Parasitology Research

, Volume 100, Issue 3, pp 487–493 | Cite as

No variation in the prevalence of point mutations in the Pfcrt and Pfmdr1 genes in isolates from Gabonese patients with uncomplicated or severe Plasmodium falciparum malaria

  • Pembe Issamou Mayengue
  • Yvonne Kalmbach
  • Saadou Issifou
  • Peter G. Kremsner
  • Francine Ntoumi
Original Paper

Abstract

In Lambaréné (Gabon), where a high level of Plasmodium falciparum resistance to chloroquine has been reported, we assessed the relationship between polymorphisms in the P. falciparum chloroquine resistance transporter (Pfcrt) and multidrug resistance-1 (Pfmdr1) genes and the clinical severity of malaria. Ninety-one and 60 P. falciparum isolates from children with uncomplicated or severe malaria were collected in 1996 and 2002, respectively. Single nucleotide mutations at codon 76 in the Pfcrt gene and at codons 86, 184, 1034, 1042, and 1246 in the Pfmdr1 gene were assessed by PCR-RFLP. All P. falciparum isolates presented the Pfcrt K76T mutation, whatever the clinical status. A high prevalence (>80%) of the Pfmdr1 86Tyr and 184Phe mutations was detected at both time points and in both clinical groups. We did not identify any specific mutation in the Pfmdr1 gene associated with the severity of disease, and the multiplicity of P. falciparum infection was also similar in both groups. Our results showed no change in the polymorphism of Pfcrt and Pfmdr1 genes in P. falciparum isolates collected in 1996 and 2002, and the severity of the disease was not associated with specific mutations neither in the Pfcrt nor in the Pfmdr1 genes in the study site.

Introduction

The resistance of Plasmodium falciparum malaria parasites to commonly used antimalarials is a major problem in most malaria endemic areas of sub-Saharan Africa. Several factors have been associated with the emergence and spread of multiple drug-resistant strains of P. falciparum, such as the drug pressure and inappropriate prevention measures and treatment (Wernsdorfer and Payne 1991; Zalis et al. 1998). P. falciparum resistance to chloroquine (CQ) has been associated with lower drug accumulation in infected erythrocytes (Krogstad et al. 1987) and to a mutation at position 76 (K76T) in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene (Fidock et al. 2000). Additionally, several studies have suggested the implication of the multidrug resistance-1 (mdr1) gene family in resistance to quinoline-containing compounds in P. falciparum (Foote et al. 1990; Wilson et al. 1993; Duraisingh et al. 1997; Reed et al. 2000; Djimde et al. 2001). Five different point mutations identified as Asn86Tyr, Tyr184Phe, Ser1034Cys, Asn1042Asp and Asp1246Tyr in the mdr1 gene of P. falciparum laboratory clones have been shown to influence the resistance to CQ (Foote et al. 1990; Wellems et al. 1990). However, epidemiological studies provided controversial results about the association between Pfmdr1 Asn86Tyr mutation and the CQ resistance both in vitro and in vivo (Basco et al. 1995; Nagesha et al. 2001; Basco and Ringwald 1998; Thomas et al. 2002). Despite the fact that the CQ resistance mutations are not absolute indicators for clinical response to CQ treatment, as other factors such as immunity may influence treatment outcome (Djimde et al. 2003), the molecular markers remain suitable tools for the assessment of CQ resistance in field isolates.

The rising incidence of malaria morbidity and mortality has been linked to an increase in the frequency of antimalarial drug-resistant parasite strains (Trape et al. 1998; Greenwood and Mutabingwa 2002). However, it is not clearly known whether this is due to the process of selection under drug pressure and increase in infection reservoir (Lines et al. 1991) or to intrinsic parasite characteristics. Studies conducted in India (Ranjit et al. 2004) and Kenya (Omar et al. 2001), where CQ was still used, have reported a selection of Pfcrt (K76T) and Pfmdr1 (Asn86Tyr, Asp1246Tyr) point mutations in P. falciparum isolates from severe rather than uncomplicated malaria. Recently, Meerman et al. (2005) found that mutant alleles of the Pfcrt and Pfmdr1 genes associated with chloroquine resistant parasites were detected with a higher frequency in Gambian patients presenting with severe malaria anaemia. By contrast, no similar findings were reported in a comparable study carried out in Sudan (Giha et al. 2006).

