Research that influences policy and practice – characteristics of operational research to improve malaria control in Mpumalanga Province, South Africa

Background

Results from numerous studies point convincingly to correlations between mutations at selected genes and phenotypic resistance to antimalarials in Plasmodium falciparum isolates. In order to move molecular assays for point mutations on resistance-related genes into the realm of applied tools for surveillance, we investigated a selection of P. falciparum isolates that were imported during the year 2001 into Europe to study the prevalence of resistance-associated point mutations at relevant codons. In particular, we tested for parasites which were developing resistance to antifolates and chloroquine. The screening results were used to map the prevalence of mutations and, thus, levels of potential drug resistance in endemic areas world-wide.

Results

337 isolates have been tested so far. Prevalence of mutations that are associated with resistance to chloroquine on the pfcrt and pfmdr genes of P. falciparum was demonstrated at high levels. However, the prevalence of mutations associated with resistance to antifolates at the DHFR and DHPS genes was unexpectedly low, rarely exceeding 60% in endemic areas.

Conclusions

Constant screening of imported isolates will enable TropNetEurop to establish a screening tool for emerging resistance in endemic areas.     

A study of the interactions between the internal larval parasites ofRhyacionia buoliana [Lepidoptera: Olethreutidae]

The interactions of 6 species of internal parasites ofRhyacionia buoliana Schiff. in a locality in northern Germany are discussed. The occurrence of super and multiparasitism is analyzed in relation to varying host densities and different rates of total parasitism. Multiparasitism betweenOrgilus obscurator Nees and 3 other species,Temelucha interruptor Grav.,Pristomerus sp. nearorbilatis Holmgr. and biting cleptoparasitic habits. Interspecific competition occurred in the form of direct physical attack between the first instar larvae. The coexistence of the internal larval parasites ofR. buoliana provides a good example of a system of “counter-balanced competition”.O. obscurator, although inferior at the level of intrinsic competition, was the dominant species at the level of extrinsic competition.     

Proteomic patterns of cervical cancer cell lines, a network perspective

Background

Systemic bacterial infections are highly regulated and complex processes that are orchestrated by numerous virulence factors. Genes that are coordinately controlled by the set of regulators required for systemic infection are potentially required for pathogenicity.

Results

In this study we present a systems biology approach in which sample-matched multi-omic measurements of fourteen virulence-essential regulator mutants were coupled with computational network analysis to efficiently identify Salmonella virulence factors. Immunoblot experiments verified network-predicted virulence factors and a subset was determined to be secreted into the host cytoplasm, suggesting that they are virulence factors directly interacting with host cellular components. Two of these, SrfN and PagK2, were required for full mouse virulence and were shown to be translocated independent of either of the type III secretion systems in Salmonella or the type III injectisome-related flagellar mechanism.

Conclusions

Integrating multi-omic datasets from Salmonella mutants lacking virulence regulators not only identified novel virulence factors but also defined a new class of translocated effectors involved in pathogenesis. The success of this strategy at discovery of known and novel virulence factors suggests that the approach may have applicability for other bacterial pathogens.     

Comparing Leishman and Giemsa staining for the assessment of peripheral blood smear preparations in a malaria-endemic region in India

Background

The first phase of malaria infection occurs in the liver and is clinically silent. Inside hepatocytes each Plasmodium sporozoite replicate into thousands of erythrocyte-infectious merozoites that when released into the blood stream result in clinical symptoms of the disease. The time between sporozoite inoculation and the appearance of parasites in the blood is defined as the pre-patent period, which is classically analysed by time-consuming and labor-intensive techniques, such as microscopy and PCR.

Methods

Luciferase-expressing Plasmodium berghei parasites were used to measure pre-patent period of malaria infection in rodents using a bioluminescence assay that requires only one microliter of blood collected from the tail-vein. The accuracy and sensitivity of this new method was compared with conventional microscopy and PCR based techniques, and its capacity to measure the impact of anti-malarial interventions against the liver evaluated.

