Low diagnostic performance of thick blood smears of 50 µl in comparison with direct examination of 10 µl blood and the leukoconcentration technique of 5ml blood among loiasis-suspected patients with low microfilaremia in Gabon, Central Africa, using the STARD-BLCM guidelines.

BACKGROUND: The aim of this study was to determine performance indicators of thick blood smears of 50 µl (TBS-50), following the Standards for the Reporting of Diagnostic Accuracy Studies-Bayesian Latent Class Model (STARD-BLCM) guidelines. TBS-50 was compared with two common parasitological techniques-direct examination of 10 µl blood and a leukoconcentration of 5 ml-for the diagnosis of microfilaremic loiasis. METHODS: The study population was recruited among patients of the Department of Parasitology-Mycology-Tropical Medicine over a period of 1 year. Age, sex, symptoms, and eosinophilia variables were recorded from laboratory registers and medical files. Direct examination of 10 µl of blood, TBS-50, and the leukoconcentration technique with 5 ml of blood were performed for each patient. The classical formula and BLCM were used to determine the diagnostic accuracy of the three techniques as well as the prevalence of microfilaremic loiasis. Three models were built within the framework of BLCM-the BLCM model I and alternative models II and III-for sensitivity analysis. RESULTS: In total, 191 patients consented to be included. The direct blood examination and TBS-50 yielded comparable qualitative and quantitative results. Hence, they are reported together. The prevalence of Loa loa microfilaremia was 9.4% (95% CI 5.7-14.5; n = 18/191) with direct blood examination/TBS-50 and 12.6% [8.2-18.1] (n = 24/191) for leukoconcentration. Comparing TBS-50 with the leukoconcentration method using the classical formula, the sensitivity was 75.0% [53.3-90.2], specificity was 100.0% [97.8-100.0], the positive predictive value was 100.0% [81.5-100.0], and the negative predictive value was 96.5% [92.6-98.7]. The prevalence of microfilaremic loiasis was estimated at 9.7% [6.2-13.7] using BLCM model I. The outputs of BLCM model I showed sensitivity of 78.9% [65.3-90.3], specificity of 100.0% [99.3-100.0], a positive predictive value of 99.1% [87.2-100.0], and a negative predictive value of 93.0% [87.3-97.7] for direct blood examination/TBS-50. CONCLUSIONS: TBS-50 demonstrates low sensitivity relative to two other techniques. In one in five cases, the result will be falsely declared negative using these methods. However, this method can be deployed with limited funds.

CLINICAL AND EPIDEMIOLOGICAL FEATURES OF MENINGOCOCCAL INFECTION AND ITS EARLY DIAGNOSIS IN RESIDENTS OF THE TRANSCARPATHIAN REGION.

OBJECTIVE: The aim: To identify clinical and epidemiological features of meningococcal infection on the initial day of a patient's medical consultation, as well as the efficacy of laboratory examinations. PATIENTS AND METHODS: Materials and methods: A retrospective analysis of 76 patients' histories diagnosed with meningococcal disease was carried out. CONCLUSION: Conclusions: Patients in the Transcarpathian region mainly develop an atypical form of meningococcal disease. Only half of all patients diagnosed with meningococcemia had a classical hemorrhagic rash. Generalized forms of meningococcal disease may proceed with normal or subfebrile temperature and without severe leukocytosis. We doubt the use of bacteriological methods of laboratory diagnosis due to their low effectiveness. The most sensitive method of laboratory diagnosis is a microscopic examination of blood smear, and cerebrospinal fluid.

Diagnostic Accuracy of a Thick Blood Smear Compared to qPCR for Malaria Associated with Pregnancy in Colombia.

