Abstracts

Background: The parasite resistance to antimalarial drugs in Central highland of Vietnam is an early warning to us that we are losing the most optimal weapons fighting the malaria. The dihydroartemisin plus piperaquine combination and chloroquin, which was listed into the essential antimalarial drugs since 2007 in Vietnam, has been used for 10 years until resistance appears in some Southern, Central of Western highland provinces.The study was conducted to evaluate the first-line drugs' efficacy in the treatment of uncomplicated Plasmodium falciparum and Plasmodium vivax patients in multicenters;

Methods: With the non-randomised controlled study design, minimum sample size, sampling, duration of follow-up, and classification of responses to treatment outcomes (WHO, 2009) in line with WHO?s protocol (2009).

Results: For DHA-PPQ regimen to P. falciparum malaria, ACPR in 3 sentinel sites were absolute cure rate (100%), except for Gia Lai sentinel with the ACPR after corrected-PCR of 95%, LCF of 1.29% and ETF of 3.71%. PCT and progression was within 48 hours o­nly; except for some cases in Gia Lai was positive asexual parasite in day 3 or ? 72 hours. The proportion of positive D₃ of 17% as clinical marker for suspected resistance. The efficacy CQ was still maintains at absolutely high level to P. vivax malaria patients, and the ACPR were 100% in 3 sentinel sites, good tolerance of DHA-PPQ and CQ was found in the treatment regimens.

Conclusions: Both DHA-PPQ and CQ regimens were highly efficacious; however, the weakness include LCF (1.29%), ETF (3.71%), and high rate of parasite existence in D₃ day (17%) in DHA-PPQ regimen as an indicator of drug resistance. Therefore, these cases should be more analyses of pharmacokinetics aspect, molecular markers to confirm the resistance more thoroughly.

Key words: Dihydroartemisinin-piperaquin, chloroquine, efficacy, resistance

INTRODUCTION

The parasite resistance to artemisinins along the Thai-Cambodia border area in the last five years is an early warning to us that we are losing the most optimal weapons fighting the parasites. Vietnam shares a border line with Cambodia, where P. falciparum is proven highly resistant to chloroquine, mefloquine, quinin and reduced responsive to various currently used drugs, including artesunate. Confronted with the warning signs, the WHO has recommended world countries to switch to the artemisinin combination therapies (ACTs). The dihydroartemisin plus piperaquine combination, which was listed into the essential antimalarial drugs since 2007 in Vietnam, has been used for 5 years until resistance appears in some Southern, Central of Western highland provinces.

Additionally, chloroquine (CQ) has long been used in Vietnam as a multi-purpose drug such as prophylaxis and treatment for both P. falciparum and P. vivax malaria for almost 60 years. Although there haven?t been any reports of CQ resistance by P. vivax in the Central highland region; many studies in the Southeast Asian countries have found resistance of the parasite to the drug at different levels. Therefore, it is necessary to evaluate the efficacy of the antimalarial drugs to contribute to the data accomplishment and to propose malaria treatment regimens that fit into the current situation and base as fundamentals for designing the national dug policy in the future. The study was conducted with objectives:

1.To evaluate the drug efficacy of dihydroartemisinine-piperaquine in the treatment of uncomplicated Plasmodium falciparum patients;

2.To evaluate the drug efficacy of chloroquine phosphate in treatment of Plasmodium vivax patients.

2. SUBJECTS AND METHODS

2.1. Locations and timing of study

The study was conducted in multi-centers in malarial hyperendemic areas at: Phu Thien district (Gialai province), Thuan Bac district (Ninh Thuan province), and Huong Hoa district (Quang Tri province) from the years 2011 to the end of 2012.

2.2. Subjects and materials

2.2.1. The uncomplicated P. falcipparum malaria patient?s group

Inclusion criteria

-Age between 6 months to under 70 years old;

-Mono-infection with P. falciparum detected by light microscopy;

-Parasitaemia of1.000 - 100.000 asexual forms/µl blood;

-Presence of axillary temperature ? 37.5°C or history of fever (past 24h);

-Ability to swallow oral medication;

-Ability and willingness to comply with the study protocol for the duration of the study and to comply with the study visit schedule;

-Informed consent from the patient or parents in the case of children;

-Not yet take any antimalarial drugs.

