Epidemiology of Plasmodium vivax Malaria in Peru
Epidemiology of Plasmodium vivax Malaria in Peru
Angel Rosas-Aguirre 1 2 3
Dionicia Gamboa 0 2 3
Paulo Manrique 2 3
Jan E. Conn 2 6 7
Marta Moreno 2 5
Andres G. Lescano 2 4
Juan F. Sanchez 2 4
Hugo Rodriguez 2 8
Hermann Silva 2 8
Alejandro Llanos-Cuentas 2 3
Joseph M. Vinetz 0 2 3 5
0 Departamento de Ciencias Celulares y Moleculares, Facultad de Ciencias y Filosofia, Universidad Peruana Cayetano Heredia , Lima , Peru
1 Research Institute of Health and Society, Université Catholique de Louvain , Brussels , Belgium
2 California San Diego, MC0760 , 9500 Gilman Drive, La Jolla, CA 92093 , and Instituto de Medicina Tropical “Alexander von Humboldt,” Universidad Peruana Cayetano Heredia , Avenida Honório Delgado 430, Lima , Peru
3 Instituto de Medicina Tropical “Alexander von Humboldt,” Universidad Peruana Cayetano Heredia , Lima , Peru
4 Facultad de Salud Pública, Universidad Peruana Cayetano Heredia , Lima , Peru
5 Division of Infectious Diseases, Department of Medicine, University of California San Diego , San Diego, California
6 Wadsworth Center, New York State Department of Health , Albany , New York
7 Department of Biomedical Sciences, School of Public Health, University at Albany (State University of New York) , Albany , New York
8 Dirección Regional de Salud Loreto, Ministerio de Salud , Iquitos , Peru
Malaria in Peru, dominated by Plasmodium vivax, remains a public health problem. The 1990s saw newly epidemic malaria emerge, primarily in the Loreto Department in the Amazon region, including areas near to Iquitos, the capital city, but sporadic malaria transmission also occurred in the 1990s-2000s in both north-coastal Peru and the gold mining regions of southeastern Peru. Although a Global Fund-supported intervention (PAMAFRO, 2005-2010) was temporally associated with a decrease of malaria transmission, from 2012 to the present, both P. vivax and Plasmodium falciparum malaria cases have rapidly increased. The Peruvian Ministry of Health continues to provide artemesininbased combination therapy for microscopy-confirmed cases of P. falciparum and chloroquine-primaquine for P. vivax. Malaria transmission continues in remote areas nonetheless, where the mobility of humans and parasites facilitates continued reintroduction outside of ongoing surveillance activities, which is critical to address for future malaria control and elimination efforts. Ongoing P. vivax research gaps in Peru include the following: identification of asymptomatic parasitemics, quantification of the contribution of patent and subpatent parasitemics to mosquito transmission, diagnosis of nonparasitemic hypnozoite carriers, and implementation of surveillance for potential emergence of chloroquine- and 8-aminoquinoline-resistant P. vivax. Clinical trials of tafenoquine in Peru have been promising, and glucose-6-phosphate dehydrogenase deficiency in the region has not been observed to be a limitation to its use. Larger-scale challenges for P. vivax (and malaria in general) in Peru include logistical difficulties in accessing remote riverine populations, consequences of government policy and poverty trends, and obtaining international funding for malaria control and elimination.
Peru, located along South America’s central Pacific coast,
is home to about 30 million inhabitants living in 1,838
municipalities in 24 administrative departments and one
constitutional province (Callao, adjacent to Lima)1; the population
is highly concentrated in the arid megacity, Lima, with
almost 10 million inhabitants. Arthropod-borne diseases
(malaria, arboviruses) generally are not important public
health threats in Lima and the southern desert regions, but
affect the humid, tropical regions where the environment
sustains arthropod vectors. Peru has the third greatest
biodiversity in the world, largely concentrated in the humid
tropical ecosystems within 84 of the 117 recognized
ecosystems found in Peru.2
Nationally, in 2015, 62,220 cases of malaria were reported
in Peru,3 which accounts for about 15% of total reported
malaria cases in the Americas and shows a continuing
increase in cases since 2012 (Figures 1 and 2). Therefore,
malaria remains an important public health problem in the
country.4 In Peru, the vast majority of malaria is
concentrated in Loreto Department (capital, Iquitos), in the
northwest Amazon region, which accounts for 95% of reported
malaria cases.3 Infection by Plasmodium vivax is more
common than that by Plasmodium falciparum (Pv/Pf ratio of 4/1
in 2015). Malaria transmission is perennial but characterized
by seasonal and epidemic increases of clinical cases detected
by routine passive case detection (PCD) in some
communities.5,6 Most affected people live in impoverished peri-urban
and rural villages sparsely distributed along an extensive
riverine network associated with tributaries feeding into the
Amazon river.6,7 Intermittent epidemic malaria as well as
sporadic cases take place in the Pacific northern coast,8 and
transmission is low intensity in the high and central jungle,9
and the gold mining department of Madre de Dios,5
accounting together for about 5% of the total malaria cases reported
Plasmodium vivax malaria transmission is maintained in
human populations by relapses from dormant liver
parasite stages (i.e., hypnozoites),10 for which no diagnostic test
exists11; treatment with primaquine (PQ) in Peru is given,
but effectiveness is unclear because administration is not
typically supervised.12 The presence of PQ resistance or
compliance has not been systemically assessed under either routine
or clinical trial conditions. Recent clinical trials of
tafenoquine (TQ) (which included substantial enrollment of
subjects in the Peruvian Amazon) for the treatment of acute
P. vivax malaria have shown that this 8-aminoquinoline is
effective not only for treatment and relapse in general,13
but also that there is a dose-dependent effect on
symptomatic homologous strain relapse (but not heterologous strain
reinfection).14 Subjects in these clinical trials were tested for
glucose-6-phosphate dehydrogenase deficiency, but clinically
significant low levels that would limit the use of
8-aminoquinolines were not found among more than 300 subjects.
