Genetic diversity of Mycobacterium tuberculosis in Madang, Papua New Guinea.

SETTING: Madang and surroundings, Papua New Guinea (PNG). OBJECTIVE: To characterise the genetic diversity and drug susceptibility of Mycobacterium tuberculosis isolates collected in Madang and surroundings. DESIGN: M. tuberculosis was isolated from sputum samples from active pulmonary tuberculosis cases. Drug resistance profiles were obtained by drug susceptibility testing. M. tuberculosis lineages were identified by single nucleotide polymorphisms and sub-typing was performed by spoligotyping. Spoligotyping and 24 locus mycobacterial interspersed repetitive units-variable number of tandem repeats were combined to identify clustered isolates. RESULTS: The 173 M. tuberculosis isolates collected belonged predominantly to the Euro-American lineage (Lineage 4) and the East-Asian lineage (Lineage 2). Multidrug-resistant M. tuberculosis were observed in 5.2% of isolates. Lineage 2 M. tuberculosis, which includes the 'Beijing' genotype, was significantly associated with any drug resistance (OR 5.2, 95%CI 1.8-15.1). Cluster analyses showed 44% molecularly clustered isolates, suggesting transmission of M. tuberculosis in the community, including transmission of primary drug-resistant M. tuberculosis. CONCLUSION: These data provide the first insight into the molecular characteristics of M. tuberculosis in the Madang area of PNG, and indicate substantial drug resistance with evidence of ongoing transmission.

[Prevalence, genetic diversity and multiplicity of Plasmodium falciparum infection in school children in central Cote d'Ivoire]. / Prevalence, diversité antigénique et multiplicité d'infections de Plasmodium falciparum en milieu scolaire au centre de la Côte d'Ivoire.

A study was carried out in the village of Taabo, located in the vicinity of a large man-made lake in central Côte d'Ivoire. The objectives were (i) to determine the level of prevalence, genetic diversity and multiplicity of Plasmodiumfakiparum infection in schoolchildren and (ii) to compare the diagnostic performance of light microscopy and polymerase chain reaction (PCR). A total of 424 schoolchildren ranging in age from 5 to 15 years underwent diagnostic testing using both light microscopy of blood smears and PCR. Multiplicity of P. falciparum infection was investigated in 196 children (46.2%). The prevalence of malaria was 54.7% based on light microscopy and 83.9% based on PCR. Genotyping based on polymorphism in the length of the restriction fragment of the gene encoding the merozoite surface protein-2 (msp2) showed that 86.5% of cases involved multiple infection with a geometric mean of 3.87 genotypes per positive child. There was a strong positive correlation between multipcity of infection and parasite density in the 56-year old age group. A total of 50 genotypes including six observed for the first time were identified and classified into families with similar-sized sequence groups: 26 x FC27 (52%) and 24 x 3D7 (48%). In comparison with PCR, the sensitivity and specificity of light microscopy for diagnosis of P. falciparum was 81.3% and 88.2% respectively. Data are discussed in the light of similar studies carried out in sub-Saharan Africa and elsewhere. These findings can serve as a basis for monitoring the longterm effect of major water resource management projects on the prevalence, genetic diversity and multiplicity of P. falciparum infection.

Molecular monitoring of parasite resistance to artemisinin in Tanzania

Artemisinin-based combination therapies (ACTs) are recommended for use against uncomplicated malaria in areas of multi-drug resistant malaria; such as sub-Saharan Africa. However; their long-term usefulness in these high transmission areas remains unclear. It has been suggested that documentation of the S769N PfATPase6 mutations may indicate an emergence of artemisinin resistance of Plasmodium falciparum in the field. The present study assessed PfATPase6 mutations (S769N and A623E) in 615 asymptomatic P. falciparum infections in Tanzania but no mutant genotype was detected. This observation suggests that resistance to artemisinin has not yet been selected in Tanzania; supporting the Ministry of Healths decision to adopt artemether+lumefantrine as first-line malaria treatment. The findings recommend further studies to assess PfATPase6 mutations in sentinel sites and verify their usefulness in monitoring emergency of ACT resistance

