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Primers and PCR conditions used for the different molecular tests evaluated
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Honduras is a tropical country with more than 70% of its population living at risk of being infected with either Plasmodium vivax or Plasmodium falciparum. Laboratory diagnosis is a very important factor for adequate treatment and management of malaria. In Honduras, malaria is diagnosed by both, microscopy and rapid diagnostic tests and to date, no...
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... multiplex and single-tube species-specific PCRs Various primers used for the different tests are provided in Table 1 and were based on a previous study [15]. Test 1 (primers AL7178/AL7142) and Test 2 (primers AL7140/AL7141) were single tube un-nested PCR tests specific to P. falciparum. ...
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Citations
... and P. malariae than previously reported 19,32,39 , which underlines the need to include them in malaria eradication strategies. This difference can be explained to the using of molecular tools, which are more sensitive than conventional methods, such as microscopy and RDTs used in previous research 40,41 . The challenge in detecting these parasites, which are typically associated with less severity and lower parasite densities, particularly with microscopy, underscores the importance of molecular tools 22 to objectively assess their impact on malaria prevalence. ...
Malaria is a significant public health challenge in Gabon, with high prevalence rates in rural and semi-urban areas. This study investigated Plasmodium infection prevalence among outpatients at a medical laboratory in Franceville, Gabon, in 2020. Data from 500 patients were analyzed, revealing an overall infection rate of 33.2% and the presence of four Plasmodium species: P. falciparum, P. malariae, P. ovale, and possibly P. vivax for the first time in Gabon. Co-infections were common, with P. falciparum and P. ovale spp. being the most prevalent at 23.5%. Asymptomatic infections accounted for 81.3% of cases, while symptomatic infections were 18.7%. P. falciparum was associated with symptomatic cases, while non-falciparum species were linked to asymptomatic infections. The findings suggest Franceville has perennial malaria transmission, highlighting the role of Plasmodium species diversity in disease severity and clinical presentation, including the first report of P. vivax infection in the Gabonese population.
... Until recent times, the distribution pattern of P. ovale was considered to be limited to tropical regions outside Ethiopia (19). However, still, it is not from health facilities treating malaria in Ethiopia, perhaps due to its tertian periodicity, typically low parasitemia and morphological resemblance with P. vivax (20), and maybe due to di culty in the diagnosis of P. ovale by microscopic examination in the study area where this pathogen was previously not known to be present and most microscopists are neither trained to detect nor aware of the presence of other species (21)(22)(23). ...
Background Malaria in children remains a major public health problem in the global, especially in sub-Saharan Africa. WHO reported an annually declining burden of malaria in Ethiopia. Despite these, different parts of the country reported a contradictory result, and malaria remains to be the main cause of morbidity among children. However, Microscopic examination of peripheral blood smear produces reliable results. Therefore, the aim of this study was to compare the performance of microscopy and nested polymerase chain reaction (nPCR) to detect malaria suspected febrile children. Methods A health institution-based cross-sectional study was conducted in northwest Ethiopia. A total of 302 malaria suspected participants were included. The capillary blood sample was collected from each study subject for the confirmation of malaria and a questionnaire was used to collect associated risk factors. Data analysis using SPSS version 23, Chi-square and multivariate analysis were performed for the comparison of categorical variables and assessment of associated risk factors, respectively. 𝑃 values less than 0.05 were taken as statistically significant. R esults: The overall prevalence of malaria was 22.2% and 18.2% by microscopy and nested polymerase chain reaction (nPCR), respectively. In nPCR malaria was found in 19.2% of males and 17.3% of female study subjects while the highest prevalence (25.6%) was observed among the age group ≥ 5 years old. Children in the age group between 1–5 years old were approximately 6 times more likely to be affected by malaria. And children who had no habited of bed net usages at all times were 12 times more likely to be contracted with malaria. Conclusion This study revealed that malaria infection among children seeks emphasis and need to specify the bottlenecks of all universal health coverage and community health awareness. In addition, the study suggested the need for adequate professional training towards clinical management of malaria to support unbiased diagnosis and treatment of childhood malaria.
