Prevalence of resistance associated polymorphisms in Plasmodium falciparum field isolates from southern Pakistan

<p>Abstract</p> <p>Background</p> <p>Scarce data are available on <it>Plasmodium falciparum </it>anti-malarial drug resistance in Pakistan. The aim of this study was, therefore, to determine the prevalence of <it>P. falciparum </it>resistance associated polymorphisms in field isolates from southern Pakistan.</p> <p>Methods</p> <p>Blood samples from 244 patients with blood-slide confirmed <it>P. falciparum </it>mono-infections were collected between 2005-2007. Single nucleotide polymorphisms in the <it>P. falciparum </it>chloroquine resistance transporter (<it>pfcrt </it>K76T), multi drug resistance (<it>pfmdr1 </it>N86Y), dihydrofolate reductase (<it>pfdhfr </it>A16V, N51I, C59R, S108N, I164L) and dihydropteroate synthetase (<it>pfdhps </it>A436S, G437A and E540K) genes and <it>pfmdr1 </it>gene copy numbers were determined using PCR based methods.</p> <p>Results</p> <p>The prevalence of <it>pfcrt </it>76T and <it>pfmdr1 </it>86Y was 93% and 57%, respectively. The prevalence of <it>pfdhfr </it>double mutations 59R + 108N/51R + 108N was 92%. The <it>pfdhfr </it>triple mutation (51I, 59R, 108N) occurred in 3% of samples. The <it>pfdhfr </it>(51I, 59R, 108N) and <it>pfdhps </it>(437G, 540E) quintuple mutation was found in one isolate. <it>Pfdhps </it>437G was observed in 51% and 540E in 1% of the isolates. One isolate had two <it>pfmdr1 </it>copies and carried the <it>pfmdr1 </it>86Y and <it>pfcrt </it>76T alleles.</p> <p>Conclusions</p> <p>The results indicate high prevalence of <it>in vivo </it>resistance to chloroquine, whereas high grade resistance to sulphadoxine-pyrimethamine does not appear to be widespread among <it>P. falciparum </it>in southern Pakistan.</p

Single nucleotide polymorphisms in Plasmodium falciparum V type H+ pyrophosphatase gene (pfvp2) and their associations with pfcrt and pfmdr1 polymorphisms

"Uncorrected proof"BACKGROUND: Chloroquine resistance in Plasmodium falciparum malaria has been associated with pfcrt 76T (chloroquine resistance transporter gene) and pfmdr1 86Y (multidrug resistance gene 1) alleles. Pfcrt 76T enables transport of protonated chloroquine out of the parasites digestive vacuole resulting in a loss of hydrogen ions (H(+)). V type H(+) pyrophosphatase (PfVP2) is thought to pump H(+) into the digestive vacuole. This study aimed to describe the geographic distribution of single nucleotide polymorphisms in pfvp2 and their possible associations with pfcrt and pfmdr1 polymorphisms. METHODS: Blood samples from 384 patients collected (1981-2009) in Honduras (n=35), Colombia (n=50), Liberia (n=50), Guinea Bissau (n=50), Tanzania (n=50), Iran (n=50), Thailand (n=49) and Vanuatu (n=50) were analysed. The pfcrt 72-76 haplotype, pfmdr1 copy numbers, pfmdr1 N86Y and pfvp2 V405I, K582R and P711S alleles were identified using PCR based methods. RESULTS: Pfvp2 was amplified in 344 samples. The pfvp2 allele proportions were V405 (97%), 405I (3%), K582 (99%), 582R (1%), P711 (97%) and 711S (3%). The number of patients with any of pfvp2 405I, 582R and/or 711S were as follows: Honduras (2/30), Colombia (0/46), Liberia (7/48), Guinea-Bissau (4/50), Tanzania (3/48), Iran (3/50), Thailand (1/49) and Vanuatu (0/31). The alleles were most common in Liberia (P=0.01) and Liberia+Guinea-Bissau (P=0.01). The VKP haplotype was found in 189/194 (97%) and 131/145 (90%) samples harbouring pfcrt 76T and pfcrt K76 respectively (P=0.007). CONCLUSIONS: The VKP haplotype was dominant. Most pfvp2 405I, 582R and 711S SNPs were seen where CQ resistance was not highly prevalent at the time of blood sampling possibly due to greater genetic variation prior to the bottle neck event of spreading CQ resistance. The association between the pfvp2 VKP haplotype and pfcrt 76T, which may indicate that pfvp2 is involved in CQ resistance, should therefore be interpreted with caution.This work was supported by Swedish International Development Cooperation Agency, Department for research Cooperation (Sida-SAREC Contribution no 75007082/03) and Sigurd och Elsa Goljes Minne Fund (project No. LA2010-0537). MIV is recipient of Post Doctoral fellowship from Fundacao para a Ciencia e Tecnologia (FCT)/Ministerio da Ciencia e Ensino Superior, Portugal - MCES (ref. SFRH/BPD/76614/2011). JU has a postdoctoral position funded by Stockholms lans landsting

