An innovative tool for moving malaria PCR detection of parasite reservoir into the field.
ABSTRACT: To achieve the goal of malaria elimination in low transmission areas such as in Cambodia, new, inexpensive, high-throughput diagnostic tools for identifying very low parasite densities in asymptomatic carriers are required. This will enable a switch from passive to active malaria case detection in the field.DNA extraction and real-time PCR assays were implemented in an "in-house" designed mobile laboratory allowing implementation of a robust, sensitive and rapid malaria diagnostic strategy in the field. This tool was employed in a survey organized in the context of the MalaResT project (NCT01663831).The real-time PCR screening and species identification assays were performed in the mobile laboratory between October and November 2012, in Rattanakiri Province, to screen approximately 5,000 individuals in less than four weeks and treat parasite carriers within 24-48 hours after sample collection. An average of 240 clinical samples (and 40 quality control samples) was tested every day, six/seven days per week. Some 97.7% of the results were available <24 hours after the collection. A total of 4.9% were positive for malaria. Plasmodium vivax was present in 61.1% of the positive samples, Plasmodium falciparum in 45.9%, Plasmodium malariae in 7.0% and Plasmodium ovale in 2.0%.The operational success of this diagnostic set-up proved that molecular testing and subsequent treatment is logistically achievable in field settings. This will allow the detection of clusters of asymptomatic carriers and to provide useful epidemiological information. Fast results will be of great help for staff in the field to track and treat asymptomatic parasitaemic cases. The concept of the mobile laboratory could be extended to other countries for the molecular detection of malaria or other pathogens, or to culture vivax parasites, which does not support long-time delay between sample collection and culture.
Project description:Although the malaria burden in the Lao PDR has gradually decreased, the elimination of malaria by 2030 presents many challenges. Microscopy and malaria rapid diagnostic tests (RDTs) are used to diagnose malaria in the Lao PDR; however, some studies have reported the prevalence of sub-microscopic Plasmodium infections or asymptomatic Plasmodium carriers in endemic areas. Thus, highly sensitive detection methods are needed to understand the precise malaria situation in these areas.A cross-sectional malaria field survey was conducted in 3 highly endemic malaria districts (Xaysetha, Sanamxay, Phouvong) in Attapeu province, Lao PDR in 2015, to investigate the precise malaria endemicity in the area; 719 volunteers from these villages participated in the survey. Microscopy, RDTs and a real-time nested PCR were used to detect Plasmodium infections and their results were compared. A questionnaire survey of all participants was also conducted to estimate risk factors of Plasmodium infection. Numbers of infections detected by the three methods were microscopy: P. falciparum (n = 1), P. vivax (n = 2); RDTs: P. falciparum (n = 2), P. vivax (n = 3); PCR: Plasmodium (n = 47; P. falciparum [n = 4], P. vivax [n = 41], mixed infection [n = 2]; 6.5%, 47/719). Using PCR as a reference, the sensitivity and specificity of microscopy were 33.3% and 100.0%, respectively, for detecting P. falciparum infection, and 7.0% and 100.0%, for detecting P. vivax infection. Among the 47 participants with parasitemia, only one had a fever (?37.5°C) and 31 (66.0%) were adult males. Risk factors of Plasmodium infection were males and soldiers, whereas a risk factor of asymptomatic Plasmodium infection was a history of ?3 malaria episodes.There were many asymptomatic Plasmodium carriers in the study areas of Attapeu province in 2015. Adult males, probably soldiers, were at high risk for malaria infection. P. vivax, the dominant species, accounted for 87.2% of the Plasmodium infections among the participants. To achieve malaria elimination in the Lao PDR, highly sensitive diagnostic tests, including PCR-based diagnostic methods should be used, and plans targeting high-risk populations and elimination of P. vivax should be designed and implemented.
