**3. Loop-mediated isothermal amplification**

This review is to introduce the principal concept of a new, advanced, and robust diagnostic method coupled with simplified visualization technique: loop-mediated isothermal amplification (LAMP) with improved sensitivity and specificity for the rapid and reliable detection of *Giardia* DNA.

The LAMP method is a one-step DNA amplification assay performed under isothermal conditions, for 60–120 min using *Bst* polymerase with strand displacement activity and three primer pairs recognizing eight distinct regions within EF-1α (elongation factor-1 alpha) gene for specific detection of *G. duodenalis* (**Figure 1**), producing a considerably high amount of DNA comparable to PCR. The LAMP reaction is carried out in a reaction mixture containing *Bst* polymerase, reaction buffer, primers, DNA template, and a fluorescent dye.

## **3.1. Primers**

LAMP employs two inner primers (FIP and BIP, with typical length of ~40–42 bp), which in turn consists of two parts each and two outer primers (F3, B3 typically length ~ 17–20 bp), which can recognize a total of six distinct regions within the target DNA (see **Figure 1**). The two loop primers employed, forward loop primer (LF) and backward loop primer (LB), were designed to accelerate the amplification reaction and to increase the detection efficiency [37]. In total, six primers recognize eight distinct sites of the target sequence, which can be seen in **Figure 1** indicated as forward (F), backward (B), and complementary (c). In detail, at the 3′ end, the F1c, F2c, and F3c sites are recognized and on the 5′ end, B1, B2, and B3 sites are recognized (**Figure 1**, **Table 1**). The role of F3 and B3 primers is similar to the ordinary and single domain primers used in PCR amplification. They recognize each one of the six regions resulting in amplification of the entire target DNA sequence.

The most common method for designing LAMP primers is the user-friendly online platform: Primer Explorer V4 software (http://primerexplorer.jp/e) running in Java Runtime Environment, a product of Eiken Chemical Co. Ltd. Additionally, Torres et al. developed an extendable LAMP signature design program called LAMP Assay Versatile Analysis (LAVA) necessary for a high-throughput informatics environment, implemented in Perl script with support modules [38]. And lastly, after the completion of the primer design, specificity of the outer primers (F3 and B3) has to be confirmed with a BLAST search (https://blast.ncbi. nlm.nih.gov/Blast.cgi) in the NCBI database. Several factors are crucial for the performance of LAMP amplification and for optimum primer combinations including GC content, melting temperature (Tm) value, distance between possible primer regions, the stability of primer ends, and ability of possible primers forming secondary structures.

#### **3.2. Mechanism behind the LAMP reaction**

The mechanism behind the LAMP reaction involves three major steps: an initial step, a cycling amplification step, and an elongation step.

**3. Loop-mediated isothermal amplification**

amplification of the entire target DNA sequence.

**3.2. Mechanism behind the LAMP reaction**

amplification step, and an elongation step.

detection of *Giardia* DNA.

114 Current Topics in Giardiasis

rescent dye.

**3.1. Primers**

This review is to introduce the principal concept of a new, advanced, and robust diagnostic method coupled with simplified visualization technique: loop-mediated isothermal amplification (LAMP) with improved sensitivity and specificity for the rapid and reliable

The LAMP method is a one-step DNA amplification assay performed under isothermal conditions, for 60–120 min using *Bst* polymerase with strand displacement activity and three primer pairs recognizing eight distinct regions within EF-1α (elongation factor-1 alpha) gene for specific detection of *G. duodenalis* (**Figure 1**), producing a considerably high amount of DNA comparable to PCR. The LAMP reaction is carried out in a reaction mixture containing *Bst* polymerase, reaction buffer, primers, DNA template, and a fluo-

