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BACKGROUND
According to the World Health Organization 2016 Malaria World Report, global control efforts have resulted in a significant reduction in the number of malaria deaths since 2000.1 Despite this success, 429,000 malaria deaths still occurred in 2015.2
A vaccine to interrupt malaria transmission would be a valuable tool for malaria eradication. In the past decade, various malaria vaccine candidates have succeeded in being implemented in phase 1 studies in endemic populations, including a few of these candidate vaccines reaching phase 2 trials.3–5 However, sustained efficacy against naturally occurring infection has been modest to none, including RTS,S, which has shown partial protective efficacy for preventing clinical malaria episodes in phase 3 studies.6
A Plasmodium falciparum transmission blocking vaccine (TBV) has been identified as a key component in the effort to eradicate malaria.7,8 TBVs induce the production of anti-sporogonic antibodies in the vaccinated human host that is then taken up by the mosquito during a blood meal and thereby halting transmission to another human host. Until the recent completion of the Pfs25H-EPA/Alhydrogel® vaccine study at our clinical study site, this type of vaccine had never been tested in the field.9,10
Epidemiological characterization of the transmission reservoir is a critical step in the design of TBV protocols. In preparation for TBV studies at our clinical site, updated parasite and gametocyte carriage rates in the targeted population were needed for sample size estimation, vaccine administration timing, and endpoint evaluation and timing. This study hypothesis was that malaria parasite carriage rates including the gametocyte carriage in various age groups in Bancoumana, Mali, is still high and will be appropriate for TBV studies.
In this study, we aimed to assess malaria infection and gametocyte carriage rate over time among various age groups of children and adults in a malaria-endemic area in Mali.
METHODS
Study site.
The study site is located in and around Bancoumana village, Mali, West Africa. Bancoumana is located 60 km southwest of Bamako and has a population of 9,000 inhabitants. The site is situated in the South-Sudanian area of Mali. The climate is hot, with daily temperatures ranging from 19°C to 40°C. The annual rainfall varies between 600 mm and 1,200 mm and the rainy season typically occurs from June to October. Malaria transmission is hyperendemic with an intense transmission season from July to November.11
Study population.
Healthy volunteers between 3 months and 50 years of age were enrolled into the study. In 2011, 250 participants were enrolled and stratified into four age groups (3–11 months, 1–4 years, 5–14 years, and 15–50 years) and followed monthly through June 2012. Starting in July 2012, only subjects 5–35 years of age (N = 121) were continued to be followed from 2012 to 2013.
The sample size calculation was based on parameter (proportion) estimation in a population. In this case, the gametocyte carriage rate per age strata was the study interest and therefore the sample size was computed on this basis.
Ethics.
The study protocol was reviewed and approved by the Ethics Committee of the Faculty of Medicine, Pharmacy, and Dentistry, University of Sciences, Techniques and Technologies in Bamako, Mali, and the National Institute of Allergy and Infectious Diseases Institutional Review Board at the National Institutes of Health.
Participant recruitment.
Bancoumana community leaders were approached by the study team prior to the start of the study and study procedures were explained. Once the community leaders’ permission was obtained, the study team invited the volunteers or their parents to come to the study clinic to obtain individual informed consent and complete screening procedures. Pregnant women were excluded from participation. Study participants underwent clinical and laboratory examinations as described below. Final eligibility was determined by the investigator prior to enrollment.
Study procedures.
Subjects were seen actively on a monthly basis for medical history, vital signs, physical examination, blood smears, and research laboratories. Unscheduled visits were completed as needed for acute illnesses with clinical evaluation and malaria diagnostics completed if indicated. Any illnesses, including malaria, were documented and treated per Mali National Guidelines.
Laboratory tests.
Giemsa-stained thick and thin films were completed using approximately10 μL of blood and examined for asexual and sexual malaria parasites and rapid diagnostic tests were used if clinically indicated. The thick and thin films were read by two certified technicians. The parasite density was calculated per 1,000 leukocytes on a thick film. Discordant results were resolved by a third read completed by a senior technician. Between the two readers, there was a concordance of 87.8% (86.4–89.2%) for asexual parasite count and a concordance of 66.4% (58.8–73.9%) for gametocyte counts. Hemoglobin levels were assessed using HemoCue® or as part of a complete blood count. A urine β-human chorionic gonadotropin test was performed at screening and as clinically indicated at any point during the study. Study physicians were able to request additional laboratory examinations related to patient care at their discretion at any time during the course of the study.
Data collection and analysis.
All data were manually reported into a case report form and faxed into iDataFax, an application used to electronically capture patient data, and analyzed using SPSS statistics software. Demographic characteristics and parasite and gametocyte carriage frequencies were summarized using descriptive statistics. Comparison between age groups and year-to-year variation regarding parasite and gametocyte carriage were done using the same SPSS statistics software.
