EVALUATION OF THE PERFORMANCE OF HISTIDINE-RICH PROTEIN-2 BASED RAPID DIAGNOSTIC TESTSAND ALLELIC DIVERSITY OF PLASMODIUM FALCIPARUM GENES IN IBADAN, SOUTHWESTERN NIGERIA

Abstract

Misdiagnosis of malaria exposes patients to drug pressure which may contribute to the emergence of anti-malarial drug resistance. The WHO recommends the use of malaria Rapid Diagnostic Tests (mRDTs) to improve malaria diagnosis in resource limited settings. However, deletion of Plasmodium falciparum Histidine Rich Protein-2 and 3 (Pfhrp-2/3) gene contribute to the proportion of false negative mRDT results. Also, information on the allelic diversity of Plasmodium falciparum genes are essential in understanding the parasite dynamics and control mechanisms. In this study the PfHRP-2-based mRDT sensitivity, Pfhrp-2/3 gene deletion and the allelic diversity of merozoite surface proteins 1 and 2 (msp1/msp2) and glutamate-rich protein (glurp) genes in Ibadan, southwest Nigeria were evaluated. Ethical approval was provided by the UI/UCH ethics committee (UI/EC/12/0279) and a written informed consent obtained from parents/guardians before patients enrolment. Finger-prick blood samples were collected from 511 febrile children age 3–59 months in a Primary Healthcare Center, Idi-Ayunre and St. Mary’s Catholic Hospital Eleta, Ibadan for mRDT screening, thick blood smears for microscopy and Dried Blood Spot (DBS) on filter paper (Whatman 3mm) for molecular genotyping. The mRDT positive children were treated with artesunate-amodiaquine at standard dosage. Giemsa-stained blood films were examined microscopically and genomic DNA was extracted from DBS using commercial kit. Nested PCR was performed using specific primers to amplify the 18SrRNA, exon-2 regions of Pfhrp-2/3, msp1, msp2 and glurp genes. The sensitivity, specificity, positive/negative predictive values (PPV/NPV), accuracy and kappa’s value of mRDT was estimated using microscopy as the gold standard. The DNA sequence of Pfhrp-2/3 genes were determined by SANGER method and the sequencesobtained were analyzed using BioEdit software. Data were analyzed using descriptive statistics and Chi-square at α0.05. Prevalence of malariausing mRDT, microscopy and PCR was 59.6%, 43.5% and 51.3 % respectively (ρ < 0.001). Based on PCR, P. falciparum accounted for 91.6% (228/249; alone/or in combination) while 23.3% (57/249) occurred as P. malariae, P. ovale or mixed infection with P. falciparum.P. vivax was not detected. The sensitivity of mRDT against microscopy was 95.2%, specificity 67%, accuracy 79%, PPV 68%, NPV 94.9% and kappa’s statistics was 0.58. Similarly, PCR performance against microscopy was; sensitivity 84%, specificity 67%, accuracy 76%, PPV 73%, NPV 80% and kappa’s statistics 0.51. Thirty one samples (12.4%) were RDT negative (false negative) but PCR positive out of which 8 (25.8%)and 4 (12.9%) had Pfhrp-2 and 3 gene deletion, respectively. The sequencedPfhrp-2/3 PCR products were successfully translated to amino acid. The Pfhrp-2/3 amino acid repeat sequences were highly diverse. Extensive allelic diversity of P. falciparummsp1,msp2 and glurp genes were also observed. The RO33 and 3D7 allelic familiesof msp1 and msp2were the most predominant. The allelic frequency for msp1, msp2 and glurp was 80.1%, 68.5% and 50.9%, respectively. The presence of parasites lacking Plasmodium falciparum histidine rich protein - 2 and 3 genes and extensive allelic diversity are contributory factors for the false negative results associated with malaria Rapid Diagnostic Tests and high transmission of malaria respectively in Ibadan.