An epidemiological survey of porcine cysticercosis in Rungwe District, Tanzania.

Taeniasis/cysticercosis caused by tapeworms belonging to the genus Taenia, poses serious veterinary and public health problems, resulting in economic burden in endemic low-income countries worldwide. However, little epidemiological data exist on infection status among pigs in many areas in Tanzania. We conducted a cross-sectional survey in Rungwe District, Mbeya Region, Tanzania, to define the prevalence and risk factors associated with porcine cysticercosis transmission. One-hundred sixty-nine pigs from 152 households were examined for circulating taeniid antigens by cysticercosis antigen (Ag) enzyme-linked immunosorbent assay (ELISA). Agarose gel electrophoresis was used to differentially diagnose Taenia species-specific cysticerci DNA bands. Structured questionnaires were administered in the surveyed households to collect information on risk factors for porcine cysticercosis transmission. Sera from eleven household pigs tested positive for porcine cysticercosis in the Ag-ELISA with an apparent prevalence of 6.5 % (95 % C.I. 3.8-11.3 %) and estimated true prevalence of 6.1 % (95 % C.I. 3.3-10.9 %). DNA Gel electrophoresis confirmed that 100 % of cysticerci isolated amongst pigs slaughtered in the study area belonged to T. solium. Of the five surveyed wards, positive household pigs were from Bulyaga, Kiwira, and Mpuguso. Lack of knowledge on porcine cysticercosis among household members was found to be significantly associated with positivity of Taenia species antigen in pigs sera (OR = 7.742, p = 0.017). Our results show that porcine cysticercosis is prevalent in Rungwe. There is a definite need to establish control measures against this potential zoonosis to safeguard veterinary and public health in the Rungwe District.

The Effect of Plasmodium falciparum (Welch) (Haemospororida: Plasmodiidae) Infection on the Susceptibility of Anopheles gambiae s.l. and Anopheles funestus (Diptera: Culicidae) to Pyrethroid Insecticides in the North-Western and South-Eastern, Tanzania.

The rapid development of insecticide resistance in malaria vectors threatens insecticide-based interventions. It is hypothesized that infection of insecticide-resistant vectors with Plasmodium parasites increases their vulnerability to insecticides, thus assuring the effectiveness of insecticide-based strategies for malaria control. Nonetheless, there is limited field data to support this. We investigated the effect of the Plasmodium falciparum infection on the susceptibility of Anopheles gambiae s.l. and Anopheles funestus to pyrethroids in south-eastern (Kilombero) and north-western (Muleba), Tanzania. The wild-collected mosquitoes were tested against 0.05% deltamethrin and 0.75% permethrin, then assessed for sporozoite rate and resistant gene (kdr) mutations. All Anopheles gambiae s.l. from Kilombero were An. arabiensis (Patton, 1905) while those from Muleba were 87% An. gambiae s.s (Giles, 1902) and 13% An. Arabiensis. High levels of pyrethroid resistance were observed in both areas studied. The kdr mutation was only detected in An. gambiae s.s. at the frequency of 100% in survivors and 97% in dead mosquitoes. The P. falciparum sporozoite rates were slightly higher in susceptible than in resistant mosquitoes. In Muleba, sporozoite rates in An. gambiae s.l. were 8.1% and 6.4% in dead mosquitoes and survivors, respectively (SRR = 1.28, p = 0.19). The sporozoite rates in Kilombero were 1.3% and 0.7% in the dead and survived mosquitoes, respectively (sporozoite rate ratio (SRR) = 1.9, p = 0.33). In An. funestus group sporozoite rates were 6.2% and 4.4% in dead and survived mosquitoes, respectively (SRR = 1.4, p = 0.54). These findings indicate that insecticides might still be effective in malaria control despite the rapid development of insecticide resistance in malaria vectors.

Anti-mosquito properties of Pelargonium roseum (Geraniaceae) and Juniperus virginiana (Cupressaceae) essential oils against dominant malaria vectors in Africa.

