Infectious Disease Reports 2020; volume 12(s1):8731

Performance comparison of higher (100 %) when using real-time PCR. two malaria rapid diagnostic Both RDTs showed a 100% agreement. RT- Correspondence: Puspa Wardhani, PCR detected higher mix infection when Department of Clinical Pathology, Faculty of test with real time polymerase compared to microscopy and RDTs. Medicine, Universitas Airlangga/Dr. Soetomo. chain reaction and gold Conclusion: RightSign and ScreenPlus General Academic Hospital, Jl. Prof. Dr. standard of microscopy RDT have excellent performance when Moestopo No. 47, 60132, Surabaya, using microscopy detection as a gold stan- Indonesia. detection method dard. Real-time PCR method can be consid- Tel: +628123176937 E-mail: [email protected], ered as a confirmation tool for malaria diag- 1,5 Puspa Wardhani, Trieva Verawaty nosis. Key words: Malaria, rapid diagnostic test, 2,5 Butarbutar, Christophorus Oetama real-time polymerase chain reaction, Adiatmaja,2,5 Amarensi Milka microscopy detection. Betaubun,3,5 Nur Hamidah,4 Aryati1,5 Contributions: Conceptualization: PW. Data 1Clinical Pathology Department; Introduction curation: PW TVB COA AMB. Formal analy- 2 Indonesia is a malaria endemic area. Clinical Pathology Specialist Study sis: PW AA NH. Funding: KEMENRIS- Programme, Clinical Pathology The province of Papua has Indonesia’s TEKDIKTI RI. Investigation: PW PW TVB Department; 3Clinical Pathology highest malaria burden. All Plasmodium COA AMB, NH, AA. Methodology: PW AA. Subspecialist Study Programme, species are present in Papua, including the Project administration: PW TVB AMB COA Plasmodium knowlesi (P. knowlesi) Clinical Pathology Department, Faculty Resources: PW AA NH. Supervision: PW AA originally discovered on Kalimantan Island. NH. Validation: PW AA. Writing–original of Medicine; 4Science and Technology The most common types of Plasmodium draft, review & editing: PW. Faculty, C Campus, Universitas 5 infection in Papua are Plasmodium Airlangga; Dr. Soetomo General falciparum (P. falciparum) and Plasmodium Conflicts of interest: The authors declare no Academic Hospital, Surabaya, Indonesia vivax (P. vivax). Plasmodium ovale (P. potential conflict of interest. ovale) and Plasmodium malariae (P. only malariae) also can be found in Papua. The Funding: The work was supported by Simlitabmas Programme by Kementretian highest cause of morbidity and mortality in 1,2 Riset Teknologi dan Pendidikan Tinggi Abstract Papua is P. falciparum. Republik Indonesia (KEMENRISTEKDIKTI Several methods for diagnosing malariauseRI), contract number: 544/UN3.14/LT/2019. Background: The diagnostic test for have been established since WHO has malaria is mostly based on Rapid confirmed how important new tests are to Acknowledgements: The authors acknowl- Diagnostic Test (RDT) and detection by diagnose Plasmodium spp rapidly, reliably, edge the funding research support of microscopy. Polymerase Chain Reaction accurately and cheaply, to address Simlitabmas programme and Merauke (PCR) is also a sensitive detection method numerous shortcomings in microscopic Hospital, Papua for sampling place support, that can be considered as a diagnostic tool. examination as the WHO’s gold standard Parasitology department Faculty of Medicine, The outcome of malaria microscopy detec- for malaria research.3,4 Brawijaya University for consultation in labo- tion depends on the examiner’s ability and Microscopic examination has ratory method. experience. Some RDT has been distributed advantages, namely allowing to identify Conference presentation: The paper had been in Indonesia, which needs to be evaluated species definitively, determine the condition presented at INSBIOMM International confer- for their results. of parasitemia, monitor malaria treatment ence on latest perspectives on Infectious Objective: This study aimed to compare response, easy execution and inexpensive3,5. the performance of RightSign RDT and Diseases, including Biotreats and Military However, the weakness of microscopy Medicine, in August 27-28th, 2019, Surabaya, ScreenPlus RDT for detection of examination is the difficulties in detecting INDONESIA. Plasmodium in human blood. We used spe- extrimely low parasitemia and mix cific real-time polymerase chain reaction