Detection Of Falciparum Research Articles

The epidemiology of malaria and anaemia in the Bonikro mining area, central Côte d'Ivoire.

BackgroundThe epidemiology of malaria and anaemia is characterized by small-scale spatial and temporal heterogeneity, which might be influenced by human activities, such as mining and related disturbance of the environment. Private sector involvement holds promise to foster public health, including the prevention and control of malaria and anaemia. Here, results from a cross-sectional epidemiological survey, conducted in communities that might potentially be affected by the Bonikro Gold Mine (BGM) in Côte d’Ivoire, are reported.MethodsIn December 2012, a cross-sectional survey was carried out in seven communities situated within a 20-km radius of the BGM in central Côte d’Ivoire. Capillary blood samples were obtained from children aged six to 59 months. Samples were subjected to a rapid diagnostic test (RDT) for Plasmodium falciparum detection, whilst haemoglobin (Hb) was measured to determine anaemia. Additionally, mothers were interviewed with a malaria-related knowledge, attitudes and practices questionnaire.ResultsA total of 339 children and 235 mothers participated in the surveys. A positive RDT for P. falciparum was found in 69% of the children, whilst 72% of the children were anaemic (Hb <11 g/dl). Plasmodium falciparum infection was significantly associated with anaemia (odds ratio (OR) 7.43, 95% confidence interval (CI) 3.97-13.89), access to a health facility (OR 5.59, 95% CI 1.81-17.32) and age (OR 0.04, 95% CI 0.01-0.12; youngest (six to 11 months) versus oldest (48-59 months) age group). Less than a quarter of mothers knew that malaria is uniquely transmitted by mosquitoes (22.3%, 95% CI 16.8-27.7%). Misconceptions were common; most of the mothers believe that working in the sun can cause malaria.ConclusionsMalaria and anaemia are highly endemic in the surveyed communities around the BGM project area in Côte d’Ivoire. The data presented here provide a rationale for designing setting-specific interventions and can be utilized as a benchmark for longitudinal monitoring of potential project-related impacts due to changes in the social-ecological and health systems.

Evaluation of a novel magneto-optical method for the detection of malaria parasites.

Improving the efficiency of malaria diagnosis is one of the main goals of current malaria research. We have recently developed a magneto-optical (MO) method which allows high-sensitivity detection of malaria pigment (hemozoin crystals) in blood via the magnetically induced rotational motion of the hemozoin crystals. Here, we evaluate this MO technique for the detection of Plasmodium falciparum in infected erythrocytes using in-vitro parasite cultures covering the entire intraerythrocytic life cycle. Our novel method detected parasite densities as low as ∼40 parasites per microliter of blood (0.0008% parasitemia) at the ring stage and less than 10 parasites/µL (0.0002% parasitemia) in the case of the later stages. These limits of detection, corresponding to approximately 20 pg/µL of hemozoin produced by the parasites, exceed that of rapid diagnostic tests and compete with the threshold achievable by light microscopic observation of blood smears. The MO diagnosis requires no special training of the operator or specific reagents for parasite detection, except for an inexpensive lysis solution to release intracellular hemozoin. The devices can be designed to a portable format for clinical and in-field tests. Besides testing its diagnostic performance, we also applied the MO technique to investigate the change in hemozoin concentration during parasite maturation. Our preliminary data indicate that this method may offer an efficient tool to determine the amount of hemozoin produced by the different parasite stages in synchronized cultures. Hence, it could eventually be used for testing the susceptibility of parasites to antimalarial drugs.

