currently , the detection and diagnosis of parasite infections rely on several laboratory methods in addition to clinical symptoms , clinical history , travel history , and geographic location of patient .
the primary tests currently used to diagnose many parasitic diseases have changed little since the development of the microscope in the 15th century by antonie van leeuwenhoek .
furthermore , most of the current tests can not distinguish between past , latent , acute , and reactivated infections and are not useful for following response to therapy or for prognosis .    
recent developments in new diagnostic tools , however , have opened new avenues for a vast improvement in parasite detection .
firstly , a number of newer serology - based assays that are highly specific and sensitive have emerged , such as the falcon assay screening test elisa ( fast - elisa ) , dot - elisa     [ 2 , 3 ] , rapid antigen detection system ( rdts ) , and luciferase immunoprecipitation system ( lips )     .
secondly , molecular - based approaches such as loop - mediated isothermal amplification ( lamp ) , real - time polymerase chain reaction , and luminex   have shown a high potential for use in parasite diagnosis with increased specificity and sensitivity .
thirdly , proteomic technology has also been introduced for the discovery of biomarkers using tissues or biological fluids from the infected host .       
the aim of this review is to highlight the potential for these new technologies in parasite diagnosis . for convenience ,
the diagnostic tools offered by the cdc ( centre for disease control , atlanta , usa ) and the nrcp ( national reference centre for parasitology , montral , canada ) are also highlighted in tables 3 and 4 .
for many years , microscopy has been the only tool available for the detection of parasites through inspection of blood smears     [ 1014 ] , tissue specimens     [ 1517 ] , feces , lymph node aspirates     [ 18 , 19 ] , bone marrow , and even cerebrospinal fluid .
however , sample preparation for direct observation is time - consuming , labour intensive , and proper diagnosis depends on qualified laboratory technicians . in the case of slide reading ,
a second independent reading is preferable , but not always required for accurate diagnosis . if need be , divided readings are resolved by a third reader . in endemic regions , where resources are limited , this proves to be difficult and misdiagnosis can significantly impact patient care . in reality ,
all major intestinal helminth infections are still solely dependent on microscopy for diagnosis . as for other parasite infections ,
many are confirmed by the use of microscopy in conjunction to other methods of diagnosis including serology - based assays and more recently molecular - based assays .
in situations where biologic samples or tissue specimens are unavailable , serology alone is the gold standard for diagnosis .
serology - based diagnosis tools can be divided into two categories : antigen - detection assays and antibody - detection assays .
these include the enzyme - linked immunosorbent assay ( elisa ) , also called enzyme immunoassay ( eia ) , and all its derived tests such as the falcon assay screening test elisa ( fast - elisa ) and the dot - elisa .
other assays include the hemagglutination ( ha ) test , indirect or direct immunofluorescent antibody ( ifa or dfa ) tests , complement fixation ( cf ) test , and immunoblotting and rapid diagnostic tests ( rdts ) .         
although the ease of use and turnaround times for serologic assays are similar to microscopy , serology - based assays are more sensitive and specific .
it becomes important for individuals whose blood smears do not permit identification of the parasite ( e.g. , differentiating between babesia and plasmodium )   or for patients exhibiting low - parasitemia and/or who are asymptomatic ( e.g. , chagasic patients )     .
classifying an infected asymptomatic patient as negative could lead to transmission of the parasite during blood transfusions or organ transplants . in the case of fasciola infection , serology tests
have also been shown to be useful in the confirmation of chronic fascioliasis when egg production is low or sporadic     .
finally , having these tests readily available allows for the monitoring of parasite clearance following therapy .  
the falcon assay screening test elisa ( fast - elisa ) consists of using synthetic and recombinant peptides to evaluate antibody responses to an antigen . in the past
, the method has been applied to the study of malaria , fasciolosis     , schistosomiasis ( reviewed in ) , and taeniasis .
however , this technique is subjected to the same drawbacks as most serology - based tests . antibodies raised against a peptide from one parasite protein may cross - react with proteins from other species .
moreover , antibodies raised against a peptide may react in some assays but not in others and some regions of a peptide may be more immunogenic than others .
no recent studies have been published on the use of the fast - elisa for the diagnosis of parasitic infections .
the main difference between the regular elisa and the dot - elisa lies in the surface used to bind the antigen of choice . in the dot - elisa ,
the plastic plate is replaced by a nitrocellulose or other paper membrane onto which a small amount of sample volume is applied .
the choice of binding matrix greatly improved the specificity and sensitivity of the assay by reducing the binding of nonspecific proteins usually observed when plastic binding matrixes are used .
