Effectiveness of combined chloroquine and primaquine treatment in 14 days versus intermittent single dose regimen, in an open, non-randomized, clinical trial, to eliminate Plasmodium vivax in southern Mexico

An open-labelled, non-randomized, clinical trial was conducted with a group of malaria patients receiving a combined dose of CQ and PQ with either an ISD schedule or the standard 14-day treatment (T14). In both cases, the first dose was given on day 0, the day of diagnosis. The treatment dosing was adjusted according to the age group of a patient, as recommended by the Mexican MCP for ISD and T14 [10]. The study was designed to evaluate two clinical and parasitological endpoints: the primary parasite blood infection clearance and clinical response at ~28 days post-diagnosis, and the incidence of recurrent blood infections during 12 months.

The study was carried out in the Tapachula municipality and surrounding communities in southern Chiapas, Mexico, a region on the Guatemala border with an altitude ranging from ~50–1200 meters above sea level. This is a hypo-endemic region showing variable malaria incidence, with an annual parasite incidence (API) of 0.579–1.523 cases/1000 inhabitants when considering only the affected villages for the period 2003–2007 (data from the Local Sanitary Jurisdiction VII). Patient recruitment was carried out in the diagnosis facility at the Regional Centre for Public Health Research-INSP (CRISP) in Tapachula City, where febrile individuals seeking malaria diagnosis and treatment can obtain free service.

Patients were recruited from February 2008 to October 2009 and the follow-up ended in September 2010. Inclusion criteria were: (1) patients over 12 months old; (2) P. vivax mono-infection, confirmed by microscopy; (3) parasite densities of 500-50,000/μl of blood; and, (4) elevated axillary temperature (≥37.5 °C) or a history of a fever episode within the previous 48 h. Patients were excluded if they: (1) lived in communities that could not be reached by travelling for 1 h by motor vehicle from the CRISP centre; (2) presented signs of severe malnutrition or anaemia; (3) had taken an anti-malaria treatment or had had a malaria infection within the previous 12 months (to reduce the probability of incorporating patients with a relapse) [20]; (4) had another possible cause for their fever or suffered from some chronic disease; (5) were allergic to CQ; or, (6) were pregnant (by patient statement and/or pregnancy test of women over 14) [7]. The pregnancy test consisted of a urine sample evaluated with Gestastrip II (Hycel, Jalisco, Mexico).

For practical purposes, patients were not randomized to form the two treatment groups (ISD and T14). Patients living in Tapachula City and the nearby area were preferred to administer the T14 treatment in order to facilitate daily supervision. On the day of diagnosis (day 0), patients were examined for axillary temperature, body weight, spleen swelling, and clinical symptoms such as fever, headache, myalgia, arthralgia, erythema, and jaundice. Capillary blood, collected by finger pricking, was used to prepare thin and thick smears and to impregnate filter paper (Whatman #2) for parasitological and serological (DNA and antibody) analysis, respectively.

For the ISD regimen, the standard single dose consisted of 10 mg/kg of CQ and 0.75 mg/kg of PQ, which the patients were scheduled to take on days 0, 30, 60, 180, 210, and 240. For the T14 treatment, the optimal dose was 25 mg/kg of CQ administered over a three-day period (10 mg/kg on days 0 and 1 and 5 mg/kg on day 2) and PQ at 0.25 mg/kg/day given during 14 days [7, 8]. However, both treatment regimens were adjusted to the age group of patients, according to the operational table prescribed by the Mexican official guidelines (Additional file 1) [10]. For small children, tablets were crushed, mixed with water and spoon fed. Treated patients were observed during 30 min after ingesting the drugs. Those who vomited the first dose were re-treated with a similar dose, and those who vomited again were treated, but excluded from the study.

The CQ and PQ tablets were provided by the local malaria control programme (drug batches used during the study are shown in Additional file 2). The first dose was administered immediately after diagnosis at CRISP (day 0), and all subsequent doses were administered under supervision by a member of the study team. If a patient was not available to receive one or two supervised doses, these were left with a family member. If the next day the patient claimed to have taken the dose and was willing to continue with the medication and follow-up, he/she remained in the study within the semi-supervised group.

In case of feeling ill (having fever or presenting one of two other malaria symptoms) at any time during the follow-up period, patients were encouraged to visit the facility at CRISP or to contact the field team by mobile phone (most patients or their parents had a mobile phone). The same recommendation was given in the event that patients had any question about the study. Whether during the treatment or follow-up period, participants unable to reach the facility were visited at their homes for treatment, clinical revision and blood sampling. All treated patients were scheduled for examination on days 2, 3, 7, 14, 21, and 28 [22]. Afterwards, parasitological and clinical examinations were implemented monthly until completing the 12-month protocol. When required, scheduling changes were made to complete the follow-up for each patient, as suggested by Ruebush et al. [23]. If patients, after the first ISD administration, were not at home on day 3, they were visited on day 4. Likewise, visits were made on day 7 ± 1, and on day 14 ± 2. For all patients (under ISD or T14), a visit was carried out on day 21 ± 2, and so on for the rest of the follow-up.

