Acta Tropica 136 (2014) 44–49
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Detection of Plasmodium vivax and Plasmodium falciparum DNA in human saliva and urine: Loop-mediated isothermal amplification for malaria diagnosis Zahra Ghayour Najafabadi a , Hormozd Oormazdi a , Lame Akhlaghi a , Ahmad Reza Meamar a , Mehdi Nateghpour b , Leila Farivar b , Elham Razmjou a,∗ a b
Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 1 November 2013 Received in revised form 29 March 2014 Accepted 31 March 2014 Available online 8 April 2014 Keywords: Plasmodium vivax P. falciparum Malaria diagnosis Saliva Urine LAMP
a b s t r a c t This study investigated loop-mediated isothermal amplification (LAMP) detection of Plasmodium falciparum and Plasmodium vivax in urine and saliva of malaria patients. From May to November 2011, 108 febrile patients referred to health centers in Sistan and Baluchestan Province of south-eastern Iran participated in the study. Saliva, urine, and blood samples were analyzed with nested PCR and LAMP targeting the species-specific nucleotide sequence of small subunit ribosomal RNA gene (18S rRNA) of P. falciparum and P. vivax and evaluated for diagnostic accuracy by comparison to blood nested PCR assay. When nested PCR of blood is used as standard, microscopy and nested PCR of saliva and urine samples showed sensitivity of 97.2%, 89.4% and 71% and specificity of 100%, 97.3% and 100%, respectively. LAMP sensitivity of blood, saliva, and urine was 95.8%, 47% and 29%, respectively, whereas LAMP specificity of these samples was 100%. Microscopy and nested PCR of saliva and LAMP of blood were comparable to nested PCR of blood (к = 0.95, 0.83, and 0.94, respectively), but agreement for nested PCR of urine was moderate (к = 0.64) and poor to fair for saliva LAMP and urine LAMP (к = 0.38 and 0.23, respectively). LAMP assay showed low sensitivity for detection of Plasmodium DNA in human saliva and urine compared to results with blood and to nested PCR of blood, saliva, and urine. However, considering the advantages of LAMP technology and of saliva and urine sampling, further research into the method is worthwhile. LAMP protocol and precise preparation protocols need to be defined and optimized for template DNA of saliva and urine. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Malaria is an important parasitic disease caused by Plasmodium, with an estimated 216 million cases annually (World Health Organization, 2012). As incorrect, delayed, or unavailable diagnosis is the most common reason for malaria death (Poschl et al., 2010), early and accurate diagnosis followed by effective and timely treatment could diminish morbidity and mortality of malaria in endemic regions (World Health Organization, 2012). According to World Health Organization (WHO) recommendations, malaria infection
∗ Corresponding author at: Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, P.O. Box 15875-6171, Tehran, Iran. Tel.: +98 21 88622653; fax: +98 21 88622653. E-mail addresses: [email protected], [email protected] (E. Razmjou). http://dx.doi.org/10.1016/j.actatropica.2014.03.029 0001-706X/© 2014 Elsevier B.V. All rights reserved.
must be parasitologically confirmed before initiating treatment, since clinical signs may be misleading (World Health Organization, 2010). Since a parasite as the cause of malaria was discovered by Laveran in 1880, the gold standard for diagnosis has been microscopic examination of thin and/or thick blood smears. Molecular methods based on DNA amplification have been developed for more sensitive diagnosis of malaria, the ability to detect low parasitemia, and greater specificity in mixed infection (Snounou et al., 1993; Kimura et al., 1997; Perandin et al., 2004; Coleman et al., 2006). One of these techniques is loop-mediated isothermal amplification (LAMP) (Notomi et al., 2000; Nagamine et al., 2002), a rapid, sensitive, specific, and simple protocol for diagnosis of parasitic diseases such as malaria (Poon et al., 2006; Han et al., 2007; Paris et al., 2007; Chen et al., 2010; Polley et al., 2010; Lee et al., 2012). The main source of the parasite for malaria diagnosis is patients’ blood. Collection of a blood sample has limitations due to its invasive nature, increasing the risk of blood-borne infectious disease,
Z. Ghayour Najafabadi et al. / Acta Tropica 136 (2014) 44–49
and invoking blood taboos in some nations (Mharakurwa et al., 2006). In order to increase community participation in malaria longitudinal surveys and to develop greater diagnostic accuracy, an alternative non-invasive approach is needed. Research has shown that trace amounts of parasite DNA are detectable in urine and saliva of infected persons (Mharakurwa et al., 2006; Nwakanma et al., 2009; Buppan et al., 2010; Putaporntip et al., 2011), and these fluids could be used as alternative sources of Plasmodium DNA for molecular detection of malaria. Unfortunately, the current PCR-based methods are laborious, costly, and available only in well-equipped laboratories, making them impractical for routine diagnosis in malaria endemic areas (Hänscheid and Grobusch, 2002). Thus, we require a feasible diagnostic test for isolation of Plasmodium DNA in saliva and urine samples. This investigation assessed the sensitivity and specificity of LAMP for detection of Plasmodium DNA in saliva and urine of malaria patients. 2. Materials and methods 2.1. Ethics statement This investigation was conducted according to the Helsinki declaration. Research protocols and informed consent arrangements were reviewed and approved by the Ethics Committee of Iran University of Medical Sciences. All patients, or the parents or guardians of participants aged 10,000 Urine
1000 < 10,000 >10,000
a b
% Agreement with microscopy (kappa)
NP : number of positives, NBP : number of positives in blood. 95% CI: the 95% confidence interval.