BACKGROUND: More than 90% of malaria cases occur in Africa where the disease is transmitted by Anopheles gambiae and Anopheles arabiensis. This study evaluated the anti-mosquito properties of Juniperus virginiana (JVO) and Pelargonium roseum (PRO) essential oils (EOs) against larvae and adults of An. gambiae sensu lato (s.l.) from East Africa in laboratory and semi-field conditions. METHODS: EOs was extracted from the aerial green parts of Asian herbs by hydrodistillation. Their constituents were characterized by gas chromatography-mass spectrometry (GC-MS). Larvicidal activities of JVO, PRO, and PRO components [citronellol (CO), linalool (LO), and geraniol (GO)] were investigated against An. gambiae sensu stricto (s.s.). The percentage of knockdown effects and mortality rates of all oils were also evaluated in the adults of susceptible An. gambiae s.s. and permethrin-resistant An. arabiensis. RESULTS: GC-MS analyses identified major constituents of JVO (sabinene, dl-limonene, ß-myrcene, bornyl acetate, and terpinen-4-ol) and PRO (citronellol, citronellyl formate, L-menthone, linalool, and geraniol). Oils showed higher larvicidal activity in the laboratory than semi-field trials. The LC50 values for JVO/PRO were computed as 10.82-2.89/7.13-0.9 ppm and 10.75-9.06/13.63-8.98 ppm in laboratory and semi-field environments, respectively at exposure time of 24-72 h. The percentage of knockdown effects of the oils were also greater in An. gambiae s.s. than in An. arabiensis. Filter papers impregnated with JVO (100 ppm) and PRO (25 ppm) displayed 100% mortality rates for An. gambiae s.s. and 3.75% and 90% mortality rates, for An. arabiensis populations, respectively. Each component of CO, LO, and GO exhibited 98.13%, 97.81%, and 87.5%, respectively, and a mixture of the PRO components indicated 94.69% adult mortality to permethrin-resistant An. arabiensis. CONCLUSIONS: The findings of this study show that PRO and its main constituents, compared to JVO, have higher anti-mosquito properties in terms of larvicidal, knockdown, and mortality when applied against susceptible laboratory and resistant wild populations of An. gambiae s.l. Consequently, these oils have the potential for the development of new, efficient, safe, and affordable agents for mosquito control.

Detection of Porcine Cysticercosis in Meat Juice Samples from Infected Pigs.

BACKGROUND: Seroprevalence of porcine cysticercosis has been generally studied using enzyme-linked immunosorbent assays (ELISA) detecting either antigens or antibodies in sera. However, serum is not always readily available. The objective of this study was to assess the diagnostic potential of meat juice in detecting porcine cysticercosis using a cysticercosis antibody ELISA. METHODS: Sera and meat juice samples from 13 different organs/tissues were collected from nine pigs naturally infected with cysticercosis and from six uninfected pigs reared under hygienic conditions. The sensitivity of the cysticercosis antibody ELISA in detecting porcine cysticercosis in meat juice samples was compared to that in serum samples from the same pigs. RESULTS: Using sera, cysticercosis was detected in all nine pigs harbouring cysticerci, but not in those reared under hygienic conditions. The sensitivity of the ELISA was highest in meat juice extracted from the diaphragm (100%), heart (89%) and neck muscle (78%) of the nine infected pigs, whereas it varied between 0 and 44% in the other samples. CONCLUSION: To the best of our knowledge, this is the first study for T. solium cysticercosis serology to use meat juice. Our results show that meat juice from pig carcass organs or muscles is a promising diagnostic specimen for the detection of porcine cysticercosis. More studies including a large sample size of pigs with varying degrees of cysticercosis infection are needed to further prove this concept.

Soil contamination by Taenia solium egg DNA in rural villages in Kongwa district, Tanzania.

The presence ofTaenia solium DNA from eggs in soils around the households in four Tanzanian villages in Kongwa district were analysed in relation to seasonal fluctuations and infection risk implications. A total of 192 pooled soil samples from five sampling points per household were examined by droplet digital Polymerase Chain Reaction (ddPCR) from 96 pig-keeping households both during the dry and rainy seasons. The pooled samples were first processed by a flotation-double sieving technique, followed by screening for worm DNA employing universal primers targeting the mitochondrial cytochrome c oxidase subunit I (cox1) gene of human taeniid species and some other helminths. All DNA positive samples were later confirmed by a specific ddPCR probe assay targeting the mitochondrial cox1 gene of T. solium. A total of 17.2% (n = 33) samples were positive with the universal ddPCR, whereas T. solium DNA was confirmed by the specific ddPCR only in 3.1% (n = 3) of the surveyed households. The detection of T. solium DNA in this study spells out a low risk of exposure to T. solium eggs from contaminated household soil. Based on our results, ddPCR seems to be a promising technology for screening T. solium eggs in soil.

