Assessment of humoral and cellular immune responses of the RTS,S/AS02D malaria vaccine candidate administered to infants living in a malaria endemic area in Mozambique

MSc (Med), Faculty of Health Sciences, University of the Witwatersrand, 2009Background: RTS,S candidate malaria vaccine has been shown to be highly immunogenic in children and infants, but the protective immune mechanisms still remain to be clearly elucidated. It is believed that RTS,S elicits a strong neutralizing humoral immune response directed against surface-exposed sporozoite proteins and cell mediated immune (CMI) responses characterized by predominantly CD4+ Th1 cells. The objective of this study was to investigate humoral and cell-mediated immune responses to the RTS,S/AS02D malaria vaccine and its association with protection against infection and disease by P. falciparum. Methodology and Principal Findings: This secondary data analysis from data of a phase I/IIb randomized, double-blind, controlled trial, included 154 healthy infants living in rural Mozambique, previously immunized with RTS,S/AS02D candidate malaria vaccine or the control Engerix-B™ vaccine. Antibodies against circumsporozoite protein (CSP) and hepatitis-B surface antigen (HBsAg) were measured with a standard ELISA. Fresh blood intracellular staining assay was performed to evaluate the expression of IL-2 and IFN-γ by CD4+ and CD8+ cells in response to in vitro stimulation of specific peptides. Data was evaluated for association with the risk of malaria detected by both active and passive case detection of infection over a period of 6 months post dose 3. Anti-HBs antibody geometric mean titers declined from 10,082 mIU/mL one month post Dose 3 to 2,751 mIU/mL at 12 months post Dose 3 in the RTS,S/AS02D group; anti-HBs v geometric mean titers were 392.4 mIU/mL and 263.9 mIU/mL, respectively in the Engerix- BTM group. Anti-CSP antibody geometric mean titers declined from 199.9 EU/mL one month post Dose 3 to 7.3 EU/mL at 12 months post Dose 3 in the RTS,S/AS02D group. Median stimulation indices of HBs-specific IL-2 and IFN-γ producing CD8+ T cells was higher in the RTS,S/AS02D group than in control group (Wilcoxon rank sum p-values for IFN-γ = 0.015, for IL-2 = 0.030) at 10.5 weeks post immunization. Median stimulation indices of anti-CSP specific IFN-γ producing CD8+ T cells at the same time point was 1.13 (IQR: 0.79 - 1.67; p=0.029). For specific IL-2-producing CD4+ T cells, the median SI was 1.14 (IQR: 0.74 – 1.60, p=0.043) at 10.5 weeks post dose three. The reduction in hazards of malaria infection were 18.3 % (95% CI: -267.9 – 81.8, p=0.793) and -12.0 % (95% CI: -295 – 68.2, p=0.86) for specific IL-2 CD4+ stimulation indices; For specific CD8+ IFN-γ stimulation indices the hazards were -103.6% (95% CI: -690.9 – 47.6; p=0.305) and 48.8% (95% CI: -97.0 – 86.7; p=0.33) at four and 10.5 weeks post immunization respectively. Conclusion: The RTS,S/AS02D vaccine was immunogenic and has elicited detectable levels of CSP specific cell mediated responses. No evidence of association was found between the antibodies anti-CSP and specific cell mediated responses and the risk of malaria

Characterization of the microbiota associated to Pecten maximus gonads using 454-pyrosequencing

A next-generation sequencing (NGS) approach was used to study the microbiota associated to Pecten maximus broodstock, applying pyrosequencing of PCR-amplified V1-V4 16S rRNA gene regions. We analysed the resident bacterial communities in female and male scallop gonads before and after spawning. DNA samples were amplified and quality-filtered reads were assigned to family and genus taxonomic levels using the Ribosomal Database Project classifier. A total of 18,520 sequences were detected, belonging to 13 phyla, including Proteobacteria (55%), Bacteroidetes (11,7%), Firmicutes (3%), Actinobacteria (2%) and Spirochaetes (1,2%), and 110 genera. The major fraction of the sequences detected corresponded to Proteobacteria, Beta- and Gammaprotebacteria being the most abundant classes. The microbiota of P. maximus gonad harbour a wide diversity, however differences on male and female samples were observed, where female gonad samples show a larger number of genera and families. The dominant bacterial genera appeared to be Delftia, Acinetobacter, Hydrotalea, Aquabacterium, Bacillus, Sediminibacterium, Sphingomonas, and Pseudomonas that were present among the four analysed samples. This next generation sequencing technique, applied for the first time in P. maximus (great scallop) gonads was useful for the study of the bacterial communities in this mollusc, unravelling the great bacterial diversity in its microbiota. [Int Microbiol 19(2): 93-99(2016)]Keywords: Pecten maximus · gonads microbiota · next-generation sequencing (NGS) · molluscs pathogens · aquacultur

