Evaluation of immunophenotypic alterations of peripheral blood lymphocytes and their sub-sets in uncomplicated P. Falciparum infection.

BACKGROUND: Malaria is a life-threatening parasitic disease typically transmitted through the bite of an infected Anopheles mosquito. There is ample evidence showing the potential of malaria infection to affect the counts of lymphocyte subpopulations in the peripheral blood, but the extent of alteration might not be consistent in all geographical locations, due to several local factors. Although Ghana is among the malaria-endemic countries, there is currently no available data on the level of alterations that occur in the counts of lymphocyte subpopulations during P. falciparum malaria infection among adults. AIM: The study was to determine the immunophenotypic alterations in the level of peripheral blood lymphocytes and their subsets in adults with uncomplicated P. falciparum malaria infection and apparently healthy participants. METHODS: The study was a cross-sectional comparative study conducted in two municipalities of the Volta region of Ghana. Blood samples were collected from study participants and taken through serology (P. falciparum/Pan Rapid Diagnostic Kits), microscopy (Thick and thin blood films) and Haematological (Flow cytometric and Full blood count) analysis. RESULTS: A total of 414 participants, comprising 214 patients with malaria and 200 apparently healthy individuals (controls) were recruited into this study. Parasite density of the malaria patients ranged from 75/µL to 84,364/µL, with a mean of 3,520/µL. It was also observed that the total lymphocytes slightly decreased in the P. falciparum-infected individuals (Mean ± SD: 2.08 ± 4.93 × 109/L) compared to the control group (Mean ± SD: 2.47 ± 0.80 × 109/L). Again, there was a significant moderate positive correlation between parasite density and haematocrit levels (r = 0.321, p < 0.001). Apart from CD45 + T-cells, more people in the control group had normal values for the lymphocyte subsets measured compared to the malaria patients. CONCLUSIONS: From the results obtained, there was high parasite density among the malaria patients suggestive of high intensity of infection in the case group. The malaria patients again showed considerable haematological alterations in lymphocyte sub-sets and the parasite density appeared to be strongly associated with CD4 + T-cell reduction. Also, the parasite density significantly associated with decreasing haematocrit levels. This indicates that lymphocyte subset enumeration can be used to effectively support malaria diagnosis.

Host-parasite interactions: a study on the pathogenicity of different Mesanophrys sp. densities and hemocytes-mediated parasitic resistance of swimming crabs (Portunus trituberculatus).

Mesanophrys sp. is a parasitic ciliate that invades and destroys the hemocytes of the swimming crab (Portunus trituberculatus). In the present study, we employed an in vitro model to elucidate how Mesanophrys sp. destroys crab hemocytes. We also evaluated the relationship between the parasite's density, the destruction rate of the hemocytes, and the rapid proliferation pattern of parasites in host crabs. We found that the survival rate and cell integrity of crab hemocytes decreased with an increase in Mesanophrys sp. density, depicting a negative correlation between hemocyte viability and parasite density. Further analyses revealed that crab hemocytes could resist destruction by a low density (10 ind/mL) of Mesanophrys sp. for a long time (60 h). Mesanophrys sp. and its culture medium (containing the ciliate secretions) destroy the host hemocytes. The natural population growth rate of Mesanophrys sp. decreased with an increase in the parasite density, but the Mesanophrys sp. density did not affect the generation time of the parasites. In summary, Mesanophrys sp. can destroy crab hemocytes, and the degree of destruction is directly proportional to the parasite density. The resistance of crab hemocytes to Mesanophrys sp. decreased gradually with an increase in the parasite density.

Performance of ultra-sensitive malaria rapid diagnostic test to detect Plasmodium falciparum infection in pregnant women in Kinshasa, the Democratic Republic of the Congo.

