Effectiveness of Malaria Treatment in Mexico
Condition: Malaria
MeSH terms: Malaria
Study type: Interventional
Phase: Phase 4

Summary: 
      In the context of malaria elimination in the Americas, solid evidence is necessary of the
      effectiveness of anti-malarial control measures delivered to the affected individuals. In the
      Americas, most P. vivax infections are sensitive to Chloroquine (CQ) and Primaquine (PQ), and
      the most effective treatment worldwide comprises administration of a total dose of 25
      milligrams (mg)/Kilogram (kg) weight of CQ distributed in three days and 3.5 mg/kg body
      weight of PQ administered during 14 days (T14). In Mexico, CQ and PQ have been administered
      since the late 50´s to treat malarious patients. In 1999 the National Malaria Control Program
      implemented an intermittent single doses treatment (ISD) as part of the overall strategy.
      After the blood sample was obtained for diagnosis of symptomatic patients, a single combined
      dose of CQ and PQ was administered, and after malaria infection confirmation, additional
      doses were administered monthly alternating each three months, during 3 years. Although, the
      number of malaria cases were reduced in most affected regions, in Southern México, many
      patients under ISD present recurrent blood infections, presumably relapse episodes were
      observed.

      Working hypothesis: the administration of ISD is low effective to eliminate relapse episodes
      and its effectiveness depends on the coincidence of the relapse episodes and the
      administration of the medication), while the T14 is highly effective to eliminate P. vivax
      primary and relapse infections.

      Objective: To determine the antimalarial drug effectiveness of the ISD and T14, based on CQ
      and PQ for the treatment of uncomplicated P. vivax infection (primary and recurrent blood
      infections) in Southern Mexico.

      Methods: The study was carried out in malaria affected communities of Southern Mexico,
      following the WHO recommendations for clinical studies. Symptomatic patients diagnosed with
      P. vivax infection that meet the inclusion criteria, were invited to participate. After they
      accepted by informed consent, patients were semi-randomized and treated with either T14
      (14-day treatment) or ISD (18 intermittent single doses of CQ-PQ). Clinical, parasitological,
      molecular and serological parameters were monitor over a 12-month follow up period to
      evaluate the treatment outcomes to cure blood infection and relapsing episodes. The study was
      conducted from February-2007 to October-2010. The results of this study will be used to
      assist the Ministry of Health of México in assessing the current national treatment
      guidelines for uncomplicated P. vivax malaria
    

Detailed description: 
      Malaria is in pre-elimination status in Mexico (Rodriguez et al., 2011; WHO, 2012) the local
      transmission of Plasmodium falciparum was interrupted since 2000, and transmission of P.
      vivax, the only causative agent in the country, is reduced to very few hot spots (Ministry of
      Health - Mexico, 1995-2005, Pan American Health Organization (PAHO), 2008, WHO, 2012).

      Cloroquine (CQ) and primaquine (PQ) are used since 1940 to treat P. vivax blood and liver
      infections in most affected areas worldwide (Coatney, 1963). However P. vivax resistant to CQ
      has been progressively reported in various geographic regions since the late 80´s, (Baird
      1995). Chloroquine, no longer recommended as mono-therapy WHO, 2010), administered (25 mg /
      Kg. body weight) during three consecutive days (10,10 and 5 mg /Kg body weight) in
      combination with PQ (0.25 - 0.75 mg/Kg body weight) during 14 days, is still used in regions
      were parasites are susceptible (White, 1998; WHO, 2010). The 14 -day regimen effectively
      cures primary blood infections and prevents relapses in at least 90% of patients
      (Galappaththy et al., 2007).

      In Mexico, CQ combined with PQ has been used as the standard treatment for P. vivax malaria
      since the 50´s of the past century. However, PQ treatment is frequently not completed due to
      undesirable side effects such as stomach upsetting. In locations of difficult access, the
      extended intervals between blood sampling, parasitological diagnosis and treatment of
      patients could take as long as 3-4 weeks. Hence, to reduce the risk of transmission, febrile
      patients in remote areas were blood sampled and immediately treated with a single dose of 10
      mg CQ and 0.75 mg PQ/kg body weight, and, only after microscopy confirmation, an intermittent
      single dose regimen (ISD) was administered (Mendez et al., 1994; Channon et al., 2003;
      NOM-032-SSA2-2002).

