DOI: 10.2478/s11686-014-0238-y © W. Stefański Institute of Parasitology, PAS Acta Parasitologica, 2014, 59(2), 267–271; ISSN 1230-2821
Evaluation of Wondfo Rapid Diagnostic Kit (Pf-HRP2/PAN-pLDH) for Diagnosis of Malaria by Using Nano-gold Immunochromatographic Assay Junlin Wu1, Yunping Peng2,3*, Xiaoyun Liu3, Wenmei Li2,3 and Shixing Tang3 1
Guangdong Institute of Microbiology; State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangzhou 510070, China; 2School of Food and Bioengineering, South China University of Technology, Guangzhou, Guangdong, 510640, China; 3Guangzhou Wondfo Biotech. Co., Ltd.; Guangzhou Accurate and Correct Test Co., Ltd. Guangzhou, Guangdong, 510643, China
Abstract Prompt and accurate diagnosis is necessary to start adequate treatment for different affecting species including P. falciparum and P. vivax. Here we described the Wondfo Rapid diagnostic Kit (Pf-HRP2/PAN-pLDH) for the detection of P. falciparum and pan-plasmodium in patient specimen by using a nano-gold immunochromatographic assay. Our rapid assay adapted nano-gold labeling techniques and the monoclonal antibodies (mAbs) against both histidine rich protein-2 (Pf HRP-2) of P. falciparum and pan plasmodium-specific pLDH (pan pLDH). The established two-antibody sandwich immunochromatographic assay could detect P. falciparum and pan-plasmodium. The sensitivity and specificity of Wondfo rapid diagnostic kit were determined by comparing with the “gold standard” of microscopic examination of blood smears. In this study1023 blood samples were collected from outpatient clinics in China and Burma, and detected by both Wondfo kit and microscopic examination. The detection sensitivity and specificity of Wondfo rapid diagnostic kit were 96.46% and 99.67% for P. falciparum (HRP2), 95.03% and 99.24% for pLDH, 96.83% and 99.74% for non-falciparum species, 96.70% and 99.74% for P. vivax, respectively. These results indicate that Wondfo rapid diagnostic assay may be useful for detecting P. falciparum and non-P. falciparum (especially P.v.) in patient specimen.
Keywords Malaria, Plasmodium falciparum, rapid diagnostic Kit, immunochromatographic assay
Introduction Malaria is one of the most serious infectious diseases in the world. It threatens the health of 3.3 billion people (half the world’s population) who are living in tropical and subtropical regions (Noppadon et al. 2009). It is estimated 3.4 billion people are at risk on malaria, of which 1.2 billion are at high risk. In high-risk areas, more than one malaria case occurs per every 1000 population. There were an estimated 207 million cases of malaria in 2012 (uncertainty range: 135–287 million) and an estimated 627 000 deaths (uncertainty range: 473 000–789 000) (WHO 2013). Although increasing efforts have been taken to control malaria, it is still the leading cause of diseases and death in the world. The WHO has recommended artemisinin combination therapies (ACT) be the first-line therapy for P. falciparum malaria worldwide (WHO 2006). However, the diagnosis of
malaria is requested for the successful treatment. Furthermore, effective diagnosis can reduce both complications and mortality from malaria. Thus, the need for effective and rapid diagnostics for global malaria control is urgent (WHO 2006). In general, screening of blood slides by microscopy is still considered to be the “gold standard”. This method is cheap and simple but labor-intensive, time consuming and requires considerable expertise to differentiate different Plasmodium species (Reyburn et al. 2004). Therefore, confirmatory diagnostic assays are highly helpful and urgently needed. The malaria rapid diagnostic tests (RDTs) are quick and easy to perform, and do not require electricity or other specific equipments. The RDTs are recommended by the WHO when reliable microscopic examination is not available (WHO 2010). There are 68 manufacturers of malaria RDT products including Guangzhou Wondfo Biotech. Co., Ltd. (WHO 2009a).
