Circulation of Highly Drug-Resistant Clostridium difficile Ribotypes 027 and 001 in Two Tertiary-Care Hospitals in Mexico

Abstract

Objective:

To assess drug susceptibility and characterize Clostridium difficile ribotypes in isolates from two tertiary-care hospitals in Mexico.

Methods:

Isolates were evaluated for genotyping, antimicrobial susceptibility testing and detection of mutations associated with drug resistance. PCR ribotyping was performed using a combination of gel-based and capillary electrophoresis-based approaches.

Results:

MIC₅₀ and MIC₉₀ were ≥128 mg/L for ciprofloxacin, erythromycin, clindamycin, and rifampicin. There was no reduced susceptibility to metronidazole or tetracycline; however, reduced susceptibility to vancomycin (≥4 mg/L) and fidaxomicin (≥2 mg/L) was detected in 50 (40.3%) and 4 (3.2%) isolates, respectively. Furthermore, the rpoB Arg505Lys mutation was more frequently detected in isolates with high minimum inhibitory concentration (MIC) to rifampicin (≥32 mg/L) (OR = 52.5; 95% CI = 5.17–532.6; p < 0.000). Of the 124 C. difficile isolates recovered, 84 (66.7%) were of ribotype 027, 18 (14.5%) of ribotype 001, and the remainder were other ribotypes (353, 255, 220, 208, 176, 106, 076, 020, 019, 017, 014, 012, 003, and 002).

Conclusion:

Ribotypes 027 and 001 were the most frequent C. difficile isolates recovered in this study, and demonstrated higher MICs. Furthermore, we found four isolates with reduced susceptibility to fidaxomicin, raising a concern since this drug is currently unavailable in Mexican Hospitals.

Introduction

Clostridium difficile infection (CDI) symptoms may range from mild diarrhea to life-threatening complications. Apart from NAP1/BI/027, other C. difficile ribotypes have been associated with severe disease, for example, ribotype 078 affects younger patients and is a frequent causative agent of community-associated disease¹; ribotype 001 is the dominant strain in eastern Europe and has higher antimicrobial resistance than other ribotypes.²

First-line treatment for mild to moderate CDI is based on oral administration of metronidazole or vancomycin, with a therapeutic efficacy >70%.³ In some patients, however, diarrheal symptoms may reappear within days or weeks after having stopped the treatment. Fidaxomicin is a relatively new narrow-spectrum macrocyclic antibiotic drug that is noninferior to vancomycin in the management of CDI and associated with lower recurrence rates than vancomycin.⁴ However, fidaxomicin is currently unavailable in Mexico, thus, vancomycin and metronidazole are still the standard treatments for CDI as recommended in the Clinical Practice Guidelines for CDI in Adults of the Society for Healthcare Epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA). Other therapeutic options that have been proposed in recent years are rifamycins (good in vitro activity against C. difficile) including rifaximin (for relapsing CDI),⁵ and linezolid (protective rather than curative activity).⁶

Resistance to erythromycin may be due to any of more than 20 classes of erythromycin ribosomal methylase (erm) genes, including ermB,⁷ which is also related to clindamycin resistance. In C. difficile, resistance to fluoroquinolones is usually due to altered DNA gyrase because of nucleotide substitutions in gyrA or gyrB genes.⁵ Resistance to rifamycins and/or fidaxomicin is mediated by mutations that lead to reduced binding to the β subunit of RNA polymerase (RpoB).^7,8 Finally, resistance to linezolid has been related to the presence of the phenicol and lincosamide resistance gene (cfr), as described for staphylococci.⁹

Although C. difficile is an important nosocomial pathogen, little is known about the epidemiology of this microorganism in Mexico. The aims of this study were to determine the drug susceptibility of Mexican C. difficile isolates, particularly to the recently licensed CDI treatment, fidaxomicin, and to study circulating ribotypes in two tertiary-care hospitals in Mexico.

Methods

Settings and study population

We designed an observational study of circulating C. difficile ribotypes, drug susceptibility, and drug resistance genes from two hospitals in Mexico: The Hospital Civil of Guadalajara “Fray Antonio Alcalde,” is a 1000-bed tertiary-care teaching hospital, in Guadalajara; and the Hospital Universitario “Dr. José Eleuterio González,” is a 450-bed tertiary-care teaching hospital in Monterrey.

