Validation of micro-chip based PCR assays for diagnosis of both Plasmodium falciparum and Plasmodium vivax

Background & objectives: Microscopy is considered as the gold standard for malaria diagnosis, however sub-microscopic infections can only be detected by Polymerase chain reaction, which demands high cost and elaborate laboratory setup. The Micro-chip PCR based Truenat Malaria Pv-Pf and Pf assay is a portable solution for detection of sub-microscopic/asymptomatic cases of malaria in the field, three lots of which were evaluated for P. falciparum and P. vivax malaria. Methods: Three lots of Truenat® Malaria Pv-Pf and Pf assay (kits) were assessed using blood samples of P. vivax and P. falciparum as well as malaria negative blood samples. DNA was extracted from the blood samples using the Trueprep Auto v2 Universal Cartridge based sample prep device and real time qPCR was performed using Truelab DUO micro PCR Analyzer with three lots of Truenat® Malaria Pv-Pf and Pf Assays. Mean, Standard deviation and one-way analysis of variance (ANOVA) was used to assess the significance of inter-lot variability in Cycle threshold values. Results: The Truenat® Malaria Pv-Pf and Pf assays identified the malaria parasites with 100% accuracy. Based on the test for variance (ANOVA) the inter-lot variability in cycle threshold values were not significant, indicating a high degree of precision. Interpretation & conclusion: Based on high accuracy and precision between different lots, the Truenat® Malaria Pv-Pf and Pf assays were found to be suitable for the diagnosis of sub-microscopic infections in field conditions to provide support in elimination of malaria.

Malaria Prevalence in a Low Transmission Area, Jazan District of Southwestern Saudi Arabia

Detailed description of malaria in low transmission areas is crucial for elimination. The current study aimed to provide a comprehensive description for malaria transmission in Jazan, a low transmission district, southwestern Saudi Arabia. Patients at a tertiary care hospital were recruited in our study between August 2016 and September 2018. Malaria diagnosis was performed through a species-specific nested polymerase chain reaction (nested PCR), microscopy and Paramax-3™ rapid detection test (RDT). Malaria was detected in 30 patients by the PCR, with point prevalence of 10.9%. Of these malaria infections, 80% was imported, 26.6% was asymptomatic and 23.3% was sub-microscopic. Malaria was reported throughout the year, with February/March and September/October peaks. Infection was significantly more in males than in females (P=0.01). Likewise, infections were detected more in febrile than in non-febrile patients (P=0.01). Adult aged 15–24 years, fever and travel were identified as high-risk factors. Malaria was primarily attributed to Plasmodium falciparum mono-infections, followed by P. vivax mono-infections and lastly to falciparum/vivax mixed infections accounting 76.6%, 16.6%, and 6.6% of PCR-confirmed malaria cases, respectively. The nested PCR was superior to the smear microscopy (sensitivity 76.6%; specificity 100%) and the RDT (sensitivity 83.3%, specificity 94.2%). The overall percent agreement between microscopy and the RDT was 92.7% (kappa=0.63). High proportion of imported malaria including sub-microscopic and sub-patent cases were described. We suggest that incorporation of molecular tool into the conventional malaria diagnosis is beneficial in Jazan district.

Comparación entre metodologías para el diagnóstico microscópico de malaria / Comparison of methodologies for microscopic malaria diagnosis

Resumen Introducción. Como parte del plan de eliminación de la malaria en Colombia, se propuso desarrollar actividades enmarcadas en la línea de trabajo: "Mejorar el acceso y la calidad del diagnóstico de malaria". Objetivo. Comparar la metodología recomendada por la Organización Panamericana de la Salus con la utilizada en Colombia para el diagnóstico de la malaria. Materiales y métodos. Se recolectaron muestras y se prepararon 88 láminas para el diagnóstico de malaria, bajo diferentes tratamientos según los parámetros evaluados. Después de la lectura microscópica por duplicado, se hicieron los respectivos cálculos de varianza para todas las posibles comparaciones de coloración con los dos métodos usados (gota gruesa y gota gruesa combinada), según la coloración (Romanowsky modificado o Giemsa) y el resultado del recuento parasitario (500, 1.000, 5.000 y 10.000 parásitos/µl de sangre). Resultados. Se obtuvo un coeficiente kappa de Cohen de concordancia entre observadores de 0,923 (IC95% 0,768-1,0). Ninguno de los factores (A: coloración, B: metodología) o interacciones (AB) tuvo un efecto estadísticamente significativo sobre los resultados, con un 95 % de nivel de confianza. Conclusión. Según los resultados obtenidos, la observación de dos gotas gruesas en una misma lámina y el uso de la tinción modificada de Romanowsky, continúa siendo una metodología adecuada para el diagnóstico de malaria en Colombia, por sus características técnicas, de almacenamiento, bajo costo y cuidados de uso.

