Mutations in Pfcrt and Pfmdr1 genes of Plasmodium falciparum isolates from two sites in Northcentral and Southwest Nigeria.

The ability of malaria parasites to develop resistance to antimalarial drugs has made it necessary to continuously survey malaria parasite populations for resistance markers. Mutations in specific malaria parasite genes confer resistance to antimalarial drugs. The study compared mutations in Pfcrt and Pfmdr1 genes of P. falciparum from two ecologically different areas of Nigeria. Plasmodium falciparum dried blood spots collected from New Bussa (Northcentral Nigeria) and Ijede (Southwest Nigeria) were analysed by PCR-RFLP for Pfcrt, K76 T, Pfmdr1, N86Y and Y184F mutations. Pfmdr1 copy number was determined by quantitative-PCR. A total of 145 blood spots [Ijede = 55; New Bussa = 90 blood spots] were analysed, but Pfcrt gene was successfully amplified in 144 samples while Pfmdr1 was amplified in 132 samples. Overall, prevalence of mutant forms of Pfcrt 76 T,Pfmdr1 86Y and 184F were 74.3% (95% CI: 66.4-81.2%), 18.2% (95% CI: 12.0-25.8%) and 35.6% (95% CI: 27.5-44.4%). The frequency of Pfcrt 76 T was similar in both study sites [Ijede: 81.8% (95%CI: 69.1-90.9%); New Bussa: 69.7% (95%CI: 59.0-79.0), p = 0.105]. However, the frequencies of Pfmdr1 86Y and 184F were significantly higher in Ijede (28.3% and 62.3%) than in New Bussa (11.4% and 17.7%), respectively (P < 0.05). Eight parasite genotypes based on three codons of the two genes were identified. The most frequent genotype was TNY 53(40.5%) while the least was KYF 1 (0.8%). The most frequent genotype in Ijede and New Bussa were TNF 18(34.0%) and TNY 40 (51.3%) respectively. The frequency of wild strain KNF in Ijede and New Bussa were 3 (5.7%) and 18 (23.1%), respectively. The distribution of the genotypes differed significantly by location. The genotypes with more than two or more mutations were more in Ijede 32 (60.4%) than in New Bussa 16 (20.5%) (p < 0.001). Amplification of Pfmdr1 copy number was not observed in the two study sites. The prevalence of Pfcrt 76 T was similar in both locations while Pfmdr1 86Y and 184F differed in both locations. Single nucleotide polymorphisms in the three codons assessed were more in Ijede than in New Bussa.

Clinical illness and outcomes in Nigerian children with persistent early-appearing anaemia following initiation of artemisinin-based combination treatments of uncomplicated falciparum malaria.

In non-anaemic children with malaria, early-appearing anaemia (EAA) is common following artemisinin-based combination treatments (ACTs) and it may become persistent (PEAA). The factors contributing to and kinetics of resolution of the deficit in haematocrit from baseline (DIHFB) characteristic of ACTs-related PEAA were evaluated in 540 consecutive children with malaria treated with artemether-lumefantrine, artesunate-amodiaquine or dihydroartemisinin-piperaquine. Asymptomatic PEAA occurred in 62 children. In a multiple logistic regression model, a duration of illness ≤3 days before presentation, haematocrit <35% before and <25% one day after treatment initiation, drug attributable fall in haematocrit ≥6%, and treatment with dihydroartemisinin-piperaquine independently predicted PEAA. Overall, mean DIHFB was 5.7% (95% CI 4.8-6.6) 7 days after treatment initiation and was similar for all treatments. Time to 90% reduction in DIHFB was significantly longer in artemether-lumefantrine-treated children compared with other treatments. In a one compartment model, declines in DIHFB were monoexponential with overall mean estimated half-time of 3.9 days (95% CI 2.6-5.1), Cmax of 7.6% (95% CI 6.7-8.4), and Vd of 0.17 L/kg (95% CI 0.04-0.95). In Bland-Altman analyses, overall mean anaemia recovery time (AnRT) of 17.4 days (95% CI 15.5-19.4) showed insignificant bias with 4, 5 or 6 multiples of half-time of DIHFB. Ten children after recovery from PEAA progressed to late-appearing anaemia (LAA). Progression was associated with female gender and artesunate-amodiaquine treatment. Asymptomatic PEAA is common following ACTs. PEAA or its progression to LAA may have implications for case and community management of anaemia and for anaemia control efforts in sub-Saharan Africa where ACTs have become first-line antimalarials. Trial registration: Pan Africa Clinical Trial Registration PACTR201709002064150, 1 March 2017 http://www.pactr.org.

Declining responsiveness of childhood Plasmodium falciparum infections to artemisinin-based combination treatments ten years following deployment as first-line antimalarials in Nigeria.

