
 panel of samples - in this study , we included 34 samples from long - term 
 residents of an endemic amazon rural community , colniza , state of mato grosso ( mt ) ; 
 subclinical malaria infections were characterised in this area during a detailed 
 descriptive epidemiology study carried out between 2003 - 2009 ( maciel 2011 ) . in that study ,
the parasitological and 
 epidemiological data from seven consecutive years identified a small community , herein 
 referred to as colniza , where 55.91% of all malaria cases were subclinical . 

consequently , malaria infection could not be excluded a priori in the 
 individuals exposed to malaria in colniza .
colniza is located inside the amazon forest 
 in the northwest of mt , roughly 1,200 km from the capital cuiab ; colniza can be 
 accessed by two paved roads , which connect mt to the states of amazonas ( br-174 ) and 
 rondnia ( mt-206 ) .
the economic activities of this municipality are based mainly on wood 
 extraction and livestock .
malaria is transmitted year - round with an annual 
 parasitological index ( api ) of 98.5 cases per 1,000 inhabitants ( maciel 2011 ) .
samples were collected in this area in 2008 during a 
 survey of asymptomatic patients in the area .
the following eligibility criteria were 
 included : ( i ) a minimum age of 15 years , ( ii ) the absence of pregnancy , ( iii ) a lifetime 
 exposure to malaria in the colniza area , ( iv ) no self - reported symptoms suggestive of 
 malaria , such as fever , myalgia , chills or headache and ( v ) negative blood parasites by 
 direct examination of giemsa - stained thick blood smears . although all samples were 
 negative by om , 14 out of 34 ( 41% ) samples were positive according to a malaria - specific 
 nested - pcr ( snounou et al .
consequently , all of the colniza samples were 
 initially considered individuals who might have submicroscopic parasitaemia and may 
 therefore be potential malaria carriers . as a positive control ,
we included 52 field 
 samples from patients who sought care at a regional reference malaria laboratory ( julio 
 muller university hospital , federal university of mato grosso , cuiab ) and whose thick 
 blood smears were positive for malaria .
based on the om results , the parasite densities 
 of the positive controls ranged from high ( > 1,000 parasites l / blood , n = 16 ) , to 
 medium ( 1,000 - 301 l / blood , n = 16 ) and low parasitaemia ( 100 - 300 parasites l / blood , n 
 = 20 ) . as a negative control ,
20 blood samples from volunteers living outside of the 
 endemic area were included ; these volunteers had never been infected or exposed to the 
 malaria parasites . 

ethics - the ethical and methodological aspects of this study were 
 approved by the ethical committee of research on human beings from the ren rachou 
 research centre / oswaldo cruz foundation ( reports 07/2006 and 01/2009 ) , according to the 
 resolution of the brazilian council on health 196/96 . 

blood examination - the giemsa - stained thick blood smear technique was 
 used for malaria diagnosis in all of the samples in the present study ; during this 
 examination , long - term experienced microscopists were in charge of examining the 
 equivalent of 0.2 l of blood ( roughly 100 microscopy fields ) . for quality assurance , 

each slide was examined by two microscopists who had over 10 years of experience in 
 reading malaria slides ; their proficiency was periodically evaluated by local / regional 
 quality assurance programs of the brazilian ministry of health ( ms ) .
parasite density 
 was estimated as the number of parasites per microlitre of blood , in accordance with the 
 standards of the ms . 

whole blood samples and preparation of the dna template - at the time 
 of the thick blood smear preparation , 5 ml of blood was drawn into sterile tubes 
 containing ethylenediamine tetraacetic acid and aliquots ( 1 ml ) of the whole blood were 
 subsequently used to purify the templates for the pcr assays .
genomic dna was extracted using a qiagen genomic dna 
 purification kit ( puregene   ) ( gentra systems , minneapolis , mn , usa ) , 
 according to the manufacturers recommendations .
the dna was eluted in a 330-l volume 
 ( 100 ng of dna /l ) and stored at -20c until it was used . 

nested - pcr - samples were amplified using a nested - pcr protocol adapted 
 from the original protocol described by snounou et al . 