All patients with confirmed CDI from February 2011 through January 2016 were included in the study. Recurrences were defined as patients with a reappearance of symptoms after resolution of the previous diarrheal episode within 8 weeks or less. As patient information was anonymized and only microbiological data were analyzed, informed consent was not required. The study was reviewed and approved by the Local Ethics Committee (Approval: 047/16).

Diagnosis of CDI

Clinical diagnosis of CDI was suspected when patients were hospitalized for more than 48 hr and had >3 loose stools in the previous 24 hr. In the Hospital Civil of Guadalajara, CDI was confirmed by real-time PCR using the Xpert^® C. difficile/Epi assay (Cepheid, Sunny Vale, CA) and in the Hospital Universitario, the diagnosis was confirmed by the use of the Meridian ImmunoCard^® Toxins A&B (Meridian Bioscience, Inc., Memphis, TN). Some patients were additionally diagnosed by the Xpert C. difficile/Epi assay in Hospital Universitario only at physicians' request.

Culture

Fecal samples from all confirmed CDI cases were treated with absolute ethanol and cultured on C. difficile agar (Neogen Corporation, MI) with cefoxitin (16 mg/L) in anaerobic conditions for up to 72 hr. Plates were cultured in either an anaerobic jar or anaerobic chamber. Identification was performed by PCR as described.¹⁰ Only one isolate per patient was included in the study.

Antimicrobial susceptibility testing

We tested antimicrobial agents used for CDI treatment such as vancomycin, metronidazole (range from 0.03 to 128 mg/L), fidaxomicin (range from 0.002 to 8 mg/L), and rifampicin (range from 0.0001 to 128 mg/L); and also antimicrobials with potential therapeutic use such as linezolid (range from 0.03 to 128 mg/L). Furthermore, we tested antimicrobials that may be associated with induction of CDI, including ciprofloxacin, moxifloxacin, erythromycin, clindamycin (range from 0.03 to 128 mg/L), and tetracycline (range from 0.008 to 128 mg/L).

Susceptibility testing was performed by the agar dilution method using Wilkins-Chalgren agar (Oxoid Limited, Basingstoke, Hampshire, England) and Schaedler's anaerobe broth (Oxoid Limited).¹¹ Briefly, overnight cultures in 5 ml of prereduced Schaedler's broth were spotted onto plates of Wilkins-Chalgren agar with different concentrations of antibiotics, using a multipoint inoculator (10⁴ colony-forming units/spot). An agar plate without an antimicrobial agent was included as a growth control in both aerobic and anaerobic atmosphere and the plates were read after 48 hr of incubation at 37°C in an anaerobic environment. C. difficile ATCC 700057 was used as quality control.

A stock solution of fidaxomicin (800 mg/L) was prepared in DMSO, then 1 ml of stock was diluted in 5 ml of DMSO and 4 ml of 10% DMSO (final concentrations 80 mg/L); further dilutions were made in 10% DMSO. Stock solutions of remaining antibiotics (2560 mg/L) and dilutions were dissolved accordingly to recommendations of the Clinical and Laboratory Standards Institute (CLSI) document M100–S27.

Mutations associated with drug resistance

To detect mutations associated with resistance to rifampicin or fidaxomicin, two regions of the rpoB gene were amplified; for rifampicin, we used previously reported primers,⁹ and for fidaxomicin, we designed the primers CdrpoB-FD-F (5′-TCATGGAAAATGGAACACCA-3′) and CdrpoB-FD-R (5′-CCAAACCTCCATCTCTCCAA-3′). We designed the primers CdrpoC-VAN-F (5′-GAATGGGTGCTGAAGCTGTA-3) and CdrpoC-VAN-R (5′-GACGGAAACGACCTTGCTTA −3′) to amplify a region in the rpoC gene that has been linked to vancomycin resistance.¹² Furthermore, the presence of the cfr gene was investigated by PCR in selected strains as previously described.¹³

Sequencing of PCR-purified products was performed by Macrogen, Inc. (Seoul, Korea). The sequences were analyzed using the NCBI Basic Local Alignment Search Tool (BLAST).