*Corresponding author: [email protected]
268
These tests are fast, easy to perform, but may be limited in sensitivity and stability. They can only detect parasitaemia levels above 200 parasites/μl blood (Ashley et al. 2009). Originally, two-band tests were used to consist of a control line and a P. falciparum specific test line by using monoclonal antibodies against either histidine – rich protein-2(HRP-2) or P. falciparum specific lactate dehydrogenase (Pf-pLDH). Most RDTs detect P. falciparum target HRP-2, only few of them are designed to detect Pf-pLDH. We have previously evaluated the two-band tests for diagnosis of Plasmodium falciparum (Peng et al. 2012). Here, we reported the evaluation results of the three-band RDTs for detecting HRP-2 and pan-pLDH in an area of intense and perennial malaria transmission in the South part of China and Burma. The aim of this study was to estimate the sensitivity, specificity, positive predictive value (PPV) and negative predictive values (NPV) of a Pf-HRP2 and PAN-pLDH based assay, using microscopic examination of blood smears as the “gold standard”.
Materials and Methods Patients and materials
A total of 1023 patients with fever were included in Hainan, Henan and Yunnan Province of China and China-Burum boarder between April 2008 and April 2010. Five milliliters of venous blood were drawn into an EDTA-coated syringe. Aliquots of these samples were frozen at –80°C until use. A thick blood film was prepared for each patient during the blood collection process and stained with Giemsa. The patients leukocytes were counted simultaneously. The species and the density of the Plasmodium parasites were determined by counting the number of parasites per 200 white blood cells. The sample was determined to be negative when no parasites were found after examination of 200 microscopic fields at × 1 000 magnification. The negative control sample was taken from an individual who was among the 1023 patients and had never been exposed to malaria. Patients were recruited to the study were axillary temperature ≥37.5°C. Pregnancy or lactating mothers were excluded from the study. Sample size is calculated according to the anticipated proportion table (Lwanga and Lemeshow 1991), assuming that anticipated population proportion of clinical failures to artemether-lumefantrine 5%, confidence level of 95%, and precision of 10%. Ethical clearance The study was approved by the ethical committee from Hainan Province’s Center for Disease Control and Prevention, Henan Province’s Center for Disease Control and Prevention and Yunnan Province’s Center for Disease Control and Prevention. All patients provided informed consent before admission into the study.
Junlin Wu et al.
Standard microscopy evaluation The microscopic blood film examinations were done according to WHO recommendations described in “Methods for surveillance of anti-malarial drug efficacy” (WHO 2009b). The malaria microscopic examination was performed by two independent experienced microscopists, for identification of the malaria parasite species if a result showed more than 20% discrepancy, an expert microscopist was requested to check the slide and the average count of the two similar results was recorded. Preparation of gold nanoparticles The 20-nm gold nanoparticles were prepared by controlled reduction of a boiling solution of 0.01% HAuCl4 with 1% sodium citrate according to a modified citrate reduction method (Huang 2006). Briefly, 200 ml of 0.01% HAuCl4 solution was brought to a rolling boil with vigorous stirring. Rapidly adding 2.6 ml of 1% sodium citrate solution to the vortex of the solution changed its color from pale yellow to burgundy. Boiling was continued for 10 min, the heating mantle was then removed, and stirring was continued for a further 15 min. After the solution reached room temperature, it was filtered through a 0.8 μm Gelman membrane filter. Conjugation of antibody and gold nanoparticles The gold nanoparticles were coated with anti-HRP-2 and antipLDH IgG respectively according to the method modified from that developed by Huang (Huang 2006). Briefly, 10 ml of gold nanoparticles was quickly added to 1 ml of anti-HRP2 or anti-pLDH IgG solution with vigorous stirring and the mixture was incubated at room temperature for 5 min. The concentration of protein in the anti-HRP-2 or anti-pLDH IgG used was increased from 15 to 125 mg/ml. After incubation the coated gold nanoparticles were stabilized by adding 10% (113 μl) of bovine serum albumin (BSA) to a concentration of 0.1% (w/v). The conjugate was centrifuged at 8000 rpm for 20 min and washed five times in 1 ml of buffer solution which contained 0.1% bovine serum albumin. The final volume of the conjugate was 1 ml. The labeled conjugate was stored at 4°C. Preparation of membrane with immobilized antibody The labeled antibody-analyte complex for signal generation was captured after the antibody had been immobilized in a particular area of the nitrocellulose membrane. The surface of the nitrocellulose was modified to improve the rinse time of the capillary. Purified antibody was diluted with 0.2% BSA in PBS to a concentration of 2.5 mg/ml. 1 μl of this solution was applied in the form of a dot to the center of strip. The strips were dried at room temperature.
269
Evaluation of Wondfo Rapid Diagnostic Kit (Pf-HRP2/PAN-pLDH)
Configuration of immunochromatographic system
then calculate the detection sensitivity in different parasite density.
