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1.
  • Elfving, Kristina, et al. (author)
  • Acute Uncomplicated Febrile Illness in Children Aged 2-59 months in Zanzibar : Aetiologies, Antibiotic Treatment and Outcome
  • 2016
  • In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:1
  • Journal article (peer-reviewed)abstract
    • BACKGROUND: Despite the fact that a large proportion of children with fever in Africa present at primary health care facilities, few studies have been designed to specifically study the causes of uncomplicated childhood febrile illness at this level of care, especially in areas like Zanzibar that has recently undergone a dramatic change from high to low malaria transmission.METHODS: We prospectively studied the aetiology of febrile illness in 677 children aged 2-59 months with acute uncomplicated fever managed by IMCI (Integrated Management of Childhood Illness) guidelines in Zanzibar, using point-of-care tests, urine culture, blood-PCR, chest X-ray (CXR) of IMCI-pneumonia classified patients, and multiple quantitative (q)PCR investigations of nasopharyngeal (NPH) (all patients) and rectal (GE) swabs (diarrhoea patients). For comparison, we also performed NPH and GE qPCR analyses in 167 healthy community controls. Final fever diagnoses were retrospectively established based on all clinical and laboratory data. Clinical outcome was assessed during a 14-day follow-up. The utility of IMCI for identifying infections presumed to require antibiotics was evaluated.FINDINGS: NPH-qPCR and GE-qPCR detected ≥1 pathogen in 657/672 (98%) and 153/164 (93%) of patients and 158/166 (95%) and 144/165 (87%) of controls, respectively. Overall, 57% (387/677) had IMCI-pneumonia, but only 12% (42/342) had CXR-confirmed pneumonia. Two patients were positive for Plasmodium falciparum. Respiratory syncytial virus (24.5%), influenza A/B (22.3%), rhinovirus (10.5%) and group-A streptococci (6.4%), CXR-confirmed pneumonia (6.2%), Shigella (4.3%) were the most common viral and bacterial fever diagnoses, respectively. Blood-PCR conducted in a sub-group of patients (n = 83) without defined fever diagnosis was negative for rickettsiae, chikungunya, dengue, Rift Valley fever and West Nile viruses. Antibiotics were prescribed to 500 (74%) patients, but only 152 (22%) had an infection retrospectively considered to require antibiotics. Clinical outcome was generally good. However, two children died. Only 68 (11%) patients remained febrile on day 3 and three of them had verified fever on day 14. An additional 29 (4.5%) children had fever relapse on day 14. Regression analysis determined C-reactive Protein (CRP) as the only independent variable significantly associated with CXR-confirmed pneumonia.CONCLUSIONS: This is the first study on uncomplicated febrile illness in African children that both applied a comprehensive laboratory panel and a healthy control group. A majority of patients had viral respiratory tract infection. Pathogens were frequently detected by qPCR also in asymptomatic children, demonstrating the importance of incorporating controls in fever aetiology studies. The precision of IMCI for identifying infections requiring antibiotics was low.
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2.
  • Morris, Ulrika, et al. (author)
  • A cluster randomised controlled trial of two rounds of mass drug administration in Zanzibar, a malaria pre-elimination setting-high coverage and safety, but no significant impact on transmission.