Parents' experiences of an abnormal ultrasound examination - vacillating between emotional confusion and sense of reality

<p>Abstract</p> <p>Background</p> <p>An ultrasound examination is an important confirmation of the pregnancy and is accepted without reflection to any prenatal diagnostic aspects. An abnormal finding often comes unexpectedly and is a shock for the parents. The aim was to generate a theoretical understanding of parents' experiences of the situation when their fetus is found to have an abnormality at a routine ultrasound examination.</p> <p>Methods</p> <p>Sixteen parents, mothers and fathers, whose fetus had been diagnosed with an abnormality during an ultrasound scan in the second or third trimester, were interviewed. The study employed a grounded theory approach.</p> <p>Results</p> <p>The core category <it>vacillating between the emotional confusion and sense of reality </it>is related to the main concern <it>assessment of the diagnosis impact on the well-being of the fetus</it>. Two other categories <it>Entering uncertainty </it>and <it>Involved in an ongoing change and adaptation </it>have each five sub-categories.</p> <p>Conclusions</p> <p>Parents are aware of that ultrasound examination is a tool for identifying abnormalities prenatally. The information about the abnormality initially results in broken expectations and anxiety. Parents become involved in ongoing change and adaptation. They need information about the ultrasound findings and the treatment without prolonged delay and in a suitable environment. The examiner who performs the ultrasound examination must be aware of how anxiety can be intensified by environmental factors. All parents should to be offered a professional person to give them <it>s</it>upport as a part of the routine management of this situation.</p

Drug resistance associated genetic polymorphisms in Plasmodium falciparum and Plasmodium vivax collected in Honduras, Central America

Background: In Honduras, chloroquine and primaquine are recommended and still appear to be effective for treatment of Plasmodium falciparum and Plasmodium vivax malaria. The aim of this study was to determine the proportion of resistance associated genetic polymorphisms in P. falciparum and P. vivax collected in Honduras. Methods: Blood samples were collected from patients seeking medical attention at the Hospital Escuela in Tegucigalpa from 2004 to 2006 as well as three regional hospitals, two health centres and one regional laboratory during 2009. Single nucleotide polymorphisms in P. falciparum chloroquine resistance transporter (pfcrt), multidrug resistance 1 (pfmdr1), dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes and in P. vivax multidrug resistance 1 (pvmdr1) and dihydrofolate reductase (pvdhfr) genes were detected using PCR based methods. Results: Thirty seven P. falciparum and 64 P. vivax samples were collected. All P. falciparum infections acquired in Honduras carried pfcrt, pfmdr1, pfdhps and pfdhfr alleles associated with chloroquine, amodiaquine and sulphadoxine-pyrimethamine sensitivity only. One patient with parasites acquired on a Pacific Island had pfcrt 76 T and pfmdr1 86Y alleles. That patient and a patient infected in West Africa had pfdhfr 51I, 59 R and 108 N alleles. Pvmdr1 976 F was found in 7/37 and two copies of pvmdr1 were found in 1/37 samples. Pvdhfr 57 L + 58 R was observed in 2/57 samples. Conclusion: The results indicate that P. falciparum from Honduras remain sensitive to chloroquine and sulphadoxine-pyrimethamine. This suggests that chloroquine and sulphadoxine-pyrimethamine should be efficacious for treatment of uncomplicated P. falciparum malaria, supporting current national treatment guidelines. However, genetic polymorphisms associated with chloroquine and sulphadoxine-pyrimethamine tolerance were detected in local P. vivax and imported P. falciparum infections. Continuous monitoring of the prevalence of drug resistant/tolerant P. falciparum and P. vivax is therefore essential also in Honduras.Swedish International Development Cooperation Agency, Department for research Cooperation (Sida-SAREC) [75007082/03]info:eu-repo/semantics/publishedVersio

