Chemotherapy Resistance Status of Common Human Pathogenic Protozoa

For thousands of years, people have used the bark, roots, and leaves of plants such as the bark of the cinchona tree, which contains quinine (QN) as remedies for malaria (Noronha et al. 2020). However, the extraction and employment of their active components as pharmaceutical drugs did not occur until the last century (Semedo et al. 2021). Throughout malaria treatment history, pharmacological research derived the most field appreciated compound, chloroquine (CQ), from QN (Berberian 1947). Incomplete patient compliance exposed Plasmodium parasites to intense drug selection pressure, leading them to develop resistance mechanisms (Waithera et al. 2023). In addition to vector control, emerging antimalarial resistance is one of the current obstacles to malaria prevention. Some of the available anti-malaria drug arsenals include QN, CQ, mefloquine (MQ), halofantrine (HF), lumefantrine (LF), quinacrine, sulfadoxine-pyrimethamine (SP), atovaquone (AQ), proguanil (PR), primaquine (PRQ), amodia quine (ADQ), piperaquine (PPQ), clindamycin, doxycycline, tetracycline, tafenoquine (TFQ), artemisinin (ART), artesunate (ARN), artemether (ARM), and other ART derivatives (Meshnick and Dobson 2001; Tse et al. 2019). Moreover, with the emergence of resistance to CQ, researchers initiated investigations on CQ resistance (CQR) in the 1960s, which led to the adoption of MQ and HF, two more drugs that met a similar fate to CQ in the 1980s (Amelo and Makonnen 2021). A genetic-cross experiment revealed that the membrane transport protein PfCRT (P. falciparum CQ resistance transporter) is essential for CQR and localizes to the food vacuole (FV), where hemoglobin is degraded (Fidock et al. 2000). Researchers have investigated the protein for its ability to transport numerous synthetic compounds. Still, they have not yet identified the physiological substrate. PvCRT, the homolog of PfCRT in P. vivax, is not believed to be connected to CQR, whereas PfCRT in falciparum is (Marques et al. 2014). Instead, PvMDR1 (P. vivax multidrug resistance protein 1), a homolog of human p-glycoprotein, is the suggested implicated gene (Schousboe et al. 2015). The ABC (ATP binding cassette) transporter PfMDR1 in P. falciparum, located on the FV membrane, is also believed to influence CQR (Reed et al. 2000). Mutations in the PfMDR1 gene (N86Y) and the PfCRT gene (K76T and A220S, respectively) confer substantial resistance to CQ (Nsobya et al. 2007). In addition to CQR, mutant PfCRT was suggested to confer resistance to PPQ and ADQ, while PfMDR1 to MQ, ART, and QN (Wicht et al. 2020). These and other changes impact the relative abundance of parasite populations by conferring fitness costs (Pulcini et al. 2015). The K76T substitution in PfCRT exhibits notable regional variability across Africa, with particularly high frequencies observed in Ethiopia (91.6%) and Mali (72.9%), compared to lower frequencies in DRC (26%) and non-African nations like India (78%) and Turkey (12.1%) (Patel et al. 2017; Hassen et al. 2022; Avcı et al. 2024; Baina et al. 2024; Dama et al. 2024). Meanwhile, the N86Y mutant in the PfMDR1 gene shows significant geographic variation as well: it occurs at 6.8% in DRC and 10.2% in Niger and rises dramatically to 78.4% in Oceania (Issa et al. 2022; Moss et al. 2022; Baina et al. 2024). Remarkably, CQ-sensitive strains have returned to countries like Ghana and Cote d’Ivoire after switching to ART-based combination therapy (ACT) instead of CQ for some years. This fact has raised the possibility of CQ returning to the field as it used to be (Asare et al. 2021). Furthermore, the parasite expresses PfMRP1, an ABC transporter located on membrane-bound vesicles and its plasma membrane. This protein promotes the transport of organic anionic substrates, such as oxidized glutathione, sulfate conjugates, and drugs. Two mutations in the PfMRP1 locus at positions Y191H and A437S were identified as being associated with resistance to QN and CQ (Gil and Fançony 2021). Although CQR remains a persistent issue, CQ is currently employed to treat uncomplicated CQ-sensitive (CQS) malaria caused by any of