Measuring antibody avidity to Plasmodium falciparum merozoite antigens using a multiplex immunoassay approach.

BACKGROUND: Antibodies (Ab) play a significant role in immunity to Plasmodium falciparum malaria. Usually, following repeated exposure to pathogens, affinity maturation and clonal selection take place, resulting in increased antibody avidity. However, some studies suggest affinity maturation may not occur to malaria antigens in endemic areas. Information on development of antibody avidity is confusing and conflicting, in part, because different techniques have been used to measure avidity. Today, bead-based multiplex immunoassays (MIA) are routinely used to simultaneously quantitate antibody levels to multiple antigens. This study evaluated the feasibility of developing an avidity MIA with 5 merozoite antigens (AMA1, EBA-175, MSP1-42, MSP2, MSP3) that uses a single chaotropic concentration. METHODS: The most common ELISA protocols that used the chaotropic reagents guanidine HCl (GdHCl), urea, and ammonium thiocyanate (NH4SCN) were adapted to a multiplex MIA format. Then, different concentrations of chaotropes and incubation times were compared and results were expressed as an Avidity Index (AI), i.e., percentage of antibody remaining bound in the presence of chaotrope. Experiments were conducted to (i) identify the assay with the widest range of AI (discriminatory power), (ii) determine the amount of chaotrope needed to release 50% of bound Ab using plasma from adults and infants, and (iii) evaluate assay repeatability. RESULTS: Overall, 4 M GdHCl and 8 M urea were weaker chaotropes than 3 M NH4SCN. For example, they failed to release significant amounts of Ab bound to MSP1-42 in adult plasma samples; whereas, a range of AI values was obtained with NH4SCN. Titration of NH4SCN revealed that 2 M NH4SCN gave the widest range of AI for the 5 antigens. Binding studies using plasma from 40 adults and 57 1-year old infants in Cameroon showed that 2.1 M ± 0.32 (mean ± SD) NH4SCN (adults) and 1.8 M ± 0.23 M (infants) released 50% of bound Ab from the merozoite antigens. CONCLUSIONS: An avidity MIA is feasible for the 5 merozoite antigens that uses a single concentration (2 M) of NH4SCN. The assay provides a simple method to quickly obtain information about Ab quantity and quality in the acquisition of immunity to malaria in endemic populations.

Seroprevalence of circulating taeniid antigens in pigs and associated risk factors in Kongwa district, Tanzania.

The aim of this study was to investigate exposure to porcine cysticercosis (PC) and associated risk factors in the Kongwa District, eastern-central Tanzania. For the first time a cross-sectional investigation of the seroprevalence in pigs using a commercial genus specific cysticercosis enzyme linked immunosorbent assay (apDia Ag-ELISA) was undertaken in eastern-central Tanzania. Moreover, the identity of suspected T. solium cysts from pigs in the study area were confirmed by sequencing parasites' mitochondrial cox1 gene. Structured questionnaires and direct observations were used to investigate risk factors associated with parasite transmission. A total of 102 pig-keeping households were surveyed during the dry season between July and August 2017 and 126 households in the rainy season between March and April 2018. Of the 447 examined pigs, 77 (17%, 95% C.I. 14%-20%) tested positive in the ELISA. Seroprevalence was higher in pigs examined during the rainy (21%, 95% C.I. 16%-26%) than dry (12%, 95% C.I. 7%-17%) season (p = 0.019). Eight cyst-positive-pigs were confirmed to be infected with T. solium by sequencing. Risk factors associated with PC seropositivity included origin of piglets or pigs (OR = 0.27, 95% C.I. 0.13-0.42, p = 0.001), socioeconomic factors and pig production system (OR = 0.22, 95% C.I. 0.07-0.37, p = 0.005) and sanitation and hygiene practices (OR = 0.19, 95% C.I. 0.04-0.34, p = 0.014). This study has recorded a high Taenia spp. seroprevalence in pigs in Kongwa suggesting the presence of people in the community carrying the adult parasite, Taenia solium. Our findings also suggest risk of infection by T. solium to people in urban centres and cities consuming pigs from rural areas in Kongwa. The high seroprevalence in Kongwa calls for further studies on taeniasis and cysticercosis in the human population in order to determine suitable control strategies.