The whole system includes sample pad, absorption pad, reagents, test line 1, test line 2 and control line. The sample pad, which controls the migration of the gold nanoparticles–antibody conjugate, was treated with 50 mmol/l borate buffer, pH 7.4, containing 1% BSA, 0.5% Tween-20, and 0.05% sodium azide. Anti-HRP-2, anti-pLDH IgG and goat anti-rabbit antibody were applied to the test line 1, test line 2 and the control line, respectively, on the nitrocellulose membrane which was dried at 35°C. An untreated absorption pad was located at the end of the strip. The nitrocellulose membrane, the absorption pad, and the pretreated sample pad were attached to the strip (Fig. 1). A sample was added into a reaction holder to start testing.
Results
Detection sensitivity and specificity with blood samples
Sample collection
A total of 1023 samples were tested by Wondfo malaria rapid diagnostic kit and microscopic examination. Sensitivity was defined as the percentage of positive results for Plasmodium falciparum or non-falciparum species (especially P.vivax) observed by Wondfo RDT divided by the total positive samples obtained by the microscopic examination. Specificity was defined as the percentage of negative test results observed by Wondfo RDT divided by the total negative samples confirmed by microscopic examination.
The study included 1023 samples obtained from 1023 patients. The interpretation of test results for P. falciparum and the nonfalciparum (including P. vivax) species is shown in Table I. Their median age was 27.22 years (range 0.8–87 years). Eighty eight patients (8.6%) were children with the age of less than five years. The male-to-female ratio was 1.58 : 1. There are 362 samples contained all four Plasmodium species at different parasite densities (Table I). For P. falciparum samples, the median parasite density was 12894 p/μl (range 120– 441600 p/μl). For P. vivax, P. malaria and P. ovale, the parasite density was 7776 p/μl (range 78–83400 p/μl), 1666.34 p/μl (300–3890 p/μl) and 480 p/μl (range 120–780 p/μl), respectively. Three out of 11 microscope-confirmed P. malariae samples had originally been diagnosed by microscopy as P. vivax, whereas 3/121 P. vivax samples as P. falciparum, 1/121 P. vivax samples as P.o. Six hundred sixty-one cases with fever (including HIV infection, tuberculosis, hepatitis, syphilis and other 29 cases of infectious diseases) were included as negative control.
Detection sensitivity with different parasite density There were 28 samples consisted of 8 P. falciparum, 10 P. vivax from Yunnan Institute of Parasitic Disease and 10 reference material from the WHO (Plasmodium falciparum in whole blood). The parasite density range of 8 P. falciparum and 15 P. vivax samples was 240–186720 p/μl and 120–30180 p/μl., respectively. The 10 reference material from the WHO was Plasmodium falciparum positive whole blood, 5 of them with parasite density of 200 p/μl and the other parasite density was 2,000 p/μl (Lwanga and Lemeshow 1991). An aliquot of each sample was thawed and made a series of 10-fold dilution or 2-fold dilution. Furthermore, a series of 2-fold dilutions were done in duplicate from the end point obtained above,
Statistical analysis The gold standard of microscopic examination, sensitivity and specificity were calculated with 95% confidence intervals (C.I.). Proportions were assessed for statistical significance using the Pearson Chi-square test, or in case of small sample size, the two-tailed Fisher’s exact test. A p-value < 0.05 was considered significant.
Detection sensitivity and specificity with blood samples Table II shows the results of 1023 specimens detected by Wondfo malaria three-bands diagnostic kit and the gold stan-
Fig. 1. The schematic description of the immunochromatographic test device
270
Junlin Wu et al.
Table I. General clinical data of samples Number of cases Samples
Age
The parasite density (parasites/µl)
Male
Female
Total
Malaria
281
81
362
27.18 ± 13.86 (0.8 ~ 87)
15313.74 ± 18069.16 (78 ~ 441600)
P. falciparum
90
23
113
25.33 ± 11.58 (1 ~ 75)
12894.37 ± 1524.68 (120 ~ 441600)
P. vivax
186
57
243
34.57 ± 15.66 (1 ~ 87)
7776.47 ± 8501.64 (78 ~ 83400)
P. malariae
9
2
11
33.73 ± 11.02 (6 ~ 62)
1666.34 ± 1032.40 (300 ~ 3890)
P. ovale
2
0
2
25.5 ± 3.0 (21 ~ 30)
480 ± 280 (180 ~ 780)
Mixed infection
6
1
7
39.25 ± 9.25 (30 ~ 53)
≈1650 ± 960 (600 ~ 2520)
Negative control
345
316
661
25.48 ± 14.22 (1 ~ 73)
–
Total
626
397
1023
26.94 ± 13.85 (0.8–87)
100% at parasite densities above 200p/µ l and 500p/µ l respectively.