In the city of Lambaréné (Gabon, Central Africa), the detection of high-grade resistance to CQ led to the change of first-line drug policy at the local Albert Schweitzer Hospital (Kremsner et al. 1993, 1994; Winkler et al. 1994). Nevertheless, in other parts of Gabon practices for the treatment of uncomplicated P. falciparum malaria remained unchanged, maintaining the excessive and often inappropriate use of CQ (Borrmann et al. 2002). The reassessment of the resistance of P. falciparum to CQ in Lambaréné showed a persistence of high-grade resistance with a failure rate reaching 100%, and Pfcrt (K76T) mutation was detected in all recrudescent infections (Binder et al. 2002; Borrmann et al. 2002).

To test the hypothesis that the progression of uncomplicated to severe manifestations of the disease may be due to the carriage/presence of resistant P. falciparum parasites to standard drugs, we proposed, in this paper, to analyse point mutations in Pfcrt and Pfmdr1 genes and to compare the prevalence of the different mutations in P. falciparum isolates collected from Gabonese children with severe or uncomplicated malaria at two different time points separated by 7 years interval.

Materials and methods

Study site

The study was carried out at the Albert Schweitzer Hospital in Lambaréné (Gabon). Lambaréné is located in an area where malaria is hyperendemic, where the predominant malarial species is P. falciparum, and the entomological inoculation rate is estimated at 50 infective bites/man/year (Sylla et al. 2000). The study was approved by the Ethics committee of the International Foundation of the Albert Schweitzer Hospital.

Recruitment of patients

Patients with uncomplicated or severe malaria were enrolled on admission to the Albert Schweitzer Hospital. Two periods of recruitment were considered: 1) from January 1995 to March 1996 during a matched pair case-control study (Kun et al. 1998); 2) from January to March 2002 by considering similar inclusion criteria. Informed consent was obtained from parents before the enrolment.

The inclusion criteria for uncomplicated malaria were: fever (≥37.5°C) or recent history of fever during the last 24–48 h preceding presentation at the hospital and presence of P. falciparum parasitemia less than 250,000 parasites/μl on admission in children older than 6 months. The matching severe malaria partner was chosen from patients of the same sex, similar age and provenance. Severe malaria was defined as severe anaemia (haemoglobin <5 g/dl) with the presence of P. falciparum parasites and/or hyperparasitemia (>250,000 parasites/μl) and other signs of severity (Kun et al. 1998). Exclusion criteria were homozygous for haemoglobin S, febrile conditions caused by other diseases than malaria and intake of anti-malarial drugs within the preceding week. Children with severe malaria were admitted to hospital and treated intravenously with quinine plus clindamycin for 4 days (Kremsner et al. 1995). Supportive treatment was given as required. Children with uncomplicated malaria were treated as outpatients, receiving a single oral dose of pyrimethamine and sulfadoxine (Schmidt-Ott et al. 1997). All children were observed until parasitological and clinical cure or death. For the present study, a subgroup of 91 out of 200 children recruited in 1996 (45 and 46 children with uncomplicated and severe malaria, respectively) was selected on the basis of the availability of blood samples from these individuals. Thirty severe malaria were matched with acute uncomplicated malaria cases in 2002.

Blood collection

At inclusion, thick blood films were prepared from each patient to determine the parasite density, and slides were examined using a light microscope covering 100 fields with the 100× objective oil immersion. The method used consisted of 10 μL of blood evenly distributed on a 10 × 18 mm area of a microscope slide (drawn on paper underneath the slide) with a micropipette. Parasite densities were recorded by multiplying the total number of parasites in 100 fields by 6, taking into account that 100 fields were equivalent to 1/6 μl of blood (Planche et al. 2001). The correct examination of blood smears was done by two staff members. The level of detection was about ten parasites per microliter of blood (Planche et al. 2001). After confirmation of malaria diagnosis, 2 ml of venous blood was collected in EDTA tubes. Blood samples were then centrifuged to separate the pellet containing the packed erythrocytes from the plasma. Packed erythrocytes were stored at −80°C and used for DNA preparation.