Typing of isolates

All isolates were typed for tcdA, tcdB, cdtA, cdtB, and for deletions in tcdC by PCR as previously described.^14,15 For ribotyping, amplification of the 16S-23S rRNA intergenic spacer region was conducted by PCR as described.¹⁶ The ATCC strain BAA-1805 (ribotype 027) was used as a control. Selected isolates were ribotyped by capillary electrophoresis at the C. difficile Ribotyping Network Reference Laboratory (CDRN) at Leeds Teaching Hospitals NHS Trust, Leeds, United Kingdom.

Results

Culture

Samples were cultured in anaerobic jars (n = 196, 57.1%) or in anaerobic chamber (n = 147, 42.9%). In total, we cultured 343 samples, of which 124 (36.1%) yielded a positive C. difficile culture (one isolate per patient). Most of the cases were from the Hospital Civil of Guadalajara (n = 76, 61.3%); the Hospital Universitario accounted for 48 of the cases (38.7%).

Antimicrobial susceptibility profiles

Four isolates (3.2%) had reduced susceptibility to fidaxomicin (MIC = 2 mg/L), whereas no isolate was resistant to either tetracycline or metronidazole. MIC₅₀ and MIC₉₀ were ≥128 mg/L to ciprofloxacin, erythromycin, clindamycin, and rifampicin (Table 1). MIC distributions of CDI treatment drugs (vancomycin, metronidazole, and fidaxomicin) are shown in Figure 1.

FIG. 1.

MIC distribution. MIC distribution and ranges of drugs used for CDI-treatment. CDI, Clostridium difficile infection; MIC, minimum inhibitory concentration.

Table 1.

Antimicrobial Susceptibilities of Mexican C. difficile Isolates

	All strains (n = 124)			Ribotype 027 (n = 84)			Ribotype 001 (n = 18)
Antimicrobial agent	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)	Geometric mean (mg/L)	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)	Geometric mean (mg/L)	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)	Geometric mean (mg/L)
CDI-associated
Ciprofloxacin	>128	>128	120.4	>128	>128	164	>128	>128	159.1
Moxifloxacin	32	64	30.6	32	64	39.3	64	64	38.4
Erythromycin	>128	>128	85.6	>128	>128	184	>128	>128	175
Clindamycin	>128	>128	89.5	>128	>128	185.6	>128	>128	176.2
Tetracycline	0.06	0.125	0.07	0.03	0.125	0.06	0.125	4	0.07
CDI treatment
Vancomycin	2	4	2.3	2	4	2.8	2	4	2.7
Metronidazole	1	2	0.9	2	2	1.4	0.25	0.5	1
Fidaxomicin	0.015	0.06	0.025	0.015	0.06	0.027	0.015	0.03	0.023
Rifampicin	>128	>128	25.2	>128	>128	99.1	>128	>128	77
Potentially therapeutic
Linezolid	4	8	3.7	4	8	4.1	8	32	4.5

CDI, Clostridium difficile infection; MIC, minimum inhibitory concentration.

Molecular analysis of drug resistance

When analyzing a region of rpoB in fidaxomicin-susceptible isolates (n = 18) and isolates with reduced susceptibility (n = 3), we found seven mutations; one of these caused an amino acid change that was not associated to reduced susceptibility strains (Table 2). Furthermore, the rpoB gene was amplified and partially sequenced in 14 rifampicin-susceptible and 22 rifampicin-resistant isolates. We detected three mutations that generated amino acid changes in both susceptible and resistant isolates; the Arg505Lys mutation was more frequently detected in resistant isolates (21/22, 95.4%) than in susceptible isolates (4/14, 28.5%) (OR = 52.5; 95% CI = 5.17–532.6; p < 0.000) (Table 2).

Table 2.

