High Levels of Sulphadoxine-pyrimethamine Resistance Pfdhfr-Pfdhps Quintuple Mutations: A Cross Sectional Survey of Six Regions in Tanzania.

In 2006, the first-line anti-malarial drug treatment in Tanzania was changed from sulphadoxine-pyrimethamine (SP) to artemether-lumefantrine (ALu), an artemisinin-based combination (ACT), since when the use of SP has been restricted for intermittent preventive treatment in pregnancy (IPTp). A number of Plasmodium falciparum mutations are known to be associated with resistance to SP, but it is not known if the prevalence of these mutations is increasing or decreasing under the conditions of reduced levels of SP use. This study reports on the current SP resistant quintuple Pfdhfr-Pfdhps mutations in six regions of Tanzania. Finger-prick blood on filter paper and rapid diagnostic test strips from P. falciparum-positive individuals of all age groups attending health facilities in six regions of Tanzania between June 2010 and August 2011 were obtained. Using chelex-100 extracted DNA, genotyping was done for mutations on codons 51, 59 and 108 of Pfdhfr and 437 and 540 of Pfdhps genes using PCR-RFLP technique. A total of 802 malaria-positive samples were screened and genotyped. The prevalence of Pfdhfr 51I, Pfdhps 437G and 540E varied between the regions (p < 0.001) whereas Pfdhfr 59R (FE 10.79, p = 0.225) and 108 N (FE 10.61, p = 0.239) did not vary between the regions. The Pfdhfr triple mutant was above 84% and close to fixation levels in all regions, whereas the Pfdhps double mutation ranged from 43.8 to 97% between the regions. The quintuple mutant (IRNGE) was the most prevalent in all regions and it varied significantly from 37.5 to 90.2% (χ2 = 1.11, p <0.001). There is evidence of persistent high levels of SP resistance markers in Tanzania with evidence of quintuple mutations that are likely to become fixed in the population. This threatens the future of SP not only in IPTp programmes, but as a combination drug for ACT. Continuous monitoring of SP-IPTp efficacy should be encouraged subsequent to searching for alternative drugs for IPTp in East Africa

Molecular monitoring of Plasmodium falciparum super-resistance to sulfadoxine-pyrimethamine in Tanzania.

BACKGROUND: Sulfadoxine-pyrimethamine (SP) is recommended for prophylactic treatment of malaria in pregnancy while artemisinin combination therapy is the recommended first-line anti-malarial treatment. Selection of SP resistance is ongoing since SP is readily available in health facilities and in private drug shops in sub-Saharan Africa. This study reports on the prevalence and distribution of Pfdhps mutations A540E and A581G in Tanzania. When found together, these mutations confer high-level SP resistance (sometimes referred to as 'super-resistance'), which is associated with loss in protective efficacy of SP-IPTp. METHODS: DNA samples were extracted from malaria-positive blood samples on filter paper, used malaria rapid diagnostic test strips and whole blood collected from eight sites in seven administrative regions of Tanzania. PCR-RFLP and SSOP-ELISA techniques were used to genotype the A540E and A581G Pfdhps. Data were analysed using SPSS version 18 while Chi square and/or Fischer Exact tests were used to compare prevalence between regions. RESULTS: A high inter-regional variation of Pfdhps-540E was observed (χ(2) = 76.8, p < 0.001). High inter-regional variation of 581G was observed (FE = 85.3, p < 0.001). Both Tanga and Kagera were found to have the highest levels of SP resistance. A high prevalence of Pfdhps-581G was observed in Tanga (56.6 %) in northeastern Tanzania and in Kagera (20.4 %) in northwestern Tanzania and the 540-581 EG haplotype was found at 54.5 and 19.4 %, respectively. Pfdhps-581G was not detected in Pwani and Lindi regions located south of Tanga region. CONCLUSIONS: Selection of SP super-resistant Pfdhps A581G is highest in northern Tanzania. Variation in distribution of SP resistance is observed across the country: northeastern Tanga region and northwestern Kagera region have highest prevalence of SP super-resistance markers, while in Pwani and Lindi in the southeast the prevalence of super-resistance was zero. More studies should be conducted to understand the factors underlying the remarkable heterogeneity in SP resistance in the country

Surveillance of artemether-lumefantrine associated Plasmodium falciparum multidrug resistance protein-1 gene polymorphisms in Tanzania.