higher sensitivity (92.2% and 73.3% (Table 2B)) than reported in previous studies (73% and 32% (Nwakanma et al., 2009) and 77% and 42% (Buppan et al., 2010)). The improvement may be related to the amplification of shorter DNA fragments. As malarial DNA may be degraded during excretion or transport to saliva or urine from blood, the possibility of detection is increased by targeting smaller fragments (Mharakurwa et al., 2006). In addition, in this study, the volume of saliva and urine was two- to five-fold the volume used in other studies (Mharakurwa et al., 2006; Buppan et al., 2010; Putaporntip et al., 2011). This suggests that the sensitivity of molecular detection of Plasmodium in saliva and urine increased following the screening showing a trace of DNA in these fluids. In this study, the use of LAMP in detection of DNA of P. vivax and P. falciparum in saliva and urine of malaria patients was demonstrated for the first time, although its effectiveness has been shown in blood samples (Han et al., 2007; Chen et al., 2010; Poschl et al., 2010; Lee et al., 2012; Lu et al., 2012). Saliva and urine nested PCR showed 92.2% and 73.3% sensitivity, respectively, whereas saliva and urine LAMP sensitivity was lower (48.5% and 30%, respectively), although both specificity and PPV were 100% (Table 2B). The low sensitivity of saliva and urine LAMP compared to nested PCR assay may indicate that the amount of parasite DNA in saliva and urine that was lower than the detection limit of LAMP (35.9 parasites/l). Previous studies have reported a minimum detection limit of LAMP and nested PCR of 30 ± 5 parasites/l and 3 ± 5 parasites/l, respectively (Chen et al., 2010; Lu et al., 2012), that are in good agreement with our study. However, the detection limit of blood LAMP in this study is somewhat worse than the best LAMP assays that reported 10–100 P. falciparum/l, 10–40 P. vivax/l (Han et al., 2007; Lucchi et al., 2010; Surabattula et al., 2013) and six P. falciparum/l (Poon et al., 2006) using primers targeting 18S rRNA. This difference may have been a consequence of reduced efficiency in DNA extraction at low parasitemia level or other factors. Quantitative real-time PCR analysis showed mean amounts of parasite DNA in the blood to be ∼600-fold and 2500-fold that in concurrent saliva and urine, respectively (Nwakanma et al., 2009). LAMP sensitivity was
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Z. Ghayour Najafabadi et al. / Acta Tropica 136 (2014) 44–49
comparable to microscopic and nested PCR assay in blood samples (98.5%), but it was decreased in saliva (48.5%) and urine (30%). This may indicate a requirement for greater integrity of the target in the LAMP assay, owing to the many primers that need to be annealed to the different regions, than for PCR (Bista et al., 2007). During DNA filtration in saliva and urine, partial degradation of the target DNA in the region of LAMP primers is more likely than in the region of PCR primers. Studies have shown that parasite density ≥1000 parasites/l influenced detection of parasite DNA in saliva and urine by nested PCR (Nwakanma et al., 2009). No correlation between parasite density and positive rates of saliva and urine nested PCR was observed in the current study, and parasite DNA was detected in saliva and urine samples by nested PCR even at low parasitemia, as was reported by A-Elgayoum et al. (2010). In the LAMP assay, higher parasitemia considerably increased the positive rates of saliva and urine samples. It seems that there is a correlation between the parasitemia and the transfer of malaria DNA to saliva and urine, through influencing on the quality of DNA. 5. Conclusions In its present form, the low sensitivity of LAMP assay for detection of malaria in saliva and urine would make it unsuitable for clinical evaluation of malaria cases. Nevertheless, if the sensitivity of the LAMP assays can be modified, such as by targeting the mitochondrial gene with a 30–100 copy number sequences per parasite (Vaidya and Mather, 2005) versus 4–8 copies for the 18s rRNA gene (Dame and McCutchan, 1983), the chance of detecting small amounts of parasite DNA in saliva and urine could be improved. Nested PCR targeting malarial mitochondrial DNA using blood samples as templates (Putaporntip et al., 2011; Hwang et al., 2012) obviously outperformed that targeting 18S rRNA based on previous studies (Snounou et al., 1993; Kimura et al., 1997; Coleman et al., 2006). Likewise, nested PCR based on mitochondrial DNA amplification of saliva and urine samples (Putaporntip et al., 2011) significantly outperformed nested PCR targeting 18S rRNA genes as reported by