BACKGROUND: Low peripheral parasitaemia caused by sequestration of Plasmodium falciparum in the placenta hampers the diagnosis of malaria in pregnant women, leading to microscopy or conventional rapid diagnostic tests (RDTs) false-negative results. Although mainly asymptomatic, maternal malaria remains harmful to pregnant women and their offspring in endemic settings and must be adequately diagnosed. Ultra-sensitive RDTs (uRDTs) are thought to be more sensitive than RDTs, and their diagnostic performance was assessed in the current study in pregnant women living in Kinshasa, a stable malaria transmission area in the Democratic Republic of the Congo. METHODS: To assess and compare the diagnostic performances of both RDTs and uRDTs, 497 peripheral blood samples were tested using microscopy and quantitative polymerase chain reaction (qPCR) as the index and the reference tests, respectively. The agreement between the different diagnostic tests assessed was estimated by Cohen's Kappa test. RESULTS: The median parasite density by qPCR was 292 p/µL of blood [IQR (49.7-1137)]. Using qPCR as the reference diagnostic test, the sensitivities of microscopy, RDT and uRDT were respectively [55.7% (95% CI 47.6-63.6)], [81.7% (95%CI 74.7-87.3)] and [88% (95% CI 81.9-92.6)]. The specificities of the tests were calculated at 98.5% (95% CI 96.6-99.5), 95.2% (95% CI 92.5-97.2) and 94.4% (95% CI 91.4-96.6) for microscopy, RDT and uRDT, respectively. The agreement between qPCR and uRDT was almost perfect (Kappa = 0.82). For parasite density (qPCR) below 100 p/µL, the sensitivity of RDT was 62% (95% CI 47.1-75.3) compared to 68% (95% CI 53.3-80.4) for uRDT. Between 100 and 200 p/µL, the sensitivity of RDT was higher, but still lower compared to uRDT: 89.4% (95% CI 66.8-98.7) for RDT versus 100% (95% CI 82.3-100) for uRDT. In both cases, microscopy was lower, with 20% (95% CI 10-33.7) and 47.3% (95% CI 24.4-71.1) respectively. CONCLUSIONS: uRDT has the potential to improve malaria management in pregnant women as it has been found to be slightly more sensitive than RDT in the detection of malaria in pregnant women but the difference was not significant. Microscopy has a more limited value for the diagnosis of malaria during the pregnancy, because of its lower sensitivity.

[Influence of white blood cells count on parasite density in Malaria in children aged 6 to 59 months in Benin]. / Influence de la numération leucocytaire sur la densité parasitaire dans le paludisme simple chez les enfants de 6 à 59 mois au Bénin.

Background: For many years, the treatment of malaria was based on clinical presumptive diagnosis, making its differential diagnosis with other causes of hyperthermia difficult. This drug pressure has led to the emergence of Plasmodium strains resistant to the most commonly used antimalarial drugs. This is why in 2004, the health authorities decided to revise the policy of malaria management by adopting a new strategy based on the rational use of artemisininbased combination therapies after the biological confirmation of suspected malaria cases. The biological diagnosis is an essential part of malaria management. The gold standard technique for diagnosis is the thick drop combined with the calculation of parasite density (PD), which is determined on the basis of the number of parasites counted in a microscopic field against a proposed standard number of leukocytes. The number of leukocytes used to calculate the parasite density should ideally be the actual number of leukocytes in the patient per cubic millimetre of blood. However, in the absence of the availability of a blood count at the time of the thick drop, an average number of 8 000 leukocytes/mm3 was used by the World Health Organisation (WHO) to estimate the parasite density. Nonetheless, in Benin the average number of leukocytes adopted by the National Malaria Control Programme (PNLP) is 6 000/mm3. The aim of our study was to determine the impact of the leukocyte count on the calculation of the parasite density in cases of uncomplicated malaria. Method: The study was a cross-sectional study with an analytical aim and took place in 2 hospitals in Benin, the Klouékanmey zone hospital in the south of Benin and the Djougou health centre in the north. It involved a population of 476 children aged between 6 and 59 months who were seen in consultation and in whom the clinical diagnosis of simple Plasmodium falciparum malaria was suspected. Children aged between 6 and 59 months, weighing at least 5 kg, with an axillary temperature ≥ 37.5°C at the time of consultation or a history of fever in the last 24 hours or other symptoms pointing to the diagnosis of malaria were included. Infestation was mono-specific for Plasmodium falciparum. Informed consent was required from the child's parents or guardian. The criteria for non-inclusion in our study were the presence of at least one sign of malaria severity, signs of severe malnutrition or a febrile state related to underlying infectious diseases other than malaria. Thick blood count and haemogram were systematically performed in all included children. Parasite density was calculated according to 3 methods, first using a weighted leukocyte count of 6 000/mm3 recommended by the Benin National Malaria Control Programme (PNLP), then a leukocyte count of 8 000/mm3 recommended by the World Health Organisation and finally the patient's actual leukocyte count obtained from the blood count. It should be noted that these different samples were respectively taken on the day of inclusion in compliance with the conditions of the pre-analytical phase in force in our medical biology laboratory. Results: At the end of our study, 313 children, i.e. 65.76% of our study population had a positive white blood cell count with a positivity rate of 62.14% in Djougou, i.e. 174 children, and 70.9% in Klouékanmey, i.e. 139 children. The average leukocyte count in these children was 11,580/mm3. Among them, 205 children had an abnormal white blood cell count, i.e. 17 cases of leukopenia (5.43%) and 188 cases of hyperleukocytosis (60.06%). Using successively the average number of 6 000 leukocytes/mm3 proposed by the Benin PNLP and that of 8 000 leukocytes/mm3 proposed by the WHO, the average parasite densities were respectively 47,943 and 63,936 trophozoïtes/µl against 92,290 trophozoïtes/µl when the real number of leukocytes of the patients was used for the calculation of the PD. By using an average of 6 000 leukocytes/mm3 for PD calculation, 60% of the calculated PDs were underestimated and 6% were overestimated. Using an average of 8 000 leukocytes/mm3 resulted in 49% of PD being underestimated and 15% being overestimated. The difference between the three calculation methods was considered statistically significant (p value <0.05). Conclusion: The use of 6 000 or 8 000 coefficients for the estimation of parasitaemia could lead to a significant underestimation of the parasite load.