infections, and it is time consuming. Received for publication: 17 February 2020. abTESTMMalaria qPCRII) and goldNon-commercial stan- Although microscopy examination is gold Accepted for publication: 1 July 2020. dard of microscopy detection method to standard in malaria diagnosis, it is better to measure diagnostic efficiency. be accompanied by RDTs or other This work is licensed under a Creative Methods: Blood specimens were evalu- methods.3,5 Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). ated using RightSign RDT, ScreenPlus Other testing methods have been RDT, Microscopy detection, and RT-PCR developed to diagnose malaria are the based ©Copyright: the Author(s), 2020 as the protocol described. The differences on proteins produced by Plasmodium, such Licensee PAGEPress, Italy on specificity (Sp), sensitivity (Sn), positive as Histidine-Rich Protein 2 (HRP-2) or Infectious Disease Reports 2020; 12(s1):8731 predictive value (PPV), and negative pre- enzymes such as Pan-Plasmodium Lactate doi:10.4081/idr.2020.8731 dictive value (NPV) were analyzed using Dehydrogenase (pLDH) and Pan-Specific McNemar and Kruskal Wallis analysis. Aldolase. This method has been widely Results: A total of 105 subjects were used and is considered an alternative to recruited. Based on microscopy test, malaria testing. This examination is also examination tools generally use serological RightSign RDT had sensitivity, Specificity, very prominent, especially in areas where techniques to detect antigens, such as Rapid PPV, NPV, 100%, 98%, 98.2%, 100%, microscopic examination is not available, or Diagnostic Tests (RDTs) or respectively. ScreenPlus showed 100% it is available but there is no laboratory staff Immunochromatography Test (ICT) and sensitivity, 98% specificity, 98.2% PPV, experienced in the examination of blood or Enzyme Linked Immunosorbent Assay 100% NPV. The sensitivity of both RDTs some other disadvantage of microscopy (ELISA)4,6,7. became lower (75%) and the specificity examination. Commercially enzyme-based The RDTs or ICT detect malaria

[page 56] [Infectious Disease Reports 2020; 12(s1):8731] Article antigens when the blood of the patient Sample collection results was carried out by two blind and passes through a membrane containing This study was a cross-sectional analyt- independent observers. Based on the Plasmodium-specific antibodies. The ic study conducted at Merauke Hospital, McNemar test between the two observers’ benefits of RDT are reliability in testing, Papua and Clinical Pathology Laboratory, results, the results were not significantly quick processing times and tests, and cheap Faculty of Medicine, Universitas Airlangga different with P<0.05. review fees, which is why it is very useful /Dr. Soetomo General Academic Hospital in endemic areas. The most commonly used Surabaya during November 2018-June Real Time PCR RDTs are methods of detection based on 2019. A total of 105 whole blood samples DNA examination of Plasmodium HRP2 and pLDH. The advantages of RDTs were collected in tubes containing Ethylene malaria using the Rotor-Gene® Q PCR are their parasitic detection limit of 50-100 Diamine Tetraacetic Acid (EDTA). from Qiagen, Tokyo, Japan with the parasites/μL. The disadvantage of RDT are abTESTMMalaria qPCR III reagent kit. reduced effectiveness and efficiency in Species identification and determi- AbTESTMMalaria qPCR III (AITbiotech examining large samples, because time nation of parasite density Pte Ltd, Singapore) can identify four consuming and a high level of Identification of Plasmodium species species of Plasmodium spp. (P. falciparum, concentration and accuracy are required.3, 8 and calculation of the parasitemia index P. vivax, P. ovale, P. malariae, P.knowlesi). Most RDTs examine two proteins at (PI) were performed microscopically on There are two main steps: DNA extraction once, such as a combination of a specific Giemsa-stained thick and thin blood from blood samples and amplification of protein P. falciparum (HRP-2) with pLDH, smears. PI was calculated using WHO DNA extracts using primers pairs and a protein that is shared by all species of guidelines Simultaneously the samples probes that hydrolyze double-dye (double- Plasmodium (non-specific) pLDH) or HRP- were using RightSign