LAMP employs two inner primers (FIP and BIP, with typical length of ~40–42 bp), which in turn consists of two parts each and two outer primers (F3, B3 typically length ~ 17–20 bp), which can recognize a total of six distinct regions within the target DNA (see **Figure 1**). The two loop primers employed, forward loop primer (LF) and backward loop primer (LB), were designed to accelerate the amplification reaction and to increase the detection efficiency [37]. In total, six primers recognize eight distinct sites of the target sequence, which can be seen in **Figure 1** indicated as forward (F), backward (B), and complementary (c). In detail, at the 3′ end, the F1c, F2c, and F3c sites are recognized and on the 5′ end, B1, B2, and B3 sites are recognized (**Figure 1**, **Table 1**). The role of F3 and B3 primers is similar to the ordinary and single domain primers used in PCR amplification. They recognize each one of the six regions resulting in

The most common method for designing LAMP primers is the user-friendly online platform: Primer Explorer V4 software (http://primerexplorer.jp/e) running in Java Runtime Environment, a product of Eiken Chemical Co. Ltd. Additionally, Torres et al. developed an extendable LAMP signature design program called LAMP Assay Versatile Analysis (LAVA) necessary for a high-throughput informatics environment, implemented in Perl script with support modules [38]. And lastly, after the completion of the primer design, specificity of the outer primers (F3 and B3) has to be confirmed with a BLAST search (https://blast.ncbi. nlm.nih.gov/Blast.cgi) in the NCBI database. Several factors are crucial for the performance of LAMP amplification and for optimum primer combinations including GC content, melting temperature (Tm) value, distance between possible primer regions, the stability of

The mechanism behind the LAMP reaction involves three major steps: an initial step, a cycling

primer ends, and ability of possible primers forming secondary structures.

**Figure 1.** Schematic representation of the three primer pairs recognizing in total eight distinct regions within the EF-1α (elongation factor-1 alpha) gene of *G. duodenalis*.

The simultaneous participation of all six primers is needed for the initial phase production of the starting structure. When the initial phase progresses and during cycling reaction, only the inner primers are used for strand displacement and DNA synthesis. Firstly, one inner FIP


**Table 1.** The sequences of the designed primers used for the EF-1α gene of *G. duodenalis* LAMP assays. (BIP) hybrid primer binds to the starting structure, producing the complementary DNA using *Bst* DNA polymerase. F3 (B3) primer binds immediately after the FIP (BIP) primer, displacing the newly synthesized DNA strand and releasing the target DNA or FIP (BIP)-linked complementary DNA strand. Because of the complementarity of F1c and F1 regions, *Bst* polymerase replaces the F3 site of target DNA sequence with F1c of newly released single strand and forms the initial stem loop-loop structure. Similarly and simultaneously, BIP and B3 primers bind to target DNA resulting in formation of single-stranded dumbbell-like starting structure with loops at both ends. The cycling amplification step uses the single-stranded dumbbell-like starting structure as starting material for further amplification in the LAMP reaction. Only the inner primers (FIP and BIP) are used during the cycling amplification step (**Figure 2**).

#### *3.2.1. Advantages and shortcomings of LAMP assay*

The LAMP assay tenders a spectrum of benefits compared to PCR. Even though PCR is sensitive, it has several intrinsic disadvantages, which limit its successful performance. For instance, the presence of inhibitors and other contents like humic acids interferes with environmental samples resulting in a negative impact on the reaction. PCR operates on the principle of denaturation, annealing, and elongation of DNA with a manifold series of repeated temperature changes. This requires an expensive electronically controlled thermal cycler. LAMP, however, runs under isothermal conditions (without complex variable), which only require a water bath or a heat block. Also, failure or not successful performance of the LAMP reaction due to inhibitors is excluded. Last but not least, the turbidity of positive reaction, which could be seen by naked eyes, obviates further visualization steps, e.g., gel electrophoresis (**Table 2**).

LAMP is considered to be field applicable as the read-out of this method is simplified and is based on naked eye visualization: (a) presence of turbidity in sample, (b) colorimetric change in the case of adding metal-ion indicators, (c) presence of fluorescence by adding DNA-intercalating dyes, and (d) confirmation by gel electrophoresis of the final LAMP products that appear as cauliflower-like structures with multiple loops. Recently, Nzelu et al. established a quick, one-step, single-tube LAMP assay combined with Flinders Technology Associates (FTA) card with pre-added malachite green as a direct sampling tool [39].