Results

The described method is very sensitive allowing the detection of parasites during the first cycles of blood stage replication. It accurately translates differences in liver load due to inoculation of different sporozoite doses as well as a result of treatment with different primaquine regimens.

Conclusions

A novel, simple, fast, and sensitive method to measure pre-patent period of malaria infection in rodents is described here. The sensitivity and accuracy of this new method is comparable to standard PCR and microscopy-based techniques, respectively.     

The use of fluorescence enhancement to improve the microscopic diagnosis of falciparum malaria

Background

The Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) are enzymes of central importance in parasite metabolism. The dhfr and dhps gene mutations are known to be associated with sulphadoxine/pyrimethamine (SP) resistance.

Objective

To investigate the effects of dhfr/dhps mutations on parasite characteristics other than SP resistance.

Method

Parasite infections obtained from 153 Sudanese patients with uncomplicated falciparum malaria treated with SP or SP + chloroquine, were successfully genotyped at nine codons in the dhfr/dhps genes by PCR-ELISA.

Results & conclusion

Mutations were detected in dhfr at N51I, S108N and C59R, and in at dhps at A/S436F, A437G, K540E and A581G, the maximum number of mutations per infection were five. Based on number of mutant codons per infection (multiplicity of mutation, MOM), the infections were organized into six grades: wild-types (grade 0; frequency, 0.03) and infections with MOM grades of 1 to 5, with the following cumulative frequency; 0.97, 0.931, 0.866, 0.719, 0.121, respectively. There was no significant association between the MOM and SP response. Importantly, immunity, using age as a surrogate marker, contributed significantly to the clearance of parasites with multiple dhfr/dhps mutations. However, these mutations have a survival advantage as they were associated with increased gametocytogenesis. The above implications of dhfr/dhps mutations were associated with MOM 2 to 5, regardless of the gene/codon locus.     

Pooled PCR testing strategy and prevalence estimation of submicroscopic infections using Bayesian latent class models in pregnant women receiving intermittent preventive treatment at Machinga District Hospital,Malawi, 2010

Background

Metarhizium anisopliae is a naturally occurring fungal pathogen of mosquitoes. Recently, Metarhizium has been engineered to act against malaria by directly killing the disease agent within mosquito vectors and also effectively blocking onward transmission. It has been proposed that efforts should be made to minimize the virulence of the fungal pathogen, in order to slow the development of resistant mosquitoes following an actual deployment.

Results

Two mathematical models were developed and analysed to examine the efficacy of the fungal pathogen. It was found that, in many plausible scenarios, the best effects are achieved with a reduced or minimal pathogen virulence, even if the likelihood of resistance to the fungus is negligible. The results for both models depend on the interplay between two main effects: the ability of the fungus to reduce the mosquito population, and the ability of fungus‐infected mosquitoes to compete for resources with non‐fungus‐infected mosquitoes.

Conclusions

The results indicate that there is no obvious choice of virulence for engineered Metarhizium or similar pathogens, and that all available information regarding the population ecology of the combined mosquito‐fungus system should be carefully considered. The models provide a basic framework for examination of anti‐malarial mosquito pathogens that should be extended and improved as new laboratory and field data become available.     

An indirect enzyme-linked immunosorbent assay for rapid and quantitative assessment of Type III virulence phenotypes of Pseudomonas aeruginosa isolates

Background

The presence of a Type III secretion system in clinical isolates of Pseudomonas aeruginosa is associated with severe disease and poor outcomes in infections caused by this pathogen. We describe an indirect enzyme-linked immunosorbent assay that rapidly and quantitatively detects two exotoxins, ExoU and ExoT, and two structural components, PopD and PcrV, of the P. aeruginosa Type III secretion system after in-vitro growth in a calcium-free minimal medium.

Methods

We used this assay to characterize the Type III secretion phenotype of 74 clinical isolates of P. aeruginosa. Findings were compared with results of standard immunoblotting and correlated with Type III secretion-dependent virulence of isolates toward cultured epithelial cells.

