Worldwide distribution, symptoms and diagnosis of the coinfections between malaria and arboviral diseases: a systematic review.

The coinfection between malaria (ML) and arboviral diseases represents a major global public health problem, particularly in tropical and subtropical countries. Despite its relevance, this topic is still insufficiently discussed in the current literature. Here, we aimed to investigate the worldwide distribution, symptoms, and diagnosis during coinfection between ML and arboviral diseases. We conducted a systematic review following the Preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement and assessed the selection and eligibility criteria, created and diagrammed maps, and analysed major symptoms with 95% confidence intervals (CI) using prevalence ratio and effect size, also performing latent class analysis. A total of 85,485 studies were retrieved, of which 56 were included: 57.14% in Asia, 25% in Africa, 14.30% in South America, and 3.56% in Europe. A total of 746 individuals were reported to be coinfected with Plasmodium and arbovirus. Concurrent ML, Dengue (DEN), Chikungunya (CHIK), and Zika (ZIK) patients are more likely to present headache and skin rash. Regarding diagnosis, 58,253 were made, of which 38,176 were positive (ML and at least one arboviral disease). The magnitude of these pathogens' coexistence points out the pressing need for improvements in public health policies towards diagnosis and prevention of both diseases, especially in endemic areas.

Challenging diagnosis of Plasmodium ovale malaria in a Colombian traveler: the importance of including P. ovale wallikeri in molecular screening.

This study reports a challenging diagnosis of Plasmodium ovale malaria in a Colombian citizen returning from Cameroon. Initial microscopy screenings conducted at two private hospitals yielded conflicting results, with the first showing negative smears and the second diagnosing P. vivax. Subsequent microscopy examinations at two government laboratories identified P. ovale, although the routine species-specific PCR strategy was negative. PCR confirmation was finally obtained when P. ovale wallikeri primers were used. Although P. ovale is not frequently found in Colombia, there is a clear need to include both P. ovale curtisi and P. ovale wallikeri in the molecular diagnostic strategy. Such need stems primarily from their extended latency period, which affects travelers, the increasing number of African migrants, and the importance of accurately mapping the distribution of Plasmodium species in Colombia.

Etiology of Acute Febrile Illnesses in Adults in the Defense Community in French Guiana.

In tropical countries, acute febrile illnesses represent a complex clinical problem for general practitioners. We describe the prevalence of different etiologies of acute febrile illnesses occurring among French service members and their families, excluding children, in general practice in French Guiana. From June 2017 to March 2020, patients with a fever ≥37.8°C with a duration of less than 15 days who sought medical care at the army medical centers in Cayenne and Kourou were prospectively enrolled. Based on clinical presentation, blood, urine, nasopharyngeal, and stool samples were collected for diagnostic testing for viruses, bacteria, and parasites (by direct examination, microscopic examination of blood smears, culture, serology, or polymerase chain reaction), and standardized biological tests were systematically performed. Among 175 patients retained for analysis, fever with nonspecific symptoms was predominant (46.9%), with 10 Plasmodium vivax malaria cases, 8 dengue infections, and 6 cases of Q fever. The second most frequent cause of acute febrile illness was upper respiratory tract infections (32.0%) due to influenza virus (n = 18) or human rhinovirus (n = 10). Among the causes of acute febrile illness in French Guiana, clinicians should first consider arboviruses and malaria, as well as Q fever in cases of elevated C-reactive protein with nonspecific symptoms and influenza in cases of signs and symptoms associated with upper respiratory tract infections. Despite an expanded microbiological search, the etiology of 51.4% of acute febrile illnesses remain unknown. Further investigations will be necessary to identify the etiology of acute febrile illnesses, including new pathogens, in French Guiana.

Detection of Plasmodium spp. in asymptomatic blood donors by the new Brazilian NAT PLUS HIV/HBV/HCV/Malaria Bio-Manguinhos kit.

BACKGROUND: Transfusion-transmitted malaria (TTM) is a public health problem in endemic and nonendemic areas. The Brazilian Ministry of Health (MH) requested the development of a nucleic acid amplification test (NAT) for the detection of Plasmodium spp. in public blood centers to increase blood safety. STUDY DESIGN AND METHODS: The new Brazilian NAT kit named NAT PLUS HIV/HBV/HCV/Malaria Bio-Manguinhos was first implemented in HEMORIO, a public blood center in the city of Rio de Janeiro. Since October 1, 2022, this blood center has been testing all its blood donations for malaria in a pool of six plasma samples to detect Plasmodium spp. by real-time polymerase chain reaction (PCR). RESULTS: Since the implementation of the NAT PLUS platform until February 2023, HEMORIO has successfully received and tested 200,277 donations. The platform detected two asymptomatic donors in the city of Rio de Janeiro, which is a nonendemic region for malaria. Our analyses suggested a malaria from the Amazon region caused by Plasmodium vivax, in the first case, while an autochthonous transmission case by Plasmodium malariae was identified in the rural area of Rio de Janeiro state. DISCUSSION: The NAT PLUS platform detects Plasmodium spp. in plasma samples with sensitivity capable of detecting subpatent infections. This is the first time worldwide that a group developed and implemented molecular diagnosis for Plasmodium spp. to be used by public blood centers to avoid TTM.