( 1993 ) ; the same primers described in the original study were used in this 
 study .
briefly , all pcr reactions were performed in 20 l volumes containing 250 m each 
 oligonucleotide primer , 10 l of master mix ( promega ) ( 0.3 units of taq polymerase , 200 
 m each deoxyribonucleotide triphosphates and 1.5 mm mgcl 2 ) and 2 l of dna 
 ( 6 l of whole blood ) .
the pcr assays were performed using an automatic thermocycler 
 ( ptc-100   v.7.0 ) ( mj research inc , usa ) and the following cycling 
 parameters were used : an initial denaturation at 95c for 5 min followed by 24 cycles of 
 annealing at 58c for 2 min , extension at 72c for 2 min and denaturation at 94c for 1 
 min followed by a final annealing incubation at 58c for 2 min and extension at 72c for 
 2 min . the temperature was then reduced to 4c until the samples were taken .
the cycling 
 parameters for the second round of pcr were the same as the first reaction , but instead 
 30 cycles of amplification were used .
the amplified products were detected by ethidium 
 bromide staining following agarose 2% gel electrophoresis ( invitrogen ) and the 
 species - specific fragment sizes were 205 bp for plasmodium falciparum , 
 120 bp for plasmodium vivax and 144 bp for plasmodium 
 malariae . because human dna represents the majority of the dna in the 
 samples , a gene present in the abo human system was amplified in the panel of samples to 
 determine the quality control of the dna extraction , as previously described ( olsson et al .
rt - pcr - identification of the malaria parasite species was performed 
 by rt - pcr amplification of the 18ssu rrna gene , as previously described ( mangold et al .
2005 ) ; based on this protocol , a 
 consensus pair of primers was used to amplify a species - specific region of the multicopy 
 18ssu rrna gene .
briefly , each 20 l reaction mix contained 2 l of genomic dna ( 6 l 
 of whole blood ) , 10 l of sybr   green pcr master mix ( applied biosystems ) , 
 2.5 mm mgso 4 ( final concentration ) and 0.5 m of each primer ( biosynthesis ) . 

the pcr conditions consisted of an initial denaturation at 95c for 10 min followed by 
 40 cycles of 90c for 30 sec and 60c for 30 sec ; fluorescence acquisition was performed 
 at the end of each extension step . after amplification , the melting curves were observed 
 from the dissociation curves and those melting curve analyses , which were based on 
 nucleotide variations within the amplicons , provided a basis for the accurate 
 differentiation of the three plasmodia : p. falciparum , p. vivax and 
 p. malariae . the amplification and fluorescence detection were 
 performed using the abi prism   7000 sequence detection system ( applied 
 biosystems ) .
the range of melting temperatures ( t 
 m 
 ) for each plasmodium was 74 - 76c for p. 
 vivax , 71 - 73c for p. falciparum and 68 - 70c for 
 p. malariae . 

reproducibility of the pcr reactions - initially , each dna sample from 
 the individuals who might harbour submicroscopic parasitaemia ( colniza ; potential 
 malaria carriers ) were submitted to three pcr reactions .
we amplified the 18ssu rrna 
 gene using nested - pcr , which is widely regarded as the  gold standard  for the pcr 
 detection of malaria parasites ( malera 2011 ) . 

additionally , the samples were also submitted to a second pcr protocol ( rt - pcr for the 
 plasmodium 18ssu rrna gene ) .
after this first round of pcr , the 
 colniza samples were considered positive or negative if all of the pcr results were 
 consistent . for the discordant results , the samples were submitted to a second round of 
 pcr , which included a re - amplification using the gold - standard nested - pcr protocol and 
 two additional replicates of the rt - pcr method ; this approach ensured that each 
 discordant sample was retested at least three times by each pcr protocol .
afterwards , 
 the discordant samples were classified as doubtful for malaria infection . for samples 
 from patients with
microscopically detectable parasitaemia ( positive controls ) , a 
 consensus result per protocol was based on the agreement between at least three pcr 
 results . 