Detection of Plasmodium vivax and Plasmodium falciparum DNA in human saliva and urine: Loop-mediated isothermal amplification for malaria diagnosis

This study investigated loop-mediated isothermal amplification (LAMP) detection of Plasmodium falciparum and Plasmodium vivax in urine and saliva of malaria patients. From May to November 2011, 108 febrile patients referred to health centers in Sistan and Baluchestan Province of south-eastern Iran participated in the study. Saliva, urine, and blood samples were analyzed with nested PCR and LAMP targeting the species-specific nucleotide sequence of small subunit ribosomal RNA gene (18S rRNA) of P. falciparum and P. vivax and evaluated for diagnostic accuracy by comparison to blood nested PCR assay. When nested PCR of blood is used as standard, microscopy and nested PCR of saliva and urine samples showed sensitivity of 97.2%, 89.4% and 71% and specificity of 100%, 97.3% and 100%, respectively. LAMP sensitivity of blood, saliva, and urine was 95.8%, 47% and 29%, respectively, whereas LAMP specificity of these samples was 100%. Microscopy and nested PCR of saliva and LAMP of blood were comparable to nested PCR of blood (к=0.95, 0.83, and 0.94, respectively), but agreement for nested PCR of urine was moderate (к=0.64) and poor to fair for saliva LAMP and urine LAMP (к=0.38 and 0.23, respectively). LAMP assay showed low sensitivity for detection of Plasmodium DNA in human saliva and urine compared to results with blood and to nested PCR of blood, saliva, and urine. However, considering the advantages of LAMP technology and of saliva and urine sampling, further research into the method is worthwhile. LAMP protocol and precise preparation protocols need to be defined and optimized for template DNA of saliva and urine.

Comparison of two isothermal amplification methods: Thermophilic helicase dependent amplification (tHDA) and loop mediated isothermal amplification (LAMP) for detection of Plasmodium falciparum

Background: Inflammation is the cells and body tissues response against an injury. Injury can be caused due to any reasons like infections, chemicals and thermal and mechanical. Though, inflammation is body’s own defense mechanism but sometimes these complex events and mediators involved in the inflammatory response can stimulate or intensify many reactions which lead to damage to body’s tissues. Non-steroidal anti-inflammatory drugs (NSAID’s) are mostly used for the treatment of inflammation and other related diseases.However, constant use of NSAID may lead to gastro-intestinal ulcers, bleedingand renal disorders. Immunomodulatory and anti-arthritic activity of Lactobacillus casei and Lactobacillus acidophilus are well known. Therefore present investigations were carried out to evaluate analgesic and antiinflammatory activity L. rahmnosus in female wistar rats. Methods &Materials: Diclofenac sodiumwas used as standard drug for comparison. L. rahmnosus, drugs andvehiclewere administered orally with feeding cannula. Analgesic activity was evaluated byacetic acid-inducedwrithing testwhile anti-inflammatory activity was tested by using carrageenan induced paw edema model. Results: Results showed that L. rahmnosus significantly decreased the paw thickness at t = 24hours in female wistar rats at P <0.05. Also, it protected the females rats from writhing induced by acetic acid. Protection provided by L. rahmnosus was more pronounced in comparison to standard drug diclofenac sodium. Conclusion: Present study clearly suggests that L. rahmnosus suppress the first phase of carrageenan induced paw edema and decreased the acetic acid induced writhings in wistar rats. Thus it can be used as a natural NSAID.

Study on validity of a rapid diagnostic test kit versus light microscopy for malaria diagnosis in Ahmedabad city, India

Light microscopy of blood smears for diagnosis of malaria in the field has several limitations, notably delays in diagnosis. This study in Ahmedabad in Gujarat State, India, evaluated the diagnostic performance of a rapid diagnostic test for malaria (SD Bioline Malaria Ag P.f/Pan) versus blood smear examination as the gold standard. All fever cases presenting at 13 urban health centres were subjected to rapid diagnostic testing and thick and thin blood smears. A total of 677 cases with fever were examined; 135 (20.0%) tested positive by rapid diagnostic test and 86 (12.7%) by blood smear. The sensitivity of the rapid diagnostic test for malaria was 98.8%, specificity was 91.5%, positive predictive value 63.0% and negative predictive value 99.8%. For detection of Plasmodium falciparum the sensitivity of rapid diagnostic test was 100% and specificity was 97.3%. The results show the acceptability of the rapid test as an alternative to light microscopy in the field setting.

