Genetic Diversity and Molecular Surveillance of Antimalarial Drug Resistance of Plasmodium falciparum among Hospitals Patients in Benue State Nigeria

Introduction: Malaria is a febrile illness caused by parasites of the genus Plasmodium and transmitted by female Anopheles mosquitoes. The genetic diversity and antimalarial drug resistance of Plasmodium falciparum are some of the major challenges of malaria control programme in Nigeria. Aim: This study was aimed at determining the genetic diversity, and molecular surveillance of antimalarial drug resistance among patients attending Government hospitals in Benue State, Nigeria. Methodology: Plasmodium falciparum deoxyribonucleic acid was extracted from dried blood spots of 60 positive malariacases among the patients. The diversity of Plasmodium falciparum was done by genotyping 3D7 and FC27 families of merozoite surface protein- 2 alleles. The Plasmodium falciparum multidrug resistance 1 and Plasmodium falciparum kelch13 genes of Plasmodium falciparum were also amplified and assessed by restriction fragment length polymorphism (RFLP) to survey molecular resistance to antimalarial drugs. Results: The results showed that the frequency of 3D7 allele 37(61.7%) was higher than FC27 allele 18(30.0%). The frequency of merozoite surface protein- 2 infections with both allelic types was 5(8.3%). There was a significant difference in the distribution of the merozoite surface protein two alleles (?2=25.9,df=2 P<.0.001). Both the Plasmodium falciparum multidrug resistance 1 Asparagine 86Tyrosine (N86Y) and Aspartic acid 1246Tyrosine (D1246Y), had 100 % mutant while the 100% while the Plasmodium falciparum kelch13 G449A had 100% wild type allele. Conclusion: The current study underscores the need for frequent monitoring of indicators of antimalaria drug resistance in Nigeria.

Advances in automatic detection technology for images of thin blood film of malaria parasite / 中国血吸虫病防治杂志

This paper reviews the computer vision and image analysis studies aiming at automated diagnosis or screening of malaria in microscope images of thin blood film smears. On the basis of introducing the background and significance of automatic detection technology,the existing detection technologies are summarized and divided into several steps,including image acqui-sition,pre-processing,morphological analysis,segmentation,count,and pattern classification components. Then,the princi-ples and implementation methods of each step are given in detail. In addition,the promotion and application in automatic detec-tion technology of thick blood film smears are put forwarded as questions worthy of study,and a perspective of the future work for realization of automated microscopy diagnosis of malaria is provided.

Use of Nigerian Medicinal Plants Protected Liver from Injury in Plasmodium berghei Infected Mice.

The effects of ethanol leaf extracts of Spilanthes uliginosa, Ocimum basilicum, Hyptis spicigera and Cymbopogon citratus on mice infected with malaria parasite was investigated. Eighty four (84) swiss mice of both sexes were used for the study. All the mice were passaged intraperitoneally with 0.2 ml parasitized blood suspension and parasitemia assessed by Geimsa stain thin blood films after seventy two hours. The mice were divided into 6 groups namely; A, B, C, D, E and F. Groups B, C, D and E were subdivided into three (3): B1, B2, B3, C1, C2, C3, D1, D2, D3, E1, E2 and E3. Both groups and subgroups contained 6 mice each. The subgroups were treated with the extracts of Spilanthes uliginosa (Sw), Ocimum basilicum, Hyptis spiligera and Cymbopogon citratus each for five (5) consecutive days with 200, 400 and 800 mg/kg body weight via oral intubation daily respectively. The results indicated a general significant (P<0.05) decrease in the average body weight of the parasitized untreated mice while the histological photomicrographs showed alterations in the liver architecture of parasitized untreated mice and restorative effects of all the plant extracts and standard drug on the liver architecture of the parasitized treated mice.

In vivo Study of Antiplasmodial Activity of Terminalia avicennioides and Its Effect on Lipid Profile and Oxidative Stress in Mice Infected with Plasmodium berghei.

Aims: To determine the antiplasmodial activity of methanolic extract of T. avicennioides and its effect on oxidative stress and the lipid profiles in mice infected with Plasmodium berghei. Study Design: Mice used for this study were grouped into five. The first group was not infected with malaria parasite (normal control), the second group was infected with the parasite but not treated with antimalarial drugs (negative control), the third group was infected with the parasite and treated with 5mg/kg body weight of artesunat (positive control), while the fourth and fifth groups were infected with malaria parasite and treated with 100 and 200mg/kg of T. avicennioides respectively. Methodology: The parasitaemia was monitored for five days. The animals were sacrificed on the fifth day and the blood was collected. The serum was used to assess the biochemical parameters using randox kits. Results: While parasite density increases in the negative control per day, there was reduction in parasite density in treated groups. The parasite clearance was significantly higher (P = .05) in those treated with 200mg/kg of T. avicennioides than those treated with 100mg/kg of T. avicennioides and 5mg/kg of artesunat. The malondialdehyde level was significantly higher in the negative control, while superoxide dismutase and catalase levels were significantly reduced when compared with group treated with 200mg/kgbdwt of T. avicennioides. HDL level was significantly higher (P = .05) in those treated with 200mg/kg than in the normal, negative and positive control. The triglycerides level was significantly higher in the negative control when compared with the group treated with the extract of T. avicennioides. Conclusion: This study showed that the methanolic extract of T. avicennioides display dose-related in vivo antiplasmodial and antioxidant activities as well as reduced the serum and liver lipoprotein cholesterol in mice infected with P. berghei.