Abstract Introduction: As part of the pre-elimination plan for malaria in Colombia, it has been proposed to develop activities within the line of work: "Improve access and quality of malaria diagnosis". Objective: To compare the methodology recommended by PAHO/WHO with that used in Colombia for the diagnosis of malaria. Materials and methods: Samples were collected and 88 slides were prepared for malaria diagnosis, under different scenarios according to the parameters to be evaluated. After duplicate mycroscopic reading, the respective variance calculations were performed for all possible staining comparisons with the two methods used (thick smear, combined thick smear), according to the staining (modified Romanowsky or Giemsa), with the result variable being the parasite density (500, 1,000, 5,000 and 10,000 parasites/µl of blood). Results: A Cohen kappa index of inter-rater agreement of 0.923 (95% CI: 0.768-1.078) was obtained. None of the factors (A: stain, B: methodology) or interactions (AB) had a statistically significant effect on the results with a 95% confidence level. Conclusion: Based on the results of the study, the preparation of two thick smears in the same slide stained with the modified Romanowsky stain is a suitable methodology for the diagnosis of malaria in Colombia, due to its technical characteristics, of storage, low cost, use and care.

An Alternative Method for Extracting Plasmodium DNA from EDTA Whole Blood for Malaria Diagnosis

Molecular techniques have been introduced for malaria diagnosis because they offer greater sensitivity and specificity than microscopic examinations. Therefore, DNA isolation methods have been developed for easy preparation and cost effectiveness. The present study described a simple protocol for Plasmodium DNA isolation from EDTA-whole blood. This study demonstrated that after heating infected blood samples with Tris–EDTA buffer and proteinase K solution, without isolation and purification steps, the supernatant can be used as a DNA template for amplification by PCR. The sensitivity of the extracted DNA of Plasmodium falciparum and Plasmodium vivax was separately analyzed by both PCR and semi-nested PCR (Sn-PCR). The results revealed that for PCR the limit of detection was 40 parasites/μl for P. falciparum and 35.2 parasites/μl for P. vivax, whereas for Sn-PCR the limit of detection was 1.6 parasites/μl for P. falciparum and 1.4 parasites/μl for P. vivax. This new method was then verified by DNA extraction of whole blood from 11 asymptomatic Myanmar migrant workers and analyzed by Sn-PCR. The results revealed that DNA can be extracted from all samples, and there were 2 positive samples for Plasmodium (P. falciparum and P. vivax). Therefore, the protocol can be an alternative method for DNA extraction in laboratories with limited resources and a lack of trained technicians for malaria diagnosis. In addition, this protocol can be applied for subclinical cases, and this will be helpful for epidemiology and control.

An Analysis Of Malaria Diagnostic Methods - Microscopy And Rapid Diagnostic Test (RDT) In Gandhinagar – Gujarat.

Abstracts: Background & Objective: Malaria is one of the major public health problems in the developing countries. Rapid diagnosis and accurate quantification of Plasmodium falciparum parasitemia are important forthe management of malaria. The objective of this study was to measure prevalence of malaria and analyse the results of malaria diagnostic methods. Methodology: RDT and microscopy was carried out to diagnose malaria. Results were simply presented as percentage positive of total number of cases under this study. Results of microscopy were compared with RDT based on antigen detection for malaria diagnosis. Results: Total 503 cases were detected having infection of malaria. Out of them 405(80.52%) were positive for P. vivax, 73 (14.51%) were positive for P. falciparum and 25 (4.97%) were having mixed infection of P. vivax and P. falciparum. Sensitivity of RDTs was excellent as compare to microscopy. Conclusion: We can conclude based on the present study that sensitivity of RDT is very good as compare to traditional microscopy. But for the confirmation microscopy remains gold standard test for malaria identification.

