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

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

10 Years of Environmental Change on the Slopes of Mount Kilimanjaro and Its Associated Shift in Malaria Vector Distributions.

INTRODUCTION: Malaria prevalence has declined in the Kilimanjaro region of Tanzania over the past 10 years, particularly at lower altitudes. While this decline has been related to the scale-up of long-lasting insecticidal nets to achieve universal coverage targets, it has also been attributed to changes in environmental factors that are important for enabling and sustaining malaria transmission. OBJECTIVES: Herein, we apply spatial analytical approaches to investigate the impact of environmental and demographic changes, including changes in temperature, precipitation, land cover, and population density, on the range of the major malaria vector species Anopheles arabiensis in two districts of Tanzania, situated on the southern slope of Mount Kilimanjaro. These models are used to identify environmental changes that have occurred over a 10-year period and highlight the implications for malaria transmission in this highland region. METHODS: Entomological data were collected from the Hai and Lower Moshi districts of Tanzania in 2001-2004 and 2014-2015. Vector occurrence data were applied alongside satellite remote sensing indices of climate and land cover, and gridded population data, to develop species distribution models for An. arabiensis for the 2004 and 2014 periods using maximum entropy. Models were compared to assess the relative contribution of different environmental and demographic factors to observed trends in vector species distribution in lowland and highland areas. RESULTS: Changes in land cover were observed in addition to increased population densities, increased warm season temperature, and decreased wetness at low altitudes. The predicted area and extent of suitable habitat for An. arabiensis declined across the study area over the 10-year period, with notable contraction at lower altitudes, while species range in higher altitude zones expanded. Importantly, deforestation and warmer temperatures at higher altitudes may have created stable areas of suitable vector habitat in the highlands capable of sustaining malaria transmission. CONCLUSION: We show that environmental changes have had an important influence on the distribution of malaria vector species in a highland area of northern Tanzania. Highland areas may be at continued risk for sporadic malaria outbreaks despite the overall range contraction of principal vector species at lower altitudes, where malaria transmission remains at low intensity

Colonization and Authentication of the Pyrethroid-Resistant Anopheles gambiae s.s. Muleba-Kis Strain; an Important Test System for Laboratory Screening of New Insecticides

Background: The emergence and spread of insecticide resistance in malaria vectors to major classes of insecticides call for urgent innovation and application of insecticides with novel modes of action. When evaluating new insecticides for public health, potential candidates need to be screened against both susceptible and resistant mosquitoes to determine efficacy and to identify potential cross-resistance to insecticides currently used for mosquito control. The challenges and lessons learned from establishing, maintaining, and authenticating the pyrethroid-resistant An. gambiae s.s. Muleba-Kis strain at the KCMUCo-PAMVERC Test Facility are described in this paper. Methods: Male mosquitoes from the F1 generation of wild-pyrethroid resistant mosquitoes were cross-bred with susceptible female An. gambiae s.s. Kisumu laboratory strain followed by larval selection using a pyrethroid insecticide solution. Periodic screening for phenotypic and genotypic resistance was done. WHO susceptibility tests and bottle bioassays were used to assess the phenotypic resistance, while Taqman™ assays were used to screen for known target-site resistance alleles (kdr and ace-1). Additionally, the strains were periodically assessed for quality control by monitoring adult weight and wing length. Results: By out-crossing the wild mosquitoes with an established lab strain, a successful resistant insectary colony was established. Intermittent selection pressure using alphacypermethrin has maintained high kdr mutation (leucine-serine) frequencies in the selected colony. There was consistency in the wing length and weight measurements from the year 2016 to 2020, with the exception that one out of four years was significantly different. Mean annual wing length varied between 0.0142–0.0028 mm compared to values obtained in 2016, except in 2019 where it varied by 0.0901 mm. Weight only varied by approximately 0.001 g across four years, except in 2017 where it differed by 0.005 g. Routine phenotypic characterization on Muleba-Kis against pyrethroids using the WHO susceptibility test indicated high susceptibility when type I pyrethroids were used compared to type II pyrethroids. Dynamics on susceptibility status also depended on the lapse time when the selection was last done. Conclusions: This study described the procedure for introducing, colonizing, and maintaining a resistant An. gambiae s.s. strain in the laboratory with leucine to serine substitution kdr allele which reflects the features of the wild-resistant population in East Africa. Challenges in colonizing a wild-resistant mosquito strain were overcome by out-crossing between mosquito strains of desired traits followed by intermittent insecticide selection at the larval stage to select for the resistant phenotype

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

Mapping clusters of chikungunya and dengue transmission in northern Tanzania using disease exposure and vector data

Background: Dengue and chikungunya are mosquito-borne viral diseases that are of public health importance throughout the tropical and subtropical regions of the world. Seasonal variations in transmission of these viruses have been suggested owing to the ecology of their mosquito vectors. However, little is known about the epidemiology of the diseases Tanzania. To address this gap, seasonal community-based cross-sectional surveys were undertaken to identify potential clusters of transmission in Hai district in northern Tanzania.Methods: Epidemiological and entomological data from two cross-sectional surveys were used to examine the spatial pattern of dengue and chikungunya transmission. Six villages namely, Boma Ng’ombe, Magadini, Rundugai, Nshara and Kware were involved in the study. Serological measures of dengue and chikungunya virus infections were derived using enzyme-linked immunosorbent assays, and all participants were geo-referenced to the household level using a global positioning system. Potential clusters of individual exposed to dengue and chikungunya virus , as well as clusters of Aedes mosquitoes in the wet and dry seasons were detected using SaTScan. All significant clusters (with p≤0.05) were mapped using ArcGIS.Results: A large, widely dispersed cluster of chikungunya exposed individuals was detected spanning Rundugai and parts of Magadini villages (RR = 2.58,  p= 0.01), while no significant clustering was observed in the dry season. Spatial clusters of Aedes aegypti were detected in Rundugai in both the wet and dry seasons (RR = 2.56, p&lt; 0.001 and RR = 2.24, p=0.05, respectively). In the dry season a small cluster was also detected in Kware (RR = 2.25, p=0.05). No significant clusters of dengue were detected in both seasons.Conclusion: Clusters of chikungunya-exposed individuals and Aedes mosquitoes indicate on-going transmission of chikungunya virus in Hai district of northern Tanzania