Standardization of DNA extraction from paraffinized spleen samples: molecular diagnosis of human malaria.

BACKGROUND: Plasmodium vivax is the main species responsible for human malaria in Brazil, and one of its manifestations is splenic malaria, though there are still challenges in its diagnosis. The present study aimed to standardize Plasmodium sp. DNA extraction from histological slices of spleen and diagnosis using real-time qPCR. METHODS: This study performed a microtomy of a paraffin-embedded spleen as a positive control for P. vivax from a patient who had been previously diagnosed with the parasite. The sample was deparaffinized with xylol and ethanol, then DNA extraction was performed with two commercial kits. qPCR was carried out with the Taqman system for detection of Plasmodium sp. and was made species-specific using PvmtCOX1 gene. From 2015 to 2019, 200 spleen samples were obtained from trauma patients subjected to splenectomy in Manaus, Amazonas. All the samples were tested for cell-free human DNA (cfDNA). RESULTS: The deparaffinization and the Plasmodium vivax DNA extraction method was successfully standardized, and the control sample was positive for P. vivax. Of the 200 samples, all qPCRs were negative, but they were positive for human PCR. CONCLUSION: Paraffinization is practical and efficient for the preservation of samples, but the formation of bonds between proteins and DNA makes extraction difficult. Despite this, in this study, it was possible to standardize a method of DNA extraction for detecting P. vivax.

Performance of the procedure for ultra-rapid extraction and loop-mediated isothermal amplification (PURE-LAMP) method to detect malaria in Haiti.

BACKGROUND: Malaria continues to cause burden in various parts of the world. Haiti, a Caribbean country, is among those aiming to eliminate malaria within a few years. Two surveys were conducted in Haiti during which we aimed to evaluate the performance of the simple and rapid procedure for ultra-rapid extraction-loop-mediated isothermal amplification (PURE-LAMP) method with dried blood spots as an alternative diagnostic method for malaria in the context of low to very low rates of transmission. METHODS: Febrile and afebrile people were recruited from three administrative divisions within Haiti: Nippes, Sud and Grand'Anse, during the summers of 2017 (early August to early September) and 2018 (late July to late August). Their blood samples were tested by microscopy, rapid diagnostic tests (RDT), PURE-LAMP and nested PCR to detect Plasmodium infection. Sensitivity, specificity, positive and negative predictive values and kappa statistics were estimated with the nested PCR results as the gold standard. RESULTS: Among 1074 samples analyzed, a positive rate of 8.3% was calculated based on the nested PCR results. Among febrile participants, the rates in 2017 and 2018 were 14.6% and 1.4%, respectively. Three positives were detected among 172 afebrile participants in 2018 by PURE-LAMP and nested PCR, and all three were from the same locality. There was no afebrile participants recruited in 2017. The PURE-LAMP, RDT and microscopy had respective sensitivities of 100%, 85.4% and 49.4%. All of the testing methods had specificities over 99%. CONCLUSIONS: This study confirmed the high performance of the PURE-LAMP method to detect Plasmodium infection with dried blood spots and recommends its use in targeted mass screening and treatment activities in low endemic areas of malaria.

Seroprevalence of viral and bacterial pathogens among malaria patients in an endemic area of southern Venezuela.