the dotted membrane is incubated first with an antigen - specific antibody followed by an enzyme - conjugated anti - antibody .
the addition of a precipitable , chromogenic substrate causes the formation of a colored dot on the membrane which can be visually read .
the benefits of this technique include its ease of use , its rapidity , and the ease of result interpretation .
it is fast , and cost - effective and more importantly can be used in the field ( e.g. , as a dipstick ) . for all these reasons ,
the dot - elisa has been and still is extensively used in the detection of human and animal parasitic diseases , including amebiasis , babesiosis , fascioliasis , cutaneous and visceral leishmaniasis , cysticercosis , echinococcosis , malaria , schistosomiasis , toxocariasis , toxoplasmosis , trichinosis , and trypanosomiasis ( all reviewed in ) . in the last few years , published studies have demonstrated the use of the dot - elisa for the detection of fasciola gigantica , haemonchus contortus , theileria equi , trypanosoma cruzi    , and trypanosoma brucei    . in the latter study
the researchers were able to demonstrate that the dot - elisa had better sensitivity and specificity than the elisa in the detection of antineurofilament and antigalactocerebrosides antibodies in cerebrospinal fluid of subjects infected with african trypanosomes .
they attributed the greater sensitivity and specificity of the dot - elisa to the use of the nitrocellulose membrane and showed that their assay was successfully reproducible in the field .
rapid antigen detection tests ( rdts ) based on immunochromatographic antigen detection have been implemented in many diagnostic laboratories as an adjunct to microscopy for the diagnosis of malaria .
rdts consist of capturing soluble proteins by complexing them with capture antibodies embedded on a nitrocellulose strip .
a drop of blood sample is applied to the strip and eluted from the nitrocellulose strip by the addition of a few drops of buffer containing a labeled antibody .
the antigen - antibody complex can then be visualized directly from the membrane .           since the appearance of the first rdts in the 990s , major improvements have been made to the technique , making the use of rdts in rural endemic regions feasible .
rdts are now rapid , stable at temperatures up to 40c , easy to use , and cost - effective thereby providing many advantages over traditional microscopic methods .
rdts are useful in the identification of p. falciparum and p. vivax infections but can not be used to identify p. malariae and p. ovale infections .
more than 80  rdts exist for the detection of either histidine - rich protein ( hrp ) specific to p. falciparum or species - specific isotypes of lactate dehydrogenase ( ldh ) .
malaria rdts have recently been introduced in african countries to help prevent misdiagnosis of malaria infections and to subsequently reduce the practice of presumptive treatment .
in fact , the tendency to treat slide - negative samples with antimalarials is still a common phenomenon .
this practice causes concern not only for the patient 's health care but also to the costs it generates in prescribing the more expensive antimalarial sulfadoxine / pyrimethamine and artemisinin - based combinations . finally , misuse of antimalarials could lead to the appearance of drug - resistant strains
the luciferase immunoprecipitation system ( lips ) is a modified elisa - based assay in which serum containing antigen - specific antibodies can be identified by measuring light production .
basically , an antigen of choice is fused to the enzyme reporter renilla luciferase ( ruc ) and expressed as a ruc - fusion in mammalian cells to allow for mammalian - specific posttranslational modifications .  
the crude protein extract is then incubated with the test serum and protein a / g beads . during the incubation ,
the ruc - antigen fusion becomes immobilized on the a / g beads , which allows the antigen - specific antibody to be quantitated by washing the beads and adding coelenterazine substrate and measuring light production     .            in recent years
, lips has been successfully applied for the identification of sera samples infected with strongyloides stercoralis ( using a ruc - nie fusion )      and loa loa ( using a ruc - llsxp-1 fusion )     .
some of the advantages of the lips technology include its rapidity and accuracy in detecting infected patients .
sensitivity is improved in part by the use of mammalian cells which produce fusion antigens free of contaminating bacterial proteins .
in addition , the strongyloides lips based on the nie antigen showed greater specificity than the elisa as no cross - reaction was observed with serum from filarial - infected subjects     .           