      The first single combined CQ-PQ is still administered and it is expected to eliminate blood
      parasites since PQ may destroy parasites surviving from the effect of CQ [Murphy et al.,
      1991), and the repeated administration of ISD was intended to suppress parasitemia
      recurrences (Mendez et al., 1994). However, its effectiveness has not been evaluated.

      Assessment and monitoring of the effectiveness of the implemented treatment strategies are
      necessary to advance towards malaria elimination in the region (malERA, 2011).

      Methodology This study was approved by the Ethical Review Committee of the National Institute
      of Public Health (INSP), Mexico. Informed consent was obtained from all patients and the
      guardians of patients under 18 years old.

      The study followed the World Health Organization (WHO) in vivo test protocol for monitoring
      antimalarial drug resistance. Since early relapses can occur after 17 days post-primary blood
      infection, for the primary blood infection clearance, patients were followed up for ≈Day 28
      and from one month and up to 12 months to monitor relapse elimination. When required, a
      flexible schedule was implemented to facilitate patients' follow up and minimized patient
      lost as suggested by others (Ruebush et al., 2003).

      Study site. The study was carried out in Tapachula municipality and surrounding communities
      in southern Chiapas, Mexico, bordering Guatemala. Patient recruitment was carried out in the
      diagnosis facility at the Regional Center for Public Health Research-INSP (CRISP) in
      Tapachula City, where febrile people seeking malaria diagnosis and treatment, obtain free
      services.

      Sample size. A minimum sample size of participants per group was expected as indicated by The
      World Health Organization for clinical studies in the Americas ((WHO, 2009; PAHO, 2010).
      However, the total patients enrolled during the study period, was determined by the number of
      cases diagnosed with P. vivax that meet the inclusion criteria and accepted to participate.

      Patient enrollment. A symptomatic patient was defined as a person that presented fever or two
      other malaria symptoms. Patients were recruited from February 2008 to October 2009 following
      the inclusion and exclusion criteria (see eligibility criteria). Enrolled patients were
      randomized to receive either, combined CQ and PQ medication in ISD or T14 schemes, but
      patients living in Tapachula City and nearby were preferred to receive T14 to facilitate
      daily treatment supervision. The same day of diagnosis and prior treatment administration
      (day 0; date annotated), patients were examined for axial temperature in celsius degree (°C),
      body weight (in kg), spleen swelling and the presence of clinical symptoms within the
      previous 48 h as fever, headache, myalgia and arthralgia. Erythema and jaundice signs were
      determined at day of enrollment. For parasitological and molecular analysis, Capillary blood
      was collected by finger pricking to prepare thin and thick smears and to impregnate filter
      papers (Whatman #2), respectively. Other demographic data were collected using a
      questionnaire e.g. age, gender, previous chloroquine and primaquine intake and malaria
      infections, etc.

      Patient treatment and follow-up. The recommended dose for one single dose consisted of 10
      mg/kg of CQ phosphate and 0.75 mg/kg of primaquine. Patients under ISD were schedule to take
      a single dose on Days 0, 30, 60, 180, 210 and 240. On the other hand, the optimal dose of T14
      included 25 mg/kg of CQ administered over a 3-day period (10 mg/kg on days 0 and 1 and 5
      mg/kg on day 2) and PQ at 0.25mg/kg/day during 14 days (WHO, 2010). However, both treatment
      regimens were adjusted to patients' age according to the operational table prescribed by the
      Mexican official guidelines (NOM-032-SSA2-2002). All treatment doses were administered under
      the supervision by a member of the study team. For small children, tablets were crushed,
      mixed with water and spoon fed. Treated patients were observed during 30 minutes after
      ingesting the drugs. Those who vomited the first dose were re-treated with a similar dose,
      but those who vomited again were excluded from the study.

      The first part of the study was designed to monitor the parasitological (primary blood
      infection clearance) and clinical responses to both treatment schedules. Treated patients
      were scheduled for examination on Days 2, 3, 7, 14, 21 and 28. The second part of the study
      was designed to monitor the occurrence of relapses by monthly parasitological and clinical
      examinations, up to 12 months.