the five human pathogenic Plasmodium species (CDC 2024b). Some compounds, such as SP, stop the production of folate by blocking enzymes called P. falciparum dihydropteroate synthase (PfDHPS) and dihydrofolate reductase-thymidylate synthase (PfDHFR-TS). Biochemical and genetic investigations of P. falciparum reveal that mutations in these genes diminish the drug sensitivity of antifolates (Pacheco et al. 2020). When the S108N mutation is present, other changes in the inhibitor binding region of PfDHFR, like A16V, N51I, C59R, and I164L, make antifolate resistance even worse. P. falciparum’s resistance to cycloguanil is linked to the PfDHFR double mutations A16V and S108T (Staines et al. 2018). In Thailand and other Southeast Asian regions, P. vivax is naturally resistant to sulfadoxine and has obtained resistance to pyrimethamine (Imwong et al. 2001). In a study, SP treatment failure occurred in over 50% of P. vivax patients due to mutations in PfDHFR and PfDHFR-TS homologs in P. vivax PvDHPS and PvDHFR-TS, which are reserved with mutations described to cause resistance in P. falciparum, suggesting a possible association (Tjitra et al. 2002). AQ is a mitochondrial electron transport inhibitor that disrupts parasite respiration by binding to cytochrome b (cyt-b) and inhibiting ubiquinol oxidation. P. falciparum isolates with a single mutation in the Pfcyt-b gene, specifically in the Y268N/S/C codon, exhibited AQ resistance (Staines et al. 2018). It is usually com bined with PR (Malarone™) for uncomplicated malaria caused by P. falciparum. Moreover, ARTs have been documented to bind to many parasite proteins and influence various cellular and organellar processes. Once activated, the carbon-centred radical of the heme drug accelerates the production of additional cytotoxic reactive oxygen species (ROS) through a cluster bomb-like effect, ultimately leading to cell death (O’Neill et al. 2010). The first-line treatment for malaria, known as ACTs, has been widely used since the early 2000s (CDC 2024b). It is based on the idea of combining two drugs, one with a shorter half-life (such as ART or an ART derivative) and one with a longer one (such as a quinolone). ACTs are most frequently administered in the following combinations: (ARM + LF), (ARN + MQ), (ARN + AM), and (dihydroartemisinin + PPQ) (Pluijm et al. 2021; CDC 2024b). Whole-genome sequencing of an ART-resistant parasite linked the P. falciparum Kelch 13 (K13) to ART resistance mutations in both clinical and field isolates of P. falciparum (Xie et al. 2020). The C580Y mutation in K13 has been strongly linked to reduced susceptibility to artemisinin-based treatments, with a particularly high prevalence in Southeast Asia, notably within the Greater Mekong Subregion, including Cambodia, Myanmar, and Thailand, where it occurs in 35.5% of cases (Takala-Harrison et al. 2015; Yoshida et al. 2021). In contrast, the K189T mutation in K13 is the dominant variant in Africa, with a prevalence of 22.8% (Hung et al. 2024). On the other hand, in vitro investigations have shown a probable link between a Y976F mutation in the PvMDR1 gene and resistance to ARN and MQ; however, additional clinical trials are necessary to understand this association fully (Cowell and Winzeler 2019). Moreover, mutations in the Na⁺/H⁺ exchanger gene Pfnhe-1 have been reported to confer resistance to QN, but this is still a debatable matter of research (Andriantsoanirina et al. 2013). For relapse prevention because of P. vivax and P. ovale liver stages, either PRQ or a newer drug called TFQ is used. While some studies indicate the effectiveness of a single dose of TFQ against P. vivax, testing on patients with blood disorders or young children remains to be done, necessitating further research (Rodrigo et al. 2020). A summary of the medications to be administered to malaria-infected patients is illustrated in Table I. The table illustrates the 1^st line of common malaria treatment regimens based on the Centers for Disease Control and Prevention (CDC) guidelines and displays the status of DR (CDC 2024b). Researchers are currently investigating, testing, or inventing various potential vaccines against malaria.