Two thick and thin blood films and a sample of blood impregnated in filter paper (Whatman #2) were prepared at every scheduled or extra visit. Patients with axillary temperatures ≥37.5 °C were symptomatically treated with paracetamol (acetaminophen) (10 mg/kg). A rapid diagnostic test (RDT) OptiMAL© [24] was applied to symptomatic patients when visited at home. Thick smears of symptomatic patients with negative RDT were analysed the same day and if a recurrent P. vivax episode was confirmed, they were immediately treated with T14 [8].

Thick and thin blood smears were stained with 10 % Giemsa (pH 7.2) for 5 min. The two thick smears were independently examined under a light microscope by two trained laboratory technicians by using oil immersion 100×. Asexual and sexual parasite densities were determined by counting the number of parasites found per 200 white blood cells (WBC) assuming 7000 WBC/μl of blood [24, 25]. In case of very light infections (less than 10 parasites/200 WBC), 500 WBC were inspected to confirm the counts. At least 500 fields were examined to record a sample as negative. One thick smear was analysed at CRISP and the second one by another microscopist at the Institute of Diagnosis and Epidemiological Reference, the national reference laboratory of quality control for malaria diagnosis in Mexico. Discrepancies on smears results (absence versus presence of parasites or counts differing by 50 %) were resolved by cross validation, examined by a third experienced microscopist. Asexual parasitaemia was quantified using the following formula: parasites/µl of blood = (number of parasites per 200/500 fields × 7000 WBC per 1 µl of blood)/number of WBC per 200/500 fields.

Molecular diagnosis has proven to be very sensitive in different regions affected by malaria [26, 27]. All blood samples (whether taken on days 2, 3, 7, 14, ~21, or ~28, or thereafter during the monthly examinations) with an increase in the anti-P. vivax IgG antibody titre (ELISA OD value), were subjected to molecular diagnosis due to suspicion of recent malaria infection. Six 5-mm punches of filter paper impregnated with dried blood were used to extract DNA, using the QIAamp^® DNA Blood Mini Kit (QIAGEN, Hilden, Germany) and following the manufacturer’s instructions. The DNA obtained was suspended in 50 µl of water and stored at −20 °C until used. The presence of the P. vivax 18S small sub-unit ribosomal RNA gene was assessed as previously reported [28]. Infected (with P. vivax: 500, 2000 and 10,000 p/µl) and uninfected blood samples were included as controls.

Antibodies against the P. vivax blood stage can last for months or years in exposed patients. However, antibody titres fade out more rapidly in treated patients [29‐31]. To reveal P. vivax re-exposure, with and without clinical symptoms and/or low parasitaemia in recurrent blood infections, retrospective research was conducted at the end of the 12-month follow-up period to look for native P. vivax blood-stage proteins in preserved blood samples by using ELISA, as previously reported [32]. Briefly, the blood samples that had been taken from each patient and smeared on filter paper were eluted in PBS and tested at 1:500 dilutions in an indirect ELISA. The reaction was revealed using goat anti-human IgG (H + L)-HRP (Pierce, Rockford, IL, USA) diluted 1:5000 and ABTS (2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) as substrate for 60 min, and then OD values were recorded in a spectrophotometer (Biotek^® model EL312) at 405 nm. Cut-off values were previously determined using unexposed individuals as the mean and 2SD as 0.25 OD values (95 % confidence). The ELISA-OD values were plotted according to time point per patient.

To determine the frequency of parasite genotypes, all P. vivax infected blood was analysed for cspr and msp3α by polymerase chain reaction and restriction fragment length polymorphism (PCR–RFLP) [20]. The homogeneity of P. vivax genotypes among groups was examined by Fisher’s exact test (α = 0.05). In addition to cspr and msp3α, parasite genotypes were analysed using msp3β after Alu I digestion to compare parasites producing primary and secondary episodes in each patient [33].

For each patient that completed the supervised treatments and the follow-up (for 28 days and 12 months post-diagnosis), parasitological and clinical data were analysed using STATA v12. To discard the possibility that a sub-dose caused delay in parasite clearance, the body weight recorded on day 0 was used to determine the accuracy of doses of CQ and PQ given to patients using age group dosing.

The first endpoint, parasitological and clinical cure of the primary blood infection, was evaluated by determining the early clinical and parasitological outcome at 2–3 days post-diagnosis, and the possible clinical and/or parasitological asexual parasite persistence or reappearance within seven to 28 days post-diagnosis. Therapeutic failure (TF) was defined as the presence of parasitaemia, while an adequate clinical and parasitological response (ACPR) was considered with the absence of parasites on days 7–28: for these analyses, both microscopic and PCR results were included.