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

Depression at antiretroviral therapy initiation and clinical outcomes among a cohort of Tanzanian women living with HIV

OBJECTIVE: The objective of the study was to assess the relationship of depression at antiretroviral therapy (ART) initiation with mortality and clinical outcomes among Tanzanian women living with HIV. DESIGN: We conducted a prospective cohort study of 1487 women who initiated ART in Dar es Salaam, Tanzania. METHODS: Symptoms of depression and anxiety were assessed using a Tanzanian-adapted and validated version of the Hopkins Symptom Checklist. Participants attended monthly clinic visits during the first 2 years of ART and CD4 T-cell counts were assessed every 4 months. Proportional hazard models were used to assess the relationship of depression with mortality and clinical outcomes. RESULTS: Symptoms consistent with depression were prevalent among 57.8% of women at ART initiation. After multivariate adjustment, including social support and stigma, depression at ART initiation was associated with increased risk of mortality [hazard ratio (HR): 1.92; 95% confidence interval (CI): 1.15-3.20; P = 0.01] and incidence of severe anemia (hemoglobin 0.05). CONCLUSION: Elimination of depression may reduce mortality during the first 2 years of ART by one-third in our study cohort. Randomized trials and rigorous implementation studies are needed to evaluate the individual and population-level effects of integrated mental health interventions and HIV treatment approaches in resource-limited settings

Colonization and Authentication of the Pyrethroid-Resistant Anopheles gambiae s.s. Muleba-Kis Strain; an Important Test System for Laboratory Screening of New Insecticides.

BACKGROUND: The emergence and spread of insecticide resistance in malaria vectors to major classes of insecticides call for urgent innovation and application of insecticides with novel modes of action. When evaluating new insecticides for public health, potential candidates need to be screened against both susceptible and resistant mosquitoes to determine efficacy and to identify potential cross-resistance to insecticides currently used for mosquito control. The challenges and lessons learned from establishing, maintaining, and authenticating the pyrethroid-resistant An. gambiae s.s. Muleba-Kis strain at the KCMUCo-PAMVERC Test Facility are described in this paper. METHODS: Male mosquitoes from the F1 generation of wild-pyrethroid resistant mosquitoes were cross-bred with susceptible female An. gambiae s.s. Kisumu laboratory strain followed by larval selection using a pyrethroid insecticide solution. Periodic screening for phenotypic and genotypic resistance was done. WHO susceptibility tests and bottle bioassays were used to assess the phenotypic resistance, while Taqman™ assays were used to screen for known target-site resistance alleles (kdr and ace-1). Additionally, the strains were periodically assessed for quality control by monitoring adult weight and wing length. RESULTS: By out-crossing the wild mosquitoes with an established lab strain, a successful resistant insectary colony was established. Intermittent selection pressure using alphacypermethrin has maintained high kdr mutation (leucine-serine) frequencies in the selected colony. There was consistency in the wing length and weight measurements from the year 2016 to 2020, with the exception that one out of four years was significantly different. Mean annual wing length varied between 0.0142-0.0028 mm compared to values obtained in 2016, except in 2019 where it varied by 0.0901 mm. Weight only varied by approximately 0.001 g across four years, except in 2017 where it differed by 0.005 g. Routine phenotypic characterization on Muleba-Kis against pyrethroids using the WHO susceptibility test indicated high susceptibility when type I pyrethroids were used compared to type II pyrethroids. Dynamics on susceptibility status also depended on the lapse time when the selection was last done. CONCLUSIONS: This study described the procedure for introducing, colonizing, and maintaining a resistant An. gambiae s.s. strain in the laboratory with leucine to serine substitution kdr allele which reflects the features of the wild-resistant population in East Africa. Challenges in colonizing a wild-resistant mosquito strain were overcome by out-crossing between mosquito strains of desired traits followed by intermittent insecticide selection at the larval stage to select for the resistant phenotype

Deletions of the Plasmodium falciparum histidine-rich protein 2/3 genes are common in field isolates from north-eastern Tanzania.

Plasmodium falciparum parasites lacking histidine-rich protein 2 and 3 (pfhrp2/3) genes have been reported in several parts of the world. These deletions are known to compromise the effectiveness of HRP2-based malaria rapid diagnostic tests (HRP2-RDT). The National Malaria Control Programme (NMCP) in Tanzania adopted HRP2-RDTs as a routine tool for malaria diagnosis in 2009 replacing microscopy in many Health facilities. We investigated pfhrp2/3 deletions in 122 samples from two areas with diverse malaria transmission intensities in Northeastern Tanzania. Pfhrp2 deletion was confirmed in 1.6% of samples while pfhrp3 deletion was confirmed in 50% of samples. We did not find parasites with both pfhrp2 and pfhrp3 deletions among our samples. Results from this study highlight the need for systematic surveillance of pfhrp2/3 deletions in Tanzania to understand their prevalence and determine their impact on the performance of mRDT