statistical analysis - a chi - squared analysis was performed to compare 
 the nested and rt - pcr protocols and to compare the single vs. multiple reactions .
the 
 agreement between the different assays was evaluated using mcnemar s chi - squared 
 analysis and the kappa (  ) coefficient of agreement .

 performance of the nested - pcr and rt - pcr protocols against a panel of reference 
 samples - as an internal control , microscopically positive ( n = 52 ) and 
 negative ( n = 20 ) control samples were evaluated using the nested and rt - pcr methods ( 
 table i ) .
both pcr protocols were specific 
 as no positive results were obtained from the blood samples of the 20 malaria nave 
 volunteers .
regardless of the pcr protocol , pcr accurately identified 98 - 100% of the 
 microscopy - positive samples ; concerning the reproducibility of the pcr amplification , no 
 significant differences were observed after retesting all 72 control samples ( 3 - 5 
 replicates per protocol ) .
additionally , for the plasmodium 
 species - specific identification , the results of both pcr protocols were highly 
 reproducible and , in general , equivalent to those obtained by om ( table ii ) .
as expected , the pcr protocols detected 
 a number of mixed infections ( p. vivax plus p. 
 falciparum ) that could not be detected by microscopy ( table ii ) .
the reproducibility of the pcr methods 
 was confirmed using  statistics , which demonstrated that no difference was observed 
 between the single and multiple pcr reactions or between the nested and rt - pcr protocols 
 ( -values of 0.70 - 0.91 ; mcnemar test , p > 0.05 ) . 

table iperformance of polymerase chain reaction ( pcr ) protocols evaluated against 
 a panel of reference samples with single or multiple assays realised per pcr 
 protocolsamplesparasites/l(n ) 
 nested - pcr positive n ( % ) 
 rt - pcr positive n ( % ) 
 single 
 multiplesinglemultiple 

 positive > 100 ( 52)51 ( 98)52 ( 100)51 ( 98)51 ( 98)negative0 ( 20)0 ( 0)0 ( 0)0 ( 0)0 ( 0 ) 
 a : parasitaemia as detected by optical microscopy ; 
 b : single , the result of the first pcr reaction , and 
 multiple , the results of three concordant pcr reactions .
there were no 
 statistical differences between results from single vs. multiple pcr assays or 
 from nested vs. real - time ( rt)-pcr for single vs. multiple reactions ( p > 
 0.05 ) . 
 

a : parasitaemia as detected by optical microscopy ; 
 b : single , the result of the first pcr reaction , and 
 multiple , the results of three concordant pcr reactions .
there were no 
 statistical differences between results from single vs. multiple pcr assays or 
 from nested vs. real - time ( rt)-pcr for single vs. multiple reactions ( p > 
 0.05 ) . 

table iipolymerase chain reaction ( pcr ) performance for the detection of 
 plasmodium falciparum and/or plasmodium 
 vivax at microscopically positive samples 
 plasmodium speciesom 
 positive samples n ( % ) 
 nested - pcrrt - pcr 

 single 
 multiplesinglemultiple 


 p. vivax 
 41 ( 79)40 ( 77)40 ( 77)40 ( 77)40 ( 77 ) 
 p. falciparum 
 11 ( 21)9 ( 17)10 ( 19)10 ( 19)10 ( 19)mixed02 ( 4)2 ( 4)1 ( 2)1 ( 2)negative01 ( 2)0 ( 0)1 ( 2)1 ( 2 ) 
 a : optical microscopy ( om ) ,
which included 52 samples 
 positive at parasite density > 100 parasites per l of blood ; 
 b : nested - pcr and real - time ( rt)-pcr were carried - out as 
 described in subjects , materials and methods ; single , the first pcr reaction , 
 and multiple , the results of three concordant pcr reactions . 
 
 a : optical microscopy ( om ) , which included 52 samples 
 positive at parasite density > 100 parasites per l of blood ; 
 b : nested - pcr and real - time ( rt)-pcr were carried - out as 
 described in subjects , materials and methods ; single , the first pcr reaction , 
 and multiple , the results of three concordant pcr reactions . 