Comparison of the Partec CyScope® Rapid Diagnostic Test with Light Microscopy for Malaria Diagnosis in Rural Tole, Southwest Cameroon.

Introduction/Aim: Malaria is a major public health problem and can lead to fatal consequences within few days if not diagnosed and promptly treated. The aim of this study was to determine the malaria parasite prevalence and assess the performance characteristics of the Partec CyScope® rapid diagnostic test (RDT) in Tole. Experimental Design, Place and Duration of Study: The study was a cross-sectional survey, carried out in Tole, Southwest Cameroon in July 2014. Methodology: A total of 231 children were studied. Information on demographic data, temperature and malaria risk factors was recorded. Capillary blood was collected by finger pricking. Thick and thin blood films were prepared for malaria parasite detection and speciation. Ten μL of blood was added unto the DAPI coated slides and read under the Partec CyScope®. Haemoglobin values were determined. Results and Conclusion: The overall prevalences of malaria parasites, fever and anaemia were 66.2%, 35.9% and 86.6% respectively. Although not statistically significant, malaria parasite prevalence was highest in children aged 1 – 5 years, higher in females, those that had stagnant water and bushes around their homes as well as those who did not use insecticide-treated bed nets and insecticide residual spraying when compared with their respective counterparts. Overall geometric mean parasite density (GMPD) was 3691 (range = 100 - 48000) parasites/μL of blood). GMPD was significantly higher (P = 0.03) in febrile than afebrile children. Prevalence of anaemia was significantly higher (P = 0.01) in malaria positive (68.5%) than negative (45.2%) children. More cases of infections were detected by light microscopy than by Partec CyScope®. The sensitivities and specificities of Partec CyScope® were 87.6% (CI = 81.4-91.1%) and 94.9% (CI = 87.5-98.0%) respectively while the positive and negative predictive values were 97.1% and 79.6% respectively. Partec CyScope® can therefore be used for mass malaria surveillance.

Low Prevalence of Plasmodium vivax - Plasmodium falciparum Mixed - Infection in Patients from Central and Eastern Part of Sudan: Implication for Case Management in Sudan.

Accurate diagnosis of malaria parasite species is crucial for rational treatment that is a key success for a malaria control and elimination programmes. The main objective of this investigation was to correct species identification and re-assessment of diagnosis method in central and eastern part of Sudan. The blood samples were collected from 71 febrile cases infected with P. vivax in Eastern and Central Sudan, diagnosed by light microscopy and also by nested-PCR assay, using 18S small sub-unit ribosomal RNA (ssrRNA) gene. The nested-PCR were detect 92.9% (66/71) and 2.8% (2/71) P. vivax and P. falciparum mono-infection, respectively. Based on microscopy method, the level of mixed - Infection was zero; however, nested-PCR assay detected 4.2% (3/71) mixed infections in collected samples. In detecting P. vivax infection, microscopy had high sensitivity (97%) and specificity (50%). In conclusion, the present data point to the need of improving microscopy diagnosis method in malaria endemic region and also suggest that although molecular techniques are not practical for diagnosis of P. vivax and P. falciparum mixed infections in any areas; these could be used to collect epidemiological facts for control and elimination of the disease in Sudan.

Malaria Diagnostic Tests: Microscopy And Rapid Diagnostic Test (RDT) Revisited.

In the prevention and control of malaria, Prompt and accurate diagnosis is the key to effective disease management. Giemsa microscopy and rapid diagnostic tests (RDTs) are the diagnostic tests each with characteristic strengths and limitations is the best way for accurate diagnosis has a key role for malaria control successfully. Reduction in morbidity and drug resistance intensity of malaria require a parasite based diagnostic methods. A parallel commitment is needed in production of antimalarial drug or malaria vaccine along with improvement in diagnostic tests and their availability to people in endemic areas.endemic areas.