Prevalence; diversite antigenique et multiplicite d'infections de Plasmodium falciparum en milieu scolaire au centre de la Cote d'Ivoire

Une etude a ete conduite a Taabo - village; localite rurale du centre de la Cote d'Ivoire; situee a proximite d'un gra n d lac artificiel. Les objectifs etaient (i) de determiner le taux d'endemicite du paludisme; la diversite antigenique et la multiplicite des infections a Plasmodium falciparum au sein d'ecoliers; et (ii) de comparer la performance du diagnostic microscopique a celle de la reaction de polymerisation en chaine (PCR). Au total; 424 eleves ages de 5 a 15 ans ont eu des examens de sang au microscope et par PCR. La multiplicite d'infection a porte sur 196 (46;2) d'entre eux. L'indice plasmodique detecte au micro s c o p e est de 54;7et de 83;9par PCR. Les typages genotypiques determines par le polymorphisme des longueurs de fragments de restriction du gene respon sable des proteines de surface-2 du mero zoite (m s p 2); ont revele 86;5de cas d'infections multipes; avec une moyenne geometrique de 3;87 genotypes par individu positif. Une correlation positive significative a ete obtenue entre a multiplicite et les densites parasitaires au sein du groupe d'age 5-6 ans. 50 genotypes dont six observes pour la premiere fois ont ete denombres puis classes en familles de tailles similaires FC27 (n=26 ; 52) et 3D7 (n=24 ; 48). Compare a la PCR; la microscopie a montre une sensibilite et une specificite respectivement de 81;3et 88;2. Nos donnees sont discutees au regard d'etudes similaires en Afrique sub-saharienne et ailleurs; et peuvent servir de base a long terme pour l'evaluation d'impact des grands amenagements d'eau sur la prevalence; la diversite antigenique et la multiplicite des infections a P. fal c i p a ru m

A DNA delivery system targeting dendritic cells for use in immunization against malaria: a rodent model.

DNA-based vaccination has emerged as a promising method of immunisation since the first demonstration of this technology. Improving the antibody responses is desirable for the protective efficacy and hence broad application of these vaccines. We examined the immunogenicity of a Plasmodium-based DNA vaccine that was targeted to antigen presenting cells by fusion to CTLA4. Fusion proteins comprising the extra-cellular domain of CTLA4, the hinge, CH2 and CH3 domains of human IgG1 and MSP-1 gene fragments were expressed in COS-7 cells. Three of the secreted proteins containing the mouse homologue of CTLA4 were shown to bind differently to the human B7-1 molecule expressed on THP-1 cells. Competition binding assays for two fusion proteins showed that binding was specific. When C57BL/6 mice were immunized with plasmids encoding the fusion proteins, antibodies against two denatured and one non-denatured MSP-1 gene fragments were successfully induced. The usefulness of this strategy in future studies of immunisaton against human malaria is discussed.

A prospective study of Plasmodium falciparum multiplicity of infection and morbidity in Tanzanian children.

Several studies suggest that in individuals with substantial previous exposure to malaria, co-infection with multiple clones of Plasmodium falciparum can protect against subsequent clinical malaria attacks. Other studies, mainly of individuals with little previous exposure, found the converse relationship. To test whether acquisition of such cross-protection tracks the acquisition of clinical immunity in general, 610 Tanzanian children aged 0-6 years were enrolled in a nine-month prospective study of the risk of morbidity in relation to parasitological status and merozoite surface protein 2 genotypes on enrolment. Prevalence of parasitaemia and multiplicity of infection increased with age. In the first year of life, the incidence of clinical malaria was almost three times higher in children with parasites at baseline than in those without. In older children, baseline P. falciparum infections appeared to protect against both parasitaemic and non-parasitaemic fever episodes. In children aged less than three years, baseline multiple infection tended to be associated with higher prospective risk of clinical malaria than single infection while in children aged more than three years the converse was found, but these effects were not statistically significant. These results provide further evidence that relationships between asymptomatic malaria infections and clinical malaria change with cumulative exposure.