Project description:Asymptomatic carriers of Plasmodium parasites hamper malaria control and eradication. Achieving malaria eradication requires ultrasensitive diagnostics for low parasite density infections (<100 parasites per microliter blood) that work in resource-limited settings (RLS). Sensitive point-of-care diagnostics are also lacking for nonfalciparum malaria, which is characterized by lower density infections and may require additional therapy for radical cure. Molecular methods, such as PCR, have high sensitivity and specificity, but remain high-complexity technologies impractical for RLS. Here we describe a CRISPR-based diagnostic for ultrasensitive detection and differentiation of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae, using the nucleic acid detection platform SHERLOCK (specific high-sensitivity enzymatic reporter unlocking). We present a streamlined, field-applicable, diagnostic comprised of a 10-min SHERLOCK parasite rapid extraction protocol, followed by SHERLOCK for 60 min for Plasmodium species-specific detection via fluorescent or lateral flow strip readout. We optimized one-pot, lyophilized, isothermal assays with a simplified sample preparation method independent of nucleic acid extraction, and showed that these assays are capable of detection below two parasites per microliter blood, a limit of detection suggested by the World Health Organization. Our P. falciparum and P. vivax assays exhibited 100% sensitivity and specificity on clinical samples (5 P. falciparum and 10 P. vivax samples). This work establishes a field-applicable diagnostic for ultrasensitive detection of asymptomatic carriers as well as a rapid point-of-care clinical diagnostic for nonfalciparum malaria species and low parasite density P. falciparum infections.
Project description:BACKGROUND:Widespread elimination of malaria requires an ultra-sensitive detection method that can detect low parasitaemia levels seen in asymptomatic carriers who act as reservoirs for further transmission of the disease, but is inexpensive and easy to deploy in the field in low income settings. It was hypothesized that a new method of malaria detection based on infrared spectroscopy, shown in the laboratory to have similar sensitivity to PCR based detection, could prove effective in detecting malaria in a field setting using cheap portable units with data management systems allowing them to be used by users inexpert in spectroscopy. This study was designed to determine whether the methodology developed in the laboratory could be translated to the field to diagnose the presence of Plasmodium in the blood of patients presenting at hospital with symptoms of malaria, as a precursor to trials testing the sensitivity of to detect asymptomatic carriers. METHODS:The field study tested 318 patients presenting with suspected malaria at four regional clinics in Thailand. Two portable infrared spectrometers were employed, operated from a laptop computer or a mobile telephone with in-built software that guided the user through the simple measurement steps. Diagnostic modelling and validation testing using linear and machine learning approaches was performed against the gold standard qPCR. Sample spectra from 318 patients were used for building calibration models (112 positive and 110 negative samples according to PCR testing) and independent validation testing (39 positive and 57 negatives samples by PCR). RESULTS:The machine learning classification (support vector machines; SVM) performed with 92% sensitivity (3 false negatives) and 97% specificity (2 false positives). The Area Under the Receiver Operation Curve (AUROC) for the SVM classification was 0.98. These results may be better than as stated as one of the spectroscopy false positives was infected by a Plasmodium species other than Plasmodium falciparum or Plasmodium vivax, not detected by the PCR primers employed. CONCLUSIONS:In conclusion, it was demonstrated that ATR-FTIR spectroscopy could be used as an efficient and reliable malaria diagnostic tool and has the potential to be developed for use at point of care under tropical field conditions with spectra able to be analysed via a Cloud-based system, and the diagnostic results returned to the user's mobile telephone or computer. The combination of accessibility to mass screening, high sensitivity and selectivity, low logistics requirements and portability, makes this new approach a potentially outstanding tool in the context of malaria elimination programmes. The next step in the experimental programme now underway is to reduce the sample requirements to fingerprick volumes.