evaluation of pcr performance for detecting submicroscopic malaria 
 infections - thirty - four samples from long - term residents of a rural amazon 
 community with frequent subclinical malaria infections ( potential malaria carriers ) were 
 assayed using both pcr - based protocols . at the time of the blood collection ,
all of 
 these individuals had a negative malaria thick blood smear , but 14 out of 34 ( 41% ) 
 individuals were positive for malaria according to the nested - pcr assay , which was 
 performed in parallel . to evaluate the reproducibility of the dna amplification by the 
 pcr - based protocols , each sample
was submitted to three additional pcr reactions ( nested 
 and rt - pcr ) ; to reach a molecular consensus , the discordant samples were re - amplified by 
 additional pcr assays ( table iii ) .
molecular 
 consensus confirmed the submicroscopic malaria infection in seven ( 21% ) individuals and 
 excluded this possibility in 14 samples ( 41% ) . in 13 ( 38% )
samples , the pcr replicates 
 led to alternating positive or negative results and these discrepancies probably reflect 
 the limitations of the molecular methods at low levels of parasitaemia . regarding the 
 identification of the plasmodium species , only two samples ( 917 and 
 930 ) were discordant ; nested - pcr identified these samples as mixed infections , however 
 these results were not confirmed by rt - pcr . on the other hand , no positive results were 
 obtained from the blood samples from five malaria nave volunteers ( data not shown ) . 

table iiireproducibility of polymerase chain reaction ( pcr)-based protocols among 34 
 long - term residents of an amazon rural community who might harbour 
 submicroscopic parasitaemia ( potential malaria carriers ) 
 codenested - pcr 
 rt - pcr 
 consensus result n ( % ) firstsecondthirdfourth  firstsecondthird 

 903negnegnegndnegndnd-906negnegnegndnegndnd-907negnegnegndnegndnd-909negnegnegndnegndnd-911negnegnegndnegndnd-912negnegnegndnegndnd-913negnegnegndnegndndnegative , 14 ( 41)918negnegnegndnegndnd-920negnegnegndnegndnd-922negnegnegndnegndnd-923negnegnegndnegndnd-934negnegnegndnegndnd-935negnegnegndnegndnd-936negnegnegndnegndnd- 

 902fvfvfvndfvndnd-904vvvndvndnd-916vvvndvndnd-921vvvndvndndpositive , 7 ( 21)924vvvndvndnd-925vvvndvndnd-929vvvndvndnd- 

 901mnegnegnegnegnegneg-908vnegnegnegvnegneg-915negnegvnegvvv-928negnegvnegnegnegneg-919negnegffnegnegneg-905fnegnegffnegnegdoubtful , 13 ( 38)910negvnegvvvv-917negfvfvfvvvv-930fvnegfvnegvvv-926vnegnegvnegnegneg-927negvvvnegnegneg-932vvvndnegnegv-933fnegnegnegnegnegf- 
 a :
initially , each sample was submitted to the first round of 
 pcr [ 3 nested - pcr ( gold - standard ) and 1 real - time ( rt)-pcr ] .
samples with 
 discordant results were resubmitted to a second round of pcr ( 1 nested - pcr and 
 2 rt - pcr ) .
this approach allowed that discordant samples were classified as 
 doubtful after being tested at least in triplicate by each pcr protocol . 

results were indicated as positive by plasmodium vivax ( v ) , 
 plasmodium falciparum ( f ) , plasmodium 
 malariae ( m ) and mixed infection by p. falciparum 
 and p. vivax ( fv ) .
a : initially , each sample was submitted to the first round of 
 pcr [ 3 nested - pcr ( gold - standard ) and 1 real - time ( rt)-pcr ] .
samples with 
 discordant results were resubmitted to a second round of pcr ( 1 nested - pcr and 
 2 rt - pcr ) .
this approach allowed that discordant samples were classified as 
 doubtful after being tested at least in triplicate by each pcr protocol . 