BACKGROUND: Malaria remains a leading public health problem worldwide. Co-infections with other pathogens complicate its diagnosis and may modify the disease's clinical course and management. Similarities in malaria clinical presentation with other infections and overlapping endemicity result in underdiagnosis of co-infections and increased mortality. Thus, the aim of this study was to determine the seroprevalence of viral and bacterial pathogens among diagnosed malaria patients in malaria-endemic areas in Venezuela. METHODS: A cross-sectional study was conducted on malaria patients attending three reference medical centres in Ciudad Bolivar, Venezuela. Clinical evaluation and laboratory tests for dengue virus (DENV), chikungunya virus (CHIKV), viral hepatitis [hepatitis A virus (HAV), hepatitis B virus (HBV), and hepatitis C virus (HCV)], and leptospirosis (LEP) were performed by enzyme-linked immunosorbent assays. Previous exposure to these pathogens was defined by the presence of specific immunoglobulin (Ig) G, and co-infection or recent exposure (CoRE) was determined by the presence of specific IgM alone or IgM + IgG. Data analysis considered descriptive statistics. Parameter distribution was statistically evaluated using Kolmogorov-Smirnov test and the necessary comparison tests. Odds ratio (OR) for complications was determined according to CoRE presence with a 95% confidence interval (CI). RESULTS: A total of 161 malaria patients were studied, 66% infected with Plasmodium vivax, 27% with P. falciparum, and 7.5% harboured P. vivax/P. falciparum mixed infection. Previous exposure to DENV (60%) and CHIKV (25%) was frequent. CoRE was confirmed in 55 of the 161 malaria patients (34%) and were more frequent in P. falciparum (49%) than in P. vivax (29%) and mixed malaria patients (25%) (OR = 2.43, 95% CI: 1.39-4.25, P = 0.018). The most frequent CoRE was DENV (15%), followed by HAV (12%), HBV (6.2%), CHIKV (5.5%), and LEP (3.7%); HCV CoRE was absent. Complicated malaria was significantly more frequent in patients with CoRE (56%) than those without CoRE (36%; OR = 2.31, 95% CI: 1.18-4.92, P = 0.013). CONCLUSIONS: We found high CoRE prevalence in malaria patients as determined by serology in the study region; cases were associated with a worse clinical outcome. Further prospective studies with samples from different infection sites and the use of molecular tools are needed to determine the clinical significance of these findings.

PET-PCR reveals low parasitaemia and submicroscopic malarial infections in Honduran Moskitia.

BACKGROUND: Malaria remains a main parasitic disease of humans. Although the largest number of cases is reported in the African region, there are still endemic foci in the Americas. Central America reported 36,000 malaria cases in 2020, which represents 5.5% of cases in the Americas and 0.015% of cases globally. Most malaria infections in Central America are reported in La Moskitia, shared by Honduras and Nicaragua. In the Honduran Moskitia, less than 800 cases were registered in 2020, considering it an area of low endemicity. In low endemicity settings, the number of submicroscopic and asymptomatic infections tends to increase, leaving many cases undetected and untreated. These reservoirs challenge national malaria elimination programmes. This study aimed to assess the diagnostic performance of Light Microscopy (LM), a nested PCR test and a photoinduced electron transfer polymerase chain reaction (PET-PCR) in a population of febrile patients from La Moskitia. METHODS: A total of 309 febrile participants were recruited using a passive surveillance approach at the Puerto Lempira hospital. Blood samples were analysed by LM, nested PCR, and PET-PCR. Diagnostic performance including sensitivity, specificity, negative and positive predictive values, kappa index, accuracy, and ROC analysis was evaluated. The parasitaemia of the positive samples was quantified by both LM and PET-PCR. RESULTS: The overall prevalence of malaria was 19.1% by LM, 27.8% by nPCR, and 31.1% by PET-PCR. The sensitivity of LM was 67.4% compared to nPCR, and the sensitivity of LM and nPCR was 59.6% and 80.8%, respectively, compared to PET-PCR. LM showed a kappa index of 0.67, with a moderate level of agreement. Forty positive cases by PET-PCR were not detected by LM. CONCLUSIONS: This study demonstrated that LM is unable to detect parasitaemia at low levels and that there is a high degree of submicroscopic infections in the Honduran Moskitia.

Imported malaria definition and minimum data for surveillance.

The mobility of malaria-infected individuals poses challenges to elimination campaigns by way of spreading parasite drug resistance, straining country-to-country collaboration, and making routine data collection difficult, especially in resource-poor settings. Nevertheless, no concerted effort has been made to develop a common framework to define the spatial and temporal components of an imported malaria case and recommend the minimum data needed to identify it. We conducted a scoping review of imported malaria literature from 2010 to 2020 which showed that definitions vary widely, and local capabilities of detecting importation are often restricted in low-income countries. Following this, we propose a common definition for imported malaria and the minimum data required to identify a case, depending on the country's capability of conducting an epidemiological investigation. Lastly, we utilize the proposed definition using data from Brazil to demonstrate both the feasibility and the importance of tracking imported cases. The case of Brazil highlights the capabilities of regular surveillance systems to monitor importation, but also the need to regularly use these data for informing local responses. Supporting countries to use regularly collected data and adopt a common definition is paramount to tackling the importation of malaria cases and achieving elimination goals set forth by the World Health Organization.

Malaria surveillance and case management in remote and indigenous communities of Panama: results from a community-based health worker pilot.