RDT and ScreenPlus dye hydrolysis probes) which are very 2 combination comnined with a species RDT.12 specific. Double-dye hydrolysis probes are specific protein species of Plasmodium fluorescent substances that are able to emit vivax (Pv-pLDH). Three proteins also can RightSign RDT and ScreenPlus light, then the light will be captured by be detected at once, such as HRP-2, non- RDT optical detectors on the Rotor-Gene® Q device, which results in an increase in specific pLDH and Pv-pLDH that can The positivity and antigen detection of only signal in the wave graph. This probe distinguish between falciparum malaria and the Plasmodium species in RightSign RDT 9 attaches to the primers of each Plasmodium non-falciparum malaria or mixed malaria. were obtained through lines or bands that species, which are compatible with the RightSign and ScreenPlus are available arise in the line test. RightSign uses the Rotor-Gene® Q optical detector channel, comercially in Indonesia. RightSign detects immunochromatography test (ICT)use method, each consisting of FAM (compatible with P. falciparum (HRP-2) and Pv-pLDH. with nitrocellulose membranes detects P. Green channels), HEX (compatible with ScreenPlus detecs P. falciparum (HRP-2) falciparum-specific anti-Histidine Rich Yellow channels), ROX (compatible with and Pan LDH. Both RDTs have not been Protein II (HRP II) on the Pf test line and Orange channels ) and Cy5 (compatible evaluated in field study in Indonesia. non-specific anti-Plasmodium Lactate with Red channel), Quasar 705 (compatible Molecular-based Polymerase Chain Dehydrogenase (pLDH) from Plasmodium with Crimson channel), VIC, TAMRA, Reaction (PCR) techniques have used for spp (P. falciparum, P. vivax, P. malariae, P. TEXAS RED. years in detecting Plasmodium spp. A study ovale) on the Pan test line. ScreenPlus on Polymerase Chain Reaction (PCR) even detected P. vivax specific anti-Plasmodium said that the sensitivity and specificity of Lactate Dehydrogenase (pLDH) antibody on the Pv test line and P. falciparum-specif- PCR are higher than microscopic Results examination. PCR has a high sensitivity and ic anti-Histidine Rich Protein II (HRP II) specificity, but has several disadvantages: it antibodies. The differences on specificity Acquisition of samples is a fairly complex examination method, it (Sp), sensitivity (Sn), positive predictive As many as 105 samples were obtained, is expensive, and requires expertise to be value (PPV), and negative predictive value which consisted of 67/105 (63.8%) were performed.3,10,11 (NPV) were analyzed using McNemar and male and 38/105 (36.2%) were female. The This study aimed to compareNon-commercial the per- Kruskal Wallis analysis. average of age of patients was 31.19 years formance of RightSign RDT and old. Median and long IQR fever obtained ScreenPlus RDT for detection Plasmodium Results confirmation from 28 malaria patients who had a history in human blood. The difference perform- Results confirmation of thick and thin of fever were five days (2-60) Microscopy ance of RDTs malaria compared to blood smears examination was carried out examination resulted in 54 (51.42%) out of microscopy detection, and Real-Time in the Clinical Pathology Laboratory 105 samples were positive of Plasmodium, Polymerase Chain Reaction (Real Time Faculty of Medicine, Universitas Airlangga and 51 (48.58%) were negative. (RT)-PCR abTESTMMalaria qPCRII). and Dr. Soetomo general academic hospital, Examination using RightSign and Surabaya. The inclusion criteria for sample ScreenPlus resulted in 51.42% were posi- acceptance were patients of all ages, males tive (Table 1). and females, clinical examinations showed Materials and Methods symptoms of fever (specific or non-specific Parasitemia index malaria) and who were willing to take part Out of 54 positive samples, 34 (63%) Ethical approval in the study by signing an informed consent were P. falciparum, 17(31.5%) were P. Ethical approval number form. The exclusion criteria were patients vivax and 3 (5.5%) were mix infections of 169/EC/KEPK/FKUA/2019 was obtained who have received malaria treatment and both species. Based on parasite density, the from the Health Research Ethics Committee patients with fever but with negative results highest parasitemia index was P. vivax of the Faculty of Medicine, Universitas of malaria microscopy examination. The which in range of 1000-10,000/µL, fol- Airlangga. interpretation of microscopy and RDTs lowed by P. falciparum of 1000-10,000/µL

