A Serological and Molecular Study of Leishmania infantum Infection in Cats from the Island of Ibiza (Spain)

Abstract

The aim of this study was to evaluate the prevalence of Leishmania infantum infection within a feline population by serologic and molecular methods and to identify associated risk factors. One hundred five cats living outdoors were studied. Sera were tested for IgG antibodies against L. infantum, Toxoplasma gondii, and feline immunodeficiency virus (FIV) and for the detection of feline leukemia virus (FeLV) p27 antigen by enzyme-linked immunosorbent assay (ELISA). L. infantum real-time polymerase chain reaction (PCR) was performed on DNA extracted from blood. L. infantum and T. gondii seroprevalence rates were 13.2% and 55.2%, respectively. The prevalence of L. infantum by PCR was 8.7%. The total rate of L. infantum infection derived from seroreactivity and/or positive PCR was 15.4%. Serology and PCR results were positively associated, and moderate agreement (kappa = 0.489) was found between Leishmania ELISA and PCR. No statistical association was found between positive Leishmania PCR results and gender, clinical status, or T. gondii seropositivity. Six of the 105 cats (5.7%) displayed clinical signs compatible with feline cutaneous leishmaniosis, and 4 out of these 6 cats (66.7%) were found to have Leishmania infection by means of serology and/or PCR. Leishmania seropositivity was associated with clinical signs of feline cutaneous leishmaniosis (p = 0.029). The prevalence of FeLV p27 antigen was 16.2% (17/105) and of FIV antibody was 20.9% (22/105), with coinfection found in 9.5% (10/105) of the cats. Leishmania ELISA seroreactivity and positive PCR results were statistically associated with FeLV infection and with coinfection of both retroviruses but not with a positive FIV status. The high seroprevalence and molecular rates of Leishmania infection observed indicate that cats are frequently infected with L. infantum, and the association with FeLV suggests a potential role for this retrovirus in feline Leishmania infection in endemic areas.

Introduction

L eishmaniosis caused by Leishmania infantum is a widespread sandfly-borne zoonotic disease with a great impact on public health. Much of the veterinary literature describes canine leishmaniosis with regard to epidemiology and pathogenesis, and this has demonstrated a high prevalence of infection in the Mediterranean Basin (Baneth et al. 2008). Feline leishmaniosis was first described in 1912 in Algeria (Sergent et al. 1912) and occurs sporadically especially in regions and countries where canine leishmaniosis is endemic (Hervas et al. 1999).

The Leishmania species shown to cause feline leishmaniosis are L. infantum in southern France (Laurelle-Magalon and Toga 1996, Ozon et al. 1998), Italy (Poli et al. 2002, Pennisi et al. 2004), Portugal (Costa Durão et al. 1994, Marcos et al. 2009), Spain (Hervas et al. 1999), Brazil (Savani et al. 2004), and Iran (Hatam et al. 2009); Leishmania mexicana in Texas (Craig et al. 1986, Barnes et al. 1993); and Leishmania amazonensis (de Souza et al. 2005) and Leishmania braziliensis (Schubach et al. 2004) in Brazil. Despite the high prevalence of L. infantum in dogs in the Mediterranean basin with common manifestations of clinical disease (Solano-Gallego et al. 2001), far fewer cases of clinical feline Leishmania infection have been described in the same region. The most frequently described lesions in feline leishmaniosis are ulcerocrusting and nodular dermatitis, alopecia, and scaling (Ozon et al. 1998, Hervas et al. 1999, Poli et al. 2002, Pennisi et al. 2004, Rufenacht et al. 2005) with the visceral form of the disease involving the spleen, liver, lymph nodes, bone marrow, eye, and kidney being less commonly described (Ozon et al. 1998, Hervas et al. 1999, 2001, Leiva et al. 2005). Although clinical cases of leishmaniosis have been reported in cats with coinfection of feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) (Pennisi 1999, Hervas et al. 2001, Poli et al. 2002, Pennisi et al. 2004, Grevot et al. 2005), the true association between feline leishmaniosis and retroviral infection remains unclear.