dard method. The 5 positive cases initially misdiagnosed by Wondfo Rapid Diagnostic Kit (Pf-HRP2/PAN-pLDH) were identified by microscopy. The sensitivity and specificity of Wondfo malaria rapid diagnostic were 96.46% and 99.67% for P. falciparum (HRP2), 95.03% and 99.24% for pLDH, 96.83% and 99.74% for non-falciparum species, 96.70% and 99.74% for P. vivax. RDT results showed very good concordance with microscopy examination when detecting P. falciparum, non-falciparum species and P. vivax.
Discussion The simple, quick, accurate, and cost-effective diagnostic tests for determining the presence of malaria parasites have been recognized as an urgent need by the World Health Organization to overcome the deficiencies of light microscopy. Numerous new malaria-diagnostic techniques have been developed (WHO 1996). This, in turn, has led to an increase in the use of RDTs for malaria, which are fast and easy to perform, and do not require electricity or specific equipment (Bell et al. 2006). Currently, 68 malaria RDTs are commercially available from 28 different manufacturers including Guangzhou Wondfo Biotech. Co., Ltd. (WHO 2008; Lwanga and Lemeshow 1991). In this study, the performance of Wondfo Rapid diagnostic Kit(Pf-HRP2/PAN-pLDH) was evaluated on a panel of
Detection sensitivity with different parasite density The detection sensitivity of Wondfo Rapid diagnostic Kit (Pf-HRP2/PAN-pLDH) increased gradually with increasing parasite density (Table III). For detection of P. falciparum, the overall sensitivity was 96.46% ranging from 10.3% at parasite densities500 p/μl
12 11 15 23
1 8 13 23
11 3 2 0
8.3 72.7 86.7 100
whole blood samples (n = 1023), obtained in Hainan, Henan and Yunnan Province of China and China-Burum border. Although the detection sensitivity was not very good for P. falciparum and P. vivax samples at low parasite densities (
Evaluation of Wondfo Rapid Diagnostic Kit (Pf-HRP2/PAN-pLDH) for Diagnosis of Malaria by Using Nano-gold Immunochromatographic Assay Junlin Wu1, Yunping Peng2,3*, Xiaoyun Liu3, Wenmei Li2,3 and Shixing Tang3 1
Guangdong Institute of Microbiology; State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangzhou 510070, China; 2School of Food and Bioengineering, South China University of Technology, Guangzhou, Guangdong, 510640, China; 3Guangzhou Wondfo Biotech. Co., Ltd.; Guangzhou Accurate and Correct Test Co., Ltd. Guangzhou, Guangdong, 510643, China
Abstract Prompt and accurate diagnosis is necessary to start adequate treatment for different affecting species including P. falciparum and P. vivax. Here we described the Wondfo Rapid diagnostic Kit (Pf-HRP2/PAN-pLDH) for the detection of P. falciparum and pan-plasmodium in patient specimen by using a nano-gold immunochromatographic assay. Our rapid assay adapted nano-gold labeling techniques and the monoclonal antibodies (mAbs) against both histidine rich protein-2 (Pf HRP-2) of P. falciparum and pan plasmodium-specific pLDH (pan pLDH). The established two-antibody sandwich immunochromatographic assay could detect P. falciparum and pan-plasmodium. The sensitivity and specificity of Wondfo rapid diagnostic kit were determined by comparing with the “gold standard” of microscopic examination of blood smears. In this study1023 blood samples were collected from outpatient clinics in China and Burma, and detected by both Wondfo kit and microscopic examination. The detection sensitivity and specificity of Wondfo rapid diagnostic kit were 96.46% and 99.67% for P. falciparum (HRP2), 95.03% and 99.24% for pLDH, 96.83% and 99.74% for non-falciparum species, 96.70% and 99.74% for P. vivax, respectively. These results indicate that Wondfo rapid diagnostic assay may be useful for detecting P. falciparum and non-P. falciparum (especially P.v.) in patient specimen.