Identification of pyrimethamine- and chloroquine-resistant Plasmodium falciparum in Africa between 1984 and 1998: genotyping of archive blood samples

<p>Abstract</p> <p>Background</p> <p>Understanding the geographical distribution of drug resistance of <it>Plasmodium falciparum </it>is important for the effective treatment of malaria. Drug resistance has previously been inferred mainly from records of clinical resistance. However, clinical resistance is not always consistent with the parasite's genetic resistance. Thus, molecular identification of the parasite's drug resistance is required. In Africa, clinical resistance to pyrimethamine (Pyr) and chloroquine (CQ) was evident before 1980 but few studies investigating the genetic resistance to these drugs were conducted before the late 1990s. In this study, genotyping of genes involved in resistance to Pyr and CQ was performed using archive blood samples from Africa between 1984 and 1998.</p> <p>Methods</p> <p>Parasite DNA was extracted from <it>P. falciparum</it>-infected blood smears collected from travellers returning to Japan from Africa between 1984 and 1998. Genotypes of the dihydrofolate reductase gene (<it>dhfr</it>) and CQ-resistance transporter gene (<it>pfcrt) </it>were determined by polymerase chain reaction amplification and sequencing.</p> <p>Results</p> <p>Genotyping of <it>dhfr </it>and <it>pfcrt </it>was successful in 59 and 80 samples, respectively. One wild-type and seven mutant <it>dhfr </it>genotypes were identified. Three <it>dhfr </it>genotypes lacking the S108N mutation (NRSI, ICSI, IRSI; amino acids at positions 51, 59, 108, and 164 with mutations underlined) were highly prevalent before 1994 but reduced after 1995, accompanied by an increase in genotypes with the S108N mutation. The <it>dhfr </it>IRNI genotype was first identified in Nigeria in 1991 in the present samples, and its frequency gradually increased. However, two double mutants (ICNI and NRNI), the latter of which was exclusively found in West Africa, were more frequent than the IRNI genotype. Only two <it>pfcrt </it>genotypes were found, the wild-type and a Southeast Asian type (CVIET; amino acids at positions 72-76 with mutations underlined). The CVIET genotype was already present as early as 1984 in Tanzania and Nigeria, and appeared throughout Africa between 1984 and 1998.</p> <p>Conclusions</p> <p>This study is the first to report the molecular identification of Pyr- and CQ-resistant genotypes of <it>P. falciparum </it>in Africa before 1990. Genotyping of <it>dhfr </it>and <it>pfcrt </it>using archive samples has revealed new aspects of the evolutionary history of Pyr- and CQ-resistant parasites in Africa.</p

The Intestinal Microbiota Plays a Role in Salmonella-Induced Colitis Independent of Pathogen Colonization

The intestinal microbiota is composed of hundreds of species of bacteria, fungi and protozoa and is critical for numerous biological processes, such as nutrient acquisition, vitamin production, and colonization resistance against bacterial pathogens. We studied the role of the intestinal microbiota on host resistance to Salmonella enterica serovar Typhimurium-induced colitis. Using multiple antibiotic treatments in 129S1/SvImJ mice, we showed that disruption of the intestinal microbiota alters host susceptibility to infection. Although all antibiotic treatments caused similar increases in pathogen colonization, the development of enterocolitis was seen only when streptomycin or vancomycin was used; no significant pathology was observed with the use of metronidazole. Interestingly, metronidazole-treated and infected C57BL/6 mice developed severe pathology. We hypothesized that the intestinal microbiota confers resistance to infectious colitis without affecting the ability of S. Typhimurium to colonize the intestine. Indeed, different antibiotic treatments caused distinct shifts in the intestinal microbiota prior to infection. Through fluorescence in situ hybridization, terminal restriction fragment length polymorphism, and real-time PCR, we showed that there is a strong correlation between the intestinal microbiota composition before infection and susceptibility to Salmonella-induced colitis. Members of the Bacteroidetes phylum were present at significantly higher levels in mice resistant to colitis. Further analysis revealed that Porphyromonadaceae levels were also increased in these mice. Conversely, there was a positive correlation between the abundance of Lactobacillus sp. and predisposition to colitis. Our data suggests that different members of the microbiota might be associated with S. Typhimurium colonization and colitis. Dissecting the mechanisms involved in resistance to infection and inflammation will be critical for the development of therapeutic and preventative measures against enteric pathogens