Validation of droplet digital Polymerase Chain Reaction for the detection and absolute quantification of Taenia solium eggs in spiked soil samples.

To enable the detection of taeniid eggs in environmental samples, a sensitive technology is required. In this study, we validated the effectiveness of a digital droplet Polymerase Chain Reaction (ddPCR) assay for detection, identification and absolute quantification of taeniid DNA from artificially contaminated soils with varying numbers of taeniid eggs using a set of universal primers, JB3 & JB4.5. The results showed that the number of cox1 copies detected increased gradually for both species with the number of taeniid eggs added to the different soil types. The highest cox1 DNA copies recovery for Taenia solium and T. lynciscapreoli was from the sand soil with lowest recovery being observed in clay soils. Therefore, ddPCR is a promising technology for screening of taeniid eggs from soil samples collected in the environment irrespective of the soil type and the number of eggs. The potential of the ddPCR protocol to detect taeniid egg DNA in spiked soil samples has great practical application for taeniid egg screening in soils from endemic areas. However, when universal primers are used in screening environmental samples, the identity of ddPCR positive samples must be confirmed by sequencing. In addition, more validation studies using species-specific primers and field soil samples is recommended.

Impact of HIV-1 infection on the IGF-1 axis and angiogenic factors in pregnant Cameroonian women receiving antiretroviral therapy.

Although mother-to-child transmission of HIV has dramatically declined, the number of in utero HIV-exposed, uninfected infants is on the increase. HIV-exposed infants are at an increased risk of mortality, morbidity and slower early growth than their non-HIV exposed counterparts. Maternal HIV increases the risk of having preterm deliveries, intrauterine growth restriction and low birth weight babies. However, the mechanism underlying dysregulation of fetal growth in HIV-infected pregnant women is unknown. We sought to determine whether maternal HIV is associated with dysregulation of the insulin-like growth factor (IGF) axis, some angiogenic factors or other related biomarkers that regulate fetal growth. A total of 102 normotensive pregnant women were enrolled in a small cross-sectional study. Amongst these were thirty-one HIV-1 positive women receiving combination antiretroviral therapy (cART) (Mean age: 30.0 ± 5.1 years; % on ART: 83.9%; median plasma viral load: 683 copies/ml; median CD4 count: 350 cells/ul) and 71 HIV uninfected women (mean age: 27.3 ± 5.8) recruited at delivery. A panel of biomarkers including IGF1 and IGF binding proteins (IGFBP1, IGFBP3), angiopoietins (ANG) 1 and 2, matrix metalloproteinases (MMP) 2 and 9, and galectin 13, was measured in plasma collected from the placental intervillous space. The levels of IGF1, IGFBP1, ANG1, ANG2, MMP2, MMP9 and Gal-13 were not affected by maternal HIV, even when adjusted for maternal factors in linear regression models (all p>0.05). It was observed that HIV-infection in pregnancy did not significantly affect key markers of the IGF axis and angiogenic factors. If anything, it did not affect women. These findings highlight the importance of the use of ART during pregnancy, which maintains factors necessary for fetal development closer to those of healthy women. However, decrease in IGF1 levels might be exacerbated in women con-infected with HIV and malaria.

Dynamics and monitoring of insecticide resistance in malaria vectors across mainland Tanzania from 1997 to 2017: a systematic review.