The increasing focal distribution of malaria transmission,5,6
the logistical difficulties in accessing remote riverine
populations,5 the specific limitations for the control of P. vivax
infections,15 together with the fact that a large proportion
of those infections are asymptomatic and not detected by
standard diagnostic methods such as light microscopy
(LM),5,6,16,17 pose major challenges to the Peruvian national
malaria control program (NMCP).
Despite the increasing research conducted in Peru over
the past decade on P. vivax malaria epidemiology,17–21
diagnostics,22,23 treatment,12,13,24 vector biology,25–28 and
molecular genetics,29–31 several knowledge gaps still need to be
addressed to support the Peruvian NMCP, so that malaria
transmission hotspots can be readily identified and that the
NMCP can more effectively implement strategies that
target such areas with effective interventions.32 In this review,
the trends in P. vivax incidence are described in relation to
major control efforts implemented in Peru over the past
70 years. The ecological context of P. vivax-endemic regions
in Peru is described, and key research findings related to
P. vivax transmission and its control in the country are
outlined. Finally, the opportunities and challenges for not
only controlling but also sustaining such measures toward
P. vivax elimination are discussed.
FIGURE 2. Annual reported cases by Plasmodium species in Peru and Loreto: 1990–2015. Information on annual malaria cases was obtained
from the Regional Health Directorate of Loreto. Numbers in the timeline point out important events that influenced malaria incidence: 1) first
report of Plasmodium falciparum in Loreto (1990); 2) first reports of chloroquine resistance (CQR) in P. falciparum (after 1994); 3) very strong
El Niño Southern Oscillation (ENSO) phenomenon (1996–1998); 4) implementation of new antimalarial treatment policy for P. falciparum and
Plasmodium vivax malaria (2001–2004); and 5) Global Fund-PAMAFRO project (2005–2010).
PERSISTENCE OF P. VIVAX TRANSMISSION
DESPITE CONVENTIONAL TEST AND TREAT
CONTROL EFFORTS IMPLEMENTED BY
PUBLIC HEALTH AGENCIES
Even though reported malaria cases were not
systematically differentiated by Plasmodium species from the 1940s to
the 1990s,4 it is likely that P. vivax dominated. Long-standing
practices for gathering malaria case data are based on
testand-treat models, which involve Ministry of Health
information systems that passively gather microscopy-confirmed
malaria cases using paper registries. The data from these
registries are reported and aggregated in a hierarchical fashion
from health post to regional to national levels. Paper
documentation is the practice at the local levels where the primary
data are gathered. After peaking with over 80,000 cases in
1944,4 malaria dropped substantially in the following years;
from 1960 to 1970, annual incidence rates remained below
one case per 1,000 inhabitants with the lowest levels
registered in 1965, when only about 1,500 total cases were
reported (Figure 1).33 These results have been due to the
use of dichloro-diphenyl-trichloroethane (DDT) in vector
control campaigns after 1946, and to the shift of malaria
control to a formal eradication strategy after 1957.4 The major
vector in the Amazon (i.e., Anopheles darlingi) was nearly
eliminated, and almost the entire coast, the inter-Andean
valleys, and the Southern Amazon were determined to be
free of malaria by 1970.34,35 However, the development
of DDT resistance, as well as reduced political and public
health interest resulting in decreased funding for control
measures and the redirection of malaria control
responsibilities toward test and treat strategies according to medical
(i.e., treating individuals) not public health (population-based
intervention) models, combined to halt progress in malaria
control.34 It must be kept in mind that changes in
governmental policies can lead to instability in the implementation
of malaria control policies as well.
In the 1980s, the Peruvian NMCP was not structured to
enable elimination activities.34 Following international
guidelines, DDT use was first halted in the Peruvian Amazon in
1988, and then stopped across all of Peru. Predictably malaria
reemerged in the 1990s, following the reintroduction and later
spread of An. darlingi in the Amazon,33 as well as the
simultaneous introduction of chloroquine (CQ)-resistant P. falciparum
strains (CQ) in the northern coast36 and CQ and sulfadoxine–
pyrimethamine (SP) resistance in the Amazon region.37,38 In
association with severe weather changes linked to the El Niño
Southern Oscillation (ENSO) climatologic phenomenon,4,39
malaria increased dramatically reaching epidemic levels
between 1997 and 1999 (Figure 2). The highest annual
malaria incidence in the country was reported in 1998 with
more than 200,000 cases, of which about 60% and 40% were
due to P. vivax and P. falciparum, respectively (Pv/Pf ratio of
1.5/1)(Figure 2). The Peruvian Amazon and the northern
coast were most affected,40 accounting for about 45% and
40%, respectively, of the total malaria cases (approximately
530,000 cases) during the 1997–1999 epidemic period.
