Author:
- Introduction
- Materials and Methods
- Clinical samples.
- Microscopic examination.
- Rapid diagnostic kit.
- DNA extraction from whole blood.
- Nested PCR assay.
- Real-time multiplex PCR.
- PlasmoNex Multiplex PCR Kit.
- Primer design.
- MSP-multiplex PCR.
- Analytical sensitivity of MSP-multiplex PCR.
- Analytical specificity of MSP-multiplex PCR.
- Clinical sensitivity and specificity.
- Statistical analysis.
- Results
- Discussion
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
World Health Organization, 2012. The World Malaria Report 2012. Geneva: World Health Organization.
-
2.
Tahar R, Boudin C, Thiery I, Bourgouin C, 2002. Immune response of Anopheles gambiae to the early sporogonic stages of the human malaria parasite Plasmodium falciparum. EMBO J 21: 6673–6680.
-
3.
Barber BE, William T, Grigg MJ, Piera K, Yeo TW, Anstey NM, 2013. Evaluation of the sensitivity of a pLDH-based and an aldolase-based rapid diagnostic test for diagnosis of uncomplicated and severe malaria caused by PCR-confirmed Plasmodium knowlesi, Plasmodium falciparum and Plasmodium vivax. J Clin Microbiol 51: 1118–1123.
-
4.
Zimmerman PA, Mehlotra RK, Kasehagen LJ, Kazura JW, 2008. Why do we need to know more about mixed Plasmodium species infections in humans? Trends Parasitol 20: 440–447.
-
5.
Mayxay M, Pukrittayakamee S, Newton PN, White NJ, 2004. Mixed-species malaria infections in humans. Trends Parasitol 20: 233–240.
-
6.
Bronzan RN, McMorrow ML, Kachur SP, 2008. Diagnosis of malaria: challenges for clinicians in endemic and non-endemic regions. Mol Diagn Ther 12: 299–306.
-
7.
O'Meara WP, Barcus M, Wongsrichanalai C, 2006. Reader technique as a source of variability in determining malaria parasite density by microscopy. Malar J 5: 118.
-
8.
Hanscheid T, 2003. Current strategies to avoid misdiagnosis of malaria. Clin Microbiol Infect 9: 497–504.
-
9.
Payne D, 1988. Use and limitations of light microscopy for diagnosing malaria at the primary health care level. Bull World Health Organ 66: 621–626.
-
10.
Singh B, Kim Sung L, Matusop A, Radhakrishnan A, Shamsull SS, Cox-Singh J, Thomas A, Conway DJ, 2004. A large focus of naturally acquired Plasmodium knowlesi infections in human beings. Lancet 363: 1017–1024.
-
11.
Shokoples SE, Ndao M, Kowalewska-Grochowska K, Yanow SK, 2009. Multiplexed real-time PCR assay for discrimination of Plasmodium species with improved sensitivity for mixed infections. J Clin Microbiol 47: 975–980.
-
12.
Mixson-Hayden T, Lucchi NW, Udhayakumar V, 2010. Evaluation of three PCR-based diagnostic assays for detecting mixed Plasmodium infection. BMC Res Notes 3: 88.
-
13.
Mens P, Spieker N, Omar S, Heijnen M, Schalling H, Kager PA, 2007. Is molecular biology the best alternative for diagnosis of malaria to microscopy? A comparison between microscopy, antigen detection and molecular tests in rural Kenya and urban Tanzania. Trop Med Int Health 12: 238–244.
-
14.
Snounou GS, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, do Rosario VE, Thaithong S, Brown KN, 1993. High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. Mol Biochem Parasitol 61: 315–320.
-
15.
Rubio JM, Benito A, Berzosa PJ, Roche J, Puente S, Subirats M, Lopez-Velez R, Garcia L, Alavar J, 1999. Usefulness of seminested multiplex PCR in surveillance of imported malaria in Spain. J Clin Microbiol 37: 3260–3264.
-
16.
Padley D, Moody AH, Chiodini PL, Saldanha J, 2003. Use of a rapid, single round, multiplex PCR to detect malarial parasites and identify the species present. Ann Trop Med Parasitol 97: 131–137.
-
17.
