INTRODUCTION
Mass spectrometry was developed as an analytical technique to determine mass to charge (m/z) ratio of chemical compounds (Aebersold and Mann, Reference Aebersold and Mann2003). With time, numerous variants of this technique evolved, which were based on different methods of ionization and detection systems. Of these, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) was found to be highly suitable for analysis of biological molecules (Momo et al. Reference Momo, Povey, Smales, O'Malley, Montague and Martin2013). In MALDI-TOF MS process, the sample or the analyte is co-crystallized within a matrix solution and ionized by irradiating with a laser beam. The matrix solution absorbs the photonic energy of laser beam, effectively desorbing and ionizing the biological molecules. The ionized biomolecules are then accelerated at a fixed potential through the fixed length of a flight tube. A detector located at the end of the flight tube measures very precisely the time of flight (TOF) of the molecular ions. The TOF of a particular molecular ion is decided by the degree of ionization, as well as its molecular mass. Based on the TOF information, a characteristic spectrum, unique for a given biomolecule is generated, which constitutes its specific fingerprint.
MALDI-TOF MS can simultaneously detect various molecules of different masses. Various researchers have therefore reported the usefulness of MALDI-TOF mass spectra of protein fragments (called peptide mass fingerprints – PMF) derived from intact cells of bacteria, viruses and fungi for their rapid and reliable identification (Fenselau and Demirev, Reference Fenselau and Demirev2001; Croxatto et al. Reference Croxatto, Prod'hom and Greub2012; Nenoff et al. Reference Nenoff, Erhard, Simon, Muylowa, Herrmann, Rataj and Gräser2013). Accordingly, the use of intact cell mass spectrometry-based molecular-phenotypic identification has been successfully reported for diagnosis of various bacterial, viral and fungal diseases (Emonet et al. Reference Emonet, Shah, Cherkaoui and Schrenzel2010; Downard, Reference Downard2013; Singhal et al. Reference Singhal, Kumar, Kanaujia and Virdi2015). Depending on the cellular composition and architecture of different groups of microorganisms, researchers have evaluated different methods of sample preparation ranging from intact-cell to protein extraction-based methodologies. Both, Gram positive and Gram negative bacteria have been identified using ‘direct bacterial profiling’, whereby a single bacterial colony spotted on the MALDI plate is over layered with matrix solution and/or a ‘preparatory extraction’ with formic acid (Ilina et al. Reference Ilina, Borovskaya, Malakhova, Vereshchagin, Kubanova, Kruglov, Svistunova, Gazarian, Maier, Kostrzewa and Govorun2009; Stephan et al. Reference Stephan, Cernela, Ziegler, Pflüger, Tonolla, Ravasi, Fredriksson-Ahomaa and Hächler2011). Specialized procedures for sample preparation have been reported for Nocardia, Actinomycetes and Mycobacteria (Clark et al. Reference Clark, Kaleta, Arota and Wolk2013). These involve boiling of Nocardia and Actinomycetes in water to promote cellular lysis, precipitation of proteins by ethanol, air drying and resuspension in a solution of formic acid and acetonitrile before spotting the sample on MALDI plate (Verroken et al. Reference Verroken, Janssens, Berhin, Bogaerts, Huang, Wauters and Glupczynski2010). Since biosafety is a major concern while working with Mycobacteria, a procedure combining inactivation and lysis was reported to be appropriate for identification of Mycobacteria (El Khéchine et al. Reference El Khéchine, Couderc, Flaudrops, Raoult and Drancourt2011). For this, bacterial colonies collected in screw-cap tubes containing water and Tween 20 were inactivated by heating and vortexed with glass beads to facilitate complete cellular disruption. These were finally suspended in a solution of formic acid-acetonitrile before spotting on the MALDI plate (El Khéchine et al. Reference El Khéchine, Couderc, Flaudrops, Raoult and Drancourt2011). For identification of fungi, various methods of sample preparation have been used based on both chemical extraction and mechanical lysis of fungal hyphae and spores (Hettick et al. Reference Hettick, Green, Buskirk, Kashon, Slaven, Janotka, Blachere, Schmechel and Beezhold2008; Lau et al. Reference Lau, Drake, Calhoun, Henderson and Zelazny2013).