Project description:INTRODUCTION:Plasmodium vivax malaria has a wide geographic distribution and poses challenges to malaria elimination that are likely to be greater than those of P. falciparum. Diagnostic tools for P. vivax infection in non-reference laboratory settings are limited to microscopy and rapid diagnostic tests but these are unreliable at low parasitemia. The development and validation of a high-throughput and sensitive assay for P. vivax is a priority. METHODS:A high-throughput LAMP assay targeting a P. vivax mitochondrial gene and deploying colorimetric detection in a 96-well plate format was developed and evaluated in the laboratory. Diagnostic accuracy was compared against microscopy, antigen detection tests and PCR and validated in samples from malaria patients and community controls in a district hospital setting in Sabah, Malaysia. RESULTS:The high throughput LAMP-P. vivax assay (HtLAMP-Pv) performed with an estimated limit of detection of 1.4 parasites/ ?L. Assay primers demonstrated cross-reactivity with P. knowlesi but not with other Plasmodium spp. Field testing of HtLAMP-Pv was conducted using 149 samples from symptomatic malaria patients (64 P. vivax, 17 P. falciparum, 56 P. knowlesi, 7 P. malariae, 1 mixed P. knowlesi/P. vivax, with 4 excluded). When compared against multiplex PCR, HtLAMP-Pv demonstrated a sensitivity for P. vivax of 95% (95% CI 87-99%); 61/64), and specificity of 100% (95% CI 86-100%); 25/25) when P. knowlesi samples were excluded. HtLAMP-Pv testing of 112 samples from asymptomatic community controls, 7 of which had submicroscopic P. vivax infections by PCR, showed a sensitivity of 71% (95% CI 29-96%; 5/7) and specificity of 93% (95% CI87-97%; 98/105). CONCLUSION:This novel HtLAMP-P. vivax assay has the potential to be a useful field applicable molecular diagnostic test for P. vivax infection in elimination settings.
Project description:Understanding naturally acquired immune responses to Plasmodium in India is key to improving malaria surveillance and diagnostic tools. Here we describe serological profiling of immune responses at three sites in India by probing protein microarrays consisting of 515 Plasmodium vivax and 500 Plasmodium falciparum proteins with 353 plasma samples. A total of 236 malaria-positive (symptomatic and asymptomatic) plasma samples and 117 malaria-negative samples were collected at three field sites in Raurkela, Nadiad, and Chennai. Indian samples showed significant seroreactivity to 265 P. vivax and 373 P. falciparum antigens, but overall seroreactivity to P. vivax antigens was lower compared to P. falciparum antigens. We identified the most immunogenic antigens of both Plasmodium species that were recognized at all three sites in India, as well as P. falciparum antigens that were associated with asymptomatic malaria. This is the first genome-scale analysis of serological responses to the two major species of malaria parasite in India. The range of immune responses characterized in different endemic settings argues for targeted surveillance approaches tailored to the diverse epidemiology of malaria across the world.
Project description:BACKGROUND:The hypotheses put forward to explain the malaria transmission cycle in extra-Amazonian Brazil, an area of very low malaria incidence, are based on either a zoonotic scenario involving simian malaria, or a scenario in which asymptomatic carriers play an important role. OBJECTIVES:To determine the incidence of asymptomatic infection by detecting Plasmodium spp. DNA and its role in residual malaria transmission in a non-Amazonian region of Brazil. METHODS:Upon the report of the first malaria case in 2010 in the Atlantic Forest region of the state of Espírito Santo, inhabitants within a 2 km radius were invited to participate in a follow-up study. After providing signed informed consent forms, inhabitants filled out a questionnaire and gave blood samples for PCR, and thick and thin smears. Follow-up visits were performed every 3 months over a 21 month period, when new samples were collected and information was updated. RESULTS:Ninety-two individuals were initially included for follow-up. At the first collection, all of them were clearly asymptomatic. One individual was positive for Plasmodium vivax, one for Plasmodium malariae and one for both P. vivax and P. malariae, corresponding to a prevalence of 3.4% (2.3% for each species). During follow-up, four new PCR-positive cases (two for each species) were recorded, corresponding to an incidence of 2.5 infections per 100 person-years or 1.25 infections per 100 person-years for each species. A mathematical transmission model was applied, using a low frequency of human carriers and the vector density in the region, and calculated based on previous studies in the same locality whose results were subjected to a linear regression. This analysis suggests that the transmission chain is unlikely to be based solely on human carriers, regardless of whether they are symptomatic or not. CONCLUSION:The low incidence of cases and the low frequency of asymptomatic malaria carriers investigated make it unlikely that the transmission chain in the region is based solely on human hosts, as cases are isolated one from another by hundreds of kilometers and frequently by long periods of time, reinforcing instead the hypothesis of zoonotic transmission.
