| 1. |
- Buwembo, William, et al.
(författare)
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Point Mutations in the folP Gene Partly Explain Sulfonamide Resistance of Streptococcus mutans
- 2013
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Ingår i: International Journal of Microbiology. - : Hindawi Limited. - 1687-918X .- 1687-9198. ; 2013, s. 367021-
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Tidskriftsartikel (refereegranskat)abstract
- Cotrimoxazole inhibits dhfr and dhps and reportedly selects for drug resistance in pathogens. Here,Streptococcus mutansisolates were obtained from saliva of HIV/AIDS patients taking cotrimoxazole prophylaxis in Uganda. The isolates were tested for resistance to cotrimoxazole and theirfolPDNA (which encodes sulfonamide-targeted enzyme dhps) cloned in pUC19. A set of recombinant plasmids carrying different point mutations in cloned folP were separately transformed intofolP-deficientEscherichia coli. Using sulfonamide-containing media, we assessed the growth offolP-deficient bacteria harbouring plasmids with differingfolPpoint mutations. Interestingly, clonedfolPwith three mutations (A37V, N172D, R193Q) derived fromStreptococcus mutans8 conferred substantial resistance against sulfonamide tofolP-deficient bacteria. Indeed, change of any of the three residues (A37V, N172D, and R193Q) in plasmid-encodedfolPdiminished the bacterial resistance to sulfonamide while removal of all three mutations abolished the resistance. In contrast, plasmids carrying four other mutations (A46V, E80K, Q122H, and S146G) infolPdid not similarly confer any sulfonamide resistance tofolP-knockout bacteria. Nevertheless, sulfonamide resistance (MIC = 50 μM) offolP-knockout bacteria transformed with plasmid-encodedfolPwas much less than the resistance (MIC = 4 mM) expressed by chromosomally-encodedfolP. Therefore,folPpoint mutations only partially explain bacterial resistance to sulfonamide.
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| 2. |
- Kamugisha, Erasmus, et al.
(författare)
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Detecting adenosine triphosphatase 6 point mutations that may be associated with Plasmodium falciparum resistance to artemisinin : prevalence at baseline, before policy change in Uganda
- 2011
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Ingår i: Tanzania Journal of Health Research. - 1821-6404. ; 13:1, s. 50-60
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Tidskriftsartikel (refereegranskat)abstract
- The artemisinin based combination therapy (ACT) of artemether and lumefantrine (Co-artem) has recently replaced chloroquine and fansidar as the first line treatment policy drug in Uganda. It is necessary to develop practical procedures to monitor the likely emergence and spread of artemisinin resistant P. falciparum strains. We have analyzed the genotypes of PfATP6 in parasites from 300 stored filter paper samples from malaria patients who were diagnosed and treated in the years 1999 to 2004 at three field sites in Uganda. This is a period just prior to introduction of Co-artem. In order to develop a simple molecular procedure for mutation detection, regions of PfATP6 encoding protein domains important in artemisinin binding was amplified by nested PCR. Three DNA products, which together contain most of the coding region of amino acids located within the putative active site of pfATP6 were readily amplified. The amplified DNA was digested by restriction enzymes and the fragments sized by agarose gel electrophoresis. For the important codons 260, 263 and 769, methods using engineered restriction sites were employed. We did not find mutations at codons for the key residues Lys 260, Leu263, Gln266, Ser769 and Asn1039. Nucleotide sequencing of pfATPase6 gene DNA from at least 15 clinical isolates confirmed the above findings and suggested that mutations at these amino acid residues have not emerged in our study sites.
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| 3. |
- Kamugisha, Erasmus, et al.