After an exhaustive study of the published literature we realized that MALDI-TOF MS has also been used for identification of protozoan parasites like Leishmania spp., Giardia spp., Cryptosporidium spp., ticks and fleas. The major aim of this review is to collate the information, howsoever limited, for applications of MALDI-TOF MS for identification of disease-causing human parasites. The usefulness and the constraints of this new technology in expanding our knowledge about identification of parasites are also discussed.
APPLICATIONS IN PARASITOLOGY
The parasites, which are known to cause diseases in human beings belong to three main categories: protozoa, helminths and ectoparasites. Protozoans, which are unicellular eukaryotes, are known to cause several diseases in humans such as malaria, leishmaniasis, amoebiasis, giardiasis, sleeping sickness etc. Helminths commonly known as worms are multicellular organisms. Many, but not all, live in the digestive tract of man and animals from where they may invade to other organs of the body, as part of their life-cycle. Ectoparasites e.g. ticks, mites and fleas are multicellular organisms, which live on the body surface of the host. The diagnosis of parasitic diseases is conventionally done by microscopic examination of blood, tissue and excreta samples of the host. The sample preparation for microscopic examination is labor-intensive, time consuming procedure and requires highly trained personnel for correct diagnosis. When tissue/biological samples are not available, or when patients exhibit low parasitaemia and/or are asymptomatic, diagnosis might be carried out using immunological assays (Ndao, Reference Ndao2009). The polymerase chain reaction (PCR)-based diagnostic assays are more specific and sensitive than both microscopic and immunological assays. These can detect parasites even when patients exhibit low parasitaemia and/or are asymptomatic (Mens et al. Reference Mens, Spieker, Omar, Heijnen, Schallig and Kager2007). The relative advantages and disadvantages of conventional and modern methods in diagnosis of various parasites have been shown in Table 1.
Table 1. Advantages and disadvantages of conventional and modern methods in clinical parasitology
With recent advances in proteomics, MALDI-TOF MS has been proposed as an alternative approach for identification of bacterial and fungal pathogens (Fenselau and Demirev, Reference Fenselau and Demirev2001; Emonet et al. Reference Emonet, Shah, Cherkaoui and Schrenzel2010; Croxatto et al. Reference Croxatto, Prod'hom and Greub2012; Downard, Reference Downard2013; Nenoff et al. Reference Nenoff, Erhard, Simon, Muylowa, Herrmann, Rataj and Gräser2013; Singhal et al. Reference Singhal, Kumar, Kanaujia and Virdi2015). The potential of this technology for accurate identification of many parasites has also been reported. The parasites which have been identified using MALDI-TOF MS have been enlisted in Table 2.
Table 2. Summary of the methods used for sample preparation of parasites for their identification by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)
MALDI-TOF MS IN IDENTIFICATION OF DIAGNOSIS OF PROTOZOAN PARASITES
Leishmania
Leishmaniasis is a vector-borne protozoan parasitic disease, the clinical spectrum of which might range from mild cutaneous leishmaniasis to life-threatening visceral leishmaniasis (Dedet and Pratlong, Reference Dedet, Pratlong, Cook and Zumla2009). Clinical presentation of the disease is influenced by the species of the genus Leishmania infecting the patient (Herwaldt et al. Reference Herwaldt, Arana and Navin1992). Also, different species of Leishmania produce disease symptoms of varied severity and respond to therapy in their own unique way. Currently, no vaccine is available for leishmaniasis, hence early identification of the infecting Leishmania species, and initiation of appropriate chemotherapy is the only way to control this disease. The conventional methods for diagnosis of infection are based on direct examination of smear and culture, which requires expertise. Culturing is labor-intensive, and the results are available only after weeks. The gold standard method for identification of Leishmania species, which has been recommended by WHO is multi-locus enzyme electrophoresis (MLEE). MLEE uses the relative electrophoretic mobilities of intracellular enzymes for characterization and differentiation of different organisms by generating their specific electromorph types. But this method is very labor-intensive, expensive and requires culturing of sufficient quantity of parasites, which may take several weeks (Pratlong et al. Reference Pratlong, Dereure, Ravel, Lami, Balard, Serres, Lanotte, Rioux and Dedet2009). Hence, this method is confined to reference laboratories only.