Keywords Malaria, Plasmodium falciparum, rapid diagnostic Kit, immunochromatographic assay
Introduction Malaria is one of the most serious infectious diseases in the world. It threatens the health of 3.3 billion people (half the world’s population) who are living in tropical and subtropical regions (Noppadon et al. 2009). It is estimated 3.4 billion people are at risk on malaria, of which 1.2 billion are at high risk. In high-risk areas, more than one malaria case occurs per every 1000 population. There were an estimated 207 million cases of malaria in 2012 (uncertainty range: 135–287 million) and an estimated 627 000 deaths (uncertainty range: 473 000–789 000) (WHO 2013). Although increasing efforts have been taken to control malaria, it is still the leading cause of diseases and death in the world. The WHO has recommended artemisinin combination therapies (ACT) be the first-line therapy for P. falciparum malaria worldwide (WHO 2006). However, the diagnosis of
malaria is requested for the successful treatment. Furthermore, effective diagnosis can reduce both complications and mortality from malaria. Thus, the need for effective and rapid diagnostics for global malaria control is urgent (WHO 2006). In general, screening of blood slides by microscopy is still considered to be the “gold standard”. This method is cheap and simple but labor-intensive, time consuming and requires considerable expertise to differentiate different Plasmodium species (Reyburn et al. 2004). Therefore, confirmatory diagnostic assays are highly helpful and urgently needed. The malaria rapid diagnostic tests (RDTs) are quick and easy to perform, and do not require electricity or other specific equipments. The RDTs are recommended by the WHO when reliable microscopic examination is not available (WHO 2010). There are 68 manufacturers of malaria RDT products including Guangzhou Wondfo Biotech. Co., Ltd. (WHO 2009a).
*Corresponding author: [email protected]
268
These tests are fast, easy to perform, but may be limited in sensitivity and stability. They can only detect parasitaemia levels above 200 parasites/μl blood (Ashley et al. 2009). Originally, two-band tests were used to consist of a control line and a P. falciparum specific test line by using monoclonal antibodies against either histidine – rich protein-2(HRP-2) or P. falciparum specific lactate dehydrogenase (Pf-pLDH). Most RDTs detect P. falciparum target HRP-2, only few of them are designed to detect Pf-pLDH. We have previously evaluated the two-band tests for diagnosis of Plasmodium falciparum (Peng et al. 2012). Here, we reported the evaluation results of the three-band RDTs for detecting HRP-2 and pan-pLDH in an area of intense and perennial malaria transmission in the South part of China and Burma. The aim of this study was to estimate the sensitivity, specificity, positive predictive value (PPV) and negative predictive values (NPV) of a Pf-HRP2 and PAN-pLDH based assay, using microscopic examination of blood smears as the “gold standard”.
Materials and Methods Patients and materials
A total of 1023 patients with fever were included in Hainan, Henan and Yunnan Province of China and China-Burum boarder between April 2008 and April 2010. Five milliliters of venous blood were drawn into an EDTA-coated syringe. Aliquots of these samples were frozen at –80°C until use. A thick blood film was prepared for each patient during the blood collection process and stained with Giemsa. The patients leukocytes were counted simultaneously. The species and the density of the Plasmodium parasites were determined by counting the number of parasites per 200 white blood cells. The sample was determined to be negative when no parasites were found after examination of 200 microscopic fields at × 1 000 magnification. The negative control sample was taken from an individual who was among the 1023 patients and had never been exposed to malaria. Patients were recruited to the study were axillary temperature ≥37.5°C. Pregnancy or lactating mothers were excluded from the study. Sample size is calculated according to the anticipated proportion table (Lwanga and Lemeshow 1991), assuming that anticipated population proportion of clinical failures to artemether-lumefantrine 5%, confidence level of 95%, and precision of 10%. Ethical clearance The study was approved by the ethical committee from Hainan Province’s Center for Disease Control and Prevention, Henan Province’s Center for Disease Control and Prevention and Yunnan Province’s Center for Disease Control and Prevention. All patients provided informed consent before admission into the study.
Junlin Wu et al.