Parasite DNA extraction

Parasite DNA was extracted from 100 μl of packed erythrocytes using the QIAamp DNA blood mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Then the DNA was recovered in 200 μl of elution buffer from the kit. DNA samples were stored at −20°C until use.

MSP-1 and MSP-2 genotyping of P. falciparum isolates and multiplicity of infection

The highly polymorphic loci, merozoite surface protein-1 (MSP-1) block 2 and merozoite surface protein-2 (MSP-2) central region were used to characterize the P. falciparum isolates by polymerase chain reaction (PCR). The K1, MAD20 and RO33 allelic families of MSP-1 and the FC27 and 3D7 allelic families of MSP-2 were identified. P. falciparum isolates were analysed using primer sequences and PCR conditions described by Ntoumi et al. (2000). Allele-specific positive controls and DNA-free negative controls were included in each round of reactions. Eight microliters of each of the PCR products were analysed by gel electrophoresis on a 1.5% agarose gel (PeqLab, Erlangen, Germany) and visualized under ultraviolet (UV) transillumination after staining with ethidium bromide.

The multiplicity of infection, defined as the mean number of parasite genotypes per infected subject, was determined using MSP-1 and MSP-2 genotyping results. The detection of one MSP-1 and MSP-2 allele was considered as one parasite genotype.

Analysis of point mutation in codon 76 in Pfcrt gene

The lysine (K) to threonine (T) mutation at codon 76 of pfcrt was detected by nested mutation-specific-restriction-enzyme digestion PCR reactions using the method described by Djimde et al. (2001) in a volume of 50 μl in a Biometra Uno II Thermal cycler (Biometra, Göttingen, Germany). Each PCR mixture contained 200 μM deoxynucleoside triphosphates, 1.5 U of Taq polymerase (Qiagen) and 2.5 mM MgCl2 (Qiagen). In this method, the second round of amplification produces a 134-bp amplicon containing codon 76. Then, 5 μl of the amplicon from the second PCR product was digested with 0.5 U of the restriction enzyme Apo1 (New England Biolabs, Beverly, MA, USA) during 6 h incubation at 50°C according to the manufacturer’s protocol. In all PCRs and digests, genomic DNA from the laboratory- adapted chloroquine-resistant strain Dd2 containing T76 mutation and chloroquine-sensitive strain HB3 without this mutation were used as positive controls, while water was used as negative control. The PCR products and the digest were run onto 2% agarose gel containing bromide ethidium and visualized under UV transillumination. The K76T mutation results in the loss of an Apo1 recognition site (Mayor et al. 2001) so that mutated (K76T) DNA samples remain undigested, while those in which the mutation is absent yield two fragments of 100 and 34 bp.

Pfmdr1 gene polymorphism analysis

Point mutations in Pfmdr1 gene were detected by different nested PCRs and successive enzymatic digestions. PCR amplifications were performed using the oligonucleotides described elsewhere (Duraisingh et al. 2000), in a final volume of 50 μl in a Biometra Uno II Thermal cycler (Biometra, Göttingen, Germany). Each PCR mixture contained 200 μM deoxynucleoside triphosphates, 1.5 units of Taq polymerase (Qiagen) and 2 mM MgCl2 (Qiagen). The K1 and 7G8 laboratory isolates, presenting different genotypes with respect to each analysed codon, were used as controls (Duraisingh et al. 2000). A negative control without parasite DNA and positive controls were run alongside the samples. For identification of different point mutations, enzymatic digestions were performed. The restriction enzymes ApoI and DraI cut the wild-type and the mutant sequence specific for asparagine at codon 86 and phenylalanine at codon 184, respectively. DdeI and AseI were used to detect the wild-type allele of serine at codon 1034 and the wild-type allele of asparagine at codon 1042, respectively. Mutation at codon 1246 (presence of tyrosine) was detected by using EcoRV. As the P. falciparum genome is haploid during the asexual blood stage, a single isolate in which both wild-type and mutant alleles were detected was considered to arise from an infection with multiple strains.

PCRs and digestion products were resolved on 1.2% agarose gel containing ethidium bromide and visualised under UV light.

