Análises de sobrevivência (AS) e regressão logística (RL) foram realizadas para verificar associações entre clareamento parasitário e tipo de tratamento. A variável de interesse em um estudo epidemiológico é a variável dependente. O modelo de Cox possibilita verificar quais são as variáveis independentes que agem de maneira intensificadora, quando avaliadas em conjunto, e considera sua significância estatística dentro de um intervalo de confiança (87).
Para análise de regressão, o desfecho avaliado foi o tempo de depuração do parasita em D4, enquanto que para a análise de sobrevivência a variável analisada foi o tempo até o calreamento
Analisamos também a prevalência de gametocitonia durante o diagnóstico (D0) e o surgimento de gametócitos entre os pacientes que não os possuíam no D0.
As curvas de Kaplan-Meier e os testes log-rank para a igualdade de funções de sobrevivência, bem como a regressão de Cox pelo método de Breslow foram utilizados na análise de sobrevivência.
Todos os testes foram realizados para um nível de significância de 5%. A análise dos dados foi realizada utilizando Stata / SE 13.0
4 RESULTADOS
O manuscrito intitulado “Effectiveness of Plasmodium falciparum treatment with artemisinin regimens in the Western Brazilian Amazon: a pragmatic study”, a ser submetido ao periódico Antimicrobial Agentes and Chemoterapy (Fator de impacto de 4.476), como artigo original (vide instruções aos autores no link http://aac.asm.org/site/misc/ifora.xhtml) compõe os resultados desta dissertação de mestrado.
Effectiveness of Plasmodium falciparum treatment with artemisinin regimens in the Western Brazilian Amazon: a pragmatic study
Raquel de Medeiros Pinto1,2, Vanderson Sampaio2,3, Marcus Vinícius Guimarães Lacerda2,4,5, Wilson Duarte Alecrim2,4, Maria das Graças Costa Alecrim2,4, Wuelton Marcelo Monteiro2,4,
Simone Schneider Weber6,7*
1- Fundação de Hematologia e Hemoterapia do Amazonas, Manaus, Amazonas, Brazil 2- Universidade do Estado do Amazonas, Manaus, Amazonas, Brazil
3- Fundação de Vigilância em Saúde, Manaus, Amazonas, Brazil
4- Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Amazonas, Brazil 5- Instituto Leônidas & Maria Deane (Fiocruz), Manaus, Amazonas, Brazil
6- Instituto de Ciências Exatas e Tecnologia, Universidade Federal do Amazonas, Itacoatiara, Amazonas, Brazil
7- Centro de Ciências Biológicas e da Saúde, Universidade Federal do Mato Grosso do Sul, Campo Grande, Mato Grosso do Sul, Brazil
*Corresponding author: Simone Schneider Weber, Centro de Ciências Biológicas e da Saúde. Universidade Federal do Mato Grosso do Sul. Cidade Universitária S/N, Caixa Postal 549, 79070-900, Campo Grande, MS, BRAZIL. Phone/fax: 55-67- 3345-7559
ABSTRACT Background
Monitoring of the sensitivity of Plasmodium to antimalarial drugs is of great importance for the therapeutic management and planning of malaria control and elimination policies. Currently, the World Health Organization (WHO) recommends artemisinin-based combination therapy (ACT) as first-line for P. falciparum malaria.
Methods
In the present study, we analyzed a cohort of patients treated in a tertiary center of Manaus, with positive microscopic diagnosis of P. falciparum malaria, under treatment with artemisinin derivatives (artesunate, artesunate plus mefloquine, artemether, arthemether plus mefloquine) and followed over 35 days after to evaluate parasite clearance and recurrences. Primary effectiveness outcome was the parasite clearance at D3. Secondary outcome was malaria recurrence until 42 days from admission.
