RISK OF RED BLOOD CELL ALLOIMMUNISATION IN RWANDA: ASSESSMENT OF PRETRANSFUSION CROSSMATCH TECHNIQUES USED IN DISTRICT HOSPITALS.

BACKGROUND: Screening of alloantibodies in patients is not yet done in district hospitals of Rwanda. The practice is to transfuse ABO/D compatible blood following an immediate spin crossmatch (IS-XM) or indirect antiglobulin test crossmatch (IAT-XM). OBJECTIVES: To assess the risk of red blood cell (RBC) alloimmunisation associated with the use of IS-XM compared to the IAT-XM in patients receiving blood transfusions in district hospitals in Rwanda. DESIGN: A cross-sectional comparative descriptive study. SETTING: Four Rwandan district hospitals. Kirehe and Nyanza hospitals used IS-XM while Muhima and Ruhengeri hospitals used IAT-XM. SUBJECTS: Blood samples were obtained from 187 patients (101 with IS-XM and 86 with IAT-XM) transfused in January, February, October, and November of 2012. RESULTS: The median age of blood recipients was 31 years (7 - 80) and 36% of them were male. Sixteen specific antibodies were identified in 12 patients: anti-RH1/D (2),anti-RH2/C (2), anti-RH3/E (2), anti-RH4/c (1), anti-RH5/e (2),anti-LE1/Lea (2),anti-JK1/Jka (1), anti-JK2/Jkb (1), anti-KEL1/K (1), anti-MNS1/M (1), and autoantibody (1).The global prevalence of redblood cell (RBC) alloimmunisationwas 6.4% (12/187). Thatprevalence was significantly higher in the IS-XM group (10.4%) than in the IAT-XM group (2.3%) with an odds ratio of 4.8; [95% CI=1.2-19.8]; and a p-value of 0.031. CONCLUSION: The prevalence of red blood cell (RBC) alloimmunisation in 187 patients receiving blood transfusions was 6.4% and was higher in recipients from hospitals using IS-XM, with Rhesus (RH) system antibodies widely predominant (56.2%).We recommend that IAT-XM be used in all district hospitals in Rwanda to minimise this risk.

Semipermeable species boundaries between Anopheles gambiae and Anopheles arabiensis: evidence from multilocus DNA sequence variation.

Attempts to reconstruct the phylogenetic history of the Anopheles gambiae cryptic species complex have yielded strongly conflicting results. In particular, An. gambiae, the primary African malaria vector, is variously placed as a sister taxon to either Anopheles arabiensis or Anopheles merus. The recent divergence times for members of this complex complicate phylogenetic analysis, making it difficult to unambiguously implicate interspecific gene flow, versus retained ancestral polymorphism, as the source of conflict. Using sequences at four unlinked loci, which were determined from multiple specimens within each of five species in the complex, we found contrasting patterns of sequence divergence between the X chromosome and the autosomes. The isolation model of speciation assumes a lack of gene flow between species since their separation. This model could not be rejected for An. gambiae and An. arabiensis, although the data fit the model poorly. On the other hand, evidence from gene trees supports genetic introgression of chromosome 2 inversions between An. gambiae and An. arabiensis, and also points to more broad scale genetic exchange of autosomal sequences between this species pair. That such exchange has been relatively recent is suggested not only by the lack of fixed differences at three autosomal loci but also by the sharing of full haplotypes at two of the three loci, which is in contrast to several fixed differences and considerably deeper divergence on the X. The proposed acquisition by An. gambiae of sequences from the more arid-adapted An. arabiensis may have contributed to the spread and ecological dominance of this malaria vector.

Patterns of DNA sequence variation in chromosomally recognized taxa of Anopheles gambiae: evidence from rDNA and single-copy loci.