Distribution of Mutations Associated to Drug Resistance Detected in Susceptible and Resistant C. difficile Isolates

Antibiotic	Gene	Polymorphism	Codon	Amino acidic change	Susceptible	Resistant	OR (95% CI), p
Rifampicin	rpoB	AGA → AAA	505	Arg505Lys	4/14 (28.5)	21/22 (95.4)	52.5 (5.17–532.6), 0.000
		CAT→AAT	502	His502Asn	0/14 (0.0)	8/22 (36.3)	NC
		ATA→ATG	548	Ile548Met	4/14 (28.5)	13/22 (59.0)	3.6 (0.85–15.10), 0.074
		Wild type			10/14 (71.5)	1/22 (4.5)
Fidaxomicin	rpoB
		GAG→CAC	1036	Glu1036Gln	15/18 (83.3)	2/3 (66.7)	0.4 (0.027–5.9), 0.489
		GGG→GGA	1020	None	15/18 (83.3)	2/3 (66.7)	0.4 (0.027–5.9), 0.489
		CTA→ TTA	965	None	15/18 (83.3)	2/3 (66.7)	0.4 (0.027–5.9), 0.489
		GAC→GAT	966	None	15/18 (83.3)	2/3 (66.7)	0.4 (0.027–5.9), 0.489
		AAC→AAT	1040	None	15/18 (83.3)	2/3 (66.7)	0.4 (0.027–5.9), 0.489
		TAC→TAT	1048	None	15/18 (83.3)	2/3 (66.7)	0.4 (0.027–5.9), 0.489
		ATT→ATC	980	None	15/18 (83.3)	2/3 (66.7)	0.4 (0.027–5.9), 0.489
		Wild type			3/18 (16.7)	1/3 (33.3)
Vancomycin	rpoC
		AAG→AAT	265	Lys266Asn	1/14 (7.1)	0/12 (0.0)	NC
		TTC→TTA	294	Phe295Leu	1/14 (7.1)	0/12 (0.0)	NC
		AAG→AAA	175	None	5/14 (35.7)	11/12 (91.7)	19.8 (1.94–201.63) 0.004
		AAG→AAA	217	None	4/14 (28.5)	11/12 (91.7)	27.5 (2.61–289.14), 0.004
		GAG→GAA	184	None	4/14 (28.5)	11/12 (91.7)	27.5 (2.61–289.14), 0.004
		GAG→GAA	193	None	4/14 (28.5)	11/12 (91.7)	27.5 (2.61–289.14), 0.004
		GAG→GAA	213	None	4/14 (28.5)	11/12 (91.7)	27.5 (2.61–289.14), 0.004
		GGG→GGA	199	None	4/14 (28.5)	11/12 (91.7)	27.5 (2.61–289.14), 0.004
		GCG→GTC	192	None	2/14 (14.2)	0/12 (0.0)	NC
		GCG→GCA	249	None	4/14 (28.5)	11/12 (91.7)	27.5 (2.61–289.14), 0.004
		CCA→CCG	234	None	4/14 (28.5)	11/12 (91.7)	27.5 (2.61–289.14), 0.004
		CAA→CAG	200	None	2/14 (14.2)	0/12 (0.0)	NC
		GCA→GCG	293	None	1/14 (7.1)	0/12 (0.0)	NC
		CTA→TTA	190	None	4/14 (28.5)	11/12 (91.7)	27.5 (2.61–289.14), 0.004
		AAC→AAT	264	None	4/14 (28.5)	11/12 (91.7)	27.5 (2.61–289.14), 0.004
		GTT→GTA	203	None	4/14 (28.5)	11/12 (91.7)	27.5 (2.61–289.14), 0.004

NC, not calculated.

We also analyzed the rpoC gene in 14 vancomycin-susceptible isolates and 12 isolates with reduced susceptibility to vancomycin and detected 22 mutations; two of them were associated with an amino acid change (Table 2). The presence of drug resistance genes was analyzed in selected isolates (i.e., isolates with the highest and lowest MIC values). The cfr gene was not amplified in any of the linezolid-susceptible (n = 17) or linezolid-resistant (n = 9) C. difficile isolates evaluated.

Ribotypes

Toxin A and toxin B genes were detected in all isolates. The binary toxin gene was detected in 89 isolates (71.8%), of which 87 (97.8%) contained the tcdC 18-bp deletion (Table 3).

Table 3.