BACKGROUND: The development and spread of artemisinin-resistant Plasmodium falciparum malaria in Greater Mekong Subregion has created impetus for continuing global monitoring of efficacy of artemisinin-based combination therapies (ACTs). This post analyses is aimed to evaluate changes in early treatment response markers 10 years after the adoption of ACTs as first-line treatments of uncomplicated falciparum malaria in Nigeria. METHODS: At 14 sentinel sites in six geographical areas of Nigeria, we evaluated treatment responses in 1341 children under 5 years and in additional 360 children under 16 years with uncomplicated malaria enrolled in randomized trials of artemether-lumefantrine versus artesunate-amodiaquine at 5-year interval in 2009-2010 and 2014-2015 and at 2-year interval in 2009-2010 and 2012-2015, respectively after deployment in 2005. RESULTS: Asexual parasite positivity 1 day after treatment initiation (APPD1) rose from 54 to 62% and 2 days after treatment initiation from 5 to 26% in 2009-2010 to 2014-2015 (P = 0.002 and P <  0.0001, respectively). Parasite clearance time increased significantly from 1.6 days (95% confidence interval [CI]: 1.55-1.64) to 1.9 days (95% CI, 1.9-2.0) and geometric mean parasite reduction ratio 2 days after treatment initiation decreased significantly from 11 000 to 4700 within the same time period (P <  0.0001 for each). Enrolment parasitaemia > 75 000 µl- 1, haematocrit > 27% 1 day post-treatment initiation, treatment with artemether-lumefantrine and enrolment in 2014-2015 independently predicted APPD1. In parallel, Kaplan-Meier estimated risk of recurrent infections by day 28 rose from 8 to 14% (P = 0.005) and from 9 to 15% (P = 0.02) with artemether-lumefantrine and artesunate-amodiaquine, respectively. Mean asexual parasitaemia half-life increased significantly from 1.1 h to 1.3 h within 2 years (P <  0.0001). CONCLUSIONS: These data indicate declining parasitological responses through time to the two ACTs may be due to emergence of parasites with reduced susceptibility or decrease in immunity to the infections in these children. TRIAL REGISTRATION: Pan African Clinical Trial Registration PACTR201508001188143 , 3 July 2015; PACTR201508001191898 , 7 July 2015 and PACTR201508001193368 , 8 July 2015 PACTR201510001189370 , 3 July 2015; PACTR201709002064150 , 1 March 2017; https://www.pactr.samrca.ac.za.

Parasite reduction ratio one day after initiation of artemisinin-based combination therapies and its relationship with parasite clearance time in acutely malarious children.

BACKGROUND: In acute falciparum malaria, asexual parasite reduction ratio two days post-treatment initiation (PRRD2) ≥ 10 000 per cycle has been used as a measure of the rapid clearance of parasitaemia and efficacy of artemisinin derivatives. However, there is little evaluation of alternative measures; for example, parasite reduction ratio one day after treatment initiation (PRRD1) and its relationship with parasite clearance time (PCT) or PRRD2. This study evaluated the use of PRRD1 as a measure of responsiveness to antimalarial drugs. METHODS: In acutely malarious children treated with artesunate-amodiaquine (AA), artemether-lumefantrine (AL) or dihydroartemisinin-piperaquine (DHP), the relationships between PRRD1 or PRRD2 and PCT, and between PRRD1 and PRRD2 were evaluated using linear regression. Agreement between estimates of PCT using PRRD1 and PRRD2 linear regression equations was evaluated using the Bland-Altman analysis. Predictors of PRRD1 > 5000 per half cycle and PRRD2 ≥ 10 000 per cycle were evaluated using stepwise multiple logistic regression models. Using the linear regression equation of the relationship between PRRD1 and PCT previously generated in half of the DHP-treated children during the early study phase, PCT estimates were compared in a prospective blinded manner with PCTs determined by microscopy during the later study phase in the remaining half. RESULTS: In 919 malarious children, PRRD1 was significantly higher in DHP- and AA-treated compared with AL-treated children (P <  0.0001). PRRD1 or PRRD2 values correlated significantly negatively with PCT values (P <  0.0001 for each) and significantly positively with each other (P <  0.0001). PCT estimates from linear regression equations for PRRD1 and PRRD2 showed insignificant bias on the Bland-Altman plot (P = 0.7) indicating the estimates can be used interchangeably. At presentation, age > 15 months, parasitaemia > 10 000/µl and DHP treatment independently predicted PRRD1 > 5000 per half cycle, while age > 30 months, haematocrit ≥31%, body temperature > 37.4 °C, parasitaemia > 100 000/µl, PRRD1 value > 1000 and no gametocytaemia independently predicted PRRD2 ≥ 10 000 per cycle. Using the linear regression equation generated during the early phase in 166 DHP-treated children, PCT estimates and PCTs determined by microscopy in the 155 children in the later phase were similar in the same patients. CONCLUSIONS: PRRD1 and estimates of PCT using PRRD1 linear regression equation of PRRD1 and PCT can be used in therapeutic efficacy studies. TRIAL REGISTRATION: Pan African Clinical Trial Registration PACTR201709002064150, 1 March 2017, http://www.pactr.org.