After the epidemic, malaria incidence in Peru dropped
to 57,712 and 68,003 cases, respectively, in 2000 and 2001,
followed by a slight increase and stabilization around
80,000–85,000 cases between 2002 and 2005.41 The
decreasing contribution of P. falciparum cases to the total malaria
cases during the period 2000–2005—as evidenced by an
increase of the Pv/Pf ratio from 2.5/1 to about 5/1—may be
an early effect of the implementation of a new malaria
treatment policy that introduced two different
artemesininbased combination therapies (ACTs) as first-line treatments
for P. falciparum, that is, mefloquine–artesunate (AS) in the
Amazon region, and AS–SP in the northern coast.37 With
P. vivax malaria, the radical treatment scheme including CQ
(3 days) and PQ were also adjusted by shortening the PQ
regimen from 14 to 7 days at increased daily dose (0.5 instead of
The new malaria treatment policy was implemented more
quickly in the northern coast than in the Amazon,
primarily because of better accessibility to malaria-endemic areas
(i.e., peri-urban communities) which facilitated training and
supervision of the local health facilities and staff.42
Test-andtreat strategies—PCD of symptomatic subjects with treatment
only of confirmed malaria infections with ACT—together with
implementation of community-based environmental
management strategies to control the predominant vector Anopheles
albimanus (such as the shift of rice cultivation patterns)
probably contributed to the steady decline in malaria incidence
observed in the northern coast from the early 2000s.8,42
In the Amazon region, the new treatment policy took
longer to implement. After 2005, the implementation
process was driven by the Global Fund’s Malaria Project
“PAMAFRO”43 by increasing accessibility and quality of
microscopy-based diagnosis, ensuring availability of effective
antimalarials, and periodic training and supervision of local
health workers, even in the most remote endemic areas. This
work did not involve intervening to reduce mosquito
breeding sites in communities as an alternative to the use of
insecticide spraying because of lack of knowledge of where such
sites lay. PAMAFRO was simultaneously applied at the
border areas of Venezuela, Colombia, Ecuador, and Peru
between October 2005 and September 2010, with common
control strategies, including early diagnosis and treatment,
active case detection (ACD) interventions in communities
of high malaria risk (as determined by a set number of cases,
and only carried out either under this PAMAFRO program
or research conditions), mass distribution of long-lasting
insecticide-treated bed nets, community participation (local
health worker involvement and empowerment to implement
campaign), and intensified health education and promotion
campaigns.44–46 During this period, malaria declined in Peru
from 87,805 reported clinical cases in 2005 to 29,339 and
23,060 cases in 2010 and 2011, respectively.41,42
Comparatively, the percentage of reduction in reported cases was
higher for P. falciparum (85%) than for P. vivax (63%),
thus keeping the increasing trend of the Pv/Pf ratio until
reaching its maximum (11.8/1) in 2010 (Figure 3). After
almost 40 years and for the second time in the known history
of malaria in Peru, the malaria incidence rate was less than
1 case/1,000 inhabitants in those 2 consecutive years (2010
and 2011) (Figure 1). The significant reduction in malaria
incidence from 1998 to 2013 led to important changes in the
malaria risk map in Peru; with each subsequent year,
lowtransmission areas predominated more and more over
highand moderate-transmission areas (Figure 3). The quantitative
contributions of PAMAFRO, sociopolitical, public health
practices and policies, and climate-related changes to these
malaria risk map changes are not known, but remain to be
explored in future work.
FIGURE 3. Malaria risk map of Peru based on annual incidence rates 1998–2013 (annual parasitological index [API]). Source: Peruvian
Ministry of Health.
However, there have been alarming developments since
2011. Since then, the number of malaria cases again began to
rise (Figures 2 and 3), with the total number of reported
P. vivax and P. falciparum cases increasing from 20,421 to
49,745 and from 2,639 to 12,475 cases, respectively, from
2011 to 2015.3 One leading hypothesis to explain the rapid
resurgence of malaria after 2011 is that the Peruvian NMCP
was politically and financially limited so that it could not keep
up with the intense control interventions in the Amazon after
the PAMAFRO project ended.6,42 Because of such a rapid
resurgence, other factors must have contributed including
weather changes (i.e., increased rain, increased river height
related to Andean snow melt related to climate change, as
well as social and political disorder in Iquitos in recent years.
Funds were reallocated to more pressing public health issues
in the region (particularly dengue), because it was assumed
that malaria was “under control,” combined with unusually
heavy rains after 2011, which damage and flooding to many
rural communities located along the Amazon river and all its
tributaries, further worsened the situation.42 Political and
financial commitment from the Peruvian government to
combat this malaria resurgence has increased since 2015, allowing
for a wider deployment of ACD interventions in the
highestrisk communities. However, the impact of these efforts might
be limited if strategies are not adapted to the specific local
malaria epidemiology, and, importantly, commitment is not
sustained. More attention to longer-term investment in
elimination strategies with development and deployment of new
malaria intervention tools will also be required.
