pfmdr1 amplification and fixation of pfcrt chloroquine resistance alleles in Plasmodium falciparum in Venezuela.

Molecular tools are valuable for determining evolutionary history and the prevalence of drug-resistant malaria parasites. These tools have helped to predict decreased sensitivity to antimalarials and fixation of multidrug resistance genotypes in some regions. In order to assess how historical drug policies impacted Plasmodium falciparum in Venezuela, we examined molecular changes in genes associated with drug resistance. We examined pfmdr1 and pfcrt in samples from Sifontes, Venezuela, and integrated our findings with earlier work describing dhfr and dhps in these samples. We characterized pfmdr1 genotypes and copy number variation, pfcrt genotypes, and proximal microsatellites in 93 samples originating from surveillance from 2003 to 2004. Multicopy pfmdr1 was found in 12% of the samples. Two pfmdr1 alleles, Y184F/N1042D/D1246Y (37%) and Y184F/S1034C/N1042D/D1246Y (63%), were found. These alleles share ancestry, and no evidence of strong selective pressure on mutations was found. pfcrt chloroquine resistance alleles are fixed with two alleles: S(tct)VMNT (91%) and S(agt)VMNT (9%). These alleles are associated with strong selection. There was also an association between pfcrt, pfmdr1, dhfr, and dhps genotypes/haplotypes. Duplication of pfmdr1 suggests a potential shift in mefloquine sensitivity in this region, which warrants further study. A bottleneck occurred in P. falciparum in Sifontes, Venezuela, and multidrug resistance genotypes are present. This population could be targeted for malaria elimination programs to prevent the possible spread of multidrug-resistant parasites.

Anti-folate drug resistance in Africa: meta-analysis of reported dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutant genotype frequencies in African Plasmodium falciparum parasite populations.

BACKGROUND: Mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes of Plasmodium falciparum are associated with resistance to anti-folate drugs, most notably sulphadoxine-pyrimethamine (SP). Molecular studies document the prevalence of these mutations in parasite populations across the African continent. However, there is no systematic review examining the collective epidemiological significance of these studies. This meta-analysis attempts to: 1) summarize genotype frequency data that are critical for molecular surveillance of anti-folate resistance and 2) identify the specific challenges facing the development of future molecular databases. METHODS: This review consists of 220 studies published prior to 2009 that report the frequency of select dhfr and dhps mutations in 31 African countries. Maps were created to summarize the location and prevalence of the highly resistant dhfr triple mutant (N51I, C59R, S108N) genotype and dhps double mutant (A437G and K540E) genotype in Africa. A hierarchical mixed effects logistic regression was used to examine the influence of various factors on reported mutant genotype frequency. These factors include: year and location of study, age and clinical status of sampled population, and reporting conventions for mixed genotype data. RESULTS: A database consisting of dhfr and dhps mutant genotype frequencies from all African studies that met selection criteria was created for this analysis. The map illustrates particularly high prevalence of both the dhfr triple and dhps double mutant genotypes along the Kenya-Tanzania border and Malawi. The regression model shows a statistically significant increase in the prevalence of both the dhfr triple and dhps double mutant genotypes in Africa. CONCLUSION: Increasing prevalence of the dhfr triple mutant and dhps double mutant genotypes in Africa are consistent with the loss of efficacy of SP for treatment of clinical malaria in most parts of this continent. Continued assessment of the effectiveness of SP for the treatment of clinical malaria and intermittent preventive treatment in pregnancy is needed. The creation of a centralized resistance data network, such as the one proposed by the WorldWide Antimalarial Resistance Network (WWARN), will become a valuable resource for planning timely actions to combat drug resistant malaria.

CD4+ T cell polyfunctional profile in HIV-TB coinfection are similar between individuals with latent and active TB infection.

CD4+ T cell counts of HIV-infected individuals with pulmonary TB (PTB) are higher than with other opportunistic infections suggesting that progression to PTB is not merely due to T cell depletion but also dysfunction. There are limited data examining T cell functional signatures in human HIV-TB co-infection particularly in PTB which accounts for about 80% of active TB disease overall. We examined a cohort of HIV-infected anti-retroviral naïve individuals in Kampala, Uganda, a TB endemic area using multiparametric flow cytometry analysis to determine IFN-γ, IL-2, IL-17, and TNF-α production in CD4+ memory T cell subsets. The cytokine frequency and polyfunctionality profile of Mycobacterium tuberculosis (MTB)-specific CD4+ T cells in HIV-infected persons with latent TB infection (LTBI) or PTB is comparable. This similarity suggests that LTBI may represent a smoldering state of persistent MTB replication rather than dormant infection. This may be a contributory mechanism to the significantly increased risk of progression to PTB in this population.

A case of cough variant asthma undiagnosed for 16 years.

A 64-year-old female patient presented with a 16-year history of persistent dry cough that was undiagnosed after workups at several healthcare facilities. The patient denies wheezing, shortness of breath or sputum production. Previous workups include chest imaging, transthoracic echocardiogram (TTE), laryngoscopy, spirometry and bronchoscopy, all of which were unremarkable. During her current evaluation, spirometry was ordered again for the patient, which showed a post-bronchodilator improvement in the FEV1 by 13%, strongly suggestive of asthma. The patient was started on pharmacological therapy for severe persistent asthma, which led to sustained symptomatic improvement per evaluation at follow-up after 2 months. Spirometric findings, clinical presentation and resolution of symptoms with adequate therapy for asthma suggest that this is a case of cough variant asthma that went undiagnosed for several years. This case report summarizes the workup for chronic cough and how the diagnosis of cough variant asthma can be missed.

Molecular analysis of chloroquine and sulfadoxine-pyrimethamine resistance-associated alleles in Plasmodium falciparum isolates from Nicaragua.