Exclusion criteria

-Presence of general danger signs in children aged under 5 years or signs of severe falciparum malaria;

-Mixed or mono-infection with another Plasmodium species;

-Presence of severe malnutrition, febrile conditions due to diseases other than malaria (acute lower respiratory tract infection, severe diarrhoea) or other known underlying chronic or severe diseases, severely vomitting, or psychological disorders;

-History of hypersensitivity reactions or contraindications to any of the medicine(s) being tested;

-A positive pregnancy test or breastfeeding women;

2.2.2. The P. vivax malaria patient?s group

Inclusion criteria

-Age over 6 months to < 70 years old;

-Mono-infection with P. vivax detected by light microscopy;

-Parasitaemia ofasexual forms ? 250/µl blood;

-Presence of axillary temperature ? 37.5°C or history of fever (past 48h);

-Ability to swallow oral medication;

-Ability and willingness to comply with the study protocol for the duration of the study and to comply with the study visit schedule;

-Informed consent from the patient or parents in the case of children;

-Not yet take any antimalarial drugs.

Exclusion criteria

-Under 6 months or ? 70 year olds;

-A positive pregnancy test or breastfeeding women;

-Presence of general danger signs in children aged under 5 years or signs of severe vivax malaria;

-Presence of severe malnutrition, febrile conditions due to diseases other than malaria (acute lower respiratory tract infection, severe diarrhoea) or other known underlying chronic or severe diseases, severely vomitting, or psychological disorders;

-Mixed or mono-infection with another Plasmodium species.

2.2.3. Antimalarial drugs to be tested in clinical trials

-Arterakine tablet (40mg dihydroartemisinin plus 320mg of piperaquin phosphate).Dosage regimen as followed:

-CQ 250mg tablet (150 mg base) with 3-day regimen as followed by day 1 (10mg base/kg bw), day 2 (10mg base/kg), and day 3 (5mg base/kg).

2.2.4. In code in patient?s clinical trials

-QTAK as Quang Tri arterakin, QTCQ as Quang Tri chloroquin ;

-NTAK as Ninh Thuan arterakin, NTCQ as Ninh Thuan chloroquin;

-GLAK as Gia Lai arterakin, GLCQ as Gia Lai chloroquin.

2.3. Study methods

2.3.1. Study design: Non-randomised controlled study design.

2.3.2. Sample size

Classical statistical methods are recommended for determining sample size, o­n the basis of an expected proportion of treatment failures, desired confi­dence interval (95%) and precision (10%).

In the DHA-PPQ regimen versus P. falciparum

In the case of a medicine with an expected failure rate of 20%, a confidence interval of 95% and a precision level of 10%, a minimum of 61 patients should be enrolled.

In the CQ regimen versus P. vivax

In the case of a medicine with an expected failure rate of 10%, a confidence interval of 95% and a precision level of 10%, a minimum of 35 patients should be enrolled

2.4. Study techniques

-Clinical evaluation and Hackett classification of spleenomegaly;

-Body temperature, body weight taking, nutrition condition evaluation;

-Urine analysis for cheking antimalarial components;

-Microscopic slide checking and parasite counting;

-Molecular markers analysis and genotyping of malaria parasites

-Measure of chloroquine and desethylchloroquine.

2.5. Clinical and laboratory assessment procedures

Studies of directly observed treatment for uncomplicated malaria are prospective evaluations of clinical and parasitological responses o­n days 0, 1, 2, 3, 7, 14, 21 and 28 (with CQ) and 35, 42 (with DHA-PPQ). The day the patient is enrolled and receives the first dose of medicine is day 0.

2.6. Loss to follow up

-Loss to follow up occurs when despite all reasonable efforts, an enrolled patient does not attend the scheduled visits;

-Patients who are lost to follow up but who subsequently return to the study site before day 28/42 will not be turned away and will be encouraged to return for check up visits.

2.7. Patient discontinuation or protocol violation

-Study patients who meet any of the following criteria will be classified as withdrawn:

+Withdrawal of consent of a patient at any time;

+Failure to complete treatment, due to persistent vomiting of the treatment, or failure to attend the scheduled visits during the first 3 days or serious adverse events necessitating termination of treatment before the full course is completed.

-Enrolment violation: severe malaria o­n D₀ or voluntary protocol violation or involuntary protocol violation occurrence during follow-up of concomitant disease, or detection of mono-infection with another malaria species during follow-up.

