Introduction_to_Protozoans-_Blood_Parasites_In_class_2023LEO.pptx

Introduction to Protozoans: Blood Parasites Debra Bramblett, PhD CVI- 2023 Objectives • Compare and the contrast four groupings of pathogenic protozoa [ Amebae, Flagellates, Ciliates, and Sporozoan] in terms of shape/structure, motility, and life cycle. • Recognize and describe important examples...

Introduction to Protozoans: Blood Parasites Debra Bramblett, PhD CVI- 2023 Objectives • Compare and the contrast four groupings of pathogenic protozoa [ Amebae, Flagellates, Ciliates, and Sporozoan] in terms of shape/structure, motility, and life cycle. • Recognize and describe important examples of human pathogenic protozoa based on structure and disease associations and know what grouping to which they belong • Compare and contrast the sporozan parasites of red blood cells [ Plasmodia species and Babesia mancroti] • Differentiate between P. vivax, P. ovale, P. malariae, P. falciparum, P. kowlesi based on appearance in a blood smear (ring forms, Schüffner dots, malarial pigment, rosette schizonts, Maurer dots, Ziemann dots, applique position, bar forms, merozoite number), variations in disease they cause, the vector of transmission and epidemiological characteristics. • Recognize a clinical presentation of Leishmaniasis (Cutaneous, Viceral and Mucocutaneous) and know the vector and geographical prevalence of Leishmania species L. donovani, L. tropica and L. braziliensis • Describe the physiology, structure the clinical syndrome, laboratory diagnosis, and life cycle of Bebesia microti • Describe physiology, structure the clinical syndrome, laboratory diagnosis, and life cycle of Trypanosoma cruzi • Recognize the risk factors for a Babesia microti and Babesia duncani infection in terms of regional prevalence and transmission vector or natural reservoir and recognize diagnostic test results for Babesia. • Compare and contrast the mechanisms of action of Chloroquine and Pyrimethamine two Protozoans: • 5 to 100μm • Eukaryotic cells with no cell wall. • Their morphology is highly variable. • They are aerobic and free living. • They are heterotrophic by phagocytosis or through cytostomes. • Asexual reproduction or some have sexual reproduction. All have a trophozoite (vegetative, feeding stage) most have a cyst (resistant, dormant) . • have a nucleus and membrane bound organelles; • microtubules and microfilaments assembled into a cytoskeleton; • genomic DNA is linear and assembled into chromosomes. • Protozoans frequently have more than one nucleus, one being for conjugation. • Diagnosis is most often accomplished by O & P (ova &parasite) examination by wet mount or permanent stains Compare and the contrast four groupings of pathogenic protozoa Four Protozoan Groupings Amoebas Flagellates causes Chagas disease Entamoeba histolytica Subphylum Mastigophora Order Kinetoplastida Family: Trypanosomatidae Causes gastrointestinal disease Phylum: Amoebozoa Subphylum: Sarcodina Giardia lamblia Ciliates Neobalantidium coli (ciliate, Ciliophora) -causes diarrhea Leishmania Trypanosoma cruzi Apicomplexans Plasmodium vivax causes malaria Babesia mancroti Phylum: Apicomplexans Class: Sporozoa Subclass: Coccidia Recognize and describe important examples of human pathogenic protozoa based on structure and disease associations and know what grouping to Protozoans Entamoeba histolytica Amebas – free living, feed by phagocytosis, move by extending pseudopodia • Apicomplexa • Intracellular living Trichomonas vaginalis Flagellates - free living, feed by cytostome, have whip-like projections called flagella. -Today’s examples include Trypanosomes and Leishmania Ciliates Move by means of hair-like projections, feed by way of a cytostome groove. • Neobalantidium coli, contains two nuclei: a large macronucleus and a small micronucleus.