Malaria and Babesiosis PDF

Chapter Malaria and Babesiosis 35 Chapter Preview „„Malaria...

Chapter Malaria and Babesiosis 35 Chapter Preview „„Malaria „„Babesiosis MALARIA or sporogony takes place) and intermediate host—man (asexual cycle or schizogony takes place) History ™™ Male Anopheles does not feed on man and feeds Malaria is one of the oldest documented diseases of exclusively on fruit juices, i.e. why male Anopheles mankind. The name “Malaria” (“Mal” means bad and “aria” mosquito does not transmit the disease. Whereas female https://t.me/docinmayking means air) was derived from the ancient false belief that Anopheles needs at least two blood meals before laying “disease is spread by air pollution through stagnant water eggs and marshy lands” ™™ Although several species of Anopheles can transmit, ™™ Sir Alphonse Laveran in 1902 and Sir Ronald Ross in 1907 the vectors of primary importance include: Anopheles won the Nobel Prize for their contributions in malaria culicifacies in rural areas, A. stephensi in urban areas and „„ Alphonse Laveran (1880) was the first to discover the A. fluviatilis in hilly areas. causative agent Plasmodium, in the red blood cell (RBC) of a patient in Algeria Human Cycle „„ Sir Ronald Ross, in 1897 had described the sexual Mode of Transmission and Infective Form cycle of the parasite in female Anopheles mosquito in ™™ Man acquires infection by the bite of female Anopheles Secunderabad, India. mosquito. Sporozoites (infective form) from the salivary ™™ Ms Tu Youyou, a chemist was awarded Nobel prize gland of the mosquito are directly introduced into the (2015), for the discovery of artemisinin, which is used cutaneous venules and enter the blood circulation for the treatment of falciparum malaria. ™™ Rarely, it can also be transmitted by blood transfusion or transplacental transmission—here, trophozoites (or Agent merozoites) act as the infective form. Although several species of Plasmodium exist infecting In humans, the asexual cycle takes place through three wide range of birds, reptiles and mammals, human stages: (1) pre-erythrocytic schizogony (2) erythrocytic infection is mainly caused by five species. schizogony, and (3) gametogony. 1. P. vivax causes benign tertian malaria (periodicity of fever is once in 48 hours, i.e. recurs every third day) Pre-erythrocytic (Hepatic) Stage 2. P. falciparum causes malignant tertian malaria (severe This stage occurs in the liver and it is so named because malaria, periodicity of fever is once in 48 hours, recurs it occurs before the invasion of RBC. It is also called every third day) exoerythrocytic stage or intrahepatic or tissue stage. 3. P. malariae causes benign quartan malaria (periodicity ™™ The motile sporozoites leave the circulation within 30 of fever is once in 72 hours, i.e. recurs every fourth day) minutes and enter the liver 4. P. ovale causes ovale tertian malaria (periodicity of fever ™™ Attachment: The circumsporozoite proteins present on is once in 48 hours, i.e. recurs every third day) the surface of sporozoites bind to the receptors present 5. P. knowlesi causes quotidian or simian malaria (fever on the surface of hepatocytes facilitating the entry of periodicity is once in 24 hours, i.e. recurs every day). It is a sporozoites parasite of monkey but can also infect humans and many ™™ Trophozoites: After entering into hepatocytes, the cases affecting man were recently reported from Asia. spindle shaped sporozoites become rounded and transform into trophozoites Life Cycle (Fig. 35.1) ™™ Schizogony: Trophozoite is the feeding stage of the Host: Plasmodium completes its life cycle in two hosts: parasite which later on undergoes several nuclear definitive host—female Anopheles mosquito (sexual cycle divisions and transforms into pre-erythrocytic schizont  Chapter 35  Malaria and Babesiosis 347 https://t.me/docinmayking Fig. 35.1: Life cycle of malaria parasite. ™™ Pre-erythrocytic schizont contains several merozoites; ™™ Merozoites bind to the glycophorin receptors on RBC released outside on rupture of hepatocyte. Merozoites surface, enter by endocytosis and are contained within then attack RBCs to initiate erythrocytic stage a parasitophorous vacuole inside the RBCs ™™ No liver injury: As only few hepatocytes are infected by ™™ Trophozoite: Soon the hepatic merozoites transform Plasmodium, so hepatic damage does not occur in malaria