RESULTS
In June 2011, 278 volunteers 3 months to 50 years of age were consented, of which 250 enrolled into the study. In 2012, 121 subjects (5–35 years of age) reenrolled into the study for an additional year. The majority of enrolled participants were females (60.2%) and of Malinke ethnicity (90%). Volunteers from 2011 were further stratified into four age groups: 3–11 months (N = 51), 1–4 years (N = 51), 5–14 years (N = 73), and 15–50 years (N = 75) (Table 1
Demographic characteristics of study population at the enrollment in June/July 2011
| Age group | Gender | ||
|---|---|---|---|
| Male | Female | Total | |
| 3–11 months | 16 | 35 | 51 |
| 1–4 years | 19 | 32 | 51 |
| 5–14 years | 35 | 38 | 73 |
| 15–50 years | 32 | 43 | 75 |
| Total | 102 | 148 | 250 |
As expected, three malaria species were detected, with P. falciparum being the most common: P. falciparum only (97.8%, 229/234), Plasmodium malariae only (1.8%, 3/234), Plasmodium ovale only (0.4%, 1/234), and mixed infections (0.4%, 1/234). Only sexual forms of P. falciparum were assessed and reported in this study. In both October 2011 and 2012, malaria infection was highest: 21.5%, 50/233 (Table 2
Malaria infection prevalence per month and age group in Bancoumana in the study cohort in 2011
| Age group | July | August | September | October | November | December |
|---|---|---|---|---|---|---|
| 3–11 months | 4.0 (2/50) | 2.1 (1/47) | 2.1 (1/48) | 13.0 (6/46) | 13.3 (6/45) | 4.4 (2/45) |
| 1–4 years | 14.0 (7/50) | 5.9 (3/51) | 12.0 (6/50) | 22.0 (11/50) | 20.0 (10/50) | 8.2 (4/49) |
| 5–14 years | 31.4 (22/70) | 31.3 (21/67) | 32.4 (22/68) | 32.4 (22/68) | 25.0 (17/68) | 13.2 (9/68) |
| 15–50 years | 11.0 (8/73) | 11.8 (8/68) | 18.6 (13/70) | 15.9 (11/69) | 17.7 (11/62) | 9.7 (6/62) |
| Total | 16.0 (39/243) | 14.2 (33/233) | 17.8 (42/236) | 21.5 (50/233) | 19.6 (44/225) | 9.4 (21/224) |
Infection prevalence is in % (n/N).
Malaria infection prevalence per month and age group in Bancoumana in the study cohort in 2012
| Age group (years) | January | February | March | April | May | June | July | August | September | October | November | December |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 5–14 | 16.4 (11/67) | 16.1 (11/68) | 10.6 (7/66) | 10.6 (7/66) | 6.0 (4/66) | 12.1 (8/66) | 13.2 (7/53) | 24.0 (12/50) | 33.9 (18/53) | 45.2 (24/53) | 28.3 (15/53) | 20.7 (11/53) |
| 15–35 | 8.3 (5/60) | 6.3 (4/63) | 5.0 (3/60) | 0 (0/55) | 5.0 (3/60) | 7.6 (4/52) | 12.5 (2/16) | 13.3 (2/15) | 13.3 (2/15) | 13.3 (2/15) | 25.0 (3/12) | 7.6 (1/13) |
| Total | 12.5 (16/125) | 11.4 (15/131) | 7.9 (10/126) | 5.7 (7/121) | 5.5 (7/126) | 9.3 (11/118) | 13.0 (9/69) | 21.5 (14/65) | 29.4 (20/68) | 38.2 (26/68) | 27.6 (18/65) | 18.1 (12/66) |
Infection prevalence is in % (n/N).
Malaria infection prevalence varied across age groups and across months both years. For example, in the age group of 5–14 years, while the malaria infection prevalence was higher in July 2011 compared with July 2012, the opposite was seen when comparing October 2011 (32%) to October 2012 (45%). Infection in adults (participants ≥ 15 years of age) was consistently > 10% during the entire transmission season both years (Tables 2 and 3, respectively).
Gametocyte carriage rates did not vary significantly between the age groups in 2011 and 2012 (Tables 4
Gametocytes carriage prevalence per month and age category in Bancoumana in study cohort in 2011
| Age group | July | August | September | October | November | December |
|---|---|---|---|---|---|---|
| 3–11 months | 0.0 (0/50) | 0.0 (0/47) | 2.0 (1/48) | 2.1 (1/46) | 4.4 (2/45) | 6.6 (3/45) |
| 1–4 years | 6.0 (3/50) | 0.0 (0/51) | 2.0 (1/50) | 0.0 (0/50) | 6.0 (3/50) | 10.2 (5/49) |
| 5–14 years | 5.7 (4/70) | 7.4 (5/67) | 7.3 (5/68) | 10.2 (7/68) | 13.2 (9/68) | 7.3 (5/68) |
| 15–50 years | 1.3 (1/73) | 2.9 (2/68) | 7.1 (5/70) | 7.2 (5/69) | 4.8 (3/62) | 3.2 (2/62) |
| Total | 3.2 (8/243) | 3.0 (7/233) | 5.0 (12/236) | 5.5 (13/233) | 7.5 (17/225) | 6.6 (15/224) |
Gametocyte carriage prevalence is in % (n/N).
Gametocytes carriage prevalence per month and age category in Bancoumana in study cohort in 2012
| Age group (years) | January | February | March | April | May | June | July | August | September | October | November | December |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 5–14 | 7.4 (5/67) | 8.8 (6/68) | 4.5 (3/66) | 1.5 (1/66) | 1.5 (1/66) | 1.5 (1/66) | 5.6 (3/53) | 8.0 (4/50) | 11.3 (6/53) | 13.2 (7/53) | 7.5 (4/53) | 7.5 (4/53) |
| 15–35 | 1.6 (1/60) | 0.0 (0/63) | 1.6 (1/60) | 0.0 (0/55) | 1.6 (1/60) | 0.0 (0/55) | 6.2 (1/16) | 6.6 (1/15) | 13.3 (2/15) | 26.6 (4/15) | 0.0 (0/12) | 7.6 (1/13) |
| Total | 4.7 (6/127) | 4.5 (6/131) | 3.1 (4/126) | 0.7 (1/136) | 1.5 (2/126) | 0.7 (1/126) | 5.7 (4/69) | 7.6 (5/65) | 11.7 (8/68) | 16.1 (11/68) | 6.1 (4/65) | 7.5 (5/66) |
Gametocytes prevalence is in % (n/N).
DISCUSSION
The characterization of a study site and associated targeted population for gametocyte carriage is a key step in designing trials of TBVs. This includes the determination of P. falciparum gametocyte carriage rates in various age groups as well as the dynamics of P. falciparum gametocyte carriage over time among infected individuals.12
At our Bancoumana site, the malaria infection rate was consistently high throughout the transmission season from July to November for two consecutive years, which was consistent with the known seasonal malaria transmission pattern in this area.11 The malaria infection variation between the two years and across the two age groups (5–14 years and 15–35 years) may be explained by the fact that many of the same individuals were followed for the 2-year period and given participation in the trial, participants may have been treated more frequently for malaria than usual. However, other factors such as the amount of precipitation, the duration of the rainy season, and other environmental conditions that may impact vector or host behaviors should be taken into account. Detection of malaria parasites (sexual and asexual) during the dry season is likely explained by persistent asymptomatic parasitemia which can last for several months11–14 as well as continued low rates of mosquito biting.15 Collectively these observations guided our team to schedule our TBV series to start during the dry season to have the last vaccination received in mid-September. It has been previously shown that gametocyte density and mosquito infectivity correlate, though it has been seen that mosquito infectivity can occur even with submicroscopic gametocytemia.15,16 This timing permitted our team to complete transmission blocking assay assessments after receipt of last vaccination during the height of gametocyte carriage.
Our study cohort demographics is consistent with the Mali general population in terms of sex ratio (0.95 male/female) but differs in terms of ethnicity, which is expected given the study population was restricted to the Bancoumana region17 and did not enroll pregnant women. Although malaria infection and gametocyte carriage rates are consistently higher in the age group of 5–14 years across the two years, the adult population is also consistently carrying gametocytes, allowing for malaria TBVs to be assessed in this age group.
The main limitation of this study is that we do not present the infectivity data of the gametocytes in the mosquito. This is still assessed during this study and will be reported in another article. Another limitation of this study is that in July 2012, only 25% of the adults followed in 2011 were reenrolled. This was due to the fact that only those subjects 18–35 years of age were targeted for reenrollment and many of our young women became pregnant and were thus withdrawn from the study. This selective withdrawal and reenrollment may influence our results, that is, toward less precise parameter estimate in that age group for 2012 compared with 2011.
Because TBVs do not directly prevent infection of vaccinated individuals and have no direct benefit to the vaccinated individual, early trials of these products to determine safety, immunogenicity, and functional activity are needed to be performed in adults. The lower infection and gametocyte carriage rates in the age group of 3–11 months are expected and consistent with available data,18–20 but the notable high carriage rates in our pediatric age groups indicate that for a licensed TBV to be successful, all age groups eventually need to be included in their assessment to achieve malaria eradication.
Acknowledgments:
This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
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