Efficacy of Artemisinin-Based Combination Treatments of Uncomplicated Falciparum Malaria in Under-Five-Year-Old Nigerian Children Ten Years Following Adoption as First-Line Antimalarials.

The efficacies of 3-day regimens of artemether-lumefantrine (AL), artesunate-amodiaquine (AA), and dihydroartemisinin-piperaquine (DHP) were evaluated in 910 children < 5 years old with uncomplicated malaria from six geographical areas of Nigeria. Parasite positivity 1 day and Kaplan-Meier estimated risk of persistent parasitemia 3 days after therapy initiation were both significantly higher, and geometric mean parasite reduction ratio 1 day after treatment initiation (PRRD1) was significantly lower in AL-treated children than in AA- and DHP-treated children. No history of fever, temperature > 38°C, enrollment parasitemia > 75,000 µL-1, and PRRD1 < 5,000 independently predicted persistent parasitemia 1 day after treatment initiation. Parasite clearance was significantly faster and risk of reappearance of asexual parasitemia after initial clearance was significantly lower in DHP-treated children. Overall, day 42 polymerase chain reaction-corrected efficacy was 98.3% (95% confidence interval [CI]: 96.1-100) and was similar for all treatments. In a non-compartment model, declines of parasitemias were monoexponential with mean terminal elimination half-life of 1.3 hours and unimodal frequency distribution of half-lives. All treatments were well tolerated. In summary, all three treatments evaluated remain efficacious treatments of uncomplicated malaria in young Nigerian children, but DHP appears more efficacious than AL or AA.

Provider and patient perceptions of malaria rapid diagnostic test use in Nigeria: a cross-sectional evaluation.

BACKGROUND: Nigeria commenced a phased programmatic deployment of rapid diagnostic tests (RDT) at the primary health care (PHC) facility levels since 2011. Despite various efforts, the national testing rate for malaria is still very low. The uptake of RDT has been variable. This study was undertaken to determine the provider and patient perceptions to RDT use at the PHC level in Nigeria with their implications for improving uptake and compliance. METHODS: A cross-sectional survey was conducted in 120 randomly selected PHCs across six states, across the six-geopolitical zones of Nigeria in January 2013. Health facility staff interviews were conducted to assess health workers (HW) perception, prescription practices and determinants of RDT use. Patient exit interviews were conducted to assess patient perception of RDT from ten patients/caregivers who met the eligibility criterion and were consecutively selected in each PHC, and to determine HW's compliance with RDT test results indirectly. Community members, each selected by their ward development committees in each Local Government Area were recruited for focus group discussion on their perceptions to RDT use. RESULTS: Health workers would use RDT results because of confidence in RDT results (95.4%) and its reduction in irrational use of artemisinin-based combination therapy (ACT) (87.2%). However, in Enugu state, RDT was not used by health workers because of the pervasive notion RDT that results were inaccurate. Among the 1207 exit interviews conducted, 549 (45.5%) had received RDT test. Compliance rate (administering ACT to positive patients and withholding ACT from negative patients) from patient exit interviews was 90.2%. Among caregivers/patients who had RDT done, over 95% knew that RDT tested for malaria, felt it was necessary and liked the test. Age of patients less than 5 years (p = 0.04) and "high" educational status (p = 0.0006) were factors influencing HW's prescription of ACT to RDT negative patients. CONCLUSION: The study demonstrated positive perception to RDT use by HW and among community members with good compliance rate among health workers at the PHC level. This positive perception should be explored in improving the current low level of malaria testing in Nigeria while addressing the influence of age on HW administration of ACT to RDT negative cases.

Factors contributing to anaemia after uncomplicated falciparum malaria in under five year-old Nigerian children ten years following adoption of artemisinin-based combination therapies as first-line antimalarials.