into trophozoites ™™ Duration of pre-erythrocytic schizogony varies from 5 ™™ Early trophozoites are called as ring forms, which are an- days to 15 days depending on the species nular or signet ring in appearance containing a central vac- ™™ Hypnozoites: Some sporozoites of P. vivax and P. uole with a peripheral thin rim of cytoplasm and a nucleus ovale do not develop further and may remain in liver ™™ Late trophozoite: Ring form enlarges and becomes as hypnozoites and cause relapse of malaria after many more irregular and transforms into late trophozoite or years amoeboid form ™™ Relapse should be differentiated from another ™™ Malarial pigment: Plasmodium feeds on hemoglobin, phenomena seen in P. falciparum and P. malariae called releasing the undigested products (hematin and iron as recrudescence (Table 35.1). porphyrin), which combine to form malarial pigment (hemozoin pigment) Erythrocytic schizogony ™™ Schizogony: Late trophozoite undergoes schizogony to The hepatic merozoites after released from pre-erythrocytic produce 6–30 daughter merozoites arranged in the form schizont, attack RBCs. of rosette. This form is known as erythrocytic schizont 348 Section 4  Bloodstream and Cardiovascular System Infections Table 35.1: Relapse and recrudescence in malaria. Relapse Recrudescence Seen in Plasmodium vivax and P. ovale infections Although seen in all species, more common in P. falciparum followed by P. malariae Few sporozoites do not develop into pre-ery­throcytic schizont, but In falciparum malaria—recrudescence is due to persistence of remain dormant (known as hypnozoites) for 3 weeks to one year drug resistant parasites, even after the completion of treatment Reactivation of hypnozoites leads to initiation of erythrocytic cycle In P. malariae infection, long-term recrudescences are seen for as and relapse of malaria long as 60 years This is due to long-term survival of erythrocytic stages at a low undetectable level in blood Concept of secondary exo or pre-erythrocytic stage: Formerly, it was postulated that relapse occurs due to secondary exo or pre-erythrocytic stage where a proportion of hepatic merozoites released from pre-erythrocytic schizont, again attack the liver cells But now, it is believed that secondary pre-erythrocytic stage does not occur and relapse occurs due to sporozoites undergoing dormancy during the primary pre-erythrocytic stage ™™ RBCs then rupture to release the daughter merozoites, ™™ Gametocytes are the infective form to mosquito. They malarial pigments and toxins into the circulation which are capable of transmission only when they are mature, result in malarial paroxysm of fever at the end of each viable, and present in sufficient density (12 gametocytes erythrocytic cycle per cubic mm of blood) to infect mosquitoes. https://t.me/docinmayking ™™ Each merozoite is potentially capable of invading a new RBC and repeating the cycle. Intra-erythrocytic life cycle Mosquito Cycle takes 48–72 hours depending upon species (Table 35.2) A female Anopheles mosquito during the blood meal, takes ™™ In P. falciparum infection, the later stages of erythrocytic both asexual and the sexual forms. The asexual forms get cycle occur in the capillaries of brain and internal organs. digested whereas the sexual forms, i.e. the gametocytes Hence, only the ring forms are found in peripheral blood undergo further development. examination, but not late trophozoites and schizonts. ™™ Each male gametocyte undergoes exflagellation and divides into eight flagellated actively motile bodies called Gametogony as male gamete or microgametes After a series of erythrocytic cycles, some merozoites after ™™ Female gametocyte does not undergo exflagellation but entering into RBCs, instead of developing into trophozoites, directly develop into one female gamete or macrogamete they transform into sexual forms called as gametocytes. ™™ Zygote: The male gamete fertilizes with the female ™™ The gametocytes are usually round in shape, except in P. gamete to form zygote falciparum in which they are crescent or banana-shaped ™™ Ookinete: Zygote transforms into a motile elongated ™™ They are of two types—(1) male gametocyte (or micro­ form called ookinete in the midgut gametocyte) and (2) female gametocyte (or macro­ ™™ Oocyst: The ookinete penetrates the stomach wall of gametocyte) the mosquito and becomes rounded, covered by a thin ™™ Gametocytes neither cause any clinical illness nor they elastic membrane to form oocyst divide, but play an important role in the transmission ™™ Sporozoites: Each oocyst undergoes sporogony (meiosis) of the disease to produce four spindle-shaped sporozoites. On rupture Table 35.2: Differences between the four malaria parasites. Properties Plasmodium vivax Plasmodium falciparum Plasmodium malariae Plasmodium ovale Relapse (Hypnozoites) Seen Not seen Not seen Seen Recrudescence Not seen Seen Seen (Up to 60 years) Not seen Erythrocytic cycle 48 hours 36–48 hours 72 hours 48 hours Prepatent period 8 days 5 days 13 days 9 days Incubation period 14 days 12 days 28 days 17 days R-G intervala 4–5 days 10–12 days 11–14 days 5–6 days Extrinsic IPb 8–10 days 9–10 days 25–28 days 14–16 days Abbreviations: aR-G interval, interval between appearance of ring form and gametocyte; bIP, incubation period. Prepatent period: It is the time between the entry of the parasite into man and demonstration of the parasite in the peripheral blood. It varies between the species. Incubation period: Time interval between the entry of the parasite into man and appearance of first clinical feature. It varies between species. Duration of erythrocytic cycle corresponds to the frequency of febrile paroxysms seen in malaria; e.g. every 48h for P. vivax.  Chapter 35  Malaria and Babesiosis 349 of the mature oocyst, the sporozoites are released and ™™ Paroxysm corresponds to the release of the successive migrate to salivary gland and the cycle is repeated broods of merozoites into the bloodstream, at the end ™™ Extrinsic incubation period: Time required to complete of RBC cycle the life cycle in mosquito varies from 1 to 4 weeks, ™™ Each paroxysm of fever is comprised of three stages depending up on the species. 1. Cold stage: Lasts for 15 minutes to 1 hour. The patient The differences in the life cycle between the four malaria feels lassitude, headache, nausea, intense cold, chills parasites have been depicted in Table 35.2. Plasmodium and rigor knowlesi has been discovered recently and therefore 2. Hot stage: Patient develops a high-grade fever of 39– discussed separately. 41°C and dry burning skin. Headache persists but nausea diminishes Plasmodium knowlesi 3. Sweating stage: Fever comes down with profuse It is a malaria parasite of monkeys, but can also rarely affect sweating. The skin becomes cold and moist. Patient humans. Anopheles leucosphyrus is the main vector. feels relieved and often asleep. This stage lasts for 2–4 Epidemiology: The first human case was documented in 1965. hours. However, cases in humans increasingly being reported from ™™ The classical paroxysm may not be present always due to Asia since 2008. ‰‰ World: Maximum cases have been reported from Malaysia maturation of generations of parasites at different times (highest), Thailand and Myanmar. The largest foci are located ™™ In P. falciparum, the fever is more irregular or even at Malaysian Borneo; 3,122 cases have been reported continuous with marked prostration, headache and between 2004–2015 nausea. ‰‰ India: The only report of P. knowlesi infection has https://t.me/docinmayking documented from Andamans. However, India has all the Anemia potential of getting cases as the vector is found in the Patient develops normocytic normochromic anemia which coastal region of Kerala and Maharashtra. may be attributed to various factors. Clinical features: P. knowlesi produces an acute illness and ™™ Parasite induced RBC destruction—Lysis of RBC due to relatively high parasitemia release of merozoites ‰‰ Paroxysms of fever occur daily (quotidian malaria) because ™™ Splenic removal of both infected RBC and uninfected of short RBC cycle (24 hours) RBC coated with the immune complexes ‰‰ Clinically it resembles P. vivax, but severe malaria is seen ™™ Bone marrow suppression leading to decreased RBC more frequently (7–10%), compared to 3% of P. vivax. However, it infects RBCs of all ages. production. ‰‰ Common complications seen are respiratory distress (most frequent) and renal failure. No cerebral malaria has been Splenomegaly reported so far. After a few weeks of febrile paroxysms, spleen gets enlarged Laboratory diagnosis: and becomes palpable. Splenomegaly is due to massive ‰‰ On blood smear examination, early trophozoite of P. knowlesi proliferation of macrophages that engulf parasitized and