Determinant of parasite clearance and density on uncomplicated falciparum malaria infections in malaria-endemic area of Lampung Province, Indonesia.

Lampung is a malaria-endemic region in Indonesia with an annual parasite incidence of 0.06 per 1,000 population. The socio-demographic factors, clinical conditions, and artemisinin combination therapy (ACT) types might affect parasite clearance and parasite density. This study aims to investigate factors that influence parasite clearance and parasite density in malaria patients. A retrospective analytic observational and a cross-sectional approach was used to conduct this study. A total of 66 malaria patients were examined to investigate parasite density and clearance, socio-demographic profiles, clinical conditions, and ACT types. To analyze data, univariate, bivariate, and multivariate tests were used. Age (P=0.045; r=0.238) and ACT type (P=0.021; r=0.273) were the only variables that had a significant correlation with parasite clearance. Age (P=0.003; r=0.345) had a significant correlation with parasite density. The most influential factors related to parasite clearance were the ACT type (dihydroartemisinin piperaquine) (P=0.017; odds ratio (OR) 0.109; 95.0% confidence interval (CI), 0.018-0.675) and age (P=0.030; OR 0.132; 95.0% CI, 0.021-0.823). Age (P=0.046; OR 0.320; 0.105-0.978, 95.0% CI) was the most significant variable associated with parasite density.

The occurrence of Sarcocystis spp. in the myocardium of alpacas (Vicugna pacos) with associated risk factors in the Peruvian Andes.

Sarcocystis masoni n. sp. (known as "S. lamacanis") infects alpacas affecting their productivity and can cause a food poisoning syndrome in humans by consuming contaminated, undercooked cardiac muscle. There are few studies estimating the prevalence of this parasite in alpacas, although this information is crucial for the control and prevention of sarcocystosis. This study aimed to determine the frequency and density of Sarcocystis masoni n. sp. in the heart of alpacas in Huancavelica, a province of the Andean region of Peru. Heart samples were taken for histopathology from 104 alpacas slaughtered at the municipal slaughterhouse of Huancavelica, the official abattoir in the Huancavelica district. No macroscopic sarcocysts were observed. All alpacas (100%) had microscopic sarcocysts of Sarcocystis masoni n. sp., with no inflammatory reactions. The alpacas showed an average sarcocyst density of 60.8 ± 23.3/mm2. Sarcocysts density was significantly higher (p < 0.05) as the age of the animals increased. In addition, sarcocysts density was significantly higher (p < 0.05) in male animals aged 4 and 5 years compared to females of the same age. These results confirmed that heart sarcocystosis is highly endemic in Peruvian alpacas. Therefore, it is recommended that alpaca hearts be well-cooked at the time of consumption. The present study showed current data and contributes to the knowledge of this parasitosis. Studies of this nature are necessary because they are the basis for developing animal health programs.