BACKGROUND: Panama is one of eight countries in Mesoamerica that aims to eliminate malaria by 2022. Malaria is concentrated in indigenous and remote regions like Guna Yala, a politically autonomous region where access to health services is limited and cases are predominately detected through intermittent active surveillance. To improve routine access to care, a joint effort was made by Guna Yala authorities and the Ministry of Health to pilot a network of community health workers (CHWs) equipped with rapid diagnostic tests and treatment. The impact of this pilot is described. METHODS: Access to care was measured using the proportion of villages targeted by the effort with active CHWs. Epidemiological impact was evaluated through standard surveillance and case management measures. Tests for differences in proportions or rates were used to compare measures prior to (October 2014-September 2016) and during the pilot (October 2016-September 2018). RESULTS: An active CHW was placed in 39 (95%) of 41 target communities. During the pilot, CHWs detected 61% of all reported cases from the region. Test positivity in the population tested by CHWs (22%) was higher than in those tested through active surveillance, both before (3.8%) and during the pilot (2.9%). From the pre-pilot to the pilot period, annual blood examination rates decreased (9.8 per 100 vs. 8.0 per 100), test positivity increased (4.2% to 8.5%, Χ2 = 126.3, p < 0.001) and reported incidence increased (4.1 cases per 1000 to 6.9 cases per 1000 [Incidence Rate Ratio = 1.83, 95% CI 1.52, 2.21]). The percent of cases tested on the day of symptom onset increased from 8 to 27% and those treated on the day of their test increased from 26 to 84%. CONCLUSIONS: The CHW network allowed for replacement of routine active surveillance with strong passive case detection leading to more targeted and timely testing and treatment. The higher test positivity among those tested by CHWs compared to active surveillance suggests that they detected cases in a high-risk population that had not previously benefited from access to diagnosis and treatment. Surveillance data acquired through this CHW network can be used to better target active case detection to populations at highest risk.

Revisión sistemática de la exactitud diagnóstica de la gota gruesa en comparación con la reacción en cadena de la polimerasa para malaria asociada al embarazo, 2010-2022 / Systematic review of the diagnostic accuracy of thick smear compared to polymerase chain reaction for pregnancy-associated malaria, 2010-2022

RESUMEN Objetivos. Evaluar la exactitud de gota gruesa (GG) frente a la reacción en cadena de la polimerasa (PCR) cuantitativa para la malaria asociada al embarazo (MAE). Materiales y métodos. Se realizó una revisión sistemática de pruebas diagnósticas en nueve bases de datos. Se evaluó la calidad metodológica con QUADAS. Se estimó sensibilidad, especificidad, cociente de probabilidad positivo (CPP) y negativo (CPN), razón de odds diagnóstica (ORD) y área bajo la curva ROC. Se determinó la heterogeneidad con el estadístico Q de Der Simonian-Laird y la incertidumbre con el porcentaje de peso de cada estudio sobre el resultado global. Resultados. Se incluyeron diez estudios con 5691 gestantes, 1415 placentas y 84 neonatos. En los estudios con nPCR (PCR anidada) y qPCR (PCR cuantitativa) como estándar, los resultados de exactitud diagnóstica fueron estadísticamente similares, con sensibilidad muy baja (50 y 54%, respectivamente), alta especificidad (99% en ambos casos), alto CPP y deficiente CPN. Usando nPCR la OR diagnóstica fue 162 (IC95%=66-401) y el área bajo la curva ROC fue 95%, mientras que con qPCR fueron 231 (IC95%=27-1951) y 78%, respectivamente. Conclusiones. Mediante un protocolo exhaustivo se demostró el bajo desarrollo de investigaciones sobre la exactitud diagnóstica de la GG en MAE. Se demostró que la microscopía tiene un desempeño deficiente para el diagnóstico de infecciones asintomáticas o de baja parasitemia, lo que afianza la importancia de implementar otro tipo de técnicas en el seguimiento y control de las infecciones por malaria en las gestantes, con el fin de lograr el control y posible eliminación de la MAE.

ABSTRACT Objective. To evaluate the accuracy of thick smear (TS) versus quantitative polymerase chain reaction (PCR) for pregnancy-associated malaria (PAM). Materials and methods. We carried out a systematic review of diagnostic tests in nine databases. Methodological quality was evaluated with QUADAS. Sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR) and area under the ROC curve were estimated. Heterogeneity was determined with the Der Simonian-Laird Q method and uncertainty with the weighted percentage of each study on the overall result. Results. We included 10 studies with 5691 pregnant women, 1415 placentas and 84 neonates. In the studies with nested PCR (nPCR) and quantitative PCR (qPCR) as the standard, the diagnostic accuracy results were statistically similar, with very low sensitivity (50 and 54%, respectively), high specificity (99% in both cases), high PLR and poor NLR. When nPCR was used, the DOR was 162 (95%CI=66-401) and the area under the ROC curve was 95%, while with qPCR it was 231 (95%CI=27-1951) and 78%, respectively. Conclusions. We demonstrated that research on the diagnostic accuracy of TS in PAM is limited. Microscopy showed poor performance in the diagnosis of asymptomatic or low parasitemia infections, which reinforces the importance of implementing other types of techniques for the follow-up and control of malaria infections in pregnant women, in order to achieve the control and possible elimination of PAM.