The recent description of subclinical Leishmania infection in cats in the Mediterranean basin countries (Martín-Sánchez et al. 2007, Maia et al. 2008) and the demonstration of infectivity of sand fly vectors from a cat (Maroli et al. 2007) raise the possibility of a reservoir host role for this species. However, the scientific literature available on feline Leishmania infection is limited (Martín-Sánchez et al. 2007, Solano-Gallego et al. 2007a, Maia et al. 2008, Hatam et al. 2009). No studies have been performed in cats living on the Island of Ibiza (Spain), where canine and human leishmaniosis are well established (Riera et al. 2004). The objective of this study was to evaluate the prevalence of L. infantum infection in an Ibizian feline population by serologic and molecular methods and to identify associated risk factors such as the animal's clinical status and concurrent infections including FeLV, FIV, and Toxoplasma gondii.

Materials and Methods

Blood was collected from 105 cats living outdoors in two shelters on the Island of Ibiza (Spain) between June and July 2008. Blood was collected into ethylenediaminetetraacetic acid and plain tubes, and full routine hematologic and clinical chemistry analyses were performed. All blood and sera were stored at −20° before usage. Breed, age, gender, and full clinical history were recorded where known but were not available for all of the cats. A full physical examination was performed by veterinarians before blood sampling. The population was composed of 49 spayed females, 4 entire females, 37 neutered males, and 11 entire males with no gender recorded for 3 of the sampled cats. All cats were domestic short-haired. Sixty of the cats displayed a variety of clinical signs. Sera from 76 cats presented for various medical reasons at the Royal Veterinary College (University of London, United Kingdom) were used for L. infantum serological evaluation by enzyme-linked immunosorbent assay (ELISA). Since L. infantum infection is not endemic in the United Kingdom (Shaw et al. 2009), these samples were used as negative controls to establish cut-off values for ELISA.

An ELISA protocol previously described for cat sera was used with some modifications (Solano-Gallego et al. 2007a). Briefly, microtiter plates were coated with 0.1 mL of L. infantum (MHOM/FR/78/LEM-75 zymodeme MON-1) antigen (1 mg/mL in 50 mL of 0.05 M carbonate-bicarbonate, pH 9.6) and incubated overnight at 4°C. One hundred microliters per well of cat sera, diluted 1:100 in phosphate-buffered saline–0.05% Tween 20 (PBST)–1% dried skimmed milk (PBST-M), was incubated for 1 h at 37°C. After three washes with PBST and one wash with PBS, 100 μL per well of anti-cat IgG (AbD; Serotec) diluted 1:7500 in PBST-M conjugated to horseradish peroxidase was added and incubated for 1 h at 37°C. Then, the plates were rewashed. The substrate solution, ortho-phenylenediamine dichloride 0.4 mg/mL, tablets plus buffer of 0.4 mg/mL urea hydrogen peroxide, and 0.05 M phosphate-citrate, pH 5.0 (SIGMAFAST™; Sigma-Aldrich), was added at 200 μL per well and developed for 10 min at 24°C. The reaction was stopped with 50 μL of 2 M H₂SO₄. Absorbance values were read at 490 nm in an automatic micro-ELISA reader (Spectra Max M2 Plate Reader, Molecular Devices). The reaction was quantified as ELISA units (EU) related to a positive cat sera used as a calibrator and arbitrarily set at 100 EU. The calibrator cat had confirmed leishmaniosis and serum from this cat has been used in a previous study (Solano-Gallego et al. 2007a). All determinations included the calibrator serum as a positive control and serum of a cat from the United Kingdom as a negative control. The cut-off was established at 69 EU for IgG (mean ± 4 standard deviations of sera of 76 cats from a United Kingdom cat population).

L. infantum polymerase chain reaction (PCR) was performed on 104 of the Ibizian cats sampled as previously described (Solano-Gallego et al. 2007b). Briefly, DNA was extracted using High Pure PCR Template Preparation Kit (Roche Applied Science) in accordance with the manufacturer's recommendations. Commercial L. infantum primers and hybridization probes LC set (TIB Molbiol) that amplify a fragment of the kinetoplast minicircle were used. Amplifications were conducted in sealed 20 μL LightCycler glass capillaries. Thermal cycling was performed according to manufacturer's instructions (TIB Molbiol). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene was used to ensure that negative results represented truly negative samples rather than a problem with DNA loading, sample degradation, PCR inhibition, or absence of feline cells (Solano-Gallego et al. 2007b). Only samples with a positive result for the GAPDH gene were evaluated for L. infantum real-time PCR.