The molecular methods based on real-time PCR, PCR amplification followed by analysis using restriction fragment length polymorphism or multilocus sequence typing, or sequencing of multiple genes (Mary et al. Reference Mary, Faraut, Lascombe and Dumon2004; Rotureau et al. Reference Rotureau, Ravel, Couppié, Pratlong, Nacher, Dedet and Carme2006; Montalvo et al. Reference Montalvo, Fraga, Monzote, Montano, De Doncker, Dujardin and Van der Auwera2010) permit investigation of clinical samples, small-sized cultures and yield results in one or 2 working days (Foulet et al. Reference Foulet, Botterel, Buffet, Morizot, Rivollet, Deniau, Pratlong, Costa and Bretagne2007).
Recently, a few studies have reported the use of MALDI-TOF MS for identification of various Leishmania species from in vitro cultures (Cassagne et al. Reference Cassagne, Pratlong, Jeddi, Benikhlef, Aoun, Normand, Faraut, Bastien and Piarroux2014; Culha et al. Reference Culha, Akyar, Yildiz Zeyrek, Kurt, Gündüz, Özensoy Töz, Östan, Cavus, Gülkan, Kocagöz, Özbel and Özbilgin2014; Mouri et al. Reference Mouri, Morizot, Van der Auwera, Ravel, Passet, Chartrel, Joly, Thellier, Jauréguiberry, Caumes, Mazier, Marinach-Patrice and Buffet2014). Mouri et al. (Reference Mouri, Morizot, Van der Auwera, Ravel, Passet, Chartrel, Joly, Thellier, Jauréguiberry, Caumes, Mazier, Marinach-Patrice and Buffet2014) described a sample preparation method for accurate and efficient identification of various Leishmania species by MALDI-TOF MS. Needle aspirates of skin lesions from infected patients were cultured in suitable medium for a few days and observed under microscope for motile promastigotes. Positive promastigote cultures were washed in pure water, re-suspended in water and ethanol and centrifuged. The residual pellet was suspended in a solution of formic acid and acetonitrile, vortexed and centrifuged. The supernatant was spotted on the MALDI plates and after the sample had air dried; it was over layered with the matrix solution (α-cyano-4- hydroxycinnamic acid, CHCA). Analysis of PMF of each sample revealed that isolates could be identified down to the subgenus level of Viannia or Leishmania, and also to the species level based on the presence/absence of mutually exclusive peaks in their PMFs. The researchers confirmed that identification of Leishmania spp. by MALDI-TOF MS was comparable with reference methods in specificity and sensitivity (Mouri et al. Reference Mouri, Morizot, Van der Auwera, Ravel, Passet, Chartrel, Joly, Thellier, Jauréguiberry, Caumes, Mazier, Marinach-Patrice and Buffet2014).
Another research group described a simple method of sample preparation for identification of human species of Leishmania by MALDI-TOF MS. The cryopreserved isolates procured from reference centers were thawed and washed with Rosewell Park Memorial Institute (RPMI) medium and incubated in the traditional Novy-MacNeal-Nicolle medium followed by incubation in RPMI medium for 1 week each. The motile promastigotes were harvested from RPMI medium, washed in saline solution and 1 µL of the saline promastigote solution was spotted on the MALDI plate and air dried. These were over layered with matrix solution (CHCA) and analysed. The researchers recorded PMFs of each isolate and constructed a reference database of main species of Leishmania which inflict humans. Applying the same procedure of washing a rich culture of promastigotes with saline, they successfully identified 66 of the 69 strains of Leishmania isolated from clinical samples (Cassagne et al. Reference Cassagne, Pratlong, Jeddi, Benikhlef, Aoun, Normand, Faraut, Bastien and Piarroux2014).
In an another study, investigators (Culha et al. Reference Culha, Akyar, Yildiz Zeyrek, Kurt, Gündüz, Özensoy Töz, Östan, Cavus, Gülkan, Kocagöz, Özbel and Özbilgin2014) employed the same sample preparation method for MALDI-TOF MS, as used by Mouri et al. (Reference Mouri, Morizot, Van der Auwera, Ravel, Passet, Chartrel, Joly, Thellier, Jauréguiberry, Caumes, Mazier, Marinach-Patrice and Buffet2014) and accurately identified Leishmania spp. which were isolated from Turkish patients after in vitro culture.
Although, all the researchers reported that MALDI-TOF MS was highly successful in identification of Leishmania spp., most of them reported that the need for cultivation of parasites before identification was a major hinderance for integration of this technique in clinical diagonostics.