Screening of febrile patients with suspected malaria from the Brazilian Amazon for virus infection.

Arthropod-borne viruses (arboviruses) are a significant public health threat, especially in tropical and subtropical regions. More than 150 arboviruses can cause febrile illness following infection in humans. The Brazilian Amazon region has the highest number of arboviruses detected worldwide. In addition to arboviruses, malaria, caused by Plasmodium vivax, is endemic in the Amazon. Patients with malaria and arboviral disease frequently show similar clinical presentation and laboratory findings, making the diagnosis of the cause of the infection challenging. The aim of this study was to evaluate the potential for viral infections in patients with suspected malaria but without Plasmodium infection in the Brazilian Amazon. We recruited 200 subjects with suspected malaria in Manaus, Brazil. First, we tested for arboviruses in serum samples from 124 of the 200 participants using an arbovirus DNA microarray platform, which did not detect any virus. Then, we mixed the serum samples of the other 76 participants in 10 pools and subjected them to next-generation sequencing. Analysis of the sequencing data revealed the presence of only one arbovirus (Zika virus) in one sample pool. This analysis also detected the presence of primate erythroparvovirus 1 and pegivirus C. These results suggest that arboviruses are not the most frequent viral infections in patients with suspected malaria but without Plasmodium infection in the metropolitan region of Manaus. Implementation of specific viral surveillance tests will help in the early detection of viruses with epidemic potential.

Frequency of placental malaria and its associated factors in northwestern Colombia, pooled analysis 2009-2020.

Knowledge about placental malaria (PM) is insufficient in the world, and incipient in Colombia where studies are few and recent. In this country, PM has been reported by Plasmodium vivax, Plasmodium falciparum, and mixed infection. The objective was to determine the frequency of PM and its associated clinical-epidemiological factors in mothers and neonates in northwestern Colombia, 2009-2020. A Retrospective pooled analysis with 602 placentas captured in five investigations. The diagnosis of PM was made with thick blood smear (TBS) and qPCR. The groups with and without PM were compared using the Chi-square test, Mann-Whitney test, and crude and adjusted prevalence ratios in a log-binomial model. The prevalence of PM was 27.7% with 92% (155/167) of submicroscopic cases; 41.3% by P. vivax, 44,3% by P. falciparum, and 14.4% by mixed infections. In the multivariate adjustment, PM was associated with the diagnosis of congenital malaria, low neonatal weight, gestational malaria, maternal anemia, previous malaria during pregnancy, and age between 25-43 years. This research is the investigation with the largest number of subjects for studying PM in Colombia, in the ecoepidemiological zone that produces more cases of malaria per year, finding a high prevalence of submicroscopic PM that caused serious maternal (anemia) and neonatal (congenital malaria and low neonatal weight) effects. The results show limitations in the timely diagnosis and treatment, given that the epidemiological surveillance program in Colombia is based on thick blood smear, which generates a substantial underestimation of the magnitude of PM, with serious effects and clinical risks. It is urgent to demand that the health authorities adopt measures such as prenatal control visits as soon as the pregnancy begins, monthly implementation of TBS, and active search for infected pregnant women in their homes and workplaces.

Evaluation of the effectiveness of the OptiMAL-IT™ rapid test in the follow up of patients diagnosed with malaria in the Peruvian Amazon / Evaluación de la efectividad de la prueba rápida OptiMAL-IT™ para el seguimiento de pacientes con diagnóstico de malaria en la Amazonía peruana

Introduction: In Peru, optical microscopy with the thick smear test continues to be performed for the follow-up of malaria patients. This test is simple but it requires microscopic equipment and suitable staff to perform the reading of the samples. Studies suggest that the rapid OptiMAL-IT™ test is an option for follow-up. Objective: To evaluate the effectiveness of OptiMAL-IT™ as a follow-up test in malaria patients in endemic areas of Perú. Materials and methods: We conducted an observational, analytical cross-sectional study of diagnostic tests performed in patients with malaria. We selected all the patients attending different health facilities in the Peruvian departments of San Martín and Loreto who met the inclusion criteria. Optical microscopy with thick smear and OptiMAL-IT™ was used on days 2, 3, 7, and 14 for Plasmodium vivax and until day 21 of follow-up for Plasmodium falciparum. Percentages of correctly classified samples and predictive values were calculated, and the results were compared between the western jungle and the eastern jungle using Chi2 or Fisher's exact tests. Results: We registered 262 patients from San Martín and 302 from Loreto. The percentage of correctly classified cases and the negative predictive value were higher than 92.0% and 93,0%, respectively, from the third day of follow-up; no statistical differences were found in the results obtained from the western jungle and those from the eastern jungle. Conclusions: The OptiMAL-IT™ test would be effective as a follow-up test in patients diagnosed with malaria in endemic areas of Perú.