This study aimed to evaluate the accuracy of the thick blood smear (TBS) versus quantitative polymerase chain reaction (qPCR) for the diagnosis of malaria associated with pregnancy (MAP) caused by P. falciparum or P. vivax in Colombia in its gestational malaria (GM), placental malaria (PM), and congenital malaria (CM) forms as well as to compare its accuracy in different subgroups of pregnant women according to the presence of fever, anemia and a history of malaria. This was a diagnostic evaluation of 829 pregnant women, 579 placentas, 381 umbilical cord samples, and 221 neonatal peripheral blood samples. Accuracy was evaluated based on the parameters of sensitivity, specificity, predictive values, likelihood ratios, and validity index, with their 95% confidence intervals. The frequency of GM was 36% (n = 297/829), PM 27% (n = 159/579), and CM 16.5% (n = 63/381) in umbilical cord samples and 2% (n = 5/221) in neonatal peripheral blood samples. For GM, the sensitivity was 55%, with higher rates in those infected with P. vivax (68%), with a history of malaria (69%), and with fever (96%). These three subgroups presented the best results in terms of the negative likelihood ratio and validity index. For PM, sensitivity was 8%; in subgroup analyses in terms of species, symptomatology (anemia and fever), and history of malaria, it was 1-18%, and the negative likelihood ratio was >0.80 in all subgroups. No false positives were recorded in any of the subgroups. The TBS did not detect any cases of CM. This study found the TBS yielded satisfactory results in terms of diagnosing GM for P. vivax, pregnant women with previous malaria and febrile. It also showed that the TBS is not useful for diagnosing PM and CM. It is necessary to conduct surveillance of MAP with molecular methods in in groups where TBS is deficient (asymptomatic GM, P. falciparum, and pregnant women without history of malaria) to optimize the timely treatment of PM and CM, avoid the deleterious effects of MAP and achieve the malaria elimination goals in Colombia.

PfSPZ-CVac malaria vaccine demonstrates safety among malaria-experienced adults: A randomized, controlled phase 1 trial.

Background: Plasmodium falciparum (Pf) Sporozoite (SPZ) Chemoprophylaxis Vaccine (PfSPZ-CVac) involves concurrently administering infectious PfSPZ and malaria drug, often chloroquine (CQ), to kill liver-emerging parasites. PfSPZ-CVac (CQ) protected 100% of malaria-naïve participants against controlled human malaria infection. We investigated the hypothesis that PfSPZ-CVac (CQ) is safe and efficacious against seasonal, endemic Pf in malaria-exposed adults. Methods: Healthy 18-45 year olds were enrolled in a double-blind, placebo-controlled trial in Bougoula-Hameau, Mali, randomized 1:1 to 2.048 × 105 PfSPZ (PfSPZ Challenge) or normal saline administered by direct venous inoculation at 0, 4, 8 weeks. Syringes were prepared by pharmacy staff using online computer-based enrolment that randomized allocations. Clinical team and participant masking was assured by identical appearance of vaccine and placebo. Participants received chloroquine 600mg before first vaccination, 10 weekly 300mg doses during vaccination, then seven daily doses of artesunate 200mg before 24-week surveillance during the rainy season. Safety outcomes were solicited adverse events (AEs) and related unsolicited AEs within 12 days of injections, and all serious AEs. Pf infection was detected by thick blood smears performed every four weeks and during febrile illness over 48 weeks. Primary vaccine efficacy (VE) endpoint was time to infection at 24 weeks. NCT02996695. Findings: 62 participants were enrolled in April/May 2017. Proportions of participants experiencing at least one solicited systemic AE were similar between treatment arms: 6/31 (19.4%, 95%CI 9.2-36.3) of PfSPZ-CVac recipients versus 7/31 (22.6%, 95%CI 29.2-62.2) of controls (p value = 1.000). Two/31 (6%) in each group reported related, unsolicited AEs. One unrelated death occurred. Of 59 receiving 3 immunizations per protocol, fewer vaccinees (16/29, 55.2%) became infected than controls (22/30, 73.3%). VE was 33.6% by hazard ratio (p = 0.21, 95%CI -27·9, 65·5) and 24.8% by risk ratio (p = 0.10, 95%CI -4·8, 54·3). Antibody responses to PfCSP were poor; 28% of vaccinees sero-converted. Interpretation: PfSPZ-CVac (CQ) was well-tolerated. The tested dosing regimen failed to significantly protect against Pf infection in this very high transmission setting. Funding: U.S. National Institutes of Health, Sanaria. Registration number: ClinicalTrials.gov identifier (NCT number): NCT02996695.