Humic colloid-borne natural polyvalent metal ions: dissociation experiment.

The natural association nature of the humic colloid-borne trace elements is investigated. Rare earth elements (REE) Th and U are chosen as naturally occurring representatives and chemical homologues for actinides of different oxidation states present in nuclear waste. Tri- and tetravalent elements in two investigated Gorleben groundwaters (Gohy-532 and -2227) almost exclusively occur as humic or fulvic colloid-borne species. Their desorption behavior from colloids is examined in the unperturbed groundwater (pH approximately 8) under anaerobic conditions (Ar/1% CO2) by addition of a chelating cation exchanger resin. Particularly, the dissociation process of naturally occurring Eu(III) in the groundwater is compared with the Eu(III) desorption from its humate complex prepared with purified Aldrich humic acid in a buffered aqueous solution at pH approximately 8. The Eu(III) dissociation from the groundwater colloids is found to be considerably slower than found for the humate complex synthesized in the laboratory. This suggests that under natural aquatic conditions the Eu(III) binding in colloids is chemically different from the simple humate complexation as observed in the laboratory experiment. The colloid characterization bythe size exclusion chromatography (SEC) and the flow field-flow fractionation (FFFF) indicates that natural colloid-borne trace elements are found predominantly in colloids of larger size (>15 nm in size), while Eu(III) in its humate complex is found mainly in colloids of hydrodynamic diameters <5 nm. The slower desorption kinetics and the larger colloid size suggest that the polyvalent metal ion binding in natural humic colloids is associated to polynucleation with other co-present trace metal ions. Radiotracer experiments reveal that isotopic equilibria with the naturally colloid-borne trace elements are not attained within a period of more than 100 days, indicating irreversible binding of at least a part of colloid-borne polyvalent trace elements. The different kinetic properties of colloid-bound Eu(III) are discussed taking the aqueous speciation based on thermodynamic data into account.

Molecular epidemiology of Plasmodium falciparum infections among asymptomatic inhabitants of a holoendemic malarious area in northern Ghana.

Age dependence of malaria infection was assessed in an age-stratified cluster sample of 308 individuals from Kassena-Nankana District of northern Ghana during June and July 2000. Overall prevalence of Plasmodium falciparum by microscopy was 70%, with the maximum among 5-9 year olds. Parasite density was highest (geometric mean 1922/microl blood) in 1-2 year olds. Eighty-two per cent of samples were positive by polymerase chain reaction (PCR), and restriction fragment length polymorphism typing of the P. falciparum msp2 revealed a mean msp2 multiplicity of 3.4 (range: 1-8) genotypes per PCR positive sample. Multiplicity increased with age until 5-9 years and then started to reduce again into adulthood. About 49.3% of infections belonged to the msp2 FC27 allelic family and 50.7% to the 3D7 family. On the day of the survey, only 3.6% of the participants had fever (axillary temperature >or= 37.5 degrees C) and 2.3% had fever associated with parasitaemia. The correlation between parasite density and msp2 multiplicity was 0.42; highest among infants, and decreased with age to a minimum among 5-9 year olds. Contrasting with results from Tanzania, this correlation increased with age in adolescents and adults. Parasite multiplicity is very high in this community, and the patterns of age dependence are similar to those in other holoendemic sites in Africa, validating the use of the age-multiplicity relationship as an indicator of malaria endemicity.

Limited polymorphism in Plasmodium falciparum sexual-stage antigens.

In areas highly endemic for malaria, individuals are frequently found to be infected simultaneously with multiple Plasmodium falciparum clones. This raises the question of whether all parasite clones produce gametocytes equally or whether gametocytogenesis is suppressed in some clones. In order to assess this in epidemiological studies, polymorphic genes specifically expressed in gametocytes could be analyzed by both amplification of genomic DNA from blood samples and by reverse transcribed polymerase chain reaction amplifying expressed gametocyte-specific genes only. Here we report the analysis of diversity in the three gametocyte-specific genes Pfs16, Pfs48/45, and Pfs230. In addition to the previously published data, limited polymorphism was found in the coding sequences of Pfs16 and Pfs48/45. Larger polymorphism was identified in Pfs230, which might allow the development of a discriminating PCR-based genotyping scheme for transmission studies. However, the limited polymorphism in Pfs16 and Pfs48/45 renders these molecules poorly useful for such studies.

