Universal newborn screening for haemoglobinopathies in Guadeloupe (French West Indies): a 27-year experience.

OBJECTIVES: In Guadeloupe, an island in the French West Indies, a universal newborn screening programme for sickle cell disease and other abnormal haemoglobins was initiated in 1984. In 1990, a comprehensive sickle cell centre was established to carry on the management programme. We here report the main results from the newborn screening programme from 1984 to 2010, and consider how the establishment of the sickle cell centre affected the programme. METHODS: All blood samples were screened for the haemoglobinopathies using two reference methods in a single reference diagnosis laboratory. DNA analyses were also performed for confirmatory tests and analysis of the globin gene status. RESULTS: Between 1 January 1984 and 31 December 2010, 178,428 newborns were screened at birth, and 585 children were diagnosed with major sickle cell syndromes (ie. an overall incidence of 1 in 304 births). Sickle cell anaemia (haemoglobin SS disease) was the most frequently observed (1 in 575 births), followed by haemoglobin SC disease (1 in 771 births) and haemoglobin Sß-thalassemia disease (1 in 4,243 births). Some other rare haemoglobin variants were also detected, the most common being HbD(Punjab). The establishment of a comprehensive sickle cell centre resulted in a significant improvement in the screening coverage (p < 0.001) and a significant reduction of the delay between diagnosis and the first medical visit (p < 0.001). CONCLUSION: The universal screening programme has made it possible to establish the incidence of the major sickle cell syndromes in Guadeloupe, and the management centre has improved its efficiency.

Alpha-thalassemia is associated with a decreased occurrence and a delayed age-at-onset of albuminuria in sickle cell anemia patients.

The aim of this study was to identify possible risk factors for albuminuria, an early marker of sickle cell anemia (SCA) glomerulopathy, in a cohort of 189 SCA adult patients followed at the Sickle Cell Center of Guadeloupe, a French Caribbean island. Biological parameters obtained at baseline, alpha-globin gene status, and beta(S) haplotypes were compared in patients stratified accordingly to graded albuminuria. Abnormal albumin excretion rate was detected in half of the studied adult patients and macroalbuminuria occurred in 21.6%. Graded albuminuria was associated with advanced age (p=0.006), systolic blood pressure (p=0.031), and worsened anemia, i.e. low hemoglobin rate (p<0.0001) and red blood cell count (p<0.0001). Alpha-thalassemia frequency was lower in microalbuminuric and macroalbuminuric patients than in normoalbuminuric patients, 12.5%, 13.75% and 26%, respectively (p=0.0057). Comparison of albuminuria-free survival curves in SCA patients without and with alpha-thalassemia showed that the median time of albuminuria onset was delayed in the later ones (p=0.021). In contrast, no association of albuminuria was detected with the Bantou beta(S) haplotype. Our results strongly suggest a protective effect of alpha-thalassemia against glomerulopathy in SCA adult patients which could be related to a decreased hemolytic rate.

Phenotypic expression and origin of the rare beta-thalassemia splice site mutation HBB:c.315 + 1G>T.

We present the hematological characteristics of five patients from Surinam and the bordering French Guyana, who are carriers of the rare beta-thalassemia (beta-thal) mutation HBB:c.315+1G>T. Analysis of the phenotype/genotype relationship shows that this allele is a beta(0)-thal variant and illustrates the modulating effect of the alpha-globin gene status on the beta-thal phenotype. The ethnic origin of the five probands, belonging to the so-called Bush Negroes Maroons of Surinam and French Guyana, strongly suggests that this beta-thal mutation has a West African origin and spread in this ethnic group because of a founder effect and/or genetic drift.

Effects of hydration and dehydration on blood rheology in sickle cell trait carriers during exercise.

This study compared the hemorheological responses of a group of sickle cell trait (SCT) carriers with those of a control (Cont) group in response to 40 min of submaximal exercise (exercise intensity, 55% aerobic peak power) performed in two conditions: one with water offered ad libitum, i.e., the hydration (Hyd) condition, and one without water, i.e., the dehydration (Dehyd) condition. Blood and plasma viscosities, as well as red blood cell rigidity, were determined at rest, at the end of exercise, and at 2 h recovery with a cone plate viscometer at high shear rate and 37 degrees C. The SCT and Cont groups lost 1 +/- 0.7 and 1.6 +/- 0.6 kg of body weight, respectively, in the Dehyd condition, indicating a significant effect of water deprivation compared with the Hyd condition, in which body weight remained unchanged. Plasma viscosity increased with exercise and returned to baseline during recovery independently of the group and condition. As previously demonstrated, resting blood viscosity was greater in the SCT carriers than in the Cont group. Blood viscosity increased by the end of exercise and returned to baseline at 2 h recovery in the Cont group in both conditions. The blood viscosity of SCT carriers did not change in response to exercise in the Dehyd condition and remained elevated at 2 h recovery. This extended hyperviscosity, in association with other biological changes induced by exercise, could be considered as a risk factor for exercise-related events in SCT carriers, similar to vasoocclusive crises, notably during the recovery. In contrast, the Hyd condition normalized the hyperviscosity and red blood cell rigidity of the SCT carriers, with blood viscosity values reaching the same lower values as those found in the Cont group during the recovery. Adequate hydration of SCT carriers should be strongly promoted to reduce the clinical risk associated with potential hyperviscosity complications.