Patterns of DNA sequence variation in the ribosomal DNA (rDNA) second internal transcribed spacer (ITS2) and five unlinked single-copy nuclear loci were examined for evidence of reproductive isolation among four chromosomally recognized taxa of Anopheles gambiae from West Africa: Savanna, Bamako, Mopti and Forest, as well as sibling species An. arabiensis and An. merus. Included among the single-copy loci were three sequence-tagged random amplified polymorphic DNA (RAPD) loci, two of which (R15 and R37) had been reported as discriminating between Mopti and other chromosomal forms. Each of the five single-copy sequences were highly polymorphic in most samples. However, the R15 and R37 loci had no diagnostic value, and therefore are not recommended as tools in recognition of field-collected An. gambiae chromosomal forms. Although pairwise comparisons between species generally revealed significant levels of differentiation at all five loci, variation was not partitioned by chromosomal form within An. gambiae at any single-copy locus examined. The few exceptions to these trends appear related to a location either inside or nearby chromosomal inversions. At the tryptophan oxygenase locus inside inversion 2Rb, variation was structured only by inversion orientation and not by taxonomic designation even between An. gambiae and An. arabiensis, providing the first molecular evidence that the 2Rb inversion was transferred between species by introgressive hybridization. By contrast, the rDNA showed fixed differences between species and a difference diagnostic for Mopti, consistent with effective, if not complete, reproductive isolation. The apparent disagreement between the data from this locus and multiple single-copy loci within An. gambiae may be explained by the much lower effective population size of rDNA, owing to concerted evolution, which confers increased sensitivity at much shorter divergence times. Taken together with the accompanying reports by della Torre et al. (2001), Favia et al. (2001) and Gentile et al. (2001), our data suggest that neutral molecular markers may not have the sensitivity required to detect isolation between these recently established taxa.

Germline transformation of the malaria vector, Anopheles gambiae, with the piggyBac transposable element.

Germline transformation of the major African malaria vector, Anopheles gambiae, was achieved using the piggyBac transposable element marked with the enhanced green fluorescent protein (EGFP) injected into mosquito embryos. Two G1 generation male mosquitoes expressing EGFP were identified among 34 143 larvae screened. Genomic Southern data and sequencing of the piggyBac insertion boundaries showed that these two males arose from one piggyBac insertion event in the injected G0 embryos. Genetic cross data suggest that the insertion site of the element either resulted in, or is tightly linked to, a recessive lethal. This was demonstrated by a deficiency in the number of EGFP-expressing offspring from inbred crosses but expected ratios in outcrosses to non-transformed individuals and failure to establish a pure-breeding line. The insertion was weakly linked to the collarless locus on chromosome 2 and was shown by in situ hybridization to be located in division 28D of that chromosome. Particularly high levels of expression were observed uniformly in salivary glands and, in most individuals, in the anterior stomach. An improvement in the injection technique at the end of the studies resulted in increased G0 hatching, transient expression and EGFP-expression rates among G1 progeny.

Mitochondrial and ribosomal internal transcribed spacer (ITS2) diversity of the African malaria vector Anopheles funestus.

The pattern of sequence variation in the mitochondrial DNA cytochrome b gene (cyt-b) and ribosomal DNA internal transcribed spacer 2 (ITS2) was examined in Anopheles funestus from Senegal and Burkina Faso in West Africa and Kenya in East Africa. From both West African countries, samples included individuals hypothesized to represent reproductively isolated taxa based upon different karyotypes and behaviours. Analysis of the cyt-b data revealed high haplotypic diversity (86%) and an average pairwise difference per site of 0.42%. Sequence variation was not partitioned by geographical origin or karyotype class. The most common haplotype was sampled across Africa (approximately 6000 km). Analysis of the ITS2 data revealed one of the longest spacers yet found in anophelines (approximately 704 bp). In common with other anopheline ITS2 sequences, this one had microsatellites and frequent runs of individual nucleotides. Also in common with data from other anopheline ITS2 studies, the An. funestus sequences were almost monomorphic, with only two rare polymorphisms detected. The results from both markers are congruent and do not support the hypothesis of reproductively isolated chromosomal taxa within An. funestus. Whether the lack of support by mitochondrial DNA (mtDNA) and ribosomal DNA (rDNA) sequences is a result of the recent origin of the presumptive taxa, or of the absence of barriers to gene flow, remains to be elucidated, using more rapidly evolving markers such as microsatellites.

