Olusola Ojurongbe

Rapid detection of Pfcrt and Pfmdr1 mutations in Plasmodium falciparum isolates by FRET and in vivo response to chloroquine among children from Osogbo, Nigeria.

BackgroundChloroquine (CQ) has been in use in Africa for a long time. Because of misuse, this drug has now lost its efficacy due to the emergence of resistance strains in most parts of Africa. Recently, it was shown that after chloroquine has been withdrawn from the market, chloroquine-sensitive Plasmodium falciparum re-emerged and chloroquine could again be used successfully as an antimalarial. Surveillance of parasite populations is, therefore, important to decide whether chloroquine could be re-introduced.MethodsTo estimate the prevalence of the most pivotal polymorphisms, including Pfcrt K76T, Pfmdr1 N86Y and Pfmdr1 Y184F mutations, and their contributions to the outcome of CQ treatment, isolates from Osogbo Western Nigeria were tested using the Fluorescence Resonance Energy Transfer (FRET) method on a real-time PCR instrument.Results116 children with acute uncomplicated P. falciparum malaria infections were treated with the standard dosage of CQ and followed-up for 28 days. Blood samples were collected on filter paper at enrollment and during follow-up for identification of parasite carrying the chloroquine resistant transporter (pfcrt) and P. falciparum-multi drug resistance (pfmdr1) gene mutations. Parasitological assessment of response to treatment showed that 62% of the patients were cured and 38% failed the CQ treatment. The presence of single mutant pfcrt (T76) alleles (P = 0.003) and in combination with mutant pfmdr1 Y86 (P = 0.028) was significantly associated with in vivo CQR. No other mutation on its own or in combinations was significantly associated with treatment outcome. Mutant pfcrt was more prevalent in both pre- and post-treatment isolates. No association was observed between age or initial level of parasitaemia and chloroquine treatment outcome.ConclusionThe result established the usefulness and accuracy of real time PCR in pfcrt and pfmdr1 mutation detection and also give further evidence to the reliability of the pfcrt T76 point mutation as a molecular marker for CQ resistance.

Ficolin-2 Levels and FCN2 Genetic Polymorphisms as a Susceptibility Factor in Schistosomiasis

BACKGROUNDnHuman ficolin-2 (L-ficolins) encoded by the FCN2 gene are pattern-recognition proteins involved in innate immunity and are associated with several infectious diseases.nnnMETHODSnA Nigerian cohort of 168 Schistosoma haematobium-infected individuals and 192 healthy controls were examined for functional single-nucleotide polymorphisms in the promoter region (-986G>A, -602G>A, -4A>G) and in exon 8 (+6424G>T) using real-time polymerase chain reaction.nnnRESULTSnThe FCN2 -986A and -4G alleles were significantly associated with the occurrence of schistosomiasis (P = .0004 for -986G>A; P = .0001 for -4A>G). The heterozygous genotypes (P = .0006 for -986G>A; P = .0002 for -4A>G) were observed to be a risk factor for susceptibility to schistosomiasis, whereas the homozygous genotypes of major alleles (P = .0002 for -986G>A; P = .0001 for -4A>G) were observed to shield against schistosomiasis. The haplotype AGGG (P = .0002) was observed to be a risk factor for susceptibility to schistosomiasis compared with controls, and the haplotype GGAG (P = .04) was observed to confer protection compared with patients. Ficolin-2 serum level was significantly higher in controls (P < .005) and in controls with GGAG haplotypes (P < .0001).nnnCONCLUSIONSnOur findings demonstrate that FCN2 promoter variants (-986G>A and -4A>G) influence ficolin-2 serum levels and susceptibility to schistosomiasis.

Mannose binding lectin and susceptibility to Schistosomiasis

Abstract Background.u2003Human ficolin 2 (encoded by FCN2) and mannose-binding lectin (encoded by MBL2) bind to specific pathogen-associated molecular patterns, activate the complement lectin cascade in a similar manner, and are associated with several infectious diseases. Our recently published study established certain FCN2 promoter variants and ficolin-2 serum levels as protective factors against schistosomiasis. Methods.u2003We used the Nigerian cohort from our recently published study, which included 163 Schistosoma haematobium–infected individuals and 183 matched healthy subjects, and investigated whether MBL deficiency and MBL2 polymorphisms are associated with schistosomiasis. Results.u2003MBL serum levels were significantly higher in controls and were associated with protection (P < .0001). The −550H minor allele was significantly associated with protection (P = .03), and the heterozygous genotypes −550HL were observed to confer protection (P = .03). The MBL2*HYPA haplotype was significantly associated with protection (P = .03), with significantly higher serum MBL levels in controls (P = .00073). The heterozygous 6-bp deletion in the promoter was observed to be a susceptibility factor in schistosomiasis (P = .03). Conclusions.u2003In agreement with findings from our recently published study, the findings reported here support the observation that MBL is also associated with protection in schistosomiasis.