Use of cluster-graphs from spoligotyping data to study genotype similarities and a comparison of three indices to quantify recent tuberculosis transmission among culture positive cases in French Guiana during a eight year period.

BACKGROUND: French Guiana has the highest tuberculosis (TB) burden among all French departments, with a strong increase in the TB incidence over the last few years. It is now uncertain how best to explain this incidence. The objective of this study was to compare three different methods evaluating the extent of recent TB transmission in French Guiana. METHODS: We conducted a population-based molecular epidemiology study of tuberculosis in French Guiana based on culture-positive TB strains (1996 to 2003, n = 344) to define molecular relatedness between isolates, i.e. potential transmission events. Phylogenetic relationships were inferred by comparing two methods: a "cluster-graph" method based on spoligotyping results, and a minimum spanning tree method based on both spoligotyping and variable number of tandem DNA repeats (VNTR). Furthermore, three indices attempting to reflect the extent of recent TB transmission (RTIn, RTIn-1 and TMI) were compared. RESULTS: Molecular analyses showed a total amount of 120 different spoligotyping patterns and 273 clinical isolates (79.4%) that were grouped in 49 clusters. The comparison of spoligotypes from French Guiana with an international spoligotype database (SpolDB4) showed that the majority of isolates belonged to major clades of M. tuberculosis (Haarlem, 22.6%; Latin American-Mediterranean, 23.3%; and T, 32.6%). Indices designed to quantify transmission of tuberculosis gave the following values: RTIn = 0.794, RTIn-1 = 0.651, and TMI = 0.146. CONCLUSION: Our data showed a high number of Mycobacterium tuberculosis clusters, suggesting a high level of recent TB transmission, nonetheless an estimation of transmission rate taking into account cluster size and mutation rate of genetic markers showed a low ongoing transmission rate (14.6%). Our results indicate an endemic mode of TB transmission in French Guiana, with both resurgence of old spatially restricted genotypes, and a significant importation of new TB genotypes by migration of TB infected persons from neighgouring high-incidence countries.

Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology.

BACKGROUND: The Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database. RESULTS: The fourth international spoligotyping database, SpolDB4, describes 1939 shared-types (STs) representative of a total of 39,295 strains from 122 countries, which are tentatively classified into 62 clades/lineages using a mixed expert-based and bioinformatical approach. The SpolDB4 update adds 26 new potentially phylogeographically-specific MTC genotype families. It provides a clearer picture of the current MTC genomes diversity as well as on the relationships between the genetic attributes investigated (spoligotypes) and the infra-species classification and evolutionary history of the species. Indeed, an independent Naïve-Bayes mixture-model analysis has validated main of the previous supervised SpolDB3 classification results, confirming the usefulness of both supervised and unsupervised models as an approach to understand MTC population structure. Updated results on the epidemiological status of spoligotypes, as well as genetic prevalence maps on six main lineages are also shown. Our results suggests the existence of fine geographical genetic clines within MTC populations, that could mirror the passed and present Homo sapiens sapiens demographical and mycobacterial co-evolutionary history whose structure could be further reconstructed and modelled, thereby providing a large-scale conceptual framework of the global TB Epidemiologic Network. CONCLUSION: Our results broaden the knowledge of the global phylogeography of the MTC complex. SpolDB4 should be a very useful tool to better define the identity of a given MTC clinical isolate, and to better analyze the links between its current spreading and previous evolutionary history. The building and mining of extended MTC polymorphic genetic databases is in progress.

Long-term population-based genotyping study of Mycobacterium tuberculosis complex isolates in the French departments of the Americas.

The three French overseas departments of the Americas are characterized both by insular (Guadeloupe and Martinique) and continental (French Guiana) settings with a tuberculosis case detection rate that varies from less than 10 per 100,000 per year in insular areas to an estimated incidence of more than 55 per 100,000 in French Guiana. Under a long-term genotyping program, more than three-fourths of all the Mycobacterium tuberculosis isolates (n = 744) received from the three settings were fingerprinted over a 10-year period (1994 to 2003) by spoligotyping and variable number of tandem DNA repeats (VNTRs) in order to understand the current trends in their detection rates, drug resistance, and groups and subpopulations at risk of contracting the disease and to pinpoint the circulating phylogeographical clades of the bacilli. The major difference in the study populations was the nationality of the patients, with a high percentage of immigrants from high-incidence neighboring countries in French Guiana and a low but increasing percentage in the French Caribbean. The rate of recent transmission was calculated to be 49.3% in French Guiana, compared to 27.2% and 16.9% in Guadeloupe and Martinique, respectively. At the phylogeographic level, 77.9% of the isolates studied belonged to four major clades (Haarlem, Latin-American and Mediterranean, T, and X) which are already reported from neighboring Caribbean islands in an international database and may underline potential interregional transmission events.