BACKGROUND: Resistance to anti-malarials is a major public health problem worldwide. After deployment of artemisinin-based combination therapy (ACT) there have been reports of reduced sensitivity to ACT by malaria parasites in South-East Asia. In Tanzania, artemether-lumefantrine (ALu) is the recommended first-line drug in treatment of uncomplicated malaria. This study surveyed the distribution of the Plasmodium falciparum multidrug resistance protein-1 single nucleotide polymorphisms (SNPs) associated with increased parasite tolerance to ALu, in Tanzania. METHODS: A total of 687 Plasmodium falciparum positive dried blood spots on filter paper and rapid diagnostic test strips collected by finger pricks from patients attending health facilities in six regions of Tanzania mainland between June 2010 and August 2011 were used. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to detect Pfmdr1 SNPs N86Y, Y184F and D1246Y. RESULTS: There were variations in the distribution of Pfmdr1 polymorphisms among regions. Tanga region had exceptionally high prevalence of mutant alleles, while Mbeya had the highest prevalence of wild type alleles. The haplotype YFY was exclusively most prevalent in Tanga (29.6%) whereas the NYD haplotype was the most prevalent in all other regions. Excluding Tanga and Mbeya, four, most common Pfmdr1 haplotypes did not vary between the remaining four regions (χ² = 2.3, p = 0.512). The NFD haplotype was the second most prevalent haplotype in all regions, ranging from 17% - 26%. CONCLUSION: This is the first country-wide survey on Pfmdr1 mutations associated with ACT resistance. Distribution of individual Pfmdr1 mutations at codons 86, 184 and 1246 varies throughout Tanzanian regions. There is a general homogeneity in distribution of common Pfmdr1 haplotypes reflecting strict implementation of ALu policy in Tanzania with overall prevalence of NFD haplotype ranging from 17 to 26% among other haplotypes. With continuation of ALu as first-line drug this haplotype is expected to keep rising, thus there is need for continued pharmacovigilance studies to monitor any delayed parasite clearance by the drug

Localization of the ATP-binding cassette (ABC) transport proteins PfMRP1, PfMRP2, and PfMDR5 at the Plasmodium falciparum plasma membrane

Contains fulltext : 76045.pdf (publisher's version ) (Open Access)BACKGROUND: The spread of drug resistance has been a major obstacle to the control of malaria. The mechanisms underlying drug resistance in malaria seem to be complex and multigenic. The current literature on multiple drug resistance against anti-malarials has documented PfMDR1, an ATP-binding cassette (ABC) protein, as an important determinant of resistance. In the Plasmodium falciparum genome, there are several ABC transporters some of which could be putative drug transporting proteins. In order to understand the molecular mechanisms underlying drug resistance, characterization of these transporters is essential. The aim of this study was to characterize and localize putative ABC transporters. METHODS: In the plasmoDB database, 16 members of the P. falciparum ABC family can be identified, 11 of which are putative transport proteins. A phylogenetic analysis of the aligned NBDs of the PfABC genes was performed. Antibodies against PfMRP1 (PfABCC1), PfMRP2 (PfABCC2), and PfMDR5 (PfABCB5) were generated, affinity purified and used in immunocytochemistry to localize the proteins in the asexual stages of the parasite. RESULTS: The ABC family members of P. falciparum were categorized into subfamilies. The ABC B subfamily was the largest and contained seven members. Other family members that could be involved in drug transport are PfABCC1, PfABCC2, PfABCG1, and PfABCI3. The expression and localization of three ABC transport proteins was determined. PfMRP1, PfMRP2, and PfMDR5 are localized to the plasma membrane in all asexual stages of the parasite. CONCLUSION: In conclusion, 11 of the 16 ABC proteins in the P. falciparum genome are putative transport proteins, some of which might be involved in drug resistance. Moreover, it was demonstrated that three of these proteins are expressed on the parasite's plasma membrane.1 p