results were indicated as positive by plasmodium vivax ( v ) , 
 plasmodium falciparum ( f ) , plasmodium 
 malariae ( m ) and mixed infection by p. falciparum 
 and p. vivax ( fv ) .
nd : not done ; neg : pcr negative for 
 plasmodium infection . of importance , the limitation of pcr amplification at low levels of parasitaemia was 
 restricted to dna from field samples because titrations of dna templates obtained from 
 the plasmodium reference isolates or species - specific plasmid 
 constructions could be detected at concentrations of less than three parasites/l of 
 blood .
figs 1 , 2 illustrate the end - point dna titrations of the rt - pcr method , in which the 
 dna templates were obtained from cultured p. falciparum or from an 
 artificially mixed infection obtained from highly - parasitaemic individuals infected with 
 p. falciparum and p. vivax ( 10,000 parasites/l of 
 blood ) .
similar results were obtained using the nested - pcr protocol ( data not 
 shown ) . 

1 : real - time polymerase chain reaction melting curve analysis obtained from 
 dna template dilutions from plasmodium falciparum continuous 
 erythrocyte culture . the p. falciparum parasite ( bhz 26/86 ) , a 
 chloroquine - resistant isolate ( carvalho et al . 

1991 ) , was maintained in culture by the candle jar method ( jensen & trager 1977 ) , with cultures 
 maintained synchronised as described ( jensen 
 1978 ) .
serial dilutions of samples included were 3,000 , 300 , 30 , 3 , 
 0.3 and 0 parasite/l of blood .
the graph was generated by using the abi prism 
  7000 sequence detection system ( applied biosystems ) . 
 

2 : real - time polymerase chain reaction melting curve analysis obtained from 
 dna template dilutions from and artificial plasmodium 
 falciparum and plasmodium vivax infection .
the 
 graph was generated by using the abi prism   7000 sequence detection 
 system ( applied biosystems ) .
serial dilutions of samples included were 3,000 , 
 300 , 30 , 3 , 0.3 and 0 parasite/l of blood . 

as part of the international effort to reduce the global incidence of malaria , it has 
 been proposed that the treatment of asymptomatic carriers should be an essential tool 
 for breaking the cycle of infection in some transmission settings ( ogutu et al .
however , improved diagnostic 
 tests are needed to reach this goal in order to accurately detect submicroscopic malaria 
 infections .
although it has been claimed that pcr - based protocols are capable of 
 detecting very low levels of parasitaemia , the reproducibility of these methods in 
 malaria field samples has been largely underestimated . here
, the main goal was to 
 analyse the reproducibility of the pcr - based protocols in detecting submicroscopic 
 malaria infections .
therefore , field samples , not standard dna templates , were used in 
 this analysis because some , if not all , of the field samples may contain inhibitors that 
 are not present in the standard dna samples ( andrews et 
 al .
2010 ) . in 
 this context , we selected the colniza area due to the high frequency of subclinical 
 malaria , which made it feasible to identify submicroscopic malaria infections .
following 
 this protocol , we demonstrated that the traditional pcr amplification protocols might 
 not be able to accurately and reproducibly identify potential submicroscopic malaria 
 carriers .
in fact , the pcr results were inconsistent in 13 out of the 34 ( ~40% ) 
 individuals , even after seven replicates of each dna template ( 4 nested - pcr replicates 
 and 3 rt - pcr replicates ) were performed .
these results were quite different from those 
 obtained from the replicate samples of the microscopic positive patients , which could be 
 confirmed with a high degree of reproducibility using both pcr - based protocols . as in the endemic areas where the cost of diagnostic tests becomes a limiting factor , we 
 decided to use the simplest and most economical format of rt - pcr in this study , which 
 relied on the detection of the fluorescent reporter sybr green i. while the sensitivity 
 of sybr green is similar to other rt chemistries , the specificity of the reporter may be 
 misleading in some few cases due to variations in the amount of template ( parida 2008 ) . in the present study , it was possible 
 to rule out this potential limitation of the sybr - based protocols because rt - pcr was 
 100% specific , with no positive results from the negative controls ( nave volunteers ) . 

