A miniPCR-Duplex Lateral Flow Dipstick Platform for Rapid and Visual Diagnosis of Lymphatic Filariae Infection

Lymphatic filariasis (LF) is a neglected major tropical disease that is a leading cause of permanent and long-term disability worldwide. Significant progress made by the Global Programme to Eliminate Lymphatic Filariasis (GPELF) has led to a substantial decrease in the levels of infection. In this limitation, DNA detection of lymphatic filariae could be useful due to it capable of detecting low level of the parasites. In the present study, we developed a diagnostic assay that combines a miniPCR with a duplex lateral flow dipstick (DLFD). The PCR primers were designed based on the HhaI and SspI repetitive noncoding DNA sequences of Brugia malayi and Wuchereria bancrofti, respectively. The limits of detection and crossreactivity of the assay were evaluated. In addition, blood samples were provided by Thais living in a brugian filariasis endemic area. The miniPCR-DLFD assay exhibited a detection limit of 2 and 4 mf per milliliter (mL) of blood for B. malayi as well as W. bancrofti, respectively, and crossamplification was not observed with 11 other parasites. The result obtained from the present study was in accordance with the thick blood smear staining for the known cases. Thus, a miniPCR-DLFD is an alternative tool for the diagnosis of LF in point-of-collection settings with a modest cost (~USD 5) per sample.

Meta-analysis of the prevalence of malaria associated with pregnancy in Colombia 2000–2020

Knowledge about malaria associated with pregnancy is scarce in Latin America, and in Colombia, little is known about the magnitude of this infection. A systematic review was conducted to determine the prevalence of malaria associated with pregnancy (MAP) and each of its three forms: gestational (GM), placental (PM), and congenital (CM) tested using thick blood smear (TBS) and PCR. Also to compare the proportion of cases due to Plasmodium falciparum and Plasmodium vivax in Colombia from the year 2000–2020. We searched in Pubmed, Science Direct, EMBASE, EMCare, Cochrane Library, Scielo, Lilacs, Google Scholar, libraries, and repositories of Colombian universities, to obtain data on prevalence of GM, PM and CM with their respective testing method. We performed a meta-analysis with a random-effects model to obtain pooled prevalence of MAP and its three forms categorized by testing methods (TBS and PCR). We used data from 14 studies (out of 258 screened) contributing 7932, 2506 women for GM and PM respectively, also data on 1143 umbilical cord blood samples, and 899 peripheral blood of neonates. We found prevalence by TBS as, MAP 4.5% (95%CI = 2.9–6.9), GM 5.8% (95%CI = 3.8–8.7), PM 3.4% (95%CI = 1.7–6.7) and CM 1.3% (95%CI = 0.6–3.0). With PCR the prevalence was, MAP 14.4% (95%CI = 7.6–25.5), GM 16.7% (95%CI = 9.0–28.8), PM 11.0% (95%CI = 4.1–26.3) and CM 16.2% (95%CI = 8.2–29.5). The prevalence of submicroscopic infection was 8.5% (95%CI = 3.4–19.7) in GM, 10.1% (95%CI = 3.5–25.5) in PM and 22.0% (95%CI = 13.2–34.3) in CM. Infections by P. vivax was dominant over P. falciparum when tested with TBS, the PCR test gave similar proportions of P. falciparum and P. vivax. This meta-analysis has demonstrated high prevalence of MAP in Colombia, and highlights the urgent need to increase attention of researchers, research funding institutions, government agencies, and health authorities to study and intervene MAP, that has currently been under investigated.