The effect of dose on the antimalarial efficacy of artemether-lumefantrine: a systematic review and pooled analysis of individual patient data

Background: Artemether-lumefantrine is the most widely used artemisinin-based combination therapy for malaria, although treatment failures occur in some regions. We investigated the effect of dosing strategy on efficacy in a pooled analysis from trials done in a wide range of malaria-endemic settings. Methods: We searched PubMed for clinical trials that enrolled and treated patients with artemether-lumefantrine and were published from 1960 to December, 2012. We merged individual patient data from these trials by use of standardised methods. The primary endpoint was the PCR-adjusted risk of Plasmodium falciparum recrudescence by day 28. Secondary endpoints consisted of the PCR-adjusted risk of P falciparum recurrence by day 42, PCR-unadjusted risk of P falciparum recurrence by day 42, early parasite clearance, and gametocyte carriage. Risk factors for PCR-adjusted recrudescence were identified using Cox's regression model with frailty shared across the study sites. Findings: We included 61 studies done between January, 1998, and December, 2012, and included 14 327 patients in our analyses. The PCR-adjusted therapeutic efficacy was 97·6% (95% CI 97·4-97·9) at day 28 and 96·0% (95·6-96·5) at day 42. After controlling for age and parasitaemia, patients prescribed a higher dose of artemether had a lower risk of having parasitaemia on day 1 (adjusted odds ratio [OR] 0·92, 95% CI 0·86-0·99 for every 1 mg/kg increase in daily artemether dose; p=0·024), but not on day 2 (p=0·69) or day 3 (0·087). In Asia, children weighing 10-15 kg who received a total lumefantrine dose less than 60 mg/kg had the lowest PCR-adjusted efficacy (91·7%, 95% CI 86·5-96·9). In Africa, the risk of treatment failure was greatest in malnourished children aged 1-3 years (PCR-adjusted efficacy 94·3%, 95% CI 92·3-96·3). A higher artemether dose was associated with a lower gametocyte presence within 14 days of treatment (adjusted OR 0·92, 95% CI 0·85-0·99; p=0·037 for every 1 mg/kg increase in total artemether dose). Interpretation: The recommended dose of artemether-lumefantrine provides reliable efficacy in most patients with uncomplicated malaria. However, therapeutic efficacy was lowest in young children from Asia and young underweight children from Africa; a higher dose regimen should be assessed in these groups. Funding: Bill and Melinda Gates Foundation

Gametocyte carriage in uncomplicated Plasmodium falciparum malaria following treatment with artemisinin combination therapy: a systematic review and meta-analysis of individual patient data

