Title: In vitro antimalarial susceptibility and molecular markers of drug resistance in Franceville, Gabon

Transcription

1 Author's response to reviews Title: In vitro antimalarial susceptibility and molecular markers of drug resistance in Franceville, Gabon Authors: Rafika ZATRA Jean Bernard LEKANA-DOUKI Faustin LEKOULOU Ulrick BISVIGOU Edgard Brice NGOUNGOU TOURE NDOUO S. Fousseyni Version: 2 Date: 22 November 2011 Author's response to reviews: see over

2 Jean Bernard Lekana-Douki Unité de Parasitologie Médicale Centre International de Recherches Médicales de Franceville B.P. 769 FRANCEVILLE GABON Dear Editor We would like to submit the revised manuscript entitled: "In vitro antimalarial susceptibility and molecular markers of drug resistance in Franceville, Gabon", Manuscript number of the first submission was MS: by Lekana Douki et al., which we would like to present for an original research. Indeed, after the first submission by July 14 th, 2011, we received reviewer comments by October 10 th, So, we have corrected the manuscript according to these comments and improved its quality. You will find the revised manuscripts within all changes are in blue color. We integrated all comments and suggestions of reviewers. See from page 2 all responses to reviewer comments. All authors have seen and approved the manuscript and have taken due care to ensure both the integrity of this work and conformity with national and institutional policies. Thank you in advance for taking the time to review this revised manuscript. Sincerely 1

3 RESPONSE TO REVIEWER S COMMENTS Reviewer 1 Reviewer's report: General Comments. This is manuscript that is describing in vitro susceptibility of Plasmodium falciparum to antimalarial drugs and the association between parasite genes polymorphisms in vitro drug response in a malaria endemic area of Gabon. The manuscript is well written and is addressing an important aspect of malaria control. The methods and data analysis are well described and appropriate. However, the sample size of the study is small and the discussion seems not to be well balanced and supported by the data. Overall, manuscript has both minor essential and major compulsory revisions that the authors must address before acceptance to publication. Minor Essential Results: 1. In vitro drug sensitivity of P. falciparum isolates. -Line 2 of this paragraph refers to figure 1. This figure does not exist, and therefore this statement should be removed. We make apologies! This figure was prepared but was forgotten during submission. So, we added it in revision version. -The number of samples is not indicated for all proportions reported in the text. N=11 is reported only for DHA and this should have been the case for all antimalarial drugs being reported in this section of the manuscript. Right! According to this good suggestion, we added these numbers: % (n=20) : line 5 page % (n= 24) : line 7 and 8 page % (n=11) : line 9 page 8 - (n=6) in CQ-resistant isolates, and 42.3% (n=11) in CQ-sensitive isolates, p=0.39). The prevalence of the Y86 mutation was respectively 37.5% (n=9) and 45.5% (n=10), 45.5% (n=5) and 34.3% (n=12), and 45.5% (n=5) and 34.3% (n=12) in MDAQ-resistant and MDAQ-sensitive. Line 21 to 24, page 8. - % in pure genotypes (n=8) and 30.4% (n=14) in CQ-sensitive isolates (23.1%, n=6) than in CQ-resistant isolates (10.0%, n=2) (p=0.05). Line 5-7 page 9. - was 26.1% (n=12) line 16 page The frequency of T2694 was respectively 25.0% (n=5) and 19.2% (n=5); 29.2% (n=7) and 22.7% (n=5); 27.3% (n=3) and 20.0% (n=7); and 27.3% (n=3) and 20.0% (n=7) in CQ-resistant and CQ-sensitive isolates; line page 9 2. Drug sensitivity and Plasmodium falciparum gene polymorphisms -The title of Table 4 should change as it gives the impression that all isolates are in the drug sensitive groups. Right! We changed title of this table to Table 4: Distribution of PfCRT polymorphism according to the drug sensitivities. - Lines 3-6 in this paragraph has to be recasted, it is quite difficult to comprehend the message being conveyed by these statements. 2

4 Text was editing again. Major Compulsory: 1. Because of the small sample size of the study presented in this manuscript, I will recommend that the manuscript be rewritten and presented as a short report for further review. Despite the small sample size, the effectives of study groups are statistically enough to take valid conclusions. Discussion: 2. The authors have reported cross-resistance between several antimalarial drugs in vitro. However, at no point during the discussion this very important aspect of the result is thoroughly discussed. Especially in the context of polymorphisms of genes that have been found to be involved in the mechanisms of resistance to various antimalrial drugs. In addition, the history of antimalarial drugs used has been demonstrated to be very important in cross-resistance to antimalarial drugs. These are important issues that are missing in the discussion of this manuscript. Right! We added followed sentences: Cross-resistance in favour of the drugs with the same mechanism was described in several antimalarial drugs. Lines 21 and 22 page 10. These data are consistent with previous data that show that the selection of MF resistance leads to the increase resistance of artemisinin in rodent model [37], despite the fact that these drugs do not belong to the same class. Indeed, MF was used before implementation of artemisinin derivates. Correlation between MF and CQ resistance is also consistent previous data showing this cross-resistance [38]. In this case CQ was introduced firstly, leading to the resistance to MF. Data of cross-resistance between these drugs warranted reinforced surveillance of antimalarial drug resistance in Gabon. Line 23 to 25 page 10 and line 1-6 page The authors found that mutant pfmdr1 Y1246 allele are higher in CQ sensitive that CQ resistant isolates, and yet in the discussion they say that they found an association between CQ resistance and pfmdr1 Y1246 allele. This discrepancy between the result and the discussion should be addressed. We make apologists! That was a writing error. So, sentence was changed to Here, we found a strong correlation between MDAQ resistance and the Pfmdr1 Y1246 genotype. Line 21 and 22, page The authors did not analyze the pfmdr1 F184 allele in the context of this manuscript. However, in the discussion of the manuscript they recommend surveillance of resistance to artemether-lumefrantrine, based on the assumption that the pfmdr1 F184 allele is important to parasite reduced susceptibility to this ACTs. The recommendation of the authors is not based on scientific evidence and should therefore be removed from the discussion of the manuscript. We changed discussion to But this mutation has been identified as an independent marker of decreased lumefantrine susceptibility [45] and it will be interesting to investigate it in our study region. Line 4-6 page A large proportion of P. falciparum from Franceville in this study was shown to harbour the pfcrt CVIET haplotype. However, at no point in the discussion of the 3

5 manuscript the high prevalence of this haplotype was discussed with susceptibility to MDAQ or the use of AQ as a partner drug in ACTs in Gabon. Right! That was a weakness of discussion. So we added followed sentences: This haplotype is associated with resistance against 4-aminoquinoline. The maintaining of the high level of frequency of CVIET, despite the withdrawal of CQ in Gabon could be due to use of amodiaquine as a partner of in ACT. Recently, CVIET was also associated with reduced sensitivity to new 4- aminoquinolines like as piperaquine, Line page The authors suggest the need for molecular surveillance with respect to in vitro sensitivity. However, an association between gene polymorphisms and in vitro susceptibility has been demonstrated only pfmdr1 Y1246 and MDAQ. I will suggest caution based on the data from this study. The recommendation of in vitro and molecular surveillance of drug resistance was based on the high prevalence of genotype associated with antimalarial drug resistance at Franceville. So, we specified in discussion In comparison with the prevalence of N86 at 2004 (15.6%) and 2009 (31.3%) this increase is gradual, calling for reinforced surveillance of artemether-lumefantrine resistance (line page 11). Discretionary Revision: It would have been great to investigate the impact of genetic background of pfmdr1 and pfcrt polymorphisms on in vitro response of P. falciparum as well as the interaction between these genes and susceptibility of parasites to various antimalarial drugs. Level of interest: An article whose findings are important to those with closely related research interests Quality of written English: Acceptable Statistical review: No, the manuscript does not need to be seen by a statistician. Declaration of competing interests: I declare that I have no competing interests. 4