A VAR2CSA:CSP conjugate capable of inducing dual specificity antibody responses

Background: Vaccine antigens targeting specific P. falciparum parasite stages are under pre-clinical and clinical development. It seems plausible that vaccine with multiple specificities will offer higher protection. With this hypothesis, we exploited the Spy- Tag/SpyCatcher conjugation system to make a, post expression, dual antigen conjugate vaccine, comprising two clinically tested antigen candidates (CSP and VAR2CSA).Methods: The DBL1x-DBL2x-ID2a region of VAR2CSA was genetically fused with SpyTag at N-terminus. The full-length CSP antigen was genetically fused to C-terminal SpyCatcher peptide. The covalent interaction between SpyTag/ SpyCatcher enables the formation of DBL1x-DBL2x-ID2a:CSP conjugate vaccine. Immunogenicity and quality of antibody responses induced by the conjugate vaccine, as well as a control CSP-SpyCatcher vaccine, was tested in BALB/c mice.Results: Serum samples obtained from mice immunized with the conjugate vaccine were able to recognize both untagged DBL1x-DBL2x-ID2a as well as CSP antigen. Moreover, the geometric mean anti-CSP antibody titer was 1.9-fold higher in serum (at day 35 and 55 post-first immunization) from mice immunized with the conjugate vaccine, as compared to mice receiving the control vaccine.Conclusion: The data obtained in this study serves as proof-of-concept for the simultaneous induction of antibodies directed against individual antigen components in a dual stage anti-malaria vaccine.Keywords: Malaria vaccine, Circumsporozoite protein, VAR2CSA, CSP SpyCatcher, SpyTag-DBL1x-DBL2x-ID2a, bacterial superglue, DBL1x-DBL2x-ID2a:CSP conjugat

Prevalence of dengue and chikungunya virus infections in north-eastern Tanzania:a cross sectional study among participants presenting with malaria-like symptoms

BACKGROUND: In spite of increasing reports of dengue and chikungunya activity in Tanzania, limited research has been done to document the general epidemiology of dengue and chikungunya in the country. This study aimed at determining the sero-prevalence and prevalence of acute infections of dengue and chikungunya virus among participants presenting with malaria-like symptoms (fever, headache, rash, vomit, and joint pain) in three communities with distinct ecologies of north-eastern Tanzania. METHODS: Cross sectional studies were conducted among 1100 participants (aged 2–70 years) presenting with malaria-like symptoms at health facilities at Bondo dispensary (Bondo, Tanga), Hai hospital (Hai, Kilimanjaro) and TPC hospital (Lower Moshi). Participants who were malaria negative using rapid diagnostic tests (mRDT) were screened for sero-positivity towards dengue and chikungunya Immunoglobulin G and M (IgG and IgM) using ELISA-based kits. Participants with specific symptoms defined as probable dengue and/or chikungunya by WHO (fever and various combinations of symptoms such as headache, rash, nausea/vomit, and joint pain) were further screened for acute dengue and chikungunya infections by PCR. RESULTS: Out of a total of 1100 participants recruited, 91.2 % (n = 1003) were malaria negative by mRDT. Out of these, few of the participants (<5 %) were dengue IgM or IgG positive. A total of 381 participants had fever out of which 8.7 % (33/381) met the defined criteria for probable dengue, though none (0 %) was confirmed to be acute cases. Chikungunya IgM positives among febrile participants were 12.9 % (49/381) while IgG positives were at 3.7 % (14/381). A total of 74.2 % (283/381) participants met the defined criteria for probable chikungunya and 4.2 % (11/263) were confirmed by PCR to be acute chikungunya cases. Further analyses revealed that headache and joint pain were significantly associated with chikungunya IgM seropositivity. CONCLUSION: In north-eastern Tanzania, mainly chikungunya virus appears to be actively circulating in the population. Continuous surveillance is needed to determine the contribution of viral infections of fever cases. A possible establishment of arboviral vector preventive control measures and better diagnosis of pathogens to avoid over-treatment of other diseases should be considered. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12879-016-1511-5) contains supplementary material, which is available to authorized users