Detection of Plasmodium falciparum or Trypanosome infection with ultrasound-based separation of red blood cells (RBCs)

Detection of Plasmodium falciparum or Trypanosome infection with ultrasound-based separation of red blood cells (RBCs)

Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis

BackgroundNucleic acid amplification is the most sensitive and specific method to detect Plasmodium falciparum. However the polymerase chain reaction remains laboratory-based and has to be conducted by trained personnel. Furthermore, the power dependency for the thermocycling process and the costly equipment necessary for the read-out are difficult to cover in resource-limited settings. This study aims to develop and evaluate a combination of isothermal nucleic acid amplification and simple lateral flow dipstick detection of the malaria parasite for point-of-care testing.MethodsA specific fragment of the 18S rRNA gene of P. falciparum was amplified in 10 min at a constant 38°C using the isothermal recombinase polymerase amplification (RPA) method. With a unique probe system added to the reaction solution, the amplification product can be visualized on a simple lateral flow strip without further labelling. The combination of these methods was tested for sensitivity and specificity with various Plasmodium and other protozoa/bacterial strains, as well as with human DNA. Additional investigations were conducted to analyse the temperature optimum, reaction speed and robustness of this assay.ResultsThe lateral flow RPA (LF-RPA) assay exhibited a high sensitivity and specificity. Experiments confirmed a detection limit as low as 100 fg of genomic P. falciparum DNA, corresponding to a sensitivity of approximately four parasites per reaction. All investigated P. falciparum strains (n = 77) were positively tested while all of the total 11 non-Plasmodium samples, showed a negative test result. The enzymatic reaction can be conducted under a broad range of conditions from 30-45°C with high inhibitory concentration of known PCR inhibitors. A time to result of 15 min from start of the reaction to read-out was determined.ConclusionsCombining the isothermal RPA and the lateral flow detection is an approach to improve molecular diagnostic for P. falciparum in resource-limited settings. The system requires none or only little instrumentation for the nucleic acid amplification reaction and the read-out is possible with the naked eye. Showing the same sensitivity and specificity as comparable diagnostic methods but simultaneously increasing reaction speed and dramatically reducing assay requirements, the method has potential to become a true point-of-care test for the malaria parasite.

A new visually improved and sensitive loop mediated isothermal amplification (LAMP) for diagnosis of symptomatic falciparum malaria

Molecular diagnosis of malaria by nucleotide amplification requires sophisticated and expensive instruments, typically found only in well-established laboratories. Loop-mediated isothermal amplification (LAMP) has provided a new platform for an easily adaptable molecular technique for molecular diagnosis of malaria without the use of expensive instruments. A new primer set has been designed targeting the 18S rRNA gene for the detection of Plasmodium falciparum in whole blood samples. The efficacy of LAMP using the new primer set was assessed in this study in comparison to that of a previously described set of LAMP primers as well as with microscopy and real-time PCR as reference methods for detecting P. falciparum. Pre-addition of hydroxy napthol blue (HNB) in the LAMP reaction caused a distinct color change, thereby improving the visual detection system. The new LAMP assay was found to be 99.1% sensitive compared to microscopy and 98.1% when compared to real-time PCR. Meanwhile, its specificity was 99% and 100% in contrast to microscopy and real-time PCR, respectively. Moreover, the LAMP method was in very good agreement with microscopy and real-time PCR (0.94 and 0.98, respectively). This new LAMP method can detect at least 5parasites/μL of infected blood within 35min, while the other LAMP method tested in this study, could detect a minimum of 100parasites/μL of human blood after 60min of amplification. Thus, the new method is sensitive and specific, can be carried out in a very short time, and can substitute PCR in healthcare clinics and standard laboratories.