... Malaria is a serious global health problem causing over 600,000 deaths in 2021 [1]. Correct diagnosis is necessary for prevention, control, and suitable treatment of malaria, and commonly performed by microscopy, rapid diagnostic tests (RDTs), and molecular methods [2]. Success of a molecular method for malaria diagnosis depends critically on the quality of genomic DNA being used for examination, which in turn depends on accuracy of the employed isolation procedure. ...
Dried blood spot (DBS) testing on confetti is one of the most common methods used to collect and extract DNA from field samples for molecular epidemiology of malaria studies. Here, we investigated whether ethanol or other solutions used to clean scissors are a source of sample contamination. DBS were prepared from 3 drops of blood from two Plasmodium falciparum cultures spotted on confetti, and 3 solutions (water, ethanol and DNase) were used to clean scissors between spots during DNA extraction. For each cleaning solution, two blank confetti were used as negative controls. Samples were analyzed by PCR-based genotyping of merozoite surface proteins 1 & 2 (msp-1 and msp-2), and P. falciparum chloroquine resistance transporter (Pfcrt). A total of 15 samples were analyzed. Based on msp-1 and msp-2 amplification, P. falciparum was detected on blank confetti when scissors were washed with ethanol. Based on Pfcrt amplification, DNA of P. falciparum was detected on blank confetti when scissors were cleaned with ethanol or water. No P. falciparum genes were detected on blank confetti when scissors were cleaned with DNase. Scissors cleaned with DNase prevented cross-contamination between samples during processing of dried blood spots, whereas ethanol (which is commonly used) fails to avert cross-contamination.
... Unlike microscopic examination, PCR examination is able to detect parasites down to <5 parasites/µL blood [13]. The better sensitivity of PCR, especially in cases with low parasite density or mixed infection [14][15][16][17][18], may reduce the error in malaria diagnosis when used appropriately. ...
... In our study, microscopic examination was only able to identify five mixed infections, while PCR found 351 mixed infections. The limitation of microscopic examination in detecting mixed infection has been well-documented [15,17,18]. A recent meta-analysis estimated that the overall sensitivity and specificity of microscopic examination against PCR when being used to diagnose malaria is 75.20% and 97.12%, respectively [16]. ...
Microscopic examination is the backbone of malaria diagnosis and treatment evaluation in Indonesia. This test has limited ability to detect malaria at low parasite density. Inversely, nested polymerase chain reaction (PCR) can detect parasites at a density below the microscopic examination’s detection limit. The objective of this study is to compare microscopic and PCR results when being used to identify malaria in suspected patients and patients who underwent dihydroartemisinin–piperaquine (DHP) therapy in the last 3–8 weeks with or without symptoms in Sumba Barat Daya, Nusa Tenggara Timur, Indonesia. Recruitment was conducted between April 2019 and February 2020. Blood samples were then taken for microscopic and PCR examinations. Participants (n = 409) were divided into three groups: suspected malaria (42.5%), post-DHP therapy with fever (4.9%), and post-DHP therapy without fever (52.6%). Microscopic examination found five cases of P. falciparum + P. vivax infection, while PCR found 346 cases. All microscopic examinations turned negative in the post-DHP-therapy group. Conversely, PCR result from the same group yielded 29 negative results. Overall, our study showed that microscopic examination and PCR generated different results in detecting Plasmodium species, especially in patients with mixed infection and in patients who recently underwent DHP therapy.
... The samples were stored at room temperature in individual sealed plastic bags with desiccant for up to five months before being transported to Tegucigalpa. The DNA was extracted using the AutoMate™ system and the PrepFiler Express Foren- A nested PCR (nPCR) was performed to amplify a segment of the ribosomal 18S gene using the primers and conditions described by Singh et al. [36,37] with modifications. Briefly, both reactions were performed in 50 µL total volume containing 2X master mix Taq polymerase (Promega Corp. Madison, WI, USA) and 2 µL of each primer 10 µM (Table 1). ...