  • 2018
  • In: BMC Medicine. - : Springer Science and Business Media LLC. - 1741-7015. ; 16:1
  • Journal article (peer-reviewed)abstract
    • BACKGROUND: Mass drug administration (MDA) has the potential to interrupt malaria transmission and has been suggested as a tool for malaria elimination in low-endemic settings. This study aimed to determine the effectiveness and safety of two rounds of MDA in Zanzibar, a pre-elimination setting.METHODS: A cluster randomised controlled trial was conducted in 16 areas considered as malaria hotspots, with an annual parasite index of > 0.8%. The areas were randomised to eight intervention and eight control clusters. The intervention included two rounds of MDA with dihydroartemisinin-piperaquine and single low-dose primaquine 4 weeks apart in May-June 2016. Primary and secondary outcomes were cumulative confirmed malaria case incidences 6 months post-MDA and parasite prevalences determined by PCR 3 months post-MDA. Additional outcomes included intervention coverage, treatment adherence, occurrence of adverse events, and cumulative incidences 3, 12, and 16 months post-MDA.RESULTS: Intervention coverage was 91.0% (9959/10944) and 87.7% (9355/10666) in the first and second rounds, respectively; self-reported adherence was 82.0% (881/1136) and 93.7% (985/1196). Adverse events were reported in 11.6% (147/1268) and 3.2% (37/1143) of post-MDA survey respondents after both rounds respectively. No serious adverse event was reported. No difference in cumulative malaria case incidence was observed between the control and intervention arms 6 months post-MDA (4.2 and 3.9 per 1000 population; p = 0.94). Neither was there a difference in PCR-determined parasite prevalences 3 months post-MDA (1.4% and 1.7%; OR = 1.0, p = 0.94), although having received at least the first MDA was associated with reduced odds of malaria infection (aOR = 0.35; p = 0.02). Among confirmed malaria cases at health facilities, 26.0% and 26.3% reported recent travel outside Zanzibar in the intervention and control shehias (aOR ≥ 85; p ≤ 0.001).CONCLUSIONS: MDA was implemented with high coverage, adherence, and tolerability. Despite this, no significant impact on transmission was observed. The findings suggest that two rounds of MDA in a single year may not be sufficient for a sustained impact on transmission in a pre-elimination setting, especially when the MDA impact is restricted by imported malaria. Importantly, this study adds to the limited evidence for the use of MDA in low transmission settings in sub-Saharan Africa.TRIAL REGISTRATION: ClinicalTrials.gov, NCT02721186 (registration date: March 29, 2016).
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3.
  • Morris, Ulrika, et al. (author)
  • Field deployment of loop-mediated isothermal amplification for centralized mass-screening of asymptomatic malaria in Zanzibar : a pre-elimination setting
  • 2015
  • In: Malaria Journal. - : Springer Science and Business Media LLC. - 1475-2875. ; 14
  • Journal article (peer-reviewed)abstract
    • BACKGROUND: Molecular tools for detection of low-density asymptomatic Plasmodium infections are needed in malaria elimination efforts. This study reports results from the hitherto largest implementation of loop-mediated isothermal amplification (LAMP) for centralized mass screening of asymptomatic malaria in Zanzibar.METHODS: Healthy individuals present and willing to participate in randomly selected households in 60 villages throughout Zanzibar were screened for malaria by rapid diagnostic tests (RDT). In 50 % of the study households, participants were asked to provide 60 μL of finger-prick blood for additional LAMP screening. LAMP was conducted in two centralized laboratories in Zanzibar, by trained technicians with limited or no previous experience of molecular methods. The LAMP assay was performed with Loopamp(TM) MALARIA Pan/Pf Detection Kit (Eiken Chemical Company, Japan). Samples positive for Plasmodium genus (Pan)-LAMP were re-tested using Plasmodium falciparum-specific LAMP kits.RESULTS: Paired RDT and LAMP samples were available from 3983 individuals. The prevalence of asymptomatic malaria was 0.5 % (CI 95 % 0.1-0.8) and 1.6 % (CI 95 % 1.1-2.2) by RDT and Pan-LAMP, respectively. LAMP detected 3.4 (CI 95 % 2.2-5.2) times more Plasmodium positive samples than RDT. DNA contamination was experienced, but solved by repetitive decontamination of all equipment and reagents.CONCLUSIONS: LAMP is a simple and sensitive molecular tool, and has potential in active surveillance and mass-screening programmes for detection of low-density asymptomatic malaria in pre-elimination settings. However, in order to deploy LAMP more effectively in field settings, protocols may need to be adapted for processing larger numbers of samples. A higher throughput, affordable closed system would be ideal to avoid contamination.
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4.