2008      were able to obtain 100% specificity and sensitivity when performing an lips assay based on the loa loa sxp-1 antigen with only a small - degree of cross - reactivity with a few onchocerca volvulus- and wuchereria bancrofti - infected patient sera . by decreasing the incubation times of a normal lips assay , they were able to minimize cross - reaction .
many of the o. volvulus sera samples tested as positive with the lips assay were negative using this 15-minute lips assay also called qlips . of interest for the application of this technique in the field
is the observation that blood obtained by finger - prick ( contaminated with red blood cells and other components ) did not interfere with the lips assay .
further studies will be useful in exploring and validating the accuracy and potential usefulness of the lips and qlips assays in the field .           
parasitic diseases such as amebiasis , cryptosporidiosis , filariasis , giardiasis , malaria ,   cysticercosis , schistosomiasis , and african trypanosomiasis do not have commercially or fda approved antibody detection tests for their diagnosis .
experimental results have been too variable due to the type of antigen preparations used ( e.g. , crude , recombinant purified , adult worm , egg ) and also because of the use of nonstandardized test procedures .
cross - reaction leading to false - positives and misdiagnosis is also a problem , especially in regions where more than one parasite is endemic . despite the fact that some parasites in south america share common epitopes , it is common to see coinfection with trypanosoma cruzi and leishmania species .
it is also a problem in africa , where cross - reactivity exists between filarial and other helminth antigens     . to a lesser extent but nonetheless important is the inability of antibody - detection tests to differentiate between past and currently active infections     .
furthermore , antibody - detection tests can not be used in parasitic infections that do not develop a significant antibody response .
this has been observed in some individuals carrying echinococcus cysts   or during cutaneous leishmaniasis ( http://www.dpd.cdc.gov/dpdx/html/leishmaniasis.htm ) .
similarly , in the case of african trypanosomiasis diagnosis , such tests are of limited use because seroconversion occurs only after the onset of clinical symptoms     .              for all these reasons ,
there is still a need to improve on the current diagnosis approaches available . since the advent of the polymerase chain reaction ( pcr )
, parasitologists have turned to molecular - based approaches in the hopes to better the existing diagnosis tools .
the many limitations of microscopy and serology - based assays have influenced parasitologists towards the use of gene amplification methods made possible with the advent of the polymerase chain reaction ( pcr ) . besides the traditional pcr , including nested and multiplexed pcr , we have seen the implementation of the real - time pcr ( rt - pcr ) for the detection of several parasitic infections .
newer technologies such as loop - mediated isothermal amplification and luminex - based assays have also emerged as possible new approaches for the diagnosis of parasitic diseases .          
molecular - based approaches based on nucleic acids offer greater sensitivity and specificity over the existing diagnostic tests .
they permit the detection of infections from very low parasitized samples including those from asymptomatic patient 's samples     .
moreover , multiplexed pcr allows for the detection of multiple sequences in the same reaction tube proving useful in the diagnosis of several parasitic infections simultaneously .
rt - pcr system unlike conventional pcr , allow for the quantification of the original template 's concentration through the use of various fluorescent chemistries , such as sybergreen , taqman probes , fluorescence resonance energy transfer ( fret ) , and scorpion primers .
this eliminates the need to visualize the amplicons by gel electrophoresis thereby greatly reducing the risk of contamination and the introduction of false - positives .
when multiplexed , rt - pcr allows for the high - throughput analysis of different sequences in one single - closed tube reaction . using multiplexed rt - pcr , shokoples et al .  
were able to identify the four human plasmodium species ( falciparum , vivax , malariae , and ovale ) in a single reaction tube even in very low parasitized samples . running
the multiplex assay not only reduced the cost per test but also allowed for a rapid turnaround time , the assay taking only three hours to complete .
it is a clear advantage over microscopy which is labour intensive and time - consuming with slow turnaround times especially during high - throughput settings .
similarly , multiplexed rt - pcr proved useful in differentiating drug - sensitive strains of malaria     .
this is important for proper antimalarial prescription . in another example , diez et al .     
were able to detect the presence of t. cruzi infection following heart transplants using pcr .
this allowed immediate treatment of the patients before reactivation of chagas disease could occur .  
these examples demonstrate that efficient and early diagnosis can directly impact patients care and that pcr - based approaches have the potential to help in making the right choice for treatment .            
although dna - based methods have shown excellent sensitivity and specificity , the introduction of these methods in daily laboratory practice is still uncommon especially in rural endemic regions .
in addition , as observed with many serology - based assays , pcr - based methods also suffer by the lack of standardization .
dna extraction , choice of primer sets , and use of various amplification protocols are all factors that may cause this diversification in results . adding an automated dna extraction step would certainly improve pcr assays for use in the diagnosis of parasitic diseases .
loop - mediated isothermal amplification ( lamp ) is a unique amplification method with extremely high specificity and sensitivity able to discriminate between a single nucleotide difference .