To evaluate the association with feline retrovirus infections, 105 of the cats were tested for FeLV antigen and for FIV antibody. Detection of FeLV antigen (p27) and FIV antibody was performed with a commercial ELISA kit (PetChek* FIV Ab/FeLV; IDEXX Laboratories). Sera were analyzed for antibodies to T. gondii with the microscopic agglutination test as described by Desmonts and Remington (1980) using a direct microagglutination commercial kit (Toxo-Screen DA; Biomerieux). Sera were diluted from 1:20 to 1:320. Positive and negative control samples were included in each plate. A titer of ≥1:40 was considered indicative of T. gondii exposure in cats (Desmonts and Remington 1980).

Cats were categorized as positive or negative for each parameter using assigned cut-off points for T. gondii and L. infantum, and positive or negative result for FeLV and FIV using data generated by the above techniques. Cats were categorized in terms of presence or absence of anemia (hemoglobin concentration <8g/dL), hyperproteinemia (total protein >8g/dL), hypoalbuminemia (albumin <2.3 g/dL), and signs compatible with feline cutaneous leishmaniosis or clinical signs not related with signs compatible with feline cutaneous leishmaniosis. Clinical signs compatible with feline cutaneous leishmaniosis included ulcerocrusted dermatitis, nodular dermatitis, alopecia, and scaling especially of face and ears (Hervas et al. 1999, Poli et al. 2002, Pennisi et al. 2004, Rufenacht et al. 2005). Clinical signs not specifically related with signs of feline cutaneous leishmaniosis included any clinical signs with the exception of the cutaneous signs described above. The most common clinical signs not specifically compatible with feline cutaneous leishmaniosis were lethargy, feline respiratory complex, gingivostomatitis, and diarrhea. Chi-square analysis and Fisher's exact test (SPSS 17.0) were used to test for associations between all of the parameters. Differences were considered significant if the p-value was <0.05. Any parameters statistically linked to the ELISA seropositivity were used in a logistic regression model (SPSS 17.0) to assess for risk factors associated with a positive ELISA result. A kappa test (GraphPad Software, Inc.) was used to establish agreement between positive Leishmania PCR and ELISA results.

Results

A seroprevalence of 13.2% was found using ELISA for Leishmania and the PCR positivity rate was 8.7%. The results are listed in Table 1. All PCR samples were positive for the GAPDH housekeeping gene. Levels of Leishmania-specific antibodies in positive cats ranged from 77% to 306% (with a mean of 116%). The parasite load ranged from 250 kinetoplast copies/mL of blood to 3.2 × 10⁷ kinetoplast copies/mL of blood with a mean and SD of 3 × 10⁶ ± 1 × 10⁷. Six cats were positive by both serology and PCR with 10 cats showing discordant results between PCR and serology. Only three cats that were PCR positive had a negative serology result (Table 2). A low Pearson correlation was found between parasite load and degree of antibody levels (r = 0.16). The serology and PCR results were positively associated by chi-square analysis (24.2, p < 0.001). In addition, logistic regression analysis identified a strong association between PCR and ELISA. A kappa value of 0.489 (with 95% CI) was found between ELISA and PCR, which demonstrated a moderate agreement (Landis and Koch 1977). No statistical association was found between PCR-positive results and gender, clinical status (specific or nonspecific signs), or T. gondii status. Six of the 105 cats (5.7%) displayed clinical signs compatible with feline leishmaniosis (Fig. 1) and 4 out of these 6 cats (66.7%) were found to have Leishmania infection by means of serology and/or PCR as shown in Table 2. Leishmania seropositivity was associated with clinical signs of feline cutaneous leishmaniosis by Fisher's exact test (p = 0.029). Leishmania-positive status based on serology and/or PCR was associated with clinical signs compatible with feline cutaneous leishmaniosis and clinical signs not related to feline cutaneous leishmaniosis by Fisher's exact test (p = 0.028, p = 0.006, respectively).

FIG. 1.

Crusted-ulcerative cutaneous lesion in a left ear pinna of cat that was positive to both Leishmania infantum polymerase chain reaction and serology (ID Ig 46).

Table 1.