  • Aydin-Schmidt, Berit, et al. (author)
  • Field Evaluation of a High Throughput Loop Mediated Isothermal Amplification Test for the Detection of Asymptomatic Plasmodium Infections in Zanzibar
  • 2017
  • In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:1
  • Journal article (peer-reviewed)abstract
    • Background New field applicable diagnostic tools are needed for highly sensitive detection of residual malaria infections in pre-elimination settings. Field performance of a high throughput DNA extraction system for loop mediated isothermal amplification (HTP-LAMP) was therefore evaluated for detecting malaria parasites among asymptomatic individuals in Zanzibar. Methods HTP-LAMP performance was evaluated against real-time PCR on 3008 paired blood samples collected on filter papers in a community-based survey in 2015. Results The PCR and HTP-LAMP determined malaria prevalences were 1.6% (95% CI 1.3-2.4) and 0.7% (95% CI 0.4-1.1), respectively. The sensitivity of HTP-LAMP compared to PCR was 40.8% (CI95% 27.0-55.8) and the specificity was 99.9% (CI95% 99.8-100). For the PCR positive samples, there was no statistically significant difference between the geometric mean parasite densities among the HTP-LAMP positive (2.5 p/mu L, range 0.2-770) and HTP-LAMP negative (1.4 p/mu L, range 0.1-7) samples (p = 0.088). Two lab technicians analysed up to 282 samples per day and the HTP-LAMP method was experienced as user friendly. Conclusions Although field applicable, this high throughput format of LAMP as used here was not sensitive enough to be recommended for detection of asymptomatic low-density infections in areas like Zanzibar, approaching malaria elimination.
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5.
  • Aydin-Schmidt, Berit, et al. (author)
  • Loop mediated isothermal amplification (LAMP) accurately detects malaria DNA from filter paper blood samples of low density parasitaemias
  • 2014
  • In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:8, s. e103905-
  • Journal article (peer-reviewed)abstract
    • BACKGROUND:Loop mediated isothermal amplification (LAMP) provides an opportunity for improved, field-friendly detection of malaria infections in endemic areas. However data on the diagnostic accuracy of LAMP for active case detection, particularly low-density parasitaemias, are lacking. We therefore evaluated the performance of a new LAMP kit compared with PCR using DNA from filter paper blood spots.METHODS AND FINDINGS:Samples from 865 fever patients and 465 asymptomatic individuals collected in Zanzibar were analysed for Pan (all species) and Pf (P. falciparum) DNA with the Loopamp MALARIA Pan/Pf kit. Samples were amplified at 65°C for 40 minutes in a real-time turbidimeter and results were compared with nested PCR. Samples with discordant results between LAMP and nested PCR were analysed with real-time PCR. The real-time PCR corrected nested PCR result was defined as gold standard. Among the 117 (13.5%) PCR detected P. falciparum infections from fever patients (mean parasite density 7491/µL, range 6-782,400) 115, 115 and 111 were positive by Pan-LAMP, Pf-LAMP and nested PCR, respectively. The sensitivities were 98.3% (95%CI 94-99.8) for both Pan and Pf-LAMP. Among the 54 (11.6%) PCR positive samples from asymptomatic individuals (mean parasite density 10/µL, range 0-4972) Pf-LAMP had a sensitivity of 92.7% (95%CI 80.1-98.5) for detection of the 41 P. falciparum infections. Pan-LAMP had sensitivities of 97% (95%CI 84.2-99.9) and 76.9% (95%CI 46.2-95) for detection of P. falciparum and P. malariae, respectively. The specificities for both Pan and Pf-LAMP were 100% (95%CI 99.1-100) in both study groups.CONCLUSION:Both components of the Loopamp MALARIA Pan/Pf detection kit revealed high diagnostic accuracy for parasite detection among fever patients and importantly also among asymptomatic individuals of low parasite densities from minute blood volumes preserved on filter paper. These data support LAMPs potential role for improved detection of low-density malaria infections in pre-elimination settings.
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6.