Genotyping of Plasmodium falciparum infections by PCR: a comparative multicentre study.

Genetic diversity of malaria parasites represents a major issue in understanding several aspects of malaria infection and disease. Genotyping of Plasmodium falciparum infections with polymerase chain reaction (PCR)-based methods has therefore been introduced in epidemiological studies. Polymorphic regions of the msp1, msp2 and glurp genes are the most frequently used markers for genotyping, but methods may differ. A multicentre study was therefore conducted to evaluate the comparability of results from different laboratories when the same samples were analysed. Analyses of laboratory-cloned lines revealed high specificity but varying sensitivity. Detection of low-density clones was hampered in multiclonal infections. Analyses of isolates from Tanzania and Papua New Guinea revealed similar positivity rates with the same allelic types identified. The number of alleles detected per isolate, however, varied systematically between the laboratories especially at high parasite densities. When the analyses were repeated within the laboratories, high agreement was found in getting positive or negative results but with a random variation in the number of alleles detected. The msp2 locus appeared to be the most informative single marker for analyses of multiplicity of infection. Genotyping by PCR is a powerful tool for studies on genetic diversity of P. falciparum but this study has revealed limitations in comparing results on multiplicity of infection derived from different laboratories and emphasizes the need for highly standardized laboratory protocols.

Molecular approaches to epidemiology and clinical aspects of malaria.

Malaria is a problem of global importance, responsible for 1-2 million deaths per year, mainly in African children, as well as considerable morbidity manifested as severe anaemia and encephalopathy in young children. Fundamental to the development of new tools for malaria control in humans is an increased understanding of key features of malaria infection, such as the diversity of outcome in different individuals, the understanding of different manifestations of the disease and of the mechanisms of immunity that allow clinical protection in the face of ongoing low-grade infection (concomitant immunity or premunition). Here, Graham Brown and colleagues review some of the ways in which molecular approaches might be used to increase our understanding of the epidemiology and clinical manifestations of malaria, as discussed at the Molecular Approaches to Malaria conference (MAM2000), Lorne, Australia, 2-5 February 2000.

Plasmodium falciparum: expression of gametocyte-specific genes in monolayer cultures and malaria-positive blood samples.

In order to facilitate molecular epidemiological studies on the transmission of the malaria parasite Plasmodium falciparum a sensitive assay for gametocyte detection based on RT-PCR was developed. The transcription of the sexual stage-specific genes Pfs16, Pfs48/45, Pfs230, and Pfs25, as well as the sexual stage- and sporozoite-specific S 18S rRNA, was detected by RT-PCR. S 18S rRNA was present in seven of nine P. falciparum-positive blood samples, despite the lack of microscopic detection of gametocytes and a parasitemia below 0.1%. Expression of the other four gametocyte-specific genes was detected less frequently in malaria-positive blood samples. These findings indicate that RT-PCR of S 18S rRNA is a highly sensitive method for gametocyte detection and, furthermore, that gametocytes are present in the peripheral blood of most malaria carriers, even if the parasitemia is below 0.1%. To determinate the expression pattern of sexual stage-specific genes in more detail, RT-PCR was performed at consecutive time points of highly synchronized monolayer cell cultures. Transcripts of all examined genes except Pfs25 were detected directly after invasion of merozoites of the strain NF54 in red blood cells.

Evolution of Pacific/Asian populations inferred from HLA class II allele frequency distributions.