Reller ME, Chen WH, Dalton J, Lichay MA, Dumler JS, 2013. Multiplex 5′nuclease quantitative real-time PCR for clinical diagnosis of malaria and species level identification and epidemiologic evaluation of malaria-causing parasites, including Plasmodium knowlesi. J Clin Microbiol 51: 2931–2938.
-
18.
Singh B, Bobogare A, Cox-Singh J, Snounou G, Abdullah MS, Rahman HA, 1999. A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies. Am J Trop Med Hyg 60: 687–692.
-
19.
Cohen J, 1960. A coefficient of agreement for nominal scales. Educ Psychol Meas 20: 37–46.
-
20.
Altman DG, 1991. Practical Statistics for Medical Research. London, United Kingdom: Chapman and Hall.
-
21.
Vafa M, Troye-Blomberg M, Anchang J, Garcia A, Migot-Nabias F, 2008. Multiplicity of Plasmodium falciparum infection in asymptomatic children in Senegal: relation to transmission, age and erythrocyte variants. Malar J 7: 17.
-
22.
Bottius E, Guanzirolli A, Trape JF, Rogier C, Konate L, Druilhe P, 1996. Malaria: even more chronic in nature than previously thought; evidence for subpatent parasitaemia detectable by the polymerase chain reaction. Trans R Soc Trop Med Hyg 90: 15–19.
-
23.
Mueller I, Widmer S, Michel D, Maraga S, McNamara DT, Kiniboro B, Sie A, Smith TA, Zimmerman PA, 2009. High sensitivity dection of Plasmodium species reveals positive correlations between infections of different species, shifts in age distribution and reduced local variation in Papua New Guinea. Malar J 8: 41.
-
24.
Mueller I, Zimmerman PA, Reeder JC, 2007. Plasmodium malariae and Plasmodium ovale– the “bashful” malaria parasites. Trends Parasitol 23: 278–283.
-
25.
Han ET, Watanabe R, Sattabongkot J, Khuntirat B, Sirichaisinthop J, Iriko H, Jin L, Takeo S, Tsuboi T, 2007. Detection of four Plasmodium species by genus and species- specific loop-mediated isothermal amplification for clinical diagnosis. J Clin Microbiol 45: 2521–2528.
-
26.
Poon LL, Wong BW, Ma EH, Chan KH, Chow LM, Abeyewickreme W, Tangpukdee N, Yuen KY, Guan Y, Looareesuwan S, Pieris JS, 2006. Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clin Chem 52: 303–306.
-
27.
Aonuma H, Suzuki M, Iseki H, Perera N, Nelson B, Igarashi I, Yagi T, Kanuka H, Fukumoto S, 2008. Rapid identification of Plasmodium-carrying mosquitoes using loop-mediated isothermal amplification. Biochem Biophys Res Commun 376: 671–676.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 88 | 88 | 11 |
| Full Text Views | 509 | 146 | 1 |
| PDF Downloads | 256 | 72 | 1 |
Comparison of Three Molecular Methods for the Detection and Speciation of Five Human Plasmodium Species
Yee Ling Lau
Yee Ling Lau
Tropical Infectious Disease Research and Education Center (TIDREC), Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
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Meng Yee Lai
Meng Yee Lai
Tropical Infectious Disease Research and Education Center (TIDREC), Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
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Claudia N. Anthony
Claudia N. Anthony
Tropical Infectious Disease Research and Education Center (TIDREC), Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
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Phooi Yee Chang
Phooi Yee Chang
Tropical Infectious Disease Research and Education Center (TIDREC), Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
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Vanitha Palaeya
Vanitha Palaeya
Tropical Infectious Disease Research and Education Center (TIDREC), Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
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Mun Yik Fong
Mun Yik Fong
Tropical Infectious Disease Research and Education Center (TIDREC), Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
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Rohela Mahmud
Rohela Mahmud
Tropical Infectious Disease Research and Education Center (TIDREC), Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
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In this study, three molecular assays (real-time multiplex polymerase chain reaction [PCR], merozoite surface antigen gene [MSP]-multiplex PCR, and the PlasmoNex Multiplex PCR Kit) have been developed for diagnosis of Plasmodium species. In total, 52 microscopy-positive and 20 malaria-negative samples were used in this study. We found that real-time multiplex PCR was the most sensitive for detecting P. falciparum and P. knowlesi. The MSP-multiplex PCR assay and the PlasmoNex Multiplex PCR Kit were equally sensitive for diagnosing P. knowlesi infection, whereas the PlasmoNex Multiplex PCR Kit and real-time multiplex PCR showed similar sensitivity for detecting P. vivax. The three molecular assays displayed 100% specificity for detecting malaria samples. We observed no significant differences between MSP-multiplex PCR and the PlasmoNex multiplex PCR kit (McNemar's test: P = 0.1489). However, significant differences were observed comparing real-time multiplex PCR with the PlasmoNex Multiplex PCR Kit (McNemar's test: P = 0.0044) or real-time multiplex PCR with MSP-multiplex PCR (McNemar's test: P = 0.0012).