BACKGROUND: Artemisinin-based combination therapies (ACTs) have remained efficacious treatments of acute falciparum malaria in many endemic areas but there is little evaluation of factors contributing to the anaemia of acute falciparum malaria following long term adoption of ACTs as first-line antimalarials in African children. METHODS: Malarious <5 year-olds randomized to artemether-lumefantrine, artesunate-amodiaquine or dihydroartemisinin-piperaquine treatments were followed up clinically for 6 weeks. Anaemia was defined as haematocrit <30%; Malaria-attributable fall in haematocrit (MAFH) as the difference between haematocrit 28-42 days post- and pre-treatment; Total MAFH (TMAFH) as the difference between days 28-42 haematocrit and the lowest haematocrit recorded in the first week post-treatment initiation; Drug-attributable fall in haematocrit (DAFH) as the difference between MAFH and TMAFH; Early appearing anaemia (EAA) as haematocrit <30% occurring within 1 week in children with normal haematocrit pre-treatment. Predictors of anaemia pre-treatment, EAA, MAFH or DAFH >4% were evaluated by stepwise multiple logistic regression models. Survival analysis and kinetics of DAFH were evaluated by Kaplan-Meier estimator and non-compartment model, respectively. RESULTS: Pre-treatment, 355 of 959 children were anaemic. Duration of illness >2 days and parasitaemia ≤10,000 µL-1 were independent predictors of anaemia pre-treatment. EAA occurred in 301 of 604 children. Predictors of EAA were age ≤ 15 months, history of fever pre-treatment and enrolment haematocrit ≤35%. The probabilities of progression from normal haematocrit to EAA were similar for all treatments. MAFH >4% occurred in 446 of 694 children; its predictors were anaemia pre-treatment, enrolment parasitaemia ≤50,000 µL-1, parasitaemia one day post-treatment initiation and gametocytaemia. DAFH >4% occurred in 334 of 719 children; its predictors were history of fever pre-and fever 1 day post-treatment initiation, haematocrit ≥37%, and parasitaemia >100,000 µL-1. In 432 children, declines in DAFH deficits were monoexponential with overall estimated half-time of 2.2d (95% CI 1.9-2.6). Area under curve of deficits in DAFH versus time and estimated half-time were significantly higher in non-anaemic children indicating greater loss of haematocrit in these children. CONCLUSION: After ten years of adoption of ACTs, anaemia is common pre-and early post-treatment, falls in haematocrit attributable to a single infection is high, and DAFH >4% is common and significantly lower in anaemic compared to non-anaemic Nigerian children. TRIAL REGISTRATION: Pan African Clinical Trial Registry (PACTR) [ PACTR201709002064150, 1 March 2017 ].

Anaemia following Artemisinin-Based Combination Treatments of Uncomplicated Plasmodium falciparum Malaria in Children: Temporal Patterns of Haematocrit and the Use of Uncomplicated Hyperparasitaemia as a Model for Evaluating Late-Appearing Anaemia.

BACKGROUND: In severe malaria, intravenous artesunate may cause delayed haemolytic anaemia but there has been little evaluation of the propensity of oral artemisinin-based combination treatments (ACTs) to cause late-appearing anaemia. METHODS: The frequency of anaemia (haematocrit <30%), and temporal changes in haematocrit were evaluated in 1,191 malarious children following ACTs. "Haematocrit conservation" was evaluated by using the fall in haematocrit/1,000 asexual parasites cleared from the peripheral blood (FIH/1,000 asexual parasites cpb), and the ratio of the average haematocrit (on the first 3 days of starting treatment):total parasitaemia cleared. RESULTS: The frequency of anaemia decreased significantly following treatment. FIH/1,000 asexual parasites cpb, average haematocrit:total parasitaemia cleared, and mean haematocrit 5 weeks after treatment began were significantly lower in hyperparasitaemic children than in children without hyperparasitaemia, suggesting haematocrit conservation during treatment followed later by a loss of haematocrit. Asymptomatic late-appearing anaemia occurred in 6% of the children. CONCLUSION: Artesunate-amodiaquine and artemether-lumefantrine contribute to haematocrit conservation at high parasitaemias but may cause late-appearing anaemia.

Therapeutic efficacy and effects of artemisinin-based combination treatments on uncomplicated Plasmodium falciparum malaria -associated anaemia in Nigerian children during seven years of adoption as first-line treatments.

BACKGROUND: Artemisinin-based combination treatments (ACTs) are the first-line treatments of uncomplicated Plasmodium falciparum malaria in many endemic areas but there are few evaluation of their efficacy in anaemic malarious children. METHODS: Therapeutic efficacy of 3-day regimens of artesunate-amodiaquine and artemether-lumefantrine was evaluated in 437 anaemic and 909 non-anaemic malarious children following treatment during a seven-year period (2008-2014). Patterns of temporal changes in haematocrit were classified based on haematocrit values <30% and ≥30%. Kinetics of the disposition of the deficit in haematocrit from 30% following treatment were evaluated using a non-compartment model. RESULTS: PCR-corrected parasitological efficacy 28 days after start of treatment was significantly higher in artesunate-amodiaquine- compared to artemether-lumefantrine-treated children [97% (95%CI: 92.8-100) versus 96.4% (95%CI: 91.3-99.4), P = 0.02], but it was similar in non-anaemic and anaemic children. Fall in haematocrit/1 000 asexual parasites cleared from peripheral blood was significantly greater at lower compared to higher parasitaemias (P < 0.0001), and in non-anaemic compared to anaemic children (P = 0.007). In anaemic children at presentation, mean anaemia recovery time (AnRT) was 15.4 days (95%CI: 13.3-17.4) and it did not change over the years. Declines in haematocrit deficits from 30% were monoexponential with mean estimated half-time of 1.4 days (95%CI: 1.2-1.6). Anaemia half-time (t½anaemia) correlated positively with AnRT in the same patients (r = 0.69, P < 0.0001). Bland-Altman analysis of 10 multiples of t½anaemia and AnRT showed narrow limit of agreement with insignificant bias (P = 0.07) suggesting both can be used interchangeably in the same patients. CONCLUSIONS: Artesunate-amodiaquine and artemether-lumefantrine remain efficacious treatments of uncomplicated P. falciparum infections in non-anaemic and anaemic Nigerian children in the last 7 years of adoption as first-line treatments. These ACTs may also conserve haematocrit at high parasitaemias and in anaemic children. TRIALS REGISTRATION: Pan African Clinical Trial Registry PACTR201508001188143 , 3 July 2015; PACTR201510001189370 , 3 July 2015; PACTR201508001191898 , 7 July 2015 and PACTR201508001193368 , 8 July 2015.

