplication or domain shuffling events while one-sixth show no similarity to polypeptides described in other organisms. Prominent among the genes that appear to have been duplicated on numerous occasions are those involved in fatty acid metabolism, regulation of gene expression, and the unusually glycine-rich PE and PPE proteins. Protein similarity analysis, coupled with inspection of the genetic neighbourhood, was used to explore possible functional relatedness. This uncovered four large mce operons whose proteins may mediate initial interactions between the tubercle bacillus and host cells, together with a cluster of genes that might encode components of a structure required for secretion of ESAT-6 like proteins. Close linkage of the mmpL genes, encoding large membrane proteins, with those required for fatty acid metabolism suggests involvement in lipid transport. Compared to free-living bacteria, M. tuberculosis has a significantly smaller transport protein repertoire and this may reflect its intracellular lifestyle.

Comparative genomics uncovers large tandem chromosomal duplications in Mycobacterium bovis BCG Pasteur.

On direct comparison of minimal sets of ordered clones from bacterial artificial chromosome (BAC) libraries representing the complete genomes of Mycobacterium tuberculosis H37Rv and the vaccine strain, Mycobacterium bovis BCG Pasteur, two major rearrangements were identified in the genome of M. bovis BCG Pasteur. These were shown to correspond to two tandem duplications, DU1 and DU2, of 29 668 bp and 36 161 bp, respectively. While DU1 resulted from a single duplication event, DU2 apparently arose from duplication of a 100 kb genomic segment that subsequently incurred an internal deletion of 64 kb. Several lines of evidence suggest that DU2 may continue to expand, since two copies were detected in a subpopulation of BCG Pasteur cells. BCG strains harbouring DU1 and DU2 are diploid for at least 58 genes and contain two copies of oriC, the chromosomal origin of replication. These findings indicate that these genomic regions of the BCG genome are still dynamic. Although the role of DU1 and DU2 in the attenuation and/or altered immunogenicity of BCG is yet unknown, knowledge of their existence will facilitate quality control of BCG vaccine lots and may help in monitoring the efficacy of the world's most widely used vaccine.