Los criterios rigidos de interpretación del deber de idoneidad frente a las acciones correctivas ofrecidas por los proveedores

La presente investigación se enmarca en uno de los deberes más relevantes del derecho de consumo, hablamos así del deber de idoneidad. Esto se ve reflejado en la finalidad de nuestro Código de Protección y Defensa del Consumidor persigue que, los consumidores finales tengan la opción prioritaria de adquirir productos y prestaciones idóneas. Sumándoles que, los usuarios usen sus derechos e instrumentos de forma tal que, alcancen una protección sistémica e integral. Nos planteamos como objetivo principal, Determinar los criterios de interpretación del deber de idoneidad dispuesto por INDECOPI, y si estos son limitantes para que los proveedores ofrezcan medidas correctivas ágiles ante la afectación del deber de idoneidad. En el desarrollo de la presente investigación, se ha propuesto como base teórica desarrollar la teoría jurídica de la “tridimensionalidad del derecho”, debido que consideramos importante establecer un marco general previo para ahondar e identificar correctamente los criterios de interpretación del deber de idoneidad. Siendo así una investigación de tipo descriptivo, con análisis cualitativo que se orientó a una muestra de las resoluciones del tribunal de INDECOPI, referentes a la interpretación del deber de idoneidad, como resultado de la revisión, análisis conjuntamente con la doctrina y jurisprudencia de tales resoluciones, se logró identificar tres criterios de interpretación del deber de idoneidad dispuestos por INDECOPI, que consisten en: (i). La infalibilidad de productos y servicios (ii). La no integración de la relación de consumo (iii). El sentido residual de las medidas correctivas. Todas adquieren una condición de rigidez, puesto que suelen presentarse como limitantes para que los proveedores ofrezcan acciones y/o medidas correctivas inmediatas en favor de los consumidores y/o usuarios ante una afectación del deber de idoneidad.Tesi

A prospective cohort study to assess the micro-epidemiology of Plasmodium falciparum clinical malaria in Ilha Josina Machel (Manhiça, Mozambique)

Background: After the decrease in clinical malaria incidence observed in Mozambique until 2009, a steady resurgence of cases per year has been reported nationally, reaching alarming levels in 2014. However, little is known about the clinical profile of the cases presented, or the possible epidemiological factors contributing to the resurgence of cases. Methods: An analysis of surveillance data collected between July 2003 and June 2013 in the high malaria-transmission area of Ilha Josina Machel (Southern Mozambique) through a paediatric outpatient morbidity surveillance system was conducted to calculate hospital-based clinical malaria rates, slide-positivity rates, and minimum community-based incidence rates (MCBIRs) and incidence rate ratios per malaria season in children younger than 15 years of age. Clinical malaria was defined as a fever ≥37.5 °C or a reported fever in the previous 24 h with a positive blood smear. Yearly mean age, geometric mean parasitaemia (GMP) and mean packed cell volume (PCV) were also described for all clinical malaria cases and compared between seasons using DID analysis or ANOVA tests. Results: During the study period, the percentage of outpatient visits presenting with confirmed clinical malaria decreased from 51 % in the 2003–2004 season to 23 % in 2008–2009, followed by an increase back to 51 % in 2012–2013. The yearly mean age of cases significantly increased from 2.9 (95 % CI 2.8–3.0) in 2003–2004 to 5.7 (95 % CI 5.6–5.7) in 2012–2013, compared to non-malaria cases. An increase in mean PCV levels was also observed (p < 0.001), as well as in GMPs: from 5778 parasites/µL in 2002–2003 to 17,316 parasites/µL in 2012–2013 (p < 0.001) mainly driven by an increase in GMP in children older than 1 year of age. MCBIRs in infants decreased by 70 % (RR = 0.3, p < 0.001) between 2003–2004 and 2012–2013. Incidence diminished by a third among children 1- to 4-years between 2003 and 2007, although such drop was unsustained as observed in 2012–2013 (RR = 1.0, 95 % CI 0.9–1.0). Finally, the incidence among children 5–14 years was 3.8 (95 % CI 3.4–4.3) times higher in 2012–2013 compared to 2003–2004. Conclusion: Since 2003, Ilha Josina Machel observed a significant reduction of clinical malaria cases which was followed by an upsurge, following the national trend. A shift in the age distribution towards older children was observed, indicating that the changes in the transmission intensity patterns resulted in a slower acquisition of the naturally acquired immunity to malaria in children