additionally , similar results were obtained with both the nested - pcr and rt - pcr methods , 
 suggesting the low variability between these two assay platforms based on the 18ssu rrna 
 gene .
it is important to emphasise that the reproducibility of the sybr green protocols 
 could be due to variations in the t 
 m that impact the melting curves , for example .
however , this does not seem to be 
 the case here , as the optimisation of the pcr reactions resulted in a very small 
 variation in the tm values . additionally , based on our positive panel 
 of samples , which included a wide - range of parasitaemia ,
finally , the low reproducibility for detecting 
 submicroscopic parasitaemia was not exclusive to the sybr green protocol , but was also 
 observed with the gold - standard nested - pcr . in conclusion , we agree with others that 
 protocols based on sybr green are more cost efficient in endemic areas than the 
 alternatives that use internal fluorogenic probes ( moreira et al .
concerning mixed - species infections , while the nested - pcr method identified five dual 
 infections of p. falciparum and p. vivax , the rt 
 method only identified two .
because the rt - pcr protocol used here was based on single 
 consensus primers , this result suggests that the mixed infection was misidentified ; 
 however , this rt - pcr protocol has been used to amplify mixed - malaria infections ( mangold et al . 2005 ) . because a comparison between 
 the pcr protocols was not the focus of this paper
the results concerning the poor performance of the pcr methods for the identification of 
 submicroscopic malaria infections corroborate previous observations of the 
 irreproducibility of parasite detection in samples with very low levels of parasitaemia 
 ( singh et al .
this result means that any 
 pcr template diluted past a certain threshold copy number will experience large 
 variations in amplification .
the absence of amplification at very low levels of 
 parasitaemia was not related to the dna extraction protocol because , as an internal 
 control , a sequence of the abo human genome was amplified in all of the tested samples . 

although relevant , this internal control did not exhibit the specific amplification 
 difficulties derived from field samples with low amounts of parasite dna .
this inherent 
 limitation of pcr amplification , previously described as the monte carlo effect ( karrer et al .
1995 ) , has been widely underestimated 
 ( stenman & orpana 2001 , soong & ladnyi 2003 ) .
in fact , specific 
 parasite pcr amplification at very low parasite densities may be hindered by several 
 distinct factors , including the biological sample itself ( guescini et al .
2007 ) 
 and the pcr reagents , even when using premade master mixes from the same manufacturer ( 
 bustin 2002 ) . regarding the scarcity of the template ,
it is clear that the likelihood of detecting 
 parasites present at very low levels decreases as the volume of blood analysed 
 decreases .
thus , for the detection of submicroscopic infections , it has been proposed 
 that the dna templates to be analysed by pcr should correspond to at least 5 l of the 
 whole blood ( proux et al .
2011 ) . in the current 
 paper , to avoid this potential limitation , both pcr protocols were performed with 2 l 
 of template , which corresponded to approximately 6 l of the whole blood collected from 
 the patient . because the use of templates isolated from substantially higher volumes of 
 blood might encounter difficulties due to the high quantities of human dna , blood 
 filtration methods for the removal of leukocytes
have been proposed to increase the pcr 
 sensitivity during malaria vaccine clinical trials ( andrews et al . 2005 , bejon et al .
2006 
 ) . after performing a multistep protocol , during which 5 ml of blood was filtered to 