Rapid diagnosis of Plasmodium falciparum malaria using a point-of-care loop-mediated isothermal amplification device.

LAMP diagnosis of malaria is simple and cost-effective with acceptable sensitivity and specificity as compared to standard diagnostic modules such as microscopy, RDTs and nested PCR, and thus its deployment for onsite screening of malaria in resource-limited regions is under consideration. However, the requirement of an electricity-operated dry bath and bulky read-out unit is still a major concern. In an effort to simplify this limitation, we have developed a portable LAMP device and fluorescence readout unit which can be used in the rapid point-of-care diagnosis of malaria. We have developed a point-of-care diagnostic LAMP device that is easy to operate by a mobile application, and the results can be quantified with a fluorescent readout unit. The diagnostic performance of the device was evaluated in 90 P. falciparum-infected clinical isolates stored at 4°C for 6-7 years and 10 freshly collected isolates from healthy volunteers. The LOD and quantitative ability of LAMP in estimating parasitemia levels were revealed with laboratory-grown P. falciparum strain (3D7). The LAMP assay performed in our device was exclusive for P. falciparum detection with sensitivity and specificity determined to be 98.89% and 100%, respectively, in clinical isolates. The LOD was documented to be 1 parasite/µl at the cut-off ADC value of 20. Parasite density estimated from ADC values showed concordance with microscopically determined parasite density of the cultured P. falciparum 3D7 strain. The LAMP assay performed in our device provides a possible portable platform for its deployment in the point-of-care diagnosis of malaria. Further validation of the quantitative ability of the assay with freshly collected or properly stored clinical samples of known parasitemia is necessary for field applicability.

Heavy Malaria Parasitaemia in Young Nigerian Infants: Prevalence, Determinants and Implication for the Health System.

BACKGROUND: Infants who are aged six months and below are often protected from malaria and usually present with light parasitaemia when infected. However, complications following heavy malaria parasitaemia in this age group are being increasingly reported. This study set out to determine the prevalence, determinants and the public health implications of heavy malaria parasitaemia in young infants (aged one to six months) at the Wesley Guild Hospital, Ilesa (a unit of the Obafemi Awolowo University Teaching Hospitals Complex). METHODS: Ill infants aged one to six months in out-patient and in-patient care were recruited over an 11-month period. Clinical examinations and blood film for malaria parasite were done for all the study participants. Heavy parasitaemia was defined as > 5000 parasites/µl. Clinical predictors of heavy parasitaemia were determined. RESULTS: Heavy parasitaemia was observed in 16(23.9%) of the sixty-seven participants with malaria infection. Presence of fever at presentation (p=0.007), excessive crying (p=0.003) and pallor (p=0.001) were associated with heavy malaria parasitaemia. However, pallor (OR = 20.653; 95%CI 2.091-203.958; p=0.010) was the only independent predictor of heavy parasitaemia among the young infants. CONCLUSION: About one-in-four ill young infants with malaria had heavy parasitaemia, which was predicted by pallor. Hence, the presence of pallor and factors related to low parental socio-economic status should increase the suspicion of heavy malaria parasitaemia in ill young infants in malaria endemic settings.

CONTEXTE: Nourrissons âgés de six mois et moins sont souvent protégés du paludisme et généralement présents avec de la lumière parasitémie lorsqu'il est infecté. Cependant, les complications qui suivent une parasitémie palustre lourde dans ce groupe d'âge est en cours de plus en plus signalés. Cette étude visait à déterminer la prévalence, les déterminants et les répercussions de l'action sur la santé publique parasitémie palustre sévère chez les jeunes nourrissons (âgés de un à six ans)mois) à l'hôpital Wesley Guild, Ilesa (une unité de l'Obafemi Complexe des hôpitaux universitaires d'Awolowo). MÉTHODES: Nourrissons malades âgés de un à six mois en ambulatoire et les soins aux patients hospitalisés ont été recrutés sur une période de 11 mois. Les examens cliniques et le film sanguin pour le parasite du paludisme ont été fait pour tous les participants à l'étude. La parasitémie lourde était défini comme > 5000 parasites/µl. Prédicteurs cliniques de lourd la parasitémie a été déterminée. RÉSULTATS: Une parasitémie sévère a été observée chez 16 (23,9%) des soixante-sept participants atteints d'une infection palustre. Présence de fièvre à la présentation (p = 0,007), pleurs excessifs (p = 0,003) et la pâleur (p = 0,001) était associée à un paludisme lourdparasitémie. Cependant, pâleur (OR = 20,653; IC à 95 % 2,091-203.958; p=0,010) était le seul prédicteur indépendant de parasitémie chez les jeunes nourrissons. CONCLUSION: Environ un jeune nourrisson malade sur quatre atteint de paludisme avait une parasitémie lourde, qui était prédite par pâleur. D'où la présence de pâleur et de facteurs liés à un faible niveau parental le statut socio-économique devrait accroître la suspicion de lourd parasitémie palustre chez les jeunes nourrissons malades dans le paludisme endémique Paramètres. Mots-clés: Jeunes nourrissons, Parasitémie palustre, Parasite lourddensité, prévalence.

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.

RTS,S vaccination is associated with reduced parasitemia and anemia among children diagnosed with malaria in the outpatient department of a district hospital in rural Malawi.

The RTS,S/AS01 malaria vaccine was recently approved by the World Health Organization, but real-world effectiveness is still being evaluated. We measured hemoglobin concentration and parasite density in vaccinated and unvaccinated children who had been diagnosed with malaria by rapid diagnostic test (mRDT) in the outpatient department of a rural hospital in Malawi. Considering all mRDT positive participants, the mean hemoglobin concentration among unvaccinated participants was 9.58 g/dL. There was improvement to 9.82 g/dL and 10.36 g/dL in the 1 or 2 dose group (p = 0.6) and the 3 or 4 dose group (p = 0.0007), respectively. Among a microscopy positive subset of participants, mean hemoglobin concentration of unvaccinated participants was 9.55 g/dL with improvement to 9.82 g/dL in the 1 or 2 dose group (p = 0.6) and 10.41 g/dL in the 3 or 4 dose group (p = 0.003). Mean parasite density also decreased from 115,154 parasites/µL in unvaccinated children to 87,754 parasites/µL in children who had received at least one dose of RTS,S (p = 0.04). In this study population, vaccination was associated with significant improvements in both hemoglobin concentration and parasite density in the setting of real-world administration of the RTS,S/AS01 vaccine.

Prevalence, Parasite Density and Determinants of Falciparum Malaria Among Febrile Children in Some Peri-Urban Communities in Southwestern Nigeria: A Cross-Sectional Study.

BACKGROUND: Malaria remains a serious public health problem worldwide, particularly in tropical and subtropical regions, including Nigeria. This study investigates the prevalence, parasite density and determinants of malaria among symptomatic children in some peri-urban communities in southwestern Nigeria. METHODS: This was a randomized cross-sectional and hospital-based study. The standard method of microscopy was employed. Thick and thin films were prepared and viewed under a light microscope to identify and quantify malaria parasites. A well-structured and pre-tested questionnaire was used to obtain the subject's information on the demographic, socio-economic and environmental variables. RESULTS: A total of 380 (71.7%) participants were infected with Plasmodium falciparum with a mean parasite density of 1857.11 parasite/µL of blood. Malaria prevalence and mean parasite density were significantly higher among male compared to their female counterparts [80.3% vs 61.4% and 2026.46 vs 1619.63 parasite/µL of blood]. Similarly, age group ≤5 years had the highest malaria prevalence (92.2%) and mean parasite density (2031.66 parasite/µL of blood) than other age groups (AOR 2.281, 95% CI: 1.187-4.384, P < 0.05). The multivariate logistic analysis showed that malaria disease is significantly associated with having mother with no formal education (AOR 12.235, 95% CI: 3.253-46.021, P < 0.05), having well and river as a major source of household water supply (AOR 13.810, 95% CI: 3.012-63.314, P < 0.05 vs AOR 5.639, 95% CI: 1.455-21.853, P < 0.05) and presence of stagnant water around home (AOR 5.22, 95% CI: 2.921-9.332, P < 0.05). Furthermore, protective factors observed include ownership of mosquito bed net (AOR 0.474, 95% CI: 0.223-1.008, P < 0.05) and distance of home to hospital (AOR 0.279, 95% CI: 0.158-0.493, P < 0.05). CONCLUSION: Malaria remains a serious public health problem in the study area. Adopting integrated malaria control measures including educating parents on malaria prevention and control strategies, distributing mosquito bed nets, and establishing larvae source management program is highly imperative.