#### **3.3. Reaction mixture and reaction conditions**

**Target** *Giardia duodenalis* EF-1α

assemblage B (AF069570)

**Primer** 

**Primer sequences**

**Sequence length**

**Source/medium**

Water, feces, surface water, and

[39, 40]

116 Current Topics in Giardiasis

sewage samples

**Ref.**

**names**

F3 B3 FIP BIP

LB LF

*G. duodenalis* EF-1α gene

F3 B3 FIP BIP FLP BLP

> **Table 1.**

5′-GACGGCCAGACGCGCGAG-3'

5′-GCGGAGGGGCTTGTCGGTC-3'

The sequences of the designed primers used for the EF-1α gene of *G. duodenalis* LAMP assays.

5′-G TACTCGAAGGAGCGCTACGAC-GCCTTCTTCCAGCCGATG-3'

5′-CTGGACCGGGGACAACA-3′

5′-ATCATCTCGCCCTTGATCTCG-3′

5′- GCCGGGATCTCGAAGGAC-3'

5′-TCGGGATGTAGTCGAACTCC-3'

5′-T GACCTGGCCGTCGTCCATCTT-GCGACGCTCGCGAACA-3'

208 bp

Feces pet dogs

[41]

5′-GAAGAAGGCCGAGGAGTTCG-TTGTCGGACCTCTCCATGA-3′

5′-ATGGACGACGGCCAGG-3′

5′-CCCTCGTACCAGGGCATC-3′

5′-AGCCGATGTTCTTGAGCTGCTT-GTACTCGAAGGAGCGCTACG-3′

178 bp

> Two reaction mixtures have been reported so far for specific detection of *Giardia duodenalis*. The first protocol uses a buffer containing reagents incorporated in the laboratory, whereas the second protocol uses supplied buffer with *Bst* polymerase. It is recommended to use HPLC-purified primers, if not all, at least FIP and BIP as the primers for purity could be crucial for rapidity and reproducibility of amplification.

> The LAMP assay developed for first time during 2009 was carried out in a 25 μl reaction mixture containing 1.6 μM each of FIP and BIP, 0.2 μM each of F3 and B3, 0.8 μM each of LF and LB, 2.8 mM of dNTP, 1.6 M of betaine, 20 mM of Tris-HCl (pH 8.8), 10 mM of KCl, 10 mM

**Figure 2.** Simplified schematic representation of the major steps in the LAMP method and localization of the eight LAMP primers on target DNA sequence for specific amplification of EF1α gene of *G. duodenalis*.


**Table 2.** Advantages and shortcomings of LAMP assay in comparison to PCR.

**Figure 2.** Simplified schematic representation of the major steps in the LAMP method and localization of the eight

LAMP primers on target DNA sequence for specific amplification of EF1α gene of *G. duodenalis*.

118 Current Topics in Giardiasis

of (NH<sup>4</sup> )2 SO4 , 16 mM of MgSO4 , 0.2% Tween 20, and the DNA template (2 μl). The reaction mixture was heated at 95°C for 2 min and then chilled on ice. Next, 8 U *Bst* DNA polymerase large fragments were added followed by incubation at 63°C for 120 min and heating at 80°C for 7 min to terminate the reaction [40]. In a consecutive report, the primer concentration was as follows: 40 pmol each of FIP and BIP primers, 20 pmol each of LF and LB primers, and 5 pmol each of F3 and B3 primers [41, 42].

The second protocol was developed in 2013 wherein the LAMP assay was carried out in a 25 μl reaction mixture containing 10× *Bst*-DNA polymerase buffer, 1.6 M betaine, 2.5 mM each deoxynucleotide triphosphates, 8 mM MgSO4 , 0.2 μM each F3 and B3 primers, 1.6 μM each FIP and BIP, 0.8 μM each loop-F and loop-B, 8 U *Bst* DNA polymerase 1 μl of 10,000× concentrated SYBR Green I, and template DNA (2 μl). In this case, the mixture was incubated at 63°C for 60 min and then heated at 80°C for 10 min [43].