Status of the use and compliance with malaria rapid diagnostic tests in formal private health facilities in Nigeria.

BACKGROUND: Nigeria has the largest number of malaria-related deaths, accounting for a third of global malaria deaths. It is important that the country attains universal coverage of key malaria interventions, one of which is the policy of universal testing before treatment, which the country has recently adopted. However, there is a dearth of data on its implementation in formal private health facilities, where close to a third of the population seek health care. This study identified the level of use of malaria rapid diagnostic testing (RDT), compliance with test results and associated challenges in the formal private health facilities in Nigeria. METHODS: A cross-sectional study that involved a multi-stage, random sampling of 240 formal private health facilities from the country's six geo-political zones was conducted from July to August 2014. Data were collected using health facility records, healthcare workers' interviews and an exit survey of febrile patients seen at the facilities, in order to determine fever prevalence, level of testing of febrile patience, compliance with test results, and health workers' perceptions to RDT use. RESULTS: Data from the 201 health facilities analysed indicated a fever prevalence of 38.5% (112,521/292,430). Of the 2077 exit interviews for febrile patients, malaria testing was ordered in 73.8% (95% CI 71.7-75.7%). Among the 1270 tested, 61.8% (719/1270) were tested with microscopy and 38.2% (445/1270) with RDT. Compliance to malaria test result [administering arteminisin-based combination therapy (ACT) to positive patients and withholding ACT from negative patients] was 80.9% (95% CI 78.7-83%). Compliance was not influenced by the age of patients or type of malaria test. The health facilities have various cadres of the health workers knowledgeable on RDT with 70% knowing the meaning, while 84.5% knew what it assesses. However, there was clearly a preference for microscopy as only 20% reported performing only RDT. CONCLUSION: In formal private health facilities in Nigeria there is a high rate of malaria testing for febrile patients, high level of compliance with test results but relatively low level of RDT utilization. This calls for improved engagement of the formal private health sector with a view to achieving universal coverage targets on malaria testing.

Assessment of Markers of Antimalarial Drug Resistance in Plasmodium falciparum Isolates from Pregnant Women in Lagos, Nigeria.

BACKGROUND: The use of antimalarial drugs for prevention and treatment is a major strategy in the prevention of malaria in pregnancy. Although sulphadoxine-pyrimethamine (SP) is currently recommended for intermittent preventive treatment of malaria during pregnancy in Nigeria, previously used drugs for prophylaxis such as chloroquine (CQ) and pyrimethamine are accessible as they are purchased over the counter. This study describes the markers of absence or presence of resistance to quinoline (Pfcrt and Pfmdr 1) and type 1 antifolate antimalarial medicines (Pfdhfr). METHODS: Plasmodium falciparum-positive dried blood spots from pregnant women attending antenatal clinics for the first time during current pregnancy were investigated for the presence of mutations at codons 72-76 of Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene by real time polymerase chain reaction (PCR) using haplotype-specific probes. PCR followed by sequence analysis was used to identify mutations at codons 86, 184, 1034, 1042 and 1246 of P. falciparum multi-drug resistance-1 (Pfmdr1) gene; and codons 16, 50, 51, 59, 108, 140 and 164 of Pfdhfr gene. RESULTS: Two haplotypes of Pfcrt (n = 54) were observed: CVMNK 13(24.2%) and CVIET 41 (75.9%) of the samples. The SVMNT haplotype was absent in this population. The Pfmdr1 (n = 28) haplotypes were NYSND 15(53.6%), YYSND 5(17.9%), NFSND 6(21.4%) and YFSND 2(7.1%). The Pfdhfr (n = 15) were ACNCSVI 4(26.7%), and ACICNSVI 1(6.7%) and ACIRNVI 10 (66.7%). The rate of occurrence of Pfcrt 76T, Pfdhfr108N, Pfmdr186Y and 184F were 75.9%, 73.3%, 25% and 28.1% respectively. The Pfmdr1 86Y was associated with low parasitaemia (median = 71 parasites/µl, P = 0.024) while Pfcrt 76T was associated with young maternal age (mean 24.1 ± 4.5 years; P = 0.006). The median parasitaemia were similar (P>0.05) in wild and mutant strains of Pfcrt 76, Pfmdr1 184 and Pfdhfr 108. There was no association between gravidity or gestational age of the women and presence of mutations in the Pfcrt, Pfmdr1 or Pfdhfr genes (P>0.05). CONCLUSION: Markers of resistance to chloroquine and pyrimethamine were high, whereas cycloguanil-resistance marker was not present in the studied population. The low level of mutations in the Pfmdr1gene indicates likely efficacy of amodiaquine against malaria in pregnancy.