Use of buffy coat thick films in detecting malaria parasites in patients with negative conventional thick films

Objective: To determine the frequency of malaria parasite detection from the buffy coat blood ilms by using capillary tube in falciparum malaria patients with negative conventional thick ilms. Methods: Thirty six uncomplicated falciparum malaria patients confirmed by conventional thick and thin films were included in the study. The patients were treated with artemisinin combination therapy at Hospital for Tropical Diseases, Bangkok, Thailand for 28 day. Fingerpricks for conventional blood films were conducted every 6 hours until negative parasitemia, then daily fingerpricks for parasite checks were conducted until the patients were discharged from hospital. Blood samples were also concurrently collected in 3 heparinized capillary tubes at the same time of fingerpricks for conventional blood films when the prior parasitemia was negative on thin films and parasitemia was lower than 50 parasites/200 white blood cells by thick film. The first negative conventional thick films were compared with buffy coat thick films for parasite identification.Results:Out of 36 patients with thick films showing negative for asexual forms of parasites, buffy coat films could detect remaining 10 patients (27.8%) with asexual forms of Plasmodium falciparum. Conclusions: The study shows that buffy coat thick films are useful and can detect malarial parasites in 27.8% of patients whose conventional thick films show negative parasitemia.

Preservation of Wild Isolates of Human Malaria Parasites in Wet Ice and Adaptation Efficacy to In Vitro Culture

Wild isolates of malaria parasites were preserved in wet ice for 2–12 days and cultivated by a candle jar method. In four isolates of <i>Plasmodium falciparum</i> collected from Myanmar and preserved for 12 days, all failed to grow. In 31 isolates preserved for 5–10 days, nine were transformed to young gametocytes, but 22 isolates grew well. From Ranong, Thailand, nine isolates preserved for 7 days were examined, and six grew well. On the other hand, all of the 59 isolates collected from eastern Indonesian islands failed to establish as culture-adapted isolates, even most of them were preserved only for 2–3 days: 10 isolates stopped to grow, and 49 isolates were transformed to sexual stages by Day 10. These results indicated that a great difference in adaptation to in vitro culture may exist between wild isolates distributed in continental Southeast Asia and in eastern Indonesia and that gametocytogenesis might be easily switched on in Indonesian isolates. In wild isolates of <i>P. vivax</i>, <i>P. malariae</i> and <i>P. ovale</i> preserved for 2–9 days, ring forms or young trophozoites survived, but adaptation to in vitro culture failed. These results indicate that wild isolates can be preserved in wet ice for 9–10 days.

Antibodies raised against hemolymph of Anopheles culicifacies reduce the fecundity and malaria parasite development.

Background & objectives: Several studies have been made to study the effect of antisera raised against different tissues (hemolymh, ovary, midgut and salivary glands) on the fecundity and malaria parasite development in the different species of mosquitoes but there are no reports on the antisera raised against the hemolymph of Anopheles culicifacies, the principal malaria vector in India accounting for 65% of malaria cases. Hence, an attempt was made to study the same and evaluate its impact on malaria parasite development. Methods: Polyclonal and multifactorial antibodies were produced in rabbits against heterogenous mixture of hemolymph proteins. Antibodies against hemolymph proteins were screened for their potential to influence reproductive performance of mosquitoes. Antibody titer in rabbit serum was determined by ELISA and putative candidate antigens were identified in the hemolymph of An. culicifacies by western blotting. Cross reactivity amongst various tissues vis-a-vis hemolymph protein was also identified. In addition, a significant reduction in oocyst development was also observed in An. culicifacies mosquitoes that ingested antihemolymph antibodies along with Plasmodium vivax. Results: The maximum reduction in fecundity (57%) was observed during fourth week, after the last booster and number of oocyts per infected mosquito reduced by 73.35% in the group of mosquitoes that ingested antihemolymph antibodies along with the infected blood meal respectively. However, the ingestion of antibodies against hemolymph proteins did not have significant influence on hatchability. Antisera raised against hemolymph proteins of An. culicifacies recognized 11 polypeptides by western blotting. Interpretation & conclusion: During the present study, 11 putative candidate antigens were identified in the hemolymph of An. culicifacies, against which antibodies produced significantly reduced the fecundity by 57%. In addition, a significant reduction in oocyst development was also observed in An. culicifacies that ingested antihemolymph antibodies along with P.vivax.