## **3.4. Specificity assessment of the LAMP assay**

The specificity of both aforementioned protocols was determined by testing DNA derived from *G. duodenalis* cysts and from phylogenetically related protozoan parasites. This includes *Cryptosporidium parvum*, *Trypanosoma brucei, Theileria parva, Toxoplasma gondii, Babesia bovis,* plankton biomass, and *G. duodenalis* assemblages A and B for the first protocol [40] and *Toxoplasma gondii, Neospora caninum, Cryptosporidium parvum, Eimeria tenella, and G. duodenalis* for the second protocol [43].

#### **3.5. Sensitivity assessment of the LAMP assay**

The sensitivity was assessed using 10-fold dilutions of genomic DNA, and the results demonstrated that LAMP successfully amplified 0.548 pg. DNA/tube (corresponding to ∼four cysts) for *G. duodenalis* assemblage B and 0.8 pg. DNA/tube (corresponding to ∼six cysts) for *G. duodenalis* assemblage A for the first protocol [40]. The detection limit for the second protocol was 10−4 ng/μl (0.1 pg/μl) and 10 times more sensitive than the PCR assay [43].

### **3.6. Sample collection and purification methods applied in combination with the LAMP**

During the development of LAMP methodology for the first time, Plutzer et al. applied it in 10 surface water samples and 15 sewage samples, all collected between 2004 and 2007 in Hungary and previously tested and identified as positive using ImmunoFluorescence Test (IFT) [40, 44]. They also used 10 human fecal samples from Hungarian human patients reported with gastroenteritis in 2007. All samples were amplified by PCRs targeting 18S rRNA [45], glutamate dehydrogenase (GDH) genes [46], triosephosphate isomerase (TPI) gene [47], and EF-1α LAMP. They found that 33 of 35 (94%) environmental and fecal samples were positive for *G. duodenalis* according to one or more of applied techniques. Here, we would like to emphasize that *G. duodenalis*-specific LAMP-amplified DNA was positive in 24 of 35 predefined positive samples, while 23 were positive for 18S rRNA, 15 for GDH, and only 3 for TPI (**Table 3**).

On a more extensive work, the same authors examined 132 aquatic bird fecal samples, collected from February to March 2008 in Hungary [41]. The fecal samples were placed in tubes using polystyrene spatulas and were homogenized in 50 ml of distilled water followed by sieving through 0.1 -mm pore size sieve. After centrifugation, 50 μl of fecal samples were subject to IFT and 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride [DAPI], whereas the remaining part underwent DNA extraction and was subject to 18S rRNA PCR and EF-1α LAMP. Altogether four fecal samples were positive for *Giardia* by IFT, five by PCR, and five by LAMP. Interestingly, *Giardia* in common was identified only in one sample with IFT. In none of the other cases was there a simultaneous/overlap identification of *Giardia* using LAMP or PCR. It is worth to mention that the quality of extracted DNA was assessed in this case with


1 Primers used according to [40].

each deoxynucleotide triphosphates, 8 mM MgSO4

**3.4. Specificity assessment of the LAMP assay**

**3.5. Sensitivity assessment of the LAMP assay**

for the second protocol [43].

120 Current Topics in Giardiasis

only 3 for TPI (**Table 3**).

at 63°C for 60 min and then heated at 80°C for 10 min [43].

each FIP and BIP, 0.8 μM each loop-F and loop-B, 8 U *Bst* DNA polymerase 1 μl of 10,000× concentrated SYBR Green I, and template DNA (2 μl). In this case, the mixture was incubated

The specificity of both aforementioned protocols was determined by testing DNA derived from *G. duodenalis* cysts and from phylogenetically related protozoan parasites. This includes *Cryptosporidium parvum*, *Trypanosoma brucei, Theileria parva, Toxoplasma gondii, Babesia bovis,* plankton biomass, and *G. duodenalis* assemblages A and B for the first protocol [40] and *Toxoplasma gondii, Neospora caninum, Cryptosporidium parvum, Eimeria tenella, and G. duodenalis*

The sensitivity was assessed using 10-fold dilutions of genomic DNA, and the results demonstrated that LAMP successfully amplified 0.548 pg. DNA/tube (corresponding to ∼four cysts) for *G. duodenalis* assemblage B and 0.8 pg. DNA/tube (corresponding to ∼six cysts) for *G. duodenalis* assemblage A for the first protocol [40]. The detection limit for the second pro-

tocol was 10−4 ng/μl (0.1 pg/μl) and 10 times more sensitive than the PCR assay [43].