Author Notes
Financial support: The study was funded by High-Impact Research Grant UM-MOHE UM.C/625/1/HIR/MOHE/MED/16 and UM.C/625/1/HIR/MOHE/CHAN/14/3 from the Ministry of Higher Education Malaysia and University Malaya Research Grant RG233/10HTM.
Authors' addresses: Yee Ling Lau, Meng Yee Lai, Claudia N. Anthony, Phooi Yee Chang, Vanitha Palaeya, Mun Yik Fong, and Rohela Mahmud, Tropical Infectious Disease Research and Education Center (TIDREC), Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia, E-mails: lauyeeling@um.edu.my, mengylai11@yahoo.com, claudia.anthony88@gmail.com, phooiyee@gmail.com, caprirox@yahoo.com, fongny@um.edu.my, and rohela@ummc.edu.my.
- Introduction
- Materials and Methods
- Clinical samples.
- Microscopic examination.
- Rapid diagnostic kit.
- DNA extraction from whole blood.
- Nested PCR assay.
- Real-time multiplex PCR.
- PlasmoNex Multiplex PCR Kit.
- Primer design.
- MSP-multiplex PCR.
- Analytical sensitivity of MSP-multiplex PCR.
- Analytical specificity of MSP-multiplex PCR.
- Clinical sensitivity and specificity.
- Statistical analysis.
- Results
- Discussion
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
World Health Organization, 2012. The World Malaria Report 2012. Geneva: World Health Organization.
-
2.
Tahar R, Boudin C, Thiery I, Bourgouin C, 2002. Immune response of Anopheles gambiae to the early sporogonic stages of the human malaria parasite Plasmodium falciparum. EMBO J 21: 6673–6680.
-
3.
Barber BE, William T, Grigg MJ, Piera K, Yeo TW, Anstey NM, 2013. Evaluation of the sensitivity of a pLDH-based and an aldolase-based rapid diagnostic test for diagnosis of uncomplicated and severe malaria caused by PCR-confirmed Plasmodium knowlesi, Plasmodium falciparum and Plasmodium vivax. J Clin Microbiol 51: 1118–1123.
-
4.
Zimmerman PA, Mehlotra RK, Kasehagen LJ, Kazura JW, 2008. Why do we need to know more about mixed Plasmodium species infections in humans? Trends Parasitol 20: 440–447.
-
5.
Mayxay M, Pukrittayakamee S, Newton PN, White NJ, 2004. Mixed-species malaria infections in humans. Trends Parasitol 20: 233–240.
-
6.
Bronzan RN, McMorrow ML, Kachur SP, 2008. Diagnosis of malaria: challenges for clinicians in endemic and non-endemic regions. Mol Diagn Ther 12: 299–306.
-
7.
O'Meara WP, Barcus M, Wongsrichanalai C, 2006. Reader technique as a source of variability in determining malaria parasite density by microscopy. Malar J 5: 118.
-
8.
Hanscheid T, 2003. Current strategies to avoid misdiagnosis of malaria. Clin Microbiol Infect 9: 497–504.
-
9.
Payne D, 1988. Use and limitations of light microscopy for diagnosing malaria at the primary health care level. Bull World Health Organ 66: 621–626.
-
10.
Singh B, Kim Sung L, Matusop A, Radhakrishnan A, Shamsull SS, Cox-Singh J, Thomas A, Conway DJ, 2004. A large focus of naturally acquired Plasmodium knowlesi infections in human beings. Lancet 363: 1017–1024.
-
11.
Shokoples SE, Ndao M, Kowalewska-Grochowska K, Yanow SK, 2009. Multiplexed real-time PCR assay for discrimination of Plasmodium species with improved sensitivity for mixed infections. J Clin Microbiol 47: 975–980.
