Efficacy of Chloroquine for the Treatment of Uncomplicated Plasmodium falciparum Malaria in Honduras

Abstract

Chloroquine (CQ) is officially used for the primary treatment of Plasmodium falciparum malaria in Honduras. In this study, the therapeutic efficacy of CQ for the treatment of uncomplicated P. falciparum malaria in the municipality of Puerto Lempira, Gracias a Dios, Honduras was evaluated using the Pan American Health Organization-World Health Organization protocol with a follow-up of 28 days. Sixty-eight patients from 6 months to 60 years of age microscopically diagnosed with uncomplicated P. falciparum malaria were included in the final analysis. All patients who were treated with CQ (25 mg/kg over 3 days) cleared parasitemia by day 3 and acquired no new P. falciparum infection within 28 days of follow-up. All the parasite samples sequenced for CQ resistance mutations (pfcrt) showed only the CQ-sensitive genotype (CVMNK). This finding shows that CQ remains highly efficacious for the treatment of uncomplicated P. falciparum malaria in Gracias a Dios, Honduras.

Full text

MEJIA TORRES AND OTHERS
TREATMENT OF UNCOMPLICATED P. FALCIPARUM MALARIA IN
HONDURAS
Efficacy of Chloroquine for the Treatment of Uncomplicated Plasmodium
falciparum Malaria in Honduras
Rosa Elena Mejia Torres,* Engels Ilich Banegas, Meisy Mendoza, Cesar Diaz, Sandra
Tamara Mancero Bucheli, Gustavo A. Fontecha, Md Tauqeer Alam, Ira Goldman,
Venkatachalam Udhayakumar, and Jose Orlinder Nicolas Zambrano
National Malaria Laboratory, National Department of Surveillance, Ministry of Health, Honduras;
National Malaria Eradication Service (SNEM), Ministry of Public Health, Ecuador; Area of Health
Surveillance and Disease Control, Pan American Health Organization, Honduras; MEIZ-Microbiology
School, National Autonomous University of Honduras (UNAH), Tegucigalpa, Honduras; Atlanta
Research and Education Foundation, Decatur, Georgia; Malaria Branch, Division of Parasitic
Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention, Atlanta,
Georgia; National Malaria Program, Department of Health Promotion, Ministry of Health, Honduras
* Address correspondence to Rosa Elena Mejia Torres, National Malaria Laboratory, National Department of
Surveillance, Ministry of Health, Honduras. E-mail: rosaelenamejiatorres@yahoo.com
Abstract.
Chloroquine (CQ) is officially used for the primary treatment of Plasmodium falciparum malaria in
Honduras. In this study, the therapeutic efficacy of CQ for the treatment of uncomplicated P.
falciparum malaria in the municipality of Puerto Lempira, Gracias a Dios, Honduras was evaluated
using the Pan American Health Organization—World Health Organization protocol with a follow-up of
28 days. Sixty-eight patients from 6 months to 60 years of age microscopically diagnosed with
uncomplicated P. falciparum malaria were included in the final analysis. All patients who were treated
with CQ (25 mg/kg over 3 days) cleared parasitemia by day 3 and acquired no new P. falciparum
infection within 28 days of follow-up. All the parasite samples sequenced for CQ resistance mutations
(pfcrt) showed only the CQ-sensitive genotype (CVMNK). This finding shows that CQ remains highly
efficacious for the treatment of uncomplicated P. falciparum malaria in Gracias a Dios, Honduras.
INTRODUCTION
Development of resistance to chloroquine (CQ) and other drug treatment in
Plasmodium falciparum malaria has led the World Health Organization (WHO) to
change its recommendations to adopt artemisinin combination therapy (ACT) as the
first-line drug for the treatment of P. falciparum malaria cases in most endemic
countries. In the Americas, CQ-resistant P. falciparum has been documented in all
South American countries endemic for malaria since the 1950s,1 including Brazil,2
Bolivia, Colombia,3 Ecuador, Peru,4 Guyana,5 Suriname,6 and Venezuela.7,8 All of
these countries have changed their drug policies in alignment with WHO
recommendations to adopt ACT.9
In contrast, for Central American countries (except Panama) and the island of
Hispaniola, CQ continues to be the first line of treatment of P. falciparum malaria,
because there is no evidence for the presence of CQ resistance in Central America
outside of Panama, Mexico, or the Caribbean islands.10 Panama is the only Central
American country with documented CQ-resistant P. falciparum malaria11 and as a
result, changed its first line of treatment to sulfadoxine-pyrimethamine (SP)
combination in 2004.
In order to provide our readers with timely access to new content, papers accepted by the American Journal of Tropical Medicine and Hygiene are posted
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http://ajtmh.org/cgi/doi/10.4269/ajtmh.12-0671The latest version is at
Accepted for Publication, Published online March 4, 2013; doi:10.4269/ajtmh.12-0671.
Copyright 2013 by the American Society of Tropical Medicine and Hygiene