Statistical analysis

Geometric mean values of parasite density were determined in both clinical groups of children (uncomplicated and severe malaria groups). The non-parametric Kruskal–Wallis and Mann–Whitney U tests were used to compare continuous variables between three (or more) and two groups, respectively. The chi-square test was used to compare nominal data. Difference was considered statistically significant when P≤0.05 (CI 95%).

Results

Characteristics of patients

Samples from 91 patients were considered in 1996. As shown in Table 1, children recruited in 1996 with severe malaria presented with a significantly higher mean parasite density (p < 0.0001) compared with uncomplicated patients.
Table 1

Characteristics of the Gabonese patients with severe or uncomplicated falciparum malaria and the multiplicity of infection

 

Severe

Uncomplicated

Pa

Year 1996

Number of patients

46

45

 

Mean age (months)

43.3 ( 13–101)

45.9 (8–140)

ns

GMPD (p/μl)

220,849 (20–900,000)

10,545 (1,000–55,000)

<0.0001

Mean haemoglobin (g/dl)

8.2 (4–13.2)

10.5 (7.9–14)

<0.0001

Female/Male

28/18

29/16

ns

Severe anaemia

6

nd

Hyperparasitaemia/other signs

40

nd

Fatal outcome

1

nd

Multiplicity of infection

2.4 (1–6)

2.2 (1–5)

ns

Pf polyclonal samples in %

68

54

ns

Year 2002

Number of patient

30

30

 

Mean age (months)

22.6 (6–54)

25.2 (6–51)

ns

GMPD (p/μl)

21,544 (300–900,000)

15,796 (10–20,4000)

ns

Mean haemoglobin (g/dl)

4 (2.7–4.9)

8.6 (5.4–12.1)

<0.0001

Female/male

17/13

16/14

ns

Severe anaemia

27

nd

Hyperparasitaemia/other signs

3

nd

Multiplicity of infection

3 (1–5)

2.9 (1–5)

ns

Pf polyclonal samples in %

97

90

ns

GMPD Geometric mean parasite density, ns not significant, nd not determined

PaComparison of continuous variable between severe and uncomplicated malaria group using Mann–Whitney U test

The multiplicity of infection reflecting the mean number of P. falciparum genotypes per infected carrier was similar in both clinical groups. However, the number of samples with more than one parasite genotype (polyclonal samples) was slightly but non-significantly higher in the severe compared with the uncomplicated group. Samples collected 7 years later in 2002 were also characterized and similar levels of multiplicity of infection and proportions of polyclonal samples were found in both clinical groups (Table 1).

Prevalence of point mutations in the Pfcrt and Pfmdr1 genes of clinical P. falciparum isolates

Genotyping of Pfcrt K76T mutation was performed on the 91 and 60 isolates from uncomplicated and severe cases collected in 1996 and 2002, respectively. All the isolates produced the expected 134 bp amplicon, and none were cut by ApoI, suggesting the presence of K76T mutation reflecting 100% of mutant type. Control HB3-strain parasites demonstrated the wild-type sequence with all sets of digestion.

Regarding the Pfmdr1 gene, as shown in Table 2, whatever the clinical group, a higher prevalence of P. falciparum isolates with the Pfmdr1 86Tyr and Pfmdr1 184 Phe mutant type alleles was detected. In both periods of recruitment, no difference in the frequency of these mutations was found neither in P. falciparum isolates from children with uncomplicated malaria nor in severe malaria. Because of the high prevalence of mutated genotype, the power to detect a difference was reasonable, despite a low sample size (e.g. a sample of 30 vs 45 subjects has a power of 84% to detect a 20% change in the prevalence of subjects with a mutation in either position 86, position 184, or both, assuming a baseline prevalence of 94%). No isolate collected in 2002 from uncomplicated cases carried the Pfmdr1 86Asn wild-type allele. Furthermore, whatever the clinical group, no mutation in P. falciparum isolates was detected at the Pfmdr1 1034 and Pfmdr1 1042 positions. About 9% of the P. falciparum isolates from uncomplicated cases had the Pfmdr1 1246Tyr mutation, while no mutation was found in isolates from severe malaria.
Table 2

Analysis of polymorphisms in pfmdr1 gene in isolates from Gabonese patients

Clinical status

Total

Number and (%) of isolates with point mutations in pfmdr1 gene

Wt

Mt

N86

Y184

S1034

N1042

D1246

Y86

F184

C1034

D1042

Y1246

UM

75 (100)