      Patients were encouraged to come back to our facilities, or if necessary to contact us by
      cell phone (most patients or their parents had a cell phone), if they felt ill at any time
      during the follow-up period or had any question about the study. Participants unable to
      return to our facilities were visited at their homes to provide treatment, clinical revision
      and blood sampling. A rapid diagnostic test (RDT) (OptiMAL©) was applied to symptomatic
      patients at their homes. The thick smears of symptomatic patients with negative RDT were
      analyzed the same day and in the case of confirming a recurrent P. vivax episode, patients
      were immediately treated with T14.

      When required, a flexible schedule was implemented to facilitate patients' follow up e.g. if
      patients after the first SD regimen were not at home on Day 3 they were visited on Day 4,
      Days 7 ± 1, and Day 14 ± 2, and for all patients (under ISD or T14) on Days 21 ± 2 and so on.
      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 (in symptomatic patients). Patients with
      axillary temperatures ≥37.5 ◦C were symptomatically treated with paracetamol (10 mg/kg).

      (see Outcome measures)

      Parasitological diagnosis. Thick and thin blood films were stained with 10% solution of
      Giemsa colorant for five min. The thick smears were examined under a light microscope using
      oil immersion 100 × by experienced microscopists. Asexual and sexual parasite densities were
      determined by counting the number of parasites against 200 white blood cells (WBC) (or 500
      WBC, if less than 10 parasites were encountered in 200 WBC fields), assuming 7,000 WBC/μl of
      blood (Gonzalez-Ceron et al., 2005).

      Anti-P. vivax blood stages IgG antibodies by ELISA. To uncover P. vivax recurrent blood
      infections; asymptomatic and/or of low parasitemia not detected by microscopy,
      immunoglobulins isotype G (IgG) against native P. vivax blood stage proteins were measured in
      all samples obtained from month 1 to 12. From each patient, blood samples preserved in filter
      papers were eluted in phosphate-buffered saline (PBS) and tested in an ELISA to detect IgG
      antibodies against P. vivax blood stages (Gonzalez-Ceron et al., 1991). Sero-conversion or
      changes in antibody titers were analyzed. A cut off value of 0.25 of absorbance value was
      previously determined.

      Molecular diagnosis . Molecular diagnosis has been shown to be very sensitive in different
      malaria affected regions (Rubio et al., 1999). All follow-up blood samples of Days 2, 3, 7,
      14, ~21 and ~28, and those samples taken during the 12-month follow up with suspicion of
      recent malaria infection, suggested by an increase in antibody anti-P. vivax IgG (by ELISA ),
      were subjected to molecular diagnosis. Six punches of 5 mm of dried blood in filter paper
      were cut and used to extract DNA using the QIAamp® DNA Blood Mini Kit (QIAGEN, Germany)
      following the manufacturer instructions. The DNA was suspended in 50 µl of water. To detect
      the P. vivax RNA ribosomal 18S subunit gene (18ssrRNA).

      Genotyping primary and recurrent P. vivax infections (Gonzalez-Ceron et al., 2013). To
      compare P. vivax genotypes of primary and secondary episodes, Polymerase chain
      reaction-restriction Fragment Length Polymorphism (PCR-RFLP) was used to analyze cspr, msp3α
      and msp3β gene markers. The homogeneity of P. vivax genotypes among groups was examined by a
      Fisher´s exact test (α = 0.05).

      Data analysis All parasitological and clinical data from each patient that completed the
      supervised treatments (for 28 days, and for 12-month) were analyzed using STATA v12. To
      discard the possibility that a sub-dose caused delay in parasite clearance, the body weights
      recorded on day 0 was used to calculate doses of CQ and PQ given to patients and compared to
      that necessary for their body weight.

      Parasitological and clinical cure of the primary blood infection:

      Classification of treatment outcomes as indicated by the methods of surveillance of
      antimalarial drug efficacy (WHO, 2009)

      Early treatment failure (ETF)

        -  danger signs or severe malaria on day 1, 2 or 3 in the presence of parasitaemia;

        -  parasitaemia on day 2 higher than on day 0, irrespective of axillary temperature;

        -  parasitaemia on day 3 with axillary temperature ≥ 37.5 ºC;

        -  parasitaemia on day 3 ≥ 25% of count on day 0. Late treatment failure (LTF)

        -  Danger signs or severe malaria in the presence of parasitaemia on any day between day 4
           and day 28 in patients who did not previously meet any of the criteria of early
           treatment failure;

        -  Presence of parasitaemia on any day between day 4 and day 28 with axillary temperature ≥
           37.5 ºC (or history of fever) in patients who did not previously meet any of the
           criteria of early treatment failure.