Assessment of competence of participants before and after 7-day intensive malaria microscopy training courses in Nigeria.

Background: The accuracy of malaria diagnosis by microscopy has been a challenge in health facilities in Nigeria due to poor competence of microscopists and inability to report on malaria species other than Plasmodium falciparum. Short microscopy courses were conducted to improve the skills of laboratory personnel to perform malaria microscopy in public health facilities in Nigeria. Materials and Methods: Seven-day malaria microscopy courses were conducted annually between 2011 and 2013 for microscopists in public health facilities. The training courses contained theoretical and practical sessions. Impact of the training was evaluated by practical and theoretical pre- and post-training assessments on malaria slide reading, parasite enumeration and basic malariology. Results: The 102 participants who completed the training consisted of medical laboratory scientists (62; 60.8%), medical laboratory technicians (24; 23.5%) and other healthcare workers (16; 15.7%). The knowledge of basic malariology (theory) at pre- and post-tests were 34% (95% CI 31.7-36.3%) and 74.9% (95% CI 71.8-78.0%), respectively (P<0.001). The mean slide reading detection, species and counting agreements in pre-training assessment were 48.9%, 27.9% and 0%, respectively, and in post-training 56.8%, 39.2% and 25%, respectively. The mean species agreements in picture test pre- and post-training were 21.9% and 55.1%, respectively. There were significant differences (P<0.05) in the median pre-test scores in picture tests and basic malariology of the three categories of participants but not in malaria slide reading and parasite counting tests. However, post-training, a significant difference in test scores of the three categories of participants was recorded only for basic malariology (P=0.0003). Conclusions: The 7-day malaria microscopy courses significantly increased the knowledge and microscopy skills of the trainees and were sufficient to bridge the significant difference in baseline microscopy skills of the different categories of trainees that participated in the training courses.

Efficacy of artemisinin-based combination treatments of uncomplicated falciparum malaria in under-five-year-old Nigerian children.

The efficacy of 3-day regimens of artemether-lumefantrine and artesunate-amodiaquine were evaluated in 747 children < 5 years of age with uncomplicated malaria from six geographical areas of Nigeria. Fever clearance was significantly faster (P = 0.006) and the proportion of children with parasitemia 1 day after treatment began was significantly lower (P = 0.016) in artesunate-amodiaquine-compared with artemether-lumefantrine-treated children. Parasite clearance times were similar with both treatments. Overall efficacy was 96.3% (95% confidence interval [CI] 94.5-97.6%), and was similar for both regimens. Polymerase chain reaction-corrected parasitologic cure rates on Day 28 were 96.9% (95% CI 93.9-98.2%) and 98.3% (95% CI 96.1-99.3%) for artemether-lumefantrine and artesunate-amodiaquine, respectively. Gametocyte carriage post treatment was significantly lower than pretreatment (P < 0.0001). In anemic children, mean time to recovery from anemia was 10 days (95% CI 9.04-10.9) and was similar for both regimens. Both treatments were well tolerated and are safe and efficacious treatments of uncomplicated falciparum malaria in young Nigerian children.

Genetic diversity and complexity of Plasmodium falciparum infections in Lagos, Nigeria.

OBJECTIVE: To analyse the genetic diversity of Plasmodium falciparum (P. falciparum) using msp-1 and msp-2 as antigenic markers. METHODS: Parasite DNA was extracted from 100 blood samples collected from P. falciparum-positive patients confirmed by microscopy, and followed by PCR-genotyping targeting the msp-1 (block2) and msp-2 (block 3) allelic families. RESULTS: All the families of msp-1 (K1, MAD20 and R033) and msp-2 (FC27 and 3D7) locus were observed. Results revealed that K1 (60/100) was the most predominant genotype of msp-1 allelic family followed by the genotypes of MAD20 (50/100) and R033 (45/100). In the msp-2 locus, FC27 genotype (62/100) showed higher frequency than 3D7 genotype (55/100). The allelic families were detected either alone or in combination with other families. However, no R033/MAD20 combination was observed. Multiplicity of infection (MOI) with msp-1 was higher in the locality of Ikorodu (1.50) than in Lekki (1.39). However, MOI with msp-2 was lower in the locality of Ikorodu (1.14) than in Lekki (1.76). There was no significant difference in the mean MOI between the two study areas (P=0.427). CONCLUSIONS: The observation of limited diversity of malaria parasites may imply that the use of antigenic markers as genotyping tools for distinguishing recrudescence and re-infections with P. falciparum during drug trials is subjective.