On October 6, 2021, the World Health Organization (WHO) recommended RTS,S/AS01 (Mosquirix™), a malaria vaccine developed by GlaxoSmithKline Biologicals, for broad usage. The sporozoite surface protein (CSP) is the basis of the subunit vaccine RTS,S/AS01. The vaccine comprises CSP fused with hepatitis B surface antigen (HBsAg) and other virus-like particles of extra HBsAg that have not been fused (Hammershaimb and Berry 2024). The WHO recommended RTS,S/AS01 for children aged 5 months and older living in regions with moderate to high malaria transmission. Currently, available malaria vaccines reduce the risk of uncomplicated malaria by approximately 40%, severe malaria by approximately 30%, and mortality by approximately 13% (WHO 2023). The CDC recommends administering malaria medicines in conjunction with other control measures.

There are only a handful of anti-Leishmania drugs available, and they all have serious drawbacks such as toxicity, expensive manufacturing costs, and low efficiency. Sodium stibogluconate (Pentostam®) and meglumine antimoniate (Glucantime®) are two examples of the several pentavalent antimonials (Sb^V) that are available as systemic therapy. Amphotericin B (AMB), pentamidine (PMD), fluconazole (FLZ), and miltefosine (MT) are additional compounds that are currently in use. Sb^V compounds are typically used in many countries as the first-line treatment (Herwaldt and Berman 1992). In Latin America, Sb^vs represent the gold standard for CL treatment (Mann et al. 2021). Several regions have seen the emergence of resistance in the past few years, including India, Europe, the Middle East, and South America (Wijnant et al. 2022). The resistance pathway is not clear yet. However, several ABC transporters, such as ABCI4 and ABCG2, were thought to contribute to drug efflux mechanisms (Ponte-Sucre et al. 2017). It was also suggested that MRP1 in Sb^v-resistant L. donovani strains reduces drug accumulation by reducing drug import functions (Mukherjee et al. 2007). The aquaglyceroporin (AQP1) transporter is another potential factor in Sb^V resistance; its inactivation may decrease Sb^V absorption, and AQP1 mutations may increase resistance (Potvin et al. 2020). The ergosterol antagonist AMB is utilized in regions where Sb^V treatment is hindered by resistance (Pinart et al. 2020). No resistance has been reported except in laboratory-pressured isolates that showed mutations in CYP51, SC5D, and SMT (Pountain et al. 2019). Moreover, an oral medication known as MT is thought to block Leishmania parasites’ phospholipid metabolism. MT resistance has been found in both in vitro and in vivo tests in L. donovani when the MT translocation pathway has mutations or deletions (Carnielli et al. 2019). Experimentally induced mutations in the L. donovani MT transporter (LdMT) and LdRos3 have been reported to affect drug transport (Srivastava et al. 2017). Another suggested resistance mechanism to MT is reduced drug accumulation by overexpression of ABCB4, ABCG4, and ABCG6 (Pérez-Victoria et al. 2011). Moreover, another drug that has shown promise against both CL and VL is paromomycin (PMM), which has a cure rate of up to 80%. The widespread application of PMM in VL treatment is challenged by the relatively high post-treatment relapse rates, which call for proper implementation and monitoring of the development of resistance (Sosa et al. 2019). The underlying causes of resistance and the specific mutations responsible remain largely unresolved and require further investigation. Table II summarizes the status of Leishmania treatment and DR status in general.