is indistinguishable from P. falciparum, sometimes shows nonparasitized coated RBCs. multiple ring forms, accole forms and double dot ring forms ‰‰ The late trophozoites (with band forms), and round gameto­ Falciparum Malaria (Malignant Tertian Malaria) cytes are morphologically similar to that of P. malariae The pathogenesis of P. falciparum is different from other ‰‰ Currently, no specific rapid diagnostic tests (RDTs) are available to detect P. knowlesi species. ‰‰ P. knowlesi specific nested PCR assays are available using Sequestration of the Parasites the primers Pmk8 and Pmkr9 targeting small subunit rRNA. Treatment: It responds well to chloroquine or primaquine. As An important feature of the pathogenesis of P. falciparum the disease rapidly progresses, treatment should be promptly is its ability to sequester (holding back) the parasites in the started. blood vessels of deep visceral organs like brain, kidney, etc. This leads to blockage of vessels, congestion and hypoxia of internal organs. Sequestration is mediated by: Pathogenesis and Clinical Feature ™™ Cytoadherence: It refers to the binding of infected eryth- Benign Malaria rocytes to endothelial cells. It is mediated by a specialized Benign malaria is milder in nature, can be caused by all four antigen called as P. falciparum erythrocyte membrane species. It is characterized by a triad of febrile paroxysm, protein-1 (PfEMP-1), which binds to specific receptors anemia and splenomegaly. present on the vascular endothelium of deep organs ™™ PfEMP-1 also helps in binding of infected RBCs to Febrile Paroxysm uninfected RBCs by a process called rosetting Fever comes intermittently depending on the species. It ™™ Since the parasites are sequestrated back in deep vessels, occurs every fourth day (72 hour cycle for P. malariae) and they can avoid frequent spleen passage, hence can escape every third day (48 hour cycle for other three species). splenic clearance 350 Section 4  Bloodstream and Cardiovascular System Infections ™™ PfEMP undergoes frequent antigenic variation, thus Chronic Complications of Malaria helps the parasite in evading the host immune response. 1. Tropical splenomegaly syndrome: It is also called as hyper-active malarial splenomegaly; occurs in malaria- Complications endemic areas in tropical Africa and Asia (including Complications of Falciparum Malaria India) P. falciparum infection is more acute and severe in nature „„ It results from an abnormal immunologic response to with more complications than the benign malaria. repeated malaria infections and is characterized by— ™™ Cerebral malaria: this is the most serious complication elevated IgM (due to polyclonal Bt cell activation) seen in falciparum malaria. It results due to plugging of and massive splenomegaly brain capillaries by the sequestered parasitized RBCs „„ Patients respond well to antimalarial chemo­ leading to vascular occlusion and cerebral anoxia prophylaxis (proguanil). (discussed in detail in Chapter 75) 2. Quartan malarial nephropathy: It is a chronic ™™ Pernicious malaria: It is characterized by blackwater complication seen with P. malariae (rarely P. knowlesi fever, algid malaria and septicemic malaria and other species). It occurs due to injury to the renal ™™ Black water fever: This syndrome is characterized glomeruli by the immune complexes, resulting in by sudden intravascular hemolysis followed by fever, nephrotic syndrome hemoglobinuria and dark urine 3. Promotes Burkitt’s lymphoma: Malaria induced severe „„ It occurs following quinine treatment to subjects immunosuppression in African children provoke Epstein- previously infected with P. falciparum Barr virus infection to develop Burkitt’s lymphoma. https://t.me/docinmayking „„ Autoimmune mechanism: Antibodies develop against parasitized and quininized RBCs. With Malaria in Special Situations subsequent infection and quinine treatment, there is Transfusion Malaria immunocomplex formation followed by complement Malaria can be transmitted by blood transfusion, needle mediated massive destruction of both parasitized stick injury, or organ transplantation. The clinical features and nonparasitized RBCs. and management of these cases are same as for naturally ™™ Algid malaria: Characterized by cold clammy skin, acquired infections (mosquito-borne) but differs in many hypotension, peripheral circulatory failure and profound