Field evaluation of the photo-induced electron transfer fluorogenic primers (PET) real-time PCR for the detection of Plasmodium falciparum in Tanzania

BackgroundAccurate diagnosis of malaria infections remains challenging, especially in the identification of submicroscopic infections. New molecular diagnostic tools that are inexpensive, sensitive enough to detect low-level infections and suitable in laboratory settings of resource-limited countries are required for malaria control and elimination programmes. Here the diagnostic potential of a recently developed photo-induced electron transfer fluorogenic primer (PET) real-time polymerase chain reaction (PCR) called PET-PCR was investigated. This study aimed to (i) evaluate the use of this assay as a method for the detection of both Plasmodium falciparum and other Plasmodium species infections in a developing country’s diagnostic laboratory; and, (ii) determine the assay’s sensitivity and specificity compared to a nested 18S rRNA PCR.MethodsSamples used in this study were obtained from a previous study conducted in the region of Iringa, Tanzania. A total of 303 samples from eight health facilities in Tanzania were utilized for this evaluation. All samples were screened using the multiplex PET-PCR assay designed to detect Plasmodium genus and P. falciparum initially in laboratory in Tanzania and then repeated at a reference laboratory at the CDC in the USA. Microscopy data was available for all the 303 samples. A subset of the samples were tested in a blinded fashion to find the sensitivity and specificity of the PET-PCR compared to the nested 18S rRNA PCR.ResultsCompared to microscopy, the PET-PCR assay was 59% more sensitive in detecting P. falciparum infections. The observed sensitivity and specificity were 100% (95% confidence interval (CI0.95) = 94-100%) and (CI0.95 = 96-100%), respectively, for the PET-PCR assay when compared to nested 18S rRNA PCR. When compared to 18S rRNA PCR, microscopy had a low sensitivity of 40% (CI0.95 = 23-61%) and specificity of 100% (CI0.95 = 96-100%). The PET-PCR results performed in the field laboratory in Tanzania were in 100% concordance with the results obtained at the reference laboratory in the USA.ConclusionThe PET-PCR is a new molecular diagnostic tool with similar performance characteristics as commonly used PCR methods that is less expensive, easy to use, and amiable to large scale-surveillance studies in developing country settings.

Evidence for perennial malaria in rural and urban areas under the Sudanian climate of Kandi, Northeastern Benin.

BackgroundIn arid settings, droughts usually lead to periods of very low or no malaria transmission. However, in rural Kandi (Sonsoro) in northeastern Benin, several malaria cases are often diagnosed during dry seasons. The underlying factors accounting for this phenomenon remain unknown.MethodsThe entomological profile of Sonsoro has been studied compared to a location in urban Kandi (Gansosso) for a period of one year. During this period, Anopheles larval habitats were investigated and populations of Anopheles gambiae s.l. were sampled by human landing catches in both areas. Enzyme-linked immunosorbent assays (ELISA) for Plasmodium falciparum circumsporozoite protein (CSP) were conducted on vector specimens and the entomological inoculation rates (EIR) were determined per season (wet versus dry) in each area. In addition, during the severe drought period, Rapid Diagnostic Tests (RDTs) were conducted on school children under the age 10 years in these areas to provide a global view of drought-malaria prevalence and to perform a crossing with entomological data in Kandi.ResultsOverall, An. gambiae s.l. was particularly abundant in rural Kandi compared to the urban area with a significant decrease of vector density in both sites during the dry season. In this period, larval sampling data identified household water sources as potential breeding sites in urban and rural Kandi. We also observed a significant seasonal variation of the infectivity rate in both areas but for each period (season), the EIR was higher in the rural site than in the urban. Data of P. falciparum detection was the reflection of entomological findings. The drought-malaria prevalence was 5.5 times higher in rural Kandi as compared to urban Kandi. The presence of a permanent water site and the low level of urbanization in rural Kandi were identified as a risk factor.ConclusionOur data showed a high level of malaria transmission in the municipality of Kandi. Household water source plays an important role in maintaining the breeding of anopheles larvae and the malaria transmission in Kandi. In rural settings, the proximity to permanent water sites could probably be the aggravating factor.