  • Elfving, Kristina, et al. (author)
  • Pathogen Clearance and New Respiratory Tract Infections Among Febrile Children in Zanzibar Investigated With Multitargeting Real-Time Polymerase Chain Reaction on Paired Nasopharyngeal Swab Samples
  • 2018
  • In: The Pediatric Infectious Disease Journal. - 0891-3668 .- 1532-0987. ; 37:7, s. 643-648
  • Journal article (peer-reviewed)abstract
    • BACKGROUND: New molecular methods have revealed frequent and often polymicrobial respiratory infections in children in low-income settings. It is not known whether presence of multiple pathogens is due to prolonged infections or to frequent exposure. The aim of this study was to analyze short-term pathogen clearance from nasopharynx and the rate of new respiratory tract infections in febrile preschool children.METHODS: Children (n = 207) with uncomplicated acute febrile illness 2-59 months of age presenting to a health center in Zanzibar, Tanzania, April-July 2011, were included. Paired nasopharyngeal swab samples, collected at enrolment and after 14 days, were analyzed by multiple real-time polymerase chain reaction for Adenovirus, bocavirus, Bordetella pertussis, Chlamydophila pneumoniae, Coronaviruses, Enterovirus, influenza A and B virus, metapneumovirus, measles virus, Mycoplasma pneumoniae, parainfluenza virus, Parechovirus, respiratory syncytial virus and Rhinovirus. An age-matched and geographically matched healthy control group (n = 166) underwent nasopharyngeal sampling on 1 occasion.RESULTS: At baseline, 157/207 (76%) patients had at least 1 pathogen detected, in total 199 infections. At follow-up (day 14), 162/199 (81%) of these infections were not detected, including >95% of the previously detected infections with Enterovirus, influenza A virus, influenza B virus, metapneumovirus or parainfluenza virus. Still 115 (56%) children were positive for at least 1 pathogen at follow-up, of which 95/115 (83%) were not found at baseline. Detection of influenza B on day 14 was significantly associated with fever during follow-up.CONCLUSION: The results suggest that children with acute febrile illness in Zanzibar rapidly clear respiratory tract infections but frequently acquire new infections within 14 days.
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7.
  • Elfving, Kristina, et al. (author)
  • Real-time PCR threshold cycle (Ct) cut-offs help to identify agents causing acute childhood diarrhea in Zanzibar.
  • 2014
  • In: Journal of clinical microbiology. - 1098-660X. ; 52:3, s. 916-923
  • Journal article (peer-reviewed)abstract
    • Molecular assays might improve identification of causes of acute diarrheal disease, but may lead to more frequent detection of asymptomatic infections. In the present study real-time PCR targeting 14 pathogens was applied on rectal swabs from 330 children aged 2-59 months in Zanzibar, 165 with acute diarrhea and 165 asymptomatic controls. At least one pathogen was detected in 94% of patients and 84% of controls, with higher rates in patients for norovirus genogroup II (20% vs. 2.4%, p<0.0001), rotavirus (10% vs. 1.8%, p=0.003) and Cryptosporidium (30% vs. 11%, p<0.0001). Detection rates did not differ significantly for enterotoxigenic Escherichia coli (ETEC)-estA (33% vs. 24%), ETEC-eltB (44% vs. 46%), Shigella (35% vs. 33%), and Campylobacter (35% vs. 33%), but for these agents Ct (threshold cycle) values were lower (pathogen loads were higher) in sick children than in controls. In multivariate analysis, Ct values for norovirus genogroup II, rotavirus, Cryptosporidium, ETEC-estA and Shigella were independently associated with diarrhea. We conclude that this real-time PCR allows convenient detection of essentially all diarrheagenic agents, and provides Ct values that may be critical for interpretation of results for pathogens with similar detection rates in patients and controls. The results indicate that assessment of pathogen load may improve identification of agents causing gastroenteritis in children.
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8.
  • Morris, Ulrika, et al. (author)
  • Rapid diagnostic tests for molecular surveillance of Plasmodium falciparum malaria -assessment of DNA extraction methods and field applicability.