Results
Of the 1.593 patients analyzed, 96,1% had negative parasitemia at D4 using artesunate monotherapy, 91,2% using rectocaps and 94,5% using artemether monotherapy, while 97,9% had negative parasitemia at D4 using artesunate plus mefloquine and 96,7% using artemether plus mefloquine. Only 1.6% of the patients analyzed had negative parasitemia between from D5 to D10. Regarding sexual parasitemia, 47% of the enrolled patients demonstrated prevalence of gametocytes. Of all patients who cleared asexual parasitemia, 1.6% of patients treated with artesunate re-presented parasitemia during follow-up, as did 0.3% of patients treated with artemether and 4.9% treated with rectocaps. Patients treated with artesunate plus mefloquine and artemether plus mefloquine did not present a return of the parasitemia during the follow-up.
Conclusion
Estimates of parasite clearance over time provide a reliable approach and can be used to detect treatment failures in field timeframes, in which incomplete treatment due to dosage errors, non-adherence and drug quality problems are possible. Real significance of this finding needs to be considered in P. falciparum elimination scenarios.
Key-words: Plasmodium falciparum; Artemisinin; Parasite clearance; Drug resistance; Treatment effectiveness; Cohort study.
INTRODUCTION
Malaria is a major health problem in tropical and subtropical regions of the world. According World Health Organization 91 countries around the world present active malaria transmission, with a total of 429,000 deaths worldwild, of wich 99% are caused by
Plasmodium falciparum (1, 2).
In the Americas, malaria occurs in 21 countries, predominantly in South America, where 4 countries (Brazil, Colombia, Venezuela and Peru) account for more than 70% of cases (2). In Brazil most of the malaria cases are concentrated in the Amazon region (Acre, Amapá, Amazonas, Maranhão, Mato Grosso, Pará, Rondônia, Roraima and Tocantins), an endemic area for the disease. In 2014, 84% of the cases were P. vivax, followed by P. falciparum with 16% according to the Epidemiological Bulletin of the Ministry of Health of 2015 (3). In 2015, around 143,000 cases were reported throughout the country.
Asexual forms of Plasmodium are responsible for the clinical manifestation of the disease, but its transmission occurs through the ingestion of sexual forms, gametocytes, by a mosquito vector (4).
Adequate and rapid treatment is the basis of disease control, aiming at rapid cure in order to avoid the disease evolve to severe forms and even death (5). In this fight there is a vast and efficient therapeutic arsenal, however, despite the proven efficacy treatment failures are frequent and may be the result of drug resistance or inadequate exposure to drugs, whether due to failure in prescription and dispensing, incorrect dosage and even if the patient does not adhere to the treatment (6,7)
Resistance to antimalarial chemotherapy is one of the major impediments in the fight against this disease. The monitoring and effectiveness of antimalarial drugs is a crucial
component in the disease´s control, as the parasite develops mechanisms to circumvent the action drugs (8,9).
Artemisinin-based combination therapies (ACTs) have recognized activity against immature gametocytes, but do not act against mature gametocytes (4). These residual gametocytes are often found in submicroscopic fractions, being a source of infection (10). In this regard, molecular monitoring is extremely important because it detects extremely low densities of gametocytes, which microscopy is not able to detect (11).
In Brazil, many studies have reported resistance of Plasmodium falciparum (Pf) to various drugs, such as chloroquine, sulfadoxine-pyrimethamine, mefloquine, quinine and amodiaquine (12, 13). Currently, artemisinin combination therapies (ACT) are recommended as the first-line therapy for treating uncomplicated falciparum malaria in all endemic country and so far there is no evidence of failure to treat such therapy in Brazil (14, 15). In this therapy, the possibility of selection of resistant parasites is decreased because most of the parasite load is eliminated by the rapid action of artemisinin and the residual parasitemia is extinguished by the partner drug, which remains longer in circulation (16).
The widespread diffusion of artemisinin treatment results in continuous pressure by the natural selection of the most resistant parasites and this is of great concern since there is no other pharmaceutical option as effective as artemisinin and its derivatives (17,18).
Several, studies already show a decrease in susceptibility of the Plasmodium
falciparum to artemisinin on the Thai-Cambodian border, in Southeast Asia. These studies
report a delay in the parasite clearance time in the blood of some patients, which suggests a change in the parasite's susceptibility pattern to artemisinin and is probably the first sign of
artemisinin resistance (19,20, 21, 22). Some reports from French Guiana also point to a similar phenomenon in Latin America (23).