(Murray 687). It is the ciliate that is a human pathogen- diarrhea • It is easy to recognize by the bean shaped macronucleus Compare and the contrast four groupings of pathogenic protozoa (Neo) Balantidium coli Entamoeba Trypanosoma Balantidium coli Compare and the contrast four groupings of pathogenic protozoa Subphylum: Mastigophora Trypanosomes: • Trypanosomes are protozoan flagellates • Four morphologic states: trypomastigote, promastigote, epimastigote, amastigote, in the mammalian hosts • Trypanosoma cruzi causes American Trypanosomiasis=Chagas disease (heart) • Different in that it multiplies in mammalian hosts as intracellular amastigotes • Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense are the causative agents of African Trypanosomiasis=African sleeping sickness • Do not have an intracellular form and multiply as trypomastigotes that circulate in the mammalian bloodstream and in other extracellular spaces trypomasti gote Amastigot Promastigote Epimastig e ote Trypanosoma cruzi • Organism: Flagellate protozoan • has a large, subterminal or terminal kinetoplast, a centrally located nucleus, an undulating membrane, and a flagellum running along the undulating membrane • Vector: Triatomine bugs also called reduviid bugs or kissing bugs • Can also be transmitted mother to baby, contaminated blood products, organ transplantation. • Disease: American Trypanosomiasis (Chagas’ Disease) • Initial infection may go unnoticed. Acute phase may involve fever of swelling around the site of inoculation. Romaña's sign, the swelling of the child's eyelid, is a marker of acute Chagas disease. The swelling is due to bug feces being accidentally rubbed into the eye • • • Regional prevalence: United States.such as abnormal rhythms, heart • Chronic infection canSouthern lead to heart diseases • failure, However primarily infectsrisk those are from endemic areas like Mexico, Central and an increased of sudden death America or South America before emigrating to the US. Diagnosis: observation of the parasite in a blood smear by microscopic examination in acute phase only. Diagnosis is generally made by testing with at least two different serologic tests. https://www.cdc.gov/dpdx/ trypanosomiasisAmerican/ index.html https://www.cdc.gov/parasites/cme/ chagas/lesson_1/2.html Trypanosoma cruzi life cycle Sporozoa/Apicomplexans  Sporozoa (apicomplexans) – obligate intracellular parasites Apicomplexa most possess a unique organelle called an apicoplast and an apical complex structure involved in penetrating a host's cell. The apical complex functions during interaction with the host cell, providing the cells with the ability to secrete enzymes and other proteins which erode extracellular material and allow the parasite to enter the host cell. Blood Plasmodium Babesia Cryptosporidium Gastrointesti nal Cyclospora Brain, muscle, eye, and other organs Cystoisospora Toxoplasma Which of the following is a diagnostic feature of Trypanosoma cruzi? A. B. C. D. Donovan bodies Terminal kinetoplast Multiple ring forms Bean shaped macronucleus E. Pyriform bodies Plasmodium • Disease: causes Malaria • 216 million cases worldwide • More than 500,000 deaths per year • Symptoms begin 10-15 days after the infective bite. • Fever, headache and chills • If not treated within 24 hours, P. falciparum malaria can progress to severe illness, often leading to death. • Infective form is called a Sporozoite • Vector: It is spread by the mosquito vector Anopheles • It is found mostly in tropical and subtropical regions • Tissue tropism is the red blood cell Anopheles • By attaching to the Duffy antigen (non-specific antigen offreeborni mosquito chemokines) pumping blood • Or by attaching to glycophorin A and B Malaria At least five different Plasmodium spp cause Malaria Two hosts are required: Human and Mosquito The life cycle Identification by blood smears The signs and symptoms of the disease: Vague influenza-like symptoms with headache, muscle pains, photophobia, anorexia, nausea and vomiting. As the disease progresses the patient begins to have a typical pattern of chills, fever, sweating & malarial rigors that appear periodically. Chronic and relapsing fevers can assist diagnosis. Some species can cause a rapidly lethal form, others can persist for 20 years P. oval and P. vivax produce hypnozoites in the liver which can remain dormant and then recur months later. P. falciparium is well known to cause kidney necrosis and failure Recognize and describe important examples of human pathogenic protozoa based on structure and disease associations and know what grouping to which they belong Epidemiology of Plasmodium (Malaria) • P. vivax is about 80% in south America and Southeast Asia • P. ovale is distributed primarily in tropical Africa, where it is often more prevalent than P. vivax • P. malariae infection occurs primarily in the same subtropical and temperate regions as the other plasmodia but is less prevalent. • Thus far, human P. knowlesi infections have been described in high numbers only in Malaysia • P. falciparum