Number (%) of Cats Positive to Different Pathogens by Serological and Molecular Techniques in Relation to Presence of Clinical Signs and Gender

	Leishmania PCR positive	Leishmania ELISA positive	Leishmania PCR+ ELISA positive	Leishmania PCR and/or ELISA positive	FeLV positive	FIV positive	FeLV/FIV coinfection	Toxoplasma gondii positive
Apparently healthy (n = 45)	2 (4.4)	3 (6.6)	2 (4.4)	3 (6.7)	6 (13.3)	5 (11.1)	3 (6.7)	24 (53.3)
Clinical signs not related with leishmaniosis (n = 60)	7 (11.7)	10 (18.5)^a	4 (7.4)^a	13 (24.1)^a	11 (18.3)	17 (28.3)^b	7 (11.7)	34 (56.7)
Leishmaniosis signs (n = 6)	2 (33.3)	3 (50)^b	1 (16.7)	4 (66.7)^b	3 (50.0)^b	2 (33.3)	1 (16.7)	4 (66.7)
Female (n = 49)	4 (8.2)	6 (12.2)	3 (6.1)	4 (8.2)	4 (8.2)	5 (10.2)	1 (2.0)	2 (55.1)
Male (n = 53)	5 (9.4)	6 (11.3)	3 (5.7)	8 (15.1)	12 (22.6)^b	17 (32.1)^b	9 (17.0)^b	31 (58.5)
Total (n = 105)	9 (8.7)^c	13 (13.2)^d	6 (6.1)^d	16 (15.4)^c	17 (16.2)	22 (20.9)	10 (9.5)	58 (55.2)

Clinical signs not related with feline cutaneous leishmaniosis (n = 54).

Statistical significant.

Total n = 104.

Total n = 98.

ELISA, enzyme-linked immunosorbent assay; FeLV, feline leukemia virus; FIV, feline immunodeficiency virus; PCR, polymerase chain reaction.

Table 2.

Gender, Retroviral Status, Toxoplasma gondii Titers, and Clinical Signs of Cats Positive by Serology and/or Polymerase Chain Reaction for Leishmania infantum

Cat ID	Gender	ELISA (% positivity)	PCR (kinetoplast/mL)	FeLV/FIV status	Toxoplasma gondii titers	Clinical signs
Ig 15	MN	+ (76.8)	−	−/+	—	Generalized dermatitis, hyperkeratosis, labial ulcer, generalized ventral alopecia^a, nasal purulent discharge, ear mites
Ig 23	MN	+ (108.8)	+ (1.2 × 10⁶)	+/+	>1:320	Feline respiratory complex, ear mites
Ig 25	FN	+ (132.9)	+ (1.4 × 10⁵)	+/+	—	Ear mites
Ig 30	FN	+ (306.1)	−	−/+	>1:320	Chronic diarrhea
Ig 32	FN	+ (147.3)	−	+/+	—	Mild gingivostomatitis
Ig 33	FN	+ (93.9)	−	−/−	>1:320	Marked gingivostomatitis, uveitis
Ig 41	MN	+ (165.1)	+ (1.1 × 10⁵)	−/−	>1:320	Marked gingivostomatitis
Ig 45	^b	+ (110.4)	−	+/−	1:320	No clinical signs noted
Ig 46	FN	+ (106.6)	+ (4.5 × 10⁵)	+/−	1:320	Crusted-ulcerative cutaneous lesion in left ear pinna^a, ear mites
Ig 51	MN	+ (80.5)	−	−/−	>1:160	Hyperkeratosis, nasal ulcer^a
Ig 53	FN	+ (123.0)	+ (9.7 × 10³)	+/+	>1:160	Chronic diarrhea, ear mites
Ig 56	MN	− (42.6)	+ (250)	−/−	>1:160	Circling, ear mites
Ig 66	FN	− (67.9)	+ (250)	+/+	<1:160	Crusted cutaneous lesions on ear tip^a, mild gingivostomatitis, ear mites
Ig 77	ME	+ (154.2)	+ (3.2 × 10⁷)	−/−	—	No clinical signs noted
Ig 73	ME	+ (78.1)	−	−/−	>1:160	Diarrhea, ear mites
Ig 95	FE	− (16.3)	+ (290)	−/−	—	No clinical signs noted

Clinical signs suggestive of feline cutaneous leishmaniosis.

Gender was not known for Ig 45.

