IDENTIFICATION OF PLASMODIUM FALCIPARUM HISTIDINE-RICH PROTEIN II AND III (PFHRP2/3) GENE DELETIONS IN TWO COMMUNITIES IN SOUTHERN GHANA: IMPLICATIONS ON RAPID DIAGNOSTIC TESTS

ABSTRACT

Malaria Rapid Diagnostic Tests (RDT’s) have improved malaria diagnosis in highly endemic rural settings. However, the increasingly high false negative rates of Plasmodiumfalciparum histidine-rich protein II (PfHRP2) based RDT kits (PfHRP2-RDT) is a major obstacle to the rapid and reliable diagnosis of malaria. This study was aimed at determining the rate of false negative RDT as well as the prevalence of P. falciparum parasites with pfhrp2 deletions in selected communities in the Southern Ghana. Whole blood was collected from volunteers living in Obom (high transmission) and Asutsuare(low transmission) and separated into plasma and cell pellet. Genomic DNA was extracted from 310 cell pellets from both sites using the ZymoDNA Kit^®. Species-specific 18srRNAPCR was used to identify P.falciparum positive samples. Pfmsp2 and glurp genotyping was used to determine recrudescence or new infection. Good quality DNA samples were then subjected to pfhrp2 exon 1 and 2 PCR as well as pfhrp3 exon 2 PCR. PfHRP2 antigen level was determined using a pfhrp2 Malaria Ag CELISA kit. Microscopy estimation of malaria parasites were 3.3 % of samples from Asutsuare against 39.8 % of Obom samples. The RDTs had 1.7 % of samples from Asutsuare while Obom had 53.4 %. Using 18srRNAPCR for P. falciparum speciation, 59.1 % of the Asutsuare samples tested positive for the malaria parasite whereas 65.8 % of the Obom samples tested positive.

Plasmodiumfalciparumparasites with deletions of both pfhrp2 and pfhrp3 gene were 1.7 % in Obom and 4.8 % in Asutsuare. An R-square value of 0.997 and 0.994 were obtained for Obom and Asutsuare Regression Analysis of ELISA respectively. Deletions of pfhrp2 and pfhrp3 genes were identified in the two study sites and there were higher quantities of PfHRP2 antigens in Obom than in Asutsuare.

CHAPTER ONE

INTRODUCTION

Background

Malaria is a disease of the poor, which significantly affect endemic countries’ economies (Kumar et al., 2013). For complete eradication of malaria to be achieved, all forms of the disease need to be diagnosed and treated. Malaria diagnosis is expensive when specificity and accuracy is desired for prompt treatment. Microscopy has been the gold standard for malaria diagnosis for many years following various staining procedures (WHO, 2010). There are other tests which have also been used in the diagnosis for the infection. Recently, prompt treatments of malaria have largely been due to the introduction of Rapid Diagnostic Tests (RDTs). The use of RDT has been a very essential component of malaria diagnosis (Hanscheid, 1999; Kumar et al., 2013; Wongsrichanalai, Barcus, Muth, Sutamihardja & Wernsdorfer, 1999; Wongsrichanalai, 2001), and has improved diagnosis in endemic and resource constraint settings (Makler, Palmer & Ager, 1998). In areas where there are no microscopists, RDTs are the major diagnostic tool for malaria infection diagnosis.

The malaria RDT principle is based on antigens or antibodies for detection. The most widely used diagnostic RDT for Plasmodium falciparum is based on detection of the histidine rich protein II (HRP2) antigen. Plasmodium falciparum has several hrp genes but only hrp2 is used in RDTs. The P. falciparum hrp (pfhrp2) gene is heat stable, abundant in the host’s blood and can persist for 28 days or more after parasite clearance (Iqbal, Siddique, Jameel & Hira, 2004; Kumar et al., 2012). The P. falciparum histidine-rich protein III (PfHRP3) antigens are known to have similar structure as the P. falciparum histidine-rich protein II (PfHRP2) antigens. Thus, they are recognised by the PfHRP2 antibodies. The PfHRP2 based RDT kits have the highest detection rate (World Health Organization-Foundation for Innovative New Diagnostics, 2015), but are also known to give inaccurate results with false positives and false negatives (Gamboa et al., 2010). One main demerit in malaria RDT diagnosis without the confirmation of microscopy and its related parasite density is false negative results. This may be due to low parasite densities or P. falciparum histidine-rich protein II (pfhrp2) gene deletions which have not been compensated for by the P. falciparum histidine-rich protein III (pfhrp3) gene (Bartoloni & Zammarchi, 2012). Adaptation of the Plasmodium parasite to fit in the constant environmental changes has led to certain P. falciparum strains having their entire pfhrp2 and pfhrp3 gene deleted (Cheng et al., 2014) or others having varying amounts of PfHRP2 antigen (Houze, Hubert, Le Pessec & Clain, 2011; Ho et al., 2014)). This, affects the test accuracy (Baiden et al., 2014; Houze, Boly, Le Bras, Deloron & Faucher, 2009; Kattenberg et al., 2012; World Health Organization, 2015; Wurtz et al., 2013).