2.8. Classification of responses to treatment outcomes (WHO, 2009)

The classification of drug response in line with World Health Organization guidelines, included of Early Treatment Failure (ETF), Late Clinical Failure (LCF), Late Parasitological Failure (LPF), and Adequate Clinical and Parasitological Response (ACPR).

2.9. Adverse events and safety profiles

-An adverse event (AE): any unfavorable and unintended sign including an abnormal laboratory finding, symptom or disease associated with the use of a medical treatment, that occurs during the course of the study;

-Serious AE as any untoward medical occurrence that results in death, is life threatening, requires inpatient hospitalization or prolongation of existing hospitalization, results in persistent or significant disability.

2.10. Data analysis and study end-points

-Data in patient were entered o­n WHO-Ringwald Pascal software version 7.1. Results should be expressed as the proportion of ACPR (or proportion of ETF, LPF or LCF) before and after adjustment by PCR;

-Parasite clearance time (PCT), fever clearance time (FCT), and the proportion of patients who are parasitemic o­n day 3.

2.11. Ethical issues in clinical trials

-Approval by the official ethical and scientific committee. Study team members must be passed GCPs and do as SOPs in protocol;

-Informed consent when patients will be included in the study;

-Confidentiality: all information o­n patients will remain confidential;

-Health-care services: free health care throughout follow-up for any illness related to malaria will be provided to the study patients regardless of treatment outcome.

3. RESULTS AND DISCUSSIONS

3.1. DHA-PPQ efficacy in the treatment of falciparum malaria

Table 3.1. Baseline clinical characteristics of patients at D0

The mean temperature at D₀ were 38.22 ± 1.04⁰C, 37.78 ± 1.2⁰C, and 38.16 ± 1.0⁰C in sentinel site of Quang tri, Gia Lai, and Ninh Thuan, respectively. Number fever days of patients at D₀ was around 1-4 days. Number of cases of enlarged spleen were 55.26%, 12.31% and 70.77% respectively in Quang Tri, Gia Lai and Ninh Thuan. These suitable for most of patients were living in hyperendemic malaria areas, hence they often get malaria and many ?reinfection attack? lead to spleen parenchyma hypertrophy and fibrosis. Especially, in this trial, 2 cases with abnormal spleen status (one case of congenital hyposplenism syndrome and o­ne of totally splenectomy due to accident.

Table 3.2. Laboratory findings and malaria parasite profile in patients

The median malaria parasite density of asexual P. falciparum at three sentinel sites around 28.125-33.197/ml. Number of cases with positive gametocyte were low (6.15-16.92%) in Quang Tri, Gia Lai, and Ninh Thuan, respectively.

B?ng 3.3. Efficacy of DHA-PPQ vs. uncomplicated falciparum malaria

Followed up to the day 42 in vivo guidelines from WHO, efficacy analysis at each sentinel site showed that in Quang Tri and Ninh Thuan with ACPR or cure rate of 100%, but in Gia Lai sentinel site with ACPR at D₄₂ only 91.67%, accompanied with ETF of 3.71%, LCF of 4.62%. In 2 cases of ETF, the case of 05GLAK with high parasite density at D₀ (99.857/ml), therefore till prolong to the day D₅ this case was completely parasite clearance. And the case of 65GLAK with parasite density of 49.673/ml, but till positive asexual P. falciparum forms after 3 days regimen and high body temperature. Both of cases are normal spleen parenchyma as above mentioned. Regarding to role of the spleen, when patients are infected with malaria, intra-erythrocytic parasite development results in remodeling of both infected and non-infected red blood cells (RBCs). The spleen filters these altered RBCs. This suggested that this splenic-pitting is responsible for a majority of the parasite clearance that is seen in patients. In fact, asplenic individuals living in falciparum endemic regions, exhibit more frequent fevers, higher parasitemias, and longer parasite clearance times following treatment with antimalarials such as artemisinin. Studies using P. berghei-infected mice, found that animals with full splenectomies had parasitemias double those found in intact and partially splenectomized mice. In other study in experiment animal, when assessed parasite clearance in DHA-treated intact and asplenic mice, it was found that the capacity to clear parasites was reduced in the asplenic population.