Introducción. En Perú, la microscopía óptica con gota gruesa continúa utilizándose en el seguimiento de los pacientes con malaria o paludismo. Esta prueba es sencilla, pero requiere de equipamiento microscópico y personal idóneo que realice la lectura de las muestras. Los estudios sugieren que la prueba rápida OptiMAL-IT™ es una opción para dicho seguimiento. Objetivo. Evaluar la efectividad de OptiMAL-IT™ como test de seguimiento en pacientes con malaria en áreas endémicas del Perú. Materiales y métodos. Se hizo un estudio observacional, transversal y analítico de pruebas diagnósticas en pacientes con malaria. Se seleccionó a todos los pacientes que cumplían con los criterios de inclusión, procedentes de diferentes establecimientos de salud de los departamentos peruanos de San Martín y Loreto. El diagnóstico se hizo mediante microscopía óptica con gota gruesa y la prueba rápida de diagnóstico inmunocromatográfico OptiMAL-IT™ en los días 2, 3, 7 y 14 para Plasmodium vivax y hasta el día 21 de seguimiento para Plasmodium falciparum. Se calculó el porcentaje de los correctamente clasificados y los valores predictivos, y se compararon los resultados de la selva occidental y la selva oriental mediante ji al cuadrado o prueba exacta de Fisher. Resultados. Se registraron 262 pacientes de San Martín y 302 de Loreto. Los porcentajes correctamente clasificados y el valor predictivo negativo fueron superiores a 92,0 y 93,0 %, respectivamente, a partir del tercer día de seguimiento; no se encontraron diferencias estadísticas en los resultados obtenidos en la Amazonía occidental y los de la oriental. Conclusiones. La prueba OptiMAL-IT™ sería efectiva como test de seguimiento en los pacientes con diagnóstico de malaria en áreas endémicas del Perú.

Image features for quality analysis of thick blood smears employed in malaria diagnosis.

BACKGROUND: The World Health Organization (WHO) provides protocols for the diagnosis of malaria. One of them is related to the staining process of blood samples to guarantee the correct parasite visualization. Ensuring the quality of the staining procedure on thick blood smears (TBS) is a difficult task, especially in rural centres, where there are factors that can affect the smear quality (e.g. types of reagents employed, place of sample preparation, among others). This work presents an analysis of an image-based approach to evaluate the coloration quality of the staining process of TBS used for malaria diagnosis. METHODS: According to the WHO, there are different coloration quality descriptors of smears. Among those, the background colour is one of the best indicators of how well the staining process was conducted. An image database with 420 images (corresponding to 42 TBS samples) was created for analysing and testing image-based algorithms to detect the quality of the coloration of TBS. Background segmentation techniques were explored (based on RGB and HSV colour spaces) to separate the background and foreground (leukocytes, platelets, parasites) information. Then, different features (PCA, correlation, Histograms, variance) were explored as image criteria of coloration quality on the extracted background information; and evaluated according to their capability to classify images as with Good or Bad coloration quality from TBS. RESULTS: For background segmentation, a thresholding-based approach in the SV components of the HSV colour space was selected. It provided robustness separating the background information independently of its coloration quality. On the other hand, as image criteria of coloration quality, among the 19 feature vectors explored, the best one corresponds to the 15-bins histogram of the Hue component with classification rates of > 97%. CONCLUSIONS: An analysis of an image-based approach to describe the coloration quality of TBS was presented. It was demonstrated that if a robust background segmentation is conducted, the histogram of the H component from the HSV colour space is the best feature vector to discriminate the coloration quality of the smears. These results are the baseline for automating the estimation of the coloration quality, which has not been studied before, but that can be crucial for automating TBS's analysis for assisting malaria diagnosis process.

An algorithm based on molecular protocols to improve the detection of Plasmodium in autochthonous malarial areas in the Atlantic Forest biome.