( 2005 ) 
 established a pcr cut - off point of 20 parasites / ml to follow the vaccinated volunteers 
 in the phase ii trials .
taken together , these results mean that the most commonly used 
 pcr methods might not accurately detect subclinical malaria infections
. these findings 
 could be particularly relevant when studying malaria in low transmission settings , where 
 a negative pcr result may indicate that the individual is truly negative or may be a 
 consequence of insufficient sensitivity of the pcr tests . at this time , it is not 
 possible to differentiate between these two possible outcomes as the validity of pcr has 
 not been adequately assessed for asymptomatic or microparasitaemic individuals ( stresman et al .
although the results described here show a clear inconsistency of the pcr results in 
 sub - patent malaria infections , the current results have limitations .
the absence of 
 similar studies performed with field samples makes it hard to compare our results with 
 other researchers ; in fact , the consistency of the pcr results in the submicroscopic 
 carriers has never been properly assessed , especially in field settings where sample 
 collection and storage may not be optimal ( stresman et 
 al . 2012 ) .
additionally , the amplification of scarce dna templates by any 
 pcr - based protocol requires a very high standard of laboratory practice . because we have 
 well - trained and qualified personnel and we followed the requirements of the 
 international organization for standardization/ international electrotechnical 
 commission 17025 , it was possible to minimise technical problems , such as those related 
 to the adequate manipulation of the devices used in the pcr assays .
consequently , at 
 present , the application of pcr - based methods is restricted to well - equipped 
 laboratories with specially trained technicians . to develop new strategies to manage the 
 parasite - negative individuals , pcr capacity building programs are underway in several 
 african countries with the help of the malaria clinical trials alliance ( malera 2011 ) .
unfortunately , an initiative of this 
 magnitude has not yet been initiated in latin america , where p. vivax 
 is the predominant species .
it is critical to achieve a sufficient sample size when studying areas with sub - patent 
 malaria infections in order to have an adequately powered study . in this context
, the 
 size of our sample set could not be increased in the colniza area ; the geographical 
 access and political conflicts , which often affect the gold - mining areas of the 
 brazilian amazon , hampered the continuation of our study there .
accordingly , the malera 
 initiative stated that sufficiently powered studies are difficult to implement in 
 settings with low malaria prevalence and this challenge could adversely impact malaria 
 control ( malera 2011 ) .
our data show that conventional pcr protocols based on the 18ssu rrna 
 plasmodial gene do not allow for the accurate detection of submicroscopic malaria 
 infections .
consequently , pcr results require careful interpretation when the population 
 of interest includes microparasitaemic individuals .
currently , no pcr protocol is able 
 to bypass the lack of pcr reproducibility at low levels of parasitaemia .
although some 
 new protocols have promise , such as loop - attenuated isothermal amplification , these 
 protocols have not yet been adequately tested in community - based studies or in the 
 detection of low - density parasitaemia ( han 2013 
 ) .
thus , studies are required to decrease the current limit of detection in the pcr 
 assays in low - transmission settings .
furthermore , we agree with researchers that these 
 protocols should be validated as a useful tool for the accurate detection of 
 submicroscopic malaria infection , through comparative multicentre studies and this step 
 is essential before these protocols are put into practice ( proux et al
 the polymerase chain reaction ( pcr)-based methods for the diagnosis of malaria 
 infection are expected to accurately identify submicroscopic parasite carriers . 
 
 although a significant number of pcr protocols have been described , few studies have 
 addressed the performance of pcr amplification in cases of field samples with 
 submicroscopic malaria infection . here , the reproducibility of two well - established 
 pcr protocols ( nested - pcr and real - time pcr for the plasmodium 18 
 small subunit rrna gene ) were evaluated in a panel of 34 blood field samples from 
 individuals that are potential reservoirs of malaria infection , but were negative for 
 malaria by optical microscopy . 
 regardless of the pcr protocol , a large variation 
 between the pcr replicates was observed , leading to alternating positive and negative 
 results in 38% ( 13 out of 34 ) of the samples . 
 these findings were quite different 
 from those obtained from the microscopy - positive patients or the unexposed 
 individuals ; the diagnosis of these individuals could be confirmed based on the high 
 reproducibility and specificity of the pcr - based protocols . 
 the limitation of pcr 
 amplification was restricted to the field samples with very low levels of 
 parasitaemia because titrations of the dna templates were able to detect < 3 
 parasites/l in the blood . in conclusion , 
 conventional pcr protocols require careful 
 interpretation in cases of submicroscopic malaria infection , as inconsistent and 
 false - negative results can occur . 
SUBJECTS, MATERIALS AND METHODS
RESULTS
DISCUSSION