M, male; F, female, N, neutered, E, entire.

Forty-four percent (7/16) of FeLV-positive cats and 25% (5/20) of FIV-positive cats were seropositive for Leishmania. Forty-four percent of cats that were coinfected with FeLV and FIV were Leishmania positive by ELISA (4/9). Leishmania seropositivity was associated with FeLV but not with FIV infection by Fisher's exact test (15.5, p = 0.001, 3.0, p = 0.132, respectively) and with coinfection of both FeLV and FIV (8.3, p = 0.017, Fisher's exact test). In addition, logistic regression identified a positive association between Leishmania ELISA and FeLV status. Positive PCR results were also associated with FeLV infection (11.077, p = 0.006, Fisher's exact test) and with coinfection of both retroviruses (13.752, p = 0.04, Fisher's exact test) but was not statistically associated with a positive FIV status.

Infection with each of the retroviruses was associated with gender by chi-square analysis (FIV: 6.2, p = 0.013; FeLV: 4.0, p = 0.05) with females more likely to be infected. The presence of clinical signs not related with feline cutaneous leishmaniosis was associated with FIV infection on chi-square analysis (4.6, p = 0.032) and with anemia (6.0, p = 0.021) by Fisher's exact Test. There was no statistical association between seropositivity for T. gondii and any of the other parameters.

Discussion

Subclinical feline infection with L. infantum and clinical feline leishmaniosis are far less frequently reported than the canine counterparts. The Leishmania seroprevalence in dogs ranges from 24% to 34% in the Balearic islands (Solano-Gallego et al. 2006b); therefore, it is much higher than the seroprevalence found in cats (13.2%) in the same area in this study. In addition, higher rates of positive PCR are reported in dogs (20%) (Tabar et al. 2008b) than in cats (8.7%) in similar geographical areas. The reported feline prevalence in Spain (established via IFAT, PCR, and/or cytology) ranges from 0.9% to 70% (Portús et al. 2002, Martín-Sánchez et al. 2007, Solano-Gallego et al. 2007a, Ayllón et al. 2008). Seroprevalence ranges from 0.9% to 28% depending on the geographical region in Spain with higher seroprevalence rates encountered in Mediterranean basin regions (Portús et al. 2002, Martín-Sánchez et al. 2007, Solano-Gallego et al. 2007a, Ayllón et al. 2008) as described in dogs (Solano-Gallego et al. 2006b, Miró et al. 2007). Fewer studies have been carried out using molecular techniques in blood with rates of positive PCR ranging from 0.4% to 30.4% (Martín-Sánchez et al. 2007, Ayllón et al. 2008, Maia et al. 2008, Tabar et al. 2008a). The role of the cat as a reservoir for L. infantum is still controversial, but it has been hypothesized (Martín-Sánchez et al. 2007, Solano-Gallego et al. 2007a) that the cat may be a secondary reservoir host rather than an incidental host by virtue of the demonstration of infectivity of sand fly vectors from a chronically infected cat in Italy (Maroli et al. 2007).

This study found a statistically significant association between Leishmania infection and clinical signs compatible with feline cutaneous leishmaniosis described in previous publications (Hervas et al. 1999, Poli et al. 2002, Pennisi et al. 2004, Rufenacht et al. 2005). In addition, a strong statistical association was found between ELISA and PCR. Discordant results can be attributed to the inherent differences between serological testing and molecular methods. For example, although PCR is a very sensitive technique, it relies upon the presence of the organism within the tissue used for molecular analysis. In PCR from blood, as in this study, low numbers or absence of the organism may decrease the sensitivity of this method. It should be taken into account that blood was found not to be the tissue of choice for Leishmania PCR detection in the dog due to the lower sensitivity when compared with other tissues (Rodríguez-Cortés et al. 2007, Solano-Gallego et al. 2007b, Maia et al. 2009). In addition, in this study, the parasite load in blood was quite variable and poorly correlated with degree of antibody levels. Other tissues that may have increased the rate of detection by PCR would be the skin, spleen, lymph nodes, or bone marrow as reported in canine Leishmania infections (Rodríguez-Cortés et al. 2007, Solano-Gallego et al. 2007b, Maia et al. 2009). In particular, this would have been interesting in view of sampling tissue from the site of cutaneous lesions from those cats with clinical signs that were compatible with feline leishmaniosis but which were negative by ELISA or PCR and to confirm Leishmania infection in suspicious lesions (Rufenacht et al. 2005).