The allele frequency distributions for the HLA class II loci, DRB1, DQB1 and DPB1, in eight Pacific/Asian populations: Hawaiian, Samoan, Malay, Papua New Guinea (PNG) Highlands, and two Indonesian and PNG Lowland groups, were determined using high-resolution polymerase chain reaction/sequence-specific oligonucleotide probe (PCR/SSOP) typing methods. The allele frequency distributions for the HLA-DRB1 locus were determined for a third Indonesian population as well as for an additional Filipino population. DRB1 alleles in the DR2 serogroup (or allelic lineage) are very common in this region; in some populations, more than 50% of the alleles belong to this serogroup. The DRB1*1502 allele is frequent in nine of the ten populations studied, reaching a frequency of 0.48 in one Indonesian population and among Filipinos. Extensive DR-DQ haplotype diversity was detected in these populations. Seven different DR2-DQB1 haplotypes were observed in the Indonesian and PNG Lowland populations, eight in the PNG Highlands and ten in Malays and Filipinos. The DRB1*0410 allele, commonly observed in Australia, is observed in the PNG Highlands at a low frequency (f=0.03) and is absent in the other populations. Two additional DRB1 alleles commonly observed in Australia, DRB1*0405 and *1407, are also observed in the PNG Highlands at high frequencies (f=0.132 and 0.126), while they are rare in the PNG Lowlands (f=0.039 and 0.013). These alleles are generally rare or absent in the other populations. The DPB1*0501 allele, common in Chinese and Japanese populations, is most frequent in the Samoan, Hawaiian, Indonesian, and Malay populations, and the *0401 allele is the most frequent DPB1 allele in the PNG Lowlands. Both of these alleles have the same very high frequency (f=0.34) in the PNG Highlands. Analyses of homozygosity (the Ewens-Watterson F statistic) in these and other populations indicate that, while most allele frequency distributions are consistent with balancing selection, values of F for the Indonesian and Javan populations may reflect positive directional selection. Phylogenetic trees constructed using the allele frequencies at the DRB1 locus of the populations reported here, as well as those for additional Pacific, Asian, and Australian populations, indicate that the PNG Highland population is more closely related to Australian populations than to PNG Lowland populations, while the PNG Lowlands are more closely related to other Melanesian populations.

Plasmodium falciparum: cloned and expressed CIDR domains of PfEMP1 bind to chondroitin sulfate A.

Adherence of erythrocytes infected with mature asexual Plasmodium falciparum parasites (iRBC) to microvascular endothelial cells contributes to the pathology of P. falciparum malaria. It has been shown that the variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) confers adhesion to a wide range of cell surface receptors. Previously, the cysteine-rich interdomain region (CIDR) of PfEMP1 has been identified as binding site to CD36. We provide evidence that the same region can also mediate binding to chondroitin sulfate A (CSA). CIDR domains of two different parasite strains were expressed in Escherichia coli as a 6xHis-tagged protein. Purified recombinant protein bound to Chinese hamster ovary (CHO) cells which naturally express chondroitin sulfate A. Treatment of wild-type CHO cells with chondroitinase ABC reduced binding up to 94.4%. Competitive binding using soluble CSA inhibited binding to CHO cells by up to 100% at 2 mg/ml and by 62.4% at 0.5 mg/ml, whereas 1 mg/ml heparan sulfate had only a little effect (18.1%). In contrast, a recombinant 6xHis-tagged DBL1 domain showed no binding to wild-type CHO cells. Such an approach of analyzing various domains of PfEMP1 as recombinant proteins may elucidate their functions and may lead to novel anti-adherence therapeutics, especially for maternal malaria infections.

Genomic distribution and functional characterisation of two distinct and conserved Plasmodium falciparum var gene 5' flanking sequences.