Effect of combining mosquito repellent and insecticide treated net on malaria prevalence in Southern Ethiopia: a cluster-randomised trial

BackgroundA mosquito repellent has the potential to prevent malaria infection, but there has been few studies demonstrating the effectiveness of combining this strategy with the highly effective long-lasting insecticidal nets (LLINs). This study aimed to determine the effect of combining community-based mosquito repellent with LLINs in the reduction of malaria.MethodsA community-based clustered-randomised trial was conducted in 16 rural villages with 1,235 households in southern Ethiopia between September and December of 2008. The villages were randomly assigned to intervention (mosquito repellent and LLINs, eight villages) and control (LLINs alone, eight villages) groups. Households in the intervention villages received mosquito repellent (i.e., Buzz-Off® petroleum jelly, essential oil blend) applied every evening. The baseline survey was followed by two follow-up surveys, at one month interval. The primary outcome was detection of Plasmodium falciparum, Plasmodium vivax, or both parasites, through microscopic examination of blood slides. Analysis was by intention to treat. Baseline imbalances and clustering at individual, household and village levels were adjusted using a generalized linear mixed model.Results3,078 individuals in intervention and 3,004 in control group were enrolled into the study. Compared with the control arm, the combined use of mosquito repellent and LLINs significantly reduced malaria infection of all types over time [adjusted Odds Ratio (aOR) = 0.66; 95% CI = 0.45-0.97]. Similarly, a substantial reduction in P. falciparum malaria infection during the follow-up surveys was observed in the intervention group (aOR = 0.53, 95% CI = 0.31-0.89). The protective efficacy of using mosquito repellent and LLINs against malaria infection of both P. falciparum/P. vivax and P. falciparum was 34% and 47%, respectively.ConclusionsDaily application of mosquito repellent during the evening followed by the use of LLINs during bedtime at community level has significantly reduced malaria infection. The finding has strong implication particularly in areas where malaria vectors feed mainly in the evening before bedtime.Trial registrationClinicalTrials.gov identifier: NCT01160809.

Alternative Malaria Diagnostic Tools: Evaluation of Plasmodium falciparum Detection along Thailand?s Border by Loop-Mediated Isothermal Amplification (LAMP) and Immunochromatographic Test (ICT)

Alternative Malaria Diagnostic Tools: Evaluation of Plasmodium falciparum Detection along Thailand?s Border by Loop-Mediated Isothermal Amplification (LAMP) and Immunochromatographic Test (ICT)

Alternative Malaria Diagnostic Tools: Evaluation of Plasmodium falciparum Detection along ThailandÂ’s Border by Loop-Mediated Isothermal Amplification (LAMP) and Immunochromatographic Test (ICT)

Introduction: Thailand has a national goal to eliminate malaria from 80 percent of the country by 2020. However, malaria-endemic areas still exist along the forested Thailand’s borders. An accurate and prompt diagnosis is the key to effective disease management. This aim of this study was to evaluate the application of LAMP as an alternative tool for falciparum malaria diagnosis of clinical suspicion of plasmodium infection in the border area, compared to those of ICT and microscopy using nested PCR as the reference detection method. Materials and Methods: One hundred and four of blood samples were obtained from patients who were diagnosed malaria positive by clinical diagnosis or subjected to suffering from malaria as observed from clinical history. The primer set used for LAMP was designed on the basis of nucleotide sequence of 18S rRNA plasmodium gene and the ICT test was performed with the parasite lactate dehydrogease (pLDH) antigen-based lateral flow test. Results: The LAMP assay has the highest agreement with the reference method (99.04%, κ=0.98) with a sensitivity (95%CI) of 98.59% (95.85-100.00) comparable to ICT and microscopy. Moreover, LAMP showed specificity 100% compared with 100% of ICT and 96.97% (91.12-100.00%) microscopy. Negative predictive value of LAMP and ICT were 97.06% and 82.50% respectively. Conclusion: LAMP is useful and more reliable for specific diagnosis in border regions where malaria is endemic but individuals are asymptomatic and hence that LAMP could be the preferred method in resource-limited laboratories to progress towards malaria elimination in Thailand.