**3.6. Sample collection and purification methods applied in combination with the LAMP**

During the development of LAMP methodology for the first time, Plutzer et al. applied it in 10 surface water samples and 15 sewage samples, all collected between 2004 and 2007 in Hungary and previously tested and identified as positive using ImmunoFluorescence Test (IFT) [40, 44]. They also used 10 human fecal samples from Hungarian human patients reported with gastroenteritis in 2007. All samples were amplified by PCRs targeting 18S rRNA [45], glutamate dehydrogenase (GDH) genes [46], triosephosphate isomerase (TPI) gene [47], and EF-1α LAMP. They found that 33 of 35 (94%) environmental and fecal samples were positive for *G. duodenalis* according to one or more of applied techniques. Here, we would like to emphasize that *G. duodenalis*-specific LAMP-amplified DNA was positive in 24 of 35 predefined positive samples, while 23 were positive for 18S rRNA, 15 for GDH, and

On a more extensive work, the same authors examined 132 aquatic bird fecal samples, collected from February to March 2008 in Hungary [41]. The fecal samples were placed in tubes using polystyrene spatulas and were homogenized in 50 ml of distilled water followed by sieving through 0.1 -mm pore size sieve. After centrifugation, 50 μl of fecal samples were subject to IFT and 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride [DAPI], whereas the remaining part underwent DNA extraction and was subject to 18S rRNA PCR and EF-1α LAMP. Altogether four fecal samples were positive for *Giardia* by IFT, five by PCR, and five by LAMP. Interestingly, *Giardia* in common was identified only in one sample with IFT. In none of the other cases was there a simultaneous/overlap identification of *Giardia* using LAMP or PCR. It is worth to mention that the quality of extracted DNA was assessed in this case with

, 0.2 μM each F3 and B3 primers, 1.6 μM

\* Modification of the manufacturer protocol: addition of ten 10-min freeze-thaw cycles after resuspension in lysis solution. \*\*Elution with 32-μl LAMP buffer [40 mmol l−1 Tris-HCL, 20 mmol l−1 KCl, 16 mmol l−1 MgSO4 , 20 mmol l−1 (NH<sup>4</sup> )2 SO4 , 0.2 v/v % Tween 20, 16 mol l−1 betaine, and 28 mmol l−1 each deoxynucleoside triphosphate].


**Table 3.** Results on evaluation studies of the sample collection and purification methods applied in combination with the LAMP in different matrices.

the inclusion of internal controls and identified that their amplification was unsuccessful in 17% of the samples of which nine were positive for *Giardia* by LAMP [41].

To clarify the role of sample inhibitors, 27 drinking water samples of 10–1000 L were collected over a 24-h time period using the ARAD filtration system and were spiked with 100 *G. duodenalis* cysts. The genomic DNA from the samples (water spiked with *G. duodenalis* cysts) was extracted and then EF-1α LAMP was performed. The results showed that LAMP reaction was not affected by inhibitors in any of the samples tested [42].

In total, 10 L Iranian surface water samples from two rivers, collected over a time period of 12 months, were filtered using 142 mm membrane filters and were comparably investigated using IFT, PCR targeting the GDH gene, and LAMP targeting the EF-1α gene. Prior to genomic DNA extraction using the QIAamp Mini Kit, all river water samples were purified through sucrose flotation. The prevalence of *G. duodenalis* cysts was 13 out of 20 water samples by IFT, 10 out of 20 by the GDH gene PCR, and 8 out of 20 by EF-1α gene LAMP assay [48]. Notably in this study, the recovery rate of the protocol was assessed in 5 L water samples, seeded with 5 and 10 cyst/L, and they reported that the mean recovery rate for *Giardia* cysts in the seeded water samples was 18% and all of them tested positive by PCR and LAMP analysis.