(författare)
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Efficacy of artemether-lumefantrine in treatment of malaria among under-fives and prevalence of drug resistance markers in Igombe-Mwanza, north-western Tanzania
- 2012
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Ingår i: Malaria Journal. - 1475-2875. ; 11, s. 58-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Drug resistance to anti-malarials is a major public health problem worldwide. This study aimed at establishing the efficacy of artemether-lumefantrine (ACT) in Igombe-Mwanza, north-western Tanzania after a few years of ACT use, and establish the prevalence of mutations in key targets for artemisinin, chloroquine and sulphadoxine/pyrimetamine (SP) drugs. Methods: A prospective single cohort study was conducted at Igombe health centre using artemether-lumefantrine combination therapy between February 2010 and March 2011. The follow-up period was 28 days and outcome measures were according to WHO guidelines. Blood was collected on Whatman filter paper for DNA analysis. DNA extraction was done using TRIS-EDTA method, and mutations in Pfcrt, Pfmdr1, Pfdhfr, Pfdhps and Pfatp6 were detected using PCR-RFLP methods established previously. Results: A total of 103 patients completed the 28 days follow-up. The mean haemoglobin was 8.9 g/dl (range 5.0 to 14.5 g/dl) and mean parasite density was 5,608 parasites/mu l. Average parasite clearance time was 34.7 hours and all patients cleared the parasites by day 3. There was no early treatment failure in this study. Late clinical failure was seen in three (2.9%) patients and late parasitological failure (LPF) was seen in two (1.9%). PCR-corrected LPF was 1% and adequate clinical and parasitological response was 96%. The majority of parasites have wild type alleles on pfcrt 76 and pfmdr1 86 positions being 87.8% and 93.7% respectively. Mutant parasites predominated at pfdhfr gene at the main three positions 108, 51 and 59 with prevalence of 94.8%, 75.3% and 82.5% respectively. Post-treatment parasites had more wild types of pfdhps at position 437 and 540 than pre-treatment parasites. No mutation was seen in pfatp6 769 in re-infecting or recrudescing parasites. Conclusion: The efficacy of artemether-lumefantrine for treatment of uncomplicated malaria is still high in the study area although the rate of re-infection is higher than previously reported. Parasite clearance after 48 hours was lower compared to previous studies. The prevalence of wild type allele pfcrt 76 K and pfmdr1 86 N was high in the study area while markers for SP resistance is still high. Artemether-lumefantrine may be selecting for wild type alleles on both positions (437 and 540) of pfdhps.
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| 4. |
- Kamugisha, Erasmus, et al.
(författare)
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Large differences in prevalence of Pfcrt and Pfmdr1 mutations between Mwanza, Tanzania and Iganga, Uganda : a reflection of differences in policies regarding withdrawal of chloroquine?
- 2012
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Ingår i: Acta Tropica. - : Elsevier BV. - 0001-706X .- 1873-6254. ; 121:2, s. 148-151
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Malaria is still a major public health problem in the world and sub-Saharan Africa is one of the most affected areas. Efforts to control malaria are highly affected by drug resistance to commonly used antimalarials. The introduction of artemisinin based combination therapy (ACT) as a first line drug seems to be a major step in treatment of uncomplicated malaria, though search for drugs to combine with artemisinins still continues. There have been reports on increased prevalence of the wild type markers Pfcrt 76K and Pfmdr1 86N in some African countries and ideas of using chloroquine (CQ) in intermittent presumptive treatment for adults (IPTa) is coming up. The common combination of artemether and lumefantrine even selects for parasites that are wild type at these positions. This study is comparing prevalence of mutation at these two positions in two East African countries with ACT as their first line drug but following somewhat different drug policies regarding CQ. In Tanzania CQ was stopped in 2001 but in Uganda CQ was retained in combination with sulfadoxine-pyrimethamine (SP) and used in home based management of fever for some time. SP is still used in IPT for pregnant women. METHODS: Blood smears and dried blood spots on Whatman filter papers were collected from 100 patients with uncomplicated malaria in Mwanza, Tanzania and 100 patients from Iganga, Uganda. DNA was extracted from all samples using Tris EDTA method. PCR and RFLP were performed and sequencing done on Pfcrt amplification products. RESULTS: The prevalence of K76T mutations at Pfcrt in samples from Mwanza, Tanzania was 40.5% (34/84) and 100% (100/100) in samples from Iganga, Uganda. Prevalence of N86Y mutations in Pfmdr1 was 16.9% (13/77) and 77.7% (63/81) in samples from Mwanza and Iganga, respectively. The re-emergence of CQ sensitive isolates in Mwanza, Tanzania showed the haplotype CVMNK typical for wild type isolates. CONCLUSIONS: The prevalence of CQ resistant parasites in Mwanza, Tanzania is low compared to the existing high level of resistant parasites in Iganga, Uganda. This could be an indication that CQ may become useful in the future in Tanzania. This study shows clearly that there is a difference in mutations at these positions in these two countries implementing similar but somewhat different drug policies. In Uganda the drug resistance has reached fixation while in Tanzania the prevalence is going down.