Mycobacterium africanum genotyping using novel spacer oligonucleotides in the direct repeat locus.

This study involves a first evaluation of 25 novel spacer oligonucleotides in addition to the 43 routine spacers for molecular characterization of a panel of 65 isolates of tubercle bacilli from different geographic origins that were initially classified as Mycobacterium africanum based on phenotypic characters. The 68-spacer format defined four additional patterns, and three groups were identified. The relatively homogeneous groups A1 and A2 included strains from West Africa, and A3-1 included strains from East Africa. The presence of deletion region RD9 confirmed the reclassification of the M. africanum subtype II spoligopattern within group A3-1 as Mycobacterium tuberculosis. These isolates may represent a diverging branch of M. tuberculosis in Africa. The use of new spacers also suggested an undergoing evolution of M. africanum subtype I in West Africa. Our results showed that the strain differentiation within the M. tuberculosis complex is improved by using novel spacers, and extensive studies using new-generation spoligotyping may be helpful to better understand the evolution of M. africanum.

Molecular epidemiology of Mycobacterium tuberculosis in western Sweden.

The genetic diversity of Mycobacterium tuberculosis isolates among patients from Sweden was determined by a combination of two PCR-based techniques (spoligotyping and variable number of tandem repeats analysis). It resulted in a clustering of 23.6% of the isolates and a rate of recent transmission of 14.1%. The clustered isolates mainly belonged to the Haarlem family (23.2%), followed by the Beijing (9.8%), Latin American and Mediterranean (LAM; 8%), and East African-Indian (EAI; 6.2%) families. A comparison of the spoligotypes with those in the international spoligotyping database showed that 62.5% of the clustered isolates and 36.6% of all isolates typed were grouped into six major shared types. A comparison of the spoligotypes with those in databases for Scandinavian countries showed that 33% of the isolates belonged to an ill-defined T family, followed by the EAI (22%), Haarlem (20%), LAM (11%), Central Asian (5%), X (5%), and Beijing (4%) families. Both the highest number of cases and the proportion of clustered cases were observed in patients ages 15 to 39 years. Nearly 10% of the isolates were resistant to one or more drugs (essentially limited to isoniazid monoresistance). However, none of the strains were multidrug resistant. Data on the geographic origins of the patients showed that more than two-thirds of the clustered patients with tuberculosis were foreign-born individuals or refugees. These results are explained on the basis of both the historical links within specific countries and recently imported cases of tuberculosis into Sweden.

Snapshot of moving and expanding clones of Mycobacterium tuberculosis and their global distribution assessed by spoligotyping in an international study.

The present update on the global distribution of Mycobacterium tuberculosis complex spoligotypes provides both the octal and binary descriptions of the spoligotypes for M. tuberculosis complex, including Mycobacterium bovis, from >90 countries (13,008 patterns grouped into 813 shared types containing 11,708 isolates and 1,300 orphan patterns). A number of potential indices were developed to summarize the information on the biogeographical specificity of a given shared type, as well as its geographical spreading (matching code and spreading index, respectively). To facilitate the analysis of hundreds of spoligotypes each made up of a binary succession of 43 bits of information, a number of major and minor visual rules were also defined. A total of six major rules (A to F) with the precise description of the extra missing spacers (minor rules) were used to define 36 major clades (or families) of M. tuberculosis. Some major clades identified were the East African-Indian (EAI) clade, the Beijing clade, the Haarlem clade, the Latin American and Mediterranean (LAM) clade, the Central Asian (CAS) clade, a European clade of IS6110 low banders (X; highly prevalent in the United States and United Kingdom), and a widespread yet poorly defined clade (T). When the visual rules defined above were used for an automated labeling of the 813 shared types to define nine superfamilies of strains (Mycobacterium africanum, Beijing, M. bovis, EAI, CAS, T, Haarlem, X, and LAM), 96.9% of the shared types received a label, showing the potential for automated labeling of M. tuberculosis families in well-defined phylogeographical families. Intercontinental matches of shared types among eight continents and subcontinents (Africa, North America, Central America, South America, Europe, the Middle East and Central Asia, and the Far East) are analyzed and discussed.

Global distribution of Mycobacterium tuberculosis spoligotypes.

We present a short summary of recent observations on the global distribution of the major clades of the Mycobacterium tuberculosis complex, the causative agent of tuberculosis. This global distribution was defined by data-mining of an international spoligotyping database, SpolDB3. This database contains 11708 patterns from as many clinical isolates originating from more than 90 countries. The 11708 spoligotypes were clustered into 813 shared types. A total of 1300 orphan patterns (clinical isolates showing a unique spoligotype) were also detected.