The Anopheles gambiae tryptophan oxygenase gene expressed from a baculovirus promoter complements Drosophila melanogaster vermilion.

An Anopheles gambiae cDNA encoding tryptophan oxygenase was placed under the control of the constitutive baculovirus promoter, ie-1. The chimeric construct, expressed transiently in vermilion (tryptophan oxygenase) mutants of Drosophila melanogaster, partially rescued adult eye color. The successful genetic complementation by this construct demonstrated both the proper function of the tryptophan oxygenase product and the effectiveness of the ie-1 promoter in directing expression of foreign genes in live insects. The functionality of An. gambiae tryptophan oxygenase in a higher fly fulfils predictions based on its structural conservation throughout millions of years of independent evolution.

Pegasus, a small terminal inverted repeat transposable element found in the white gene of Anopheles gambiae.

Pegasus, a novel transposable element, was discovered as a length polymorphism in the white gene of Anopheles gambiae. Sequence analysis revealed that this 535 bp element was flanked by 8 bp target site duplications and 8 bp perfect terminal inverted repeats similar to those found in many members of the Tc1 family. Its small size and lack of long open reading frames preclude protein coding capacity. Southern analysis and in situ hybridization to polytene chromosomes demonstrated that Pegasus occurs in approximately 30 copies in the genomes of An. gambiae and its sibling species and is homogenous in structure but polymorphic in chromosomal location. Characterization of five additional elements by sequencing revealed nucleotide identities of 95% to 99%. Of 30 Pegasus-containing phage clones examined by PCR, only one contained an element exceeding 535 bp in length, due to the insertion of another transposable element-like sequence. Thus, the majority, if not all, extant Pegasus elements may be defective copies of a complete element whose contemporary existence in An. gambiae is uncertain. No Pegasus-hybridizing sequences were detected in nine other anophelines and three culicines examined, suggesting a very limited taxonomic distribution.

The Tryptophan oxygenase gene of Anopheles gambiae.

The Anopheles gambiae gene encoding tryptophan oxygenase, a homolog of the Drosophila melanogaster vermilion gene, has been molecularly cloned and characterized. Unlike Drosophila, where it is X-linked, the A. gambiae gene maps to chromosome 2R, subdivision 12E, by in situ hybridization to the polytene chromosomes. Of the six introns present, four are positioned identically to those of the Drosophila homolog, one is similarly positioned, and one is novel. A 1 955 nt cDNA potentially encodes a 392 amino acid protein of an estimated 45 kDa. Amino acid comparisons between the deduced protein and previously known tryptophan oxygenases revealed 74% identity between Anopheles and Drosophila, and 53% identity between Anopheles and nematode or mammalian proteins. Northern analysis detected a developmentally regulated transcript about 2 kb in length. Since this gene is known to control adult eye color in other flies, its cloning from A. gambiae provides the basis for a dominant phenotypic marker for germline transformation, one whose expression, unlike that of white, is not cell autonomous.

Pantropic retroviral vectors integrate and express in cells of the malaria mosquito, Anopheles gambiae.

The lack of efficient mechanisms for stable genetic transformation of medically important insects, such as anopheline mosquitoes, is the single most important impediment to progress in identifying novel control strategies. Currently available techniques for foreign gene expression in insect cells in culture lack the benefit of stable inheritance conferred by integration. To overcome this problem, a new class of pantropic retroviral vectors has been developed in which the amphotropic envelope is completely replaced by the G glycoprotein of vesicular stomatitis virus. The broadened host cell range of these particles allowed successful entry, integration, and expression of heterologous genes in cultured cells of Anopheles gambiae, the principle mosquito vector responsible for the transmission of over 100 million cases of malaria each year. Mosquito cells in culture infected with a pantropic vector expressing hygromycin phosphotransferase from the Drosophila hsp70 promoter were resistant to the antibiotic hygromycin B. Integrated provirus was detected in infected mosquito cell clones grown in selective media. Thus, pantropic retroviral vectors hold promise as a transformation system for mosquitoes in vivo.