A single-round multiplex PCR assay for the identification of Anopheles minimus related species infected with Plasmodium falciparum and Plasmodium vivax

This study aimed to develop a single-round multiplex PCR method for the identification of Anopheles minimus complex (An. minimus and Anopheles harrisoni) and Anopheles aconitus subgroup (An. aconitus and Anopheles varuna), and for the simultaneous detection of Plasmodium falciparum and Plasmodium vivax in these vectors. Five primers were created for a single-round multiplex PCR assay to identify four anopheline mosquitoes combined with three Plasmodium primers for the detection of P. falciparum and P. vivax in vectors. The four species of anopheline vectors and two Plasmodium species, P. falciparum and P. vivax, could be identified by the combination of eight primers in the single-round multiplex PCR assay. The amplified species-specific products were 380bp for An. minimus, 180bp for An. harrisoni, 150bp for An. aconitus, 310bp for An. varuna, 276bp for P. falciparum, and 300bp for P. vivax. The sensitivities were 0.5pg/μl (25sporozoites/μl) for P. falciparum DNA and between 0.5 and 5pg/μl (25–250sporozoites/μl) for P. vivax DNA. Furthermore, this developed method could be used to identify field caught An. minimus complex, An. aconitus subgroup from Thailand and Lao PDR. Also, it was successfully used to identify the species An. minimus, An. harrisoni, An. aconitus and An. varuna and to detect and identify P. falciparum and P. vivax in caught anopheline mosquitoes. The sensitivity of this method was high for simultaneous detection of P. falciparum and P. vivax in anopheline mosquitoes.

Performances of Malaria P.f/Pan Rapid Test Device Acon® (Pf HRP2/pan aldolase) and Malaria Pf Rapid Test Device Acon® (Pf HRP2) for the diagnosis of malaria in adults and children living in Gabon, Central Africa

The Malaria Pf Rapid Test Device Acon® (Acon Labs) and the pan HRP2/aldolase RDT, Malaria P.f/Pan Rapid Test Device Acon® (Acon Labs), performances were evaluated for malaria species diagnosis in 592 febrile patients living in Gabon using microscopy as gold standard. Sensitivities were equal or above 96.0% for Plasmodium falciparum detection, of 62.5% for non–P. falciparum malaria species detection and higher in younger children (100%). Negative predictive values were greater than 97.0%. Acon®HRP2 had a higher specificity (96.6%) and lower false-positive (FP) rate (9.3%) compared to Acon®Pf/Pan, which had a specificity of 87.3% and a FP rate of 27.1% (P < 0.01). Overall, 32.5% of all Acon® Pf/Pan tests resulted in a “faint band” with only 2 resulted from samples with a parasitemia below 100 p/μL. The accuracy of Acon®HRP2 RDT for the diagnosis of P. falciparum infection is confirmed. However, the high FP rate observed with Acon®Pf/Pan is a limitation for its use.

Routine parallel diagnosis of malaria using microscopy and the malaria rapid diagnostic test SD 05FK60: the experience of Médecins Sans Frontières in Myanmar