other ways: shock ™™ The infective form can be intraerythrocytic forms such as ™™ Septicemic malaria: Characterized by high-grade fever merozoites, trophozoites or schizonts but not gametocytes with the dissemination of the parasite to various organs ™™ There is no pre-erythrocytic stage of development and leading to multiorgan failure no relapse ™™ Pulmonary edema and adult respiratory distress ™™ The incubation period is often short syndrome: Severe falciparum malaria in adults may lead ™™ Radical chemotherapy with primaquine is unnecessary to noncardiogenic pulmonary edema, often aggravated as there is no relapse. by over hydration. It does not usually respond to antimalarial therapy; mortality rate is more than 80% Malaria in Pregnancy ™™ Hypoglycemia: It is associated with a poor prognosis Malaria during pregnancy increases the risk of fetal distress and is particularly problematic in children and pregnant and can result in premature labor low birth weight and still women and following quinine therapy birth. In areas with high malaria transmission, pregnant ™™ Renal failure: It occurs due to erythrocyte sequestration women are particularly vulnerable to severe anemia, in renal microvasculature leading to acute tubular hypoglycemia and acute pulmonary edema. necrosis. It is common among adults than children ™™ Bleeding/disseminated intravascular coagulation: Malaria in Children Patient presents with significant bleeding and hemor- Nearly one million children die of falciparum malaria each rhages from the gums, nose and intestine with or without year in endemic countries. evidence of disseminated intravascular coagulation Certain complications are relatively common among ™™ Severe jaundice: More common among adults than children like convulsions, coma, hypoglycemia, metabolic children; it results from hemolysis, hepatocyte injury acidosis and severe anemia; whereas other complications and cholestasis like jaundice, acute renal failure, and acute pulmonary ™™ Severe normochromic, normocytic anemia: Character- edema are unusual in children. ized by hematocrit of less than 15% or hemoglobin level of less than 5 g/dL with parasitemia level >105/µL (>2%) Immunity against Malaria ™™ Acidosis: Results from the accumulation of organic acids Both innate and acquired immunity contribute to the like lactic acid. resistance against malaria.  Chapter 35  Malaria and Babesiosis 351 Innate Immunity ™™ Optimum temperature (20–30°C), humidity (60%) and This refers to the inherent mechanisms of host resistance rainfall. against malaria parasite. This depends upon various factors. Situation in World (WHO Malaria Report, 2018) ™™ Age of RBCs: P. falciparum attacks RBCs of any age, P. vivax and P. ovale attack the young RBCs and In 2018, 228 million cases of malaria with about 4 lakh reticulocytes; whereas P. malariae attacks older RBCs deaths occurred worldwide; Africa affected the worst ™™ Nature of hemoglobin: Sickle cell disease, hemoglobin (93%), followed by South-East Asia (3.4%). C and E, fetal hemoglobin and thalassemia hemoglobin ™™ Incidence rate: It is about 57 cases per 1000 population are resistant to falciparum malaria at risk. The South-East Asia recorded the largest decline ™™ Hereditary ovalocytosis: In this condition, the rigid (70%) in incidence rate compared to 2010 RBCs are resistant to falciparum malaria ™™ P. falciparum is the most common species worldwide; ™™ G6PD deficiency: RBCs with glucose-6-phosphate accounting for 99.7% of malaria cases in African region, dehydrogenase deficiency are resistant to falciparum 50% in South-East Asia malaria ™™ Globally, 53% of the P. vivax burden is in South-East ™™ Duffy negative red blood cells: Duffy blood group region, with 47% of the vivax cases being reported from antigens present on RBC membrane act as receptors for India. P. vivax is the predominant species in America P. vivax. So, people with Duffy negative RBCs (e.g. West (75%). Africans) are resistant to vivax malaria ™™ Age: Children are more prone to infection and Malaria Situation in India (NVBDCP Report) According to National Vector Borne Disease Control https://t.me/docinmayking complications. However, newborn are protected from falciparum malaria because of the high concentration Programme (NVBDCP), 3.3 lakh malaria cases were of fetal hemoglobin in first few months of life reported from India in 2019, with 73 