Distribution of T1, Q, Pegasus and mariner transposable elements on the polytene chromosomes of PEST, a standard strain of Anopheles gambiae.

The chromosomal locations of four families of transposable elements, T1, Q, Pegasus and mariner, have been determined by in situ hybridization to polytene chromosomes of ovarian nurse cells of the mosquito Anopheles gambiae. As part of this effort, we have developed a vigorous pink-eyed laboratory strain of A. gambiae (PEST), rendered homozygous standard for chromosomal inversions on all autosomes. Ten different individuals of this strain were studied with each transposable element probe. The average number of hybridization sites per genome was 83.9 for T1, 63.4 for Q, 31.5 for Pegasus and 64.7 for mariner, excluding pericentric and centromeric regions. However, some degree of polymorphism was observed within each family such that, considering all ten individuals, 94 different sites were detected for T1, 82 sites for Q, 45 sites for Pegasus and 71 sites for mariner. The mean occupancy per site varied from 0.70 (Pegasus) to 0.91 (mariner), which, while significantly higher than that seen for transposable elements in natural populations of Drosophila melanogaster, is comparable to that seen in established laboratory stocks. In addition, these element families were not randomly distributed. All but Pegasus were concentrated in centromeric heterochromatin and centromere-proximal euchromatin, most showed a deficit of hybridization sites in the distal section of chromosomes, and a significant proportion of sites were coincident between families. These results provide the first detailed examination of the cytogenetic location of transposable elements in a nondrosophilid insect, and, through comparison with the behavior of transposable elements in Drosophila, may provide insight into the interaction between elements and host. The mapped elements are also expected to serve as landmarks useful in integrating the developing physical map of the PEST strain with the chromosomal banding pattern.

Cloning and characterization of the white gene from Anopheles gambiae.

A 14 kb region of genomic DNA containing the X-linked Anopheles gambiae eye colour gene, white, was cloned and sequenced. Genomic clones containing distinct white+ alleles were polymorphic for the insertion of a small transposable element in intron 3, and differed at 1% of nucleotide positions compared. Sequence was also determined from a rare 2914 bp cDNA. Comparison of cDNA and genomic sequences established an intron-exon structure distinct from Drosophila white. Despite a common trend in Anopheles and Drosophila of weak codon bias given low levels of gene expression, codon usage by Anopheles gambiae white was strongly biased. Overall amino acid identity between the predicted mosquito and fruitfly proteins was 64%, but dropped to 14% at the amino terminus. To correlate phenotypically white-eyed strains of A. gambiae with structural lesions in white, five available strains were analysed by PCR and Southern blotting. Although these strains carried allelic mutations, independently generated by gamma radiation (three strains) or spontaneous events (two strains), no white lesions were detected. Significantly, another non-allelic X-linked mutation, causing an identical white-eyed phenotype, has been correlated with a structural defect in the cloned white gene (Benedict et al., 1995). Taken together, these observations suggest that the white-eyed mutants analysed in the present study carry mutations in a second eye colour gene and are most likely white+.

Q: a new retrotransposon from the mosquito Anopheles gambiae.

A new family of retrotransposons (RTPs) without long terminal repeats (LTRs), designated Q, has been isolated from the malaria vector Anopheles gambiae. The nucleotide sequence of a complete element Q-22, was determined and analysed. Approximately 4.5 kb long, Q-22 contains two long overlapping open reading frames (ORFs) that potentially encode proteins with nucleic acid binding and reverse transcriptase domains similar to those of non-LTR RTPs previously described. The 3' end is characterized by variable numbers of the triplet repeat TAA, immediately following a polyadenylation signal. In situ hybridization of nurse cell polytene chromosomes revealed about twenty labelled sites distributed over all arms and diffuse hybridization to the chromocentre. Cross-hybridizing sequences with the same internal structure occur in all members of the A. gambiae complex. Genomic Southerns of wild A. gambiae specimens probed with Q suggest that Q is or recently was capable of retrotransposition.