BackgroundMalaria rapid diagnostic tests (RDTs) are commonly used in Médecins Sans Frontières (MSF) programmes to detect acute malaria infection. Programmes in regions with both Plasmodium falciparum and non-falciparum malaria (i.e. Plasmodium ovale, Plasmodium malariae and Plasmodium vivax) use a three-band P. falciparum/Pan test such as the SD Bioline Malaria Ag P.f/Pan 05FK60 (Standard Diagnostics, Kyonggi, Republic of Korea), hereafter referred to as SD 05FK60, as used by the MSF-Holland clinics in Rakhine state, Myanmar. In spite of published reports of generally good test performance, medical and paramedical staff on the ground often doubt the diagnostic accuracy of these RDTs.MethodsParallel testing with malaria microscopy and RDT was conducted at two clinics in Rakhine state, Myanmar, for a period of 14 months as a programmatic response due to doubts and concerns of medical and paramedical staff into malaria RDTs.ResultsA total of 2,585 blood samples from non-pregnant suspected malaria patients were examined by the SD 05FK60 RDT and microscopy at two clinics in Myanmar from October 2010 to December 2011. The reference standard microscopy diagnosed 531 P. falciparum and 587 P. vivax or P. malariae mono-infections. The overall sensitivity for P. falciparum detection by the SD 05FK60 was 90.2% (95% CI: 87.4-92.6) and for P. vivax/P. malariae 79.4% (95% CI: 75.9-82.6). The overall specificity for P. falciparum detection by the SD 05FK60 was 98.5% (95% CI: 97.7-99.1) and for P. vivax/P. malariae 98.7% (95% CI: 97.9-99.2). The sensitivity for P. falciparum was >91% for parasitaemia levels of >100-1,000 parasites/μl and increased for P. vivax/P. malariae with the parasitaemia level but was overall lower than for P. falciparum 25/408 and 13/420 cases, respectively, of P. falciparum and non-falciparum malaria were missed by the RDT.ConclusionIn field conditions in Myanmar, the SD 05FK60 malaria RDT performed consistent with other reports. The test detected malaria caused by P. vivax/P. malariae to a lesser extent than P. falciparum infection. Sensitivity improved with increasing parasitaemia level, however even at higher levels some infections were missed. The SD 05FK60 is adequate for use in settings where high quality microscopy is not available.

Comparative evaluation of microscopy, OptiMAL® and 18S rRNA gene based multiplex PCR for detection of Plasmodium falciparum &Plasmodium vivax from field isolates of Bikaner, India

ObjectiveTo evaluate microscopy, OptiMAL® and multiplex PCR for the identification of Plasmodium falciparumm (P. falciparum) and Plasmodium vivax (P. vivax) from the field isolates of Bikaner, Rajasthan (Northwest India). MethodsIn this study, a multiplex PCR (P. falciparum and P. vivax) was further developed with the incorporation of Plasmodium malariae (P. malariae) specific primer and also a positive control. The performance of microscopy, plasmodium lactate dehydrogenase (pLDH) based malaria rapid diagnostic test OptiMAL® and 18S rRNA gene based multiplex PCR for the diagnosis of P. falciparum and P. vivax was compared. ResultsThe three species multiplex PCR (P. falciparum, P. vivax and P. malariae) with an inbuilt positive control was developed and evaluated. In comparison with multiplex PCR, which showed the sensitivity and specificity of 99.36% (95%CI, 98.11%–100.00%) and 100.00% (95%CI, 100.00%–100.00%), the sensitivity and specificity of microscopy was 90.44% (95%CI, 88.84%–95.04%) and 99.22% (95%CI, 97.71%–100.00%), and OptiMAL® was 93.58% (95%CI, 89.75%–97.42%) and 97.69% (95%CI, 95.10%–100.00%). The efficiencies were 99.65%, 95.10% and 95.45% for multiplex PCR, microscopy and OptiMAL®, respectively. ConclusionsOur results raise concerns over the overall sensitivities of microscopy and OptiMAL®, when compared to the multiplex PCR and thus stress the need for new molecular interventions in the accurate detection of the malarial parasites. This further highlights the fact that further developments are needed to improve the performance of rapid diagnostic tests at field level.

