Fixation of pfcrt chloroquine resistance alleles in Plasmodium falciparum clinical isolates collected from unrest tribal agencies of Pakistan

Abstract Plasmodium falciparum resistance to Chloroquine (CQ) is a significant cause of mortality and morbidity worldwide. There is a paucity of documented data on the prevalence of CQ-resistant mutant haplotypes of Pfcrt and Pfmdr1 genes from malaria-endemic war effected Federally Administered Tribal Areas of Pakistan. The objective of this study was to investigate the prevalence of P. falciparum CQ-resistance in this area. Clinical isolates were collected between May 2017 and May 2018 from North Waziristan and South Waziristan agencies of Federally Administrated Trial Area. Subsequently, Giemsa-stained blood smears were examined to detect Plasmodium falciparum. Extraction of malarial DNA was done from microscopy positive P. falciparum samples, and P. falciparum infections were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes). All PCR confirmed P. falciparum samples were sequenced by pyrosequencing to find out mutation in Pfcrt gene at codon K76T and in pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y. Out of 121 microscopies positive P. falciparum cases, 109 samples were positive for P. falciparum by nested PCR. Pfcrt K76T mutation was found in 96% of isolates, Pfmdr1 N86Y mutation was observed in 20%, and 11% harboured Y184F mutation. All samples were wild type for Pfmdr1 codon N1042D and D1246Y. In the FATA, Pakistan, the frequency of resistant allele 76T remained high despite the removal of CQ. However, current findings of the study suggest complete fixation of P. falciparum CQ-resistant genotype in the study area.


Fixation of pfcrt chloroquine resistance alleles in Plasmodium falciparum clinical isolates collected from unrest tribal agencies of Pakistan
The current study is designed to investigate CQ-resistance patterns based on Pfcrt-76T and Pfmdr1 N86Y/Y184F markers among P. falciparum isolates collected from North and South Waziristan agencies of FATA, KP.

Study locations and ethics
FATA is a mountainous region of northwest Pakistan, bordering Afghanistan. Unrest and political destabilization in this region had destroyed public health infrastructure and health care system leading to the emergence of different infectious and vector-borne diseases like malaria. This study's objective is to check the pattern of CQ-resistance in P. falciparum from malaria symptomatic patients between May 2017 and May 2018, irrespective of age and gender. About 5 mL of anti-coagulated blood was collected by the venipuncture technique after taking proper consent from parents/guardians. The exclusion criteria were non-consent and other species of Plasmodium. The study was approved by the Advanced Studies and the Research Board University of Gujrat, Pakistan.

Sample collection and microscopy
About 5 mL of anti-coagulated venous blood specimen was collected from malaria symptomatic patients. Thin and thick blood smears were prepared and stained with 10% Giemsa stain. Smears were examined under 1000X magnification of light microscope by trained microscopist, and remaining blood specimen was stored in -20 °C for molecular drug resistance analysis.

DNA extraction and speciation
Parasitic DNA was extracted from 121 microscopy confirmed P. falciparum positive samples using DNA mini kit (Qiagen) using blood samples according to the manufacturer instructions. P. falciparum specie was reconfirmed by two rounds of nested polymerase chain reaction (nPCR). Amplification of Plasmodium (genus) DNA was done in the primary round nPCR using genus-specific primers and speciation in the second round of nPCR. Published Primers, ingredients concentration, and thermal cycler conditions were used (Snounou et al., 1993;Soares-Pinheiro et al., 2017). Nested PCR of 121 samples confirmed 109 samples positive for P. falciparum; 4 were with mixed-species infection, and the rest were not amplifiable. MR4 clones MRA-340G, MRA-343G, 3D7, and HB3, were used as positive, whereas sterile water was used as a negative control, as shown in Figure 1.

Pyrosequencing of Pfcrt and Pfmdr1 genes
Previously published pyrosequencing protocol was used to analyze CQ resistance mutations in Pfcrt and Pfmdr1 genes of P. falciparum (Khattak et al., 2013a). The amplified product was resolved on QIAxcel capillary electrophoresis (Qiagen) with a 50 bp DNA ladder, as shown in Figure 2.