Diagnostic performance and comparison of ultrasensitive and conventional rapid diagnostic test, thick blood smear and quantitative PCR for detection of low-density Plasmodium falciparum infections during a controlled human malaria infection study in Equatorial Guinea.

BACKGROUND: Progress towards malaria elimination has stagnated, partly because infections persisting at low parasite densities comprise a large reservoir contributing to ongoing malaria transmission and are difficult to detect. This study compared the performance of an ultrasensitive rapid diagnostic test (uRDT) designed to detect low density infections to a conventional RDT (cRDT), expert microscopy using Giemsa-stained thick blood smears (TBS), and quantitative polymerase chain reaction (qPCR) during a controlled human malaria infection (CHMI) study conducted in malaria exposed adults (NCT03590340). METHODS: Blood samples were collected from healthy Equatoguineans aged 18-35 years beginning on day 8 after CHMI with 3.2 × 103 cryopreserved, infectious Plasmodium falciparum sporozoites (PfSPZ Challenge, strain NF54) administered by direct venous inoculation. qPCR (18s ribosomal DNA), uRDT (Alere™ Malaria Ag P.f.), cRDT [Carestart Malaria Pf/PAN (PfHRP2/pLDH)], and TBS were performed daily until the volunteer became TBS positive and treatment was administered. qPCR was the reference for the presence of Plasmodium falciparum parasites. RESULTS: 279 samples were collected from 24 participants; 123 were positive by qPCR. TBS detected 24/123 (19.5% sensitivity [95% CI 13.1-27.8%]), uRDT 21/123 (17.1% sensitivity [95% CI 11.1-25.1%]), cRDT 10/123 (8.1% sensitivity [95% CI 4.2-14.8%]); all were 100% specific and did not detect any positive samples not detected by qPCR. TBS and uRDT were more sensitive than cRDT (TBS vs. cRDT p = 0.015; uRDT vs. cRDT p = 0.053), detecting parasitaemias as low as 3.7 parasites/µL (p/µL) (TBS and uRDT) compared to 5.6 p/µL (cRDT) based on TBS density measurements. TBS, uRDT and cRDT did not detect any of the 70/123 samples positive by qPCR below 5.86 p/µL, the qPCR density corresponding to 3.7 p/µL by TBS. The median prepatent periods in days (ranges) were 14.5 (10-20), 18.0 (15-28), 18.0 (15-20) and 18.0 (16-24) for qPCR, TBS, uRDT and cRDT, respectively; qPCR detected parasitaemia significantly earlier (3.5 days) than the other tests. CONCLUSIONS: TBS and uRDT had similar sensitivities, both were more sensitive than cRDT, and neither matched qPCR for detecting low density parasitaemia. uRDT could be considered an alternative to TBS in selected applications, such as CHMI or field diagnosis, where qualitative, dichotomous results for malaria infection might be sufficient.

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 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.

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.

USAGE OF A RAPID DIAGNOSTIC TEST FOR MALARIA IN CHILDREN.