Chloroquine (CQ) is used as a first-line therapy for the treatment of Plasmodium falciparum malaria in Nicaragua. We investigated the prevalence of molecular markers associated with CQ and sulfadoxine-pyrimethamine (SP) resistance in P. falciparum isolates obtained from the North Atlantic Autonomous Region of Nicaragua. Blood spots for this study were made available from a CQ and SP drug efficacy trial conducted in 2005 and also from a surveillance study performed in 2011. Polymorphisms in P. falciparum CQ resistance transporter, dihydrofolate reductase, and dihydropteroate synthase gene loci that are associated with resistance to CQ, pyrimethamine, and sulfadoxine, respectively, were detected by DNA sequencing. In the 2005 dataset, only 2 of 53 isolates had a CQ resistance allele (CVIET), 2 of 52 had a pyrimethamine resistance allele, and 1 of 49 had a sulfadoxine resistance allele. In the 2011 dataset, none of 45 isolates analyzed had CQ or SP resistance alleles.

Historical shifts in Brazilian P. falciparum population structure and drug resistance alleles.

Previous work suggests that Brazilian Plasmodium falciparum has limited genetic diversity and a history of bottlenecks, multiple reintroductions due to human migration, and clonal expansions. We hypothesized that Brazilian P. falciparum would exhibit clonal structure. We examined isolates collected across two decades from Amapá, Rondônia, and Pará state (n = 190). By examining more microsatellites markers on more chromosomes than previous studies, we hoped to define the extent of low diversity, linkage disequilibrium, bottlenecks, population structure, and parasite migration within Brazil. We used retrospective genotyping of samples from the 1980s and 1990s to explore the population genetics of SP resistant dhfr and dhps alleles. We tested an existing hypothesis that the triple mutant dhfr mutations 50R/51I/108N and 51I/108N/164L developed in southern Amazon from a single origin of common or similar parasites. We found that Brazilian P. falciparum had limited genetic diversity and isolation by distance was rejected, which suggests it underwent bottlenecks followed by migration between sites. Unlike Peru, there appeared to be gene flow across the Brazilian Amazon basin. We were unable to divide parasite populations by clonal lineages and pairwise FST were common. Most parasite diversity was found within sites in the Brazilian Amazon, according to AMOVA. Our results challenge the hypothesis that triple mutant alleles arose from a single lineage in the Southern Amazon. SP resistance, at both the double and triple mutant stages, developed twice and potentially in different regions of the Brazilian Amazon. We would have required samples from before the 1980s to describe how SP resistance spread across the basin or describe the complex internal migration of Brazilian parasites after the colonization efforts of past decades. The Brazilian Amazon basin may have sufficient internal migration for drug resistance reported in any particular region to rapidly spread to other parts of basin under similar drug pressure.

Genetic backgrounds of the Plasmodium falciparum chloroquine resistant transporter (pfcrt) alleles in Pakistan.

Chloroquine (CQ) resistance in Plasmodium falciparum has been associated with point mutations in the P. falciparum CQ resistance transporter gene (pfcrt). Previous studies have shown 4-5 independent origins for CQ resistant pfcrt alleles globally, two in South America, one each in Southeast Asia, Papua New Guinea (PNG) and Philippines. In Asia, at least two different alleles corresponding to amino acids 72-76 (CVIET and SVMNT) have been found. The CVIET allele originated in Southeast Asia and then spread to Asia and Africa as well. The SVMNT allele, originating from PNG, has been found in India. This study was undertaken to investigate the genetic background of the CQ resistant pfcrt haplotypes in Pakistan. We genotyped microsatellite markers surrounding the pfcrt gene (six different markers at -12.3, -4.8, -1, 1.5, 3.9, 18.8 kb) in 114 clinical isolates of P. falciparum collected from different regions in Pakistan. Microsatellite analysis showed a significant reduction in genetic variation among the mutant SVMNT pfcrt alleles when compared to wild type alleles. The predominant SVMNT haplotype found in this study shared the same microsatellite haplotype found in both PNG and India. Two isolates with CVIET haplotypes showed similar microsatellite background to those found in Africa and Asia. In conclusion, this study suggests that CQ resistant SVMNT haplotypes in India and Pakistan have a common ancestral origin similar to that of Papua New Guinean isolates.

South American Plasmodium falciparum after the malaria eradication era: clonal population expansion and survival of the fittest hybrids.

Malaria has reemerged in many regions where once it was nearly eliminated. Yet the source of these parasites, the process of repopulation, their population structure, and dynamics are ill defined. Peru was one of malaria eradication's successes, where Plasmodium falciparum was nearly eliminated for two decades. It reemerged in the 1990s. In the new era of malaria elimination, Peruvian P. falciparum is a model of malaria reinvasion. We investigated its population structure and drug resistance profiles. We hypothesized that only populations adapted to local ecological niches could expand and repopulate and originated as vestigial populations or recent introductions. We investigated the genetic structure (using microsatellites) and drug resistant genotypes of 220 parasites collected from patients immediately after peak epidemic expansion (1999-2000) from seven sites across the country. The majority of parasites could be grouped into five clonal lineages by networks and AMOVA. The distribution of clonal lineages and their drug sensitivity profiles suggested geographic structure. In 2001, artesunate combination therapy was introduced in Peru. We tested 62 parasites collected in 2006-2007 for changes in genetic structure. Clonal lineages had recombined under selection for the fittest parasites. Our findings illustrate that local adaptations in the post-eradication era have contributed to clonal lineage expansion. Within the shifting confluence of drug policy and malaria incidence, populations continue to evolve through genetic outcrossing influenced by antimalarial selection pressure. Understanding the population substructure of P. falciparum has implications for vaccine, drug, and epidemiologic studies, including monitoring malaria during and after the elimination phase.