Enteric protozoans
Water-borne protozoan parasites are usually diagnosed by microscopic examination of stool or water samples, which can neither predict the species nor indicate the viability of the infecting protozoans (U.S. Environmental Protection Agency, 1999). In research laboratories, the application of MALDI-TOF MS to human intestinal parasites has been limited to obtaining general parasitic proteome data and discovery of novel biomarkers (Thézénas et al. Reference Thézénas, Huang, Njie, Ramaprasad, Nwakanma and Fischer2013). The first study which reported the use of MALDI-TOF MS for identification of Cryptosporidium parvum – a chlorine resistant protozoan parasite was carried out in 2000 (Magnuson et al. Reference Magnuson, Owens and Kelty2000). Cryptosporidium parvum is found in surface waters and causes cryptosporidiosis, a parasitic infection known characterized by intractable or persistent diarrhoeal illness and potential mortality among immunocompromised individuals. Magnuson et al. (Reference Magnuson, Owens and Kelty2000) investigated two types of C. parvum oocysts for analysis by MALDI-TOF MS – whole-oocysts and freeze-thawed oocysts.The oocysts were obtained from fecal samples of experimentally infected mice. For whole-oocyst analysis, the samples were extensively washed with de-ionized water and spotted on the MALDI plate. For samples subjected to MALDI analysis after freeze-thaw, washed oocyst samples from above were alternately frozen (in liquid nitrogen) and thawed (at 60 °C) for 1 min each for five times. The sample was centrifuged and the supernatant was spotted on the MALDI plate. Before the sample dried, it was over layered with matrix solution containing 3, 5-dimethoxy-4-hydroxy-cinnamic acid and left for air-drying. Another 0·25 ml of matrix solution was applied over it and mass spectra were acquired. The researchers observed that the PMFs of Cryptosporidium oocysts, which were both washed and freeze-thawed were highly reproducible and had increased sensitivity. They further compared the PMFs of C. parvum with that of Cryptosporidium muris – another member of the same genus and found that PMFs of C. parvum were quite distinct from those of C. muris. They further investigated oocysts from various lots and found that the same washing and freeze-thaw procedure gave highly reproducible mass spectra. They suggested that MALDI-TOF MS was a simple and rapid supplement method for quality controls for production of C. parvum oocysts (Magnuson et al. Reference Magnuson, Owens and Kelty2000). Later, Glassmeyer et al. (Reference Glassmeyer, Ware, Schaefer, Shoemaker and Kryak2007) described an improved method of sample preparation for acquiring mass spectra of C. parvum oocysts by MALDI-TOF MS. Intact oocysts obtained from fecal samples of experimentally infected mice washed thrice in high performance liquid chromatography (HPLC) grade water and finally suspended in it were used as the sample for analysis. They used 3, 5-dimethoxy-4-hydroxy-cinnamic acid as the matrix solution and suggested that if the oocyst sample was mixed with the matrix solution and allowed to stand for 45 min before spotting on the MALDI plate, it was easier to obtain spectra with more number of clear peaks.
Members of the genus Giardia are protozoan parasites, which cause giardiasis, characterized by asymptomatic illness to severe abdominal pain, acute diarrhea and sometimes death. Currently, the Giardia cysts are detected in water by microscopic examination, which can neither predict the species nor indicate the viability of the cysts (U.S. Environmental Protection Agency, 1999). An independent research group described a MALDI-TOF MS-based approach for identification of Giardia lamblia and Giardia muris from intact cysts (Villegas et al. Reference Villegas, Glassmeyer, Ware, Hayes and Schaefer2006). The intact cysts were washed thrice in distilled water and suspended in distilled water. An equal volume of the matrix solution (3, 5-dimethoxy-4-hydroxy-cinnamic acid) was mixed with the sample and incubated for 60 min, spotted on the MALDI plate and air-dried. Mass spectra were acquired for each isolate and it was observed that the mass spectral fingerprints of G. lamblia and G. muris were sufficiently distinct to allow their identification by MALDI-TOF MS (Villegas et al. Reference Villegas, Glassmeyer, Ware, Hayes and Schaefer2006).
