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Elfving K.,Gothenburg University | Andersson M.,Gothenburg University | Msellem M.I.,Zanzibar Malaria Elimination Programme | Welinder-Olsson C.,Gothenburg University | And 5 more authors.
Journal of Clinical Microbiology | Year: 2014

Molecular assays might improve the identification of causes of acute diarrheal disease but might lead to more frequent detection of asymptomatic infections. In the present study, real-time PCR targeting 14 pathogens was applied to rectal swabs from 330 children aged 2 to 59 months in Zanzibar, including 165 patients with acute diarrhea and 165 asymptomatic control subjects. At least one pathogen was detected for 94% of the patients and 84% of the controls, with higher rates among patients for norovirus genogroup II (20% versus 2.4%; P<0.0001), rotavirus (10% versus 1.8%; P=0.003), and Cryptosporidium (30% versus 11%; P<0.0001). Detection rates did not differ significantly for enterotoxigenic Escherichia coli (ETEC)-estA (33% versus 24%), ETEC-eltB (44% versus 46%), Shigella (35% versus 33%), and Campylobacter (35% versus 33%), but for these agents threshold cycle (CT) values were lower (pathogen loads were higher) in sick children than in controls. In a 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 the interpretation of results for pathogens with similar detection rates in patients and controls. The results indicate that the assessment of pathogen loads may improve the identification of agents causing gastroenteritis in children. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Xu W.,Karolinska Institutet | Xu W.,Dalian University of Technology | Morris U.,Karolinska Institutet | Aydin-Schmidt B.,Karolinska Institutet | And 6 more authors.
PLoS ONE | Year: 2015

A prerequisite for reliable detection of low-density Plasmodium infections in malaria preelimination settings is the availability of ultra-sensitive and high-throughput molecular tools. We developed a SYBR Green real-time PCR restriction fragment length polymorphism assay (cytb-qPCR) targeting the cytochrome b gene of the four major human Plasmodium species (P. falciparum, P. vivax, P. malariae , and P. ovale) for parasite detection and species determination with DNA extracted from dried blood spots collected on filter paper. The performance of cytb-qPCR was first compared against four reference PCR methods using serially diluted Plasmodium samples. The detection limit of the cytb-qPCR was 1 parasite/μl (p/μl) for P. falciparum and P. ovale, and 2 p/μl for P. vivax and P. malariae, while the reference PCRs had detection limits of 0.5 -10 p/μl. The ability of the PCR methods to detect low-density Plasmodium infections was then assessed using 2977 filter paper samples collected during a cross-sectional survey in Zanzibar, a malaria pre-elimination setting in sub-Saharan Africa. Field samples were defined as 'final positive' if positive in at least two of the five PCR methods. Cytb-qPCR preformed equal to or better than the reference PCRs with a sensitivity of 100% (65/65; 95%CI 94.5-100%) and a specificity of 99.9%(2910/2912; 95% CI 99.7- 100%) when compared against 'final positive' samples. The results indicate that the cytb-qPCR may represent an opportunity for improved molecular surveillance of lowdensity Plasmodium infections in malaria pre-elimination settings. © 2015 Xu et al.

Maliti D.,Ifakara Health Institute | Maliti D.,University of Glasgow | Magesa S.,Rti International | Kisinza W.,National Institute for Medical Research | And 3 more authors.
PLoS ONE | Year: 2014