During 2015, Çiçek and Şakru used effectively *Giardia* LAMP assay in 39 human fecal samples obtained from Turkey [49]. They primarily screened the patient's fecal material microscopically in native and stained with lugol iodine method to determine the cyst density. After that, samples were subject to DNA extraction using QIAamp DNA Stool Mini Kit and tested for EF-1α gene using LAMP for *Giardia* and beta-giardin (bg) PCR. EF-1α gene LAMP and bg gene region PCR for detection of *G. intestinalis* were found positive in 32 (82%) and 19 (48.7%) of the cases, respectively. Interestingly, the authors stated a significant difference between patients with higher cyst density and lower cyst density (p = 0.0001) through the PCR positivity rate [49].

An existing literature documents the performance of EF-1α gene LAMP for detection of *Giardia* in environmental water samples in Germany [50]. The investigators of this study examined a wide palette of different water types and compared the effectiveness of three detection methods: IFT, PCR, and LAMP. A total number of 185 samples originated from influent and effluent wastewater treatment plants (WWTPs), surface waters, a recreational area, groundwater, untreated water from a drinking water plant, and tap water were analyzed during the period from July 2009 to January 2011. For the extraction of the genomic DNA of all sample types, QIAamp Mini Kit was used [50]. All the samples were investigated by three detection assays: IFT, 16S rRNA by PCR, and EF-1α gene by LAMP. The comparison of the three methods indicated better results with IFT compared with the DNA-based assays, among which the LAMP assay was more sensitive than the applied PCR for detection of *Giardia*. The ranking results were as follows: IFA over LAMP and LAMP over nested PCR (56.8 > 42.7 > 33.5%, respectively). Despite nonconcordance of the methods resulting from statistical calculations, the authors outlined differences considering analytical steps such as sample preparation, DNA extraction, and analytical targets. A further explanation closely related to the variable detection capabilities of the assays according to authors is that the samples might contain *G. duodenalis* assemblages other than A and B, which might not be detected by LAMP but may be detected by PCR and/or IFT. The authors in this case speculated a little further over data interpretation and concluded that another unambiguous factor for the superiority of IFT over the other methods is also possible as IFT detects at the taxonomic level of respective *Giardia* genera and the assemblages cannot be discriminated by this method.

Between 2012 and 2014, Koloren et al. collected 420 environmental and 120 drinking water samples from Turkey [51]. All samples were collected by Al<sup>2</sup> (SO4 ) 3 flocculation and were purified by sucrose flotation technique. DNA isolation was conducted in the purified samples according to QIAamp DNA Mini Kit protocol, and they investigated all samples using EF-1α gene LAMP, small subunit (SSU) rRNA, and GDH PCR. A total of 141 (58.7%), 125 (52.1%), and 120 (50%) were identified positive by each of the aforementioned methods, respectively [51].

Li et al. developed an alternative protocol, including new primer pairs detecting the EF-1α gene of *Giardia*, with potential application for clinical diagnosis of *G. lamblia* from dogs' feces. They collected feces from dogs and processed them by flotation technique with saturated zinc sulfate and purification by sucrose gradient solution. To obtain the genomic DNA template, purified cysts from all fecal samples were subject to QIAamp DNA Stool Mini Kit. The results of microscopy, PCR (performed with the outer primers B3 and F3 of the LAMP assay), and EF-1α gene LAMP for *Giardia* were compared and the results showed that 5 (6.9%) of the 72 dog fecal samples tested positive by microscopy, and 7 (9.7%) and 8 (11.1%) tested positive by PCR and LAMP, respectively [43].

Thoughtfully, we are describing the results of an investigation of Nago et al. (unfortunately, whose contents, preparation steps, and details of the full text are not at our disposal) [52]. They reported that they developed a LAMP assay capable of detecting 3.12 × 10−1 *G. lamblia* cysts per reaction in spiked fecal specimens. Out of the 19 spiked samples, 16 (84%) were successfully amplified by LAMP assay and resulted in positive readings. Furthermore, they attempted to ascertain the negative reaction result in three fecal samples, which is likely due to inhibition. For this, they investigated two specific parameters: dilutions of extracted DNA and addition of bovine serum albumin (BSA) to the LAMP reaction mixture. This modification seemed to yield positive results and to have positive effect on the occurrence of falsenegative readings.