An association was found between a positive Leishmania status (by both serologic and molecular methods) and FeLV. These findings are in contrast to other studies where no such association was found between retroviral infections and Leishmania infection in cats (Martín-Sánchez et al. 2007, Ayllón et al. 2008) with the exception of one study from Italy where a significant higher L. infantum seropositivity rate in FIV-positive cats was reported (Pennisi et al. 1998). However, several clinical leishmaniosis cases have been reported in cats with concurrent retroviral infections (Pennisi 1999, Hervas et al. 2001, Poli et al. 2002, Pennisi et al. 2004, Grevot et al. 2005). These findings suggest that a retroviral infection, in particular FeLV, is a potential risk factor for feline L. infantum infection. A positive association between Leishmania and retroviral infections may suggest the role of immunosuppression in susceptibility of cats to Leishmania as has been recognized in human beings where Leishmania subclinical infection can develop to disease and even become disseminated and predispose to the visceral form (Alvar et al. 2008). It should also raise the clinical suspicion for L. infantum in cats with FeLV in endemic areas especially if presenting with consistent clinical signs compatible with feline leishmaniosis.

Previous studies using experimental infection of cats with L. braziliensis species showed that peak antibody level occurred on resolution of skin lesions (Simoes-Mattos et al. 2005). The immune response mediated by antibodies is not considered protective against Leishmania and the period during which cats showed active skin lesions that harbored parasites could not be determined by serology. Lesion size was also not correlated to antibody titer (Simoes-Mattos et al. 2005). These findings suggest the possibility of a misdiagnosis in a cat with skin lesions but negative serology. Therefore, serological titers are not frequently correlated with clinical signs as demonstrated in experimental L. infantum infection in cats (Kirkpatrick et al. 1984). The pathogenic evolution of L. braziliensis and L. infantum in the vertebrate host is very different as well as the type of immune responses elicited by the two parasites (Murray et al. 2005, Castro et al. 2007, Dantas-Torres 2007, 2009). Further investigation is needed to determine how similar are these experimental models with L. braziliensis and L. infantum to natural L. infantum infection in cats. This raises another consideration, which is the importance of screening of feline blood donors for L. infantum infection. Studies among canine (Tabar et al. 2008a) and human (Riera et al. 2004) blood donors have demonstrated a high rate of infection by PCR in blood samples, often despite being seronegative.

The prevalence of FeLV antigenemia and FIV antibodies were similar to other studies (Hartmann 2006, Sellon and Hartmann 2006, Solano-Gallego et al. 2006a). Statistical association was found between sick and FIV antibodies as previously described (Hartmann 2006, Sellon and Hartmann 2006, Solano-Gallego et al. 2006a). In disagreement with previous studies (Hartmann 2006, Sellon and Hartmann 2006), female cats were more likely to have FIV antibodies and FeLV antigenemia.

Recent studies have demonstrated T. gondii seroprevalence rates of 45% in Barcelona (Gauss et al. 2003), 30.8% in Madrid, and 33.7% in La Rioja (Miró et al. 2004) similar to the high seroprevalence found in the Ibiza island. No association was found between positive serology and/or PCR for Leishmania and T. gondii antibodies in this study. Although other studies have described an immunologic relationship between FIV infection and failure to control T. gondii infection (Levy et al. 2004), no association between T. gondii seroreactivity and FIV infection was found in this study. However, this cannot be directly compared to presence or absence of T. gondii antibodies.

In conclusion, the high seroprevalence and molecular rates of Leishmania infection observed indicate that cats are frequently infected with L. infantum, and the association with FeLV suggests a potential role for this retrovirus in the development of Leishmania infection in cats in endemic areas.

Footnotes

Acknowledgments

The authors would like to thank Dr. Alhelí Rodríguez Cortés from Facultat de Veterinària, Universitat Autònoma de Barcelona, for the supplying the positive feline serum controls for setting up the ELISA and Aviva Petrie for assistance with the statistical analysis. Molecular testing was kindly supported by Laboratorio Privato Veterinario San Marco (Padova, Italy).

Disclosure Statement

The authors do not have potential conflict of interest.

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