Distribution of Ribotypes Among C. difficile Strains (n = 124)

N	Genotype	Ribotype
84	tcdA+, tcdB+, cdtA+, cdtB+, tcdCΔ18+	027
18	tcdA+, tcdB+, cdtA−, cdtB−, tcdCΔ18−	001
5	tcdA+, tcdB+, cdtA−, cdtB−, tcdCΔ18−	106
3	tcdA+, tcdB+, cdtA+, cdtB+, tcdCΔ18	176
3	tcdA+, tcdB+, cdtA−, cdtB−, tcdCΔ18−	003
1 from each ribotype	tcdA+, tcdB+, cdtA+, cdtB+, tcdCΔ18−	019, 208
1 from each ribotype	tcdA+, tcdB+, cdtA−, cdtB−, tcdCΔ18−	002, 012, 014, 017, 020, 076, 220, 255, 353

Eighty-four isolates (67.7%) demonstrated the same ribotype banding patterns of the control strain BAA-1805 (ribotype 027) (Table 2) and we randomly selected twelve strains that were all confirmed to be ribotype 027 by the CDRN (Leeds, United Kingdom). Similarly, of 18 isolates (14.5%) that demonstrated similar banding pattern to ribotype 001 (90% of similarity), four randomly selected isolates were confirmed to be ribotype 001 by the CDRN. The ribotypes and presence of toxin genes of the other isolates are summarized in Table 3.

Discussion

In our study, ribotype 027 was the predominant strain, accounting for 67.7% of the cases; this ribotype is considered epidemic and has been reported worldwide.¹⁷ In previous publications we have found 027 strain as the predominant ribotype in our settings,^18,19 the latter is in contrast to diverse studies where there is a high diversity of ribotypes and 027 stains account for less than 30%.^20–22 The second most frequent ribotype was 001, accounting for 14.5% of the cases; this ribotype is the main ribotype circulating in Korea,²³ Czech Republic,² Croatia,²⁴ and Slovakia,²⁵ however, this is the first report on ribotype 001 circulation in Mexico. This strain has been associated with high drug resistance, including resistance to ciprofloxacin, erythromycin, and clindamycin.²⁴

Fidaxomicin is an FDA-approved antibiotic for the treatment of CDI.⁸ A previous study that included 1,323 isolates showed an MIC₉₀ of 0.5 mg/L against C. difficile²⁶; similarly, Snydman et al. reported 925 isolates that were inhibited at a fidaxomicin concentration ≤1 mg/L, and the MIC₉₀ was 0.5 mg/L.²⁰ In the present study, we observed an MIC₉₀ of 0.06 mg/L; however, we detected four ribotype 027 isolates with a fidaxomicin MIC of 2 mg/L; these isolates were recovered from patients with recurrent CDI. This finding is of interest since fidaxomicin is unavailable in Mexican hospitals and none of the patients included in the study were exposed to this drug. However, we performed susceptibility testing only once; nevertheless, the MIC of control strain were reproducible and the four strains with MIC = 2 mg/L were detected in a batch of 36 isolates being tested at the same time. MICs of the remaining 32 isolates were ≤0.125 mg/L.

Reduced susceptibility to fidaxomicin may be due to point mutations in the rpoB gene (RNA polymerase subunit β).¹² We detected one point mutation (Glu1036Gln) in RpoB, but it was not associated with drug resistance (p = 0.489). Leeds et al. identified two mutations in rpoB that were associated with reduced susceptibility to fidaxomicin; one of them coded a Gln1073Arg substitution and the second was a frameshift after amino acid 117 of a homolog of the MarR family of transcriptional regulators¹²; however, we did not detect these mutations in our strains. Other mutations associated with fidaxomicin reduced susceptibility have been reported, but all of them have been obtained through the serial passage of strains into media containing fidaxomicin.²⁷ Goldstein et al. isolated a strain with an MIC of 16 mg/L to fidaxomicin from a patient with an episode of recurrence during a clinical trial²⁸; this isolate harbored a Val1143Gly substitution in rpoB. However, the authors did not consider that resistance had developed during the clinical trial and did not explain the clinical relevance of this finding. To our knowledge, there is no report of clinical resistance to fidaxomicin. It is widely known that fidaxomicin reaches high levels in the gut (1,000 μg/g of faeces)²⁸; thus, the actual implication of the high MIC in the strains is unknown, considering the lack of reports on clinical resistance and particularly in Mexico, where this drug is not available.

In our study, we detected reduced susceptibility to vancomycin of 40.3% with 48.8% of reduced susceptibility in 027 strains and 33.3% in 001 strains; proportions as high as 87.7% have been reported in ribotype 027 strains.²⁹ Similarly, to fidaxomicin, it is unlikely that reduced susceptibility impacts on clinical response, due to the high levels of vancomycin reached in the gut (>2,000 mg/L); however, among patients from whom we recovered isolates with reduced susceptibility (50 patients), nine died because of CDI and five of these patients received vancomycin. Nevertheless, other factors may have acted in these patients' response to treatment.

