Improved detection of mixed P. falciparum-P. vivax infection at a rural health centre in Ethiopia using PCR.

This study aims to this study is to compare the co-infection Plasmodium falciparum + Plasmodium vivax and compare the detection of cases of mixed-species malaria using light microscopy versus semi-nested multiplex PCR (sPCR). Investigators collected 3060 samples at a rural health centre in Ethiopia from December 2010 to October 2011. Two capillary blood specimens were taken from each patient, one for diagnosis of Plasmodium infection by light microscopy and the other for sPCR-based diagnosis. LM detected 627 positive cases; these samples, together with 582 negatives by LM, were also subjected to sPCR testing. Of the 627 positive samples by LM, 68.4% were positive for P. vivax, 30.5% for P. falciparum, and 1.1% for P. falciparum + P. vivax co-infection. Using the sPCR technique, we identified 788 samples positive for Plasmodium: 33.0% for P. vivax, 26.5% for P. falciparum, 3.7% for P. falciparum + P. vivax co-infection, 2.0% for P. ovale and 0.8% for P. vivax + P. ovale co-infection. In the case of P. falciparum + P. vivax co-infection, light microscopy diagnosis showed a sensitivity of 11.1%, a specificity of 99.8%, a positive predictive value of 71.4% and a negative predictive value of 96.6%. The concordance rate for identifying P. falciparum + P. vivax co-infection (kappa statistic) with microscopy and sPCR was 0.184. The LM approach has low sensitivity for the detection of mixed-species infections, while sPCR is more useful.

Efficacy of Artesunate + Sulphadoxine-Pyrimethamine (AS + SP) and Amodiaquine + Sulphadoxine-Pyrimethamine (AQ+ SP) for Uncomplicated falciparum Malaria in Equatorial Guinea (Central Africa)

The objectives of the study were (i) to evaluate the efficacy of combination drugs; such as artesunate + sulphadoxinepyrimethamine (AS + SP) and amodiaquine + sulphadoxine-pyripethamine (AQ+ SP) in treatment of uncomplicated falciparum malaria (ii) to differentiate recrudescence from reinfection by analysing msp-1 and msp-2 genes of Plasmodium falciparum in treatment failure cases. Methods. We carried out an in vivo study in the year 2005 in 206 children between 6 to 59 months age groups. Of the 206; 120 received AQ+ SP; and 86 received AS + SP. A clinical and parasitological followup during 14 days was undertaken. Finger-prick blood sample from each patient was taken onWhatman filter paper (no. 3) on days 0; 7; 14 and also the day when the parasite and symptoms reappeared for PCR analysis. Results. Late treatment failure was observed in 3.5(4/114) with AQ+ SP; and 2.5(2/79) with AS + SP. The success rate was 96.5with AQ+ SP and 97.5with AS + SP. No deaths and severe reactions were recorded. Out of the 6 treatment failure cases; one was reinfection as observed by PCR analysis of msp-1 and msp-2 genes on day 14. Discussion. Both the combinations found to be efficacious and safe and could be used as a first-line treatment for uncomplicated falciparum malaria in Equatorial Guinea

Trypanosoma brucei gambiense in domestic livestock of Kogo and Mbini foci (Equatorial Guinea).

OBJECTIVE: To evaluate Trypanosoma brucei gambiense infection in peri-domestic livestock from Kogo and Mbini foci (Equatorial Guinea) in order to investigate its possible implication in the sleeping sickness transmission cycle in these hypoendemic foci. METHODS: Samples from 698 domestic animals (goats, sheep and pigs) from trypanosomiasis-endemic localities of Kogo and Mbini foci were tested for animal trypanosomes and T. b. gambiense (group I) by species-specific polymerase chain reaction. RESULTS: Trypanosoma brucei s.l., the predominant trypanosome species, was detected in 182 (52.6%) samples from Mbini and in 127 (36.1%) samples from Kogo. T. b. gambiense was only identified in seven (2%) of the Mbini samples and one co-infection (with T. vivax) was observed. CONCLUSION: The occurrence of T. b. gambiense in peri-domestic livestock in Mbini and its absence in Kogo could explain the epidemiological differences between the two foci and could have significant implications for sleeping sickness control in Equatorial Guinea.