Fisher’s exact test was used to compare two or more independent proportions between treatment groups, considering gender, PCR positivity, parasite persistence, the presence of symptoms, and the distribution of the P. vivax genotype on day 2 or 3. The Wilcoxon rank sum test was used for non-parametric comparisons: age, asexual and sexual parasitaemia, drug doses, number of days of symptoms, and axial temperature. All tests of significance were two-tailed and P values < 0.05 were considered statistically significant.

During the recruitment period, the diagnostic facility at CRISP received 1,820 symptomatic patients, of which 343 were diagnosed with P. vivax by microscopy. Of the 153 self-reporting malaria-symptomatic patients that satisfied the inclusion criteria, 86 received the T14 treatment and 67 the ISD regimen (Fig. 1). More patients were assigned to T14, expecting that several patients would not complete all supervised T14 doses. The proportion of gender and median age of participants, median and interquartile range (IQR) of the duration of symptoms (in days), and parasite densities were similar in both groups (Table 1). There was no difference in the proportion of patients indicating certain malaria symptoms, such as fever, headache, myalgia, arthralgia, chills, jaundice, blisters, and erythema (Table 1).

Most patients recruited for the study were living in an area of 18 × 10 sq km and they were comprised of mostly indigenous and Mexican mestizo populations. Although a higher proportion of T14 patients lived in the Tapachula City and its surrounding areas (zone 1), and a higher proportion of ISD patients lived in the foothills (zone 2) (Additional file 3), ISD and T14 patient groups had similar proportions of P. vivax cspr-msp3α genotypes (p = 0.248; Chi square test; Additional file 4). There was a significantly lower CQ dose in male patients when comparing the ISD group (median dose 0.88/IQR 0.16) to the T14 group (median dose 1.0/IQR 0.29, p = 0.01).

Out of 67 participants that received the initial dose of ISD, 18 (26.8 %) and 23 (34.3 %) completed the programmed follow-up visits and the flexible-schedule visits, respectively. Patients missing three continuous scheduled visits were excluded from the follow-up. Thirteen patients were not accessible for one visit during the first 14 days, and another four missed two follow-up visits. There were nine patients who withdrew, including four on day 2, two on day 3, one on day 7, and two on day 14. Six patients were not willing to be subjected to finger pricking, two moved to inaccessible communities, and another took an extra medication of CQ-PQ on day 3.

Out of 86 patients with the T14 treatment, 49 (56.9 %) completed the 14 supervised doses. Of these, 44 completed all follow-up visits (24 as programmed and 20 with the flexible schedule option, needed only for days 21 and 28). Three patients missed one visit, on day 21 or 28. There were two withdrawals of patients afraid of finger pricking on day 21.

Of the T14 group, 81 and 75 were sampled during supervised treatment on day 2 and 3, respectively. For the ISD group, 57 patients were sampled on these 2 days. There were no differences in the clinical outcome between the two treatment groups (Table 2). In seven of these patients (three in the T14 group and four in the ISD group), small blisters on the lips and/or outer edges of the mouth were detected. In most of these patients, temperatures above 38 °C had been registered at recruitment/diagnosis.

No difference in parasitaemia clearance was observed between treatments. Few patients had asexual blood parasites detected by microscopy on days 2 or 3 (Table 2). The reduction in the number of patients with asexual parasitaemia on day 3 with respect to day 2 was significant and similar for both treatments (T14 p = 0.000; SD p = 0.016). The number of PCR-positive samples on days 2 and 3 was higher than that determined by microscopy. On day 2, 41.3 % (1.87-fold higher than microscopy) and 34.1 % (2.3-fold higher than microscopy) of samples were PCR-positive for the ISD and T14 treatments, respectively. Persistent parasitaemia, detected by microscopy and/or PCR on days 2 and 3, was inversely correlated to the parasite count on day 0. That is, a higher parasite density (median 5068/IQR 2,067) was found on day 0 in patients that earlier cleared blood parasites, compared to the density observed (median 2891/IQR 1,357, also on day 0) in patients that on days 2 and 3 still harboured blood parasites (p = 0.0375). On the other hand, parasite persistence was not associated with parasite genotype, lower CQ doses, or patient gender or age.

All samples from days 7 to 21 were negative by microscopy and PCR, regardless of the treatment (Table 2). A light yellowish jaundice was observed in some ISD and T14 patients. In some ISD patients, this symptom persisted through day 14, while among T14 patients, the proportion with light jaundice increased with PQ administration (representing 40 % on day 14). No patient reported dark urine or any other clinical symptoms of haemolysis. In all patients, the jaundice disappeared by day 28, and no additional treatment was necessary. Erythema, present in some patients of both treatment schedules, disappeared after completion of the treatment. Light jaundice, erythema and/or blisters occurred concomitantly in only three patients.