6 Reviewer 2 Reviewer's report: This is a neatly written report by Zatra R. and colleagues that provide useful information on current status of in vitro P. falciparum response to antimalarial drugs and on the prevalence of molecular markers of resistance. Major Compulsory Revisions The cut-off for resistance to Monodesethyl amodiaquine is apparently wrong. The authors used 30nM. However, the exact cut-off for this drug is not known but most authors use nm (Basco L et al., Am. J. Trop. Med. Hyg., 76(1), 2007, pp and Kaddouri et al., Int J Parasitol Jun;38(7):791-8). We make apologist! During data analysis we cut-off of MDAQ IC 50 was confounded with amodiaquine s. So, we started again analysis with resistance cut-off known of 60 nm according to Basco L et al We changed text, tables and figure 1. - and monoadhesylamodiaquine were 100 nm, 20 nm, 60 nm Line 7 page 7. - and 52.2% (n= 24) line 6 and 7 page 8. - Tables 2, 3 and 4 were modified. See these tables. - respectively 37.5% (n=9) and 45.5% (n=10) line page 8 - only 3 MDAQ-resistant isolates (12.5%), (p=0.03). Lines 6 and 7 page 9 - The frequency of T2694 was ; 29.2% (n=7) and 22.7% (n=5); for MDAQ-resistant and MDAQ-sensitive isolates. Line page 9. The cut-off for DHA is not known either. It is not appropriate to talk about "resistance to DHA" after such a classical in vitro test. Because of the very fast activity of artemisinin derivatives, classical in vitro assays are now viewed as not appropriate for testing the in vitro activity of this class of compounds on P. falciparum. The authors should state the values they obtained with their method without qualifying the IC50 as being resistant or sensitive. Right! According to this good reviewer s comment we removed term resistance for DHA and we used: - In abstract: diminished DHA sensitivity lines page 2. - The IC 50 cut-off values for resistance to chloroquine, mefloquine and monoadhesylamodiaquine were 100 nm, 20 nm, 60 nm respectively and cut-off for decreased susceptibility of DHA was 10 nm. (line 7 to 8 page 7). - DHA-low susceptible (line 9-10 page 8). - Low susceptibility (in figure 1). - - DHA-low susceptible (line 1 page 9). - DHA-low susceptible (line 11 page 9). - DHA-low susceptible (line 21, page 9). - DHA low susceptibility isolates (line page 10). - decrease susceptibility to DHA (Line 23 page 10). The statistics need to be reviewed by a Statistician. Right! According to reviewer comments numbers were revised and statistics were reviewer by Dr E. Ngoungou who is statistician of our team. He has an excellent skill. Minor Essential Revisions 5

7 No Figure 1 was provided We make apologist! This figure was prepared but was forgotten during submission. So, we added it in revision version. In the Discussion nM is not a narrow range. Right! Term narrow was cancelled and sentence becomes Our results are in keeping with data for the capital, Libreville, where the prevalence of artemether resistance was 14%, with a range of 0.8 to 34.8 nm (mean IC nm) [35]. Line page 10. The sentence --- seven isolates (11.3%). is not complete. Right! Terms possible owing to were cancelled and sentence becomes Seven isolates (11.3%) could not be farmed, consistently with manufacture informations. Line 10-11, page 11. Table 2: There must be something wrong with the calculation of P values. r2 = 0.54 is not significant while r2 of 0.45 is. According to reviewer s comment about the cut-off of MDAQ resistance, the analysis of correlations was started again and values in table 2 were changed. See table 2 revised. Table 3: The results with MDAQ are probably flawed by the wrong cut-off of resistance used. Right! Because of correction of cut-off of MDAQ, tables 2, 3, and 4 were modified (see revised tables). Level of interest: An article of limited interest Quality of written English: Acceptable Statistical review: Yes, but I do not feel adequately qualified to assess the statistics. Declaration of competing interests: 'I declare that I have no competing interests' 6