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Random Distribution of Mixed Species Malaria Infections in Papua New Guinea
Random Distribution of Mixed Species Malaria Infections in Papua New Guinea
Random Distribution of Mixed Species Malaria Infections in Papua New Guinea
225231
Copyright 2000 by The American Society of Tropical Medicine and Hygiene
AND
Division of Geographic Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio;
Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea
Abstract. Plasmodium falciparum (Pf), P. vivax (Pv), P. malariae (Pm), and P. ovale (Po) infections are endemic
in coastal areas of Papua New Guinea. Here 2,162 individuals living near Dreikikir, East Sepik Province, have been
analyzed for complexity of malaria infection by blood smear and polymerase chain reaction (PCR) diagnoses. According to blood smear, the overall prevalence of Plasmodium infection was 0.320. Most individuals (0.283) were
infected with a single species only. The prevalence of mixed species infections was low (0.037). Further analysis of
a 173-sample subset by nested PCR of small subunit ribosomal DNA resulted in an overall 3.0-fold increase in
prevalence of infection, with a 17.5-fold increase in the frequency of mixed species infections. Among mixed species
infections detected by PCR, the frequency of double species was 0.364, and that of triple species was 0.237. Nine
individuals (0.052) were infected with all 4 species. To determine if infection status (uninfected, single, and multiple
infections) deviates from an independent random distribution (null hypothesis), observed versus expected frequencies
of all combinations of Plasmodium species infections, or assemblages (Pf-, Pv-, Pm-, Po-, to Pf, Pv, Pm, Po),
were compared using a multiple-kind lottery model. All 4 species were randomly distributed whether diagnosed by
blood smear or PCR in the overall population and when divided into age group categories. These findings suggest
that mixed species malaria infections are common, and that Plasmodium species appear to establish infection independent of one another.
Four species of Plasmodium cause human malaria: P. falciparum (Pf), P. vivax (Pv), P. malariae (Pm), and P. ovale
(Po). They differ greatly with respect to their biology and
clinical manifestations.1 Sympatric combinations of these
species occur in human populations and within infected individuals. Knowledge of their prevalence and transmission
dynamics within a given geographic region is key to the
design of effective control measures. An understanding of
their interactions will be necessary to interpret thoroughly
the outcome of vaccine trials. Previous studies have observed complex relationships among Plasmodium species in
patients treated for syphilis,24 and in surveys of naturally
infected individuals living in malaria-endemic regions
around the world.510 Experimental infections involving nonendemic adults have shown that susceptibility to individual
Plasmodium species may be influenced by the species of
previous or current infections.24 Natural infection relationships among human malaria species appear to vary based
upon geographic differences, which are certain to involve
not only bio-climatic variation but also genetic differences
in human and vector populations.510
Microscopic examination of Giemsa-stained thick and thin
blood smears has been the diagnostic method of choice for
species identification in epidemiologic studies and medical
diagnosis.11 The major limitations of microscopy include
lack of trained personnel and the length of time required for
blood smear examination. Consequently, species identification becomes ambiguous when parasitemia is low and in
cases of mixed species infections, leading to incorrect or
failed species identification.7,9 Several efficient molecularlevel detection methods have been developed to overcome
these limitations, and subsequently validated using field
samples. These include antigen-12,13 or nucleic acid-based detection.1420 With the advent of these methods, significant increases in the reported prevalence of mixed species infections have been observed in various geographic regions.16,2028
225
226
Province, Papua New Guinea. Natural vegetation is rain forest. Residents are predominantly subsistence farmers and
have cleared away many areas to make gardens. Samples
were collected from July to September, 1996, which corresponds to the relatively dry season (July to November, rainfall 50150 mm/month; December to June, rainfall 80
290 mm/month).37 The highest human exposure rates to sporozoite-infected mosquitoes occur during this time period37
(Bockarie M, unpublished data). Blood samples from all villagers 5 years old were collected as part of a filariasis
control project.38 More than 85% of eligible subjects agreed
to participate. Ethical approval for this study and the procedure for oral informed consent were obtained from the
Medical Research and Advisory Council of Papua New
Guinea and the Case Western Reserve University/University
Hospitals of Cleveland Human Investigation Committee.
Blood smear examination. Blood films were prepared
and examined as described previously.33 Briefly, thick and
thin smears were stained with 4% Giemsa and examined
under oil-immersion (100) for 100 fields. Parasite species
were identified using both thick and thin film preparations.
Preparation of DNA template. The DNA was extracted
from whole blood (200 l) from study subjects or infected
chimpanzees using a QIAamp 96 or individual spin blood
kits (QIAGEN, Valencia, CA) according to the manufacturers protocol. Plasmodium falciparum (FCB) genomic DNA
was kindly provided by Dr. Xin-zhuan Su (Laboratory of
Malaria Genetics, National Institutes of Health, Bethesda,
MD). Blood samples from chimpanzees infected with P. vivax (type II), P. malariae, or P. ovale were provided by Dr.
W. E. Collins (Centers for Disease Control and Prevention,
Atlanta, GA).
Polymerase chain reaction-based diagnosis. Amplification of ssu rDNA was performed following nested PCR strategies, using genus-specific (nest 1) and species-specific (nest
2) primers (Research Genetics, Huntsville, AL) identical to
those described.16 All reactions (25 l) were performed in
buffer containing 3 pmoles of appropriate upstream and
downstream primers, 67 mM Tris-HCl, pH 8.8, 6.7 mM
MgSO4, 16.6 mM (NH4)2SO4, 10 mM 2-mercaptoethanol,
100 M dATP, dGTP, dCTP, and dTTP, and 2.5 units of
thermostable DNA polymerase. Nest 1 amplification conditions were 95C for 5 min (1); 95C for 1 min, 63C for 2
min, 72C for 2 min (5); 95C for 1 min, 58C for 2 min,
72C for 2 min (35); 95C for 1 min, 55C for 2 min, 72C
for 2 min (5); and 95C for 1 min, 58C for 2 min, 72C
for 2 min (1). A 3-l aliquot of the nest 1 reaction was
then used as a template in nest 2 species-specific reaction.
For control reactions, nest 1 products, amplified from cloned,
species-specific ssu rDNAs, were diluted 1:1,000 or 1:
10,000 in sterile distilled water prior to nest 2 amplification.
Nest 2 amplification conditions were 95C for 5 min (1);
95C for 1 min, 63C for 2 min, 72C for 2 min (5); 95C
for 1 min, 58C for 2 min, 72C for 2 min (30); 95C for
1 min, 55C for 2 min, 72C for 2 min (5); and 72C for
5 min (1). The PCR assays were performed using a Peltier
Thermal Cycler, PTC-225 (MJ Research, Watertown, MA).
The PCR products from nest 1 and nest 2 amplifications
were loaded on 2% Agarose I (Amresco, Solon, OH) gels,
and electrophoresis was performed in 1 TBE buffer (8.9
mM Tris, 8.9 mM boric acid, 2.0 mM EDTA). The gels were
stained for 30 min with SYBR Gold (Molecular Probes, Eugene, OR), diluted 1:10,000 in 1 TBE buffer, and DNA
products were visualized on a Storm 860 using ImageQuant
software (instrumentation and software developed by Molecular Dynamics, Sunnyvale, CA).
To assess the possibility of cross-contamination, a series
of study samples representing the full spectrum of infection
(no infection to all 4 species infection) were subjected to
PCR with negative controls spaced between each sample. No
evidence of PCR amplification of species-specific amplicons
was observed in any of the negative controls.
Polymerase chain reaction-based cloning and DNA sequence analysis of ssu rDNA. Nest 1 amplicons (approximately 1,200 basepairs) from each of the parasites described
above were cloned into the pCR2.1-TOPO plasmid vector
using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA)
according to the manufacturers protocol. The clones were
amplified using extended M13 forward (5-GTT TTC CCA
GTC ACG ACG TTG TAA AAC GAC GGC CAG-3) and
M13 reverse (5-TGA GCG GAT AAC AAT TTC ACA
CAG GAA ACA GCT ATG AC-3) primers. Temperature
cycling conditions were 95C for 30 sec, 45C for 30 sec,
72C for 2 min (5); and 95C for 30 sec, 65C for 30 sec,
72C for 2 min (30). Genus-specific nest 1 amplicons were
purified using a QIAquick PCR purification kit and recommended protocol (QIAGEN). Sequencing of DNA was performed using fluorescence-based sequencing protocols on an
ABI377 automated sequencer (Applied Biosystems, Foster
City, CA). Sequences were analyzed using Sequencher software (Version 3.0.1 Demo; Gene Codes Corporation, Ann
Arbor, MI). Plasmodium falciparum, P. vivax, P. malariae,
and P. ovale sequences were assigned GenBank accession
numbers AF145334, AF145335, AF145336, and AF145337,
respectively. Bacterial clones for plasmids containing each
species-specific ssu rRNA nest 1 amplicon are available
through the Malaria Research and Reference Reagent Resource Center (MR4) at: http://www.malaria.mr4.org.
Statistical analysis. Plasmodium species prevalence was
determined by enumerating infected individuals and dividing
by the total population (2,162) or population subset (173)
size. The multiple-kind lottery model34 was used to calculate
the expected distribution of parasite species assemblages in
the population. Chi-square values were calculated using heterogeneity tests (number of rows number of columns) to
compare observed versus expected values.
RESULTS
227
TABLE 1
Summary of not infected and all single, double, triple, and quadruple
infections detected by blood smear diagnosis*
Parasite assemblage
Pf
Pv
Pm
Po
Pf Pv
Pf Pm
Pf Po
Pv Pm
Pv Po
Pm Po
Pf Pv Pm
Pf Pv Po
Pf Pm Po
Pv Pm Po
Pf Pv Pm Po
Not infected
Observed
361
176
75
0
55
19
0
5
0
0
1
0
0
0
0
1,470
Expected
Chi-square
370.25
180.45
71.08
0.00
45.58
17.96
0.00
8.75
0.00
0.00
2.21
0.00
0.00
0.00
0.00
1,465.71
Chi-square (df 15)
0.23
0.11
0.22
1.95
0.06
1.61
0.66
0.01
4.85
228
TABLE 2
Summary of not infected and all single, double, triple, and quadruple
infections detected by polymerase chain reaction diagnosis*
Parasite assemblage
Pf
Pv
Pm
Po
Pf Pv
Pf Pm
Pf Po
Pv Pm
Pv Po
Pm Po
Pf Pv Pm
Pf Pv Po
Pf Pm Po
Pv Pm Po
Pf Pv Pm Po
Not infected
Observed
Expected
32
30.34
15
10.13
3
4.04
0
1.27
40
44.64
16
17.81
4
5.61
3
5.95
0
1.87
0
0.75
27
26.21
8
8.25
5
3.29
1
1.10
9
4.85
10
6.88
Chi-square (df 15)
Chi-square
0.09
2.34
0.27
1.27
0.48
0.18
0.46
1.46
1.87
0.75
0.02
0.01
0.88
0.01
3.56
1.41
15.08
FIGURE 2. Comparison of Giemsa-stained blood smear- and polymerase chain reactionbased diagnosis of mixed Plasmodium species
infections. Each panel (top to bottom) contains nest 2 products amplified using species-specific primers indicated on the right (Pf Plasmodium
falciparum; Pv P. vivax; Pm P. malariae; Po P. ovale). Organization of samples within each panel (left to right) includes DNA size
marker (100-basepair [bp]) bp ladder), a negative control to which no DNA was added, nest 2 products from all 4 species-specific plasmidbased positive controls (Pf, Pv, Pm, and Po), and 17 individual study subjects (lanes 117). Below each panel the Giemsa-stained blood smear
status is indicated for each species infection: infected; not infected.
229
TABLE 3
Comparison of Plasmodium species infection prevalence diagnosed
by blood smear and small subunit ribosomal RNA DNA-based
polymerase chain reaction (PCR) methods*
TABLE 4
Comparison of observed and expected frequencies of parasite assemblages by age group-blood smear analysis*
510 years (n 37)
510 years
11 years
Parasite
infection
Blood smear
PCR
Pf
Pv
Pm
Po
Not infected
Pf
Pv
Pm
Po
Not infected
0.432
0.270
0.216
0.000
0.297
0.228
0.162
0.125
0.000
0.551
0.892
0.838
0.297
0.162
0.054
0.794
0.529
0.390
0.154
0.059
Parasite assemblage
Observed Expected
Chisquare
11 years (n 136)
Observed Expected
Chisquare
Pf
9
9.15 0.00
22
22.74 0.02
Pv
6
4.45 0.54
18
14.86 0.66
Pm
4
3.31 0.14
12
11.00 0.09
Po
0
0.00
0
0.00
Pf Pv
3
3.39 0.04
4
4.39 0.03
Pf Pm
3
2.52 0.09
5
3.25 0.94
Pf Po
0
0.00
0
0.00
Pv Pm
0
1.23 1.23
0
2.12 2.12
Pv Po
0
0.00
0
0.00
Pm Po
0
0.00
0
0.00
Pf Pv Pm
1
0.93 0.00
0
0.63 0.63
Pf Pv Po
0
0.00
0
0.00
Pf Pm Po
0
0.00
0
0.00
Pv Pm Po
0
0.00
0
0.00
Pf Pv Pm Po
0
0.00
0
0.00
Not infected
11
12.01 0.09
75
77.01 0.05
Chi-square (df 15) Chi-square (df 15)
2.14
4.56
* For definitions of abbreviations, see Table 1.
TABLE 5
Comparison of observed and expected frequencies of parasite assemblages by age grouppolymerase chain reaction analysis*
510 years (n 37)
Parasite assemblage
Observed Expected
Chisquare
11 years (n 136)
Observed Expected
Chisquare
Pf
4
3.15 0.23
28
26.23 0.12
Pv
2
1.97 0.00
13
7.65 3.74
Pm
0
0.16 0.16
3
4.34 0.41
Po
0
0.07 0.07
0
1.24 1.24
Pf Pv
16
16.28 0.00
24
29.51 1.03
Pf Pm
0
1.33 1.33
16
16.75 0.03
Pf Po
0
0.61 0.61
4
4.79 0.13
Pv Pm
0
0.83 0.83
3
4.88 0.73
Pv Po
0
0.38 0.38
0
1.40 1.40
Pm Po
0
0.03 0.03
0
0.79 0.79
Pf Pv Pm
7
6.89 0.00
20
18.84 0.07
Pf Pv Po
2
3.15 0.42
6
5.39 0.07
Pf Pm Po
0
0.26 0.26
5
3.06 1.23
Pv Pm Po
0
0.16 0.16
1
0.89 0.01
Pf Pv Pm Po
4
1.33 5.34
5
3.44 0.71
Not infected
2
0.38 6.86
8
6.80 0.21
Chi-square (df 15)
Chi-square (df 15)
16.69
11.93
* For definitions of abbreviations, see Table 1.
230
human pathogen, it is mandatory that the efforts to characterize the magnitude and complexity of Plasmodium species
infections be improved. The importance of characterizing
overall malaria infection accurately has been the focus of a
number of thorough reviews.710
Overall, this study has found that infections in the Dreikikir study population by Pf, Pv, and Pm are approximately
3-fold higher, and that Po is frequently observed when PCRbased diagnosis is compared with conventional blood smear
methods. Furthermore, as a result of the increased sensitivity
of PCR, mixed infections, including those caused by all four
human malaria parasite species, are more common than reported by blood smear diagnosis. Since the observations
from this study are consistent with others based upon molecular diagnostic methodologies, it is important to re-assess
how the sensitivity and specificity of these assays are determined. Recent discussion has suggested that numerous factors related to blood smear diagnosis raise the question as to
whether this methodology should be considered as the true
gold standard.4042 If, in this study, the PCR-based assay is
considered as the gold standard, sensitivity and specificity
of the blood smear-based assay are as follows: 0.31 (sensitivity) and 0.91 (specificity) for Pf, 0.22 and 0.87 for Pv,
and 0.30 and 0.94 for Pm. This evaluation of the two diagnostic assays reflects widely recognized methodologic differences leading to more sensitive detection of malaria parasites by PCR.
Results from this study provide insight beyond the technical advances identified above. Increased sensitivity for detecting infection improves the estimation of the parasite reservoir size and the characteristics of species-to-species interactions within endemic populations. With the PCR-based
prevalence of infection at 0.940 and of mixed infection at
0.653 in this study population, it is likely that all individuals
may be infected by more than one Plasmodium species at
any given time. Furthermore, the prevalence of infection detected by PCR was not observed to decrease with age as was
diagnosed by blood smear. In fact, the PCR prevalence of
P. malariae is observed to increase in the 11-year-old age
group compared with the 510-year-old age group, which is
consistent with a previous report.32 These observations
should be considered in attempts to understand age-acquired
immunity to Plasmodium species parasites more completely.
Since heterologous or cross-species factors have been considered to influence the acquisition of immunity to malaria
parasites,4,8 it is important to monitor species-to-species interactions. In this study, comparisons between observed and
expected prevalence of mixed infection, detected by both
blood smear and PCR, suggest that individual species establish infection independently of the others. Since this finding
was obtained in both age group categories considered, results
suggest that acquisition of immunity does little to influence
the independent distribution of parasites in infected individuals. When these findings were compared with PCR-based
point-prevalence studies specifying the parasites involved in
single and each mixed malaria infection, mixed species infections were randomly distributed in one African-based (Pf-,
Pm-, Po-endemic23) population, two Thai-based (Pf-, Pv-endemic17 and Pf-, Pv-, Pm-, Po-endemic22) populations, and
one South American-based (Pf-, Pv-endemic24) study population. Therefore, findings of an independent distribution of
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