The potential of MALDI-TOF MS has also been reported for identification of intestinal protozoan parasites of the genus Blastocystis from clinical samples (Martiny et al. Reference Martiny, Bart, Vandenberg, Verhaar, Wentink-Bonnema, Moens and van Gool2014). The pathogenicity of Blastocystis in humans has been related to different subtypes of this parasite (Souppart et al. Reference Souppart, Moussa, Cian, Sanciu, Poirier, Alaoui, Delbac, Boorom, Delhaes, Dei-Cas and Viscogliosi2010). Martiny et al. (Reference Martiny, Bart, Vandenberg, Verhaar, Wentink-Bonnema, Moens and van Gool2014) described the use of MALDI-TOF MS for identification and differentiation of various subtypes of Blastocystis, which are frequently present in clinical samples. Two methods of sample preparation were evaluated–ethanol/formic acid extraction and direct deposition. In ethanol/formic acid extraction procedure, parasite colonies were suspended in distilled water and absolute ethanol and centrifuged. The pellet was washed with distilled water and re-suspended in formic acid and pure acetonitrile, centrifuged and spotted onto the MALDI plate. In direct deposition method, parasite colonies were directly deposited on the MALDI plate without any pre-treatment. When air-dried, deposit in each method was covered with the matrix solution (CHCA). The researchers reported that the mass spectra of Blastocystis isolates, which were obtained after ethanol/formic acid extraction were better than direct deposition method. Their results indicated that MALDI-TOF MS might serve as a valuable tool for identification and determination of various subtypes of Blastocystis (Martiny et al. Reference Martiny, Bart, Vandenberg, Verhaar, Wentink-Bonnema, Moens and van Gool2014).
Entamoeba histolytica causes amebiasis, which ranks second after malaria as a common protozoan parasitic disease. Conventionally, the parasite is detected by microscopy and culture, which detect pathogenic Entamoeba histolytica and nonpathogenic Entamoeba dispar. Entamoeba dispar is a non-pathogenic species which is morphologically identical to E. histolytica (Tanyuksel and Petri, Reference Tanyuksel and Petri2003). The PCR and real-time PCR-based molecular methods can differentiate between the two Entamoeba species (Calderaro et al. Reference Calderaro, Gorrini, Bommezzadri, Piccolo, Dettori and Chezzi2006). Recently Calderaro et al. (Reference Calderaro, Piergianni, Buttrini, Montecchini, Piccolo, Gorrini, Rossi, Chezzi, Arcangeletti, Medici and De Conto2015) described the applicability of MALDI-TOF MS for identification and differentiation of E. histolytica and E. dispar grown both axenically and in xenic cultures from clinical samples. Proteins were extracted from cultures containing 106 trophozoites mL−1 using ethanol/formic acid extraction procedure and spotted on the MALDI plate. Mass spectral analysis of E. histolytica and E. dispar isolates showed presence of at least five peaks capable of clearly discriminating E. histolytica and E. dispar. The researchers reported that the main advantage of MALDI-TOF MS (detection limit 106 trophozoites g−1 of feces) over molecular methods such as real-time PCR (detection limit 10 trophozoites g−1 of feces) is that it is less laborious and inexpensive (excluding the instrument cost) for diagnosis of E. histolytica in using in vitro xenic cultures (Calderaro et al. Reference Calderaro, Piergianni, Buttrini, Montecchini, Piccolo, Gorrini, Rossi, Chezzi, Arcangeletti, Medici and De Conto2015).
MALDI-TOF MS IN IDENTIFICATION OF ECTOPARASITES
Ticks
Ticks are obligate parasites, which serve as vectors for many bacterial (Parola and Raoult, Reference Parola and Raoult2001), viral (Hubálek and Rudolf, Reference Hubálek and Rudolf2012) and protozoan pathogens (Gray et al. Reference Gray, Zintl, Hildebrandt, Hunfeld and Weiss2010). Since, certain tick species are specific vectors for certain microbial pathogens, an early identification of the tick species, which might have bitten an individual may give an early clue to the disease transmitted by it and initiation of post exposure therapy. Only, expert and experienced entomologists do morphological identification of the ticks’ species, and that too not in damaged specimens or specimens in immature stage of life-cycle (Parola and Raoult, Reference Parola and Raoult2001). Currently, PCR assays for distinguishing tick species in damaged specimens or in different stages of the development are not available (Yssouf et al. Reference Yssouf, Flaudrops, Drali, Kernif, Socolovschi, Berenger, Raoult and Parola2013). An elaborate review on advantages and disadvantages of morphological, molecular and MALDI-TOF MS-based techiques in identification of ticks has been recently published (Yssouf et al. Reference Yssouf, Almeras, Raoult and Parola2016).