Honduras has the highest malaria burden in Central America.12 Honduras
accounted for 69%, 84%, and 91% of all P. falciparum cases reported in Central
America in 2007, 2008, and 2009, respectively.13 A remarkable decrease in the
transmission of the disease has been observed in the country in the last decade: from
35,125 cases in 2000 to 8,368 cases by 2008.13 Most of malaria cases are found in the
northeastern region of the country, particularly the state of Gracias a Dios, which
reported 33% (2,798 cases) of all malaria cases in 2008 and accounted for 69–94% of
P. falciparum cases over 2007–2009.14
In Honduras, CQ efficacy was determined using in vivo protocol only in 1981, and
since that time, no additional studies have been conducted.15 To confirm the efficacy
of CQ for the treatment of P. falciparum malaria in Honduras, an in vivo study was
conducted using Pan American Health Organization (PAHO) –WHO protocol with a
28-day follow-up. In addition, P. falciparum isolates collected from this study were
tested for the presence of CQ-associated molecular markers in the pfcrt gene.
MATERIALS AND METHODS
Study location and patient enrolment.
The study was carried out from September of 2008 to September of 2009 in the
municipality of Puerto Lempira, Gracias a Dios, Honduras (Figure 1), a highly
endemic area for P. falciparum malaria. This study was approved by the institutional
ethical review committee of Ethics Committee of the Medical Sciences Faculty of the
National University of Honduras (UNAH-IRB 00003070). All participants provided
written consent to participate in the study. Patients residing within a radius of 30–45
minutes of travel time (car or boat) were included, because this area was convenient
for the study team to conduct follow-up observations. Criteria for patient inclusion
were uncomplicated P. falciparum malaria as described by national and international
definitions.10 This criteria included participants ages between 6 months and 60 years
with microscopically detectable monoinfection with P. falciparum, parasitemia levels
greater than 250 but less than 50,000 asexual forms/µL, willingness to attend follow-
up visits for 28 days, and willingness to participate in the study and provide informed
written consent. The exclusion criteria included pregnancy or lactation (pregnancy
test was performed for women of childbearing age), signs of severe malaria,16
underlying chronic disease (cardiac, renal, or hepatic disease or malnutrition), history
of allergy to CQ, and presence of mixed infection with different species of malaria
parasites.
Sample size.
The sample size was determined according to the proportion of treatment failures
expected in this population. Assuming a CQ treatment failure rate of 5% in a
population of infinite size, a power of 80%, and a significance level of 5%, 42 patients
per treatment would be required. If allowing for 15% attrition, 49 patients would be
needed. A total of 69 patients who were determined to have only P. falciparum
infection based on microscopic diagnosis was enrolled for this study, and 68 of the
patients completed the follow-up until day 28.
Microscopy and sample collection.
Blood smears were stained with 3% Giemsa for 30 minutes at room temperature.
Smears were analyzed by experienced microscopists. The parasite density was