In-vitro studies on the sensitivity pattern of Plasmodium falciparum to antimalarial drugs and local herbal extracts

BackgroundThe resistance of human malaria parasites to anti-malarial compounds has become considerable concern, particularly in view of the shortage of novel classes of anti-malarial drugs. One way to prevent resistance is by using new compounds that are not based on existing synthetic antimicrobial agents.ResultsSensitivity of 100 Plasmodium falciparum isolates to chloroquine, quinine, amodiaquine, mefloquine, sulphadoxine/pyrimethamine, artemisinin, Momordica charantia (‘Ejirin’) Diospyros monbuttensis (‘Egun eja’) and Morinda lucida (‘Oruwo’) was determined using the in vitro microtest (Mark III) technique to determine the IC50 of the drugs. All the isolates tested were sensitive to quinine, mefloquine and artesunate. Fifty-one percent of the isolates were resistant to chloroquine, 13% to amodiaquine and 5% to sulphadoxine/pyrimethamine. Highest resistance to chloroquine (68.9%) was recorded among isolates from Yewa zone while highest resistance to amodiaquine (30%) was observed in Ijebu zone. Highest resistance to sulphadoxine/pyrimethamine was recorded in Yewa and Egba zones, respectively. A positive correlation was observed between the responses to artemisinin and mefloquine (P<0.05), artemisinin and quinine (P<0.05) and quinine and mefloquine (P<0.05). A negative correlation was observed between the responses to chloroquine and mefloquine (P>0.05). Highest anti-plasmodial activity was obtained with the ethanolic extract of D. monbuttensis (IC50 = 3.2nM) while the lowest was obtained from M. lucida (IC50 =25nM).ConclusionsNatural products isolated from plants used in traditional medicine, which have potent anti-plasmodial action in vitro, represent potential sources of new anti-malarial drugs.

Occult Hepatitis B Virus Infection in Nigerian Blood Donors and Hepatitis B Virus Transmission Risks

Background Occult hepatitis B virus infection (OBI) characterized by the absence of detectable HBsAg remains a potential threat in blood safety. We investigated the actual prevalence, viral factors and genotype of OBI infections in Nigerian blood donors. Methods Serum collected from two blood banks were reconfirmed as HBsAg seronegative by ELISA. Forty HBsAg positive samples were employed as controls. HBV-DNA was amplified from all donors and viral loads were determined using quantitative real-time PCR. Antibodies to the HBV core, surface and HBe antigen (anti-HBc,anti-HBs,HBeAg) were measured. The PreS/S and PreC/C regions of the HBV genome were sequenced. Results Of the 429 blood donors, 72(17%) were confirmed as OBI by DNA detection in different reference labs and excluded the concern of possible contamination. Of the 72 OBI samples, 48(67%) were positive for anti-HBc, 25(35%) positive for anti-HBs, and 2(3%) positive for HBeAg. Of the 72 OBI samples, 31(43%) were seropositive for either anti-HBc, anti-HBs or HBeAg, 21 (30%) positive for both anti-HBc and anti-HBs,one positive for both anti-HBc and HBeAg. None of the OBI samples were positive for all three serological markers. The viral load was <50copies/ml in the OBI samples and genotype E was predominant. The L217R polymorphism in the reverse transcriptase domain of the HBV polymerase gene was observed significantly higher in OBI compared with HBsAg positive individuals (P<0.0001). Conclusion High incidence of OBI is relevant in high endemic areas worldwide and is a general burden in blood safety. This study signifies the high prevalence of OBI and proposes blood donor samples in Nigeria should be pre-tested for OBI by nucleic acid testing (NAT) and/or anti-HBc prior to transfusion to minimize the HBV infection risk.