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| 5. |
- Reddy, Sreenivasulu B., et al.
(författare)
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High Affinity Antibodies to Plasmodium falciparum Merozoite Antigens Are Associated with Protection from Malaria
- 2012
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:2
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Tidskriftsartikel (refereegranskat)abstract
- Background: Malaria kills almost 1 million people every year, but the mechanisms behind protective immunity against the disease are still largely unknown. Methodology/Principal Findings: In this study, surface plasmon resonance technology was used to evaluate the affinity (measured as kd) of naturally acquired antibodies to the Plasmodium falciparum antigens MSP2 and AMA1. Antibodies in serum samples from residents in endemic areas bound with higher affinities to AMA1 than to MSP2, and with higher affinities to the 3D7 allele of MSP2-3D7 than to the FC27 allele. The affinities against AMA1 and MSP2-3D7 increased with age, and were usually within similar range as the affinities for the monoclonal antibodies also examined in this study. The finding of MSP2-3D7 type parasites in the blood was associated with a tendency for higher affinity antibodies to both forms of MSP2 and AMA1, but this was significant only when analyzing antibodies against MSP2-FC27, and individuals infected with both allelic forms of MSP2 at the same time showed the highest affinities. Individuals with the highest antibody affinities for MSP2-3D7 at baseline had a prolonged time to clinical malaria during 40 weeks of follow-up, and among individuals who were parasite positive at baseline higher antibody affinities to all antigens were seen in the individuals that did not experience febrile malaria during follow up. Conclusions/Significance: This study contributes important information for understanding how immunity against malaria arises. The findings suggest that antibody affinity plays an important role in protection against disease, and differs between antigens. In light of this information, antibody affinity measurements would be a key assessment in future evaluation of malaria vaccine formulations.
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| 6. |
- Venkatesan, Meera, et al.
(författare)
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Polymorphisms in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes : parasite risk factors that affect treatment outcomes for P. falciparum malaria after artemether-lumefantrine and artesunate-amodiaquine.
- 2014
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Ingår i: The American journal of tropical medicine and hygiene. - : American Society of Tropical Medicine and Hygiene. - 1476-1645 .- 0002-9637. ; 91:4, s. 833-43
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Tidskriftsartikel (refereegranskat)abstract
- Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 - 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36-17.97, P < 0.001 : were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine.
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| 7. |
- William, Buwembo, et al.
(författare)
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Cotrimoxazole Prophylaxis Specifically Selects for Cotrimoxazole Resistance in Streptococcus mutans and Streptococcus sobrinus with Varied Polymorphisms in the Target Genes folA and folP
- 2012
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Ingår i: International Journal of Microbiology. - : Hindawi Limited. - 1687-918X .- 1687-9198. ; , s. 916129-
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Tidskriftsartikel (refereegranskat)abstract
- The selection of antibiotic resistance by cotrimoxazole prophylaxis was evaluated, and we characterized the mechanism of cotrimoxazole resistance in Streptococcus mutans and Streptococcus sobrinus. In vitro susceptibility to six antibiotics was evaluated on 64 mutans streptococci group (MSG) isolates from a cotrimoxazole prophylaxis group and compared to 84 MSG isolates from a nonprophylaxis group. The folA and folP genes were sequenced and compared with reference sequences at NCBI. Only resistance to cotrimoxazole was significantly higher in the prophylaxis group (54.7% versus 15.5%, OR = 6.59, 95% CI: 2.89-15.3, P < 0.05). Resistance to amoxicillin, ceftriaxone, chloramphenicol, erythromycin, and tetracycline was 1.4%, 25.5%, 6.2%, 6.5%, and 29.6% of the isolates, respectively. Considerable polymorphisms were found in the folP gene in S. mutans, but this could not be linked to sulfonamide drug resistance. No variation was seen in folP or folA genes of S. sobrinus. Genetic transfer of folate pathway genes seems unlikely in these isolates.
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