Molecular surveillance of pfhrp2 and pfhrp3 deletions in Plasmodium falciparum isolates from Mozambique

BACKGROUND: Malaria programmes use Plasmodium falciparum histidine-rich protein-2 (PfHRP2) based rapid diagnostic tests (RDTs) for malaria diagnosis. The deletion of this target antigen could potentially lead to misdiagnosis, delayed treatment and continuation of active transmission. METHODS: Plasmodium falciparum isolates (n = 1162) collected in Southern Mozambique were assessed by RDTs, microscopy and/or 18SrRNA qPCR. pfhrp2 and pfhrp3 deletions were investigated in isolates from individuals who were negative by RDT but positive by microscopy and/or qPCR (n = 69) using gene-specific PCRs, with kelch13 PCR as the parasite DNA control. RESULTS: Lack of pfhrp2 PCR amplification was observed in one of the 69 isolates subjected to molecular analysis [1.45% (95% CI 0.3-7.8%)]. CONCLUSIONS: The low prevalence of pfhrp2 deletions suggests that RDTs will detect the vast majority of the P. falciparum infections. Nevertheless, active surveillance for changing deletion frequencies is required

Dynamics of Afebrile Plasmodium falciparum Infections in Mozambican Men

Background: Afebrile Plasmodium falciparum infections usually remain undetected and untreated in the community and could potentially contribute to sustaining local malaria transmission in areas aiming for malaria elimination. Methods: Thirty-two men with afebrile P. falciparum infections detected with rapid diagnostic test (RDTs) were followed for 28 days. Kaplan-Meier estimates were computed to estimate probability of parasite positivity and of reducing parasitaemia by half of its initial level by day 28. Trends of parasite densities quantified by microscopy and qPCR were assessed using Poisson regression models, and the microscopy to qPCR positivity ratio was calculated at each time point. Three survival distributions (Gompertz, Weibull, and gamma) were used to evaluate their strength of fit to the data and to predict the median lifetime of infection. Results: The cumulative probability of parasite qPCR positivity by day 28 was 81% (95% CI 60.2-91.6). Geometric mean parasitemia at recruitment was 516.1 parasites/muL and fell to <100 parasites/muL by day 3, reaching 56.7 parasites/muL on day 28 (p-value<0.001). The ratio of P. falciparum positive samples by microscopy to qPCR decreased from 0.9 to 0.52 from recruitment to day 28. The best model fit to the data was obtained assuming a Gompertz distribution. Conclusions: Afebrile P.falciparum infections detectable by RDT in semi-immune adults fall and stabilize at low-density levels during the first four days since detection, suggesting a rapid decline of potential transmissibility in this hidden parasite reservoir

Under treatment of pneumonia among children under 5 years of age in a malaria-endemic area: population-based surveillance study conducted in Manhica district- rural, Mozambique

BACKGROUND: Integrated Management of Childhood Illness (IMCI) guidelines were developed to decrease morbidity and mortality, yet implementation varies across settings. Factors associated with poor adherence are not well understood. METHODS: We used data from Manhica District Hospital outpatient department and five peripheral health centers to examine pneumonia management for children <5 years old from January 2008 to June 2011. Episodes of IMCI-defined pneumonia (cough or difficult breathing plus tachypnea), severe pneumonia (pneumonia plus chest wall in-drawing), and/or clinician-diagnosed pneumonia (based on discharge diagnosis) were included. RESULTS: Among severe pneumonia episodes, 96.2% (2,918/3,032) attended in the outpatient department and 70.0% (291/416) attended in health centers were appropriately referred to the emergency department. Age<1 year, malnutrition and various physical exam findings were associated with referral. For non-severe pneumonia episodes, antibiotics were prescribed in 45.7% (16,094/35,224). Factors associated with antibiotic prescription included age <1 year, abnormal auscultatory findings, and clinical diagnosis of pneumonia; diagnosis of malaria or gastroenteritis and pallor were negatively associated with antibiotic prescription. CONCLUSION: Adherence to recommended management of severe pneumonia was high in a hospital outpatient department, but suboptimal in health centers. Antibiotics were prescribed in fewer than half of non-severe pneumonia episodes, and diagnosis of malaria was the strongest risk factor for incorrect management