Reliable and rapid characterization of functional FCN2 gene variants reveals diverse geographical patterns

BackgroundFicolin-2 coded by FCN2 gene is a soluble serum protein and an innate immune recognition element of the complement system. FCN2 gene polymorphisms reveal distinct geographical patterns and are documented to alter serum ficolin levels and modulate disease susceptibility.MethodsWe employed a real-time PCR based on Fluorescence Resonance Energy Transfer (FRET) method to genotype four functional SNPs including -986u2009Gu2009>u2009A (#rs3124952), -602u2009Gu2009>u2009A (#rs3124953), -4Au2009>u2009G (#rs17514136) and +6424u2009Gu2009>u2009T (#rs7851696) in the ficolin-2 (FCN2) gene. We characterized the FCN2 variants in individuals representing Brazilian (nu2009=u2009176), Nigerian (nu2009=u2009180), Vietnamese (nu2009=u2009172) and European Caucasian ethnicity (nu2009=u2009165).ResultsWe observed that the genotype distribution of three functional SNP variants (−986u2009Gu2009>u2009A, -602u2009Gu2009>u2009A and -4Au2009>u2009G) differ significantly between the populations investigated (pu2009<u20090.0001). The SNP variants were highly linked to each other and revealed significant population patterns. Also the distribution of haplotypes revealed distinct geographical patterns (pu2009<u20090.0001).ConclusionsThe observed distribution of the FCN2 functional SNP variants may likely contribute to altered serum ficolin levels and this may depend on the different disease settings in world populations. To conclude, the use of FRET based real-time PCR especially for FCN2 gene will benefit a larger scientific community who extensively depend on rapid, reliable method for FCN2 genotyping.

Molecular surveillance of drug-resistant Plasmodium falciparum in two distinct geographical areas of Nigeria

ZusammenfassungDie Entwicklung von therapie-resistenter Plasmodium falciparum-Malaria ist seit langem als Haupthindernis zur Bekämpfung von Mortalität und Morbidität erkannt. Wir haben daher die Verbreitung von Genveränderungen, die mit der Resistenz gegen Chloroquine und Pyrimethamin assoziiert sind, in P. falciparum-Isolaten aus zwei geographisch distinkten Gegenden in Nigeria untersucht. Mit Hilfe von RT-PCR und DNA-Sequenzierung wurde die Prävalenz dieser Mutationen bestimmt. Die Prävalenz der pfcrt T76-Mutation in den beiden Gegenden war 92.3 % gegen 86 % in Osogbo verglichen mit 93 % in Lafia (P = 0.4453). Sequenzanalyse des pfcrt Haplotyps (Aminosäuren 72–76) ergab CVIET als einzigen resistenten Haplotyp an beiden Orten. Die Häufigkeit von pfmdr1-Polymorphismen war höher in Lafia (39 %) als in Osogbo (35 %); die kombinierte Prävalenz in beiden Orten war 45.5 % (P = 0.6604). Die Prävalenz der pfdhfr-Triplemutante war hoch in beiden Gegenden: in Osogbo 84 % gegenüber 91 % in Lafia für I51, sowie 88 % gegen 87 % und 96 % gegen 96 % für die R59 and N108 Mutationen. Die kombinierte Prävalenz von pfcrt- und pfmdr1–Mutationen in Osogbo und Lafia war 44.2 % mit einem Risiko von 0.4164; die kombinierte Prävalenz aller Genveränderungen in pfcrt, pfmdr1 and pfdhfr war 40.4 % mit einem Risiko von 1.081. Diese Ergebnisse zeigen die weite Verbreitung der Resistenz gegen Chloroquin und Pyrimethamin in beiden untersuchten Regionen.SummaryDrug resistance against P. falciparum has been recognized as the crucial obstacle to curbing mortality and morbidity from malaria. We therefore determined the baseline distribution of pfcrt and pfmdr1 genes associated with resistance to chloroquine and pfdhfr gene associated with resistance to pyrimethamine in P. falciparum isolates collected from two geographically distinct areas of Nigeria. We use RT-PCR assays and sequencing to determine the prevalence of these mutations. The combined prevalence of pfcrt T76 mutation in the two sites was 92.3% with 86% from Osogbo compared to 93% from Lafia. Sequencing analysis of the (Pfcrt) K76T haplotype (amino acids 72–76) revealed CVIET as the only resistance haplotype present in the two areas. The frequency of pfmdr1 polymorphisms was higher in Lafia (39%) compared to that in Osogbo (35%) and the combined prevalence from the two sites was 45.5%. The prevalence of the pfdhfr triple mutant alleles was high in both locations. The Osogbo vs Lafia prevalence for pfdhfr mutations was 84% vs 91%, 88% vs 87% and 96% vs 96% for I51, R59 and N108, respectively. None of the samples from the two locations had the T108 mutation. The combined prevalence of pfcrt and pfmdr1 in Osogbo and Lafia was 44.2% with a risk ratio of 0.4164 while the combined prevalence of pfcrt,pfmdr1 and pfdhfr was 40.4% with a risk ratio of 1.081. These results strongly suggest the widespread distribution of CQ and pyrimethamine resistance without any marked distinction between the two locations.