-
12.
Mixson-Hayden T, Lucchi NW, Udhayakumar V, 2010. Evaluation of three PCR-based diagnostic assays for detecting mixed Plasmodium infection. BMC Res Notes 3: 88.
-
13.
Mens P, Spieker N, Omar S, Heijnen M, Schalling H, Kager PA, 2007. Is molecular biology the best alternative for diagnosis of malaria to microscopy? A comparison between microscopy, antigen detection and molecular tests in rural Kenya and urban Tanzania. Trop Med Int Health 12: 238–244.
-
14.
Snounou GS, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, do Rosario VE, Thaithong S, Brown KN, 1993. High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. Mol Biochem Parasitol 61: 315–320.
-
15.
Rubio JM, Benito A, Berzosa PJ, Roche J, Puente S, Subirats M, Lopez-Velez R, Garcia L, Alavar J, 1999. Usefulness of seminested multiplex PCR in surveillance of imported malaria in Spain. J Clin Microbiol 37: 3260–3264.
-
16.
Padley D, Moody AH, Chiodini PL, Saldanha J, 2003. Use of a rapid, single round, multiplex PCR to detect malarial parasites and identify the species present. Ann Trop Med Parasitol 97: 131–137.
-
17.
Reller ME, Chen WH, Dalton J, Lichay MA, Dumler JS, 2013. Multiplex 5′nuclease quantitative real-time PCR for clinical diagnosis of malaria and species level identification and epidemiologic evaluation of malaria-causing parasites, including Plasmodium knowlesi. J Clin Microbiol 51: 2931–2938.
-
18.
Singh B, Bobogare A, Cox-Singh J, Snounou G, Abdullah MS, Rahman HA, 1999. A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies. Am J Trop Med Hyg 60: 687–692.
-
19.
Cohen J, 1960. A coefficient of agreement for nominal scales. Educ Psychol Meas 20: 37–46.
-
20.
Altman DG, 1991. Practical Statistics for Medical Research. London, United Kingdom: Chapman and Hall.
-
21.
Vafa M, Troye-Blomberg M, Anchang J, Garcia A, Migot-Nabias F, 2008. Multiplicity of Plasmodium falciparum infection in asymptomatic children in Senegal: relation to transmission, age and erythrocyte variants. Malar J 7: 17.
-
22.
Bottius E, Guanzirolli A, Trape JF, Rogier C, Konate L, Druilhe P, 1996. Malaria: even more chronic in nature than previously thought; evidence for subpatent parasitaemia detectable by the polymerase chain reaction. Trans R Soc Trop Med Hyg 90: 15–19.
-
23.
Mueller I, Widmer S, Michel D, Maraga S, McNamara DT, Kiniboro B, Sie A, Smith TA, Zimmerman PA, 2009. High sensitivity dection of Plasmodium species reveals positive correlations between infections of different species, shifts in age distribution and reduced local variation in Papua New Guinea. Malar J 8: 41.
-
24.
Mueller I, Zimmerman PA, Reeder JC, 2007. Plasmodium malariae and Plasmodium ovale– the “bashful” malaria parasites. Trends Parasitol 23: 278–283.
-
25.
Han ET, Watanabe R, Sattabongkot J, Khuntirat B, Sirichaisinthop J, Iriko H, Jin L, Takeo S, Tsuboi T, 2007. Detection of four Plasmodium species by genus and species- specific loop-mediated isothermal amplification for clinical diagnosis. J Clin Microbiol 45: 2521–2528.
-
26.
Poon LL, Wong BW, Ma EH, Chan KH, Chow LM, Abeyewickreme W, Tangpukdee N, Yuen KY, Guan Y, Looareesuwan S, Pieris JS, 2006. Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clin Chem 52: 303–306.
-
27.
Aonuma H, Suzuki M, Iseki H, Perera N, Nelson B, Igarashi I, Yagi T, Kanuka H, Fukumoto S, 2008. Rapid identification of Plasmodium-carrying mosquitoes using loop-mediated isothermal amplification. Biochem Biophys Res Commun 376: 671–676.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 88 | 88 | 11 |
| Full Text Views | 509 | 146 | 1 |
| PDF Downloads | 256 | 72 | 1 |