Results

Results of the ELISA assay were concordant with immunoblot detection of the secreted antigens for 73 of 74 isolates. The Type III secretion phenotype assessed by this immunoassay predicted bacterial virulence toward epithelial cells in vitro for all but five of the clinical isolates.

Conclusion

The availability of an ELISA assay for rapid detection of Type III secreted virulence factors will facilitate large clinical studies to examine whether the Type III secretion phenotype of a P. aeruginosa isolate predicts the course of clinical disease in a patient and should be taken into account in determining optimal treatment strategies for infected patients.     

Biological control of pathogen communication in the rhizosphere: A novel approach applied to potato soft rot due to Pectobacterium atrosepticum

Background and aims

Recent basic knowledge on the regulation of virulence in pectinolytic bacteria revealed pathogen communication via quorum sensing signals as a crucial event for the expression of virulence and the onset of disease symptoms. In this paper, we present and discuss advances in a new biocontrol approach based on the interference of microbial communication involved in the cellular density and microenvironment sensoring.

Methods

This emerging strategy consists in the characterization of the signaling molecules used by the target pathogen, then the use of harmless structural analogs to stimulate plant associated-microflora able to degrade both molecule families.

Results

The biocontrol method has been applied for the first time for the control of Pectobacterium atrosepticum. This psychrotrophic bacterium synthesizes N-acyl-homoserine lactones involved in cell-to-cell communication that triggers soft rot and blackleg of potato. The use of the gamma-caprolactone stimulant promotes the emergence and catabolic activity of Rhodococcus erythropolis antagonistic populations in the potato rhizosphere.

Conclusions

Rhodococcus bacteria have the ability to disrupt the quorum sensing-based communication of P. atrosepticum by degrading N-acyl-homoserine lactone signaling molecules and prevent disease.     

Roll Back Malaria? The scarcity of international aid for malaria control

Background

Intraerythrocytic malaria parasites actively import obligate nutrients from serum and export proteins and lipids to erythrocyte cytoplasm and membrane. The import of macromolecules in the malaria parasite has been the subject of many debates. To understand the import of macromolecules by the parasite, we studied the uptake of proteins by Plasmodium falciparum infected human erythrocyte.

Methods

Proteins were biotin labelled individually, purified on a gel filtration column and added to uninfected and infected asynchronized culture. The uptake of these proteins by malaria parasites was determined by western blot analysis of parasite pellet and their different fractions using streptavidin-horseradish conjugate. To further confirm this import, we studied the uptake of¹²⁵I-labelled proteins by western blot analysis as well as used direct immunofluorescence method.

Results

Here we show that biotin labelled and radio-iodinated polypeptides of molecular sizes in the range of 45 to 206 kDa, when added in the culture medium, get direct access to the parasite membrane through a membrane network by by-passing the erythrocyte cytosol. The import of these polypeptides is ATP-dependent as sodium azide treatment blocks this uptake. We also show that malaria parasites have the ability to take up and degrade biotin labelled human serum albumin, which has been shown to be essential for the parasite growth.

Conclusions

These results can be used, as a basis to explore the role of human serum albumin in the intraerythrocytic development of parasites, and this in turn can be an important adjunct to the development of novel antimalarial drugs.     

Gene duplications contribute to the overrepresentation of interactions between proteins of a similar age

Background

Disruptive selection has been documented in a growing number of natural populations. Yet, its prevalence within individual systems remains unclear. Furthermore, few studies have sought to identify the ecological factors that promote disruptive selection in the wild. To address these issues, we surveyed 15 populations of Mexican spadefoot toad tadpoles, Spea multiplicata, and measured the prevalence of disruptive selection acting on resource-use phenotypes. We also evaluated the relationship between the strength of disruptive selection and the intensity of intraspecific competition??an ecological agent hypothesized to be an important driver of disruptive selection.

Results

Disruptive selection was the predominant mode of quadratic selection across all populations. However, a directional component of selection favoring an extreme ecomorph??a distinctive carnivore morph??was also common. Disruptive selection was strongest in populations experiencing the most intense intraspecific competition, whereas stabilizing selection was only found in populations experiencing relatively weak intraspecific competition.

Conclusions

Disruptive selection can be common in natural populations. Intraspecific competition for resources may be a key driver of such selection.     

Interspecific competition in Arabidopsis thaliana: root hairs are important for competitive effect, but not for competitive response

Background and aims

The role of root hairs in intraspecific competition for Phosporus (P) is well examined, but their importance during interaction with other plant species is unknown, as is the differential meaning for competitive effect and response. This study aims to fill this gap of knowledge.

Methods

Competitive abilities of Arabidopsis thaliana wildtype and mutants with aberrant root hair phynotypes (root hair deficient, rhd2-1 or excessive root hair density, prc1-1) were examined in a pot-experiment with P-deficient sand. Competitive effects on a phytometer (Hieracium pilosella) or on A. thaliana itself were assessed as well as competitive responses to species mixtures.

Results

In intraspecific interaction, the competitive effect of wildtype was superior to that of rhd2-1 or prc1-1. This was much less pronounced in interspecific interaction. Competitive response was entirely uniform between Arabidopsis root phenotypes.

Conclusions

The notion that root hairs are important for competition for P should be differentiated. With A. thaliana root hairs less important in inter- than in intraspecific interaction and with root hairs entirely unimportant for competitive response, functional mechanisms of competition for P appear quite complex. Such differential importance of root traits in different facets of competition might well be more common than previously thought.     

To what extent can traditional medicine contribute a complementary or alternative solution to malaria control programmes?

Background

Sulphadoxine and pyrimethamine are anti-folate drugs that show synergistic anti-malarial effect. Point mutations in dihydrofolate reductase (dhfr) and dihydropteorate synthatase (dhps) cause anti-folate drug resistance phenotype in human malaria parasites. This study presents pattern of point mutations in dhfr/dhps genes among Indian sub-continent.

Methods

Microscopically diagnosed one hundred Plasmodium vivax field isolates were collected from five widely separated geographical regions of India. Dhfr and dhps genes were PCR amplified and sequenced. Previously published mutations data were collected and analyzed using Chi square test to identify geographical cluster of mutant/wild type genotypes.

Results

Sequence analysis revealed single (S58R), double (S58R/S117N) and quadruple (F57L/S58R/T61M/S117T/) point mutations at dhfr and single (A383G) to double (A383G/A553G) mutations at dhps in P. vivax field isolates. In addition, three new mutations were also observed at dhfr. Both, dhfr and dhps genes revealed tandem repeat variations in field isolates. Dhps revealed very low mutation frequency (14.0%) compared to dhfr (50.70%). Comparative analysis revealed a progressive increase in frequency of quadruple mutant dhfr genotype (p < 0.001) within five years in north-eastern state (Kamrup, Assam). Frequency of dhfr genotypes revealed three distinct geographical clusters of wild (northern India), double mutant (southern India), and quadruple mutant (north-eastern and island regions of India) on the Indian sub-continent.

Conclusion

Study suggests that SP may be susceptible to P. vivax in India, except Andaman and north-eastern state. The distinction of geographical regions with sensitive and resistant parasite phenotypes would be highly useful for designing and administering national anti-malarial drug policy.     

Effects of human and porcine bile on the proteome of Helicobacter hepaticus

Background

Helicobacter hepaticus colonizes the intestine and liver of mice causing hepatobiliary disorders such as hepatitis and hepatocellular carcinoma, and has also been associated with inflammatory bowel disease in children. In its habitat, H. hepaticus must encounter bile which has potent antibacterial properties. To elucidate virulence and host-specific adaptation mechanisms of H. hepaticus modulated by human or porcine bile, a proteomic study of its response to the two types of bile was performed employing two-dimensional gel electrophoresis (2-DE) and mass spectrometry.

Results

The 2-DE and mass spectrometry analyses of the proteome revealed that 46 proteins of H. hepaticus were differentially expressed in human bile, 18 up-regulated and 28 down-regulated. In the case of porcine bile, 32 proteins were differentially expressed of which 19 were up-regulated, and 13 were down-regulated. Functional classifications revealed that identified proteins participated in various biological functions including stress response, energy metabolism, membrane stability, motility, virulence and colonization. Selected genes were analyzed by RT-PCR to provide internal validation for the proteomic data as well as provide insight into specific expressions of motility, colonization and virulence genes of H. hepaticus in response to human or porcine bile.

Conclusions

Overall, the data suggested that bile is an important factor that determines virulence, host adaptation, localization and colonization of specific niches within host environment.     

Recurrent and founder mutations in the Netherlands: cardiac Troponin I (TNNI3) gene mutations as a cause of severe forms of hypertrophic and restrictive cardiomyopathy

Background

About 2-7% of familial cardiomyopathy cases are caused by a mutation in the gene encoding cardiac troponin I (TNNI3). The related clinical phenotype is usually severe with early onset. Here we report on all currently known mutations in the Dutch population and compared these with those described in literature.

Methods

TheTNNI3 gene was screened for mutations in all coding exons and flanking intronic sequences in a large cohort of cardiomyopathy patients. All Dutch index cases carrying a TNNI3 mutation that are described in this study underwent extensive cardiological evaluation and were listed by their postal codes.

Results

In 30 families, 14 different mutations were identified. Three TNNI3 mutations were found relatively frequently in both familial and non-familial cases of hypertrophic cardiomyopathy (HCM) or restrictive cardiomyopathy (RCM). Haplotype analysis showed that p.Arg145Trp and p.Ser166Phe are founder mutations in the Netherlands, while p.Glu209Ala is not. The majority of Dutch TNNI3 mutations were associated with a HCM phenotype. Mean age at diagnosis was 36.5 years. Mutations causing RCM occurred less frequently, but were identified in very young children with a poor prognosis.

Conclusion

In line with previously published data, we found TNNI3 mutations to be rare and associated with early onset and severe clinical presentation.     

Characterization of Sr9h,a wheat stem rust resistance allele effective to Ug99

Key message

Wheat stem rust resistance gene SrWeb is an allele at the Sr9 locus that confers resistance to Ug99.

Abstract

Race TTKSK (Ug99) of Puccinia graminis f. sp. tritici, the causal fungus of stem rust, threatens global wheat production because of its broad virulence to current wheat cultivars. A recently identified Ug99 resistance gene from cultivar Webster, temporarily designated as SrWeb, mapped near the stem rust resistance gene locus Sr9. We determined that SrWeb is also present in Ug99 resistant cultivar Gabo 56 by comparative mapping and an allelism test. Analysis of resistance in a population segregating for both Sr9e and SrWeb demonstrated that SrWeb is an allele at the Sr9 locus, which subsequently was designated as Sr9h. Webster and Gabo 56 were susceptible to the Ug99-related race TTKSF+ from South Africa. Race TTKSF+ possesses unique virulence to uncharacterized Ug99 resistance in cultivar Matlabas. This result validated that resistance to Ug99 in Webster and Gabo 56 is conferred by the same gene: Sr9h. The emergence of pathogen virulence to several resistance genes that are effective to the original Ug99 race TTKSK, including Sr9h, suggests that resistance genes should be used in combinations in order to increase resistance durability.     

Acquisition of rifabutin resistance by a rifampicin resistant mutant of Mycobacterium tuberculosis involves an unusual spectrum of mutations and elevated frequency

Background

Mutations in a small region of the rpoB gene are responsible for most rifamycin resistance in Mycobacterium tuberculosis. In this study we have sequentially generated resistant strains to first rifampicin and then rifabutin. Portions of the rpoB gene were sequenced from 131 randomly selected mutants. Second round selection resulted in a changed frequency of specific mutations.

