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.

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.

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 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.

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.

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.

Prevalence of malaria in pregnant women in Lagos, South-West Nigeria.

Prevalence rates reported for malaria in pregnancy in Nigeria vary considerably. The accuracy of results of malaria diagnosis is dependent on training, experience, and motivation of the microscopist as well as the laboratory facility available. Results of training programmes on malaria microscopy have shown low levels of sensitivity and specificity of those involved in malaria diagnosis routinely and for research. This study was done to ascertain the true prevalence of malaria in pregnancy in Lagos, South-West Nigeria. A total of 1,084 pregnant women were recruited into this study. Blood smears stained with Giemsa were used for malaria diagnosis by light microscopy. Malaria infection during pregnancy presents mostly as asymptomatic infection. The prevalence of malaria in this population was 7.7% (95% confidence interval; 6.2-9.4%). Factors identified to increase the risk of malaria infection include young maternal age (< 20 years), and gravidity (primigravida). In conclusion, this study exposes the over-diagnosis of malaria in pregnancy and the need for training and retraining of laboratory staffs as well as establishing the malaria diagnosis quality assurance programme to ensure the accuracy of malaria microscopy results at all levels.

Congenital Toxoplasmosis: a Review of its Pathology; Immune Response and Current Treatment Options

Toxoplasmosis; caused by the protozoan parasite Toxoplasma gondii; is one of the most common parasites of man and other warm-blooded animals. Humans are infected through contaminated food; water; and blood transfusion; organ transplantation or from mother to foetus through the placenta. Severe congenital infections occur as a result of primary T. gondii infection in early pregnancy. Transmission of T.gondii to the foetus can result in serious health problems; including mental retardation; seizures; blindness and death. Frequency of foetal infection is higher when maternal infection occurs later in pregnancy and sequelae are more severe when maternal infections occur early in the first trimester of pregnancy. The ability of the parasite to survive intracellularly largely depends on the blocking of different proapoptotic signaling cascades of the host cells. During pregnancy; however; alterations in the incidence of apoptosis are associated with abnormal placental morphology and function. Both cellular and humoral immune responses control T.gondii infection. Toxoplasma is asymptomatic; infected women can only be detected by serological testing. In many instances; congenital toxoplasmosis can be prevented by educating pregnant women and women of childbearing age about the route of transmission. The need for screening suspected cases of T. gondii will help reduce transmission to the foetus