      Adequate clinical and parasitological response (ACR)

      - Absence of parasitemia within days 7-28 without any TF signal. For the ACR analysis,
      microscopic parasitological results were included with and without PCR adjustment.

      Fisher's exact test was used to compare two or more independent proportions: gender, presence
      of symptoms, distribution of P. vivax genotype, PCR positivity and parasite persistence on
      days 2 and 3 between treatment groups. The Wilcoxon rank sum test was used for non-parametric
      comparisons: age, asexual and sexual parasitemia, drug doses, days of symptoms, and axial
      temperature. All tests of significance were two tailed and P values < 0.05 were considered
      statistically significant.

      Recurrent blood infections, when detected during the 12 month follow-up were registered as
      well as the patients retiring. The effectiveness was calculated as the percent of the
      accumulated number of patients without P. vivax blood infection (first recurrent case)
      detected at each month of sampling, divided by the total number of patients followed up. Z
      test was used to determine differences in the proportion of patients or samples with a
      recurrent infection. To plot cumulative incidence of patient with first recurrence in
      percent, the Kaplan -Meier failure estimates was used.

      To evaluate the clinical and parasitological outcome on primary blood infection, samples were
      obtained for days 2. 3, 7, 14, 21 and 28 in a flexible schedule and for recurrent blood
      infections were continue to sample each month up to 12 months. Blood samples from Day 2 to
      Day 28 were analyzed by microscopy of the thick smear and by molecular diagnosis. Samples
      from patient with symptoms were immediately analyzed and if a recurrent infection detected,
      patients were rescued by the 14-Day treatment. To uncover asymptomatic infections, all other
      samples within 1-12 months were analyzed by microscopy and tested antibody IgG titers and
      only those suspected of a recurrent blood infection were assayed by molecular diagnosis.

      Only data from patients that completed the supervise treatments were include in the analysis.
      Parasitological and clinical cure of the primary blood infection: The analysis of response to
      treatment considered two outcomes based on 1) the early clinical and parasitological outcome
      was evaluated after 2-3 days post-treatment, 2) and therapeutic failure (TF) if clinical
      and/or parasitological asexual parasite persistence or reappearance occurred within 7-28
      days. An adequate clinical and parasitological response (ACPR) was defined as (1) absence of
      parasitemia within days 7-28 without any TF signal. For the ACR analysis, microscopic
      parasitological results were included with and without PCR adjustment. The effectiveness for
      relapse elimination was evidence by the monthly follow up to one year by detecting the
      presence of symptoms and /or the parasite in the blood stream and the P. vivax genotype
      matching of primary and recurrent infections.

      Classification of treatment outcomes for recurrent blood infections detected beyond 28 days
      and up to 12 months:

      Recurrent blood infection. The recurrence of symptomatic or asymptomatic asexual
      parasitaemia, after or during treatment administration, can be produced by a relapse episode
      or a new infection. A relapse is caused by a hypnozoite, dormant stage at the liver present
      in s proportion of patients after a primary blood infection.

      The gender, age of patients with recurrent blood infections are indicated, as well as the
      timing, parasitemia, serological changes , parasite genotype, presence or absence of
      symptoms. The proportion of recurrent episodes matching or not the genotype were compared for
      both treatment regimens and statistical significance calculated (p value).

      SUPERVISION, FORMATS AND PROCEDURES TO AVOID MISSING DATA:

      Patient registry: all documents about a patient had its name, village (and home number/
      address) and the unique code. The PI was regularly supervising the procedures for diagnosis
      and patient recruitment and follow up at lab and field setting. The registry procedures and
      sheets of information per patient are kept in individual files by the PI:

        -  Form 1: Patient diagnosed with P. vivax: patient name, age, gender, any medication taken
           before diagnosis, patient address, living, asexual and sexual parasitemia per µl of
           blood. Name of the tutor in case it is under 18 years old patient.

        -  Form 2: Informed consent: one patient has met the inclusion criteria, it was asked to
           participate by using the informed consent of the study, approved by the ethical
           committee of the National Institute for Public Health in Mexico.