Impact of asymptomatic Plasmodium falciparum infection on the risk of subsequent symptomatic malaria in a longitudinal cohort in Kenya.

Background: Asymptomatic Plasmodium falciparum infections are common in sub-Saharan Africa, but their effect on subsequent symptomaticity is incompletely understood. Methods: In a 29-month cohort of 268 people in Western Kenya, we investigated the association between asymptomatic P. falciparum and subsequent symptomatic malaria with frailty Cox models. Results: Compared to being uninfected, asymptomatic infections were associated with an increased 1 month likelihood of symptomatic malaria (adjusted hazard ratio [aHR]: 2.61, 95% CI: 2.05 to 3.33), and this association was modified by sex, with females (aHR: 3.71, 95% CI: 2.62 to 5.24) at higher risk for symptomaticity than males (aHR: 1.76, 95% CI: 1.24 to 2.50). This increased symptomatic malaria risk was observed for asymptomatic infections of all densities and in people of all ages. Long-term risk was attenuated but still present in children under age 5 (29-month aHR: 1.38, 95% CI: 1.05 to 1.81). Conclusions: In this high-transmission setting, asymptomatic P. falciparum can be quickly followed by symptoms and may be targeted to reduce the incidence of symptomatic illness. Funding: This work was supported by the National Institute of Allergy and Infectious Diseases (R21AI126024 to WPO, R01AI146849 to WPO and SMT).

Parasitaemia estimation and prediction of hepatocellular dysfunction among Ghanaian children with acute malaria using haemoglobin levels.

Malaria is an important global health disease which puts individuals, particularly children, at a greater risk of mortality. Plasmodium falciparum is distinguished from the rest of the Plasmodia by its high level of parasitaemia. They infect liver cells (hepatocytes), and multiply into merozoites and rupture liver cells in the process, prior to infection of red blood cells. This study sought to estimate the extent to which P. falciparum parasitaemia correlates with hepatocellular dysfunction among Ghanaian children suffering from acute malaria in three malaria endemic districts in Ashanti Region and to predict liver dysfunction from the estimation of haemoglobin (HB) levels. A prospective uncontrolled before- and after study was conducted among under five years children with acute malaria (n = 300) and a control group (n = 20) within the same age brackets. The serum activities of liver enzymes such as aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and gamma glutamyl transferase (GGT) were measured in patients and control subjects. The study observed an inverse relationship between mean HB and parasitaemia (mean HB level of 10.34 ± 0.14 versus parasitaemia <10,000 parasites/µL as against 8.06 ± 0.16 versus parasitaemia ≥10,000 parasites/µL). The mean levels of AST, ALT, ALP and GGT were higher (p < 0.0001) in the serum of the infected children before treatment compared with post treatment. Moreover, the receiver operating characteristics (ROC) curve was applied to establish that HB level at 10.9 g/dL predicted liver dysfunction with the area under the curve (AUC) being 0.75 ± 0.03 (P < 0.0001). The parasitaemia estimation and prediction of hepatocellular dysfunction in Ghanaian children with acute malaria could be done via HB levels.

Genetic diversity and distribution patterns of PfEMP1 in Plasmodium falciparum isolates along the Thai-Myanmar border.

Duffy binding-like domain (DBL) and cysteine-rich interdomain region (CIDR) domain genes of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) encode malaria virulence proteins. The variants of these genes have been reported to be associated with severe/complicated malaria. The present study investigated the prevalence and distribution patterns of DBLα0.6/9, DBLα1.1, DBLα1 not var3 genes, DBLα2/α1.1/2/4/7, DBLß12 & DBLß3/5, DBLε8, CIDRα1.4, and CIDRα1.6 of P. falciparum isolates along the Thai-Myanmar border. The association between PfEMP1 variants and parasite density was also investigated. Two hundred and thirteen finger-prick dried blood spot (DBS) or whole blood samples were collected in 2007 and 2015, from patients with acute uncomplicated P. falciparum in Tak, Kanchanaburi, and Ranong provinces. Analysis of the variant genes was performed using polymerase chain reaction (PCR). The DBLs variant which was found at the highest and lowest frequencies in the three provinces were DBLα1 not var3 (72.77%), and DBLε8 (17.37%). The two CIDR domain variants were found at relatively lower frequencies compared with DBL domain variants (9.9% and 30.1%). P. falciparum isolates carrying the four PfEMP1 variants, i.e., DBLα0.6/9, DBLα1.1, DBLα2/α.1.1/2/4/7, and DBLε8 were found to be significantly associated with low parasitemia. Both DBLα0.6/9 and DBLα2/α1.1/2/4/7 variant genes which were present at high frequencies in this border area could be potential candidate markers for predicting P. falciparum hyperparasitemia and in this border area. Furthermore, the information could be exploited as candidate proteins for the development of an effective malaria vaccine in specific malaria-endemic areas.