Anti-toxoplasma medications primarily target the folate pathway, an enzyme complex including the dihydrofolate reductase (DHFR) and the dihydropteroate synthetase (DHPS). This complex is involved in DNA synthesis (Lapinskas and Ben-Harari 2019). It is noteworthy to mention that no medication available today can eliminate T. gondii tissue cysts from the infected host; instead, they remain dormant within the host as long as the immune system is robust enough to prevent the cysts from reactivating and becoming tachyzoites (Konstantinovic et al. 2019). Spiramycin (SPI) is the preferred treatment for acute T. gondii infection during pregnancy (at least for the 1^st trimester) due to the potential for PYR to result in congenital abnormalities (Bogacz 1954). In cases when fetal toxoplasmosis is identified beyond week 16 of gestation, the treatment is typically substituted with PYR and sulfadiazine (SDZ) because SPI has trouble crossing the placenta barrier (Robert-Gangneux et al. 2011). Regrettably, there are significant adverse effects associated with the combination. Both PYR and SDZ suppress the DNA synthesis in tachyzoites of T. gondii and may have a similar impact on specific host organs, even bone marrow. Hence, incorporating folinic acid (FA) into the drug combination can prevent these negative consequences, which are restored when the medication is stopped, as seen in previous studies (Prusa et al. 2015). Various combinations are being investigated to find a better treatment option with fewer adverse effects since treatment failure has been linked to either DR, malabsorption, or intolerance of the current combination (Silva et al. 2017). Clarithromycin, cotrimoxazole, AQ, dapsone, and azithromycin are other medications used to treat toxoplasmosis. However, these treatments do not work against the bradyzoites form (Dunay et al. 2018). Because of the lack of conclusive evidence from in vitro research, identifying genes imparting resistance to PYR and SDZ is an area of dispute (Meneceur et al. 2008; Doliwa et al. 2013). However, treating clinical toxoplasmosis with AQ is possible if SPI is unavailable (SA Maternal & Neonatal Clinical Network 2015). Like P. falciparum, AQ kills Toxoplasma at its chronic bradyzoite stage by blocking the mitochondrial electron transport chain. Evidence suggests that AQ binds to the Qo domain of cyt-b, hence targeting the Toxoplasma cyt-bc1 enzyme (Alday et al. 2017). Significant mutational alterations in M129L and I254L on the Qo domain are thought to be responsible for T. gondii’s resistance to AQ. However, this theory has not been validated by further research (McFadden et al. 2000). Furthermore, the treatment of immunocompromised individuals is based on the same medications used to treat congenital toxoplasmosis, demonstrating how limited our existing ammunition is and emphasizing the importance of searching and testing for more and safer compounds (Hajj et al. 2021). Table III summarizes the status of T. gondii’s standard treatment and DR status in general.

Several anti-trypanosomal drugs are available to use in clinics, including PMD, suramin (SUR), melarsoprol (Mel^b), eflornithine (EFL), fexinidazole (FXZ), and nifurtimox (NFX). PMD is a drug administered to treat the initial phase of the illness caused by T.b.g (Bouteille et al. 2003). There are several routes via which the medication enters parasite cells. Partial transport is facilitated by the aminopurine transporter P2, also known as T.b. aminopurine transporter 1 (TbAT1). This has been demonstrated by observing reduced sensitivity to PMD when the TbAT1 gene is knocked out and partial suppression of PMD transport by adenine, an established substrate of TbAT1 (de Koning et al. 2004). In 2001, the presence of two additional channels, a high-affinity PMD transporter (HAPT1) and a low-affinity PMD transporter (LAPT1), were identified, which facilitated the majority of PMD transport (Bridges et al. 2007). The synthesis of DNA and RNA is believed to be inhibited by PMD, which interferes with nuclear mechanisms (Sands et al. 1985). It is reported that PMD resistance in HAT results from the loss of TbAT1 function (Matovu et al. 2003). Even with PMD resistance, treatment effectiveness is over 93% (WHO 2024b). If PMD is not available to treat the initial stage of T.b.g, FXZ is recommended (CDC 2024d). Also, NECT (NFX/EFL Combination Therapy), a therapy created in 2009, works just as well at treating gambiense types of disease in Central