occurs almost exclusively in tropical and subtropical regions of Africa. Co-infection with human immunodeficiency virus (HIV) is common in these regions and may pose a risk factor for severe malaria. • Malaria was considered eliminated from the United state in 1951 by vector control but was a big problem in the 30’s and 40’s Differentiate between P. vivax, P. ovale, P. malariae, P. falciparum, P. kowlesi based on appearance in a blood variations in disease they cause, the vector of transmission and epidemiological characteristics. P. falciparu m P. vivax The life cycles of parasites often suggest useful clues for diagnosis • The life cycle of Apicomplexans includes • Sporogony -parasite is injected into the host by the vector. Asexual reproduction occurs, resulting in sporozoites • Merogony-another round of asexual reproduction resulting in merozoites; this can occur many times in various tissues of the host. • Gametogony- sexual reproduction occurs leading to the production of gametocytes. followed by zygote (diploid) production, followed by transition into a structure that produces more sporozoites called a schizont Plasmodium life cycle Exoerythrocytic cycle 1. Bite of mosquito 2. Delivers Sporozoites 3. Which travel to Liver and reproduce in Schizonts(3) 4. Hepatocyte rupture (4) 5. Plasmodial merozoites are released 6. Merozoites Infect erythrocytes (5) to produce ring forms Erythrocytic cycle (825d) Meiosis 7. Trophozoites develop into Schizonts 8. Rupture of RBCs 9. Merozoites infect more RBCs 10. Some develop into Gametocytes 11. Blood meal by mosquito Asexual reproductio n Incubation period(s) of Malaria • Primary attacks generally occurs 8-25 days after bite. • Includes time required to migrate through the liver, schizogony and symptom production by propagation in the blood. • BUT time differs by immune status or species of the plasmodium, dose of sporozoites, or even partially effective chemoprophylaxis (Atovaquone, Chloroquine, Doxycycline). • Relapses may develop months or years after mosquito bites from the activation of latent hypnozoites in the liver. • Hypnozoites are produced only certain species: • P. vivax and P. ovale • The hypnozoites are not eradicated by standard antimalarials and can awaken days to months to years after the last bout Malaria and Chemoprophylaxis Type of drug • characterized by high fevers, chills and rigors, nausea, vomiting and diarrhea • a consequence, in part, of ruptured erythrocytes and the cytokine response to the sudden release of parasites, hemoglobin and erythrocyte membranes • Anemia can be profound and the spleen can enlarge markedly as it filters remnants and disposes of ruptured erythrocytes. Blood schizontocid e Target Trophozoite in blood Clinical application Prophyla xis Licensed drug(s) Treatment of acute malaria Suppressiv Chloroquine, e quinine, mefloquine, doxycycline, atovaquoneproguanil (Malarone)AV-PG, Primary tissue schizontocid e Active schizont in liver None Causal Primaquine Hypnozoitoci Dormant Prevention of None de hypnozoite in relapse liver Primaquine Gametocytoc Gametocyte ide in blood Prevention of None transmission Primaquine Sporontocide Forms in mosquito including sporozoite Prevention of Causal Primaquine transmission prophylaxi s Cannot be used in patients with glucose-6-phosphatase dehydrogenase (G6PD) deficiency d l Identification of Plasmodium o d r G a • Microscopic examination of blood films is a direct and useful means of detecting malarial parasites • Unfortunately, the concentration of organisms often fluctuates, so collection of multiple specimens over 6, 12, 24 hrs and even several days is required • Several antigen detection tests are also available for detection of Malarial parasites in conjunction with microscopic examination of thick and thin blood smears. • There is a PCR test for Plasmodium vivax which has 91-96% sensitivity d n a t s What is the object surrounded by RBCs in this picture? A. B. C. D. E. Ring form of P. falciparum Trophozoite of P. ovale Gametocyte of P. ovale Schizont of P. malariae Gametocyte of P. falciparum P. vivax P. ovale P. malariae P. falciparum Young trophozoites =Ring-forms Appliqué position Growing Trophozoites Bar form Mature trophozoites 12-25 merozoites 6-12 Mature Schizonts Sausage shaped gametocyte = Cell with many merozoites Macrogametocytes 8 merozoites Schüffner dots Ragged cell walls Microgametocytes Round gametocytes From Strickland GT: Hunter’s Tropical Medicine. 