#### **3.7. The current momentum toward LAMP**

the inclusion of internal controls and identified that their amplification was unsuccessful in

To clarify the role of sample inhibitors, 27 drinking water samples of 10–1000 L were collected over a 24-h time period using the ARAD filtration system and were spiked with 100 *G. duodenalis* cysts. The genomic DNA from the samples (water spiked with *G. duodenalis* cysts) was extracted and then EF-1α LAMP was performed. The results showed that LAMP reaction was

In total, 10 L Iranian surface water samples from two rivers, collected over a time period of 12 months, were filtered using 142 mm membrane filters and were comparably investigated using IFT, PCR targeting the GDH gene, and LAMP targeting the EF-1α gene. Prior to genomic DNA extraction using the QIAamp Mini Kit, all river water samples were purified through sucrose flotation. The prevalence of *G. duodenalis* cysts was 13 out of 20 water samples by IFT, 10 out of 20 by the GDH gene PCR, and 8 out of 20 by EF-1α gene LAMP assay [48]. Notably in this study, the recovery rate of the protocol was assessed in 5 L water samples, seeded with 5 and 10 cyst/L, and they reported that the mean recovery rate for *Giardia* cysts in the seeded

During 2015, Çiçek and Şakru used effectively *Giardia* LAMP assay in 39 human fecal samples obtained from Turkey [49]. They primarily screened the patient's fecal material microscopically in native and stained with lugol iodine method to determine the cyst density. After that, samples were subject to DNA extraction using QIAamp DNA Stool Mini Kit and tested for EF-1α gene using LAMP for *Giardia* and beta-giardin (bg) PCR. EF-1α gene LAMP and bg gene region PCR for detection of *G. intestinalis* were found positive in 32 (82%) and 19 (48.7%) of the cases, respectively. Interestingly, the authors stated a significant difference between patients with higher cyst density and lower cyst density (p = 0.0001) through the PCR positiv-

An existing literature documents the performance of EF-1α gene LAMP for detection of *Giardia* in environmental water samples in Germany [50]. The investigators of this study examined a wide palette of different water types and compared the effectiveness of three detection methods: IFT, PCR, and LAMP. A total number of 185 samples originated from influent and effluent wastewater treatment plants (WWTPs), surface waters, a recreational area, groundwater, untreated water from a drinking water plant, and tap water were analyzed during the period from July 2009 to January 2011. For the extraction of the genomic DNA of all sample types, QIAamp Mini Kit was used [50]. All the samples were investigated by three detection assays: IFT, 16S rRNA by PCR, and EF-1α gene by LAMP. The comparison of the three methods indicated better results with IFT compared with the DNA-based assays, among which the LAMP assay was more sensitive than the applied PCR for detection of *Giardia*. The ranking results were as follows: IFA over LAMP and LAMP over nested PCR (56.8 > 42.7 > 33.5%, respectively). Despite nonconcordance of the methods resulting from statistical calculations, the authors outlined differences considering analytical steps such as sample preparation, DNA extraction, and analytical targets. A further explanation closely related to the variable detection capabilities of the assays according to authors is that the samples might contain *G. duodenalis* assemblages other than A and B, which might not be detected by LAMP but may be detected by PCR and/or IFT. The authors in this case speculated

water samples was 18% and all of them tested positive by PCR and LAMP analysis.

17% of the samples of which nine were positive for *Giardia* by LAMP [41].

not affected by inhibitors in any of the samples tested [42].

ity rate [49].

122 Current Topics in Giardiasis

*G. duodenalis* is one of the most prominent waterborne parasite worldwide and causative agent for several outbreaks in developing, developed, and industrialized countries with fatal consequences, mostly affecting the weakest of the population [12, 13]. The lack of sanitation and health care in Third World nations where malnutrition due to scarcity of food is common leads to highest prevalence of giardiasis in the population. As is often the case, the most vulnerable population groups are also the worst affected: children under the age of 5 years, elderly, and immunocompromised people. Particularly, the mortality rate is correspondingly and shatteringly high among these groups. As a result, scientists and politicians should be encouraged to counteract this dilemma at all levels. Key measures not only include the establishment of appropriate hygiene measures and sanitary facilities and access to clean water but also, or in particular, the setting up of surveillance systems and monitoring programs.