Introduction
Malaria still ranks as the third-highest morbidity and mortality, causing disease globally. About 3.2 billion peoples in 97 countries and territories are at risk of malaria. About 214 million malaria cases have been reported, with an estimated 0.4 million deaths mostly caused by P. falciparum. Malaria is the 6 th biggest threat of morbidity, and Pakistan's mortality could affect 205 million people, with 0.3 million cases each year (WHO, 2020;DMC, 2019;Khattak et al., 2013a). According to the World Health Organization (WHO), the Federally Administrated Tribal Area (FATA) is the second-highest malaria-endemic region (23%) of Pakistan after Khyber Pakhtunkhwa (KP) province (31%). However, FATA is on top of all provinces and territories regarding annual parasite incidence, blood examination, and test positivity rate (DMC, 2019).
Chloroquine (CQ) remained the standard treatment for P. falciparum malaria for more than five decades. In Pakistan, the first clinical case of CQ-resistance in P. falciparum was reported in 1984; however, many in vivo and in vitro CQ resistance reports are available (Bouma et al., 1996;Ghanchi et al., 2011;Khattak et al., 2013a, b;Rawasia et al., 2012;Yaqoob et al., 2018).
CQ resistance in P. falciparum results from mutations in two genes Plasmodium falciparum CQ-resistant transporter (Pfcrt) and Plasmodium falciparum multidrug resistance transporter 1 (Pfmdr1) gene. Both encode trans-membrane CQ transporter proteins present on the membrane of P. falciparum digestive vacuole. These transporter proteins are responsible for the transport and gathering of CQ in the parasite's food vacuole, which is the site for CQ action, while mutations in these genes alter the efflux of CQ from the intracellular digestive vacuole (Plowe, 2009;Vieira-Neta et al., 2021). Resistance to CQ is mediated by mutations at codons 72-76 (C72S/R, M74I/T, N75E/D/K/I, and K76T/I/N) P. falciparum Pfcrt gene, results in amino acid substitutions from wild type CVMNK to two most prevalent mutant haplotypes CVIET and SVMNT present in African and South-East Asian countries respectively. These mutations have been associated with CQ reduced sensitivity; however, mutation at positive 76 with substitution of K with T is the key determinant mutation for in vitro (Fidock et al., 2000;Wellems and Plowe, 2001) and in vivo CQ-resistance (Djimde et al., 2001). Similarly, mutations at N86Y, Y184F, S1034C, N1042D, and D1246Y in the Pfmdr1 gene interfere with CQ transportation to food vacuole resulting in reduced CQ susceptibility to P. falciparum (Foote et al., 1990;Mallick et al., 2012;Petersen et al., 2011).

Results
A total of 121 microscopy confirmed P. falciparum clinical isolates were collected from North and South Waziristan agencies of FATA, KP. Nested PCR revealed 109 P. falciparum microscopy positive samples, and 04 samples were mixed for P. falciparum plus P. vivax, and the rest were not amplifiable. Mixed species and negative isolates were excluded from the study after the re-extraction of DNA and re-amplification ( Figure 3). All 109 P. falciparum positive samples were analyzed for drug resistance polymorphism in Pfcrt and Pfmdr1. CQ coffering drug resistance mutation K76T was found in 105 (96%) samples. This mutation in the Pfcrt gene is associated with reduced CQ sensitivity in P. falciparum (Bray et al., 2005;Fidock et al., 2000), as shown in Figure 4. In N86Y, a mutant allele was found in 22 (20%), and 12 (11%) isolates harbored Y184F mutation. Not a single sample showed Pfmdr1 S1034C and N1042D mutation, as shown in Table 1.