Persistence of markers of chloroquine resistance among P. falciparum isolates recovered from two Nigerian communities.

Background: A recovery in chloroquine efficacy following a period of cessation has raised the possibility of its reintroduction for malaria chemotherapy. We investigated the prevalence of the major markers of chloroquine resistance years after the withdrawal of the drug in Nigeria. Materials and Methods: Finger prick blood samples were collected from participants presenting with symptoms of malaria in two selected health centres each representing Lekki and Ijede communities of Lagos, Nigeria. Thick and thin blood smears were prepared for microscopy and dry blood spots made from malaria-positive participants for parasite DNA extraction. The detection of mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance (pfmdr1) genes was performed by nested polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Results: Of the 1527 blood samples that were confirmed by PCR to be P. falciparum positive, 412 and 344 were typed for the molecular detection of pfcrt and pfmdr1 gene mutations, respectively. The mutant alleles of pfcrt were present among 290 (70%) parasite carriers while the pfmdr1 mutant allele was found in 117 (34%) of the total population. There were higher distributions of the mutant alleles for the two loci in Ijede than in Lekki. The observed frequencies of pfcrt mutant alleles in the two parasite populations were in agreement with the expected frequencies predicted by Hardy-Weinberg. In comparing data with studies conducted between 2000 and 2002 in Ijede, we observed an increase in the prevalence of mutant type pfcrt against a marginal decline in the pfmdr1 mutant type. Conclusion: The high frequencies of pfcrt mutation are suggestive of a persistent drug pressure and continuing inefficacy of chloroquine as an antimalarial drug.

Factors associated with risk of malaria infection among pregnant women in Lagos, Nigeria.

BACKGROUND: Pregnant women living in an area of stable malaria transmission such as Lagos, Nigeria, have been identified as being at an increased risk of the effects of malaria infection. In this area, most of the infections are asymptomatic which means they are overlooked and untreated much to the detriment of the mother and her foetus. The reality of scaled-up malaria interventions with long-lasting insecticide treated nets, vector control, artemisinin combination therapy (ACT) and intermittent preventive treatment of malaria pregnancy (IPTp) using sulphadoxine pyrimethamine (SP) is that it is also essential to determine the risk factors at play in these kinds of circumstances. This study was aimed at identifying the factors associated with risk of malaria infection in pregnant women in Lagos, Southwest Nigeria. METHODS: Demographic information and malaria prevention practices of the pregnant women studied were captured using structured questionnaire. Microscopy was used to establish malaria infection, species identification and parasite density. Relative risk and multivariate logistic regression analysis were used to compare factors associated with malaria in pregnant women. RESULTS: Malaria microscopy details, demographic information and malaria prevention practices of the pregnant women were obtained using a structured questionnaire. The prevalence of malaria using peripheral blood from 1,084 pregnant women that participated in the study was 7.7%. Plasmodium falciparum (P. falciparum) was seen in 95.2% of the cases as either mixed infection with P. malariae (3.6%) or as a mono infection (91.6%). Malaria preventive practices associated with a significant reduction (P<0.05) in the malaria infection was the use of insecticide sprays (RR = 0.36, 95 C.I. 0.24-0.54), and the combined use of insecticide spray and insecticide-treated nets (ITN) (RR= 6.53, 95% C.I. 0.92-46.33). Sleeping under ITN alone (RR = 1.07, 95% C.I. 0.55-2.09) was not associated with significant reduction in malaria infection among the study participants with malaria parasitaemia. Young maternal age (<20years) (RR = 2.86, 95% C.I. 1.48 - 5.50), but not primigravidity (RR = 1.36, 95% C.I. 0.90-2.05), was associated with an increased risk of malaria infection during pregnancy. After a multivariate logistic regression, young maternal age (OR = 2.61, 95% C.I. 1.13 - 6.03) and the use of insecticide spray (OR = 0.38, 95% C.I. 0.24-0.63) were associated with an increase and a reduction in malaria infection, respectively. CONCLUSION: Malaria prevalence was low among the pregnant women studied. Young maternal age and non-usage of insecticidal spray were the main factors associated with an increased risk of malaria infection among pregnant women in Lagos, Nigeria.