Table 3.4. Analysis of early treatment failure cases by in vivo test

In two cases of ETF, the case of 05GLAK with extremely high parasite density at D₀ (99.857/ml), therefore till prolong to the day D₅, and the case of 65GLAK with parasite density of 49.673/ml, but till positive asexual P. falciparum forms after 3 days. The cases of LCF at the D₂₆ or D₄₂ must be done in PCR analysis.

Table 3.5. Discrimination of reinfection or recrudescence by PCR

The result of adjusted-PCR showed that 2 cases of LCF were reinfection or new infection of P. falciparum (29GLAK and 48GLAK) at the day D₄₂. Specially, the case of 62GLAK was recrudescence o­n the D₂₆. This patient has parasite cleared after 3 days treatment of DHA-PPQ, then reappeared of P. falciparum alone at D₂₆. With the results of LCF in Gia Lai sentinel, after corrected-PCR, efficacy was changed increasing the ACPR rate from 91.67% to 95%, the LCF rate reducing from 4.62% to 1.29%, two cases of reinfection or new infection, and the rest 2 cases of ETF were 3.71%.

Table 3.6. PCR-adjusted and unadjusted treatment outcomes

Figure 3.1. Efficacy of DHA-PPQ versus P. falciparum at 3 sentinel sites

With the results PCR corrected in discrimination of recrudescence and reinfection, DHA-PPQ efficacy was changed increasing the ACPR rate from 91.67% to 95%, the LCF rate reducing from 4.62% to 1.29%.

Table 3.7. Efficacy of DHA-PPQ in parasite and fever clearance

The differences in the baseline median parasite density between the treatment groups are not significant at the day D₀. However, compared to Quang Tri and Ninh Thuan, Gia Lai sentinel showed that parasite clearance time (61.5h) much longer than Quang Tri (37.8h) and 37.3h (Ninh Thuan). In paralell with rapid PCT, the clinical symptomes would be improved better at Quang Tri and Ninh Thuan, but in Gia Lai seemed to be longer in fever clearance time as some patients who are positive asexual parasite at 72 hours or more.

Table 3.8. Proportion of positive asexual P. falciparum at  D₃

Analysis of all cases of asexual parasite existence, the positivity rate of 17% at day 3 (72 hours) after treatment with DHA-PPQ in Gia Lai as an important indirect clinical indicator of treatment failure or resistance.

Table 3.9. Progression and asexual parasite clearance from D₀ to D₃

Figure 3.2. Progression of parasite clearance day by day (24h)

In total of 65 cases in Gia Lai sentinel site, there was 17% rate of positive parasite at day D₃ or D_4.This mean parasite clearance data by step every 24h from D₀ to D₅ in detail of 44.121/mL decreased by 9.633/mL, 358/mL, 66/mL, 8/mL, and 0/mL, respectively. This is currently the best available indicator here, need to be more analysis of pharmacokinetics.

Analysis of all cases of asexual parasite existence, the positivity rate of 17% at day 3 (72 hours) after treatment by DHA-PPQ regimen in Gia Lai as an important indirect clinical indicator of treatment failure or resistance, and the parasitemic o­n day 3 is currently the best available indicator used in routine monitoring to measure P. falciparum sensitivity to artemisinins. With an increase in parasite clearance time (61.5h) and evidence 17% of cases with parasites detectable o­n day 3 after treatment with DHA-PPQ. To make sure exact resistance, need to be done more pharmacokinetic and molecular marker analysis in the next time.

Table 3.10.Proportions of adverse events in DHA-PPQ treated group

Using consecutive 3 days of DHA-PPQ therapy, some adverse events were observed. Several other mild side-effects including headache, dizziness, nausea, itching and rash. It is very difficult to differentiate these adverse events with malaria disease symptoms.

3.2. Chloroquin efficacy in the treatment of vivax malaria patients

Table 3.11. Baseline clinical characteristics of patients before trials

Some baseline clinical and paraclinical characteristics of patient profile showed that most of patients were fever or history of fever during past 48hs before study enrollment, and mean body temperature at 3 sentinel sites around 38.51 ± 1.15⁰C; 38.29 ± 1.2⁰C and 38.29 ± 1.20⁰C in Quang Tri, Gia Lai and Ninh Thuan, respectively. Because of living in hyperendemic malaria areas, the patients had enlarged spleen of 33.93%; 25.81% and 40.30% in Quang Tri, Gia Lai, and Ninh Thuan, respectively. Majority in spleenomegaly cases, grade I and II.