Standard microscopy evaluation The microscopic blood film examinations were done according to WHO recommendations described in “Methods for surveillance of anti-malarial drug efficacy” (WHO 2009b). The malaria microscopic examination was performed by two independent experienced microscopists, for identification of the malaria parasite species if a result showed more than 20% discrepancy, an expert microscopist was requested to check the slide and the average count of the two similar results was recorded. Preparation of gold nanoparticles The 20-nm gold nanoparticles were prepared by controlled reduction of a boiling solution of 0.01% HAuCl4 with 1% sodium citrate according to a modified citrate reduction method (Huang 2006). Briefly, 200 ml of 0.01% HAuCl4 solution was brought to a rolling boil with vigorous stirring. Rapidly adding 2.6 ml of 1% sodium citrate solution to the vortex of the solution changed its color from pale yellow to burgundy. Boiling was continued for 10 min, the heating mantle was then removed, and stirring was continued for a further 15 min. After the solution reached room temperature, it was filtered through a 0.8 μm Gelman membrane filter. Conjugation of antibody and gold nanoparticles The gold nanoparticles were coated with anti-HRP-2 and antipLDH IgG respectively according to the method modified from that developed by Huang (Huang 2006). Briefly, 10 ml of gold nanoparticles was quickly added to 1 ml of anti-HRP2 or anti-pLDH IgG solution with vigorous stirring and the mixture was incubated at room temperature for 5 min. The concentration of protein in the anti-HRP-2 or anti-pLDH IgG used was increased from 15 to 125 mg/ml. After incubation the coated gold nanoparticles were stabilized by adding 10% (113 μl) of bovine serum albumin (BSA) to a concentration of 0.1% (w/v). The conjugate was centrifuged at 8000 rpm for 20 min and washed five times in 1 ml of buffer solution which contained 0.1% bovine serum albumin. The final volume of the conjugate was 1 ml. The labeled conjugate was stored at 4°C. Preparation of membrane with immobilized antibody The labeled antibody-analyte complex for signal generation was captured after the antibody had been immobilized in a particular area of the nitrocellulose membrane. The surface of the nitrocellulose was modified to improve the rinse time of the capillary. Purified antibody was diluted with 0.2% BSA in PBS to a concentration of 2.5 mg/ml. 1 μl of this solution was applied in the form of a dot to the center of strip. The strips were dried at room temperature.
269
Evaluation of Wondfo Rapid Diagnostic Kit (Pf-HRP2/PAN-pLDH)
Configuration of immunochromatographic system
then calculate the detection sensitivity in different parasite density.
The whole system includes sample pad, absorption pad, reagents, test line 1, test line 2 and control line. The sample pad, which controls the migration of the gold nanoparticles–antibody conjugate, was treated with 50 mmol/l borate buffer, pH 7.4, containing 1% BSA, 0.5% Tween-20, and 0.05% sodium azide. Anti-HRP-2, anti-pLDH IgG and goat anti-rabbit antibody were applied to the test line 1, test line 2 and the control line, respectively, on the nitrocellulose membrane which was dried at 35°C. An untreated absorption pad was located at the end of the strip. The nitrocellulose membrane, the absorption pad, and the pretreated sample pad were attached to the strip (Fig. 1). A sample was added into a reaction holder to start testing.
Results
Detection sensitivity and specificity with blood samples
Sample collection
A total of 1023 samples were tested by Wondfo malaria rapid diagnostic kit and microscopic examination. Sensitivity was defined as the percentage of positive results for Plasmodium falciparum or non-falciparum species (especially P.vivax) observed by Wondfo RDT divided by the total positive samples obtained by the microscopic examination. Specificity was defined as the percentage of negative test results observed by Wondfo RDT divided by the total negative samples confirmed by microscopic examination.
The study included 1023 samples obtained from 1023 patients. The interpretation of test results for P. falciparum and the nonfalciparum (including P. vivax) species is shown in Table I. Their median age was 27.22 years (range 0.8–87 years). Eighty eight patients (8.6%) were children with the age of less than five years. The male-to-female ratio was 1.58 : 1. There are 362 samples contained all four Plasmodium species at different parasite densities (Table I). For P. falciparum samples, the median parasite density was 12894 p/μl (range 120– 441600 p/μl). For P. vivax, P. malaria and P. ovale, the parasite density was 7776 p/μl (range 78–83400 p/μl), 1666.34 p/μl (300–3890 p/μl) and 480 p/μl (range 120–780 p/μl), respectively. Three out of 11 microscope-confirmed P. malariae samples had originally been diagnosed by microscopy as P. vivax, whereas 3/121 P. vivax samples as P. falciparum, 1/121 P. vivax samples as P.o. Six hundred sixty-one cases with fever (including HIV infection, tuberculosis, hepatitis, syphilis and other 29 cases of infectious diseases) were included as negative control.