Discussion
This is the first molecular assessment of P. falciparum CQ-resistance in this war-affected North and South Waziristan agencies of FATA Pakistan. In earlier 2008, CQ and SP were the only choice of drugs for malaria treatment in Pakistan, after the anti-malarial drug policy for uncomplicated P. falciparum was changed to (AS+SP) (artesunate plus sulphadoxine-pyrimethamine). However, the use of CQ as mono-therapy is still there in many health care setups (Ghanchi et al., 2011;Khattak et al., 2013a;Koenderink et al., 2010).
Pfcrt K76T mutation associated with CQ resistance was found in 96% of isolates, and similar findings have been reported in other studies (Khan et al., 2020;Yaqoob et al., 2018;Khattak et al., 2013a;Khatoon et al., 2009). Pfcrt K76T mutation and SVMNT haplotype in all samples are in accordance with results reported on samples collected from Pakistan, India, Iran, and Saudi Arabia (Ocan et al., 2019;Zomuanpuii et al., 2020;Khan et al., 2020; Bin Dajem and Al-Qahtani, 2010) Studies from Papua New Guinea and South American also reported SVMNT haplotype that was found in our isolates, whereas CVIET haplotype is more prevalent in Asia/Africa (Yang et al., 2007;Wootton et al., 2002) Pfcrt K76T mutation and SVMNT haplotype had been associated with CQ resistance (Plowe, 2009).
These findings indicate complete fixation of CQ-resistance in P. falciparum is might possibility because of very high CQ-resistance in Pakistan could be due to the co-existence of P. falciparum with P. vivax infections, and CQ is the recommended anti-malarial treatment for P. vivax malaria treatment (WHO, 2020, Mallmann et al., 2018. Misdiagnosis of co-infection by conventional light microscopy is widespread in resource-limited areas (Mayxay et al., 2001). Different studies have reported mixed species malaria infections in Pakistan (Khatoon et al., 2009;Khattak et al., 2013b).
Many studies have uncovered the role of the mdr1 gene family in modulating the sensitivity of different anti-malarial, including CQ (Duraisingh et al., 1997;Price et al., 2004;Wilson et al., 1993;Warhurst, 2001). Mutations in pfmdr1 and pfcrt genes have been associated with malarial infection severity as reports from India, the Gambia, Iranian, Angola, Tanzania, and eastern Sudan (Vathsala et al., 2004;Figueiredo et al., 2008;Khalil et al., 2005;Meerman et al., 2005;Zakeri et al., 2008). Mutations that further increase CQ-resistance are N86Y and Y184F; these were observed in 15% and 7% isolates, respectively. Isolates carrying wild type and mutant allele were counted as mutant.
Our results are similar to the studies carried out in Pakistani, Iranian, and Indianan isolates (Khatoon et al., 2009;Pathak et al., 2014;Jalousian et al., 2008). Few mutations in the Pfmdr1 gene and its single copy suggest that artesunate and (AS+SP) partner drugs such as mefloquine and lumefantrine are likely to have high efficacy in Pakistan. The presence of Pfmdr1 mutations at codons N86Y and Y184F of Pfmdr1 and Pfcrt mutation at K76T leads to an elevated level of CQ-resistance as investigated previously (Mayengue et al., 2007;Babiker et al., 2001;Reed et al., 2000). Warhurst (2001) believed that the Pfcrt gene mutation imparts a basic level of CQ-resistance; however, an additive effect in CQ resistance is observed with Pfmdr1 mutations.

Conclusion
Execution of complete withdrawal of CQ drug after WHO anti-malarial policy revised was not fully adopted; thus, its continued access and use could have contributed to the persistence of CQ resistance alleles in this region. As K76T pfcrt mutation has been fixed in P. falciparum so proper Plasmodium species detection should be done, CQ for uncomplicated P. falciparum malaria should be used strongly be discouraged as it is a first-line treatment for P.  Malaria Journal, vol. 7, no. 1, pp. 236. http://dx.doi.org/10.1186/1475-2875-7-236. PMid:19014684. FOOTE, S., KYLE, D., MARTIN, R., ODUOLA, A., FORSYTH, K., KEMP, D. and COWMAN, A., 1990.