Current trends in the use of RDT has resulted in significant improvements in infection rate reporting from different countries in Africa. However, to date, a number of all these countries, including Democratic Republic of Congo, Kenya,

Mali, Senegal and Zambia have reported pfhrp2/3 gene deletions (Deme et al., 2014; Kabayinze et al., 2008; Parr et al., 2016; Wurtz et al., 2013).

The demand for malaria RDT kits in malaria diagnosis have increased since the WHO recommended its usage in 2009 (WHO, 2015). Presently, most National Malaria Control Programs (NMCPs) in endemic countries are using RDTs as the initial step of disease diagnosis. To monitor the use of RDTs, WHO instituted the Foundation for Innovative New Diagnostics (FIND) for quality assurance of RDT, which includes Lot Testing (WHO-FIND, 2015). This has helped in taking out most of the unapproved RDTs from the health system (Cunningham, 2013). There are many different tests which can be used for diagnosis of malaria infection aside RDTs. However, improvement of RDTs is very essential for clinical diagnosis in the communities where there are no microscopists and good equipment for infection diagnosis.

The basis of any disease control scheme for community and individual level is the availability of suitable diagnostic tools, which are highly sensitive (200 p/µL) and 99 % specific (WHO, 2010). Without these, interventions put in place cannot be tracked and suitable treatment not provided. PfHRP2 RDTs have been used in recent times as the most readily available and fast diagnostic method for malaria infections (Cohen, Dupas & Schaner, 2015; Baker et al., 2010). However, their false negative and positive rates have given much room for criticisms (Kumar et al., 2013). It has also brought about treatment burdens (Cohen, et al., 2015), since most false positives may be given treatment for malaria where there is actually no infection. This can cause hepatotoxicity leading to liver damage from drug burden. False negatives also bring about no treatments of the disease and so persons living with the infection become reservoirs for the spread of the parasite as well as give the parasite more time for building drug resistance. This could be the reason why deaths of malaria are still recorded in this age when Artemisinin-based Combination Therapies (ACTs) have proven to be more effective for the treatment of malaria. This can cause low productivity and thus affect economies where pfhrp2/pfrhp3 deletions are higher.

Low transmission areas have very few malaria cases as well as more clonal parasite infections and so other diseases which present similar symptoms are looked at and treated when PfHRP2 RDTs are negative for individuals living in the area. However, the results may be false negative which may be due to pfhrp2/pfhrp3 gene deletions or low antigen levels of the PfHRP2 (Gamboa et al., 2010). As a result, studies on gene deletions in low transmission areas are necessitated. On the other hand, high transmission areas were often seen as malaria prone and thus individuals presenting symptoms of malaria were still treated for malaria even when there were no parasites in the body (WHO, 2010). However, the current standard treatment guidelines require treatment only after individuals have tested positive for malaria RDT or microscopy (WHO, 2017).

In rural health facilities, malaria is often ruled out after RDT is tested negative, however, the patient may still be harbouring the parasites in his/her body. A study in Uganda determined that while 73% of febrile patients received antimalarials, only 35% had positive RDT results, and overall appropriate treatment was only 34% (Mbonye, Lal, Cundill, Hansen, Clarke & Magnussen, 2013). This may be due to gene deletions and false positives which made the RDT unable to capture the P. falciparum infection at the time as well as antigen persistence in the blood. These pfhrp2/pfhrp3 deletants should be extensively studied in low transmission and high transmission areas to be able to find appropriate diagnostic method for high and low transmissions alike.

For more Molecular Biology & Biotechnology Projects Click here

===================================================================

Item Type: Ghanaian Topic  |  Size: 123 pages  |  Chapters: 1-5

Format: MS Word  |  Delivery: Within 30Mins.

===================================================================

DOWNLOAD THIS PROJECT MATERIAL NOW! CLICK HERE!

Share: