Prevalence and patterns of antifolate and chloroquine drug resistance markers in Plasmodium vivax across Pakistan.

BACKGROUND: Plasmodium vivax is the most prevalent malaria species in Pakistan, with a distribution that coincides with Plasmodium falciparum in many parts of the country. Both species are likely exposed to drug pressure from a number of anti-malarials including chloroquine, sulphadoxine-pyrimethamine (SP), and artemisinin combination therapy, yet little is known regarding the effects of drug pressure on parasite genes associated with drug resistance. The aims of this study were to determine the prevalence of polymorphisms in the SP resistance-associated genes pvdhfr, pvdhps and chloroquine resistance-associated gene pvmdr1 in P. vivax isolates collected from across the country. METHODS: In 2011, 801 microscopically confirmed malaria-parasite positive filter paper blood samples were collected at 14 sites representing four provinces and the capital city of Islamabad. Species-specific polymerase chain reaction (PCR) was used to identify human Plasmodium species infection. PCR-positive P. vivax isolates were subjected to sequencing of pvdhfr, pvdhps and pvmdr1 and to real-time PCR analysis to assess pvmdr1 copy number variation. RESULTS: Of the 801 samples, 536 were determined to be P. vivax, 128 were P. falciparum, 43 were mixed vivax/falciparum infections and 94 were PCR-negative for Plasmodium infection. Of PCR-positive P. vivax samples, 372 were selected for sequence analysis. Seventy-six of the isolates (23%) were double mutant at positions S58R and S117N in pvdhfr. Additionally, two mutations at positions N50I and S93H were observed in 55 (15%) and 24 (7%) of samples, respectively. Three 18 base pair insertion-deletions (indels) were observed in pvdhfr, with two insertions at different nucleotide positions in 36 isolates and deletions in 10. Ninety-two percent of samples contained the pvdhps (S382/A383G/K512/A553/V585) SAKAV wild type haplotype. For pvmdr1, all isolates were wild type at position Y976F and 335 (98%) carried the mutation at codon F1076L. All isolates harboured single copies of the pvmdr1 gene. CONCLUSIONS: The prevalence of mutations associated with SP resistance in P. vivax is low in Pakistan. The high prevalence of P. vivax mutant pvmdr1 codon F1076L indicates that efficacy of chloroquine plus primaquine could be in danger of being compromised, but further studies are required to assess the clinical relevance of this observation. These findings will serve as a baseline for further monitoring of drug-resistant P. vivax malaria in Pakistan.

Genetic loci associated with delayed clearance of Plasmodium falciparum following artemisinin treatment in Southeast Asia.

The recent emergence of artemisinin-resistant Plasmodium falciparum malaria in western Cambodia could threaten prospects for malaria elimination. Identification of the genetic basis of resistance would provide tools for molecular surveillance, aiding efforts to contain resistance. Clinical trials of artesunate efficacy were conducted in Bangladesh, in northwestern Thailand near the Myanmar border, and at two sites in western Cambodia. Parasites collected from trial participants were genotyped at 8,079 single nucleotide polymorphisms (SNPs) using a P. falciparum-specific SNP array. Parasite genotypes were examined for signatures of recent positive selection and association with parasite clearance phenotypes to identify regions of the genome associated with artemisinin resistance. Four SNPs on chromosomes 10 (one), 13 (two), and 14 (one) were significantly associated with delayed parasite clearance. The two SNPs on chromosome 13 are in a region of the genome that appears to be under strong recent positive selection in Cambodia. The SNPs on chromosomes 10 and 13 lie in or near genes involved in postreplication repair, a DNA damage-tolerance pathway. Replication and validation studies are needed to refine the location of loci responsible for artemisinin resistance and to understand the mechanism behind it; however, two SNPs on chromosomes 10 and 13 may be useful markers of delayed parasite clearance in surveillance for artemisinin resistance in Southeast Asia.

Clinical manifestations of new versus recrudescent malaria infections following anti-malarial drug treatment.

BACKGROUND: Distinguishing new from recrudescent infections in post-treatment episodes of malaria is standard in anti-malarial drug efficacy trials. New infections are not considered malaria treatment failures and as a result, the prevention of subsequent episodes of malaria infection is not reported as a study outcome. However, in moderate and high transmission settings, new infections are common and the ability of a short-acting medication to cure an initial infection may be outweighed by its inability to prevent the next imminent infection. The clinical benefit of preventing new infections has never been compared to that of curing the initial infection. METHODS: Children enrolled in a sulphadoxine-pyrimethamine efficacy study in Blantyre, Malawi from 1998-2004 were prospectively evaluated. Six neutral microsatellites were used to classify new and recrudescent infections in children aged less than 10 years with recurrent malaria infections. Children from the study who did not experience recurrent parasitaemia comprised the baseline group. The odds of fever and anaemia, the rate of haemoglobin recovery and time to recurrence were compared among the groups. RESULTS: Fever and anemia were more common among children with parasitaemia compared to those who remained infection-free throughout the study period. When comparing recrudescent vs. new infections, the incidence of fever was not statistically different. However, children with recrudescent infections had a less robust haematological recovery and also experienced recurrence sooner than those whose infection was classified as new. CONCLUSIONS: The results of this study confirm the paramount importance of providing curative treatment for all malaria infections. Although new and recrudescent infections caused febrile illnesses at a similar rate, recurrence due to recrudescent infection did have a worsened haemological outcome than recurrence due to new infections. Local decision-makers should take into account the results of genotyping to distinguish new from recrudescent infections when determining treatment policy on a population level. It is appropriate to weigh recrudescent malaria more heavily than new infection in assessing treatment efficacy.

Bacillus anthracis comparative genome analysis in support of the Amerithrax investigation.

Before the anthrax letter attacks of 2001, the developing field of microbial forensics relied on microbial genotyping schemes based on a small portion of a genome sequence. Amerithrax, the investigation into the anthrax letter attacks, applied high-resolution whole-genome sequencing and comparative genomics to identify key genetic features of the letters' Bacillus anthracis Ames strain. During systematic microbiological analysis of the spore material from the letters, we identified a number of morphological variants based on phenotypic characteristics and the ability to sporulate. The genomes of these morphological variants were sequenced and compared with that of the B. anthracis Ames ancestor, the progenitor of all B. anthracis Ames strains. Through comparative genomics, we identified four distinct loci with verifiable genetic mutations. Three of the four mutations could be directly linked to sporulation pathways in B. anthracis and more specifically to the regulation of the phosphorylation state of Spo0F, a key regulatory protein in the initiation of the sporulation cascade, thus linking phenotype to genotype. None of these variant genotypes were identified in single-colony environmental B. anthracis Ames isolates associated with the investigation. These genotypes were identified only in B. anthracis morphotypes isolated from the letters, indicating that the variants were not prevalent in the environment, not even the environments associated with the investigation. This study demonstrates the forensic value of systematic microbiological analysis combined with whole-genome sequencing and comparative genomics.

Bacillus anthracis in China and its relationship to worldwide lineages.

BACKGROUND: The global pattern of distribution of 1033 B. anthracis isolates has previously been defined by a set of 12 conserved canonical single nucleotide polymorphisms (canSNP). These studies reinforced the presence of three major lineages and 12 sub-lineages and sub-groups of this anthrax-causing pathogen. Isolates that form the A lineage (unlike the B and C lineages) have become widely dispersed throughout the world and form the basis for the geographical disposition of "modern" anthrax. An archival collection of 191 different B. anthracis isolates from China provides a glimpse into the possible role of Chinese trade and commerce in the spread of certain sub-lineages of this pathogen. Canonical single nucleotide polymorphism (canSNP) and multiple locus VNTR analysis (MLVA) typing has been used to examine this archival collection of isolates. RESULTS: The canSNP study indicates that there are 5 different sub-lineages/sub-groups in China out of 12 previously described world-wide canSNP genotypes. Three of these canSNP genotypes were only found in the western-most province of China, Xinjiang. These genotypes were A.Br.008/009, a sub-group that is spread across most of Europe and Asia; A.Br.Aust 94, a sub-lineage that is present in Europe and India, and A.Br.Vollum, a lineage that is also present in Europe. The remaining two canSNP genotypes are spread across the whole of China and belong to sub-group A.Br.001/002 and the A.Br.Ames sub-lineage, two closely related genotypes. MLVA typing adds resolution to the isolates in each canSNP genotype and diversity indices for the A.Br.008/009 and A.Br.001/002 sub-groups suggest that these represent older and established clades in China. CONCLUSION: B. anthracis isolates were recovered from three canSNP sub-groups (A.Br.008/009, A.Br.Aust94, and A.Br.Vollum) in the western most portion of the large Chinese province of Xinjiang. The city of Kashi in this province appears to have served as a crossroads for not only trade but the movement of diseases such as anthrax along the ancient "silk road". Phylogenetic inference also suggests that the A.Br.Ames sub-lineage, first identified in the original Ames strain isolated from Jim Hogg County, TX, is descended from the A.Br.001/002 sub-group that has a major presence in most of China. These results suggest a genetic discontinuity between the younger Ames sub-lineage in Texas and the large Western North American sub-lineage spread across central Canada and the Dakotas.

Pre-Columbian origins for North American anthrax.

Disease introduction into the New World during colonial expansion is well documented and had a major impact on indigenous populations; however, few diseases have been associated with early human migrations into North America. During the late Pleistocene epoch, Asia and North America were joined by the Beringian Steppe ecosystem which allowed animals and humans to freely cross what would become a water barrier in the Holocene. Anthrax has clearly been shown to be dispersed by human commerce and trade in animal products contaminated with Bacillus anthracis spores. Humans appear to have brought B. anthracis to this area from Asia and then moved it further south as an ice-free corridor opened in central Canada approximately 13,000 ybp. In this study, we have defined the evolutionary history of Western North American (WNA) anthrax using 2,850 single nucleotide polymorphisms (SNPs) and 285 geographically diverse B. anthracis isolates. Phylogeography of the major WNA B. anthracis clone reveals ancestral populations in northern Canada with progressively derived populations to the south; the most recent ancestor of this clonal lineage is in Eurasia. Our phylogeographic patterns are consistent with B. anthracis arriving with humans via the Bering Land Bridge. This northern-origin hypothesis is highly consistent with our phylogeographic patterns and rates of SNP accumulation observed in current day B. anthracis isolates. Continent-wide dispersal of WNA B. anthracis likely required movement by later European colonizers, but the continent's first inhabitants may have seeded the initial North American populations.

Real-time PCR assays of single-nucleotide polymorphisms defining the major Brucella clades.

Members of the genus Brucella are known worldwide as pathogens of wildlife and livestock and are the most common organisms of zoonotic infection in humans. In general, brucellae exhibit a range of host specificity in animals that has led to the identification of at least seven Brucella species. The genomes of the various Brucella species are highly conserved, which makes the differentiation of species highly challenging. However, we found single-nucleotide polymorphisms (SNPs) in housekeeping and other genes that differentiated the seven main Brucella species or clades and thus enabled us to develop real-time PCR assays based around these SNPs. Screening of a diverse panel of 338 diverse isolates with these assays correctly identified each isolate with its previously determined Brucella clade. Six of the seven clade-specific assays detected DNA concentrations of less than 10 fg, indicating a high level of sensitivity. This SNP-based approach places samples into a phylogenetic framework, allowing reliable comparisons to be made among the lineages of clonal bacteria and providing a solid basis for genotyping. These PCR assays provide a rapid and highly sensitive method of differentiating the major Brucella groups that will be valuable for clinical and forensic applications.

Global genetic population structure of Bacillus anthracis.

Anthrax, caused by the bacterium Bacillus anthracis, is a disease of historical and current importance that is found throughout the world. The basis of its historical transmission is anecdotal and its true global population structure has remained largely cryptic. Seven diverse B. anthracis strains were whole-genome sequenced to identify rare single nucleotide polymorphisms (SNPs), followed by phylogenetic reconstruction of these characters onto an evolutionary model. This analysis identified SNPs that define the major clonal lineages within the species. These SNPs, in concert with 15 variable number tandem repeat (VNTR) markers, were used to subtype a collection of 1,033 B. anthracis isolates from 42 countries to create an extensive genotype data set. These analyses subdivided the isolates into three previously recognized major lineages (A, B, and C), with further subdivision into 12 clonal sub-lineages or sub-groups and, finally, 221 unique MLVA15 genotypes. This rare genomic variation was used to document the evolutionary progression of B. anthracis and to establish global patterns of diversity. Isolates in the A lineage are widely dispersed globally, whereas the B and C lineages occur on more restricted spatial scales. Molecular clock models based upon genome-wide synonymous substitutions indicate there was a massive radiation of the A lineage that occurred in the mid-Holocene (3,064-6,127 ybp). On more recent temporal scales, the global population structure of B. anthracis reflects colonial-era importation of specific genotypes from the Old World into the New World, as well as the repeated industrial importation of diverse genotypes into developed countries via spore-contaminated animal products. These findings indicate humans have played an important role in the evolution of anthrax by increasing the proliferation and dispersal of this now global disease. Finally, the value of global genotypic analysis for investigating bioterrorist-mediated outbreaks of anthrax is demonstrated.

Strain-specific single-nucleotide polymorphism assays for the Bacillus anthracis Ames strain.

Highly precise diagnostics and forensic assays can be developed through a combination of evolutionary analysis and the exhaustive examination of genomic sequences. In Bacillus anthracis, whole-genome sequencing efforts revealed ca. 3,500 single-nucleotide polymorphisms (SNPs) among eight different strains and evolutionary analysis provides the identification of canonical SNPs. We have previously shown that SNPs are highly evolutionarily stable, and the clonal nature of B. anthracis makes them ideal signatures for subtyping this pathogen. Here we identified SNPs that define the lineage of B. anthracis that contains the Ames strain, the strain used in the 2001 bioterrorist attacks in the United States. Sequencing and real-time PCR were used to validate these SNPs across B. anthracis strains, including (i) 88 globally and genetically diverse isolates; (ii) isolates that were shown to be genetic relatives of the Ames strain by multiple-locus variable number tandem repeat analysis (MLVA); and (iii) several different lab stocks of the Ames strain, including a clinical isolate from the 2001 letter attack. Six SNPs were found to be highly specific for the Ames strain; four on the chromosome, one on the pX01 plasmid, and one on the pX02 plasmid. All six SNPs differentiated the B. anthracis Ames strain from the 88 unique B. anthracis strains, while five of the six separated Ames from its close genetic relatives. The use of these SNPs coupled with real-time PCR allows specific and sensitive (<100 fg of template DNA) identification of the Ames strain. This evolutionary and genomics-based approach provides an effective means for the discovery of strain-specific SNPs in B. anthracis.

Use of single nucleotide polymorphisms in the plcR gene for specific identification of Bacillus anthracis.

A TaqMan-minor groove binding assay designed around a nonsense mutation in the plcR gene was used to genotype Bacillus anthracis, B. cereus, and B. thuringiensis isolates. The assay differentiated B. anthracis from these genetic near-neighbors and determined that the nonsense mutation is ubiquitous across 89 globally and genetically diverse B. anthracis strains.