BACKGROUND: Malaria still claims substantial lives of individuals in Tanzania. Insecticide-treated nets (ITNs) and indoor residual spray (IRS) are used as major malaria vector control tools. These tools are facing great challenges from the rapid escalating insecticide resistance in malaria vector populations. This review presents the information on the dynamics and monitoring of insecticide resistance in malaria vectors in mainland Tanzania since 1997. The information is important to policy-makers and other vector control stakeholders to reflect and formulate new resistance management plans in the country. METHODS: Reviewed articles on susceptibility and mechanisms of resistance in malaria vectors to insecticides across mainland Tanzania were systematically searched from the following databases: PubMed, Google scholar, HINARI and AGORA. The inclusion criteria were articles published between 2000 and 2017, reporting susceptibility of malaria vectors to insecticides, mechanisms of resistance in the mainland Tanzania, involving field collected adult mosquitoes, and mosquitoes raised from the field collected larvae. Exclusion criteria were articles reporting insecticide resistance in larval bio-assays, laboratory strains, and unpublished data. Reviewed information include year of study, malaria vectors, insecticides, and study sites. This information was entered in the excel sheet and analysed. RESULTS: A total of 30 articles met the selection criteria. The rapid increase of insecticide resistance in the malaria vectors across the country was reported since year 2006 onwards. Insecticide resistance in Anopheles gambiae sensu lato (s.l.) was detected in at least one compound in each class of all recommended insecticide classes. However, the Anopheles funestus s.l. is highly resistant to pyrethroids and DDT. Knockdown resistance (kdr) mechanism in An. gambiae s.l. is widely studied in the country. Biochemical resistance by detoxification enzymes (P450s, NSE and GSTs) in An. gambiae s.l. was also recorded. Numerous P450s genes associated with metabolic resistance were over transcribed in An. gambiae s.l. collected from agricultural areas. However, no study has reported mechanisms of insecticide resistance in the An. funestus s.l. in the country. CONCLUSION: This review has shown the dynamics and monitoring of insecticide resistance in malaria vector populations across mainland Tanzanian. This highlights the need for devising improved control approaches of the malaria vectors in the country.

The impact of Anopheles gambiae egg storage for mass rearing and production success.

BACKGROUND: Mass rearing requires a large colony from which male individuals can be harvested for sterilization and release. Attention is needed when monitoring life parameters of the reared population, knowing that any variations within the target population would lead to mismatching between two populations. The aim of this study was to assess the impact of Anopheles gambiae sensu stricto (s.s.) egg storage on hatchability and life history traits. For each parameter, comparison was made between freshly laid and stored eggs in three densities (40, 80, 120 eggs). METHODS: Anopheles gambiae s.s. freshly laid eggs were collected from the Tropical Pesticide Research Institute (TPRI) insectary. Eggs to be stored were kept at - 20 °C for 10 min and then transferred to refrigerators at 4 °C for intervals of 5, 10, 15, 20, and 25 days. After respective storage days, the eggs were transferred from refrigerators to ambient temperature of (25 ± 2) °C for 24 h and then placed in incubators for 24 h. Thereafter eggs were hatched. The egg hatchability, emerged larvae development, larvae survival and emerged adult sex ratios were monitored. RESULTS: This study found that hatching rates decreased with increase in storage time. The difference was significant in eggs stored for 10 and 15 days (P < 0.05). There were no significant differences in hatching rates between An. gambiae eggs stored for 5 days and freshly hatched eggs (P > 0.05). Anopheles larvae development (L1 to pupae) was not significantly affected by storage time across all hatching densities. The study also found that larvae survival decreased with increase in egg storage time. However, there was no significant difference between larvae from freshly hatched eggs and those from eggs at 5 and 10 storage days (P > 0.05) but not for eggs stored for 15 days. Furthermore, there was a decrease in emerged adult males and increase in females relative to increased time of egg storage. The difference was significant (P < 0.05) at 15 storage days but not for eggs stored for 5 and 10 days (in triplicate densities). CONCLUSION: From this study it was concluded that storing An. gambiae eggs at 4 °C and 48 ± 2% relative humidity (RH) for 5 days is the optimal condition and time that did not affect egg hatching rates, larval development and survivorship and emerged adult mosquito sex ratio.

Monitoring Compliance and Acceptability of Intermittent Preventive Treatment of Malaria Using Sulfadoxine Pyrimethamine after Ten Years of Implementation in Tanzania.