Rapid diagnostic tests for molecular surveillance of Plasmodium falciparum malaria -assessment of DNA extraction methods and field applicability

BackgroundThe need for new malaria surveillance tools and strategies is critical, given improved global malaria control and regional elimination efforts. High quality Plasmodium falciparum DNA can reliably be extracted from malaria rapid diagnostic tests (RDTs). Together with highly sensitive molecular assays, wide scale collection of used RDTs may serve as a modern tool for improved malaria case detection and drug resistance surveillance. However, comparative studies of DNA extraction efficiency from RDTs and the field applicability are lacking. The aim of this study was to compare and evaluate different methods of DNA extraction from RDTs and to test the field applicability for the purpose of molecular epidemiological investigations.MethodsDNA was extracted from two RDT devices (Paracheck-Pf® and SD Bioline Malaria Pf/Pan®), seeded in vitro with 10-fold dilutions of cultured 3D7 P. falciparum parasites diluted in malaria negative whole blood. The level of P. falciparum detection was determined for each extraction method and RDT device with multiple nested-PCR and real-time PCR assays. The field applicability was tested on 855 paired RDT (Paracheck-Pf) and filter paper (Whatman® 3MM) blood samples (734 RDT negative and 121 RDT positive samples) collected from febrile patients in Zanzibar 2010. RDT positive samples were genotyped at four key single nucleotide polymorphisms (SNPs) in pfmdr1 and pfcrt as well as for pfmdr1 copy number, all associated with anti-malarial drug resistance.ResultsThe P. falciparum DNA detection limit varied with RDT device and extraction method. Chelex-100 extraction performed best for all extraction matrixes. There was no statistically significant difference in PCR detection rates in DNA extracted from RDTs and filter paper field samples. Similarly there were no significant differences in the PCR success rates and genotyping outcomes for the respective SNPs in the 121 RDT positive samples.ConclusionsThe results support RDTs as a valuable source of parasite DNA and provide evidence for RDT-DNA extraction for improved malaria case detection, molecular drug resistance surveillance, and RDT quality control.

Improved assay to detect Plasmodium falciparum using an uninterrupted, semi-nested PCR and quantitative lateral flow analysis

BackgroundA rapid, non-invasive, and inexpensive point-of-care (POC) diagnostic for malaria followed by therapeutic intervention would improve the ability to control infection in endemic areas.MethodsA semi-nested PCR amplification protocol is described for quantitative detection of Plasmodium falciparum and is compared to a traditional nested PCR. The approach uses primers that target the P. falciparum dihydrofolate reductase gene.ResultsThis study demonstrates that it is possible to perform an uninterrupted, asymmetric, semi-nested PCR assay with reduced assay time to detect P. falciparum without compromising the sensitivity and specificity of the assay using saliva as a testing matrix.ConclusionsThe development of this PCR allows nucleic acid amplification without the need to transfer amplicon from the first PCR step to a second reaction tube with nested primers, thus reducing both the chance of contamination and the time for analysis to < two hours. Analysis of the PCR amplicon yield was adapted to lateral flow detection using the quantitative up-converting phosphor (UCP) reporter technology. This approach provides a basis for migration of the assay to a POC microfluidic format. In addition the assay was successfully evaluated with oral samples. Oral fluid collection provides a simple non-invasive method to collect clinical samples.

Persistent detection of Plasmodium falciparum, P. malariae, P. ovale curtisi and P. ovale wallikeri after ACT treatment of asymptomatic Ghanaian school-children

Two hundred and seventy four asymptomatic Ghanaian school-children aged 5 to 17years were screened for malaria parasites by examination of blood films. One hundred and fifty five microscopically-positive individuals were treated with dihydroartemisinin-piperaquine and followed for 3weeks. Retrospective species-specific PCR of all 274 screened samples identified an additional 60 children with sub-patent parasitaemia, and a substantial proportion of co-infections with Plasmodium malariae, Plasmodium ovale curtisi and Plasmodium ovale wallikeri. One hundred individuals harboured at least one non-falciparum parasite species. Using standard double-read microscopy, the 21-day efficacy of treatment against Plasmodium falciparum was 91.4% among the 117 children seen at all 5 visits. Using nested PCR to test 152 visit 5 blood samples, 22 were found to be parasite-positive. Twenty individuals harboured P. falciparum, four harboured P. ovale spp. and two P. malariae, with four of these 22 isolates being mixed species infections. The persistent detection of low density Plasmodium sp. infections following antimalarial treatment suggests these may be a hitherto unrecognised obstacle to malaria elimination.