it is characterised by the use of six different primers specifically designed to recognise eight distinct regions on a target gene , with amplification only occurring if all primers bind and form a product . in the past , lamp has been successfully applied for the rapid detection of both dna and rna viruses such as the west nile   and sars viruses .
recently , parasitologists have adapted the lamp approach for the detection of several parasitic diseases including the human parasites entamoeba , trypanosoma    , taenia    , plasmodium    , and cryptosporidium    , the animal parasites theilera     and babesia    [ 178 , 179 ] , and even to the identification of vector mosquitoes carrying plasmodium     and dirofilaria immitis   parasites .
most of these studies have brought to light the many advantages of this method over the common pcr technique .            unlike a regular pcr reaction ,
lamp is carried out at a constant temperature ( usually in the range of 6065c ) .
this unique feature not only results in higher yields , but also eliminates the need to buy a thermal cycler and shortens the reaction time by eliminating time lost during thermal changes .
in addition , the reaction can be carried out without extracting the dna from the collected samples as shown in the case of rime , a nonautonomous retroelement found in trypanosoma brucei rhodesiense and t. b. gambiense    . in 35 minutes , using a simple water bath , rime lamp was able to detect both t. b. gambiense and t. b.   rhodesiense directly from blood , serum , and csf samples .
in addition to the above advantages , lamp reactions are easy to set up , and results can readily be assessed . the sample of interest is mixed with primers , substrates , and a dna polymerase capable of strand displacement in a microcentrifuge tube . during the reaction ,
large amounts of pyrophosphate ions are produced , leading to the formation of a white precipitate .
this turbidity is proportional with the amount of dna synthesized therefore one can assess the reaction by real - time measurement of turbidity or more importantly , simply through the naked - eye .             for all these reasons , the future adoption of lamp
furthermore , as more groups apply lamp to the field of parasitology , we will see the appearance of lamp - modified assays that meet specific detection needs .
for example , in a recent study on bovine babesia     , a multiplex - lamp ( mlamp ) assay was developed to simultaneously detect b. bovis and b. bigemina from dna extracted from blood spotted on filter paper .
similarly , han et al .      implemented a lamp assay based on the 18s rrna gene for the detection of the four human plasmodium species ( falciparum , vivax , malariae , and ovale ) .
lamp had a similar sensitivity and a greater specificity than nested pcr , yielding similar results but at a faster turnaround time .
their results are consistent with other studies demonstrating the rapidity and the improved specificity and sensitivity obtained using the lamp assay .
luminex technology is a bead - based flow - cytometric assay that allows the detection of various targets simultaneously ( http://www.luminexcorp.com/ ) .
the microsphere beads can be covalently bound to antigens , antibodies , or oligonucleotides that will serve as probes in the assay . up to 100 microspheres are available each emitting unique fluorescent signals when excited by laser therefore allowing the identification of different targets . adapted to the study of parasites ,
the luminex assay could identify multiple organisms or different genotypes of one particular organism during the same reaction utilizing very low volume .
the approach could prove useful in the study of antigenic diversity and drug - resistance alleles and for the diagnosis of parasitic diseases .          
luminex was applied to the study of cryptosporidium    . c. hominis and c. parvum can not be distinguished using antigen detection or serology assays .
only dna - based approaches have been successful in doing so by exploiting the single nucleotide difference in the microsatellite-2 region ( ml-2 ) of both species     .
ultimately dna sequencing is the diagnosis tool of choice but it is costly , labour - intensive and time - consuming . in a recent study , bandyopadhyay et al .      successfully detected and distinguished c. hominis and c. parvum in 143 dna extracts using luminex technology by using oligonucleotide probes specific to the ml-2 regions of each species .
turnaround time was about 5 hours making this assay not only much faster but also less expensive than pcr followed by dna sequencing .
it also proved to be 100% specific and more sensitive than a direct fluorescent antibody ( dfa ) test , a method routinely used to identify cryptosporidium and giardia species .
note that dfa can not differentiate between c.   hominis and c.   parvum .        
similarly in other research , luminex technology was able to detect all - blood stage parasite levels of the four human plasmodium species ( falciparum , vivax , malariae , and ovale ) simultaneously     .
this study demonstrated that luminex technology can improve the speed , the accuracy , and the reliability of other pcr methods .
for example , the need for gel electrophoresis to differentiate the ldr products representing the four human plasmodium species is eliminated .
second , all samples are handled simultaneously and continuously through a 96-well plate format from dna extraction all through data analysis .