Development of Monoclonal Antibodies That Target 1-Cys Peroxiredoxin and Differentiate <i>Plasmodium falciparum</i> from <i>P. vivax</i> and <i>P. knowlesi</i>

Prompt and accurate diagnosis of malarial patients is a crucial factor in controlling the morbidity and mortality of the disease. Effective treatment decisions require a correct diagnosis among mixed-species malarial patients. Differential diagnosis is particularly important in cases of <i>Plasmodium vivax</i>, a species that shares endemicity with <i>P. falciparum</i> in most endemic areas. Moreover, it is difficult to identify <i>P. knowlesi</i> on the basis of morphology alone, and rapid diagnostic tests are still not available for this malaria species. Therefore, the development of diagnostic tests applicable to the field is urgently needed. 1-Cys peroxiredoxin (1-Cys-Prx) in <i>P. falciparum</i> is abundantly expressed in the mature asexual stages, making it a promising candidate as a diagnostic antigen. In this study, we produced five monoclonal antibodies (mAbs) against <i>P. falciparum</i> 1-Cys-Prx (Pf1-Cys-Prx) by immunizing BALB/c mice with recombinant Pf1-Cys-Prx and subsequent hybridoma production. Cross reactivity of established mAbs with the orthologous molecule of Pf1-Cys-Prx in <i>P. vivax</i> (Pv1-Cys-Prx) and <i>P. knowlesi</i> (Pk1-Cys-Prx) was examined. Western blot analyses showed that three mAbs reacted with Pv1-Cys-Prx and Pk1-Cys-Prx but two mAbs did not. These results indicate that the two mAbs were effective in differentiating <i>P. falciparum</i> from <i>P. vivax</i> and <i>P. knowlesi</i> and could be used in differential diagnosis as well as comparative molecular studies of human <i>Plasmodium</i> species.

A comparative laboratory diagnosis of malaria：microscopy versus rapid diagnostic test kits

Objective: To compare the two methods of rapid diagnostic tests (RDTs) and microscopy in the diagnosis of malaria. Methods: RDTs and microscopy were carried out to diagnose malaria. Percentage malaria parasitaemia was calculated on thin films and all non-acute cases of plasmodiasis with less than 0.001% malaria parasitaemia were regarded as negative. Results were simply presented as percentage positive of the total number of patients under study. The results of RDTs were compared to those of microscopy while those of RDTs based on antigen were compared to those of RDTs based on antibody. Patients' follow-up was made for all cases. Results:All the 200 patients under present study tested positive to RDTs based on malaria antibodies (serum) method (100%). 128 out of 200 tested positive to RDTs based on malaria antigen (whole blood) method (64%), while 118 out of 200 patients under present study tested positive to visual microscopy of Lieshman and diluted Giemsa (59%). All patients that tested positive to microscopy also tested positive to RDTs based on antigen. All patients on the second day of follow-up were non-febrile and had antimalaria drugs. Conclusions: We conclude based on the present study that the RDTs based on malaria antigen (whole blood) method is as specific as the traditional microscopy and even appears more sensitive than microscopy. The RDTs based on antibody (serum) method is unspecific thus it should not be encouraged. It is most likely that Africa being an endemic region, formation of certain levels of malaria antibody may not be uncommon. The present study also supports the opinion that a good number of febrile cases is not due to malaria. We support WHO’s report on cost effectiveness of RDTs but, recommend that only the antigen based method should possibly, be adopted in Africa and other malaria endemic regions of the world.

Evaluation of Rapid Diagnostics for Plasmodium falciparum and P. vivax in Mae Sot Malaria Endemic Area, Thailand

Prompt and accurate diagnosis of malaria is the key to prevent disease morbidity and mortality. This study was carried out to evaluate diagnostic performance of 3 commercial rapid detection tests (RDTs), i.e., Malaria Antigen Pf/Pantrade mark, Malaria Ag-Pftrade mark, and Malaria Ag-Pvtrade mark tests, in comparison with the microscopic and PCR methods. A total of 460 blood samples microscopically positive for Plasmodium falciparum (211 samples), P. vivax (218), mixed with P. falciparum and P. vivax (30), or P. ovale (1), and 124 samples of healthy subjects or patients with other fever-related infections, were collected. The sensitivities of Malaria Ag-Pftrade mark and Malaria Antigen Pf/Pantrade mark compared with the microscopic method for P. falciparum or P. vivax detection were 97.6% and 99.0%, or 98.6% and 99.0%, respectively. The specificities of Malaria Ag-Pftrade mark, Malaria Ag-Pvtrade mark, and Malaria Antigen Pf/Pantrade mark were 93.3%, 98.8%, and 94.4%, respectively. The sensitivities of Malaria Ag-Pftrade mark, Malaria Antigen Pf/Pantrade mark, and microscopic method, when PCR was used as a reference method for P. falciparum or P. vivax detection were 91.8%, 100%, and 96.7%, or 91.9%, 92.6%, and 97.3%, respectively. The specificities of Malaria Ag-Pftrade mark, Malaria Ag-Pvtrade mark, Malaria Antigen Pf/Pantrade mark, and microscopic method were 66.2%, 92.7%, 73.9%, and 78.2%, respectively. Results indicated that the diagnostic performances of all the commercial RDTs are satisfactory for application to malaria diagnosis.