Pharmacokinetic profile of amodiaquine and its active metabolite desethylamodiaquine in Ghanaian patients with uncomplicated falciparum malaria.

BACKGROUND: Accurate measurement of anti-malarial drug concentrations in therapeutic efficacy studies is essential to distinguish between inadequate drug exposure and anti-malarial drug resistance, and to inform optimal anti-malarial dosing in key target population groups. METHODS: A sensitive and selective LC-MS/MS method was developed and validated for the simultaneous determination of amodiaquine and its active metabolite, desethylamodiaquine, and used to describe their pharmacokinetic parameters in Ghanaian patients with uncomplicated falciparum malaria treated with the fixed-dose combination, artesunate-amodiaquine. RESULTS: The day-28 genotype-adjusted adequate clinical and parasitological response rate in 308 patients studied was > 97% by both intention-to-treat and per-protocol analysis. After excluding 64 patients with quantifiable amodiaquine concentrations pre-treatment and 17 with too few quantifiable concentrations, the pharmacokinetic analysis included 227 patients (9 infants, 127 aged 1-4 years, 91 aged ≥ 5 years). Increased median day-3 amodiaquine concentrations were associated with a lower risk of treatment failure [HR 0.87 (95% CI 0.78-0.98), p = 0.021]. Amodiaquine exposure (median AUC0-∞) was significantly higher in infants (4201 ng h/mL) and children aged 1-5 years (1994 ng h/mL) compared to older children and adults (875 ng h/mL, p = 0.001), even though infants received a lower mg/kg amodiaquine dose (median 25.3 versus 33.8 mg/kg in older patients). Desethylamodiaquine AUC0-∞ was not significantly associated with age. No significant safety concerns were identified. CONCLUSIONS: Efficacy of artesunate-amodiaquine at currently recommended dosage regimens was high across all age groups. Reassuringly, amodiaquine and desethylamodiaquine exposure was not reduced in underweight-for-age young children or those with high parasitaemia, two of the most vulnerable target populations. A larger pharmacokinetic study with close monitoring of safety, including full blood counts and liver function tests, is needed to confirm the higher amodiaquine exposure in infants, understand any safety implications and assess whether dose optimization in this vulnerable, understudied population is needed.

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.

Malaria parasite density and plasma apolipoprotein A1 in symptomatic and asymptomatic infections in Nigerian children.

BACKGROUND & OBJECTIVES: Alterations in plasma apolipoproteins in individuals with malaria infection and their potential roles in the pathogenesis are known but the link between the malaria parasite density and apolipoprotein A1 (apo-A1) level is insufficiently understood. This study was conducted to determine whether the plasma apo-A1 level is influenced by the degree of parasitaemia in malaria infections. METHODS: In a case-control study, a convenient sample of children aged 2-10 years with uncomplicated malaria cases (UMC), asymptomatic parasitaemia cases (APC) and healthy children without parasitaemia (HCP) was recruited. The cases consisted of 61 UMC and 21 APC, while the controls consisted of 24 HCP. Levels of apo-A1 was determined using immunoturbidimetric assay and compared among the different degrees of parasite density. RESULTS: Of the 82 participants with parasitaemia, density was ≤1000/µL in 12, 1001-10000/µL in 21 and >10000/µL in 49 children. There was significant difference among the mean values of apolipoprotein A1 of the three groups, viz: UMC [91.4 (95% CI: 81.3, 101.5) mg/dL], APC [67.0 (95% CI: 48.9, 84.9) mg/dL] and HCP [99.0 (95% CI: 76.6, 121.3) mg/dL], p=0.029. Post-hoc analysis revealed that the mean plasma level of apo-A1 in HCP was significantly higher than APC by 32.0±12.4 mg/dL and UMC by 7.5±4.2 mg/dL. However, there were no differences in the mean apolipoprotein A1 levels among the three groups of parasite density. INTERPRETATION & CONCLUSION: The presence of parasitaemia causes a remarkable reduction in apolipoprotein A1 level that was not influenced by the degree of parasitaemia.