determined by using the PAHO–WHO criteria assuming 6,000 leukocytes/µL. A total
of 137 (as one failed to complete follow-up) blood spots (Whatman number 3),
including 69 samples collected at the time of enrolment and 68 samples collected at
the 28-day follow-up, was dried and stored at −20°C until use for molecular analysis.
Drug treatment and follow-up.
The antimalarial drug and treatment schedule used was CQ at 25 mg/kg body
weight divided into daily doses over 3 days: 10 mg/kg on day 1, 10 mg/kg on day 2,
and 5 mg/kg on day 3. SP and quinine were chosen as alternative drugs if CQ was not
effective. Supervised treatment was administered to all patients followed by clinical
and parasitological evaluation on days 0, 1, 2, 3, 7, 14, 21, and 28. Therapeutic
response was classified in patients who completed the follow-up using the definitions
described in the standard PAHO–WHO protocol.17
To facilitate the administration of proper dosage, tables of weight-based dosing
were available. Treatment choice and dosage were administered by the same team
under the supervision of clinical personnel. Patients were monitored for 30 minutes
after drug administration for adverse reactions or intolerance. Paracetamol was
administered for fever (axillary temperature higher than 38.5°C) with doses of 10
mg/kg (maximum = 500 mg) four times per day conditional on the presence of fever.
Patient follow-up.
Patients enrolled before treatment with CQ received supervised treatment in the
Health Center for follow-up until day 28. Because only patients with uncomplicated
malaria treated with drugs with established safety profiles were included in the study,
daily monitoring was not required. Both legal guardians (parents of children under 18
years) and patients were informed about the significance of treatment and the
importance of the 28-day follow-up.
Molecular diagnosis and genotyping for CQ resistance markers.
Genomic DNA from P. falciparum-positive filter paper blood spots was isolated
using the Tris–ethylenediaminetetraacetic acid (EDTA) method as described earlier.18
To confirm the parasite species present in the samples, an 18s rRNA nested
polymerase chain reaction (PCR) was performed with primers and cycling conditions
as described by Singh and others.19 PCR results confirmed presence of P. falciparum
infection in 68 patients, and 1 patient was found to have P. vivax but not P.
falciparum infection as reported recently.20
To determine if there is any parasite with CQ-resistant genotype, the DNA was
subjected to the PCR amplification of the partial regions of pfcrt (covering codons
72–76) using a nested PCR approach. The primary PCR was performed with 5′-
AGCAAAAATGACGAGCGTTATAG-3′ (F) and 5′-
ATTGGTAGGTGGAATAGATTCTC-3′ (R) primers with the following cycling
parameters: initial denaturation at 94°C for 10 minutes followed by 35 cycles of
denaturation at 94°C for 30 seconds, annealing at 59°C for 30 seconds, and extension
at 72°C for 30 seconds followed by a final extension at 72°C for 10 minutes.21
Secondary PCR was done using 5′-TTTTTCCCTTGTCGACCTTAAC-3′ (F) and 5′-
AGGAATAAACAATAAAGAACATAATCATAC-3′ (R) primers. The cycling
parameters for secondary PCR were the same as for the primary PCR, except that
annealing was set at 56°C for 30 seconds and the number of cycles was reduced to 30.

Sequencing of gene fragments was carried out on both strands with their respective
nested primers using the standard sequencing protocols on an ABI 3130xl Genetic
Analyzer (Applied Biosystems, CA) as described earlier.21
Statistics.
Double entry of data was executed and then analyzed using Excel. Median and
geometric means were used for analysis of continuous variables and percentages for
categorical and nominal variables.
RESULTS
A total of 4,827 febrile patients attended two Health Centers, of whom 3,714
patients were negative for malaria according to light microscopy (Figure 2). These
patients were referred to medical consultation to investigate other diseases; 1,113
patients were diagnosed with malaria, of whom 791 (71%) patients had P. vivax, 263
(24%) patients had P. falciparum, and 59 (5%) patients showed a mixed infection
with both species. Of the total cases of P. falciparum malaria captured in the sentinel
sites, only 69 cases were included in this study (26%): the remainder of cases did not
meet protocol inclusion criteria or the patient refused to participate. Among these 69
patients included in the study, 1 patient was excluded from the final analysis, because
this patient was found to have only P. vivax but not P. falciparum infection based on
subsequent PCR test.20 Demographic data of study participants are given in Table 1.
All patients selected for the study were clinically evaluated according to height,
weight, physical examination, and blood pressure on day 0, whereas temperature and
blood smear examination were done on days 0, 1, 2, 3, 7, 14, 21, and 28 (Figure 3). At
the end of evaluations on day 7, all patients were parasite-negative (absence of
microscopically detectable asexual stages in blood), and none developed parasitemia
through day 28 of the trial, indicating 100% efficacy of CQ (Figure 4). None of the
patients reported having taken antimalarial drugs on their own (CQ, primaquine, or
SP); 57 patients (95%) took analgesic, antipyretic, and/or anti-inflammatory
medications to reduce symptoms and discomfort.
Molecular markers associated with CQ resistance.
The pfcrt gene sequencing data for 68 samples showed no mutations within amino
acid residues 72–76. All the samples had the CVMNK amino acid sequence (CQ-
sensitive ancestral genotype) in this region, confirming the absence of parasites with
CQ-resistant pfcrt allele.
DISCUSSION
The emergence of drug-resistant strains of P. falciparum has contributed to
worldwide resurgence of malaria in recent decades,22 and it is associated with
increased mortality and morbidity.23 Drug susceptibility usually relies on various
factors, such as the intensity of infection, immune status, plasma concentrations of the
drug, and duration of drug application24–26; however, the inherent capacity of the
parasite to tolerate drugs is mostly based on its genotype.27,28 We conducted this study
to evaluate in vivo effectiveness of CQ against P. falciparum malaria infections and
determine the pfcrt genotypes of the P. falciparum strains currently circulating in a
highly endemic region of Honduras.

Although Honduras has not reported resistance to the current first-line antimalarial
drugs CQ and primaquine, it remained unclear if the efficacy of CQ has changed over
the time, and there have been occasional local anecdotal reports of treatment failure
by both physicians and patients. Another concern was whether any potential misuse of
CQ in the local population has altered the clinical efficacy of this drug. Recently, the
state of Gracias a Dios has become a major hub for human migration from South
America, where CQ resistance is fixed in P. falciparum parasites. This finding has
raised concern about potential importation of CQ-resistant parasites through human
migrants. Therefore, continuous monitoring for the CQ efficacy and molecular
surveillance for CQ-resistant pfcrt genotype in this region are essential for the support
of current drug policy.
Because there are different parameters of clinical and parasitological assessment
to evaluate resistance to antimalarial drugs, standard protocols were used in this
study.29 We used the PAHO–WHO standard methodology and followed patients for
28 days to assess the efficacy of CQ for the treatment of uncomplicated P. falciparum
infection. In this study, the most important finding is the 100% efficacy of CQ
treatment of clearing P. falciparum infection during the 28 days of follow-up. This
finding is further confirmed by the exclusive presence of only CQ-sensitive ancestral
codons of the pfcrt gene (CVMNK genotype) in all the parasite samples tested. This
study is consistent with the research conducted 3 decades ago,15 which showed, at the
regional level, the usefulness of CQ as a first-line drug. Our study provides strong
support for the continuation of CQ for the primary treatment of malaria in Honduras
for now.
This study was carried out in the area with the highest incidence of malaria in
Honduras and did not include patients from other regions of the country. Although the
data from this study cannot be extrapolated to the rest of the country, recent results
obtained by Jovel and others30 confirm that P. falciparum isolates from other regions
of the country also possessed a susceptible wild-type pfcrt genotype. Furthermore,
they showed that CQ-resistant alleles of pfmdr1, sulphadoxine-resistant pfdhps alleles,
and pyrimethamine-resistant pfdhfr alleles were also not detectable in this region.
Because conducting in vivo drug efficacy trials can be expensive, it is reasonable to
continuously monitor the parasite populations from different parts of the country for
the presence of CQ-resistant pfcrt allele as an early warning signal for the potential
emergence of CQ resistance.
The findings from this study are consistent with recent reports indicating that CQ
remains efficacious for the treatment of falciparum malaria in Nicaragua31 and Haiti.32
Collectively, these findings are consistent with continuation of CQ as the primary
drug of choice for the treatment of malaria in this region. It is still unknown why CQ-
resistant parasites have failed to establish in Central American regions outside of
Panama, although CQ-resistant parasites are fixed in South America and have been
found in Panama.11 Implementation of an active molecular surveillance program to
detect emergence of any CQ-resistant P. falciparum parasites will help to develop
appropriate measures to prevent the spread of resistant parasites and evaluate the
efficacy of CQ for continued treatment of falciparum malaria in this region
In conclusion, this study showed evidence that there is no resistance to CQ, which
is the first-line drug used in the country, according to the National Guidelines for
malaria.10 However, it is necessary to maintain regular monitoring of therapeutic
efficacy of antimalarial drugs with standardized methodology and rigorous quality