Malaria is the most important vector-borne disease in the world and a challenge for control programs. In Brazil, 99% of cases occur in the Amazon region. In the extra-Amazonian region, a non-endemic area, epidemiological surveillance focuses on imported malaria and on autochthonous outbreaks, including cases with mild symptoms and low parasitemia acquired in the Atlantic Forest biome. In this scenario, cases are likely to be underreported, since submicroscopic parasitemias are not detected by thick blood smear, considered the reference test. Molecular tests are more sensitive, detecting asymptomatic individuals and mixed infections. The aim of this study was to propose a more efficient alternative to detect asymptomatic individuals living in areas of low malaria endemicity, as they are reservoirs of Plasmodium that maintain transmission locally. In total, 955 blood samples from residents of 16 municipalities with autochthonous malaria outbreaks in the Sao Paulo State were analyzed; 371 samples were collected in EDTA tubes and 584 in filter paper. All samples were initially screened by a genus-specific qPCR targeting ssrRNA genes (limit of detection of 1 parasite/µL). Then, positive samples were subjected to a nested PCR targeting ssrRNA and dihydrofolate reductase-thymidylate synthase genes (limit of detection of 10 parasites/µL) to determine Plasmodium species. The results showed a statistically significant difference (K = 0.049; p < 0.0001) between microscopy positivity (6.9%) and qPCR (22.9%) for EDTA-blood samples. Conversely, for samples collected in filter paper, no statistical difference was observed, with 2.6% positivity by thick blood smear and 3.1% for qPCR (K = 0.036; p = 0.7). Samples positive by qPCR were assayed by a species-specific nested PCR that was in turn positive in 26% of samples (16 P. vivax and 4 P. malariae ). The results showed that molecular protocols applied to blood samples from residents in areas with autochthonous transmission of malaria were useful to detect asymptomatic patients who act as a source of transmission. The results showed that the genus-specific qPCR was useful for screening positives, with the subsequent identification of species by nested PCR. Additional improvements, such as standardization of blood plotting on filter paper and a more sensitive protocol for species determination, are essential. The qPCR-based algorithm for screening positives followed by nested PCR will contribute to more efficient control of malaria transmission, offering faster and more sensitive tools to detect asymptomatic Plasmodium reservoirs.

Evaluación de la efectividad de la prueba rápida OptiMAL-IT™ para el seguimiento de pacientes con diagnóstico de malaria en la Amazonía peruana / Evaluation of the effectiveness of the OptiMAL-IT™ rapid test in the follow up of patients diagnosed with malaria in the Peruvian Amazon

Introducción. En Perú, la microscopía óptica con gota gruesa continúa utilizándose en el seguimiento de los pacientes con malaria o paludismo. Esta prueba es sencilla, pero requiere de equipamiento microscópico y personal idóneo que realice la lectura de las muestras. Los estudios sugieren que la prueba rápida OptiMAL-IT™ es una opción para dicho seguimiento. Objetivo. Evaluar la efectividad de OptiMAL-IT™ como test de seguimiento en pacientes con malaria en áreas endémicas del Perú. Materiales y métodos. Se hizo un estudio observacional, transversal y analítico de pruebas diagnósticas en pacientes con malaria. Se seleccionó a todos los pacientes que cumplían con los criterios de inclusión, procedentes de diferentes establecimientos de salud de los departamentos peruanos de San Martín y Loreto. El diagnóstico se hizo mediante microscopía óptica con gota gruesa y la prueba rápida de diagnóstico inmunocromatográfico OptiMAL-IT™ en los días 2, 3, 7 y 14 para Plasmodium vivax y hasta el día 21 de seguimiento para Plasmodium falciparum. Se calculó el porcentaje de los correctamente clasificados y los valores predictivos, y se compararon los resultados de la selva occidental y la selva oriental mediante ji al cuadrado o prueba exacta de Fisher. Resultados. Se registraron 262 pacientes de San Martín y 302 de Loreto. Los porcentajes correctamente clasificados y el valor predictivo negativo fueron superiores a 92,0 y 93,0 %, respectivamente, a partir del tercer día de seguimiento; no se encontraron diferencias estadísticas en los resultados obtenidos en la Amazonía occidental y los de la oriental. Conclusiones. La prueba OptiMAL-IT™ sería efectiva como test de seguimiento en los pacientes con diagnóstico de malaria en áreas endémicas del Perú.

Introduction: In Peru, optical microscopy with the thick smear test continues to be performed for the follow-up of malaria patients. This test is simple but it requires microscopic equipment and suitable staff to perform the reading of the samples. Studies suggest that the rapid OptiMAL-IT™ test is an option for follow-up. Objective: To evaluate the effectiveness of OptiMAL-IT™ as a follow-up test in malaria patients in endemic areas of Perú. Materials and methods: We conducted an observational, analytical cross-sectional study of diagnostic tests performed in patients with malaria. We selected all the patients attending different health facilities in the Peruvian departments of San Martín and Loreto who met the inclusion criteria. Optical microscopy with thick smear and OptiMAL-IT™ was used on days 2, 3, 7, and 14 for Plasmodium vivax and until day 21 of follow-up for Plasmodium falciparum. Percentages of correctly classified samples and predictive values were calculated, and the results were compared between the western jungle and the eastern jungle using Chi2 or Fisher's exact tests. Results: We registered 262 patients from San Martín and 302 from Loreto. The percentage of correctly classified cases and the negative predictive value were higher than 92.0% and 93,0%, respectively, from the third day of follow-up; no statistical differences were found in the results obtained from the western jungle and those from the eastern jungle. Conclusions: The OptiMAL-IT™ test would be effective as a follow-up test in patients diagnosed with malaria in endemic areas of Perú.