Malaria prevalence and its sociodemographic determinants in febrile children - a hospital-based study in a developing community in South-East Nigeria.

BACKGROUND: Malaria remains one of the major contributors of child mortality in many developing countries in Africa. Identifying its determinants will help in prevention and prompt intervention in these settings. METHODS: This cross-sectional descriptive study was conducted over an eight-month period. It enrolled 382 children who were presented with fever to the children outpatient and emergency unit of a tertiary hospital in South-east Nigeria. A structured questionnaire was used to collect information on socio-demographic factors. Blood film microscopy for malaria and parasite density was done on all subjects that tested positive for malaria. RESULT: The malaria prevalence rate was 16.7%, 26.7%, 29.9% and 46.2% in children < 5 years, 5 to < 10 years, 10 to < 15 years and 15-17 years respectively. Logistic regression analysis showed that malaria was more prevalent in older children but children under the age of 5 years were more prone to higher parasite density. Also, children of mothers with lower educational attainment, children from families of lower socio-economic class and resident in rural settings had higher likelihood of malaria infection. CONCLUSIONS: Sustained improvement in strategies to prevent malaria infection is still imperative in children of all ages, especially those under 5 years, children from families of low socio-economic class and those residents in rural communities.

Sensitivity and specificity of malaria rapid diagnostic test (mRDT CareStatTM) compared with microscopy amongst under five children attending a primary care clinic in southern Nigeria.

BACKGROUND: Malaria diagnosis using microscopy is currently the gold standard. However, malaria rapid diagnostic tests (mRDTs) were developed to simplify the diagnosis in regions without access to functional microscopy. AIM: The objective of this study was to compare the diagnostic accuracy of mRDT CareStatTM with microscopy. SETTING: This study was conducted in the paediatric primary care clinic of the Federal Medical Centre, Asaba, Nigeria. METHODS: A cross-sectional study for diagnostic accuracy was conducted from May 2016 to October 2016. Ninety-eight participants were involved to obtain a precision of 5%, sensitivity of mRDT CareStatTM of 95% from published work and 95% level of confidence after adjusting for 20% non-response rate or missing data. Consecutive participants were tested using both microscopy and mRDT. The results were analysed using EPI Info Version 7. RESULTS: A total of 98 children aged 3-59 months were enrolled. Malaria prevalence was found to be 53% (95% confidence interval [CI] = 46% - 60%), whilst sensitivity and specificity were 29% (95% CI = 20% - 38%) and 89% (95% CI = 83% - 95%), respectively. The positive and negative predictive values were 75% (95% CI = 66.4% - 83.6%) and 53% (95% CI = 46% - 60%), respectively. CONCLUSION: Agreement between malaria parasitaemia using microscopy and mRDT positivity increased with increase in the parasite density. The mRDT might be negative when malaria parasite density using microscopy is low.

High malaria parasitemia among outpatient febrile children in low endemic area, East-Central Tanzania in 2013.

OBJECTIVE: This study investigated the prevalence and distribution patterns of malaria in Kilosa district as part of non-malaria causes of febrile illnesses in children study. We enrolled febrile patients aged 2-13 years presenting at the outpatient department during the rainy and dry seasons, in 2013. For each participant, we tested for malaria parasites and identified parasite species using microscopy. We then calculated parasite density and estimated geometric mean parasite density. RESULTS: The overall malaria prevalence in febrile children was 23.7% (n = 609). Plasmodium falciparum accounted for 98.6% of malaria positives. There was a heterogeneous distribution of malaria cases among the 17 wards constituting the catchment area. A high proportion (69.4%, n = 144) of malaria positive individuals had high parasite densities. Individuals who were enrolled in the rainy season had higher geometric mean parasite density (15415.1 parasites/µl, 95% CI 10735.3-22134.9) compared to the dry season (6115.3 parasites/µl, 95% CI 4237.8-8824.6). The relatively high malaria prevalence recorded in Kilosa, an area considered low endemicity, calls for concerted effort in documenting malaria burden at fine geographical scales and tailor preventive and control strategies that target hotspots of high malaria transmission.