1 (1.3)

18 (24)

75 (100)

75 (100)

68 (90.7)

74 (98.7)

57 (76)

0 (0)

0 (0)

7 (9.3)

1996

45 (100)

1 (2.2)

12 (26.7)

45 (100)

45 (100)

41 (91)

44 (97.8)

33 (73.3)

0 (0)

0 (0)

4 (8.8)

2002

30 (100)

0 (0)

6 (20)

30 (100)

30 (100)

27 (90)

30 (100)

24 (80)

0 (0)

0 (0)

3 (10)

Severe

76 (100)

8 (10.5)°

23 (30)

76 (100)

76 (100)

76 (100)

65 (85.5)°

53 (70)

0 (0)

0 (0)

0 (0)

1996

46 (100)

6 ( 13)°

14 (30)

46 (100)

46 (100)

46 (100)

39 (84.8)°

32 ( 69.5)

0 (0)

0 (0)

0 (0)

2002

30 (100)

2 (6.6)°

9 (30)

30 (100)

30 (100)

30 (100)

26 (86.6)°

21 (70)

0 (0)

0 (0)

0 (0)

N86, Y184, S1034, N1042 and D1246 correspond to Asn86, Tyr184, Ser1034, Asn1042 and Asp1246, respectively; Y86, F184, C1034, D1042, Y1246 correspond to 86Tyr, 184Phe, 1034Cys, 1042Asp and 1246Tyr, respectively. Total represented the total number of subjects in each group.

UM Uncomplicated malaria group (as reported in Materials and methods), Severe severe malaria group as reported in (Materials and methods), Wt wild type, Mt mutant type

°The prevalence without considering mixed infections

Mixed genotypes were found only at the Pfmdr1 86 position in the severe malaria group in one and two isolates from 1996 and 2002 cohorts, respectively.

Discussion

In the present study, Pfcrt and Pfmdr1 gene polymorphisms were analysed in isolates from Gabonese patients living in a place where resistance to CQ remains high. We have characterized P. falciparum isolates collected from children suffering from uncomplicated and severe malaria over two periods of time in 1996 and 2002 to investigate if there was any difference in the distribution of mutations within the Pfcrt and Pfmdr1 genes in relation to the severity of the disease. Overall, P. falciparum isolates from both clinical groups, whatever the period of time, harboured the Pfcrt mutation. As the assessment of in vivo efficacy of CQ for the treatment of uncomplicated P. falciparum malaria in Lambaréné showed high-grade resistance with a failure rate reaching 100%, the presence of this mutation in all isolates was expected.

Regardless of the time period and the clinical status of children, we observed a high prevalence of Pfmdr1 86Tyr and Pfmdr1 184Phe mutations. This is in concordance with previous findings in Central Africa, particularly in Gabon and Cameroon (Grobusch et al. 1998; Basco and Ringwald 2002; Binder et al. 2002; Mawili-Mboumba et al. 2002; Uhlemann et al. 2005). As expected in the P. falciparum isolates from Central Africa (Basco and Ringwald 2002; Mawili-Mboumba et al. 2002), no mutation at the Pfmdr11034 and Pfmdr1 1042 positions and a low prevalence of mutation at the Pfmdr1 1246 codon were detected in the present study.

Several studies have suggested an involvement of the multidrug resistance (mdr) gene family in the modulation of the susceptibility to several antimalarials, including CQ (Wilson et al. 1993; Duraisingh et al. 1997; Djimde et al. 2001; Warhurst 2001; Price et al. 2004). An association of point mutations in the Pfmdr1 and Pfcrt genes with the severe manifestation of the disease, which might be due to progression of uncomplicated to severe disease after treatment failure or to increased virulence of CQ resistant parasites, has been reported from Kenya, India and The Gambia (Omar et al. 2001; Ranjit et al. 2004; Meerman et al. 2005). In our work, we did not find any selection of Pfmdr1 point mutations associated with the severity of the disease, as was also recently shown in Sudan (Giha et al. 2006).

The absence of difference in the prevalence of Pfmdr1 mutations between isolates from severe and uncomplicated malaria may confirm the lack of any direct association between Pfmdr1 point mutations and in vivo CQ resistance in our study site (Binder et al. 2002). However, the detection of high prevalence of the Pfmdr1 86Tyr and Pfmdr1 184Phe in combination with Pfcrt K76T mutation may indicate higher levels of CQ resistance as reported by Reed et al. (2000) and Babiker et al. (2001). It has been suggested by Warhurst (2001), that a mutation in the Pfcrt gene is required to confer a basic level of resistance before mutations in Pfmdr1 can have an effect, and the presence of both mutations may, however, be responsible for class II or III levels of CQ resistance (i.e. a level indicated by persistent parasitemia). Polymorphisms in the Pfmdr1 gene have also been associated with a modulation of the susceptibility to quinine, halofantrine, mefloquine and artemisinin (Duraisingh et al. 2000; Sidhu et al. 2005). As in Gabon, the official recommendation for the first-line drug policy changed to artemisinin-based combination therapy, and in Lambarene the severe malaria cases are treated with quinine. The data presented in this paper are not in favour of using polymorphism in the Pfmdr1 gene as a suitable molecular marker for the surveillance of resistance to quinine and artemisinin.

We found a slightly higher multiplicity of infection and proportion of polyclonal infections in 2002 compared with those in 1996. This is not in accordance with results from Senegal (Konate et al. 1999), showing a stable level in multiplicity of infection and polyclonality over time. As the 91 isolates from 1996 were tested at the same time as the 2002 samples, this discrepancy might simply reflect a possible DNA degradation due to a long period of sample storage; however, we cannot rule out an influence of fluctuating transmission intensity and/or differences in the pool of parasite populations.

In our study, we found a similar multiplicity of infection in isolates from children with uncomplicated and severe malaria whatever the period of blood collection. This contrasts with findings in Senegal (Robert et al. 1996), where the multiplicity of infection was found to be lower in severe cases, probably reflecting the non-detection of minor parasite genotypes (Contamin et al. 1995).

The no-difference in terms of both multiplicity of infection and prevalence of mutations in Pfcrt and Pfmdr1 genes between uncomplicated and severe malaria cases may suggest that other changes in intrinsic parasite and/or host factors that enhance parasite multiplication and virulence may lead to the severity of the malaria disease in our study site.

In conclusion, our results showed: i) no change in the polymorphism of Pfcrt and Pfmdr1 genes in P. falciparum isolates collected in 1996 and 2002, and ii) the severity of the disease was not associated with specific mutations in the Pfcrt and Pfmdr1 genes in our study site in Gabon.

Notes

Acknowledgements

We are grateful to the children who participated in this study and to the staff of the Albert Schweitzer Hospital for their cooperation. We thank Dr. Jürgen F. Kun for his advice, Dr. Adrian Luty for his critical reading of the manuscript, and Dr. Bertrand Lell for the statistical analysis. PIM was a fellow of Deutscher Akademischer Austausch Dienst (DAAD). FN received financial support from the Alexander von Humboldt Foundation and is a member of the MIMPAC network. This study received financial support under the Multilateral Initiative on Malaria (MIM) project A30031 through the UNICEF/UNDP/World Bank/WHO Special Programme for Research.

Conflict of interest statement

The authors declare no conflict of interest with any company or organisation.

Author’s contribution

Pembe Issamou Mayengue was involved in the study design, responsible for the analysis of malaria parasites, analysis and interpretation of the data and for writing the paper.

Dr. Francine Ntoumi was involved in the supervision of the study, analysis and interpretation of the data, and writing the manuscript.

Dr. Saadou Issifou was involved in the study design and was responsible for the blood collection.

Prof. Peter G. Kremsner contributed to the study design and to the critical revision of the manuscript.

Yvonne Kalmbach was involved in the analysis of malaria parasites and in drafting the manuscript.

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Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Pembe Issamou Mayengue
    • 1
    • 2
  • Yvonne Kalmbach
    • 2
  • Saadou Issifou
    • 1
  • Peter G. Kremsner
    • 1
    • 2
  • Francine Ntoumi
    • 1
    • 2
  1. 1.Medical Research Unit, Albert Schweitzer HospitalLambarénéGabon
  2. 2.Department of Parasitology, Institute for Tropical MedicineUniversity of TübingenTübingenGermany

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