Estudos apontam alta eficácia das ACTs contra a malária por P. falciparum e ausência de marcadores genéticos associados à resistência aos fármacos no Brasil (15). Segundo a Worldwide Antimalarial Resistance Network in Latin America até o momento não há evidência de que a resistência do protozoário tenha chegado ao Brazil.
The principal objective of our study was to determine the proportion of patients who are parasitemic on day 4 (D4) over time, as an indicator of suspected artemisinin tolerance in
METHODS
Study site
Our study was carried out at Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, in Brazilian Northern region. FMT-HVD is a hospital reference for the diagnosis and treatment of tropical, infectious, parasitic and dermatological diseases. On average, 27 thousand users use the institution's outpatient and hospital services monthly. In 2016, a total of 49145 positive cases of malaria were detected, of which only 7% were P.
falciparum, 92% were P. vivax and 0.42% mixed malaria (P. falciparum + P. vivax),
according to data from the SIVEP. Study design
This was a retrospective cohort study performed to assess the responses to artemisinin-based therapy, over time. Approval for the study design was obtained by the institution’s ethics committee (CEP/FMT-HVD CAAE number: 46481215.2.0000.0005).
Data collection
The data were collected by retrospectively reviewing of the registration forms over 11-year period (September 1991 to December 2002). Enrolled data of positive malaria, including
P. falciparum or mixed (P. falciparum plus P. vivax), who received treatment with
artemisin-based therapies: artesunate (AS); artesunate/mefloquine (ASMQ); artemether (AM); artemether/mefloquine (AMMQ). All data with incomplete or illegible information were excluded from the study.
Database was constructed in Microsoft Excel program (Microsoft Corp.). Information was gathered on positive diagnosis for P. falciparum, age, sex, artemisinin-based therapies,
day follow-up (D1 to D7, D14, D21, D28 and D35). The individuals were stratified into four groups according ages: <1 year (0–11 months), 1 to less than five years (1 to <5), 5 to less than 15 years (5 to <15) and older than 15 years (≥15).
Artemisinin-based therapy and follow-up evaluations
Two artemisinin-based therapeutic schemes to P. falciparum were found as recommendation during study period: (1) monotherapy with artesunate (IV) or artemether (IM) as 1.5 mg kg−1/day, for 35 day; (2) combined therapy with artesunate (IV) or artemether (IM) 1 mg kg−1/day, followed by oral mefloquine, starting in D2, at a dose 20 mg kg−1/day for 35 day. Mixed malaria (concomitance Pf +Pv) was treated with same therapeutic scheme described to P. falciparum.
The parasite densities were quantified from thick blood films by counting the number of ring forms until 200 leukocytes also counted, then multiplying found number by 30 (which assumes an average number of 6,000 leukocytes/µL). During treatment, the parasitemia were daily registered until day 7 (D0 to D7) throughout hospitalization, after that, weekly until day 35 (D14, D21, D28 and 35).
The data were divided into two periods of six years (sexenium), and then compared to determine the percentage of positive parasitemia on D4 to each evaluated artemisinin-based therapy. The ´day-4´ parasitemia measure was considered a surveillance tool for artemisinin tolerance, guaranteeing the current WHO-recommended cut-off value (of 72 hours) (7, 26). Gametocyte monitoring
For malaria transmission to occur, it is necessary the existence of carriers of the sexual form of the parasite (gametocytes), the presence of anopheline vectors and susceptible individuals. Due to this fact it is very important monitoring the gametocytes in order to
develop actions aimed at reducing transmission. In the present study we analyzed the prevalence of gametocytemia in the diagnosis (D0) and appearance of gametocytes among the patients that did not have gametocytes in the D0.