Uso de paneles de láminas estandarizadas para la evaluación de competencias en el diagnóstico microscópico de malaria en la Amazonía Peruana / Use of standardized blood smear slide sets for competency assessment in the malaria microscopic diagnosis in the Peruvian Amazon

Objetivos. Evaluar la competencia de los microscopistas en el diagnóstico de la malaria mediante paneles de láminas estandarizados en la Amazonía peruana. Materiales y métodos. Estudio transversal, realizado entre los meses de julio y septiembre de 2007, en 122 establecimientos de salud de primer nivel de atención de la Amazonía peruana. En el marco del Proyecto PAMAFRO, se evaluó las competencias en el diagnóstico de malaria en 68 microscopistas sin experiencia (

Objectives. To assess the competency of microscopists for malaria diagnosis using standardized slide sets in the Peruvian Amazon. Material and methods. Cross-sectional study carried out in 122 first level health facilities of the Peruvian Amazon, between July and September 2007. Within the frame of the project "Control Malaria in the border areas of the Andean Region: A community approach" (PAMAFRO), we evaluated the malaria diagnosis performance in 68 microscopists without expertise (< 1 year of expertise) and 76 microscopists with expertise (> 1 year) using standardized sets of 20 blood smear slides according to the World Health Organization (WHO) recommendations. A correct diagnosis (correct species identification) was defined as "agreement", a microscopist was qualified as an "expert" if they have an agreement ≥90 percent (≥ 18 slides with correct diagnosis), as a "referent" with an agreement between 80 percent and <90 percent, "competent" if they are between 70 and <80 percent and "in training" if they have <70 percent. Results. Microscopists with expertise (68.6 percent) had more agreement than those without expertise (48.2 percent). The competency assessment was acceptable (competent, referent, or experts levels) in 11.8 percent of the microscopists without expertise and in 52.6 percent from those with expertise. The agreement was lower using blood smear slides with P. falciparum with low parasitaemia, with P. malariae and with mixed infections. Conclusions. Is the first assessment, we found only one of three microscopists from the Peruvian Amazon is competent fro malaria diagnosis according to the WHO standards. From this baseline data, we have to continue working in order to improve the competency assessment of the microscopists within the frame of a quality assurance system.

To evaluate the diagnostic yield and cost of rapid diagnostic test plasmodium falciparum (paracheck F test) in the malarial hyperendermic areas of the central Vietnam

Background: In 2006, the project of global fund for malaria prevention in Vietnam provided a large number of rapid diagnostic test Paracheck F for Vietnam for the purpose of rapid malaria diagnose. However, there is no study on evaluation the effect of rapid diagnostic test compared with microscopy method. Objective: To evaluate the diagnostic yield and cost of paracheck F test and microscopy in malaria diagnosis and treatment. Subject and Method: The study was carried out in 6 communes belongs to Quang Tri and Quang Binh provinces from September to November - 2006. The study was divided into 3 phases. Phase 1: diagnoses and treatments are based on clinical symptoms, phase 2: diagnoses and treatments are based on the results of paracheck and phase 3: diagnoses and treatments are based on the results of microscopy. All phases, both the common patients and malarial patients and the amount of anti-malarial drugs were treated, the amount of money was spent on transport and days work off of malarial patients and their relatives were calculated. Result: The investigation data on expenditure of malaria patients showed that: the average direct cost of malaria patient in phase 1 is VND 116.100; phase 2: VND 119.400 and phase 3: VND 120.800 per 1 treatment course. There is no significant difference between direct costs in three phases (p > 0.05). Conclusion: The expense efficiency for finding out a case of malaria by paracheck and microscopy is equivalent and lower than the expense of diagnosis based on clinical symptoms.