[Infectious Disease Reports 2020; 12(s1):8731] [page 57] Article and 10,001-200,000/µL, while the mixed Table 1. Basic characteristics of subjects and study samples. infection was evenly distributed as <1000, Variable (n) Total % 1000- 10,000 and 10,001-200,000/µL. In this study, a high parasitemia index Total malaria 105 52.4 was obtained with each dominance of 1000- Positive microscopy test 54/105 51.42 10,000 parasites/µL was 41.2% in P.falci- Negative microscopy test 51/105 48.58 Positive rightsign test 54/105 51.42 parum and 52.9% in P.vivax, while the par- Positive screenplus test 54/105 51.42 asitemia index<1000 parasites/µL was 5.9% in P. falciparum and P. vivax (Table 1). Parasitemia index P. vivax per µL blood* 34/54 63 <1000 2/34 5.9 1000-10,000 18/34 52.9 The negative samples 10,001-200,000 14/34 41.2 Fifty negative Plasmodium based on >200,000 0/34 0 RDT examination, consisted of 25 (50%) Parasitemia index P. falciparum per µL blood* 17/54 31.5 samples with Dengue fever, 4 (8%) samples <1000 1/17 5.9 with urinary tract infections, 3 (6%) samples 1000-10,000 7/17 41.2 with pneumonia, and 4 (8%) samples with 10,001-200,000 7/17 41.2 local infection, 3 (6%) samples with sepsis >200,000 2/17 11.7 and 11 (22%) samples with hepatitis B. Parasitemia index mixed (P. falciparum & P. vivax) per µL blood* 3/54 5.5 <1000 Comparison of microscopy, two 1000-10,000 1/3 33.3 RDTs and RT-PCR 10,001-200,000 1/3 33.3 The diagnostic value of Right Sign and >200,000 1/3 33,3 ScreenPlus against microscopy consisted Age (Mean±SD) years 0/3 0 Sn = 100% and Sp = 98%, PPV = 98, 2%, Gender 31.19 ± 17.97 - NPV = 100% (Table 2). The results of the Male only 67/105 63.8 sensitivity and specificity of both tests on Female 38/105 36.2 gold standard microscopy in this study Day of fever (Median (IQR)) days (n=28) 5 (2-60) - obtained high, namely 100% and 98%. The results showed a good compatibility use between RightSign, ScreenPlus and microscopy. The diagnostic value of Table 2 Diagnostic performance of RightSign and ScreenPlus compared to microscopic RightSign and ScreenPlus against Real- and real-time PCR Examination. Time PCR Sn = 75.3 % and Sp = 100 %, PPV = 100%, NPV = 64% (Table 2). Diagnostic performance RightSign ScreenPlus McNemar test showed that no signifi- Microscopic RT-PCR Microscopic RT-PCR cance different between RightSign with Sn (%) (CI 95%) 100 75.3 100 75.3 microscopic examination with P=1. There Sp (%) (CI 95%) 98 100 98 100 was a significant difference between PPV (%) (CI 95%) 98.2 100 98.2 100 RightSign and RT-PCR Results (Tables 3 and 4). Table 5 shows the variety of NPV (%) (CI 95%) 100 64 100 64 Plasmodium species detected by RightSign vs. microscopic and Table 6 showed the dis- tribution of Plasmodium species against RT- PCR. RT-PCR detected more mix infection Table 3. Comparison of RightSign vs. microscopic examination. compared to microscopic, 24.8% vs. 2.9 %. Tables 7 and 8 shows the comparisonNon-commercial of Right Sign Microscopic results Total P-value* the species of Plasmodium detected by Positive Negative ScreenPlus, microscopic and RT-PCR. The Positive 54 (98.2%) 1 (1.8%) 55 (100%) 1.000 percentage was the same as the results of Negative 0 (0%) 50 (100%) 50 (100%) RightSign. McNemar statistical analysis on the performance differences between Total 54 (51.4%) 51 (48.6%) 105 (100%) RightSign and ScreenPlus showed that *Statistical analysis using McNemar test. N =105, significance at P<0.05. there was no significant difference with P>0.05 (Tables 9 and 10). Mann Whitney analysis revealed no significant difference on parasitemia index between ScreenPlus Table 4. Comparison of RightSign vs. PCR results. and RightSign (P>0.05). Right Sign PCR Results Total P-value* Positive Negative Positive 55 (100%) 0 (0%) 55 (100%) < 0,001 Discussion Negative 18 (36%) 32 (64%) 50 (100%) The location for the sampling popula- Total 73 (69.5%) 32 (30.5%) 105 (100%) tion in this study was Merauke, one of the * Statistical analysis using McNemar test. N =105, significance at P<0.05 .