Heterogeneity of G6PD deficiency prevalence in Mozambique: a school-based cross-sectional survey in three different regions

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked hereditary enzymatic abnormality that affects more than 400 million people worldwide. Most deficient individuals do not manifest any symptoms; however, several precipitant agents—such as fava intake, infections, or several drugs—may trigger acute haemolytic anaemia. Countries should be informed of the prevalence of this enzymatic anomaly within their borders, in order to make safe and appropriate national decisions regarding the use of potentially unsafe drugs for G6PD deficient individuals. Methods: A school-based cross-sectional survey was conducted in three districts in Mozambique, namely Manhiça, located in the south; Mocuba in the centre; and Pemba in the northern tip of the country. G6PD deficiency was evaluated using the CareStart™ diagnostic test, and enzyme activity levels were measured through fluorescence spectrophotometry in deficient individuals. Chi squared and ANOVA tests were used to assess prevalence and mean enzyme activity differences, and logistic regression was used to identify risk factors associated to the deficiency. Results: G6PD deficiency prevalence estimates were lowest in the northern city of Pemba (8.3%) and among Emakhuwas and Shimakondes, and higher in the centre and southern regions of the country (16.8 and 14.6%, respectively), particularly among Elomwes and Xichanganas. G6PD deficiency was significantly more prevalent among male students than females (OR = 1.4, 95% CI 1.0–1.8, p = 0.02), although enzyme activity levels were not different among deficient individuals from either gender group. Finally, median deficiency levels were found to be more severe among the deficient students from the north (0.7 U/gHg [0.2–0.7] p < 0.001) and south (0.7 U/gHg [0.5–2.5]), compared to those from the centre (1.4 U/gHg [0.6–2.1]). Conclusion: These findings suggest that Mozambique, as a historically high malaria-endemic country has considerable levels of G6PD deficiency, that vary significantly across the country. This should be considered when planning national strategies for the use of licensed drugs that may be associated to haemolysis among G6PD individuals, or prior to the performance of future trials using primaquine and other 8-aminoquinolines derivatives

Malaria in rural Mozambique. Part II: children admitted to hospital

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In-Vivo Efficacy of Chloroquine to Clear Asymptomatic Infections in Mozambican Adults: A Randomized, Placebo-controlled Trial with Implications for Elimination Strategies

Recent reports regarding the re-emergence of parasite sensitivity to chloroquine call for a new consideration of this drug as an interesting complementary tool in malaria elimination efforts, given its good safety profile and long half-life. A randomized (2:1), single-blind, placebo-controlled trial was conducted in Manhica, Mozambique, to assess the in-vivo efficacy of chloroquine to clear plasmodium falciparum (Pf) asymptomatic infections. Primary study endpoint was the rate of adequate and parasitological response (ACPR) to therapy on day 28 (PCR-corrected). Day 0 isolates were analyzed to assess the presence of the PfCRT-76T CQ resistance marker. A total of 52 and 27 male adults were included in the CQ and Placebo group respectively. PCR-corrected ACPR was significantly higher in the CQ arm 89.4% (95%CI 80-98%) compared to the placebo (p < 0.001). CQ cleared 49/50 infections within the first 72 h while placebo cleared 12/26 (LRT p < 0.001). The PfCRT-76T mutation was present only in one out of 108 (0.9%) samples at baseline, well below the 84% prevalence found in 1999 in the same area. This study presents preliminary evidence of a return of chloroquine sensitivity in Mozambican Pf isolates, and calls for its further evaluation in community-based malaria elimination efforts, in combination with other effective anti-malarials. TRIAL REGISTRATION: www.clinicalTrials.gov NCT02698748