Genetic Sequence Variation in the Plasmodium falciparum Histidine-Rich Protein 2 Gene from Field Isolates in Tanzania: Impact on Malaria Rapid Diagnosis

Malaria rapid diagnosis test (RDT) is crucial for managing the disease, and the effectiveness of detection depends on parameters such as sensitivity and specificity of the RDT. Several factors can affect the performance of RDT. In this study, we focused on the pfhrp2 sequence variation and its impact on RDTs targeted by antigens encoded by Plasmodium falciparum histidine-rich protein 2 (pfhrp2). Field samples collected during cross-sectional surveys in Tanzania were sequenced to investigate the pfhrp2 sequence diversity and evaluate the impact on HRP2-based RDT performance. We observed significant mean differences in amino acid repeats between current and previous studies. Several new amino acid repeats were found to occur at different frequencies, including types AAY, AHHAHHAAN, and AHHAA. Based on the abundance of types 2 and 7 amino acid repeats, the binary predictive model was able to predict RDT insensitivity by about 69% in the study area. About 85% of the major epitopes targeted by monoclonal antibodies (MAbs) in RDT were identified. Our study suggested that the extensive sequence variation in pfhrp2 can contribute to reduced RDT sensitivity. The correlation between the different combinations of amino acid repeats and the performance of RDT in different malaria transmission settings should be investigated further

СПЕКТРОСКОПІЯ ЯДЕРНОГО МАГНІТНОГО РЕЗОНАНСУ В АНАЛІЗІ ОБ'ЄКТІВ ДОВКІЛЛЯ

Additional file 3. Multiplication rate

Cellulose filtration of blood from malaria patients for improving <i>ex vivo</i> growth of <i>Plasmodium falciparum</i> parasites

BACKGROUND: Establishing in vitro Plasmodium falciparum culture lines from patient parasite isolates can offer deeper understanding of geographic variations of drug sensitivity and mechanisms of malaria pathogenesis and immunity. Cellulose column filtration of blood is an inexpensive, rapid and effective method for the removal of host factors, such as leucocytes and platelets, significantly improving the purification of parasite DNA in a blood sample. METHODS: In this study, the effect of cellulose column filtration of venous blood on the initial in vitro growth of P. falciparum parasite isolates from Tanzanian children admitted to hospital was tested. The parasites were allowed to expand in culture without subcultivation until 5 days after admission or the appearance of dead parasites and parasitaemia was determined daily. To investigate whether the filtration had an effect on clonality, P. falciparum merozoite surface protein 2 genotyping was performed using nested PCR on extracted genomic DNA, and the var gene transcript levels were investigated, using quantitative PCR on extracted RNA, at admission and 4 days of culture. RESULTS: The cellulose-filtered parasites grew to higher parasitaemia faster than non-filtered parasites seemingly due to a higher development ratio of ring stage parasites progressing into the late stages. Cellulose filtration had no apparent effect on clonality or var gene expression; however, evident differences were observed after only 4 days of culture in both the number of clones and transcript levels of var genes compared to the time of admission. CONCLUSIONS: Cellulose column filtration of parasitized blood is a cheap, applicable method for improving cultivation of P. falciparum field isolates for ex vivo based assays; however, when assessing phenotype and genotype of cultured parasites, in general, assumed to represent the in vivo infection, caution is advised. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12936-017-1714-2) contains supplementary material, which is available to authorized users