[page 58] [Infectious Disease Reports 2020; 12(s1):8731] Article districts in the Papua Province. Riskesdas in Table 5. Comparison of species detection between RightSign and microscopic. 2013 stated that Papua Province was one of Right Sign Microscopic Total the provinces with the highest malaria bur- Negative P. falciparum P. vivax Mix den in Indonesia with Annual Parasites Incidence (API) of 45.85% in 2016. High Negative 50 (100%) 0 (0%) 0 (0%) 0 (0%) 50 (100%) malaria burden areas will affect the back- P. falciparum 1 (5.6%) 16 (88.9%) 0 (0%) 1 (5.6%) 18 (100%) ground of malaria exposure in the popula- Pan 0 (0%) 1 (2.7%) 34 (91.9%) 2 (5.4%) 37 (100%) tion, thus providing higher parasitic densi- Total 51 (48.6%) 17 (16.2%) 34 (32.4%) 3 (2.9%) 105 (100%) ties compared to non-endemic regional pop- ulations.4 Parasitic density will produce a high antigenemia protein resulting in high positivity in the detection of RDT protein Table 6. Comparison of species detection between RightSign and RT-PCR. antigens, as well as the positivity of Right Sign RT-PCR Total Plasmodium detection by microscopy. Negative P. falciparum P. vivax Mix RDT sensitivity was influenced by var- ious factors which included location and Negative 32 (64%) 3 (6%) 10 (20%) 5 (10%) 50 (100%) population sampling, antigenemia protein in P. falciparum 0 (0%) 5 (27.8%) 0 (0%) 13 (72.2%) 18 (100%) the sample itself and optimal temperature Pan 0 (0%) 0 (0%) 29 (78.4%) 8 (21.6%) 37 (100%) 13, 14 stability of the reagent kit. Total 32 (30.5%) 8 (7.6%) 39 (37.1%) 26 (24.8%) 105 (100%) Optimal temperature stability influ- ences Rapid diagnostic test (RDT) sensitiv- ity (4-30ºC). If the optimal temperature is Table 7. Comparison of species detection between ScreenPlus and microscopic results. stable, during delivery transportation or during examination, the RDT performance ScreenPlus Microscopic examination Total will run well and provide valid results.14 In Negative P. falciparum P.only vivax Mix this study, the temperature stability of the Negative 50 (100%) 0 (0%) 0 (0%) 0 (0%) 50 (100%) reagent kit was maintained at 4-30oC with the storage of reagent kits carried out in the P. falciparum 1 (5.9%) 17 (94.4%) 0 (0%) 0 (0%) 18 (100%) refrigerator before being used. RDT sensi- P. vivax 0 (0%) 1 (2.7%) 34 (91.9%) 2 (5.4%) 37 (100%) tivity was also influenced by the suitability Total 51 (48.6%) 18 (16.2%)use 34 (32.4%) 3 (2.9%) 105 (100%) between observers in reading RDT results. Two observer concordance was analyzed by the McNemmar test with P-value>0.05 and no significant difference was found in the Table 8. Comparison of species detection between ScreenPlus and RT-PCR. results. Arum I et al. showed similar results ScreenPlus PCR Results Total of RDT with this study. The diagnostic Negative P. falciparum P. vivax Mix value of RDT against microscopy with Sn 100%, Sp 96.99%, PPV 83.2%, and NPV Negative 32 (64%) 3 (6%) 10 (20%) 5 (10%) 50 (100%) 100%.15 P. falciparum 0 (0%) 5 (29.4%) 0 (0%) 13 (72.2%) 18 (100%) One false positive result of P. falci- P. vivax 0 (0%) 0 (0%) 29 (78.4%) 8 (21.6%) 37 (100%) parum in RightSign and ScreenPlus in this Total 32 (30.5%) 8 (7.6%) 39 (37.1%) 26 (24.8%) 105 (100%) study could be attributed to protein antigen- emia HRP-2 malaria parasite that could still be identified in the blood of the patient for up to 30 days after antimalarial therapy due Table 9. Comparison of ScreenPlus and RightSign. to slow clearance of HRP-2 inNon-commercial the blood. Another reason is the presence of gameto- RightSign results Screen plus results Total P-value* cytes in the blood following antimalarial Positive Negative therapy. Gametocytes in the blood continue Positive 55 (100%) 0 (0%) 55 (100%) 1,000 to produce all