Approximately 50 highly diverse var genes distributed throughout the haploid genome of the malaria parasite Plasmodium falciparum code for PfEMP1 variants located on the surface of infected erythrocytes. PfEMP1 is involved in cytoadherence of parasitised red blood cells and undergoes antigenic variation through differential expression of var genes. Members of the var gene family are located in chromosome-internal positions on chromosomes 4, 7, 8 and 12, and in subtelomeric regions of all chromosomes. Here we show that there are two distinct and conserved types of 5' upstream regions (var17-type and 5B1-type) of var genes, and suggest that most subtelomeric var genes are flanked by a var17-type 5' upstream sequence. In contrast, 5B1-type 5' upstream are localised to chromosomes that have been shown to contain var genes within chromosome-internal regions. Transcriptional analysis using RT-PCR revealed that var genes flanked by either type of 5' upstream sequence are transcribed in in vitro cultured trophozoite stage parasites. In addition, we have shown that the 5' flanking sequences of four different var genes are able to drive transient expression of the cat reporter gene. Our results suggest that at least the minimal regulatory sequences required for transcription of var genes are conserved among both subgroups of the var gene family. Furthermore, these sequences provide new markers for the investigation of the chromosomal organisation of var genes.

Genetic analysis of IgG subclass responses against RESA and MSP2 of Plasmodium falciparum in adults in Papua New Guinea.

Contributions of environmental and genetic factors to IgG subclass responses against Plasmodium falciparum antigens RESA and MSP2 were investigated among adults in a highly endemic area of Papua New Guinea. Heritabilities were estimated using variance component analysis. Familial aggregation of several responses was found, including IgG1, IgG2 and IgG3 responses against RESA, IgG1 and IgG3 responses against the 3D7 form of MSP2 and IgG1, IgG2 responses against the FC27 form of MSP2. Allowance for sharing of houses explained some of the non-genetic variance but not the familial aggregation. The variance of IgG3 responses against RESA and IgG1, IgG2 against MSP2 (FC27) was partly explained by sharing of HLA class II genotypes, although heritability was low. Segregation analyses indicated that any genetic regulation was more complex than governed by a single major gene. Such host genetic variation in responses to specific malaria antigens has implications for immuno-epidemiology and vaccine development.

Detection of nanocolloids with flow-field flow fractionation and laser-induced breakdown detection.

A new nanosize colloid detection method comprised of flow-field flow fractionation (FFFF) with laser-induced breakdown detection (LIBD) is presented, which is capable of characterizing the colloid size distribution as well as determining the number density of each size fraction in very low concentrations. The method facilitates the detection of aquatic colloids particularly in the lower range of nanometer size (< 50 nm) with the sensitivity much higher than a laser light-scattering method (LLS), i.e., the lower ppb range. The method is tested with a mixture of polystyrene colloids in three different nominal sizes, 19, 50, and 102 nm, and the results are compared with those of the LLS method. For colloids of 19-nm diameter, the present method demonstrates the detection sensitivity over 3 orders of magnitude better than that of the LLS method. The limitation of the detection sensitivity arises from the surface bleeding of a ceramic frit overlying the separation channel of the used FFFF instrument.

Analysis of Plasmodium falciparum infections in a village community in Northern Nigeria: determination of msp2 genotypes and parasite-specific IgG responses.

The genetic diversity of P. falciparum and multiplicity of infection has been studied in a village in Northern Nigeria at the end of the rainy season, when transmission is high. We analysed blood samples from 104 individuals aged 5-70 years by polymerase chain reaction (PCR) amplifying the gene for the merozoite surface protein MSP2 followed by genotyping based on restriction fragment length polymorphism (RFLP). 94.2% of all samples were parasite positive by PCR and over 80% of those had multiple infections. The age distribution of the average number of parasite clones present in P. falciparum infections showed an initial increase, then reached a peak multiplicity in children 8-10 years of age, and afterwards decreased significantly with age. Mean multiplicity in those 8-10-year-old children was 5.4 clones per carrier. Peak multiplicity and parasite diversity in Nigerian individuals is compared to findings from other study sites in Africa and PNG. The prevalence of IgG antibodies against the circumsporozoite protein (CSP), an indicator for malaria exposure, was over 85% in all age groups showing a high exposure of villagers to P. falciparum. OD values in ELISA were positively correlated with age. There was no correlation between the level of IgG against CSP and the multiplicity of P. falciparum infections determined by PCR of msp2. These results imply that in highly endemic areas multiplicity of infection is not directly correlated with exposure to P. falciparum.