6 th Ed. WB Saunders Philidelphia , 1984 Plasmodium diagnosis and species identification by blood smear  P. vivax:  solitary ring-forms, 12-25 merozoites per schizont, and enlarged RBCs with Schüffner dots, round gametocyte (48- hour fever cycle, 2-days)  P. falciparum:  multiple ring forms per RBC, sausage shaped mature gametocytes, appliqué position of the ring form. Kidney damage (blackwater fever). Shorter fever cycles (36-hours that shorten to continuous)  P. ovale:  Infected cells have Schüffner dots and ragged cell walls. (48-hour fever cycle, 2 days)  P. malariae:  Bar and band forms. Schizonts composed of eight merozoites in a rosette surrounding a pigment granule. (72- hour fever cycles, 3-day). • P. knowlesi (Malaysia): • Initially thought to be specific to Monkeys. Monkey to Human by blood transfusion proven but thought to be rarely a natural infection. Now known to cause human cases and accounts for most (70%) human malaria infections requiring hospitalization in Sarawak, Malaysian Borneo, and is widespread throughout Malaysia. Hard to distinguish from P. malariae …but is potentially deadly while P. malariae is considered benign. P. vivax Invades only young immature erythrocytes Round Gametocyte Ring form Trophozoite Schizont Malarial pigment P. vivax High peripheral blood parasitemias are not observed with the severe pathology of vivax malaria like they are with the P. falciparum. P. vivax parasites selectively infect reticulocytes and accumulate as developing sexual stages ( gametocytes) and mature replicative stages (schizonts) in the bone marrow and liver Ring-form trophozoites of P. vivax in thin blood smears. Ring-form trophozoites of P. vivax usually have a thick cytoplasm with a single, large chromatin dot. http://www.dpd.cdc.gov/DPDx/HTML/ImageLibrary/M-R/Malaria/vivax/body_Malaria_vivax_il5.htm P. vivax Macrogametocytes of P. vivax in a thin blood smear. From : http://www.dpd.cdc.gov/DPDx/HTML/ImageLibrary/M-R/Malaria/vivax/body_Malaria_vivax_il11.htm P. vivax Developing Schizont of P. vivax. Mature Schizonts contain 12-24 merozoites each of which contain a dot of chromatin and a mass of cytoplasm. http://www.dpd.cdc.gov/DPDx/HTML/ImageLibrary/M-R/Malaria/vivax/ Trophozoite Ring form P. falciparum invades any RBC at any stage. Multiple merozoites can infected the same cell. Gametocyte P. falciparum P. falciparum 1.Early trophozoites/ringforms 2. Developing trophozoites 3. Immature Schizonts (asexual replication) 4. Mature Schizonts 5. Microgametocytes 6. Macrogametocytes Notes: High number of parasitized cells Multiple Trophozoite rings per cell Appliqué position of trophozoites Sausage shaped gametocyte P. Falciparum can infect erythrocytes of all ages, promoting heavy parasite burdens. High parasite densities, increased parasite multiplication rates, and evidence of high parasite biomass on peripheral blood smears which are associated with increased severity of malaria and death. P. ovale trophozoites in thin blood smear Invades only young immature erythrocytes. Fimbriation (ragged edges) and Schüffner’s dots. Schuffner dots are common features of P. vivax and P. ovale but not P. falciparum, or P. malariae. The life cycle of P. ovale includes hypnozoites, which are dormant stages in the liver. These stages can be reactivated in weeks, months, or years after the initial infection, causing disease relapse P. malariae Parasitemia often below the level of detection by microscopy Patients can remain infected an asymptomatic for years before presenting with fevers and splenomegaly decades after. Life cycle involved hypnozoites Trophozoite Band form Schizonts with eight merozoites The band form is a diagnostic characteristic of this species. It is due to the preference for development in older erythrocytes which have ridged cell membranes as P. ovale • P. ovale schizonts have 6 to 14 merozoites with large nuclei, clustered around a mass of dark-brown pigment. • The schizonts will appear oval. P. knowlesi Giemsa-stained thin blood films of patient infected with Plasmodium knowlesi, showing a ring form (A), a trophozoite with Sinton and Mulligan stippling (B), a band form resembling P. malariae (C), and an early schizont (D). Original magnification ×100. http://wwwnc.cdc.gov/eid/ article/16/4/pdfs/09-1624.pdf Plasmodium falciparium • Disease: • Although any malarial infection may be fatal, P. falciparum is the most likely to result in death if left untreated. • Involvement of the brain (cerebral malaria) resulting in coma and death is most often seen in P. falciparum. • Kidney damage is also associated with P. falciparum malaria, resulting in an illness called black water fever • rapid destruction of RBCs produces a marked hemoglobinuria and can result in acute renal failure, tubular necrosis, nephrotic syndrome, and death • P. falciparum does not produce hypnozoites in the liver. Relapses from the liver are not known to occur.