        -  Form 3: a clinical record sheet was fill in with patient´s name and unique code,
           clinical data from the symptomatic patient as presence of fever (within 48 h), headache,
           myalgias, arthralgias, symptoms suggesting paroxysm, clinical revision for the search of
           erythema, jaundice, and patient weight were collected.

        -  Form 4: follow up sheet was also implemented to register at all visits (schedule and
           unscheduled) the date of patient interview, clinical symptoms and blood samples
           collection. In case of absence of patient at home, it was annotated in the form and
           visit was re-schedule for the next day (in case of follow up within 28 days, but could
           be re-schedule within one week, or for monthly follow up (within month one to month 12).

        -  An Excel-data sheet data base was regularly up date (each month) and double check with
           the lab, field team and PI to prevent loss of information and inaccuracies.

        -  The slides and other samples were delivered to the laboratory wrapped in a paper with
           patient unique code, date of collection. Every weekday there was a reception by the same
           lab technician to check out all sample codes and dates with formats per patient. As the
           information was constantly revised, any inconsistency was resolved by samples both lab
           and field teams. One of two slides with thick and thin smear per time per patient were
           prepared and send regularly to the malaria laboratory at the National Institute for
           Diagnosis and Epidemiological Reference-Mexico (regularly every month), they were
           deliver in person by the PI as well as collection of results and any inquiry about
           sample´s code and results was revised and resolved. At the end of the study all samples
           were collected by the PI and in case of inconsistencies, samples were crossed between
           microscopists of the two institutions, these procedures were coordinated by the PI.

             -  A copy of the follow up form was kept at the patient's home. Data checks, Source
                data verification to assess the accuracy: Standard Operating Procedures to address
                registry operations and analysis activities, such as patient recruitment, data
                collection, data management, data analysis, reporting for adverse events, etc.

      The personnel involved in this project had specific tasks, and the PI regularly supervised
      all different activities at lab and field personnel of the study. For each patient recruited
      there was a schedule follow up and dates of visit programmed (one copy was provided to each
      patient), and when possible the cellphone of the patient or parent was useful to warn one or
      two prior days the next visit arrival.

      Field team: comprised by two people, one clinician and one biologist. Sample reception in the
      Lab: was done by the same technician all over the study, separate the slides from the filter
      paper, and deliver and/or preserve the samples.

      Microscopists: high-skilled experienced personnel, one checking samples from symptomatic
      patients prior to recruitment. Other microscopist was examining the slides from the study,
      prioritizing those samples from patients under follow up and clinically suspicious. After the
      examination, the slides were concentrated by the lab-supervisor and kept by the PI.

      Sample size necessary to demonstrate an effect: the recommendations of the WHO for malaria
      treatment evaluation in the Americas were followed, but limited by the number of patients
      with inclusion criteria that accepted to participate during the 2 year-period. The project
      was also extended to nine months.

      Data or samples with unclear information were discarded. They were not included in the
      analysis, to avoid patient lost a flexible schedule was implemented on site for missing
      patients Patient retirement (comprises voluntary or involuntary retirement, or protocol
      violation). The patient itself or patient´s tutor could retire from the study at any time, or
      the PI retires the patient if;

        1. Presence of any concomitant disease

        2. Detection of mixed Plasmodium species infection

        3. Administration of other anti-malarials or extra doses of treatment during follow up

        4. Patient does not wish to continue

        5. Get pregnant

        6. Received incomplete supervise treatment

        7. Other adverse effects: severe allergies, presence of other disease by an infectious
           agent

        8. Turn into severe malaria

        9. Receiving rescue treatment
    

Intervention name(s): Chloroquine phosphate; primaquine
Intervention type(s): Drug; Drug
Intervention description(s): use at operational doses by age group according to the Mexican guidelines for vector borne diseases; use at operational doses by age group according to the Mexican guidelines for vector borne diseases
Other intervention name(s): aralen
Intervention MeSH terms: Chloroquine; Chloroquine diphosphate; Primaquine
Allocation: Randomized
Intervention model: Single Group Assignment
Masking: None (Open Label)
Primary purpose: Treatment
Keywords: Plasmodium vivax; Mexico; treatment; chloroquine; primaquine; 14-day treatment (T14); intermittent single doses (ISD)