Statistical analysis
Survival analysis (SA) and logistic regression (LR) were performed to verify associations between parasite clearance and type of treatment. For regression analysis, the endpoint evaluated was the time of parasite clearance in D4, while for the survival analysis the analyzed variable was the time until clearance. We also analyzed the prevalence of gametocytemia during diagnosis (D0) and the emergence of gametocytes among patients who did not have gametocytes in the D0. Kaplan-Meier curves and log-rank tests for equality of survival functions as well as Cox regression by the Breslow method were used in the survival analysis. For the regression analysis, stepwise backward procedure was applied. All the tests were performed for a significance level 5%. Data analysis was conducted using Stata/SE 13.0.
RESULTS
Patients’ characteristics and treatments
Between September 1991 and December 2002, 2702 patients were screened, but 1109 individuals were not eligible, according flowchart summarized in Figure 1. A total 1593 patients were considered enrolled and followed up until 35 days (97.6% of those enrolled). During cohorted period, the main treatment described was artemisinin monotherapy (AS or AM) in 85.6% (1364/1593) (Table 1).
Asexual parasite clearance analysis
Considering all analyzed artemisinin-based treatments, 95.8% presented asexual parasitemia clearance until D4 and only 1.6% between D5-D10 (Suplementary Table S1). The results revealed only three patients (0.1%) with negative parasitemia between D7 and D10. We also assessed the efficiency of artemisinin-based therapies under first six years (first sexenium) and the last six years (second sexenium), which demonstrated an advanced performance on second sexenium (Figure 2).
The asexual parasitemia was rapidly undetectable in the blood in most of evaluated treatments according survival analysis presented in Figure 3. Because asexual parasite clearance rates showed correlation with different artemisinin-based therapies, future comparisons were performed to AS versus ASMQ, AS versus AM and AS versus AMMQ (Figure 4). To investigate whether parasitemia clearance depends on others factors, we used a linear regression model (Cox regression) that evaluated the proportional risk using artesunate as a comparison to outcome of asexual stages clearance, as well the effects of host age and sexenium (Table 2). According this model, artesunate was the most effective on clearance of asexual parasitemia, with better performance in the second sexenium (about 134 times more).
In addition, age was inversely correlated with clearance of asexual forms, a 5% of reduction for every 1-year increase in age (95% CI, 0.9304 - 0.9719).
Sexual parasite emergence and clearance
The sexual stage analyze indicated prevalence of gametocytes in 47% (748/1593) of enrolled patients (Table 3). Among the individuals who exhibited sexual parasitemia (Figure 5), statistical analyzes indicated that only individuals treated with artemether, when compared with artesunate, revealed significant correlation to gametocyte clearance (p-value < 0.0289) (Figure 5B). Note that enrolled patients did not used primaquine, an exclusion criteria applied in this study (Figure 1). Then, it is important highlight that all artemisinin-based therapies exhibited an effect gametocide until D4, independent if was alone or combined posology (Figure 5A-C). In other hand, between patients with sexual parasitemia, 26.2 % (196/748) presented gametocytes at enrollment (day 0), compared to 73.7% (552/748) who exhibited sexual stage at least once during the 35-day follow-up (Table 3).
Recurrences analysis
Regarding parasitemia recurrence, 1.6% of patients treated with artesunate re-presented parasitemia during follow-up, as did 0.3% of patients treated with artemether and 4.9% treated with rectocaps. Patients treated with artesunate plus mefloquine and artemether plus mefloquine did not present a return of the parasitemia during the follow-up.
The multivariate analysis presented in Table 4 was performed considering the variables treatment and presence of gametocytes in D0. Findings revealed artemether monotherapy about 20% more efficient (95% CI, 0.9366 - 1.5528, p-value 0.146) to sexual stage. In addition, the presence of gametocytes at inclusion also seems to have an influence on clearance (95% CI, 0.958981 - 1.528274, p-value 0.108).
DISCUSSION
This is a retrospective analysis on the susceptibility of P. falciparum to artemisinin based-therapies at Brazilian north region, after its introduction in 1991. Herein, we have shown that monotherapy (artesunate and artemether) was more efficient than combined with mefloquine, inclusive the second analyzed sexenium. Artemisinin based-therapies rapidly clear parasitemia and are currently the drugs of choice for the treatment of severe malaria. Herein, we were able to find more of 75% of reduction on asexual parasite clearance rates during first two days of artesunate treatment in comparison with the others, and more than 90% until D4, suggesting no therapeutic failure to artesunate monotherapy during analyzed period. This data indicate there are no clinical evidence of declining AS efficacy in Northern Brazil during first decade of artesunate use, contradict the claim of ACTs introduction, in response to the emergence of therapeutic failure with artemisinin monotherapy.