  • 2013
  • In: Malaria journal. - : Springer Science and Business Media LLC. - 1475-2875. ; 12
  • Journal article (peer-reviewed)abstract
    • The need for new malaria surveillance tools and strategies is critical, given improved global malaria control and regional elimination efforts. High quality Plasmodium falciparum DNA can reliably be extracted from malaria rapid diagnostic tests (RDTs). Together with highly sensitive molecular assays, wide scale collection of used RDTs may serve as a modern tool for improved malaria case detection and drug resistance surveillance. However, comparative studies of DNA extraction efficiency from RDTs and the field applicability are lacking. The aim of this study was to compare and evaluate different methods of DNA extraction from RDTs and to test the field applicability for the purpose of molecular epidemiological investigations.
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9.
  • Msellem, Mwinyi I (author)
  • Efficacy of artemisinin based combination therapy and effectiveness of rapid diagnostic test for management of patients with plasmodium falciparum malaria in Zanzibar
  • 2009
  • Licentiate thesis (other academic/artistic)abstract
    • Background: Due to rapid spread of antimalarial drug resistance to Plasmodium falciparum malaria many African countries have recently changed their first-line treatment policy to artemisinin-based combination therapy (ACT). Zanzibar was one of the first regions in Africa to implement ACT free of charge to all age groups through public health facilities in 2003. The introduction of ACT, a highly efficacious but also considerably more expensive treatment than previously used mono-therapies, puts a heavy extra burden on malaria control programmes in Africa. This highlights the need of improved diagnostics to ensure targeting of ACT to patients with parasitologically confirmed malaria infection, particularly in settings beyond the reach of high quality microscopy service. Rapid diagnostic tests (RDT) for malaria represent such a potential candidate, considering they are simple to use and do not require highly trained staff, advanced laboratory facilities or electricity. The aim of this thesis was to study the efficacy of ACT in children with uncomplicated P. falciparum malaria and the effectiveness of RDT for management of fever patients in rural health facilities in Zanzibar. The studies included in the thesis are a part of the scientific evaluation of the ongoing integrated, wide scale, high coverage malaria control interventions in Zanzibar. Methods: Study I was a comparative clinical trial assessing the efficacy of artesunate+amodiaquine (ASAQ) and artemether-lumefantrine (AL) in children with microscopically confirmed uncomplicated P. falciparum malaria. Some 408 patients were enrolled in this study, which was conducted between November 2002 and February 2003 in Kivunge and Micheweni Cottage Hospitals, i.e. prior to the of deployment of the drugs as first- and second line treatment for uncomplicated malaria in Zanzibar. Follow-up was extended to 42 days. Primary end-point was polymerase chain reaction (PCR) adjusted cure rates by day 42 in the respective treatment arms. Study II was a cross-over validation study assessing the influence of RDT aided diagnosis on ACT prescription compared to clinical diagnosis (CD) only in patients with fever within the last 48 hours attending 4 primary health care facilities in Zanzibar. Some 1887 patients were enrolled between February and August 2005. Follow-up was 14 days. ACT was to be prescribed to patients diagnosed with malaria in both groups. Results: Study I. PCR adjusted cure rates by day 42 were 188/206 (91%) for ASAQ and 185/197 (94%) for AL, [Odds ratio (OR) 1.48 95% CI 0.69-3.15; p=0.314]. However, AL provided a stronger protection against reinfections during follow-up. Study II. RDT was associated with lower prescription rate of ACT than CD alone, 361/1005 (36%) compared with 752/882 (85%) [OR 0.04 (95%CI 0.03-0.05) p<0.001]. In contrast, prescription of antibiotics was higher after RDT than CD alone, i.e. 372/1005 (37%) and 235/882 (27%) [OR 1.8 (95%CI 1.5-2.2) p<0.001]. Conclusion: Both ASAQ and AL were highly efficacious, which justify their introduction as new malaria treatment policy in Zanzibar. RDTs resulted in improved adequate treatment and may therefore represent an important tool for management of patients with fever in peripheral health care settings in Zanzibar.
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