Malariometric survey of ibeshe community in ikorodu, lagos state: dry season.

Malariometric surveys generate data on malaria epidemiology and dynamics of transmission necessary for planning and monitoring of control activities. This study determined the prevalence of malaria and the knowledge, attitude, and practice (KAP) towards malaria infection in Ibeshe, a coastal community. The study took place during the dry season in 10 villages of Ibeshe. All the participants were screened for malaria. A semistructured questionnaire was used to capture sociodemographic data and KAP towards malaria. A total of 1489 participants with a mean age of 26.7 ± 20.0 years took part in the study. Malaria prevalence was 14.7% (95% CI 13.0-16.6%) with geometric mean density of 285 parasites/µL. Over 97% of participants were asymptomatic. Only 40 (2.7%) of the participants were febrile, while 227 (18.1%) were anemic. Almost all the participants (95.8%) identified mosquito bite as a cause of malaria, although multiple agents were associated with the cause of malaria. The commonest symptoms associated with malaria were hot body (89.9%) and headache (84.9%). Window nets (77.0%) were preferred to LLIN (29.6%). Malaria is mesoendemic in Ibeshe during the dry season. The participants had good knowledge of symptoms of malaria; however, there were a lot of misconceptions on the cause of malaria.

Application of stool-PCR for the diagnosis of Helicobacter pylori from stool in Nigeria- a pilot study.

There are various methods for detection of Helicobacter pylori and the gold standard for non-invasive detection is the urea breath test (UBT). The aim of the study is therefore to detect H. pylori from the stool of patients with dyspepsia by PCR and compare results obtained with UBT. A total of 97 stool samples from patients presenting with dyspeptic symptoms in Lagos University Teaching Hospital (LUTH) were screened for urea breath test (UBT) and the presence of H. pylori DNA using stool-PCR. Out of 97 stool samples analysed, 38 (39.2%) were positive for Helicobacter spp. and 20 (20.6%) positive for H. pylori by PCR, through amplification of 16S rRNA and glmMgenes respectively. Of the 20 positive by glmM gene, the cagAgene was detected in 8 (40%) samples, while 47 (48.5%) out of 97 stool samples were positive for H. pylori by UBT. The sensitivity and specificity of the glmM gene compared with UBT as the gold standard is 42.6% and 100% respectively. The positive predictive value (PPV) was 100% while the negative predictive value (NPV) was 60%.The method may be useful for detecting H. pylori from stool amongst children especially where most hospitals lack endoscope for children although the method is expensive.

Scaling up of intermittent preventive treatment of malaria in pregnancy using sulphadoxine-pyrimethamine: prospects and challenges.

Intermittent preventive treatment of malaria during pregnancy with sulphadoxine-pyrimethamine (IPTpSP) is one of the major strategies of malaria control in most African countries where malaria is endemic. The use of sulphadoxine-pyrimethamine (SP) for intermittent preventive treatment of malaria during pregnancy was adopted when proof of its superiority to weekly prophylactic dosing with either chloroquine or pyrimethamine became evident from studies in different malaria endemic countries. The administration of 2 and 3 treatment doses of SP for HIV-negative and HIV-positive pregnant women respectively, given after quickening and at an interval not less than 4 weeks was recommended. The prospects of this control strategy lies on the efficacy of SP, convenient treatment dose and high compliance rate. However, the implementation of this strategy and the efficacy of SP are faced with challenges such as: timing of SP administration, rising levels of parasite resistance to SP in the general population, effect of folate supplementation, adequacy of the recommended doses with regards to malaria endemicity and HIV status, interactions between SP and antiretroviral drugs and low coverage in the bid to scale-up its use. This review highlights the prospects and challenges of scaling up IPTp-SP.

Parasitologic Assessment of Two-Dose and Monthly Intermittent Preventive Treatment of Malaria during Pregnancy with Sulphadoxine-Pyrimethamine (IPTP-SP) in Lagos, Nigeria.

Intermittent preventive treatment of malaria with sulphadoxine-pyrimethamine (IPTP-SP) is a key strategy in the control of malaria in pregnancy. However, reports of increasing level of resistance to SP using nonpregnant populations have made it imperative for the continuous monitoring of the efficacy of SP in pregnant women. This study assessed using microscopy, monthly dosing and the standard two-dose regimen among 259 pregnant women attending antenatal clinics in Lagos, Nigeria that consented 122 in the two-dose arm (Arm A) and 137 in the monthly dose arm (Arm B). Baseline parasitaemia in the two groups was 5 (4.1%) and 3 (2.2%) in Arms A and B, respectively. Few of the women developed parasitaemia after the initial SP dose in Arms A 4 (3.3%) and B 2 (1.5%). However, none of the women had malaria infection after the second dose in both Arms. Although IPTP-SP is suggestive of protecting the women from malaria infection, there was no significant difference observed between the two dosing schemes.