Intermittent preventive treatment using SP (IPTp-SP) is still a superior interventional approach to control malaria during pregnancy. However its rate of use has gone down tremendously in malaria endemic areas. This study forms part of a larger study aimed at monitoring the compliance of IPTp-SP policy in malaria endemic areas of Tanzania. Two cross-sectional studies were conducted in Dar es Salaam and Njombe Regions of Tanzania. Overall, 540 pregnant women and 21 healthcare workers were interviewed using structured questionnaires. This study revealed that 63% of women were not willing to take SP during pregnancy while 91% would only take it if they tested positive for malaria during antennal visits. 63% of the interviewed women did not know the recommended dose of SP required during pregnancy, despite the fact that 82% of the women were aware of the adverse effect of malaria during pregnancy. It was found out that 54% of pregnant women (30-40 weeks) took single dose, 34% took two doses, and 16% did not take SP at the time of interview. It was also found that SP was not administered under direct observed therapy in 86% of women. There was no significant relationship between number of doses received by pregnant women and antenatal clinic (ANC) start date (r2 = 0.0033, 95% CI (-0.016 to 0.034)). However positive correlation between drug uptake and drug availability was revealed (p = 0.0001). Knowledge on adverse effects of placental malaria among pregnant women was significantly associated with drug uptake (OR 11.81, 95% CI (5.755-24.23), p = 0.0001). We conclude that unavailability of drugs in ANC is the major reason hindering the implementation of IPTp-SP.

Optimization of BeWo model to investigate placental responses to Plasmodium falciparum infected erythrocytes.

Background: Establishment of an in vitro model to study placental malaria is essential for understanding the biology and pathogenesis of placental malaria. We defined experimental variables for obtaining responses of BeWo cells to placental binding Plasmodium falciparum infected erythrocytes (IE, CS2 parasites). Materials and methods: Experimental variables included i) concentration of forskolin, a cyclic adenosine monophosphate inducer important in the induction of syncytialisation of BeWo, ii) suitable period of incubating BeWo with forskolin, and iii) ratio of IE to BeWo cells and length of incubation to induce physiological changes in BeWo cells, including the vasculogenic factors vascular endothelial growth factor A (VEGFA), endoglin, and angiopoietin-2; an anti-angiogenic factor (inhibin A); a regulator of cell growth, mammalian target of rapamycin (mTOR); a chemokine (IL-8); and the cytokine macrophage inhibition factor. The human hormone, chorionic gonadotrophin was used as a marker for syncytialisation. Results: We showed that 72 hrs incubation of BeWo with 10 µm forskolin resulted in higher levels of syncytialisation and hCG secretion. Overall, the best condition was to co-culture syncytialised BeWo with a 10:1 ratio of IE for 48 hours. Under these conditions, when co-cultured with IE, BeWo produced increased amounts of IL-8 (p=0.0001), VEGF (p=0.001) and endoglin (p=0.001). Conclusion: The model can be used to evaluate the impact of IE, inflammatory cytokines and other factors associated with placental malaria on syncytiotrophoblast function.

Syncytiotrophoblast Functions and Fetal Growth Restriction during Placental Malaria: Updates and Implication for Future Interventions.

Syncytiotrophoblast lines the intervillous space of the placenta and plays important roles in fetus growth throughout gestation. However, perturbations at the maternal-fetal interface during placental malaria may possibly alter the physiological functions of syncytiotrophoblast and therefore growth and development of the embryo in utero. An understanding of the influence of placental malaria on syncytiotrophoblast function is paramount in developing novel interventions for the control of placental pathology associated with placental malaria. In this review, we discuss how malaria changes syncytiotrophoblast function as evidenced from human, animal, and in vitro studies and, further, how dysregulation of syncytiotrophoblast function may impact fetal growth in utero. We also formulate a hypothesis, stemming from epidemiological observations, that nutrition may override pathogenesis of placental malaria-associated-fetal growth restriction. We therefore recommend studies on nutrition-based-interventional approaches for high placental malaria-risk women in endemic areas. More investigations on the role of nutrition on placental malaria pathogenesis are needed.