Several research studies have shown the potential of MALDI-TOF MS for rapid and reliable identification of the tick species (Karger et al. Reference Karger, Kampen, Bettin, Dautel, Ziller, Hoffmann, Süss and Klaus2012; Yssouf et al. Reference Yssouf, Flaudrops, Drali, Kernif, Socolovschi, Berenger, Raoult and Parola2013, Reference Yssouf, Almeras, Terras, Socolovschi, Raoult and Parola2015a , Reference Yssouf, Almeras, Berenger, Laroche, Raoult and Parola b ). Karger et al. (Reference Karger, Kampen, Bettin, Dautel, Ziller, Hoffmann, Süss and Klaus2012) described the use of whole-animal mass spectrometry for reliable determination of various species of ticks. Adult ticks and nymphs homogenized separately in a plastic pestle were treated with guanidinium chloride solution and subject to sonification in a water bath. The sample was centrifuged and the extract was acidified with trifluoroacidic acid (TFA). The acidified extracts were concentrated and spotted directly on the MALDI plate and allowed to dry. Dried spots were overlaid with the matrix solution (CHCA) and again allowed to dry. Mass spectra were acquired for each sample and a reference database of spectra was constructed. Comparison and analysis of mass spectra of unknown samples with the reference database allowed determination of the species of the ticks. The method was reproducible and capable of identifying the correct species of the ticks when adults or nymphs or even larvae were used as the starting material. Cluster analysis indicated that the primary determinant of the MALDI mass spectra was the species. The developmental stages formed distinct clusters within the given species. It was observed that species identification of ticks was also possible using body parts and engorged ticks. Only a small, but substantial part of the tick's body (except leg specimens) was required to yield spectra sufficient for species determination.
Yssouf et al. (Reference Yssouf, Flaudrops, Drali, Kernif, Socolovschi, Berenger, Raoult and Parola2013) further described the use of MALDI-TOF MS for rapid identification of tick species using only leg specimens. The legs of the ticks were homogenized in formic acid and acetonitrile solution, centrifuged and the supernatant over layered with the matrix solution (CHCA), was deposited on the MALDI plate. Mass spectra were acquired and a reference database of spectra was constructed. Comparison of test samples against the spectral database revealed that all organisms obtained from wild or removed from patients were accurately identified using MALDI-TOF MS. The researchers observed that with the help of MALDI-TOF MS, identity of tick species could be established in less than an hour. They proposed a procedure by which MALDI-TOF MS could be used to identify ticks species and determine the presence of Rickettsia in them. Interestingly, the PMF profiles of protein extracts prepared from tick legs were successful in discriminating the infected and uninfected ticks (Yssouf et al. Reference Yssouf, Almeras, Terras, Socolovschi, Raoult and Parola2015a ). In another study the same research group reported that protein mixture of hemolymph recovered from distal portion of amputated leg of ticks mixed in a solution of formic acid and acetonitrile, spotted on the MALDI plate and overlaid with the matrix solution (CHCA) was mixed with the samples by pipetting was also useful for identification of tick species and the associated pathogens (Yssouf et al. Reference Yssouf, Almeras, Berenger, Laroche, Raoult and Parola2015b ).
Recently Rothen et al. (Reference Rothen, Githaka, Kanduma, Olds, Pflüger, Mwaura, Bishop and Daubenberger2016) reported the usefulness of MALDI-TOF MS for reliable identification of species of closely related afrotropical ticks. Two to eight legs of a tick were detached, placed in a microcentrifuge tube containing formic acid, homogenized and centrifuged. The supernatant was mixed with the matrix solution and spotted on the MALDI plate. The mass spectra profiles were highly successful in identifying the various tick species. The researchers reported that MALDI-TOF MS proved to be a reliable tool in identifying morphologically and genetically, highly similar tick species.
Fleas
Fleas are small insects, which serve as vectors for several pathogens in animals and humans like, Yersinia pestis (causes plague), Rickettsia typhi (causes murine typhus), Rickettsia prowazekii (causes rural epidemic typhus), Rickettsia felis (causes flea-borne spotted fever) and many species of human-disease causing Bartonella (Bitam et al. Reference Bitam, Dittmar, Parola, Whiting and Raoult2010). Fleas are identified at the species level by morphological examination with the help of reference (identification) keys. This requires great expertise and is a time consuming process, hence not suitable for identification of large population of fleas. Molecular methods like sequencing of the 18S rRNA gene are available (Michael, Reference Michael2002), but a PCR assay, which can distinguish flea species, or ideal PCR primers, which can identify the relevant gene fragments are still unavailable (Yssouf et al. Reference Yssouf, Flaudrops, Drali, Kernif, Socolovschi, Berenger, Raoult and Parola2013).