PERUVIAN P. VIVAX-ENDEMIC REGIONS
About half the total population of Peru is potentially
exposed to malaria or lives in areas with ecological factors
favorable to transmission,42 both of which are found in the
northern and southern Amazon region, and in the Pacific
northern coast (Table 1).
The northern Amazon region comprises predominantly the
Loreto Department, located in the northeast of Peru and
covering almost 30% of total territory in Peru and most of
the Amazon region. Loreto is home to about 1 million
inhabitants.1 Its climate is warm and humid with a heavy
rainy season from November to May and a mild rainy season
from June to October.33 Malaria transmission is perennial,
peaking between February and July,47 but with some
variations across districts. The highly anthropophilic mosquito,
An. darlingi, is the primary malaria vector, and a recent
report showed a trend in this vector for increasing outdoor
biting compared with previous reports of near-equal indoor
and outdoor biting.28 Plasmodium vivax and P. falciparum
infections occur at a ratio of 4/1 mainly in people living in
open or semiclosed wooden houses located in rural and
remote villages along the main Amazon river and its
tributaries.7 All age groups are at risk of infection, though adults
more so than children.
Despite the fact that Loreto has been considered to be
hypoendemic for malaria transmission,16 routine surveillance
data and epidemiological studies suggest that malaria
transmission in the region is highly heterogeneous, with some
areas in relatively remote Amazonia having entomological
inoculation rates (EIRs) rivaling those found in holoendemic
regions of Africa.19 More than 80% of the total malaria
clinical cases detected by PCD in Loreto are concentrated among
only 10–20% of the total communities of the department.6
Moreover, cross-sectional studies using LM for malaria
diagnosis have also showed that malaria parasite prevalence
varied considerably across communities around Iquitos
(capital of Loreto),16,47 and those differences remained or even
P. vivax (99%), P. falciparum (< 1%)
Closed houses with completed walls
Commerce, fishing, agriculture
Perennial transmission with
Tropical rainforest, heavy rains
Tropical rainforest, heavy rains
Accessibility to health services
ENSO = El Niño Southern Oscillation.
increased when using more sensitive diagnostic tests.16,48
Indeed, the use of polymerase chain reaction (PCR) in
epidemiological studies has shown that an important proportion
of the total infections are asymptomatic and with low and
submicroscopic (subpatent) parasite densities.5,6,16,48 Similarly,
entomological studies have also shown evidence for
transmission heterogeneity by finding a wide variation of EIR estimates
at the microgeographical level in riverine campsites about
50 km of Iquitos and along the only paved highway in Loreto
(the Iquitos–Nauta road).19,26,49 Among the potential
explanations for the spatial heterogeneity of malaria are ecological
factors that facilitate mosquito breeding and resting sites are
associated with deforestation, natural and man-made water
bodies (e.g., fish ponds), and surrounding secondary
vegetation are among the potential explanations for the spatial
heterogeneity of malaria. In addition to vector-related
ecological factors, human socioeconomic differences such as
poorer housing conditions (open or semiclosed houses)50
and outdoor economic activities (such as farming, logging,
or fishing) and travel along riverine routes among endemic
sites of transmission likely play an important role in the
maintenance of malaria transmission, often with unexpected,
The southeastern jungle of the Peruvian Amazon region,
that is, the Madre de Dios Department, has long
experienced intermittently epidemic and sporadic malaria,
virtually all of which is due to P. vivax.51 The primary driver
of malaria in this region is illegal gold mining, with
attendant ecological, political, and socioeconomic change.
Conventional wisdom is that malaria is repeatedly reintroduced
into the region by migrants who acquired infection in
endemic areas, so that small outbreaks occur, then
disappear for unknown reasons. The analysis of PCD data
between 2001 and 2012 (J. F. Sanchez, J. M. Vinetz, A. G.
Lescano, unpublished data), showed that of 203,773 febrile
cases, 30,811 (15%) were confirmed cases of malaria, all
due to P. vivax. The 10 P. falciparum cases identified
during this period were likely imported from Brazil. Health
facilities located in areas of intense illegal gold mining
reported 30-fold more malaria cases than those in nonmining
areas (ratio = 31.54, 95% confidence interval [CI] = 19.28,
51.60); health facilities located more than 1 km from the
InterOceanic Highway reported significantly more cases
than those within this distance (ratio = 16.20, 95% CI =
Transmission of malaria in Madre de Dios is unstable and
geographically heterogeneous. The primary malaria vector is
An. darlingi, but to date it has proven difficult to identify
breeding sites of this mosquito species within environmental
contamination and degradation related to human
settlements and mining-related chemical discharges in the
malariaaffected areas. Anopheles (Nyssorhynchus) benarrochi B and
Anopheles (Nyssorhynchus) rangeli were also found among
anopheline specimens obtained and analyzed in 2011 by
human landing catch, Shannon and CDC traps (Centers for
Disease Control and Prevention, Atlanta, GA) from the
malaria endemic localities of Santa Rosa and San Pedro in
Madre de Dios Department, Peru; neither of these potential
secondary vectors were found to be positive for Plasmodium
infection by cytochromb b (CytB)-PCR, although the sample
size was relatively small.52 Research findings in Madre de
Dios highlight the unpredictable nature of malaria
transmission in areas of Peru where public health policies are difficult
to implement, and only control measures based on PCD
have been carried out. These observations underscore the
importance of continued stochastic reintroductions of malaria,
that timely, surveillance-based interventions are needed to
control malaria in neglected and politically challenging regions,
and the need for new tools for monitoring and predicting
newly emerging malaria in areas where anophelism remains,
for example, in illegal gold mining camps.
The northwestern coast, which includes Tumbes, Piura,
Lambayeque, and La Libertad departments, has historically
been the second most important region for malaria
transmission after the Amazon rainforest.4 This region is
characterized by the presence of coastal valleys conducive to
propagation of anopheline vectors (primarily An. albimanus)
as well as by human migration for labor-intense agricultural
activities.53 The region typically has hot and humid summers
with moderate rains from December to March and winter
seasons from April to November with cool and dry days.
However, this seasonal climate pattern has changed in the
past by the ENSO phenomenon,39 with torrential rains and
strong winds causing flooding and landslides, events that
have been associated with malaria outbreaks (e.g., malaria
epidemic in 1997–1998).
Malaria has significantly declined in north-coastal Peru
over the past decade.3 Currently, reported malaria cases
are exclusively P. vivax and concentrated in a few localities
of Piura and Tumbes.3 Transmission occurs in epidemic and
sporadic patterns between February and June, the months
of highest density of the main vector, An. albimanus.53 The
most affected people live in peri-urban localities in close
proximity to irrigated fields and irrigation canals which
enable anopheline mosquitoes to propagate and survive.20,53
Although houses in those localities have complete walls
(closed houses), they are mainly made of poor materials that
do not exclude mosquitoes.
PLASMODIUM VIVAX MALARIA IN PERU
Recent years have seen the increased recognition that
P. vivax malaria may present as a severe disease according
to newly published World Health Organization (WHO)
criteria.54–57 Severe malaria has been observed in Peru,
both in the northern coast58 and related to travelers who
presented in Lima (a nonendemic area) with severe malaria
due to P. vivax (and P. falciparum) acquired in known
malarious regions.9 Severe vivax malaria in Peru appears to
have similar characteristics compared with elsewhere in
Amazonia,59 but appears to be less common in Amazonia
than in India.60 In the Loreto region, more than two-thirds
of cases of severe malaria arriving at referral hospitals
are due to P. vivax and belong to Group I of the new WHO
classification, meaning that, because of the possibility of
P. falciparum coinfection (without parasitological proof),61
such cases must be treated with intravenous AS in addition
to standard P. vivax therapy. Although such cases present
with profound prostration, the level of consciousness is
generally preserved. Consistent with this observation, recent
autopsy information from Brazil indicates that P. vivax is not
found in brain in patients with coma.62 Limitations of
studies of severe vivax malaria include lack of investigation
of comorbid illness or coinfection, and their retrospective
nature. Further, mechanisms of severe P. vivax malaria
pathogenesis, whether from the host response or parasite
virulence mechanisms, remain to be explored, particularly with
regard to pulmonary and multiorgan failure syndromes.
DETECTION OF P. VIVAX INFECTION
In Peru, governmental policy dictates that LM remains
the main approach to diagnose acute malaria and guide
appropriate treatment.63 The Peruvian Ministry of Health
has established a strong National Network of Public Health
laboratories classified into four levels: local, intermediate,
regional, and national level. This network is designated to
perform both diagnosis and quality control of thick blood
smears,64 according to international norms published by
Microscopy is routinely performed on samples from all
febrile cases who present to health facilities and/or hospitals,
local and intermediate laboratory levels, respectively, with
signs and symptoms suggestive of acute malaria. In 2015 in
Loreto, 321,723 thick blood smears were read at health
facilities as part of this PCD, and 48,987 thick blood smears
were read as part of reactive ACD, from collaterals,
individuals who live in close proximity to passively detected cases
(Directorate of Public Health Reference Laboratory, Iquitos,
Peru, personal communication). After PAMAFRO ended
in 2010, ACD activities carried out by the Regional Health
Office (DIRESA) substantially declined in the region.
Directly related to current resurgent P. vivax and
P. falciparum malaria in the Loreto Department of Peru,
access to timely and effective malaria diagnostics remains a
challenge in the region. During the PAMAFRO project,
the coverage of the National Network of Public Health
laboratories increased with purchase and deployment of new
microscopes combined with intensive training of health
personnel.46 There are still areas where, because of
logistical challenges, other diagnostic tools should be used, such
as point-of-care deployable rapid diagnostic tests (RDTs).
RDTs were introduced in Peru in 1998 as an alternative
to microscopy in remote areas of the Peruvian Amazon.
However, the clinical performance of RDTs has not been
consistently useful in Peru. RDTs were last reported to be
used in Loreto for confirmatory diagnosis in 2006, within
the PAMAFRO project, with an unacceptable performance
(sensitivity of 77% and 54% for non-falciparum and
P. falciparum, respectively); results were observed to be even
worse when parasitemia was less than 100/μL on LM65 In
addition, many false-negative RDTs related to
histidinerich protein (HRP)-2 have been observed. Published data
from Peru indicate that P. falciparum field isolates
commonly lack the pfhrp2 gene (the main target used by
previously used RDTs) limiting further the use of some RDTs.66
Another study with RDTs in this region, also showed
crossreactions of P. vivax samples with the Pf-pLDH or PfHRP2
tests; P. falciparum-positive samples with high parasitemias
cross-reacted with Pv-pLDH.23 Based on these observations,
the ideal RDT to be used in remote areas of the Peruvian
Amazon should target Pf-pLDH and Pv-pLDH separately in
the same device, with good heat stability and better
sensitivity. Currently, these RDTs are not able effectively to identify
very low parasitemia—either of P. vivax or P. falciparum—to
identify asymptomatic reservoirs of transmission.
Serological tests, unlike LM and RDTs, cannot be used for
the diagnosis of acute malaria because antibodies can persist
for months or years after infection has resolved.67
However, combining serological responses with geospatial
analysis enables the accurate identification of hotspots for
targeting with appropriate interventions.6,11 In two studies
with contrasting epidemiological settings of Peru, the use of
PCR to detect current malaria infections due to P. vivax and
P. falciparum, and serology to identify past malaria exposure
to both species, have repeatedly proved their added value in
accurately understanding the local epidemiology,
determining risk factors for malaria, and describing spatial
heterogeneity in transmission.6,11 In a third study conducted in the
northern coast, spatial analysis using specific IgG responses
to PvMSP-119 and PvAMA-1 of individuals enrolled in a
cross-sectional survey proved to be reliable for detecting
hotspots of clinical malaria cases detected by PCD in the
following year.42 A number of knowledge and technical gaps
need still to be addressed for developing and/or
optimizing serological tests for programmatic surveillance.68 These
research gaps are larger for P. vivax than for P. falciparum,
since less information is available about P. vivax antigens for
use in serosurveillance.68
TREATMENT EFFICACY FOR P. VIVAX DISEASE
AND FOR PREVENTING RECURRENT EPISODES
The widespread use of CQ plus PQ, in combination, may
be the reason for the few, isolated cases of CQ-resistant
P. vivax (CQRPV) reported in Peru. Indeed, Peru is one of
the few countries in the Americas with reports of CQ
resistance,69 but with no new data reported since 2003. A 28-day
drug efficacy trial in the Amazon region and the northern
Coast between 1998 and 2001, including 242 P. vivax-infected
individuals who received CQ alone (25 mg/kg, over a 3-day
period), found four individuals with recurrence of P. vivax
parasitemia on days 21 and 28 after treatment. All were from
the Amazon, and in two of them, CQRPV was confirmed by
measuring drug levels at the time of recurrence.38 More
recently, a drug effectiveness study also in the Amazon
found that four of 540 patients treated with the CQ–PQ
combination had a symptomatic recurrence of P. vivax
parasitemia within 35 days of treatment; however, in only one of
them, the recurrence occurred against normally therapeutic
blood levels of CQ, suggesting a probable case of CQRPV.70
Evaluating the contribution of relapses in the P. vivax
burden of endemic regions, as well as assessing the efficacy of
drugs to prevent P. vivax relapse, is challenging because a
recurrent P. vivax infection cannot easily be distinguished
between recrudescence (i.e., failure to treat the initial
infection), reinfection (i.e., new infection), or relapse (i.e.,
hypnozoite reactivation).10 Nevertheless, three studies conducted
in the Peruvian Amazon with strict follow-up of individuals
with confirmed P. vivax infections after receiving radical cure
treatment, showed that P. vivax recurrences are relatively
common in the Amazon region. The first study conducted
between 2006 and 2008 was a 6-month follow-up of patients
who were randomly assigned to treatment schemes
including CQ plus three different PQ regimens (5 days of PQ at
0.5 mg/kg/day, 7 days of PQ at 0.5 mg/kg/day, and 14 days
of PQ at 0.25 mg/kg/day).24 Schemes with 7 and 14 days of
PQ did not show significant differences in recurrence rates
(10% versus14%) during the follow-up period, but they
were more effective in preventing them than the scheme
with only 5 days (28.4% of recurrences). The second study
was a 2-year cohort of P. vivax-infected individuals from
29 Amazonian communities (2008–2011) aimed to evaluate
the efficacy of current national treatment of the prevention
of recurrent infections. Of 270 individuals who completed
the 2-year follow-up, 53% (144) had P. vivax recurrent
infections, most of them (70%) had several with a median of
three recurrences (range, 211 recurrences).71 The third study
was a phase 2b multicenter clinical trial (2011–2013) that
followed up 136 infected individuals in the Peruvian Amazon
for 6 months to assess dose response and safety of
singledose TQ plus CQ, PQ plus CQ, and CQ alone for P. vivax
radical cure.13 Findings showed high efficacy of regimens
including single-dose TQ of 300 mg and 600 mg with,
respectively, 81% and 84% recurrence-free efficacy at 6 months, in
comparison with 59% and 12% recurrence-free efficacy at
6 months for recommended WHO scheme based on 14-day
PQ plus CQ and CQ alone, respectively. Adverse events were
similar between treatments, and there was a dose-dependent
effect on symptomatic homologous strain relapse (but not
heterologous strain reinfection); 50% of recurrent infections
were homologous (i.e., same molecular genotype).14
GENETIC DIVERSITY AND POPULATION
STRUCTURE OF P. VIVAX
Population genetics studies in the Peruvian Amazon have
revealed high heterogeneity in P. vivax transmission, with
low or high population diversity at community level, and
high diversity among populations. Recent large-scale whole
genome surveys of P. vivax from all endemic regions
confirm these findings, and demonstrate that Peruvian isolates
have generally diverged from global populations except for
some imported cases from Brazil.72,73 Studies conducted in
communities along Iquitos–Nauta road, or with high
mobility of inhabitants, showed high diversity accompanied by a
considerable proportion of polyclonal infections and high
multiplicity of infection (MOI) (i.e., average number of
parasite lineages per infected individual). Study findings in 2006
in the riverine community Mazan (north of Iquitos), where
people have high mobility because of their economic
activities (farming, logging, and fishing), and in peri-Iquitos-urban
Moronococha, both showed high heterozygosity (He 0.66 and
0.69, respectively) and high proportion of polyclonal
infections (44–70%).74 Similar results were found in another
peri-Iquitos village, Zungarococha, between 2003 and2004,
within urban areas in Iquitos and in rural communities
along the Iquitos–Nauta road.30,75 A recent longitudinal
study conducted over 25 different villages (among five study
areas) confirmed these findings, showing that study areas
along the highway from Iquitos to Nauta (cluster A2 and
A3) had higher MOI (1.5–2) than isolated communities with
low mobility (cluster A1, A4, and A5). However, He was
high in all communities (He 0.66–0.76).76 On the other
hand, in San Carlos, a relatively small and isolated village, at
10 km from the Iquitos–Nauta road, genetic diversity and
MOI were constrained (He = 0.49, MOI = 1.1).71,74 Similar
patterns were reported in other peri-Iquitos villages: Fray
Martin, Santa Rita, San Jose de Lupuna, and San Pedro,
rural communities at about 3–7 km north of Iquitos, on the
other bank of Nanay river, only accessible from Iquitos by
boat.74 These observations collectively suggest that in this
setting of isolated settlements, where inhabitants carry out
most of their activities in and around their communities and
seldom visit or travel to other places, P. vivax diversity seems
to be restricted with few polyclonal infections and low MOI.
Although P. vivax populations in Iquitos and neighboring
areas are diverse, there is strong genetic differentiation among
geographically isolated sites, suggesting that transmission tends
to be local and clustered.
Genetic analysis of P. vivax infections from four of the
total 29 communities (2008–2011) included in the previously
mentioned 2-year cohort study was done to evaluate the
efficacy of current national treatment of the prevention of
recurrent infection,71 and showed that 82% of recurrent infections
carried at least one different allele from those at day 0 (day
of treatment). However, a different pattern was found in
other communities (e.g., San Carlos) where most recurrent
infections were caused by similar haplotypes, suggesting true
relapse rather than reinfection.17,29,75,77
The high genetic diversity of P. vivax in Peru might be
considered unusual given the relatively low transmission
intensity in the region, and probably relates to
accumulation of genetically heterogeneous hypnozoites over time, a
biological feature of P. vivax that makes it impossible to
definitively differentiate relapse from reinfection.78 The high
level of genetic diversity of P. vivax in Peru (and elsewhere),
as demonstrated in recent global genomic surveys72 as well
as by the use of other molecular markers,30 is a major
feature of P. vivax biology, and is key for understanding the
transmission epidemiology of this malaria parasite.79
Anopheles darlingi remains the most widespread,
aggressive, and intractable malaria vector in Amazonia, including
Peru. Invasion of both newly created (anthropic) and natural
breeding sites is often swift and likely linked to high
adaptability/plasticity.80,81 Where fishponds are the main breeding
site, An. darlingi abundance82 and fishpond presence near
human habitation are significantly associated with increased
numbers of malaria cases. The identification of such new
risk multipliers reveals lacunae in our understanding of
An. darlingi’s success, that is, patterns of dispersal, survival, and
genetic adaptability. Anopheline survival, one of the key
factors in determining vectorial capacity and
spatiotemporal transmission patterns, is understudied in An. darlingi in
To our knowledge, only one study in Peru (along the
Iquitos–Nauta Highway) has characterized and analyzed
breeding sites in relation to malaria risk.79 Heterogeneity
of An. darlingi feeding behavior is well documented and
includes endophagy, exophagy, anthropophily, and
opportunism, depending on habitat (landscape), host availability,
and quality of breeding sites. Behavioral variability has
been posited as an explanation for An. darlingi’s broad
ecological success, but we have found (J. Conn and others,
unpublished data) genetically differentiated exophagic and
endophagic An. darlingi populations. A critical knowledge
gap—currently being experimentally addressed in ongoing
studies—is whether these An. darlingi populations differ in
vector competence and can be specifically targeted for more
effective vector control and elimination.
EXPERIMENTAL STUDIES OF P. VIVAX
TRANSMISSION TO AN. DARLINGI IN PERU
It has long been established that human host immune
factors modulate P. vivax transmission from humans to
quitoes83; experimental studies in the Peruvian Amazon
using wild-caught, F1-generation An. darlingi mosquitoes
have confirmed and extended these observations.84,85
Understanding mechanisms by which the human host response may
impair parasite infectivity for mosquitoes has direct
implications for the development of transmission-blocking vaccines.
Experiments using standard membrane feeding assays found
that only about half (52/102) of P. vivax parasitemic subjects
successfully infected mosquitoes.78,79 Transmitters were more
likely to have microscopically visible gametocytes, higher
parasitemia, and, in terms of basic clinical parameters, a
slower pulse rate than nontransmitters. Quantitative
assessment of gametocytemia, using the first-reported real-time
reverse transcriptase Pvs25 PCR assay to quantify P. vivax
gametocytes, was significantly and positively correlated with
oocyst counts. These experiments were the first to establish
a system of determining transmission patterns in
experimental infection of outbred natural neotropical malaria vectors
in the Amazon region and concluded that P. vivax-infected
subjects inefficiently infected outbred F1 An. darlingi
mosquitoes. The results raised the possibility that some degree
of naturally occurring transmission-blocking immunity is
present on a population basis in the Peruvian Amazon, an area
of low intensity of malaria transmission.84,85
Toward the goal of determining mechanisms that modulate
the efficiency by which Plasmodium spp. infect An. darlingi
mosquitoes, recent reports describe the establishment of
laboratory colonies of An. darlingi in Peru.86 The availability of
these colonies sets the stage for further experimental study
of the transmission biology of malaria in Amazonia, both
with regard to P. vivax and P. falciparum, whether from
infected humans to mosquitoes, or conversely with regard to
obtaining P. vivax sporozoites for use in in vitro
experimental infections, and potentially in vivo in controlled human
challenge infections, as is being carried out in Australia and
Colombia.87 Peruvian Ministry of Health in Loreto approval
has been obtained to carry out direct feeds on
malariainfected individuals as well, the safety of which is enhanced
by using the colonized mosquitoes (currently stable and
propagating well at generation 40) which do not have known
transovarially transmitted pathogens. Therefore, in Peru, there is
the infrastructure and capacity to carry out future
experiments to more precisely determine the infectivity efficiency
of different P. vivax parasitemic individuals for An. darlingi,
for example, comparing symptomatic (who have higher and
patent parasitemia) and asymptomatic (who have lower
parasitemias, whether patent or subpatent) subjects.
CONCLUSIONS: KEY PUBLIC HEALTH
CHALLENGES FOR CONTROLLING AND
ELIMINATING P. VIVAX INFECTION
Strategies both to control and eliminate P. vivax in
Amazonia will have to take into account the complex
interactions of parasite biology (P. vivax liver-infecting dormant
hypnozoites), human behavior, and highly variable
environments that drive changes in mosquito vector behaviors.
These factors have led to continued low transmission and
residual malaria in Peru, and underlie challenges to the
sustainability of conventional malaria control strategies, as
illustrated by repeated malaria resurgences over decades
whenever control measures are not sustained. The
complexity of Amazonian malaria is augmented by intense human
movement related to work and social interactions, which
combined with asymptomatic infections lead to “silent”
reservoirs of malaria parasites moving across space and time
and maintain endemic malaria transmission.
To overcome these challenges in coming years, research
should focus on development, evaluation, and deployment
of new molecular and biomarker tools for the rapid and
efficient identification of individual with hypnozoites, and
asymptomatic, subpatent human reservoirs of continuing transmission.
Epidemiology studies need to focus on and target human
behaviors that serve to maintain and move malaria parasites
around endemic regions. Further, as all effective malaria
control measures in the past have intervened against the
vectors, understanding the plasticity, breeding, host tropisms,
and genetic factors that drive An. darlingi-transmitted malaria
Finally, it is important to emphasize that academic research,
such as described herein, has important potential to inform
public health officials at local, national, and international
levels. In Peru, such interactions have increasingly taken place
at these different levels, and academic researchers from Peru
have been participating in meetings with the Loreto Ministry
of Health, the NMCP, and the U.S. Agency for
International Development–organized Amazonian Malaria
Initiative in which the Pan American Health Organization is a
partner. In venues such as these, it is important for
datadriven research to inform policy-making toward the goal of
regional elimination, a major goal in Peru. Developing and
maintaining policies toward making long-term commitments
to malaria elimination means incorporating new data and
tools emerging from field- and laboratory-based studies
toward novel elimination strategies.
Published online October 31, 2016.
Financial support: This work was supported by the following: U.S.
Public Health Service grants U19AI089681, D43TW007120, and
K24AI068903 (Joseph M. Vinetz), and D43TW007393 (Andres G.
Lescano) from the U.S. National Institutes of Health; “Strengthening
the diagnosis, treatment and surveillance strategies for malaria
control and elimination in the Peruvian Amazon” Agency: L’Académie
Belge de Recherche et d’Enseignement supérieur- Commission de la
Coopération au Développement (ARES-CCD); “Círculo de
Investigación Nro. 4: Hacia la eliminación de la malaria en el Perú,”
(Convenio de Financiamiento N° 008-2014-FONDECYT); and
“Producción-Evaluación de antígenos candidatos a vacunas y
desarrollo de pruebas diagnósticas rápidas para Plasmodium
falciparum en la Amazonía peruana” (178-FINCyT-IB-2013).
This is an open-access article distributed under the terms of the
Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the
original author and source are credited.
The authors alone are responsible for the views expressed in this
article and they do not necessarily represent the views, decisions or
policies of the institutions with which they are affiliated.
Received April 6 , 2016 . Accepted for publication September 29 , 2016 .
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