three antigenemia proteins Negative 0 (0%) 50 (100%) 50 (100%) (HRP-2, p-LDH and aldolase). Antigenemia p-LDH and aldolase proteins have faster Total 55 (52,4%) 50 (47,6%) 105 (100%) clearance time from the blood after anti- *Statistical analysis using McNemar test. N =105, significance at P<0.05. malarial therapy.16,17 Rheumatoid factor and heterophilic antibodies in the patient’s blood are other cause of false positivity in Table 10. Comparison of species detection between ScreenPlus and RightSign the RDT.18 RightSign Results ScreenPlus Results Total The results of the comparison between Negative P. falciparum P. vivax RDTs and gold standard microscopy exam- ination were not statistically significantly Negative 50 (100%) 0 (0%) 0 (0%) 50 (100%) different. The examiners involved in the P. falciparum 0 (0%) 18 (94.4%) 0 (0%) 18 (100%) microscopy examination may have influ- Pan 0 (0%) 0 (0%) 37 (100%) 37 (100%) enced the results, however, the trained and Total 50 (47.6%) 17 (16.2%) 37 (35.2%) 105 (100%) certified malaria microscopy examiner

[Infectious Disease Reports 2020; 12(s1):8731] [page 59] Article should produce a true and reliable 12. World Health Organisation. Malaria results.10,12 Conformity between the results References microscopy quality assurance manual – of RDTs and microscopy was also insepara- 1. Surjadjaja C, Surya A, Baird JK. Ver. 2. Who. 2016;140. ble from the density of parasitemia. The Epidemiology of Plasmodium vivax in 13. Boyle MJ, Wilson DW, Beeson JG. population of malaria species in this study Indonesia. Am J Trop Med Hyg. 2016; New approaches to studying was dominated by P. vivax and then fol- 95:121–32. Plasmodium falciparum merozoite lowed by P. falciparum, so that although 2. WHO. Indonesia South-East Asia invasion and insights into invasion biol- there was a high parasitemia, but because of Region - Malaria Profile. 2018; ogy. Int J Parasitol 2013:43:1–10. the dominance of malaria species occupied Available from: https://www.who.int/ 14. Ashley EA, Touabi M, Ahrer M, et al. by P. vivax, the chance to produce false pos- malaria/publications/country- Evaluation of three parasite lactate itives in pLDH was small. Patients with profiles/profile_idn_en.pdf?ua=1 dehydrogenase-based rapid diagnostic high P. falciparum parasitemia could give 3.Tangpukdee N, Duangdee C, tests for the diagnosis of falciparum and false positive results in pLDH and end with Wilairatana P, Krudsood S. Malaria vivax malaria. Malar J 2009;8:1–12. high P. vivax findings on RDT diagnosis : A brief review. 2009;47:93– 15. Arum IL, Purwanto AP, Arfi S, et al. Uji examination.19 The sensitivity of RightSign 102. diagnostik Plasmodium malaria meng- and ScreenPlus to RT-PCR was lower than 4. Global WHO, Programme M. World the sensitivity to microscopy as the gold gunakan metode imunokromatografi Health Organization. Good practices for diperbandingkan dengan pemeriksaan standard for malaria research, but the selecting and procuring rapid diagnostic specificity is higher. A research in Flores mikroskopis. Indones J Clin Pathol Med tests for malaria. 2011: pp.1–71 Lab 2018;12:118. reported that real-time PCR found that RT 5. Joanny F, Löhr SJ, Engleitner T, et al. 16. Hawkes M, Katsuva JP, Masumbuko PCR was able to detect almost 8 times more Limit of blank and limit of detection of CK. Use and limitations of malaria cases of Plasmodium infection compared to Plasmodium falciparum thick blood rapid diagnostic testing by community microscopic examination as the gold smear microscopy in a routine setting in health workers in war-torn Democratic standard for malaria testing. This is due to Central Africa Malar J 2014;13:1–7. the ability of RT PCR to detect 6. Balaghaleh MR, Zarean M, Aghaee Republiconly