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      Comparison of three diagnostic methods (microscopy, RDT, and PCR) for the detection of malaria parasites in representative samples from Equatorial Guinea

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          Abstract

          Background

          Malaria in Equatorial Guinea remains a major public health problem. The country is a holo-endemic area with a year-round transmission pattern. In 2016, the prevalence of malaria was 12.09% and malaria caused 15% of deaths among children under 5 years. In the Continental Region, 95.2% of malaria infections were Plasmodium falciparum, 9.5% Plasmodium vivax, and eight cases mixed infection in 2011. The main strategy for malaria control is quick and accurate diagnosis followed by effective treatment. Early and accurate diagnosis of malaria is essential for both effective disease management and malaria surveillance. The quality of malaria diagnosis is important in all settings, as misdiagnosis can result in significant morbidity and mortality. Microscopy and RDTs are the primary choices for diagnosing malaria in the field. However, false-negative results may delay treatment and increase the number of persons capable of infecting mosquitoes in the community. The present study analysed the performance of microscopy and RDTs, the two main techniques used in Equatorial Guinea for the diagnosis of malaria, compared to semi-nested multiplex PCR (SnM-PCR).

          Results

          A total of 1724 samples tested by microscopy, RDT, and SnM-PCR were analysed. Among the negative samples detected by microscopy, 335 (19.4%) were false negatives. On the other hand, the negative samples detected by RDT, 128 (13.3%) were false negatives based on PCR. This finding is important, especially since it is a group of patients who did not receive antimalarial treatment.

          Conclusions

          Owing to the high number of false negatives in microscopy, it is necessary to reinforce training in microscopy, the “Gold Standard” in endemic areas. A network of reference centres could potentially support ongoing diagnostic and control efforts made by malaria control programmes in the long term, as the National Centre of Tropical Medicine currently supports the National Programme against Malaria of Equatorial Guinea to perform all of the molecular studies necessary for disease control. Taking into account the results obtained with the RDTs, an exhaustive study of the deletion of the hrp2 gene must be done in EG to help choose the correct RDT for this area.

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          Most cited references34

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          First case of a naturally acquired human infection with Plasmodium cynomolgi

          Since 1960, a total of seven species of monkey malaria have been reported as transmissible to man by mosquito bite: Plasmodium cynomolgi, Plasmodium brasilianum, Plasmodium eylesi, Plasmodium knowlesi, Plasmodium inui, Plasmodium schwetzi and Plasmodium simium. With the exception of P. knowlesi, none of the other species has been found to infect humans in nature. In this report, it is described the first known case of a naturally acquired P. cynomolgi malaria in humans. The patient was a 39-year-old woman from a malaria-free area with no previous history of malaria or travel to endemic areas. Initially, malaria was diagnosed and identified as Plasmodium malariae/P. knowlesi by microscopy in the Terengganu State Health Department. Thick and thin blood films stained with 10% Giemsa were performed for microscopy examination. Molecular species identification was performed at the Institute for Medical Research (IMR, Malaysia) and in the Malaria & Emerging Parasitic Diseases Laboratory (MAPELAB, Spain) using different nested PCR methods. Microscopic re-examination in the IMR showed characteristics of Plasmodium vivax and was confirmed by a nested PCR assay developed by Snounou et al. Instead, a different PCR assay plus sequencing performed at the MAPELAB confirmed that the patient was infected with P. cynomolgi and not with P. vivax. This is the first report of human P. cynomolgi infection acquired in a natural way, but there might be more undiagnosed or misdiagnosed cases, since P. cynomolgi is morphologically indistinguishable from P. vivax, and one of the most used PCR methods for malaria infection detection may identify a P. cynomolgi infection as P. vivax. Simian Plasmodium species may routinely infect humans in Southeast Asia. New diagnostic methods are necessary to distinguish between the human and monkey malaria species. Further epidemiological studies, incriminating also the mosquito vector(s), must be performed to know the relevance of cynomolgi malaria and its implication on human public health and in the control of human malaria. The zoonotic malaria cannot be ignored in view of increasing interactions between man and wild animals in the process of urbanization.
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            Use and limitations of light microscopy for diagnosing malaria at the primary health care level.

            Recent developments in diagnostic techniques for malaria, particularly DNA probes and sero-immunology, have raised questions as to how these techniques might be used to facilitate malaria diagnosis at the most peripheral levels of the primary health care system. At present, malaria diagnosis is based on the light microscope and is likely to remain so in the immediate future. This article describes how the diagnosis of malaria by light microscopy has been improved over the years, and expresses the need for further improvement while concomitant research into other standardized and simplified techniques for the diagnosis of malaria is vigorously pursued.
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              Impact of microscopy error on estimates of protective efficacy in malaria-prevention trials.

              Microscopy is an imperfect reference standard used for malaria diagnosis in clinical trials. The purpose of this study was to provide an assessment of the accuracy of basic microscopy, to compare polymerase chain reaction (PCR)-based diagnosis with microscopy results, and to assess the effect of microscopy error on apparent protective efficacy. The sensitivity and specificity of basic, compared with expert, microscopy was determined to be 91% and 71%, respectively. In a clinical trial, agreement between PCR and microscopy results improved with expert confirmation of initial results. In a simulated 12-week trial with weekly routine malaria smears, a very high specificity (>99%) for each malaria smear was found to be necessary for an estimate of protective efficacy to be within 10%-25% of the true value, but sensitivity had little effect on this estimate. Microscopy error occurs and can affect clinical trial results.
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                Author and article information

                Contributors
                +34 91 822 22 23 , pberzosa@isciii.es
                +34 91 822 22 23
                +34 91 822 22 23
                +34 91 822 22 23
                +34 91 822 22 23
                +34 91 822 22 23
                +34 91 822 22 23
                +34 91 822 22 23
                Journal
                Malar J
                Malar. J
                Malaria Journal
                BioMed Central (London )
                1475-2875
                17 September 2018
                17 September 2018
                2018
                : 17
                : 333
                Affiliations
                [1 ]ISNI 0000 0000 9314 1427, GRID grid.413448.e, Malaria Laboratory, National Centre of Tropical Medicine, , Institute of Health Carlos III, ; C/Monforte de Lemos 5, 28029 Madrid, Spain
                [2 ]Network Collaborative Research in Tropical Diseases, RICET, Madrid, Spain
                [3 ]Reference Centre for Control of Endemic Diseases (CRCE), Malabo, Equatorial Guinea
                [4 ]Ministry of Health and Social Welfare of Equatorial Guinea, Malabo, Equatorial Guinea
                [5 ]ISNI 0000000121060879, GRID grid.10041.34, Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de la Laguna, ; Tenerife, Spain
                Author information
                http://orcid.org/0000-0002-8768-717X
                Article
                2481
                10.1186/s12936-018-2481-4
                6142353
                30223852
                741731ae-8323-4b56-976f-0656c7ec99d7
                © The Author(s) 2018

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 22 February 2018
                : 10 September 2018
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100004587, Instituto de Salud Carlos III;
                Award ID: TRPY 1282/15
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2018

                Infectious disease & Microbiology
                malaria,diagnosis,microscopy,rdts,snm-pcr
                Infectious disease & Microbiology
                malaria, diagnosis, microscopy, rdts, snm-pcr

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