BACKGROUND: Malaria is still the primary cause of pediatric deaths. The efficient management of pediatric malaria requires its rapid and accurate diagnosis. To fulfill this requirement, rapid diagnostic tests have been developed, but their evaluation before commercialization is never exhaustive. The aim of this study was to evaluate the performance of a rapid diagnostic test (SD Bioline Malaria Antigen P.f/Pan) to diagnose malaria in children. MATERIALS AND METHODS: Testing was conducted on children aged between 6 months and 15 years who were examined at the "Centre Mère Enfant (CME) of the "Chantal Biya" Foundation (FCB). as a result of fever. Enrollment took place from April to October 2014. All children presenting with fever were sampled (3ml of blood). These blood samples were tested for malaria using microscopy on a thick blood smear and by a rapid diagnostic test (RDT) SD Bioline Malariae Antigen P.f/Pan. RESULTS: A total of 249 children were enrolled in this study. Malaria presence as determined by microscopy and by RDT was 30.9% and 58.2% respectively. The sensitivity, specificity, positive and negative predictive values compared to microscopy were: 75; 48.8; 39, and 81.6%. With these performances, the malaria SD Bioline rapid test presents lower values compared to WHO recommendations for rapid tests (sensitivity > 95%) in children. CONCLUSION: SD Bioline Malaria Antigen P.f/Pan test should only be used in peripheral health structures that lack resources, and should be aided by clinical diagnosis.

Performance of parallel test in detecting malaria infection for returnees from malaria endemic area / 国际检验医学杂志

Objective To evaluate the performance of parallel test in detecting malaria infection for returned person from malaria endemic area.Methods The blood samples of 484 returnees from malaria endemic area were analyzed and detected by thick blood smear,rapid diagnostic test (RDT) and nest PCR in four companies involving the African labor dispatching.Results The sensitivi ty of thick blood smear and RDT was 0.628 and 0.744 respectively,which of the parallel test was 0.930.On the other hand,the area under the curve (AUC) of parallel test was 0.930 (95％CI:0.895-0.986),which was higher than thick blood smear[0.814 (95％CI:0.724-0.904)]and RDT[0.847 (95％CI:0.769-0.926)].Conclusion Thick blood smear and RDT,which consist of parallel test,could improve the detection sensitivity and accuracy for returnees from malaria epidemical area effectively.This approach is worthy of popularization and application.

Plasmodium malariae in the Colombian Amazon region: you don't diagnose what you don't suspect.

BACKGROUND: Malaria is a worldwide public health problem; parasites from the genus Plasmodium spp. are the aetiological agent of this disease. The parasite is mainly diagnosed by microscope-based techniques. However, these have limited sensitivity. Many asymptomatic infections are sub-microscopic and can only be detected by molecular methods. This study was aimed at comparing nested PCR results to those obtained by microscope for diagnosing malaria and to present epidemiological data regarding malaria in Colombia's Amazon department. METHODS: A total of 1392 blood samples (taken by venepuncture) from symptomatic patients in Colombia's Amazon department were analysed in parallel by thick blood smear (TBS) test and nested PCR for determining Plasmodium spp. infection and identifying infecting species, such as Plasmodium vivax, Plasmodium malariae and/or Plasmodium falciparum. Descriptive statistics were used for comparing the results from both tests regarding detection of the disease, typing infecting species and their prevalence in the study region. Bearing the microscope assay in mind as gold standard, PCR diagnosis performance was evaluated by statistical indicators. CONCLUSION: The present study revealed great differences between both diagnostic tests, as well as suggesting high P. malariae prevalence from a molecular perspective. This differed profoundly from previous studies in this region of Colombia, usually based on the TBS test, suggesting that diagnosis by conventional techniques could lead to underestimating the prevalence of certain Plasmodium spp. having high circulation in this area. The present results highlight the need for modifying state malaria surveillance schemes for more efficient strategies regarding the detection of this disease in endemic areas. The importance of PCR as a back-up test in cases of low parasitaemia or mixed infection is also highlighted.