Population genetic structures of the two major malaria vectors Anopheles gambiae s.s. and An. arabiensis, differ markedly across Sub-Saharan Africa, which could reflect differences in historical demographies or in contemporary gene flow. Elucidation of the degree and cause of population structure is important for predicting the spread of genetic traits such as insecticide resistance genes or artificially engineered genes. Here the population genetics of An. gambiae s.s. and An. arabiensis in the central, eastern and island regions of Tanzania were compared. Microsatellite markers were screened in 33 collections of female An. gambiae s.l., originating from 22 geographical locations, four of which were sampled in two or three years between 2008 and 2010. An. gambiae were sampled from six sites, An. arabiensis from 14 sites, and both species from two sites, with an additional colonised insectary sample of each species. Frequencies of the knock-down resistance (kdr) alleles 1014S and 1014F were also determined. An. gambiae exhibited relatively high genetic differentiation (average pairwise FST = 0.131), significant even between nearby samples, but without clear geographical patterning. In contrast, An. arabiensis exhibited limited differentiation (average FST = 0.015), but strong isolation-by-distance (Mantel test r = 0.46, p = 0.0008). Most time-series samples of An. arabiensis were homogeneous, suggesting general temporal stability of the genetic structure. An. gambiae populations from Dar es Salaam and Bagamoyo were found to have high frequencies of kdr 1014S (around 70%), with almost 50% homozygote but was at much lower frequency on Unguja Island, with no. An. gambiae population genetic differentiation was consistent with an island model of genetic structuring with highly restricted gene flow, contrary to An. arabiensis which was consistent with a stepping-stone model of extensive, but geographicallyrestricted gene flow. © 2014 Maliti et al.

Elfving K.,Gothenburg University | Shakely D.,Karolinska Institutet | Andersson M.,Gothenburg University | Baltzell K.,University of California at San Francisco | And 14 more authors.
PLoS ONE | Year: 2016

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 Xray (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. © 2016 Elfving et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Haji K.A.,Zanzibar Malaria Elimination Programme | Thawer N.G.,Rti International | Khatib B.O.,Zanzibar Malaria Elimination Programme | Mcha J.H.,Zanzibar Malaria Elimination Programme | And 8 more authors.
Parasites and Vectors | Year: 2015

Background: Indoor residual spraying (IRS) of households with insecticide is a principal malaria vector control intervention in Zanzibar. In 2006, IRS using the pyrethroid lambda-cyhalothrine was introduced in Zanzibar. Following detection of pyrethroid resistance in 2010, an insecticide resistance management plan was proposed, and IRS using bendiocarb was started in 2011. In 2014, bendiocarb was replaced by pirimiphos methyl. This study investigated the residual efficacy of pirimiphos methyl (Actellic® 300CS) sprayed on common surfaces of human dwellings in Zanzibar. Methods: The residual activity of Actellic 300CS was determined over 9 months through bioassay tests that measured the mortality of female Anopheles mosquitoes, exposed to sprayed surfaces under a WHO cone. The wall surfaces included; mud wall, oil or water painted walls, lime washed wall, un-plastered cement block wall and stone blocks. Insecticide susceptibility testing was done to investigate the resistance status of local malaria vectors against Actellic 300CS using WHO protocols; Anopheline species were identified using PCR methods. Results: Baseline tests conducted one-day post-IRS revealed 100 % mortality on all sprayed surfaces. The residual efficacy of Actellic 300CS was maintained on all sprayed surfaces up to 8 months post-IRS. However, the bioassay test conducted 9 months post-IRS showed the 24 h mortality rate to be ≤&80 % for lime wash, mud wall, water paint and stone block surfaces. Only oil paint surface retained the recommended residual efficacy beyond 9 months post-IRS, with mortality maintained at ≥97 %. Results of susceptibility tests showed that malaria vectors in Zanzibar were fully (100 %) susceptible to Actellic 300CS. The predominant mosquito vector species was An. arabiensis (76.0 %) in Pemba and An. gambiae (83.5 %) in Unguja. Conclusion: The microencapsulated formulation of pirimiphos methyl (Actellic 300CS) is a highly effective and appropriate insecticide for IRS use in Zanzibar as it showed a relatively prolonged residual activity compared to other products used for the same purpose. The insecticide extends the residual effect of IRS thereby making it possible to effectively protect communities with a single annual spray round reducing overall costs. The insecticide proved to be a useful alternative in insecticide resistance management plans. © 2015 Haji et al.

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