finally , the high - throughput capability of the luminex system confers it a clear advantage over the use of labour - intensive microscopy for large scale studies . since proteins are the main catalysts , structural elements , signalling messengers , and molecular machines of biological tissues , proteomic studies are able to provide substantial clinical relevance .
proteins can be utilized as biomarkers for tissues , cell types , developmental stages , and disease states as well as potential targets for drug discovery and interventional approaches .
the next generation of diagnostic tests for infectious diseases will emerge from proteomic studies of serum and other body fluids .
recent advances in this area are attributable largely to the introduction of mass spectrometry platforms capable of screening complex biological fluids for individual protein and peptide  biomarkers .
 proteomic strategy can identify proteins in two ways : bottom - up and top - down approaches . in the former
, the proteins in a biological fluid are proteolytically shattered into small fragments that can be easily sequenced and the resultant spectra are compared with those in established peptide databases .
bottom - up strategies are difficult to quantitate and can not identify modified molecules ( e.g. , alternately spliced , glycosylated ) .
since each open reading frame in the human genome is thought to generate at least 10 modified proteins , this issue is a major limitation .          
top - down strategies seek to identify proteins and peptides ( and their natural variants ) in complex biological fluids .
two - dimensional ( 2d ) gel electrophoresis was first described in 1975 . with this method ,
proteins are resolved in the first dimension based on ph ( a process called isoelectric focusing ) and in the second dimension by their molecular weight .
this technique is labor intensive , and low throughput and requires large amounts of sample .
other techniques used for the expression analysis of proteins are matrix - assisted laser desorption ionization time - of - flight mass spectrometry ( maldi - tof ms ) , surface - enhanced laser desorption ionization time of flight mass spectrometry ( seldi - tof ms ) , liquid chromatography combined with ms ( lc  ms  ms ) , isotope - coded affinity tags ( icat ) , and isotope tags for relative and absolute quantification ( itraq ) .        
the development of automated , high - throughput proteomic technologies such as maldi - tof and seldi - tof ms has enabled large numbers of clinical samples to be analyzed simultaneously in a short time .
all proteomics - based diagnostic efforts seek to identify biomarkers that , alone or in combination , can distinguish between  case  and  control  groups .         the main limitation of seldi compared to maldi resides in the fact that seldi has lower resolution and lower mass accuracy . in addition , seldi is unsuitable for high molecular weight proteins ( > 100  kda ) and is limited to the detection of bound proteins on to the proteinchip array .      
the seldi , a derivation of maldi , allows sample binding to chemically active proteinchip surfaces .
several types of proteinchip arrays are available with differing abilities to bind proteins with different chemical ( anionic , cationic , hydrophobic , metallic , and normal phase ) or biological ( antibody , enzymes , receptors ) properties , thereby allowing the direct analysis of proteins from complex biological samples without the need for prior separation by 2d gel electrophoresis .
the output of the seldi is a spectrum of mass - to - charge ratios ( m : z values ) with their corresponding relative intensities ( approximating to relative abundance ) .       
seldi analyses were initially applied to the discovery of early diagnostic or prognostic biomarkers of cancer ( reviewed in ) .
recently , this technique has been applied to the study of serum biomarkers of infectious diseases such as severe acute respiratory syndrome     , african trypanosomiasis     , fascioliasis     , cysticercosis     , and chagas diseases ( ndao et al . , submitted ) .
such studies have focused on identifying a distinctive configuration of circulating serum proteins that are indicative of a specific pathophysiological state , a so - called  proteomic fingerprint .       
the real potential of proteomic fingerprinting is in its use as a discovery tool for novel biomarkers that can then be incorporated into simple bedside diagnostics based on affordable technologies such as immunologically based antigen - detection tests that could be implemented in dipstick or cassette formats .
 methods for the diagnosis of infectious diseases have stagnated in the last 2030 years . 
 few major advances in clinical diagnostic testing have been made since the introduction of pcr , although new technologies are being investigated . 
 many tests that form the backbone of the  modern  microbiology laboratory are based on very old and labour - intensive technologies such as microscopy for malaria . pressing needs include more rapid tests without sacrificing sensitivity , value - added tests , and point - of - care tests for both high- and low - resource settings . in recent years 
 , research has been focused on alternative methods to improve the diagnosis of parasitic diseases . 
 these include immunoassays , molecular - based approaches , and proteomics using mass spectrometry platforms technology . 
 this review summarizes the progress in new approaches in parasite diagnosis and discusses some of the merits and disadvantages of these tests . 
1. Introduction
2. Microscopy
3. Serology-Based Assays
4. Molecular-Based Approaches