Although artesunate monotherapy was highly effective in clearing asexual blood-stage parasitemia detected by microscopy until D4, we also described high cure rates (98%, Table S1) to ASMQ treatment. Similar clearance rates to this combination therapy have been reported previously in Peruvian Amazon (24). In addition, Ladeia-Andrade support the use of ASMQ fixed-dose combination as a first-line regimen to treat P. falciparum in Juruá Valley, region with high transmission of P. falciparum in Brazil (15), where authors showed no molecular evidence of resistance to AS and MQ during 2010 to 2013 period. Data reinforce that artemisinin and its derivatives remain highly effective in the treatment of falciparum malaria in Brazil (15).
It is worth mentioning that, these results reflect the first decade of artemisinin-based therapies use on North Brazil, and current monitoring could be necessary to perform a
comparison of the actual situation of the resistance to antimalarial in region. Since a study, using artesunate monotherapy in Suriname, inflamed an alert after of suspicion of resistance in this neighboring region (25). Several articles have reported P. falciparum resistance to artemisinin derivatives in Southeast Asia, which is of great concern, as there is no alternative as effective as artemisinin and the spread of resistance throughout the world would be catastrophic (26, 27, 28). In 2013, a published article indicated increased incidence of parasitemia persistence in D3 in Suriname (29). This study compared the efficacy of artemether-lumenfantrine, evaluating the clearance of parasitemia up to D3, with another study conducted in 2005 to 2006 to detect the appearance of resistance to artemether. Findings may represent emerging resistance of Plasmodium to artemether in a country outside the emerging region of resistance, Asia, which causes great concern, since it is an area with a history of malaria infection and also because it is a gold mining area (29).
Regarding the analysis of parasitemia recurrence, several factors may have contributed to the occurrence of such a fact. Prescribing and dispensing failures may be involved, because the patient's weight should be taken into consideration for dose calculations, as well as the patient's insertion into the groups such as the elderly, pregnant women or children. Non-adherence to the drug would also contribute to treatment failures, but in this study the dispensing of drugs was assisted, since the patients were hospitalized.
Although the focus is asexual parasitemia, due to being responsible for the clinical symptoms of the disease, we also analyzed the presence of gametocytes. We observed that the proportion of individuals with gametocytes was relatively high, 47% of the individuals enrolled. The presence of gametocytes is essential to infection transmission to other mosquitoes (11). Therefore, understanding the influence of gametocytemia and gametocidal properties of antimalarial drugs are great relevance for actions aimed to reducing the
transmission of the disease. Herein, we shown that artemether monotherapy was more efficient in clearing sexual blood-stage parasite, as well gametocyte presence at early phase of treatment (day 0) also seems influence this clearance.
In spite of the limitations because it is a retrospective study, involving observation for a long period of time, and with information retrieved from medical record books, it is the largest series of cases with patients treated with artemisinin-based therapies already described in the Amazon region. It is essential to continue this work longitudinally, in order to determine the incidence of infection more precisely, and to understand the dynamics of malaria transmission in the state of Amazonas.
CONCLUSION
This study showed stable levels of P. falciparum sensitivity to artemisinin derivatives in the Amazon region over a period of 11 years. This is due to campaign efforts and actions to contain and eradicate malaria. But this is not an easy task. In the last decade, the world has seen significant declines in the number of P. falciparum malaria cases, improvements in case management, scaling up of vector control interventions and other preventive strategies are among the key factors that have contributed to this improvement.
The findings do not evidence the occurrence of P. falciparum resistance to artemisinin derivatives in the assessed period, however, routine monitoring of artemisinin should continue so that the first signs of resistance are detected early.
Acknowledgements The authors wish to thank
The authors’ contributions are as follows: