Professional Documents
Culture Documents
Malaria Journal
DOI 10.1186/s12936-015-0615-5
RESEARCH
Open Access
Background
Malaria remains a serious public health problem in
Indonesia. Approximately 45% of the populations across
the archipelago are at risk for malaria infection [1], with
417,819 confirmed malaria cases in 2012. However, the
national annual parasite incidence (API) i.e., the number
of people per 1,000 populations that experienced at least
one case of malaria in a 12-month period, had decreased
from 4.68 in 1990 to 1.69 in 2012. This is an encouraging development towards an overall national target API of below 1 by year 2030 [2].
While several areas have witnessed significant reductions in malaria prevalence, other regions (e.g., eastern
Indonesia) have remained problematic for performing effective control strategies because of remoteness, lack of
adequate resources and sufficient budgets to combat
both vectors and parasites. Papua and West Papua provinces of Indonesia, located on the western half of the island of New Guinea, have the highest malaria burdens
in the country, with recent province-wide APIs of 60.6
and 52.3, respectively [3]. All four species of Plasmodium parasites are present in Papua, with Plasmodium
falciparum and Plasmodium vivax as the most common
infections, followed with far less frequency by Plasmodium ovale and Plasmodium malariae. In high transmission areas, mixed species infections are not uncommon.
Mimika Regency, covering a vast area of the southern
part of Papua Province, had an API of 531.3 in 2012
with an overall P. falciparum/P. vivax case infection ratio of 1.3:1 [4]. Other report had estimated the average
API closer to 876 in the immediate Timika area, the
capital of Mimika, and where the vast majority of the
population resides [5].
The World Health Organization (WHO) recommends
all clinically suspected malaria cases have parasitological
confirmed diagnosis, using either a malaria-specific rapid
diagnostic test (RDT) or direct visualization of parasites
using microscopy, before treatment [6]. For more than a
century, use of microscopy has been considered the gold
standard for malaria diagnosis, species identification,
and to quantify parasitaemia [7]. Various public and private health care facilities in the Timika area can perform
standard microscopic diagnosis of malaria, but this is
often compromised by the poor condition and maintenance of the microscope and the irregular availability of a
trained laboratory technician. In many of the remote
villages in the Mimika Regency (particularly those without electricity, skilled staff, or microscopist) and most
public-run clinics, only RDT is used for malaria diagnosis. INDEC Diagnostics (Jakarta, Indonesia) manufactures a multi-panel malaria RDT Plasmotec Malaria-3
(hereafter referred to using the product catalog number
XW-P07) that meets ISO 13485:2003 standards [8]. Including the companys internal assessment of the RDT,
Page 2 of 11
Methods
Page 3 of 11
The study sampling continued in a consecutive manner until the target sample size was obtained. The minimum sample size (n = 400) for accurately estimating
sensitivity and specificity [18] was based on the reported
lowest RDT sensitivity value (84.4% for P. vivax), the
Mimika 2012 estimated malaria incidence of 0.53 infections per person-year in the resident population, and an
absolute precision value of 0.05. Those cases with incomplete information regarding symptom presentations
or laboratory findings, and infections with only P. ovale
or P. malariae parasites based on qPCR were excluded
from the final analysis.
Microscopy and qPCR were used as the reference standards in this study. With each RDT, a matching thick
and thin blood film (one slide per sample) was prepared
and stained with Giemsa solution (1:10 dilution for approximately 20 min). Slides were examined using a compound light microscope under x1,000 oil-immersion
magnification by a qualified laboratory technician in the
clinic. All blood films were examined for a minimum of
100 high-magnification fields before being recorded as
either negative for malaria parasites and for the detection of low density mixed species infections. The parasite densities were estimated for each sample counted
separately by parasite species. Parasite numbers were reported per 200 white blood cells (WBC) to estimate
Page 4 of 11
Quantitative real-time PCR (qPCR) amplification was performed using a Rotor-Gene Q (Qiagen, Hilden, Germany).
Data was analyzed using Stata 12.0 software (Stata Corporation, College Station, Texas, USA, license no.
08762859510). RDT performance was calculated compared with matched microscopy and qPCR results with
95% confidence intervals (CI) for the following values:
sensitivity (Sn), specificity (Sp), positive predictive value
(PPV), negative predictive value (NPV), positive likelihood
ratio (PLR), negative likelihood ratio (NLR), and Kappa
score. RDT sensitivity was also calculated based on parasite density. Odds ratios and confidence intervals were calculated for correlation between low parasite counts
(100/l blood) and RDT false negative results based on
matching microscopy and qPCR results; as well as between low parasite counts and body temperature at the
time of blood sampling. For statistical inference, the
Fishers exact test was used for interpretation of false
negative RDT results between malaria infections with
densities above and below 100 parasites/l blood with significance set at p-value of <0.05.
Ethical review
Results
Between 10 April and 14 May 2014, 428 suspected malaria patients attending a local clinic in Kuala Kencana,
Mimika, voluntarily provided blood samples (Figure 2).
Of these initial samples, eight had incomplete data, one
Page 5 of 11
Suspected Malaria
Patients (n=428)
RDT
(n=428)
Microscopy
1st reading (n=428)
Microscopy
2nd reading (n=428)
Excluded no
sample for
PCR (n=4)
Microscopy
3rd reading (n=71)
PCR
(n=424)
Excluded Pm-only
qPCR result (n=1)
Excluded incomplete
result/data (n=8)
Data Analysis
(n=415)
Figure 2 Distribution of blood samples for determining XW-P07
performance.
Page 6 of 11
302/415 (72.8%)
197/398 (49.5%)
11,388 (4068,040)
1-100/l
5/88 (5.7%)
101-1,000/l
18/88 (20.5%)
1,001-10,000/l
34/88 (38.6%)
10,001-100,000/l
31/88 (35.2%)
3,143 (4027,000)
1-100/l
14/85 (16.5%)
101-1,000/l
25/85 (29.4%)
1,001-10,000/l
42/85 (49.4%)
10,001-100,000/l
4/85 (4.7%)
Positive samples were divided between P. falciparum and P. vivax infections, and
those with mixed species infections were counted separately as P. falciparum and
P. vivax.
repeated with a new cassette using the same blood sample. Laboratory test findings are shown in Table 3. RDT
results found 21.0% (87/415) of samples reactive for P.
falciparum, 14.5% (60/415) for P. vivax, and 1.2% (5/
415) with mixed P. falciparum/P. vivax infections. Microscopy produced a total SPR of 40.5% (168/415); with
P. falciparum slightly more common than P. vivax. To
determine RDT performance using matching microscopy, positive slides were divided between P. falciparum
and P. vivax; and those with mixed species infections
Table 2 Signs and symptoms of suspected malaria cases
with and without parasitaemia based on microscopic
diagnosis
Signs and symptoms
Parasitaemia
Parasitaemia
Present
(n = 168)
Absent
(n = 247)
Fever
154 (91.7%)
177 (71.7%)
Chills
68 (40.5%)
52 (21.0%)
Sweating
13 (7.7%)
12 (4.9%)
Nausea
71 (42.3%)
60 (24.3%)
Vomiting
35 (20.8%)
32 (13.0%)
Diarrhea
24 (14.3%)
41 (16.6%)
Headache
71 (42.3%)
57 (23.1%)
Myalgia
52 (31.0%)
55 (22.3%)
12 (7.1%)
5 (2.0%)
3 outcomes
97 (57.7%)
77 (31.2%)
4 outcomes
60 (35.7%)
26 (10.5%)
Temp 37.5C
112 (66.7%)
85 (34.4%)
Page 7 of 11
Table 3 Summary RDT results compared with matching microscopy and qPCR in 33 table
RDT
Microscopy Pf
Microscopy Pf, Pv
Microscopy Pf, Pm
Microscopy Pv
Result
PCR
PCR
PCR
PCR
PCR
PCR
PCR
PCR
PCR
Microscopy NP
PCR
PCR
Pf
Pf, Pv
NP
Pf, Pv
Pf, Pm
Pv
Pv, Pm
NP
Pf
Pv
NP
Pf
32
Pf, Pan
40
Pv
14
Pv, Pan
43
Pan
No parasites
18
227
observations
77
78
237
NP = No parasites. Pf: Plasmodium falciparum, Pv: Plasmodium vivax, Pm: Plasmodium malariae.