(Murray 731) • Growing trophozoite stages and schizonts of P. falciparum are rarely seen in blood films because their forms are sequestered in the liver and spleen.(Murray 731) • Thus, peripheral blood smears from patients with P. falciparum malaria characteristically contain only young ring forms and occasionally gametocytes. Anemia in Malaria • Intravascular lysis and phagocytic removal of infected erythrocytes. • But this accounts for a fraction of the reduction in RBCs • Excess removal of uninfected erythrocytes that have accelerated senescence accounts for 90% of the loss. • Release of inflammatory cytokines is associated with impaired production of erythropoietin, decreased responsiveness of erythroid progenitor cells and increased erythrophagocytic activity • All accounting for normochromic normocytic anemia Leishmaniasis • Obligate intracellular parasites that are transmitted to humans by bites from an infected female sand fly. • Cutaneous form is more widespread through the middle east (Afganistan, Algeria, Iran, Iraq, Saudi arabia, Syria) • Leishmania tropica • Red papule at fly’s bite site . Become irritated and intensely pruritic enlarges and ulcerates • Viceral leishmaniasis is seen in Bangladesh, Brazil India, Nepal and Sudan. Cutaneous leishmaniasis. (Left) Skin macrophage containing Leishman-Donovan bodies (note the nucleus and bar-shaped kinetoplast, arrows) • L. donovani and L. infantum • Can be 1. fulminating rapidly fatal disease or 2. more chronic or 3. symptomatic self-limiting • Febrile illness can resemble malaria • The organism proliferates and invade the cells of the reticuloendothelial system, marked enlargement of the liver and spleen, weight loss, and emaciation occur. Kidney damage may occur • With persistent infection there may be deeply pigmented granulomatous areas of skin referred to as post –Kala-azar dermal leishmaniasis. • Mucocutaneous leishmaniais in most often in Bolivia, Brazil, and Peru • L. braziliensis most often the species • Cutaneous lesions plus destruction of mucous membranes. The mucosal lesions do not heal well and secondary bacterial infection are common producing severeof and disfiguring facial mutilation and and Mucocutaneous) and know the vector and Recognize a clinical presentation Leishmaniasis (Cutaneous, Viceral occasionally death.of Leishamania species L. donovani, L. tropica and L. braziliensis geographical prevalence Donovan bodies in macrophage Macrophage containing multiple Leishmania amastigotes. Note that each amastigote has a nucleus (red arrow) and a rodshaped kinetoplast (black arrow). https:// www.cdc.gov/ parasites/ leishmaniasis/ health_profession als/index.html#tx Recognize the risk factors for a Babesia microti and Babesia duncani infection in terms of regional prevalence and transmission vector or natural reservoir and recognize diagnostic test results for Babesia. Babesia microti and Babesia duncani  Vector: Transmitted by Tick (Ixodes scapularis)  Diagnosis  In Blood smears it tends to form ring-forms in tetrads   Pyriform bodies (pairs) and cruciform merozoites No pigment production  Fluorescent Antibody detection (IFA)   East coast Babesia microti West coast Babesia duncani  Procleix – for the NAAT detection of RNA in whole blood https://doi.org/ 10.1128/ mSphere.00928-20  Regional prevalence of Babesia  Northeast and upper mid-west of US  Disease: Babesiosis More than 200 cases of transfusion-transmitted babesiosis have been reported since 1980 with a fatality rate of approximately 20% prompting the U.S. Food and  Treatment: Drug Administration (FDA) to  Atovaquone, Azithromycin, Clindamycin, Quinine (for severely ill patients) finalize recommendations for Describe the physiology, structure the clinical syndrome, laboratory diagnosis, and life cycle  Initial disease: malaise, headache, chills, sweating, fatigue without periodicity  Later hemolytic anemia develops  Renal failure, Hepatomegaly, splenomegaly can develop in advanced disease.  