Analysis of malaria clinical-epidemiological predictors in individuals from Brazilian Amazon.

This research aims to determine whether the combination of epidemiological and clinical features can predict malaria. Diagnostic investigation detected 22.3% of individuals with Plasmodium vivax (P. vivax) malaria, with significant predominance of the male gender. The malaria triad (fever, chills and headache) had a more expressive frequency (81.1%) in individuals with positive thick blood than those with negative thick blood smear (65.1%), although there was no statistical significance. Among the variables analysed as predictive for positive thick blood smear, it was observed that personal history of travel to an endemic malaria area and past malaria infection (PMI) were significantly associated with malaria, even in multiple logistic regression. Fever had the higher sensitivity (94.6%) and past malaria history had the greater specificity (68.2%), with accuracy of 23.5% and 67.5%, respectively. In combined analysis, fever with chills had the highest sensitivity (91.9%), but low accuracy (38.5%). High specificity (91.5%) was found in the association of malaria triad, PMI and history of travel to endemic malaria area (which along with anorexia, was higher 94.6%), with good accuracy (80.7%), suggesting that the screening of patients for performing thick blood smear can be based on these data. The epidemiological features and the malaria triad (fever, chills and headache) can be predictors for identification of malaria patients, concurring to precocious diagnosis and immediate treatment of individuals with malaria.

Malaria prevalence in Commune 5 in Tumaco (Nariño, Colombia).

Background Urban malaria is a public health problem in Colombia and there is still lack of knowledge about its epidemiological characteristics, which are key to the implementation of control measures. The presence of urban malaria cases and disease diagnosis are some of the challenges faced by malaria elimination programs. The objective of this research was to estimate malaria prevalence, explore associated factors and detect pfhrp 2/3 genes, in the urban area of Tumaco between July and December 2019. Methods A prevalence study was conducted by using a stratified random probability sample. Structured surveys were administered and blood samples were taken and examined through optical microscopy, rapid diagnostic tests (RDT) and polymerase chain reaction (PCR). A logistic regression model was used to explore associated factors. Results 1,504 people living in 526 households were surveyed. The overall prevalence was 2.97% (95% CI: 2.1 - 4.3%). It was higher in males, in the 10-19 age group and in asymptomatic cases. The prevalence of pfhrp2 amplification was 2.16% (95% CI: 1.6 - 2.9%). Households with three or more people had a higher risk of malaria infection (adjusted odds ratio (ORa) 4.05; 95% confidence interval (CI) 1.57-10.43). All cases were due to P. falciparum. Conclusions The prevalence of urban malaria was low. Strategies to eliminate malaria in urban areas should be adjusted considering access to early diagnosis, asymptomatic infection, and the RDTs used to detect the presence of the pfhrp2 gene.

Human-centered design process and solutions to promote malaria testing and treatment seeking behavior in Guyana hinterlands.

BACKGROUND: Malaria is a persistent public health challenge among miners and other hard-to-reach populations in Guyana's hinterland, specifically in Regions 1, 7, 8, and 9. Despite an overall decrease in malaria prevalence throughout Guyana, it remains common among mining populations whose work conditions both contribute toward malaria transmission and make it difficult to seek timely, Ministry of Health (MoH) approved malaria testing and treatment services. In an effort to develop innovative approaches to address this public health challenge, an interdisciplinary team of public health professionals, designers, and mining organizations collaborated using a human-centered design (HCD) process facilitated by the USAID-funded Breakthrough ACTION Guyana project in partnership with the MoH. METHODS: This paper describes two phases: [1] Define and [2] Design & Test. In the Define phase, following a literature review, we conducted 108 qualitative interviews with miners, camp managers, trained malaria testers, health workers, and other key stakeholders to understand experiences and challenges when seeking malaria testing and treatment services. These interviews were synthesized into 11 insights on issues such as risk perception, malaria knowledge, preventive behaviors, traditional and self-treatment, adherence to the correct treatment, testing, and coordination and communication gaps. From these insights, during the Design & Test phase, we developed 33 "How might we…?" questions which led to 792 ideas, of which eight emergent concepts were prototyped and refined in the field with 145 miners, camp managers, and stakeholders. RESULTS: The five final prototypes included: "Little Mosquito, Big Problem" social behavior change campaign; rapid counseling cards; branded malaria testing and treatment services; innovations in treatment adherence; and a participants, content, and logistics approach. CONCLUSION: When applying HCD to public health issues, there are both opportunities and challenges to reconcile gaps that may exist between the two disciplines. However, HCD provides additional tools and mindsets to generatively work with migrant and mobile mining communities to encourage malaria testing and treatment services.