Methods

Mycobacterium tuberculosis (strain Mtb72) rifamycin resistant mutants were selected in vitro with either rifampicin or rifabutin. One mutant R190 (rpoB S522L) selected with rifampicin had a rifampicin MIC of 32 μg/ml but remained sensitive to rifabutin (MIC<0.8 μg/ml). This mutant was subjected to a second round of selection with rifabutin.

Results

All 105 first round resistant mutants derived from the parent strain (Mtb72) screened acquired mutations within the 81 bp rpoB hotspot. When the rifampicin resistant but rifabutin sensitive S522L mutant was subjected to a second round of selection, single additional rpoB mutations were identified in 24 (92%) of 26 second round mutants studied, but 14 (54%) of these strains contained mutations outside the 81 bp hotspot (codons 144, 146, 148, 505). Additionally, spontaneous rifabutin resistant mutants were produced at >10 times the frequency by the S522L mutant than the parent strain.

Conclusion

First round selection of mutation S522L with rifampicin increased the frequency and changed the spectrum of mutations identified after selection with rifabutin.     

Monitoring access to nationally commissioned services in England

Background

Deficiency of complex II (succinate dehydrogenase, SDH) represents a rare cause of mitochondrial disease and is associated with a wide range of clinical symptoms. Recently, mutations of SDHAF1, the gene encoding for the SDH assembly factor 1, were reported in SDH-defective infantile leukoencephalopathy. Our goal was to identify SDHAF1 mutations in further patients and to delineate the clinical phenotype.

Methods

In a retrospective data collection study we identified nine children with biochemically proven complex II deficiency among our cohorts of patients with mitochondrial disorders. The cohort comprised five patients from three families affected by SDH-defective infantile leukoencephalopathy with accumulation of succinate in disordered cerebral white matter, as detected by in vivo proton MR spectroscopy. One of these patients had neuropathological features of Leigh syndrome. Four further unrelated patients of the cohort showed diverse clinical phenotypes without leukoencephalopathy. SDHAF1 was sequenced in all nine patients.

Results

Homozygous mutations of SDHAF1 were detected in all five patients affected by leukoencephalopathy with accumulated succinate, but not in any of the four patients with other, diverse clinical phenotypes. Two sisters had a mutation reported previously, in three patients two novel mutations were found.

Conclusion

Leukoencephalopathy with accumulated succinate is a key symptom of defective complex II assembly due to SDHAF1 mutations.     

Multilocus sequence typing method for identification and genotypic classification of pathogenic Leptospira species

Background

Multi-drug resistant Pseudomonas aeruginosa nosocomial infections are increasingly recognized worldwide. In this study, we focused on the virulence of multi-drug resistant clinical strains P. aeruginosa against the intestinal epithelial barrier, since P. aeruginosa can cause lethal sepsis from within the intestinal tract of critically ill and immuno-compromised patients via mechanisms involving disruption of epithelial barrier function.

Methods

We screened consecutively isolated multi-drug resistant P. aeruginosa clinical strains for their ability to disrupt the integrity of human cultured intestinal epithelial cells (Caco-2) and correlated these finding to related virulence phenotypes such as adhesiveness, motility, biofilm formation, and cytotoxicity.

Results

Results demonstrated that the majority of the multi-drug resistant P. aeruginosa clinical strains were attenuated in their ability to disrupt the barrier function of cultured intestinal epithelial cells. Three distinct genotypes were found that displayed an extreme epithelial barrier-disrupting phenotype. These strains were characterized and found to harbor the exoU gene and to display high swimming motility and adhesiveness.

Conclusion

These data suggest that detailed phenotypic analysis of the behavior of multi-drug resistant P. aeruginosa against the intestinal epithelium has the potential to identify strains most likely to place patients at risk for lethal gut-derived sepsis. Surveillance of colonizing strains of P. aeruginosa in critically ill patients beyond antibiotic sensitivity is warranted.