deaths. ™™ Nutritional status: It has a paradoxical effect. Severe ™™ Over the last decade, Eastern Indian states such as malaria is rare in children suffering from malnutrition. Odisha, Chhattisgarh and Jharkhand have accounted for maximum malaria cases; P. falciparum being the Acquired Immunity predominant species Both cellular and humoral immunity contribute to the ™™ However, in 2018 and 2019, Uttar Pradesh accounted resistance against malaria. for highest malaria burden in India, where majority of ™™ Humoral immunity: Circulating antibodies against cases were due to P. vivax. As a result, P. vivax became asexual forms give protection by inhibiting the RBC the most common species in India (>50%), followed by invasion and sequestration, whereas antibodies against P. falciparum (46%) sexual forms help in reducing the transmission of ™™ P. malariae infections are 7 days) (Table 35.3). 352 Section 4  Bloodstream and Cardiovascular System Infections Table 35.3: Stratification of States/UTs in India based on annual Peripheral Blood Smear parasite incidence (API). Peripheral smear study still remains the simple and Categories Definition gold standard confirmatory test for detection of malarial Category 0 States/UTs with zero indigenous cases of parasites. (Prevention of re- malaria establishment phase) Specimen Category 1 States/UTs including their districts with Peripheral blood is the specimen of choice, collected from (Elimination phase) API of < 1/1,000 population at risk ear lobe or by finger prick in older children and adults and Category 2 States/UTs with API 200 parasites per μL of blood. ¾¾ P. falciparum: Schizonts are not seen in the peripheral smear Speciation of Malaria Parasites ‰‰ Gametocytes: In P. falciparum, the gametocyte is crescentic The speciation by thin smear is based on the detection of the or banana-shaped and larger than RBCs, whereas for other ring forms, schizonts, gametocytes, type of pigments produced species, it is spherical and almost occupies the RBC (Figs and RBC size (Fig. 35.3). 35.4D, E and 35.5B and C). https://t.me/docinmayking ‰‰ RBC size: Parasitized RBC is normal in size and shape for ‰‰ Ring forms: It is the most important form that helps in P. falciparum and P. malariae; enlarged in size for P. vivax, accurate speciation. It comprises of a vacuole in the center, peripheral thin rim of blue cytoplasm, surrounding the red Contd... nucleus. ¾¾ P. vivax: Rings are 2.5 μm size, occupies 1/3 rd of the RBC size. Cytoplasm opposite to the nucleus is thick. Late trophozoites are amoeboid-shaped (Figs 35.4A and B). ¾¾ P. falciparum: Rings are 1.5 μm size, smaller than in P. vivax, occupying 1/6th of RBC. Three variants of ring forms may be seen such as (Fig. 35.5A):  Multiple ring forms (inside the same RBC)  Accole (appliqué) form: Ring form attached to RBC membrane  Double dot/head phone shaped ring form: Ring form with fragmented nucleus ¾¾ P. malariae: Early trophozoite is similar to that of P. vivax, but late trophozoite is band-shaped (called band forms) (Fig. 35.5D) ¾¾ P. ovale: Ring forms are similar to that of P. vivax, but present inside oval-shaped RBC (Fig. 35.5E) Note: In falciparum malaria, only the gametocytes and ring forms are demonstrated in peripheral blood but not schizonts and late trophozoites (as the later stages of erythrocytic cycle Fig. 35.3: Morphological forms of malaria parasites seen in the occurs in deep vessels, not in peripheral blood). peripheral smear. A B C D E Figs 35.4A to E: Thin blood smear showing different forms of Plasmodium vivax: A. Ring form; B. Amoeboid form; C. Schizont; D. Male gametocyte; E. Female gametocyte. Source: DPDx Image Library, Centre for Disease Control and Prevention (CDC), Atlanta (with permission). 