To compare the frequencies of recurrent infections for the T14 and ISD groups, only patients that completed at least 3 months of follow-up were included. Thirty-seven and 49 patients under supervised T14 and ISD treatment, respectively, met this criterion. The number of recurrent infections that occurred in patients recruited to T14 and ISD groups was truthful; the recurrences  were not associated with a particular geographic location (Additional file 5). Moreover, no differences were observed between the 18 patients that completed the semi-supervised T14 treatment and those whose doses were all supervised. All samples from these 18 patients (101 blood samples obtained from day 3 to 28) were negative by microscopy and PCR, similar to the supervised group.

The comparison between the number and timing of recurrent blood infections in the T14 versus ISD treatment group is shown in Table 3 and Fig. 2. Three patients (P3, P47, P57) under supervised T14 had a symptomatic recurrent blood infection, after asymptomatic periods of about 7–8 months (late recurrences). All these patients were affected by different P. vivax genotypes, but the genotypes of parasites during relapses were similar to those of their primary infections (homologous recurrent infections) (Table 4). Similarly, another three patients under semi-supervised T14 (P70, P113, P149) had late symptomatic recurrent blood infections (detected by microscopy) that were genetically homologous to their primary infection. No difference in the proportion and timing of recurrent episodes between supervised versus semi-supervised T14 patients was identified (p = 0.54; Additional file 6). A significantly higher number of recurrent blood infections was detected in patients under the ISD regimen (Z value 3.85; p = 0.000) (Table 3). Eighteen of the 49 patients in this group had at least one recurrent blood infection, detected by microscopy and/or PCR. The majority of the recurrent episodes were symptomatic (17 of 23), whether confirmed by microscopy and/or PCR (Table 5; Additional file 7). Upon confirmation of the first symptomatic recurrent infection, patients were immediately treated with T14. Because blood samples in asymptomatic patients were not processed immediately after sampling, some cases of recurrent parasitaemia were not opportunely detected. The recurrent cases in this group were distributed at similar frequencies of 23.3, 20, 30, and 27.7 % within the first, second, third, and fourth quarters of the year. Only 33 % (13 of 29) of recurrent blood infections coincided with the administration of the scheduled ISD dose.

For 16 of the 20 samples from ISD patients in which the P. vivax genotype could be obtained, the primary blood infections matched their respective recurrent parasites (Table 5; Additional file 7). No association existed between the genotype and the time interval between primary and recurrent infections. That is, five patients having a primary infection with genotype cspvk210-msp3α C/msp3β V relapsed after quite different time intervals (days 32, 66, 95, 178, 287, and 352), and three patients with genotype cspvk210-msp3α B/msp3 β III also relapsed (with homologous parasites) after distinct intervals (days 74/183, 98 and 236). With all recurrent infections treated with supervised T14, the patients had no further detectable recurrent infections, with one exception (patient P44, 65 days after this treatment).

IgG antibodies against the P. vivax blood stage were detected by ELISA in 151 of 153 samples, with OD values from 0.3 to >3.0 (mean 1.14 ± 0.73) on day 7 after the initial treatment. Antibody OD values decreased post-treatment, causing nearly 50 % of the patients to be ELISA-negative within 3 months. The antibody response in two patients of the ISD group remained positive, with low OD values, until the end of the study. However, there were no clinical symptoms of a recurrent blood infection or parasitaemia (Fig. 3).

In the T14 group, recurrent P. vivax blood infections were accompanied by a drastic increase in the antibody OD value, which decreased after the infection was treated with T14 (Fig. 3a). Antibody titres increased prior to the onset of clinical symptoms and/or parasitaemia. In addition, one patient (P61) likely presented asymptomatic recurrent infections, since an increased and persistently high level of antibody titres was detected on day 95–313. The sample on day 313 was also PCR-positive (Fig. 3a; Table 4). Patient P149 (semi-supervised group) had high antibody titres on days 307 and 322 (on the latter day parasite infection was detected by PCR), suggesting a long period of asymptomatic blood infection. In the ISD group, antibody responses against symptomatic and asymptomatic recurrent infections were observed (Fig. 3b; Table 5; Additional file 7), but in three patients with increased antibody OD values (P22, 0.186_D315 → 0.57_D341; P87, 0.55_D121 → 1.73_D149; P115, 0.12_D115 → 0.90_D158), it was not possible to demonstrate malaria blood infection by microscopy or PCR. No correlation was documented between the low PQ dosage in T14 or ISD and the presence of recurrent blood infections (Table 6). Interestingly, subjects affected with genotype cspvk247-msp3α/A had a serological response before their recurrent parasitaemia was detected (T14 patients P47 and P61; ISD patients P45 and P80).