Table 3.12. The haematology and malaria parasite profile in patients

Median asexual form of P. vivax parasite were 3.185; 3.256 and 2.810/ml in Quang Tri, Gia Lai, and Ninh Thuan sentinel sites, respectively. Especially, number of vivax malaria cases had positive gametocyte of 75%, 80.65% and 89.36%.

Table 3.13. The efficacy of CQ regimen vs. P. vivax malaria

Figure 3.3. Efficacy of CQ regimen vs. P. vivax malaria

The data showed that the ACPR were 100% in three sentinel sites.

Table 3.14. Efficacy in parasite clearance and fever clearance of CQ

Concerning parasite and fever clearance time, after 3 days of CQ therapy, PCT were 39.26 ± 7.68h; 31.22 ± 6.28h and 41.20 ± 4.69 h in sentinel of Quang Tri, Gia Lai and Ninh Thu?n, respectively. Although mean body temperature of patients were 38.5 ± 1.5⁰C, 37.5 ± 1.7⁰C and 38.5 ± 1.0⁰C, the FCT were about 48 hours. These does mean stable CQ-sensitive P. vivax.

However, with the literature of drug resistance all over the world, especially in the neighboring countries of the Mekong subregion, and with the fact that CQ has been used for over 60 years with various purposes, the warning signs of reduced susceptibility or resistance may occur in Vietnam and Central-Western highlands in particular in the near future. Routine supervisions of the drug efficacy in some areas, especially in endemic areas with higher proportion of P. vivax of the parasite formula, is really necessary.

Due to difficulties in recording the symptoms reported or exclusively relating to P. vivax infection, these studies showed that some mild and low proportion adverse events by every 12 hours interval examination in first 3 consecutive days, such as nausea, abdomen pain, itching, headache, dizziness, rash, or vision blurred.

CONCLUSIONS AND RECOMMENDATIONS

1. Conclusions

1.1. The efficacy of DHA-PPQ regimen to P. falciparum malaria

-Adequate clinical and parasitological responses (ACPR) in 3 sentinel sites were from 95-100%, including 100% of absolute cure rate in Quang Tri, Ninh Thuan; except for Gia Lai sentinel with the ACPR after corrected-PCR of 95%, LCF of 1.29% and ETF of 3.71%;

-PCT and progression was very quick, within 48 hours o­nly; except for some cases in Gia Lai was positive asexual parasite in day 3 or ? 72 hours. FCT in parallel with PCT was just within 48 hours in Quang Tri & Ninh Thuan, but longer in Gia Lai;

-The proportion of positive D₃ of 17% as clinical marker for suspected resistance.

1.2. The drug efficacy of chloroquine in treatment of P. vivax malaria

-The efficacy CQ was still maintains at absolutely high level to P. vivax malaria patients, and the ACPR were 100% in 3 sentinel sites;

-The chloroquin?s PCT and FCT as well were quick, within 48 hours since CQ was given;

-Generally, good tolerance of DHA-PPQ and CQ was found in the treatment of uncomplicated P. falciparum and P. vivax malaria patients. Some side-effects occurred at mild level, with low proportion, no serious reactions that require no interventions with first aid or drug withdrawals, especially these symptoms have improved remarkably after drug withdrawals.

2. Recommendations

At present, the combined DHA-PPQ regimen is highly efficacious; however, the weakness include LCF (1.29%) and ETF (3.71%) in 2 patients with abnormal spleens, and high rate of parasite existence in D₃ day (17%) in DHA-PPQ regimen as an indicator of drug resistance. Therefore, it is recommended that:

-The role of the spleen is very obvious in parasite clearing intervention, so that it is necessary to supplement this criterion into ?exclusive criteria? of the proposals to assess the P. falciparum and P. vivax drug efficacy in vivo when conducting the research in the field or evaluating the effectiveness at the hospital treatment system;

-All the cases with parasite existence until D₃ after completing DHA-PPQ therapy, there should be more analyses of pharmacokinetics aspect, molecular markers to confirm the susceptibility/ resistance of P. falciparum in the treatment with DHA-PPQ more thoroughly;

-The CQ regimen as recommended by the MoH still remains effective; therefore, CQ is considered the first of choice for P. vivax malaria treatment, but need to monitoring of their efficacy routinely.

References

Other news »

---