Knockdown resistance mutations (kdr) and insecticide susceptibility to DDT and pyrethroids in Anopheles gambiae from Equatorial Guinea.

OBJECTIVES: To determine the frequency of knockdown resistance (kdr) mutations in the malaria vector Anopheles gambiae s.s. from continental Equatorial Guinea; and to relate kdr genotypes with susceptibility to DDT and pyrethroid insecticides in this vector. METHODS: Female mosquitoes were collected in two villages, Miyobo and Ngonamanga, of mainland Equatorial Guinea. Insecticide susceptibility tests were performed following WHO procedures. Anopheles gambiae complex specimens were identified to species and molecular form by PCR. Genotyping of the kdr locus was performed by allele-specific PCR and direct sequencing in a subset of samples. RESULTS: Both M and S molecular forms of A. gambiae were found in Ngonamanga whereas only the S-form was identified in Miyobo. The two kdr mutations were detected in S-form samples of both villages, with a higher frequency of the kdr-e (Leu-1014-Ser) allele (Miyobo: 16%; Ngonamanga: 40%). The kdr-w (Leu-1014-Phe) mutation was also detected in 3% of the M-form. All individuals tested for pyrethroids were susceptible. A mortality rate of 86% was obtained for DDT. An overall kdr allele frequency (i.e. kdr-e + kdr-w) of 22% was detected in DDT resistant individuals, whereas susceptible individuals had a kdr frequency of 6%. CONCLUSION: The co-occurrence of both kdr mutations and reduced susceptibility to DDT found in A. gambiae highlights the importance of implementing efficient surveillance of insecticide resistance in Equatorial Guinea.

Malaria vectors in the Bioko Island (Equatorial Guinea): estimation of vector dynamics and transmission intensities.

The current study was performed on the Bioko Island (Equatorial Guinea) with the aim of establishing a rapid assessment technique for mapping malaria risk and measuring vector densities. Human bait collection, tent traps, light traps, indoor resting collection, and window exit traps were used to collect Anopheles gambiae s.s. and Anopheles funestus, the two anopheline species involved in malaria transmission in this island. Capture data were used to compare differences in the behavior and vectorial capacity of An. gambiae s.s. and An. funestus. Differences in the two species of mosquitoes were found in relation to the season and trapping methods used. Entomological inoculation rates (EIR) for Plasmodium falciparum were calculated using a polymerase chain reaction (PCR) test with individual anopheline mosquitoes from human bait collections in two villages during the dry and rainy seasons. P. falciparum sporozoites were detected from both dissected heads/thorax and abdomens of both species.

Malaria vectors in Bioko Island (Equatorial Guinea): PCR determination of the members of Anopheles gambiae Giles complex (Diptera: Culicidae) and pyrethroid knockdown resistance (kdr) in An. gambiae sensu stricto.

Anopheles gambiae sensu lato Giles, 1902 and Anophelesfunestus Giles, 1900 are the main malaria vectors on the island of Bioko (Equatorial Guinea). This study was carried out to determine: a) members of the An. gambiae complex that may be present on the island of Bioko and, b) the sensitivity of An. gambiae sensu stricto to pyrethroids. The analysis by PCR detected the presence of An. gambiae s.s. as the major vector of the complex and the "forest chromosomal form" was demonstrated by cytogenetic analysis. The presence of Anopheles melas in the southwest, north and southeast of the island justifies its study as a vector. The molecular characterization of pyrethroid knockdown resistance (kdr) showed that the populations of An. gambiae s.s. were sensitive and no mutations were found. This fact justifies the implementation on a large scale of pyrethroid-impregnated bednets within the framework of the Malaria Control Program of Equatorial Guinea.

Amplified fragment length polymorphism (AFLP) protocol for genotyping the malarial parasite Plasmodium falciparum.