Yssouf et al. (Reference Yssouf, Socolovschi, Leulmi, Kernif, Bitam, Audoly, Almeras, Raoult and Parola2014) described the use of MALDI-TOF MS for rapid identification of various species of fleas using specimens from legs, heads only or body without abdomen. The specimens were homogenized in a solution of formic acid and acetonitrile, and centrifuged. Equal volumes of supernatant and matrix were spotted on the MALDI plate and mass spectra of each sample were acquired. Since the spectral profiles obtained from flea body without abdomen provided consistent and reproducible data, these were used to create the database of PMFs. Comparison of the test samples against the library of spectral database confirmed a rapid and reliable discrimination of flea species (Yssouf et al. Reference Yssouf, Socolovschi, Leulmi, Kernif, Bitam, Audoly, Almeras, Raoult and Parola2014).
Concluding remarks
Until sometimes back, for most microbiologists it was difficult to comprehend that MALDI-TOF MS might be suitable for identification of whole microorganisms, and replace the conventional methods used in routine clinical microbiological laboratories. But, the development of microorganism-databases, paved the way for progression of MALDI-TOF MS from a protein identification tool to a diagnostic technique. The regulatory approval of MALDI-TOF MS by government agencies like US Food and Drug Administration and China Food and Drug Administration for in vitro diagnosis has provided the required impetus for development and commercialization of this exciting new technology (Luo et al. Reference Luo, Siu, Yeung, Chen, Ho, Leung, Tsang, Cheng, Guo, Yang, Ye and Yam2015; Patel, Reference Patel2015).
It is quite obvious that research on application of MALDI-TOF MS in identification of various parasites has not advanced as it has for identification of bacteria, viruses and fungi. It would be worthwhile to recount some of the reasons underlying it. One of the primary reasons for this might be the complex biological nature of the parasite life-forms (oocyst/promastigote/trophozoites etc.) from which the protein sample may be extracted for MALDI-TOF MS analysis. Another reason might be the complex biology of the life cycles of the parasites as well as the diseases caused by them. For identification of parasites, the need to culture parasites in vitro is a major bottleneck for rapid MALDI-TOF-based identification of parasites (Mouri et al. Reference Mouri, Morizot, Van der Auwera, Ravel, Passet, Chartrel, Joly, Thellier, Jauréguiberry, Caumes, Mazier, Marinach-Patrice and Buffet2014). The MALDI-TOF MS method is currently not available for field diagnosis and is carried out only in laboratories due to the high costs involved in initial procurement of the instrument.
Despite such constraints, MALDI-TOF MS has nevertheless significant advantages over the morphological, immunological and molecular detection methods. The main advantages of MALDI-TOF MS are the rapidity and accuracy associated with this technology. Also, it does not require highly trained laboratory personnel (Graça et al. Reference Graça, Volpini, Romero, Oliveira Neto, Hueb, Porrozzi, Boité and Cupolillo2012). From the literature it is also clear that the technology is not constrained by sample size and contamination by the host proteins. Since, MALDI-TOF MS has a wide spectrum of applications, cheaper versions of this instrument would definitely increase its affordability and application in field diagnosis. The efforts are already underway to develop simplified versions of the instrument using microfluidics technology (Yang et al. Reference Yang, Chao, Nelson and Ros2012). There is no denying the fact that the proof of the principle for application of MALDI-TOF MS for identification of parasites has already been demonstrated.
ACKNOWLEDGEMENTS
The authors acknowledge the financial support received from Science & Engineering research Board of Department of Science & Technology, DU-DST-PURSE II grant and University R & D research grant to University of Delhi South Campus.
FINANCIAL SUPPORT
Financial assistance was received as Start up Research Grant for Young Scientists (SB/YS/LS-156/2014) from Science & Engineering Research Board of Department of Science & Technology (NS), DU-DST-PURSE II grant and University R & D research grant for doctoral and postdoctoral studies to University of Delhi South Campus (JSV).