control. An early indicator is the presence of CQ-resistant molecular markers, which
can be monitored through the application of routine molecular surveillance of P.
falciparum isolates in the country.
Received October 31, 2012.
Accepted for publication November 30, 2012.
Acknowledgments:
The authors thank Efrain Burgos, Director of the Department of Health of Gracias a Dios, and Edgardo
L. Barahona, technician in environmental health of the state, for providing support in carrying out this
study. Rosalba Quintero Montana trained the microscopists in the study in diagnostics. Prabhjot Singh
helped in drafting and revised the final paper. We also thank the members of the Department of Health
of Gracias a Dios and the Hospital of Puerto Lempira, where this study was carried out.
Financial support: This work was funded by the US Agency for International Development (USAID)
under the USAID Pan American Health Organization agreement for the Amazon Malaria Initiative
Network for Surveillance of Anti-Malarial Drug Resistance Project 527A-00-08-00026-00 and the
Ministry of Health of Honduras.
Authors’ addresses: Rosa Elena Mejia Torres, Engels Ilich Banegas, and Meisy Mendoza, National
Malaria Laboratory, National Department of Surveillance, Ministry of Health, Honduras, E-mails:
rosaelenamejiatorres@yahoo.com, engelsbanegas@yahoo.com, and elimeis@hotmail.com. Cesar Diaz,
National Malaria Eradication Service (SNEM), Ministry of Public Health, Ecuador, E-mail:
diazcortezert@gmail.com. Sandra Tamara Mancero Bucheli, Pan American Health Organization,
Honduras, E-mail: mancerot@paho.org. Gustavo A. Fontecha, MEIZ-Microbiology School, National
Autonomous University of Honduras (UNAH), Tegucigalpa, Honduras, E-mail:
gustavo.fontecha@unah.edu.hn. Md Tauqeer Alam, Malaria Branch, Division of Parasitic Diseases and
Malaria, Center for Global Health, Centers for Disease Control and Prevention, Atlanta Research and
Education Foundation, Decatur, GA, E-mail: hsf1@cdc.gov. Ira Goldman and Venkatachalam
Udhayakumar, Malaria Branch, Division of Parasitic Diseases and Malaria, Center for Global Health,
Centers for Disease Control and Prevention, Atlanta, GA, E-mails: ifg1@cdc.gov and vxu0@cdc.gov.
Jose Orlinder Nicolas Zambrano, National Malaria Program, Department of Health Promotion,
Ministry of Health, Honduras, E-mail: jnicolaszambrano@yahoo.es.
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FIGURE 1. Map of Honduras showing the study site for in vivo drug efficacy study in
Puerto Lempira, Honduras.
FIGURE 2. Flowchart of the enrolment process for in vivo drug efficacy
study based on microscopic diagnosis in Puerto Lempira, Honduras.
FIGURE 3. Number of study participants with fever (> 37.5°C) by day of observation in the
in vivo drug efficacy study in Puerto Lempira, Honduras.
FIGURE 4. Geometric means of asexual parasite density by day of observation in
the in vivo drug efficacy study in Puerto Lempira, Honduras.
TABLE 1
Characteristics of study participants enrolled in the in vivo
drug efficacy study in Puerto Lempira, Honduras
Characteristic N = 68*
Median age in years (range) 17 (1–40)
Children under 5 years (%) 7, 10
Children 5–15 years (%) 21, 31
Adults 40, 59
Males (%) 39, 57
Axillary temperature ≥ 37.5°C (day 0; %) 46
Geometric mean parasite density (µL−1; day 0) 6,268
* Although 69 patients were enrolled based on the microscopic diagnosis of P.
falciparum, 1 patient was excluded from the final analysis, because subsequent PCR
experiments determined presence of P. vivax infection but not P. falciparum infection
in that patient.

Figure 1

Figure 2

Figure 3

Figure 4