Discussion
Definitive diagnosis and confirmation of disease status is
the cornerstone of evidence-based medicine. Many malariaendemic areas of the world lack sufficient capacity and
resources to accurately diagnose the infection and where
reliance on the presentation of clinical signs and symptoms alone are inadequate and imprecise indicators of
specific disease. The WHO recommendations for procurement of malaria RDTs are currently based on the
Table 4 RDT performance compared with matching microscopy and qPCR (n = 415) for all infections regardless of
parasite density
RDT performance
Plasmodium falciparum
(n = 88 microscopy +)
Prevalence
Sn*
Plasmodium vivax
(n = 85 microscopy +)
Plasmodium falciparum
(n = 88 qPCR +)
Plasmodium vivax
(n = 94 qPCR +)
21.2%
20.5%
21.2%
22.6%
92% (84.3-96.7%)
72.9% (62.2-82%)
92% (84.3-96.7%)
66% (55.5-75.4%)
Sp*
96.6% (94.1-98.3%)
99.1% (97.4-99.8%)
96.6% (94.1-98.3%)
99.1% (97.3-99.8%)
PPV*
88% (79.6-93.9%)
95.4% (87.1-99%)
88% (79.6-93.9%)
95.4% (87.1-99.0%)
NPV*
97.8% (95.6-99.1%)
93.4% (90.3-95.8%)
97.8% (95.6-99.1%)
90.9% (87.3-93.7%)
PLR*
27.4 (15.3-49.1)
80.2 (25.8-249)
27.4 (15.3-49.1)
70.6 (22.7-220)
NLR*
0.08 (0.04-0.17)
0.27 (0.19-0.39)
0.08 (0.04-0.17)
0.34 (0.26-0.46)
Kappa
0.87
0.79
0.87
0.73
Sn = sensitivity; Sp = specificity; PPV = positive predictive value; NPV = negative predictive value; PLR = positive likelihood ratio; NLR = negative likelihood ratio.
*95% CI.
Page 8 of 11
Table 5 RDT percent test sensitivity by parasite density based on microscopy for Plasmodium falciparum and Plasmodium
vivax
Parasite density in blood
Plasmodium falciparum
Plasmodium vivax
n = RDT+/microscopy+
Sn (%)
n = RDT+/microscopy+
Sn (%)
1-100/l
3/5
60
3/14
21.4
101-1,000/l
14/18
77.8
13/25
52
1,001-10,000/l
33/34
97
42/42
100
10,001-100,000/l
31/31
100
4/4
100
4,800 parasites/l
640 parasites/l
Sn = sensitivity.
False
True
Negative
Positive
Pf 100/l
Pf >100/l
78
Pv 100/l
11
Pv >100/l
12
59
qPCR
False
True
Negative
Positive
Pf 100/l
Pf >100/l
78
Pv 100/l
10
Pv >100/l
12
59
p-value
10.4 (1.4-77.2)
0.05
18.0 (4.4-74.5)
<0.001
p-value
6.5 (0.5-77.3)
0.2
16.4 (3.9-68.6)
<0.001
Page 9 of 11
Conclusions
As a point-of-care device, the XW-P07 provided good test
sensitivity, specificity, positive and negative predictive
values and likelihood ratios, as well as inter-procedure
agreement for detecting P. falciparum infections. For P.
vivax infections, the test provided acceptable specificity
and positive likelihood ratio, but with lower sensitivity,
negative likelihood ratio (below test acceptance criteria),
and inter-procedure agreement comparison. Low parasite
blood densities (100 parasites/l), especially with P. vivax,
increased the probability of false negative test results.
Normal body temperature was strongly associated with
the incident of lower parasitaemia, further complicating
diagnosis. The RDT meets WHO minimum performance
criteria for test specificity (>90%) and invalid rate (<5%).
As vivax malaria is a very common and important parasitic
infection in the population studied, all primary health centers in the Mimika Regency should begin, or continue
using, expert-level and quality assured standard microscopy in a sustainable manner for greater accuracy in malaria detection.
Page 10 of 11
Competing interests
The authors received no product or financial support outside of normal
operating budgets. The authors declare that they have no competing
interests.
Authors contribution
The study was conceptualized and designed by LF and MJB. LF, BS, S, and
EA organized sample collection and performed data entry. LF performed
statistical analysis. LF, MJB, BS analyzed and interpreted the results and
drafted the manuscript. JHK, TBTS, and MJB provided major inputs for the
manuscript revision and finalization. All authors approved the final
manuscript.
Acknowledgements
We thank Haripurnomo Kushadiwijaya, Edi Harianto, Vonny Hartana, and
laboratory staff for their kind contributions to data collection and technical
support. We are also grateful to PT. Freeport Indonesia, an affiliate of
Freeport-McMoRan Inc., for the continued support towards improvement of
the health and welfare in their workforce and local communities.
Author details
1
Public Health & Malaria Control, International SOS, PT. Freeport Indonesia,
Kuala Kencana, Papua, Indonesia. 2Center for Tropical Medicine, Faculty of
Medicine, Gadjah Mada University, Yogyakarta, Indonesia. 3Public Health
Department, Faculty of Medicine, Gadjah Mada University, Yogyakarta,
Indonesia.
Received: 1 October 2014 Accepted: 20 February 2015
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