Although this bacterial species has a vanG homolog inducible by vancomycin, it does not promote vancomycin resistance.³⁰ Leeds et al. identified an Asp244Tyr substitution in rpoC that was associated with reduced susceptibility to vancomycin.¹² Despite our efforts to detect this mutation, it was not found. Leeds et al. reported additional mutations: a Pro108Leu substitution in a transferase encoded by murG/CD2725, a stop codon after amino acid 326 in an exonuclease encoded by CD3659, and a single amino acid deletion in an L-serine dehydrogenase (sdaB). Therefore, it seems that diverse mechanisms are responsible for reduced susceptibility to vancomycin, particularly those involved in cell wall biosynthesis.

On the other hand, we observed no reduced susceptibility to metronidazole; similar findings have been reported in other studies.^21,31 Clinical failures with metronidazole treatments have been attributed to the development of heteroresistance and deficiencies in the pharmacokinetics of the drug resulting in low luminal concentrations following oral administration. Reduced susceptibility to metronidazole is known to be unstable, with the loss of levels of resistance due to laboratory manipulation.⁵

High MICs to linezolid have been occasionally described in C. difficile.^9,32 Interestingly, the isolates of ribotype 001 showed higher MICs (8–32 mg/L) than 027 isolates (8 mg/L). Although linezolid is not used for the treatment of CDI, linezolid is widely used in the Hospital Civil of Guadalajara for the treatment of nosocomial pneumonia, surgical wound infections, and bloodstream infections not associated with a catheter. Marin et al. found nine isolates resistant to linezolid and cfr-positive that belonged to the same clonal cluster, suggesting possible horizontal transmission of these strains among patients in their hospital setting.⁹ However, we did not detect the cfr gene in any of the selected C. difficile isolates.

We also found a high proportion of isolates with elevated MICs to rifampicin in 027 strains (95.2%) and 001 strains (83.3%). In contrast, Tenover et al. found lower proportions (27.5%) of isolates with high MIC to rifampicin.²¹ For this antimicrobial agent, a bimodal distribution of MICs has been reported; Noren et al. found 80% of strains with low MIC (>0.016 mg/L) or high MIC (>256 mg/L).³³ In our isolates, we detected three previously reported amino acid substitutions in RpoB associated with rifampicin resistance: Arg505Lys, His502Asn, and Ile548Met. Curry et al.¹³ reported all three changes, including Arg505Lys, which was present in isolates with MICs >32 mg/L. We also confirmed the importance of this mutation in rifampicin-resistant isolates (OR = 52.5, CI 5.17–532.6, p = 0.000). Similarly, the authors reported an Ile548Met change in isolates with MICs >32 mg/L, however, in the C. difficile strains evaluated in the present study, this change was not associated with rifampicin resistance (p = 0.074).

Our study has some limitations. First, we were unable to recover all isolates from all samples, in fact, the recovery rate was low. The low recuperation can be attributed to the medium used, which does not incorporate sodium taurocholate as spore germinant; and the use of ethanol to eliminate any vegetative organisms that survived freezing. Consequently, the isolates are not homogeneously distributed throughout the study period, making it difficult to study distribution over time, and perhaps generating bias on ribotype prevalence; second, clinical diagnosis was not confirmed in a uniform way. Apart from differences in diagnosis, this may have contributed to the low recovery of isolates; and finally, data of ribotyping in 027 and 001 strains were mainly extrapolated from primary results of conventional electrophoresis.

In conclusion, this is the first report on drug susceptibility of C. difficile ribotypes circulating in Mexico. Ribotypes 027 and 001 were the most frequent and highly drug resistant; furthermore, we found four isolates with reduced susceptibility to fidaxomicin, raising a concern since this drug is unavailable in Mexican Hospitals. The clinical relevance of these findings needs to be addressed to fully understand the epidemiology of CDI in Mexican hospitals.

Footnotes

Acknowledgment

The authors thank Lucy Acevedo for technical support.

Disclosure Statement

No competing financial interests exist.

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