Some results of malaria parasite species collected from Daknong province and analysis of drug resistance in P.Palciparum by the polymerase chain reaction

Background: In many years, National Institute of Malariology, Parasitology and Entomology conducted collection, storage and preservation of malaria parasites species \r\n', u'Objective: to evaluate some results of malaria parasite species collected from Daknong province and analysis of drug resistance in P.Palciparum by the polymerase chain reaction.\r\n', u'Subject and method: Malaria parasite species collected from Daknong province in 2006. Thirty-five isolates were confirmed to be resistant with chloroquin by in vitro test. Polymerase chain reaction-restriction fragment leng polymorphism were used. \r\n', u'Results: 55 Plasmodium jalciparum. 7 Plasmodium vivax. 4 Plasmodium malariae. 1 Plasmodium ovale samples were collected from the malaria patients. A preliminary analysis of drug-resistant mutations in the Plasmodium jalciparum chloroquine resistance transpory (pfcrt) and P Jalciparummulti-drug resistant genes showed that the change of the order of amino-acid of Plasmodium jalciparum was closely correlated to chloroquine resistance in 35 isolates at the mutant allele 76 of pfcrt gene of chloroquine resistant Plasmodiuntjalciparum isolates. \r\n', u'Conclusion: These results contributed to supplement malaria parasite species that were stored in National Institute of Malariology, Parasitology ad Entomology.\r\n', u'

Some remarks on the quality of malaria parasite examination in Thanh Hoa province in 2002

In 2002, 99.807 blood slides were taken and examined in Thanh Hoa province, of which 33.464 slides were taken from fever patients (33.52%). 196 positive cases were detected. 52 microscopic points were checked, of which 10 microscopic points were very good functioning (19.23%), 27 points were good (59.9%), 12 were average and 3 were not good (5.76%). The quality of slides taken by the commune and village health workers was not so good, the slides were too thick or too thin with blood. In some villages, the slides were taken massively in order to fulfill the target. Checking the quality of microscopy showed that the mistake rate of microscopy was 13.16% as compared with the total slides sent for rechecking. In some microscopic points, the mistake rate was high (the provincial hospital had the mistake of 6/19 positive slides)

Rapid diagnostic test in early detecting P.falciparum and controlling malaria in epidemic prevention

To introduce laboratory or testing techniques in detecting malaria parasite with 4 Plasmodium strains is P.vivax, P.falciparum, P.malariae and P.ovale, includes: Microscopic tests as Peripheral smear; Quantiative Buffy Coat test (QBC test); Detecting malaria pigment in monocytic leukemia or macrophage. Non-microscopic tests as Parasight-F test, Optimal assay, Malaria Immune Chromatography test - P.falciparum, Polymerase Chain Reaction; Detecting antibodies by RIA or ELISA

Vectorial role of An.dirus S.L. in deep forest in Khanh Phu

Research on vetorial role on forest malaria at 4 different ecological arears such as in the villages, forest fringe, deep forest and plot huts in Khanh Phu from Janruary to December, 2002. The results: The Anopheles dirus species component in the plot hut areas and deep forest are not more difference than in the villages. An.dirus account for 83.1% in plot huts and 96.9% in deep forest. The malaria transmission of An.dirus is very high in the plot huts areas and deep forests. The malaria infection risk is gradually increased from villages to forest fringe, then the plot huts in deep forests. Especially, the malaria infection risk in the plot hut areas and deep forests is over 20 fold higher than that in the villages

New Channels and Transporters as Anti-malaria Drug Targets / 中国寄生虫学与寄生虫病杂志

In order to get more nutrition from outside the erythrocyte,new channels were induced by malaria par-asite.These channels play an important role in physiology of the parasitized cell.They are of interest both as potential targets in their own right and as potential drug targeting routes capable of mediating the entry of cytotoxic drugs into the app-ropriate compartment of the infected cell.It is hoped that this new anti-malarial strategy will help to create a sustainable anti-malaria-drug-development portfolio for the treatment of malaria.

Conditional Inhibition of Hemozoin Formation by Chloroquine in vitro / 中国寄生虫学与寄生虫病杂志

Objective To study the characteristics of inhibition on hemozoin formation by chloroquine under in vitro condition.Methods Under different concentrations(0.5-2 mol/L) of sodium acetate(NaAc) and at the pH range of 4.0-5.0, chloroquine was tested for inhibition of ?-hematin(hemozion) formation by using the HPIA(heme polymerization inhibitory activity) assay.The morphology of ?-hematin crystals was determined by light microscopy.Ultraviolet spectrophotometry was employed to measure ?-hematin content, and the size of ?-hematin crystal was analyzed by X-ray diffraction(XRD) .Results Chloroquine exhibited varied effect on ?-hematin formation, depending on pH value and Na+ concentration.When the NaAc concentration increased from 0.5 mol/L(pH 4.2) to 2 mol/L(pH 4.8), the chloroquine inhibitory effect also increased.Results suggested that there exists a threshold pH, below which the ?-hematin formation escalates and chloroquine inhibition declines, and at or above which chloroquine exerts a stronger inhibitory effect on ?-hematin formation.With the increase of pH from 4.4 to 4.8, the crystallinity and the size of crystal changed from 6.93% and 357  to 6.32% and 264 , respectively.When pH reached to 5, no more ?-hematin formed.Chloroquine could reduce the crystallinity and crystal size of ?-hematin at same pH value.Morphology analysis on the samples was consistent with the above results.Conclusion Chloroquine inhibits hemozoin formation only when the pH value is at or above threshold pH.