Lectin Complement Protein Collectin 11 (CL-K1) and Susceptibility to Urinary Schistosomiasis

Background Urinary Schistosomiasis is a neglected tropical disease endemic in many sub Saharan -African countries. Collectin Kidney 1 (CL-K1, encoded by COLEC11 on chromosome 2p25.3), a member of the vertebrate C-type lectin super family, has recently been identified as pattern-recognition molecule (PRR) of the lectin complement pathway. CL-K1 is preferentially expressed in the kidneys, but also in other organs and it is considered to play a role in host defense to some infectious agents. Schistosome teguments are fucosylated and CL-K1 has, through its collagen-like domain, a high binding affinity to fucose. Methodology/Principal Findings We utilized a Nigerian study group consisting of 167 Schistosoma haematobium infected individuals and 186 matched healthy subjects, and investigated the contribution of CL-K1 deficiency and of COLEC11 polymorphisms to infection phenotype. Higher CL-K1 serum levels were associated with decreased risk of schistosome infection (Pcorr = 0.0004). CL-K1 serum levels were differentially distributed between the COLEC11 genotypes and haplotypes observed. The non-synonymous variant p.R216H was associated with the occurrence of schistosomiasis (OR = 0.44, 95%CI = 0.22–0.72, Pcorr = 0.0004). The reconstructed COLEC11*TCCA haplotypes were associated with higher CL-K1 serum levels (P = 0.002) and with decreased schistosomiasis (OR = 0.38, 95%CI = 0.23–0.63, Pcorr = 0.0001). Conclusions In agreement with findings from our earlier published study, our findings support the observation that CL-K1 and their functional variants may be host factors associated with protection in schistosomiasis and may be a useful marker for further investigations.

Correlation of Interleukin-6 levels and lectins during Schistosoma haematobium infection.

Urogenital schistosomiasis caused by Schistosoma haematobium induces a Th2 immune response, including expression of Interleukin-6. IL-6 confers protection from experimental Schistosoma-induced pulmonary hypertension and modulates production of mannose-binding lectin (MBL) and other lectins. We studied IL-6 levels in schistosomiasis and its effect on lectins production. Elevated IL-6 levels occurred in cases, compared to controls. IL-6 correlated with the lectins MBL, ficolin-2 and Collectin Kidney-1 (CL-K1) in cases, but correlated inversely in controls. The study shows that IL-6 levels are elevated in individuals infected with urogenital schistosomiasis. IL-6 was also found to be correlated with the production of lectins in S. haematobium infection. A similar correlation between IL-6 and MBL was observed during visceral leishmaniasis.

High prevalence of dihydrofolate reductase gene mutations in Plasmodium falciparum parasites among pregnant women in Nigeria after reported use of sulfadoxine-pyrimethamine

Abstract This study assesses the prevalence of asymptomatic Plasmodium falciparum parasitemia positivity and P. falciparum dihydrofolate reductase (pfdhfr) mutations in parasite isolates among pregnant women in Southwest Nigeria. Plasmodium falciparum parasitemia was confirmed by microscopy and nested PCR in 200 pregnant women attending antenatal care. The prevalence of pfdhfr polymorphisms was determined by direct sequencing of the gene fragments containing the C50R, N51I, C59R, S108N, and I164L mutations. Information on the use of antimalarial drugs and methods applied to prevent malaria were obtained by a questionnaire. The prevalence of asymptomatic P. falciparum infection was 30% (60/200). The frequency of the pfdhfr triple-mutant alleles (N51I, C59R, and S108N) was 63% (38/60); none of the isolates carried the I164L mutation. Among the investigated pregnant women, 40% used un-prescribed antimalarials such as dihydroartemisinin (18%), chloroquine (14%) or pyrimethamine (9%), while only 20.5% used sulfadoxine-pyrimethamine for prevention and 39.5% did not use any drug. The prevalence of P. falciparum parasitemia (37%) was higher among pregnant women who had not taken any antimalarial drugs. A significant difference in the prevalence of the pfdhfr triple-mutant alleles was observed among women who took SP (90%) compared to those who did not take any drug (82%) and women who took dihydroartemisinin (67%) p = 0.007). Poor adherence to the World Health Organisation (WHO) strategies for malaria prevention among pregnant women was observed in addition to high prevalence of pfdhfr mutations. These findings underline the need to improve control of malaria among pregnant women in the study area.