Detection sensitivity with different parasite density There were 28 samples consisted of 8 P. falciparum, 10 P. vivax from Yunnan Institute of Parasitic Disease and 10 reference material from the WHO (Plasmodium falciparum in whole blood). The parasite density range of 8 P. falciparum and 15 P. vivax samples was 240–186720 p/μl and 120–30180 p/μl., respectively. The 10 reference material from the WHO was Plasmodium falciparum positive whole blood, 5 of them with parasite density of 200 p/μl and the other parasite density was 2,000 p/μl (Lwanga and Lemeshow 1991). An aliquot of each sample was thawed and made a series of 10-fold dilution or 2-fold dilution. Furthermore, a series of 2-fold dilutions were done in duplicate from the end point obtained above,
Statistical analysis The gold standard of microscopic examination, sensitivity and specificity were calculated with 95% confidence intervals (C.I.). Proportions were assessed for statistical significance using the Pearson Chi-square test, or in case of small sample size, the two-tailed Fisher’s exact test. A p-value < 0.05 was considered significant.
Detection sensitivity and specificity with blood samples Table II shows the results of 1023 specimens detected by Wondfo malaria three-bands diagnostic kit and the gold stan-
Fig. 1. The schematic description of the immunochromatographic test device
270
Junlin Wu et al.
Table I. General clinical data of samples Number of cases Samples
Age
The parasite density (parasites/µl)
Male
Female
Total
Malaria
281
81
362
27.18 ± 13.86 (0.8 ~ 87)
15313.74 ± 18069.16 (78 ~ 441600)
P. falciparum
90
23
113
25.33 ± 11.58 (1 ~ 75)
12894.37 ± 1524.68 (120 ~ 441600)
P. vivax
186
57
243
34.57 ± 15.66 (1 ~ 87)
7776.47 ± 8501.64 (78 ~ 83400)
P. malariae
9
2
11
33.73 ± 11.02 (6 ~ 62)
1666.34 ± 1032.40 (300 ~ 3890)
P. ovale
2
0
2
25.5 ± 3.0 (21 ~ 30)
480 ± 280 (180 ~ 780)
Mixed infection
6
1
7
39.25 ± 9.25 (30 ~ 53)
≈1650 ± 960 (600 ~ 2520)
Negative control
345
316
661
25.48 ± 14.22 (1 ~ 73)
–
Total
626
397
1023
26.94 ± 13.85 (0.8–87)
100% at parasite densities above 200p/µ l and 500p/µ l respectively.
dard method. The 5 positive cases initially misdiagnosed by Wondfo Rapid Diagnostic Kit (Pf-HRP2/PAN-pLDH) were identified by microscopy. The sensitivity and specificity of Wondfo malaria rapid diagnostic were 96.46% and 99.67% for P. falciparum (HRP2), 95.03% and 99.24% for pLDH, 96.83% and 99.74% for non-falciparum species, 96.70% and 99.74% for P. vivax. RDT results showed very good concordance with microscopy examination when detecting P. falciparum, non-falciparum species and P. vivax.
Discussion The simple, quick, accurate, and cost-effective diagnostic tests for determining the presence of malaria parasites have been recognized as an urgent need by the World Health Organization to overcome the deficiencies of light microscopy. Numerous new malaria-diagnostic techniques have been developed (WHO 1996). This, in turn, has led to an increase in the use of RDTs for malaria, which are fast and easy to perform, and do not require electricity or specific equipment (Bell et al. 2006). Currently, 68 malaria RDTs are commercially available from 28 different manufacturers including Guangzhou Wondfo Biotech. Co., Ltd. (WHO 2008; Lwanga and Lemeshow 1991). In this study, the performance of Wondfo Rapid diagnostic Kit(Pf-HRP2/PAN-pLDH) was evaluated on a panel of
Detection sensitivity with different parasite density The detection sensitivity of Wondfo Rapid diagnostic Kit (Pf-HRP2/PAN-pLDH) increased gradually with increasing parasite density (Table III). For detection of P. falciparum, the overall sensitivity was 96.46% ranging from 10.3% at parasite densities500 p/μl
12 11 15 23
1 8 13 23
11 3 2 0
8.3 72.7 86.7 100
whole blood samples (n = 1023), obtained in Hainan, Henan and Yunnan Province of China and China-Burum border. Although the detection sensitivity was not very good for P. falciparum and P. vivax samples at low parasite densities (