others (Mharakurwa et al., 2006; Nwakanma et al., 2009; Buppan et al., 2010; Pooe et al., 2011); thereby, the advent of LAMP targeting malarial mitochondrial DNA (Polley et al., 2010; Hopkins et al., 2013; Polley et al., 2013) along with an increase in volume of samples (as shown in this study) will substantially improved diagnostic performance in terms of sensitivity as well as species identification of all human malaria species. LAMP technology (Mabey et al., 2004; Njiru, 2012) matches the WHO recommendations for an ideal diagnostic test for developing countries. The simplicity, easy availability, and non-invasiveness of saliva or urine sampling that can be performed even by patients with limited training, ought to encourage further extensive studies, with a larger number of clinical samples. Acknowledgments The authors are grateful to all patients who donated blood, urine, and saliva samples for this study in Chabahar District and Sarbaz District. We would like to thank the staff of the Public Health Department, Sistan and Baluchestan Province, especially Mohammad Sakeni, Khodadad Gorgij, Mohammad Rafi Parastar, Yaghoob Jadgal in Chabahar District, and Habiballah Berah in Sarbaz District for their valuable assistance in sample collection and field work. We are extremely grateful to Zahra Rampisheh for her great assistance in analyzing data. This study was supported by Iran University of Medical Sciences, Tehran, Iran grant P/999.
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Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Detection of Plasmodium vivax and Plasmodium falciparum DNA in human saliva and urine: Loop-mediated isothermal amplification for malaria diagnosis Zahra Ghayour Najafabadi a , Hormozd Oormazdi a , Lame Akhlaghi a , Ahmad Reza Meamar a , Mehdi Nateghpour b , Leila Farivar b , Elham Razmjou a,∗ a b
Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 1 November 2013 Received in revised form 29 March 2014 Accepted 31 March 2014 Available online 8 April 2014 Keywords: Plasmodium vivax P. falciparum Malaria diagnosis Saliva Urine LAMP
a b s t r a c t This study investigated loop-mediated isothermal amplification (LAMP) detection of Plasmodium falciparum and Plasmodium vivax in urine and saliva of malaria patients. From May to November 2011, 108 febrile patients referred to health centers in Sistan and Baluchestan Province of south-eastern Iran participated in the study. Saliva, urine, and blood samples were analyzed with nested PCR and LAMP targeting the species-specific nucleotide sequence of small subunit ribosomal RNA gene (18S rRNA) of P. falciparum and P. vivax and evaluated for diagnostic accuracy by comparison to blood nested PCR assay. When nested PCR of blood is used as standard, microscopy and nested PCR of saliva and urine samples showed sensitivity of 97.2%, 89.4% and 71% and specificity of 100%, 97.3% and 100%, respectively. LAMP sensitivity of blood, saliva, and urine was 95.8%, 47% and 29%, respectively, whereas LAMP specificity of these samples was 100%. Microscopy and nested PCR of saliva and LAMP of blood were comparable to nested PCR of blood (к = 0.95, 0.83, and 0.94, respectively), but agreement for nested PCR of urine was moderate (к = 0.64) and poor to fair for saliva LAMP and urine LAMP (к = 0.38 and 0.23, respectively). LAMP assay showed low sensitivity for detection of Plasmodium DNA in human saliva and urine compared to results with blood and to nested PCR of blood, saliva, and urine. However, considering the advantages of LAMP technology and of saliva and urine sampling, further research into the method is worthwhile. LAMP protocol and precise preparation protocols need to be defined and optimized for template DNA of saliva and urine. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Malaria is an important parasitic disease caused by Plasmodium, with an estimated 216 million cases annually (World Health Organization, 2012). As incorrect, delayed, or unavailable diagnosis is the most common reason for malaria death (Poschl et al., 2010), early and accurate diagnosis followed by effective and timely treatment could diminish morbidity and mortality of malaria in endemic regions (World Health Organization, 2012). According to World Health Organization (WHO) recommendations, malaria infection
∗ Corresponding author at: Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, P.O. Box 15875-6171, Tehran, Iran. Tel.: +98 21 88622653; fax: +98 21 88622653. E-mail addresses: [email protected], [email protected] (E. Razmjou). http://dx.doi.org/10.1016/j.actatropica.2014.03.029 0001-706X/© 2014 Elsevier B.V. All rights reserved.