354 Section 4  Bloodstream and Cardiovascular System Infections A B C D E Figs 35.5A to E: Thin blood smear showing: A. P. falciparum ring forms such as multiple rings (blue arrow), accole form (red arrow) and head phone-shaped ring form (black arrow); B. Female gametocyte of P. falciparum; C. Male gametocyte of P. falciparum; D. Band form of P. malariae; E. Ring form of P. ovale. Source: DPDx Image Library, Centers for Disease Control and Prevention (CDC), Atlanta (with permission). Quantification of Parasites Quantitative Buffy Coat Examination Thick smear is preferred to thin smear for quantification of The quantitative buffy coat (QBC) is an advanced parasitemia. microscopic technique for malaria diagnosis. It consists of ™™ Previously, the “plus system” was used, which is simple three basic steps: but far less accurate for establishing parasite density in 1. Blood (60 µL) is collected in a capillary tube coated https://t.me/docinmayking thick blood films. Now this is obsolete internally with acridine orange (Fig. 35.6A) ™™ Currently, the quantification is done by calculating the 2. Capillary tube is centrifuged, which causes separation number of parasites counted compared to number of of components of blood according to their densities, white blood cells (WBCs) in the thick smear or number forming discrete layers as RBCs, WBCs, lymphocytes and of RBCs counted in the thin smear platelets (Fig. 35.6B) Quantification by thick smear = 3. Examination of capillary tube at the buffy coat No. of parasites counted region under ultraviolet (UV) light source (Figs 35.6C × Total WBC count No. of WBC counted and D). ™™ Quantification is helpful for: Interpretation „„ Assessing the severity of infection Acridine orange has a property of staining the nuclear DNA „„ Monitoring the response to the treatment fluorescent brilliant green. Normal RBCs don’t take up the „„ Detecting drug resistance of P. falciparum. stain (as they are a nucleated). However, parasitized RBCs appear as brilliant green dots. WBCs also take up the stain Fluorescence Microscopy (Figs 35.6C and D). Kawamoto technique is a fluorescent staining method for demonstrating malaria parasites. Blood smears are Advantages prepared on a slide and are stained with acridine-orange QBC is faster (the entire tube can be screened within and examined under a fluorescence microscope. Nuclear minutes), more sensitive (at least as good as a thick film), DNA is stained green. and quantification is possible. A B C D Figs 35.6A to D: A. QBC capillary tube; B. Magnified view of QBC capillary tube after centrifugation; C. Crescent shaped gametocyte of Plasmodium falciparum; D. Ring forms of Plasmodium falciparum seen as fluorescent dots. Source: C and D. Department of Microbiology, Sri Siddhartha Medical College, Tumkur, Karnataka (with permission).  Chapter 35  Malaria and Babesiosis 355 Disadvantages along with NCM to meet its corresponding antibodies It is expensive, less specific and speciation is difficult. at different test lines (Fig. 35.7) ™™ Interpretation is based on immobilization of malarial Antigen Detection by Rapid Diagnostic Tests antigens at test lines 1 and/or 2 forming colored bands Rapid diagnostic tests (RDTs) have revolutionized the (Figs 35.7A and B) diagnosis of malaria. „„ If band is formed at only test line 1: indicates P. ™™ Antigens: Several malarial antigens can be detected: falciparum infection „„ Parasite lactate dehydrogenase (pLDH): It is „„ If band is formed at only test line 2: indicates produced by all Plasmodium species. Currently Plasmodium species other than P. falciparum available test kits can differentiate pan-LDH common infection to all species and Pf-LDH specific to P. falciparum „„ If bands are formed at both test lines 1 and 2: indicates „„ Parasite aldolase: Produced by all Plasmodium P. falciparum or mixed infection. species Note: The band at control line must come to validate the „„ Histidine rich protein-2 (HRP-II): It is produced test; if it does not come, test is considered invalid. Control only by P. falciparum. line is coated with antibody against polyclonal malarial ™™ Principle: Test kits currently available use a nitrocellulose antibody present in buffer. membrane (NCM) strip with two parasite detection lines Advantages of Rapid Diagnostic Tests and a control line (Fig. 35.7A): „„ Test line-1: Coated with capture antibodies specific Rapid diagnostic tests are simple to perform, do not need for P. falciparum (e.g. HRP-II or Pf-LDH) extra equipment or trained microscopist. https://t.me/docinmayking ™™ Sensitivity: Rapid diagnostic tests are more than 90% „„ Test line-2: Coated with capture antibodies com- mon to all Plasmodium spp. (e.g. pan-LDH or sensitive at >100 parasites/µL. But the sensitivity is aldolase). markedly reduced at 100 parasites/µL, sensitivity >90% 5 parasites/µL in thick film good as a thick film 3 mg/dL) hypnozoites of P. vivax (to prevent relapse) Jaundice (Serum bilirubin >3 mg/dL) Contraindicated in infants, pregnant women* Severe anemia (Hb