BACKGROUND: Gametocytes are responsible for transmission of malaria from human to mosquito. Artemisinin combination therapy (ACT) reduces post-treatment gametocyte carriage, dependent upon host, parasite and pharmacodynamic factors. The gametocytocidal properties of antimalarial drugs are important for malaria elimination efforts. An individual patient clinical data meta-analysis was undertaken to identify the determinants of gametocyte carriage and the comparative effects of four ACTs: artemether-lumefantrine (AL), artesunate/amodiaquine (AS-AQ), artesunate/mefloquine (AS-MQ), and dihydroartemisinin-piperaquine (DP). METHODS: Factors associated with gametocytaemia prior to, and following, ACT treatment were identified in multivariable logistic or Cox regression analysis with random effects. All relevant studies were identified through a systematic review of PubMed. Risk of bias was evaluated based on study design, methodology, and missing data. RESULTS: The systematic review identified 169 published and 9 unpublished studies, 126 of which were shared with the WorldWide Antimalarial Resistance Network (WWARN) and 121 trials including 48,840 patients were included in the analysis. Prevalence of gametocytaemia by microscopy at enrolment was 12.1 % (5887/48,589), and increased with decreasing age, decreasing asexual parasite density and decreasing haemoglobin concentration, and was higher in patients without fever at presentation. After ACT treatment, gametocytaemia appeared in 1.9 % (95 % CI, 1.7–2.1) of patients. The appearance of gametocytaemia was lowest after AS-MQ and AL and significantly higher after DP (adjusted hazard ratio (AHR), 2.03; 95 % CI, 1.24–3.12; P = 0.005 compared to AL) and AS-AQ fixed dose combination (FDC) (AHR, 4.01; 95 % CI, 2.40–6.72; P < 0.001 compared to AL). Among individuals who had gametocytaemia before treatment, gametocytaemia clearance was significantly faster with AS-MQ (AHR, 1.26; 95 % CI, 1.00–1.60; P = 0.054) and slower with DP (AHR, 0.74; 95 % CI, 0.63–0.88; P = 0.001) compared to AL. Both recrudescent (adjusted odds ratio (AOR), 9.05; 95 % CI, 3.74–21.90; P < 0.001) and new (AOR, 3.03; 95 % CI, 1.66–5.54; P < 0.001) infections with asexual-stage parasites were strongly associated with development of gametocytaemia after day 7. CONCLUSIONS: AS-MQ and AL are more effective than DP and AS-AQ FDC in preventing gametocytaemia shortly after treatment, suggesting that the non-artemisinin partner drug or the timing of artemisinin dosing are important determinants of post-treatment gametocyte dynamics

A systematic review and meta-analysis of evidence for correlation between molecular markers of parasite resistance and treatment outcome in falciparum malaria

<p>Abstract</p> <p>Background</p> <p>An assessment of the correlation between anti-malarial treatment outcome and molecular markers would improve the early detection and monitoring of drug resistance by <it>Plasmodium falciparum</it>. The purpose of this systematic review was to determine the risk of treatment failure associated with specific polymorphisms in the parasite genome or gene copy number.</p> <p>Methods</p> <p>Clinical studies of non-severe malaria reporting on target genetic markers (SNPs for <it>pfmdr1</it>, <it>pfcrt</it>, <it>dhfr</it>, <it>dhps</it>, gene copy number for <it>pfmdr1</it>) providing complete information on inclusion criteria, outcome, follow up and genotyping, were included. Three investigators independently extracted data from articles. Results were stratified by gene, codon, drug and duration of follow-up. For each study and aggregate data the random effect odds ratio (OR) with 95%CIs was estimated and presented as Forest plots. An OR with a lower 95^thconfidence interval > 1 was considered consistent with a failure being associated to a given gene mutation.</p> <p>Results</p> <p>92 studies were eligible among the selection from computerized search, with information on <it>pfcrt </it>(25/159 studies), <it>pfmdr1 </it>(29/236 studies), <it>dhfr </it>(18/373 studies), <it>dhps </it>(20/195 studies). The risk of therapeutic failure after chloroquine was increased by the presence of <it>pfcrt </it>K76T (Day 28, OR = 7.2 [95%CI: 4.5–11.5]), <it>pfmdr1 </it>N86Y was associated with both chloroquine (Day 28, OR = 1.8 [95%CI: 1.3–2.4]) and amodiaquine failures (OR = 5.4 [95%CI: 2.6–11.3, p < 0.001]). For sulphadoxine-pyrimethamine the <it>dhfr </it>single (S108N) (Day 28, OR = 3.5 [95%CI: 1.9–6.3]) and triple mutants (S108N, N51I, C59R) (Day 28, OR = 3.1 [95%CI: 2.0–4.9]) and <it>dhfr</it>-<it>dhps </it>quintuple mutants (Day 28, OR = 5.2 [95%CI: 3.2–8.8]) also increased the risk of treatment failure. Increased <it>pfmdr1 </it>copy number was correlated with treatment failure following mefloquine (OR = 8.6 [95%CI: 3.3–22.9]).</p> <p>Conclusion</p> <p>When applying the selection procedure for comparative analysis, few studies fulfilled all inclusion criteria compared to the large number of papers identified, but heterogeneity was limited. Genetic molecular markers were related to an increased risk of therapeutic failure. Guidelines are discussed and a checklist for further studies is proposed.</p