Most severe disease in asplenic and immunocompromised Recognize the risk factors for a Babesia microti and Babesia duncani infection in terms of regional prevalence and transmission vector or natural reservoir and recognize diagnostic test results for Babesia. http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6127a2.htm Peromyscus leucopus Describe the physiology, structure the clinical syndrome, laboratory diagnosis, and life cycle Incidence of cases of babesiosis2011 On January 1, 2011, babesiosis became a nationally notifiable condition. Can babesiosis be transmitted by transfusion in areas where it’s not spread by ticks? Yes. Because individuals who donate blood travel and blood products are shipped around the country, Are U.S. blood donors being tested for babesiosis? Yes. On March 6, 2018, the Food and Drug Administration (FDA) approved the first two tests for screening blood Incidence of cases of babesiosisdonors as well as organ and tissue donors for Babesia infection 2013 Does Babesia pose a risk to people who receive blood products? Yes. Although bloodborne transmission is thought to be uncommon, babesiosis is the most frequently reported transfusiontransmitted parasitic infection in the U.S. It remains an important concern. https://www.cdc.gov/parasites/ babesiosis/resources/ Recognize the risk factors for a Babesia microtibabesiosis_policy_brief.pdf and Babesia duncani infection in terms of regional prevalence and transmission vector or natural reservoir and recognize diagnostic test results for Babesia. https://www.cdc.gov/ parasites/babesiosis/ resources/ babesiosis_surveillance_sum mary_2018b.pdf Some Anti-Protozoa Drugs Please review but I will not be testing you on the drugs Drugs for Chagas • benznidazole • Inhibits the synthesis of DNA, RNA, and proteins within the T. cruzi parasite • Benznidazole is thought to be reduced to various electrophilic metabolites by nitroreductases present in Trypanosoma cruzi . These metabolites likely bind to proteins, lipids, DNA, and RNA resulting in damage to these macromolecules. https://www.drugbank.ca/drugs/DB11989 • Nifurtimox • One hypothesis for the MOA involves the ability of this agent to form a nitro-anion radical metabolite, which reacts with the nucleic acids of the parasite, causing a significant breakage in the deoxyribonucleic acid (DNA). Drugs for Plasmodium Protozoa • Chloroquine is still the drug of choice against all five species of Plasmodium although drug resistance is becoming an issue. • Proposed mechanisms of action 1. binding to deoxyribonucleic acid (DNA) and interfering with DNA replication, 2. binding to ferriprotoporphyrin IX released from hemoglobin in infected erythrocytes, producing a toxic complex 3. raising the pH of the parasite's intracellular acid vesicles, thus interfering with its ability to degrade hemoglobin. • 4-aminoquinolines like Chloroquine destroy the erythrocytic stage of malaria and thus may be used prophylactically to suppress clinical illness or therapeutically to terminate an acute attack. • The 8-aminoquinolines (e.g., primaquine) accumulate in tissue cells and destroy the extra-erythrocytic (hepatic) stages of malaria, resulting in a radical cure of the infection.(Murray 710) Compare and contrast the mechanisms of action of Chloroquine and Pyrimethamine two different drugs Folic acid Antagonist for Malaria and Toxoplasmosis. • diaminopyrimidines (pyrimethamine and trimethoprim) and sulfonamides are folic acid antagonist • Protozoans are not able to synthesize folate required for DNA synthesis • Sulfonamides inhibit the conversion of aminobenzoic acid to dihydropteroic acid • The diaminopyrimidines inhibit dihydrofolate reductase, which results in the blocking of formation of purines, pyrimidines, and certain amino acids. • effective at concentrations far below those needed to inhibit the mammalian enzyme • Used together, Sulfonamide + diaminopyrimidine, a synergistic effect is achieved via the blockade of two steps in the same metabolic pathway Protein Synthesis inhibitors • Clindamycin, Spiramycin, Tetracycline, and doxycycline • All act to inhibit protein synthesis in bacteria as well as Plasmodium, Bebesia and Amebae • Doxycycline is used for chemoprophylaxis of chloroquine-resistant P. falciparum malaria, and Tetracycline may be used with quinine for the treatment of chloroquine-resistant P. falciparum infection