Detección de casos submicroscópicos de Plasmodium spp, utilizando técnicas clásicas y moleculares en pacientes gestantes de Córdoba, Colombia / Detection of submicroscopic infection with Plasmodium spp, using classical and molecular techniques in pregnant patients from Córdoba, Colombia

Introducción: la malaria gestacional afecta a las madres y al embrión o feto en desarrollo; requiere diagnóstico rápido y tratamiento oportuno y efectivo para evitar las complicaciones y muertes. Objetivo: comparar las técnicas de gota gruesa, PCR anidada y PCR en tiempo real (qRT-PCR), para diagnosticar infecciones submicroscópicas por Plasmodium falciparum y P. vivax. Metodología: se estudiaron 21 mujeres con manifestaciones clínicas de malaria, incluyendo gestantes y no gestantes, en Puerto Libertador, Córdoba, Colombia; de todas se obtuvieron muestras de sangre periférica y, en las gestantes, de placenta y cordón umbilical. Se extrajo el ADN y se lo amplificó por PCR anidada y cuantitativa (qRT-PCR). Para el análisis estadístico se usaron los programas Graphpad PRISM y EPIDAT. Resultados: las tres técnicas diagnosticaron satisfactoriamente la presencia de P. falciparum y P. vivax en sangre periférica, cordón y placenta. Las pruebas moleculares presentaron sensibilidad y especificidad del 100%; dos casos de infección por P. falciparum no identificados por gota gruesa (submicroscópicos) se diagnosticaron con las dos técnicas de PCR. Conclusión: la qRT-PCR es ventajosa en comparación con la PCR anidada porque su estandarización es más corta, requiere menos infraestructura y permite cuantificar el ADN.

Introduction: Gestational malaria affects both the mother and the development of her embryo or fetus. Rapid diagnosis and timely and effective treatment are required to prevent complications and deaths. Objective: To compare thick blood smear with nested PCR and real-time PCR (qRT-PCR) for the diagnosis of submicroscopic infections with Plasmodium falciparum and P. vivax. Methodology: 21 women with clinical manifestations of malaria, including both pregnant and non-pregnant, were studied in Puerto Libertador, Córdoba, Colombia. Peripheral blood specimens were obtained from all of them; umbilical cord and placenta blood specimens were taken in the pregnant ones. DNA was extracted and amplified for nested PCR or qRT-PCR. Statistical analysis was done using Graphpad PRISM and EPIDAT softwares. Results: The three techniques were satisfactory for the detection of Plasmodium falciparum and P. vivax in peripheral blood and in the umbilical cord and placenta specimens. Molecular tests were 100% sensitive and specific. Two submicroscopic cases of P. falciparum infection were detected with the two PCR techniques. Conclusion: qRT-PCR is advantageous over nested PCR because its standardization is shorter, it requires lesser infrastructure and it allows the quantification of DNA.

Síndrome de dificultad respiratoria aguda del adulto por Plasmodium vivax / Adult acute respiratory distress syndrome by Plasmodium vivax

El síndrome de dificultad respiratoria aguda del adulto en pacientes con malaria está asociado a infección por Plasmodium falciparum, ocasionalmente manifestado en pacientes infectados por Plasmodium vivax, por lo que han sido pocos los casos reportados en la literatura (1). Reportamos el caso de un paciente de 43 años quien estuvo en área endémica y desarrolló síndrome de dificultad respiratoria aguda del adulto (SDRA) por Plasmodium vivax. El diagnóstico fue realizado por métodos microscópicos. Concluimos que el SDRA asociado a Plasmodium vivax puededesarrollarse antes de iniciar terapia antimalárica, condición con una alta morbimortalidad. (Acta Med Colomb 2014; 39: 211-215).

The adult acute respiratory distress syndrome in patients with malaria is associated with Plasmodium falciparum infection, and only occasionally manifested in patients infected with Plasmodium vivax, so few cases have been reported in the literature. 1 The case of a 43 year old patient who was in an endemic area and developed acute adult respiratory distress syndrome (ARDS) by Plasmodium vivax is reported. The diagnosis was made by microscopic methods. It was concluded that ARDS associated with Plasmodium vivax can develop before starting antimalarial therapy, a condition with high morbidity and mortality. (Acta Med Colomb 2014; 39: 211-215).