must be parasitologically confirmed before initiating treatment, since clinical signs may be misleading (World Health Organization, 2010). Since a parasite as the cause of malaria was discovered by Laveran in 1880, the gold standard for diagnosis has been microscopic examination of thin and/or thick blood smears. Molecular methods based on DNA amplification have been developed for more sensitive diagnosis of malaria, the ability to detect low parasitemia, and greater specificity in mixed infection (Snounou et al., 1993; Kimura et al., 1997; Perandin et al., 2004; Coleman et al., 2006). One of these techniques is loop-mediated isothermal amplification (LAMP) (Notomi et al., 2000; Nagamine et al., 2002), a rapid, sensitive, specific, and simple protocol for diagnosis of parasitic diseases such as malaria (Poon et al., 2006; Han et al., 2007; Paris et al., 2007; Chen et al., 2010; Polley et al., 2010; Lee et al., 2012). The main source of the parasite for malaria diagnosis is patients’ blood. Collection of a blood sample has limitations due to its invasive nature, increasing the risk of blood-borne infectious disease,
Z. Ghayour Najafabadi et al. / Acta Tropica 136 (2014) 44–49
and invoking blood taboos in some nations (Mharakurwa et al., 2006). In order to increase community participation in malaria longitudinal surveys and to develop greater diagnostic accuracy, an alternative non-invasive approach is needed. Research has shown that trace amounts of parasite DNA are detectable in urine and saliva of infected persons (Mharakurwa et al., 2006; Nwakanma et al., 2009; Buppan et al., 2010; Putaporntip et al., 2011), and these fluids could be used as alternative sources of Plasmodium DNA for molecular detection of malaria. Unfortunately, the current PCR-based methods are laborious, costly, and available only in well-equipped laboratories, making them impractical for routine diagnosis in malaria endemic areas (Hänscheid and Grobusch, 2002). Thus, we require a feasible diagnostic test for isolation of Plasmodium DNA in saliva and urine samples. This investigation assessed the sensitivity and specificity of LAMP for detection of Plasmodium DNA in saliva and urine of malaria patients. 2. Materials and methods 2.1. Ethics statement This investigation was conducted according to the Helsinki declaration. Research protocols and informed consent arrangements were reviewed and approved by the Ethics Committee of Iran University of Medical Sciences. All patients, or the parents or guardians of participants aged 10,000 Urine
1000 < 10,000 >10,000
a b
% Agreement with microscopy (kappa)
NP : number of positives, NBP : number of positives in blood. 95% CI: the 95% confidence interval.