Project description:BACKGROUND: The mechanisms by which humans regulate pro- and anti-inflammatory responses on exposure to different malaria parasites remains unclear. Although Plasmodium vivax usually causes a relatively benign disease, this parasite has been suggested to elicit more host inflammation per parasitized red blood cell than P. falciparum. METHODOLOGY/PRINCIPAL FINDINGS: We measured plasma concentrations of seven cytokines and two soluble tumor necrosis factor (TNF)-? receptors, and evaluated clinical and laboratory outcomes, in Brazilians with acute uncomplicated infections with P. vivax (n?=?85), P. falciparum (n?=?30), or both species (n?=?12), and in 45 asymptomatic carriers of low-density P. vivax infection. Symptomatic vivax malaria patients, compared to those infected with P. falciparum or both species, had more intense paroxysms, but they had no clear association with a pro-inflammatory imbalance. To the contrary, these patients had higher levels of the regulatory cytokine interleukin (IL)-10, which correlated positively with parasite density, and elevated IL-10/TNF-?, IL-10/interferon (IFN)-?, IL-10/IL-6 and sTNFRII/TNF-? ratios, compared to falciparum or mixed-species malaria patient groups. Vivax malaria patients had the highest levels of circulating soluble TNF-? receptor sTNFRII. Levels of regulatory cytokines returned to normal values 28 days after P. vivax clearance following chemotherapy. Finally, asymptomatic carriers of low P. vivax parasitemias had substantially lower levels of both inflammatory and regulatory cytokines than did patients with clinical malaria due to either species. CONCLUSIONS: Controlling fast-multiplying P. falciparum blood stages requires a strong inflammatory response to prevent fulminant infections, while reducing inflammation-related tissue damage with early regulatory cytokine responses may be a more cost-effective strategy in infections with the less virulent P. vivax parasite. The early induction of regulatory cytokines may be a critical mechanism protecting vivax malaria patients from severe clinical complications.
Project description:BACKGROUND:Population-based studies conducted in Latin America have shown a high proportion of asymptomatic and submicroscopic malarial infections. Considering efforts aiming at regional elimination, it is important to investigate the role of this asymptomatic reservoir in malaria transmission in peri-urban areas. This study aimed to estimate the prevalence of Plasmodium spp. and gametocyte burden on symptomatic and asymptomatic infections in the Brazilian Amazon. RESULTS:Two cross-sectional household surveys (CS) were conducted including all inhabitants in a peri-urban area of Manaus, western Amazonas State, Brazil. Malaria parasites were detected by light microscopy (LM) and qPCR. Sexual stages of Plasmodium spp. were detected by LM and RT-qPCR. A total of 4083 participants were enrolled during the two surveys. In CS1, the prevalence of Plasmodium vivax infections was 4.3% (86/2010) by qPCR and 1.6% (32/2010) by LM. Fifty percent (43/86) of P. vivax infected individuals (qPCR) carried P. vivax gametocytes. In CS2, 3.4% (70/2073) of participants had qPCR-detectable P. vivax infections, of which 42.9% (30/70) of infections were gametocyte positive. The P. vivax parasite density was associated with gametocyte carriage (P?<?0.001). Sixty-seven percent of P. vivax infected individuals and 53.4% of P. vivax gametocyte carriers were asymptomatic. CONCLUSIONS:This study confirms a substantial proportion of asymptomatic and submicroscopic P. vivax infections in the study area. Most asymptomatic individuals carried gametocytes and presented low asexual parasitemia. This reservoir actively contributes to malaria transmission in the Brazilian Amazon, underscoring a need to implement more efficient control and elimination strategies.