As is often said, prevention is better than cure. However, scanning the objective slides with a microscope is time consuming and exhausting. Cysts could be covered in debris or if at all when available for examination, each cyst will have to be checked for different morphological characteristics, and therefore, skilled technicians are needed. Due to the visualization difficulties of microscopic readings from samples, significant progress has been made in molecular methods such as PCR and PCR-RFLP aiming at proper characterization of *G. duodenalis* into its different assemblages and subassemblages. Therefore, researchers are frequently confronted with the challenge of defining new methods, specific for rapid and accurate diagnosis and for tracking the source of contamination. This is necessary in order to provide efficient treatment and prevent grievance. Even though we have managed to overcome some of the upcoming obstacles, the presence of inhibitors, low sensitivity of molecular methods, and lack of interand in some cases intralaboratory standardization in PCR methods are the main reasons that urge scientists to develop further methods.

Water is worth protecting and is the most important nutrient. Contamination of water by *G. duodenalis* is a health risk to all of us. Infective stages of *Giardia* species are able to persist in the aquatic environment for months, which is also the major route of infection. The fast and reliable detection of the parasites and ability to trace its origin can curb the occurrence of larger outbreaks or epidemics premature or better, even avoid one.

With this chapter, we would like to emphasize how effective the innovative LAMP process is. It is worth to be presented to a large specialist audience: one because it offers many advantages over other detection methods and secondly as it is very efficient and easy to carry out without the need for expensive equipment. Moreover, in this case, it is irrelevant that test matrix available for analysis. The detection is easy in stool and tissue samples as well as in environmental samples, mud, and water.

The chapter summarizes all relevant information on the detection of *G. duodenalis* with the LAMP procedure and gives a comprehensive overview of the current state of the art. This is a collection of all available protocols related to the development and application of a simple field-usable method that can meet the needs for a quicker and objective readout for the diagnosis of giardiasis in water and feces. The LAMP assay is ranked among the most accurate molecular tools thanks to its high diagnostic sensitivity and specificity. The future utility of a simple portable device (tube scanner) in which both the amplification platform (heating block) and fluorescent detection unit for end point use (with the ability to acquire real time data) has been envisioned to be combined into a single unit for LAMP assay for the detection of *Giardia* infections.

At present, LAMP is entering the ranks of the recognized detection methods among the World Health Organization (WHO) collaborating centers on foodborne trematode infections. This has been achieved mainly after the reported diagnosis of *Opisthorchis viverrini* infection in stool samples by the use of the LAMP technique [53]. The establishment and the use of a commercial LAMP assay (TB-LAMP) for the detection of tuberculosis was the subject of the expert group meeting organized by the WHO in Geneva in May 2013, and they certified LAMP as a potential diagnostic test. During the last year, CDC-UGA had financially supported the development of RealAmp-LAMP platform for the accurate detection of *Plasmodium vivax* infections [54]. The LAMP is considered as a technology under development with potential for future application and is currently undergoing large-scale evaluation by the Foundation for Innovative new Diagnostics (FIND) [55] and Centers for Disease Control (CDC) [55]. The proposed method can be expanded to be a quick and specific alternative screening technique for other life-threatening pathogens such as Ebola virus, human papilloma virus (HPV), human immunodeficiency virus (HIV), hepatitis C, etc. Moreover, in case of outbreaks, it could help prevent progression to active disease through early detection in saliva and examine the distribution of pathogens in different body fluids during infectious and noninfectious phases.

The establishment of surveillance activities is the most important step for health care professionals in prevention and in case of outbreaks tracking the source of contamination as fast as possible. Therefore, we advocate for LAMP as a suitable tool, in which given this expense and the large number of ongoing projects addressing, there is clearly a need for the development of a fast, economic assay, and user-friendly approach to detect *G. duodenalis* by fastest possible processing.