of Congo. Malar J 2009;8:1– submicroscopic infections, with or without MA, et al. Comparison of PfHRP-2 / 8. clinical malaria, that are not detected 17. Mogeni P, Williams TN, Omedo I et al. 10,20 pLDH RDTs with Light Microscopy in microscopically. This condition may be a Low Prevalence Setting in Detecting malaria hotspots : A compari- due to the ability of RT PCR to detect DNA Southeastern Iran, Sistan use and son of rapid diagnostic test, microscopy, fragments of malaria parasites when Baluchestan: Due to Implementation of and polymerase chain reaction. J Infect microscopically detecting the existence of Dis. 2017;216:1091–8. 21 Malaria Elimination Program. 2018; Plasmodium malaria. 5:1–7. 18. Wongsrichanalai C, Barcus MJ, Muth S, Molecular methods are universally 7. Moody, A. Rapid diagnostic tests for et al. A review of malaria diagnostic accepted to be more sensitive than malaria parasites. Clin Microbiol Rev tools: Microscopy and rapid diagnostic microscopy, however PCR (multiplex PCR, 2002;15:66–78. test (RDT). Am J Trop Med Hyg real time PCR, or conventional) requires 8. Berzosa P, De Lucio A, Romay-Barja 2007;77:119–27. more sophisticated laboratories, trained M, et al. Comparison of three diagnostic 19. Harris I, Sharrock WW, Bain LM, et al. personnel, longer times, and higher costs methods (microscopy, RDT, and PCR) A large proportion of asymptomatic because PCR examination is not to be a for the detection of malaria parasites in Plasmodium infections with low and routine examination of malaria. Malaria representative samples from Equatorial sub-microscopic parasite densities in examination results can be more accurate if Guinea 11 Medical and Health Sciences PCR is used as a reference standard for the low transmission setting of Temotu 1108 Medical Microbiology. Malar J Province, Solomon Islands: Challenges malaria testing because PCR method is able 2018;17:1–12. Available from: to detect parasitemia below the microscopic for malaria diagnostics in an elimina- https://doi.org/10.1186/s12936-018- limit. tion setting. Malar J 2010;9:1–8. 2481-4 20. Khairnar K, Martin D, Lau R, et al. 9. WHO. Evaluations P. Recommended Non-commercial Multiplex real-time quantitative PCR, selection criteria for procurement of microscopy and rapid diagnostic Conclusions malaria rapid diagnostic tests. 2018. immuno-chromatographic tests for the RightSign and ScreenPlus RDT have a 10. Han TZ, Han KT, Aye KH, et al. detection of Plasmodium spp: very good performance. The analysis of Comparison of microscopy and PCR for Performance, limit of detection analysis Plasmodium antigen detection by the detection of human Plasmodium RightSign, ScreenPlus, and microscopy species and Plasmodium knowlesi in and quality assurance. Malar J 2009; research was not significantly different. southern Myanmar. Asian Pac J Trop 8:1–17. Further research is needed to find out the Biomed 2017;7:680–5. Available from: 21. Fransisca L, Kusnanto JH, Satoto TBT, diagnostic value of non-falciparum and http://dx.doi.org/10.1016/j.apjtb.2017.0 et al. Comparison of rapid diagnostic non-vivax Plasmodium in RightSign and 6.004 test Plasmotec Malaria-3, microscopy, ScreenPlus. Real time-PCR detected higher 11. Nakatsu M, Kato Y, Komaki-yasuda K, and quantitative real-time PCR for diag- mix infection when compared to Perpe J. A novel PCR-based system for noses of Plasmodium falciparum and microscopy and RDTs, therefore real-time the detection of four species of human Plasmodium vivax infections in PCR method can be considered as an effec- malaria parasites and Plasmodium Mimika Regency, Papua, Indonesia. tive confirmation tool for malaria diagnosis. knowlesi 2018;1–15. 2015;1–11.

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