The diagnosis of malaria in Honduras is based mainly on microscopic observation of the parasite in thick smears or the detection of parasite antigens through rapid diagnostic tests when microscopy is not available. The specific treatment of the disease depends exclusively on the positive result of one of these tests. Given the low sensitivity of conventional methods, new diagnostic approaches are needed. This study evaluates the in-field performance of a device (Gazelle™) based on the detection of hemozoin. This was a double-blind study evaluating symptomatic individuals with suspected malaria in the department of Gracias a Dios, Honduras, using blood samples collected from 2021 to 2022. The diagnostic performance of Gazelle™ was compared with microscopy and nested 18ssr PCR as references. The sensitivity and specificity of Gazelle™ were 59.7% and 98.6%, respectively, while microscopy had a sensitivity of 64.9% and a specificity of 100%. The kappa index between microscopy and Gazelle™ was 0.9216 using microscopy as a reference. Both methods show similar effectiveness and predictive values. No statistical differences were observed between the results of the Gazelle™ compared to light microscopy (p = 0.6831). The turnaround time was shorter for Gazelle™ than for microscopy, but the cost per sample was slightly higher for Gazelle™. Gazelle™ showed more false-negative cases when infections were caused by Plasmodium falciparum compared to P. vivax. Conclusions: The sensitivity and specificity of Gazelle™ are comparable to microscopy. The simplicity and ease of use of the Gazelle™, the ability to run on batteries, and the immediacy of its results make it a valuable tool for malaria detection in the field. However, further development is required to differentiate Plasmodium species, especially in those regions requiring differentiated treatment.
... than the detection P. falciparum. Nucleic acid tests developed for malaria diagnosis have repeatedly shown better performance and accuracy in detecting malaria parasites [3]. ...
... falciparum/malariae, P. falciparum/ovale) than microscopy (n=42) or 18S-rRNA-qPCR (n=39). Many methods are molecular based methods have been developed to detect mixed infections [3] but the present study shows that the non-18S-rRNA-qPCR is more efficient technique. Furthermore, the 18S-rRNA target gene has been pointed in previous studies that it is not fully satisfactory because of its ability to cross-hybridize with human DNA for P. malariae [35]. ...
Background: Microscopy is the gold standard for Malaria diagnosis with shortcomings such as false positives, false negatives,errors in species identification,and errors in enumeration of parasites.Quantitative real-time PCR (qPCR) has improved submicroscopic malaria diagnosis. This study evaluated the performance, concordance, correlation and methods agreement of two monoplex qPCR assays against expert malaria microscopy for the detection and enumeration of malaria parasites. Methods: This was a cross sectional study utilizing 127 archived blood samples collected from five provinces in Kenya. Malaria microscopy was conducted by two independent microscopists then 18S-rRNA-qPCR and non-18S-rRNA-qPCR assays were done to identify and quantify the infecting species.The sensitivity,specificity,and predictive values.Cohen Kappa value was used to quantify the method agreement and Bland Altman test was used to assess the bias and limits of agreement.Correlation between microscopy and qPCR parasite densities was determined by the Spearman's rank test. Statistical significance was taken at p<0.05. Results: A higher sensitivity and a lower specificity were observed in all the three plasmodium species in non 18SrRNA-qPCR compared to 18S-rRNA-qPCR. The sensitivity and specificity of 18S-rRNA-qPCR was 91.3% and 75% in detection of P.falciparum,67.6% and 88.1% in detection of P.malariae,and 55.8% and 91.4% in detection of P.ovale.The sensitivity and specificity of non 18S-rRNA-qPCR was 99.1% and 66.7% in detection of P.falciparum,77.9% and 88.1% in detection of P. malariae, and 79.4% and 90.3% in detection of P. ovale. All the positive and negative predictive values were above 70% except the negative predictive value for 18S-rRNA-qPCR (47.4%).Kappa of more than 0.5 was observed between microscopy and both18S-rRNA-qPCR and non-18S-rRNA-qPCR in the detection of all three malaria parasites. The non-18S-rRNA-qPCR method had higher kappa > 0.65,in all the three species compared to 18S-rRNA-qPCR method (kappa < 0.55).There was a clear positive correlation between microscopy parasite density and the parasite densities estimated by the 18S-rRNA-qPCR and Non-18S-rRNA-qPCR (P<0.001). Conclusion: The results showed that both monoplex realtime PCR methods demonstrated a high performance compared to microscopy proving to be better methods in the identification and speciation of malaria parasites especially of low parasitemia.The realtime PCR methods also had a positive correlation with parasite density and hence can be used in accurate determination of parasite densities when compared to microscopy. Therefore, this study recommends the utilization of realtime PCR methods in the detection,speciation and quantification of both microscopic and submicroscopic malaria parasites.