Malaria parasite detection in thick blood smear microscopic images using modified YOLOV3 and YOLOV4 models

Background Manual microscopic examination of Leishman/Giemsa stained thin and thick blood smear is still the “gold standard” for malaria diagnosis. One of the drawbacks of this method is that its accuracy, consistency, and diagnosis speed depend on microscopists’ diagnostic and technical skills. It is difficult to get highly skilled microscopists in remote areas of developing countries. To alleviate this problem, in this paper, we propose to investigate state-of-the-art one-stage and two-stage object detection algorithms for automated malaria parasite screening from microscopic image of thick blood slides. Results YOLOV3 and YOLOV4 models, which are state-of-the-art object detectors in accuracy and speed, are not optimized for detecting small objects such as malaria parasites in microscopic images. We modify these models by increasing feature scale and adding more detection layers to enhance their capability of detecting small objects without notably decreasing detection speed. We propose one modified YOLOV4 model, called YOLOV4-MOD and two modified models of YOLOV3, which are called YOLOV3-MOD1 and YOLOV3-MOD2. Besides, new anchor box sizes are generated using K-means clustering algorithm to exploit the potential of these models in small object detection. The performance of the modified YOLOV3 and YOLOV4 models were evaluated on a publicly available malaria dataset. These models have achieved state-of-the-art accuracy by exceeding performance of their original versions, Faster R-CNN, and SSD in terms of mean average precision (mAP), recall, precision, F1 score, and average IOU. YOLOV4-MOD has achieved the best detection accuracy among all the other models with a mAP of 96.32%. YOLOV3-MOD2 and YOLOV3-MOD1 have achieved mAP of 96.14% and 95.46%, respectively. Conclusions The experimental results of this study demonstrate that performance of modified YOLOV3 and YOLOV4 models are highly promising for detecting malaria parasites from images captured by a smartphone camera over the microscope eyepiece. The proposed system is suitable for deployment in low-resource setting areas.

A timed tally counter for microscopic examination of thick blood smears in malaria studies

Background Despite many technological advances for malaria parasite detection (e.g. high resolution image acquisition), microscopic reading of thick blood smear (TBS) remains the gold standard. Even though available in low technology environment, the microscopy of TBS is slow and time consuming. Moreover microscopy may induce errors at many levels and has no quality control. Methods A electronic extension of the mechanical tally counter is proposed. In addition to the counting process it includes the process of counting itself that relies on the time elapsed between two successive pressures of the counting button leading to a timed tally counter (TTC). The microscopist performs the reading with the specific instruction starting by counting, in each high power fields, leucocytes first and then parasites. The time-stamp of all pressures of counting buttons are recorded along with the nature of the count. The data are recorded internally in CSV format and are exportable. The detection of HPFs locations and leukocyte/parasite counts per HPFs is performed through a hidden semi-Markov model (with outliers) allowing both to take into account the known distribution of leukocyte per HPFs (using a negative binomial distribution) and the pauses and hesitation of the microscopist during the reading. Parameters are estimated via the expectation-maximization algorithm. Hyper-parameters are calibrated using expert annotations. Forward/backward recursions are used to obtain the HPFs locations. Results This approach provides richer data at no extra cost. It has been demonstrated that the method can derive parasites per HPF, leukocytes per HPF, and parasite/leukocyte ratio with robust non-parametric confidence intervals. Moreover a direct digital data entry leads to a less expensive process and decreased time-consuming and error-prone manual data entry. Lastly the TTC allows detecting possible protocol break during reading and prevents the risk of fraud. Discussion and conclusion Introducing a programmed digital device in the data acquisition of TBS reading gives the opportunity to develop easily new (possible adaptive) reading protocols that will be easily followed by the reader since they will be embedded directly in the device. With the TTC the reader only has to read HPFs, counting leukocytes first and parasites second, and the counter will beep when the protocol is completed.

Global Threshold Using Otsu and Active Contour for Detection of Malaria Parasites in Thick Blood Smear

Malaria is a disease caused by the plasmodium parasite and has caused many fatalities. In general, identifying malaria parasite infection can be done by visually observing thick and thin blood smears through microscopic devices. Identification of parasites in thick blood preparations has a higher level of difficulty than thin blood preparations. In thick blood preparations, various objects such as artefacts and noise have a structure similar to the structure of parasitic objects. This paper aims to develop a parasite detection method based on image processing in thick blood smears, consisting of two main stages. First is to improve image quality by applying contrast value stretching, converting green channels, and refining each image. Second is to segment the plasmodium parasite using global threshold Otsu and active contour followed by several morphological operations. The proposed method produces a high sensitivity of 98.06% with an average negative false rate of 1.4%. With the sensitivity level obtained, it can be interpreted that most of the parasitic objects have been detected correctly in one blood sample image.