and West African countries. It is considered much safer for patients and is usually used for second-stage gambiense disease (CDC 2024d). Moreover, since the 1920s, SUR has been employed as the primary therapy for the hemolymphatic early phases of HAT caused by T.b.r (Zoltner et al. 2020). Research indicates that it is conveyed by the invariant surface glycoprotein 75 (ISG75) and the major facilitator superfamily transporter (MFST). SUR interacts synergistically with the second-stage medicines EFL, NFX, and Mel^b (Makarov et al. 2023). By contrast, PMD’s action is inhibited by SUR (Guimaraes and Lourie 1951). Apart from having a half-life of around 44–54 days and being in use for nearly a century, there have been no instances of SUR resistance in human pathogenic trypanosomes (Babokhov et al. 2013). Moreover, Mel^b is an organic medicine containing melaminophenyl arsenic. It was developed in the late 1940s as a treatment for second-stage HAT because of its capacity to cross the BBB. It continues to be the primary treatment for second-stage T.b.r infection (Nok 2003). TbAT1, as well as aquaglyceroporin 2 (AQP2), are suggested as the transporters responsible for the selective uptake of Mel^b by the parasite (Graf et al. 2013). Research indicates that each of TbAT1 and TbAQP2 have significant involvement in the transport of Mel^b and PMD (Ungogo et al. 2022). Furthermore, Mel^b and PMD cross-resistance (MPXR) are two of the most distinct patterns from T.b. DR research (Munday et al. 2015). This phenomenon has since been linked to the decreased uptake of these two drugs from MPXR Trypa nosoma cells, which may result from genetic modifications or the loss of essential transporter proteins (Munday et al. 2014). Table IV summarizes the status of T.b. current treatment and DR status in general.

Metronidazole (MTZ), albendazole (ALB), and tinidazole (TNZ) are the most commonly prescribed drugs for giardiasis treatment (CDC 2024e). MTZ monotherapy and ALB have shown effectiveness in most cases, but there have been reported cases of treatment failure (Krakovka et al. 2022). Although the precise process by which MTZ kills anaerobic microbes is still unresolved, a potential explanation is that it disrupts their double-strand DNA. The compound is also thought to inhibit the function of thioredoxin reductase, a redox enzyme, in G. lamblia by targeting its disulfide reductase activity. These mechanisms induce severe oxidative stress (Riches et al. 2020). The reduced cellular concentrations of pyruvate, ferredoxin oxidoreductase and downregulation of ferredoxin pathways in G. lamblia are believed to contribute to its resistance to MTZ. Resistance leads to decreased MTZ uptake into the protozoa lumen due to the low-redox-potential anaerobic metabolism. The proportion of treatment failures attributable to actual resistance is unknown (Adam 2021). ALB affects β-tubulin, which is a cytoskeleton subunit. The resistance to ALB is believed to be caused by changes in the cytoskeleton of G. lamblia, specifically in the ROD domain of the β-tubulin structure (Lagunas-Rangel et al. 2021). Alternatively, other research suggests an efflux mechanism where an ABC-C1 actively transports less ALB, resulting in a lower ALB concentration (Ángeles-Arvizu et al. 2021). Another theory for ALB resistance is to reduce ROS generated by ALB by activating an antioxidant response (Argüello-García et al. 2015). Table V summarizes the general treatment and DR status of Giardia.

Although several medications are available to treat amoebiasis, MTZ has long been considered the gold standard for E. histolytica. It has been well-established that it is safe and effective against amoebiasis (CDC 2019). Thus far, there has been no evidence of resistance from E. histolytica. Other systems, including bacteria such as E. coli, suggest the drug exerts its effects through DNA damage and oxidative stress. However, the in vitro mechanisms of resistance and the existence of resistant clones have yet to be fully established (Jackson et al. 1984). A recent investigation found that treating clinical isolates with MTZ increased their IC⁵⁰ (Singh et al. 2023). Moreover, like MTZ, TNZ is a second-generation nitroimidazole that acts on several protozoa. In addition to effectively eliminating amoebiasis, its greater half-life (12 to 14 hours vs. 8 hours) makes it possible to shorten the duration of treatment (Sawyer et al. 1976). Table VI summarizes the current Entamoeba treatment status.