... was performed in samples from 30 non-human primates nine samples (30%) of them were found to be positive for Plasmodium (11) . Different PCR forms have been reported including: two single-PCR reactions which are nested 1PCR and nested 2PCR designed to detect Plasmodium falciparum infections, one single PCR to detect Plasmodium vivax infections, and one multiplex one-step PCR reaction to detect both parasite species (12) . ...
Background: Malaria remains a global leading cause of morbidity and mortality. The absence of effectivevaccines is still the vital hindrance to the management and elimination of malaria. From that standpoint,accurate laboratory diagnosis could be the right hand for disease management. Objective: This reviewintended to assess the recent perspectives and upcoming directions in molecular diagnosis of malaria.Methods: This review was conducted by using internet searching tools where 35 published papers wereretrieved from the credible online publishers and among them, 27 papers that satisfy the inclusion criteriawere profoundly reviewed.Results: Among the 27 articles, 22(81.48 %) papers focus on contribution of PCR based-method in malariadiagnosis, 4(14.8%) report on comparison between polymerase chain reaction(PCR) and other moleculartechniques, 7(30%) emphasize on advantages and disadvantages of PCR, 4(14.8%) represent relationshipamong PCR and LAMP, 3(11%) discuss the most promising molecular diagnostic tools, 5(18%) focus oncomparative designs of different PCR methods, whereas 1(3.7%) emphasizes on parasite density and 2(7%)on pigment containing monocyte.Conclusion: This review conclude that microscopy remains the gold standard method for malaria diagnosisand speciation in limited resource settings but also molecular based-methods provide significant alternativeswith superior sensitivity and specificity.
... Detection of malaria parasites was based on amplification of the 18s rRNA gene of the genus Plasmodium spp. through a nested PCR approach as described in previous studies [27]. Briefly, the first PCR was carried out in a volume of 25 µL, with 12.5 µL of Taq Master Mix 2X, 1 µL of primers rPLU1 and rPLU5 (10 µM), 5 µL of DNA, and nuclease-free water. ...
Malaria remains a life-threatening disease in many tropical countries. Honduras has successfully reduced malaria transmission as different control methods have been applied, focusing mainly on indoor mosquitoes. The selective pressure exerted by the use of insecticides inside the households could modify the feeding behavior of the mosquitoes, forcing them to search for available animal hosts outside the houses. These animal hosts in the peridomicile could consequently become an important factor in maintaining vector populations in endemic areas. Herein, we investigated the blood meal sources and Plasmodium spp. infection on anophelines collected outdoors in endemic areas of Honduras. Individual PCR reactions with species-specific primers were used to detect five feeding sources on 181 visibly engorged mosquitoes. In addition, a subset of these mosquitoes was chosen for pathogen analysis by a nested PCR approach. Most mosquitoes fed on multiple hosts (2 to 4), and 24.9% of mosquitoes had fed on a single host, animal or human. Chicken and bovine were the most frequent blood meal sources (29.5% and 27.5%, respectively). The average human blood index (HBI) was 22.1%. None of the mosquitoes were found to be infected with Plasmodium spp. Our results show the opportunistic and zoophilic behavior of Anopheles mosquitoes in Honduras.
... Detection of malaria parasites was based on amplification of the 18s rRNA gene of the genus Plasmodium spp. through a nested PCR approach as described in previous studies [26]. Briefly, the first PCR was carried out in a volume of 25 µl with 12.5 µl of Taq Master Mix 2X, 1 µl of primers rPLU1 and rPLU5 (10 µM), 5 µl of DNA, and nuclease-free water. ...
Malaria remains a life-threatening disease in many tropical countries. Honduras has successfully reduced malaria transmission as different control methods have been applied focusing mainly on indoor mosquitoes. The selective pressure exerted by the use of insecticides inside the households could modify the feeding behavior of the mosquitoes forcing them to search for available animal hosts outside the houses. These animal hosts in the peridomicile could consequently become an important factor in maintaining vector populations in endemic areas. Herein, we investigated the blood meal sources and Plasmodium spp. infection on anophelines collected outdoors in endemic areas of Honduras. Individual PCR reactions with species-specific primers were used to detect five feeding sources on 181 visibly engorged mosquitoes. In addition, a subset of these mosquitoes where chosen for pathogen analysis by a nested PCR approach. Most mosquitoes fed on multiple hosts (2 to 4), and 24.9% of mosquitoes were fed on a single host, animal or human. Chicken and bovine were the most frequent blood meal sources (29.5% and 27.5% respectively). The average human blood index (HBI) was 22.1%. None of the mosquitoes was found to be infected with Plasmodium spp. Our results show the opportunistic and zoophilic behavior of Anopheles mosquitoes in Honduras.
... Reactions were amplified by an initial denaturation at 95°C for 2 min, 35 cycles of 95°C for 30 sec, 54ºC for 30 sec, and 72°C for 45 sec, with a final extension at 72°C for 5 min. Amplicons were visualized by 1.5% agarose gel electrophoresis with ethidium bromide (14). Positive and negative controls were included in all PCR reactions. ...
... vivax and P. falciparum). This method has demonstrated a sensitivity comparable to the classical nested PCR targeting ribosomal sequences 18S, under conditions of moderate and high parasitaemia (14). However, it is not possible to affirm that the current approach is able to detect very low parasitaemias that are typical of some asymptomatic infections, and this would be the most important limitation of our study. ...
Background: According to the World Health Organization, 219 million cases of malaria were reported in 2017 worldwide. During the last 8 years, the region of the Americas has experienced an average decrease in the incidence of malaria. Honduras has reported a reduction of 96.3% in the incidence of malaria between 2000 and 2017. Detection of submicroscopic infections is a great challenge for countries with low-endemic settings due to their relevance in the transmission of the parasite to the mosquito. Pregnant women are one of the populations most vulnerable to the complications of malaria and asymptomatic infections are considered as potential reservoirs of infection. The present study estimated the presence of Plasmodium asymptomatic infections in pregnant women and their newborns in an area of low endemicity in Honduras. Methods: Blood samples were collected from 300 asymptomatic mothers, from the umbilical cord of their newborns, and placentas. The DNA was extracted from dried blood spots using the Whatman FTA® purification reagent and the molecular diagnosis of the parasite was performed through two un-nested single tube species-specific PCR tests. Results: Nine hundred DNA samples successfully amplified the human beta globin partial sequence. None of the samples analysed revealed the presence of the parasite through this methodological approach. Conclusion: No asymptomatic malaria infections were detected among 300 pregnant women and their children in an area of low endemicity of Honduras. Implementation of more sensitive diagnostic techniques will contribute significantly on preventing transmission in order to eliminate malaria in the Central American sub region.