Cost-effectiveness of rapid diagnostic tests, compared to microscopic tests, for the diagnosis and treatment of gestational malaria in Colombia from an institutional perspective

Background Gestational malaria is associated with negative outcomes in maternal and gestational health; timely diagnosis is crucial to avoid complications. However, the limited infrastructure, equipment, test reagents, and trained staff make it difficult to use thick blood smear tests in rural areas, where rapid testing could be a viable alternative. The purpose of this study was to estimate the cost-effectiveness of rapid tests type III (Plasmodium falciparum/Plasmodium spp P.f/pan) versus microscopic tests for the diagnosis and treatment of gestational malaria in Colombia. Methods Cost-effectiveness analyses of gestational malaria diagnosis from an institutional perspective using a decision tree. Standard costing was performed for the identification, measurement and assessment phases, with data from Colombian tariff manuals. The data was collected from Health Situation Analysis, SIVIGILA and meta-analysis. Average and incremental cost-effectiveness ratio were estimated. The uncertainty was assessed through probabilistic sensitivity analysis. Results The cost of rapid diagnostic tests in 3,000 pregnant women with malaria was US$66,936 and 1,182 disability adjusted life years (DALYs) were estimated. The cost using thick blood smear tests was US$50,838 and 1,023 DALYs, for an incremental cost-effectiveness of US$ 101.2. The probabilistic sensitivity analysis of rapid diagnostic tests determined that they are highly cost-effective in 70% of the cases, even below the US$1,200 threshold; also, they showed an incremental net monetary benefit of $150,000 when payer’s willingness is US$1,000. Conclusion The use of rapid diagnostic tests for timely diagnosis and treatment of gestational malaria is a highly cost-effective strategy in Colombia, with uncertainty analyses supporting the robustness of this conclusion and the increased net monetary benefit that the health system would obtain. This strategy may help in preventing the negative effects on maternal health and the neonate at a low cost.

Impacts of Seasonal Malaria Chemoprevention on Malaria Burden among under Five-Year-Old Children in Borno State, Nigeria

Malaria disproportionately affects all ages with a high burden among children below five years. Thus, control measures are deployed including Seasonal Malaria Chemoprevention (SMC). The present study assessed the impacts of SMC on malaria burden among subjects aged 3–59 months in Borno State, Nigeria. Twenty (20) clusters were randomly selected from accessible 16 Local Government Areas (LGAs) of Borno State, Nigeria, and SMC was deployed in 10 of the clusters by administering a full dose of amodiaquine plus sulfadoxine-pyrimethamine at monthly intervals for 4 months consecutively. Three hundred and ninety-nine children were enrolled in the study. A structured questionnaire was used to obtain demographic and malaria-related data. Thick blood smear, thin blood smear, and capillary sample were collected two weeks after the 4th cycle of SMC. The prevalence of malaria and anaemia was determined among the subjects and for the clusters. The proportions of the female (46.4%; 185/399) and male (53.6%; 214/399) subjects were similar (p>0.05) with subjects aged 24–47 months (35.8%; 143/399) accounting for the highest proportion (p<0.05). Malaria prevalence was 10.3% (41/399) and was higher among non-SMC subjects (15.9%; 31/195) than among SMC subjects (4.9%; 10/204) (p<0.05, df = 1, χ2 = 10.8). Malaria prevalence was higher in non-SMC clusters (80.0%; 8/10) than in SMC clusters (30.0%; 3/10) (p<0.05, df = 1, χ2 = 40.5). The mean haematocrit of the 399 subjects was 34.0 ± 5.3% with an anaemia prevalence of 18.1% (72/399). The mean haematocrit was higher among SMC subjects (35.4 ± 5.0% vs. 33.1 ± 4.2%; p<0.05) while anaemia prevalence was higher among non-SMC subjects (21.5% vs. 14.6%; p<0.05, df = 1, χ2 = 2.8). Of the SMC subjects, 4.9% reported adverse drug reactions. SMC is safe and significantly reduced malaria burden among children in Borno State, and thus, the measure could be deployed in the state for effective malaria control.