Project description:A substantial proportion of Plasmodium species infections are asymptomatic with densities too low to be detectable with standard diagnostic techniques. The importance of such asymptomatic plasmodium infections in malaria transmission is probably related to their duration and density. To explore the duration of asymptomatic plasmodium infections and changes in parasite densities over time, a cohort of participants who were infected with Plasmodium parasites was observed over a 2-year follow-up period.In this open cohort study, inhabitants of four villages in Vietnam were invited to participate in baseline and subsequent 3-monthly surveys up to 24 months, which included the collection of venous blood samples. Samples were batch-screened using ultra-sensitive (u)PCR (lower limit of detection of 22 parasites per mL). Participants found to be infected by uPCR during any of these surveys were invited to join a prospective cohort and provide monthly blood samples. We estimated the persistence of Plasmodium falciparum and Plasmodium vivax infections and changes in parasite densities over a study period of 24 months.Between Dec 1, 2013, and Jan 8, 2016, 356 villagers participated in between one and 22 surveys. These study participants underwent 4248 uPCR evaluations (11·9 tests per participant). 1874 (32%) of 4248 uPCR tests indicated a plasmodium infection; 679 (36%) of 1874 tests were P falciparum monoinfections, 507 (27%) were P vivax monoinfections, 463 (25%) were co-infections with P falciparum and P vivax, and 225 (12%) were indeterminate species of Plasmodium. The median duration of P falciparum infection was 2 months (IQR 1-3); after accounting for censoring, participants had a 20% chance of having parasitaemia for 4 months or longer. The median duration of P vivax infection was 6 months (3-9), and participants had a 59% chance of having parasitaemia for 4 months or longer. The parasite densities of persistent infections oscillated; following ultralow-density infections, high-density infections developed frequently.Persistent largely asymptomatic P vivax and P falciparum infections are common in this area of low seasonal malaria transmission. Infections with low-density parasitaemias can develop into much higher density infections at a later time, which are likely to sustain malaria endemicity.The Wellcome Trust, Bill & Melinda Gates Foundation.
Project description:Background:Indicators of successful malaria control interventions include a reduction in the prevalence and densities of malaria parasites contained in both symptomatic and asymptomatic infections as well as a reduction in malaria transmission. Individuals harboring malaria parasites in asymptomatic infections serve as reservoirs for malaria transmission. This study determined the prevalence of asymptomatic malaria parasite carriage in afebrile children attending six different schools in two districts, the Cape Coast Metropolitan Assembly (CCMA) and the Komenda Edina Eguafo Abirem (KEEA) of the Central Region of Ghana. Methods:This cross sectional study recruited afebrile children aged between 3 and 15 years old from six randomly selected schools in the Central Region of Ghana. Finger-pricked blood was collected and used to prepare thick and thin blood smears as well as spot a strip of filter paper (Whatman #3). Nested PCR was used to identify Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax in DNA extracted from the filter paper spots. The multiplicity of P. falciparum infection was determined using merozoite surface protein 2 genotyping. Results:Out of the 528 children sampled, PCR identified 27.1% to harbor Plasmodium parasites in asymptomatic infections, whilst microscopy identified malaria parasites in 10.6% of the children. The overall PCR estimated prevalence of P. falciparum and P. malariae was 26.6% and 1.3%, respectively, with no P. ovale or P. vivax identified by PCR or microscopy. The RDT positivity rate ranged from 55.8% in Simiw to 4.5% in Kuful. Children from the Simiw Basic School accounted for 87.5% of all the asymptomatic infections. The multiplicity of P. falciparum infection was predominantly monoclonal and biclonal. Conclusions:The low prevalence of asymptomatic malaria parasite carriage by the children living in the Cape Coast Metropolis suggests that the malaria control interventions in place in CCMA are highly effective and that additional malaria control interventions are required for the KEEA district to reduce the prevalence of asymptomatic malaria parasite carriers. No molecular evidence of P. ovale and P. vivax was identified in the afebrile children sampled from the selected schools.