higher sensitivity (92.2% and 73.3% (Table 2B)) than reported in previous studies (73% and 32% (Nwakanma et al., 2009) and 77% and 42% (Buppan et al., 2010)). The improvement may be related to the amplification of shorter DNA fragments. As malarial DNA may be degraded during excretion or transport to saliva or urine from blood, the possibility of detection is increased by targeting smaller fragments (Mharakurwa et al., 2006). In addition, in this study, the volume of saliva and urine was two- to five-fold the volume used in other studies (Mharakurwa et al., 2006; Buppan et al., 2010; Putaporntip et al., 2011). This suggests that the sensitivity of molecular detection of Plasmodium in saliva and urine increased following the screening showing a trace of DNA in these fluids. In this study, the use of LAMP in detection of DNA of P. vivax and P. falciparum in saliva and urine of malaria patients was demonstrated for the first time, although its effectiveness has been shown in blood samples (Han et al., 2007; Chen et al., 2010; Poschl et al., 2010; Lee et al., 2012; Lu et al., 2012). Saliva and urine nested PCR showed 92.2% and 73.3% sensitivity, respectively, whereas saliva and urine LAMP sensitivity was lower (48.5% and 30%, respectively), although both specificity and PPV were 100% (Table 2B). The low sensitivity of saliva and urine LAMP compared to nested PCR assay may indicate that the amount of parasite DNA in saliva and urine that was lower than the detection limit of LAMP (35.9 parasites/l). Previous studies have reported a minimum detection limit of LAMP and nested PCR of 30 ± 5 parasites/l and 3 ± 5 parasites/l, respectively (Chen et al., 2010; Lu et al., 2012), that are in good agreement with our study. However, the detection limit of blood LAMP in this study is somewhat worse than the best LAMP assays that reported 10–100 P. falciparum/l, 10–40 P. vivax/l (Han et al., 2007; Lucchi et al., 2010; Surabattula et al., 2013) and six P. falciparum/l (Poon et al., 2006) using primers targeting 18S rRNA. This difference may have been a consequence of reduced efficiency in DNA extraction at low parasitemia level or other factors. Quantitative real-time PCR analysis showed mean amounts of parasite DNA in the blood to be ∼600-fold and 2500-fold that in concurrent saliva and urine, respectively (Nwakanma et al., 2009). LAMP sensitivity was
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comparable to microscopic and nested PCR assay in blood samples (98.5%), but it was decreased in saliva (48.5%) and urine (30%). This may indicate a requirement for greater integrity of the target in the LAMP assay, owing to the many primers that need to be annealed to the different regions, than for PCR (Bista et al., 2007). During DNA filtration in saliva and urine, partial degradation of the target DNA in the region of LAMP primers is more likely than in the region of PCR primers. Studies have shown that parasite density ≥1000 parasites/l influenced detection of parasite DNA in saliva and urine by nested PCR (Nwakanma et al., 2009). No correlation between parasite density and positive rates of saliva and urine nested PCR was observed in the current study, and parasite DNA was detected in saliva and urine samples by nested PCR even at low parasitemia, as was reported by A-Elgayoum et al. (2010). In the LAMP assay, higher parasitemia considerably increased the positive rates of saliva and urine samples. It seems that there is a correlation between the parasitemia and the transfer of malaria DNA to saliva and urine, through influencing on the quality of DNA. 5. Conclusions In its present form, the low sensitivity of LAMP assay for detection of malaria in saliva and urine would make it unsuitable for clinical evaluation of malaria cases. Nevertheless, if the sensitivity of the LAMP assays can be modified, such as by targeting the mitochondrial gene with a 30–100 copy number sequences per parasite (Vaidya and Mather, 2005) versus 4–8 copies for the 18s rRNA gene (Dame and McCutchan, 1983), the chance of detecting small amounts of parasite DNA in saliva and urine could be improved. Nested PCR targeting malarial mitochondrial DNA using blood samples as templates (Putaporntip et al., 2011; Hwang et al., 2012) obviously outperformed that targeting 18S rRNA based on previous studies (Snounou et al., 1993; Kimura et al., 1997; Coleman et al., 2006). Likewise, nested PCR based on mitochondrial DNA amplification of saliva and urine samples (Putaporntip et al., 2011) significantly outperformed nested PCR targeting 18S rRNA genes as reported by others (Mharakurwa et al., 2006; Nwakanma et al., 2009; Buppan et al., 2010; Pooe et al., 2011); thereby, the advent of LAMP targeting malarial mitochondrial DNA (Polley et al., 2010; Hopkins et al., 2013; Polley et al., 2013) along with an increase in volume of samples (as shown in this study) will substantially improved diagnostic performance in terms of sensitivity as well as species identification of all human malaria species. LAMP technology (Mabey et al., 2004; Njiru, 2012) matches the WHO recommendations for an ideal diagnostic test for developing countries. The simplicity, easy availability, and non-invasiveness of saliva or urine sampling that can be performed even by patients with limited training, ought to encourage further extensive studies, with a larger number of clinical samples. Acknowledgments The authors are grateful to all patients who donated blood, urine, and saliva samples for this study in Chabahar District and Sarbaz District. We would like to thank the staff of the Public Health Department, Sistan and Baluchestan Province, especially Mohammad Sakeni, Khodadad Gorgij, Mohammad Rafi Parastar, Yaghoob Jadgal in Chabahar District, and Habiballah Berah in Sarbaz District for their valuable assistance in sample collection and field work. We are extremely grateful to Zahra Rampisheh for her great assistance in analyzing data. This study was supported by Iran University of Medical Sciences, Tehran, Iran grant P/999.
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