We have established an amplified fragment length polymorphism (AFLP) protocol for identifying anonymous polymorphic loci of the malarial parasite, Plasmodium falciparum. The method consists of the following steps (i) digestion and ligation in one reaction; (ii) selective fluorescence forward primers labelled; (iii) PCR products resolved in polyacrylamide gels using the ABIPRISM 377 XL DNA sequencer and, (iv) the use of Genescan software to size the fragments. This standardized protocol distinguished between 2 standard reference clones of P. falciparum from West African and Southeast Asian and 2 Central African isolates from patients with clinical malaria. The AFLP protocol resulted in evenly distributed and reproducible band patterns for amplified fragments ranking from 163 to 489 bp long +/-0.5 S.D. The primer Tru ACA labelled with the phosphoramidite 6-carboxifluorescein (FAM-blue) was easy to interpret, with a maximum of 53 bands per clone and of 81 per isolate (mixed falciparum populations) whereas the primer Tru AG labelled with the hexachlorinated analogue (HEX-green) showed a less clear pattern of bands and reproducibility than Tru ACA.

The potential utility of the Semi-Nested Multiplex PCR technique for the diagnosis and investigation of congenital malaria.

We report three cases of congenital malaria involving two malarial immune mothers living in Spain. Diagnostic PCR and Genotyping PCR for merozoite surface proteins 1 and 2 were essential to show that mothers and new-borns had different Plasmodium population parasites at the moment of the delivery, and that the infection was acquired earlier in gestation by transplacental transmission. In the first case the Plasmodium species founded in both, mother and child were different. Malaria in the twins showed a mixed infection (P. falciparum plus P. malariae) while the mother presented a P. falciparum infection. These facts were confirmed studying the polymorphisms for MSP1 and MSP2. Blood samples of the newborns were analyzed an half hour after delivery excluding the possibility of re-infection by mosquito bite and indicating a vertical transmission during pregnancy.

Usefulness of seminested multiplex PCR in surveillance of imported malaria in Spain.

The use of a new PCR-based method for the diagnosis of malaria in the Spanish Malaria Reference Laboratory has promoted an increase in confirmed cases of malaria. From August 1997 to July 1998, a total of 192 whole-blood samples and 71 serum samples from 168 patients were received from the hospitals of the Spanish National Health System. Most of the patients came from west-central African countries (85%). This molecular method showed more sensitivity and specificity than microscopy, detecting 12.4% more positive samples than microscopy and 13% of mixed infections undetectable by Giemsa stain. Plasmodium falciparum was the main species detected, with 68% of the total positive malaria cases, followed by Plasmodium malariae (29%), Plasmodium vivax (14%), and Plasmodium ovale (7%), including mixed infections in all cases. This report consists of the first wide, centralized survey of malaria surveillance in Spain. The reference laboratory conducted the analysis of all imported cases in order to detect trends in acquisition. The use of a seminested multiplex PCR permitted confirmation of the origins of the infections and the Plasmodium species involved and confirmation of the effectiveness of drug treatments. This PCR also allowed the detection of the presence in Spain of primaquine-tolerant P. vivax strains from west-central Africa, as well as the detection of a P. falciparum infection induced by transfusion.

Semi-nested, multiplex polymerase chain reaction for detection of human malaria parasites and evidence of Plasmodium vivax infection in Equatorial Guinea.

A semi-nested, multiplex polymerase chain reaction (PCR) based on the amplification of the sequences of the 18S small subunit ribosomal RNA (ssrRNA) gene was tested in a field trial in Equatorial Guinea (a hyperendemic focus of malaria in west central Africa). The method uses a primary PCR amplification reaction with a universal reverse primer and two forward primers specific for the genus Plasmodium and to mammals (the mammalian-specific primer was included as a positive control to distinguish uninfected cases from inhibition of the PCR). The second amplification is carried out with the same Plasmodium genus-specific forward primer and four specific reverse primers for each human Plasmodium species. The PCR amplified products are differentiated by fragment size after electrophoresis on a 2% agarose gel. Four villages from three regions of the island of Bioko (Equatorial Guinea) and two suspected Plasmodium vivax-P. ovale infections from the hospital of Malabo were tested by microscopy and PCR. The PCR method showed greater sensitivity and specificity than microscopic examination and confirmed the existence of a focus of P. vivax infections in Equatorial Guinea suspected by microscopic examination. It also provided evidence of several mixed infections, mainly P. falciparum and P. malariae, the two predominant species causing malaria in Equatorial Guinea.