INTRODUCTION Hemoflagellates are the flagellated protozoa that are found in peripheral blood circulation. Examples include Leishmania and Trypanosoma; both are transmitted by the bite of the insect vector. !ey have an oval to elongated body, nucleus, and a single "agellum arising from kinetoplast. Kinetoplast represents multiple copies of mitochondrial DNA. !e intracellular portion (root) of the "agellum is called as the axoneme Based upon the arrangement of the "agellum, they exist in four morphological stages (Figs 36.1A to D) 1. Amastigote form: Round to oval, lacks "agellum 2. Promastigote form: Lanceolate shaped; kinetoplast is anterior to nucleus and "agellum arises from the anterior end 3. Epimastigote form: Elongated, kinetoplast is placed close to the nucleus. Flagellum arises from the lateral side and traverses the body as a short undulating membrane and comes out from the anterior end 4. Trypomastigote form: Elongated, kinetoplast lies near the posterior end. Flagellum arises posteriorly and runs as a long undulating membrane. In Leishmania amastigote and promastigote forms are seen. Amastigotes are diagnostic form, found in man Promastigotes are infective stage to man, found in insect vector. In Trypanosoma, epimastigote and trypomastigote forms are seen. In man, trypomastigotes are diagnostic form In insect vector, both epimastigotes and trypomastigotes are found, the latter being the infective stage to man. LEISHMANIASIS Leishmaniasis is caused by an obligatory intracellular protozoa of the genus Leishmania, which primarily a#ects the reticulo-endothelial system of the host. Vector: Leishmaniasis is transmitted by the bite of the female sand"y vector Clinical forms: Leishmaniasis presents in various clinical forms Visceral leishmaniasis (VL) Post–kala-azar dermal leishmaniasis (PKDL): It occurs few months to years following VL Cutaneous forms: Occurs in various forms such as cutaneous leishmaniasis (CL), diffuse cutaneous leishmaniasis (DCL), leishmaniasis recidivans (LR) and mucocutaneous leishmaniasis (MCL) (Chapter 57). Subgenera: Leishmania has two subgenera, L. Leishmania (L.L.) and L. Viannia (L.V.). Both of the subgenera comprise of nearly 20 species Old and New World: Depending up on the geographical distribution, leishmaniasis is classi$ed into two groups https://t.me/docinmaykingA B C D Figs 36.1A to D: Various morphological forms of !agellates (schematic diagrams): A. Amastigote; B. Promastigote; C. Epimastigote; D. Trypomastigote. CHAPTER 36 Visceral Leishmaniasis and Trypanosomiasis 361 Old World Leishmaniasis Seen in Asia, Africa and less frequently in Europe; transmitted by sand!y of genus Phlebotomus. L.L. donovani: Causes VL and PKDL L.L. infantum: Causes VL and PKDL L.L. tropica complex: Causes CL (Chapter 57) New World Leishmaniasis (Chapter 57) Seen in Central and South America; transmitted by sand!y of genus Lutzomyia. L.L. chagasi: Causes VL and CL L.L. mexicana complex: Causes CL L. Viannia braziliensis complex: Causes MCL and CL In Indian subcontinent, leishmaniasis is anthropophilic a#ecting only humans; whereas in other parts of the world, it is a zoonotic disease, transmitted from various animals. Visceral leishmaniasis (VL) and PKDL are discussed here. !e various cutaneous and mucocutaneous forms are discussed in Chapter 57. VISCERAL LEISHMANIASIS Visceral leishmaniasis (VL) is mainly caused by the old world species L. donovani and L. infantum, together known as L. donovani complex and also by new world species, L. chagasi. L. donovani is the most common species causing VL. It is found in India and Africa. It was named after Sir William Leishman and Sir Donovan, who simultaneously contributed to the discovery of this parasite in a patient from Kolkata and Chennai respectively in the same year 1903. !e subsequent discussion in this chapter is con$ned to VL due to L.donovani. !e di#erences from VL due to L. infantum and L. chagasi is described in Table 36.1. Epidemiology of VL Leishmaniasis is endemic in 97 countries; most of them are developing countries of tropical and temperate regions. Table 36.1: Various forms of visceral leishmaniasis. World: About 50-90 thousand new cases of VL occur worldwide each year, out of which >90% of cases are from three regions—(i) South-East Asia: India, Bangladesh, and Nepal; (ii) East Africa: Ethiopia, Sudan, and Kenya; and (iii) Brazil India: India is one of the worst a#ected country. In 2019, about 3,139 cases of VL and 821 cases of PKDL have been reported from India with nil death. Maximum cases were reported from Bihar (76% of VL, and 52% of PKDL) and followed by Jharkhand. Life Cycle (Leishmania donovani, Fig. 36.2) L. donovani completes its life cycle in two hosts —(i) Vertebrate host (man, dog, rodents, etc.) (ii) Invertebrate host (female sand"y, of genus Phlebotomus argentipes). Infective form: Promastigote forms found in alimentary canal of female sand"y serve as the infective form Mode of transmission: By the bite of an infected female sand"y, discharging promastigotes (infective forms) into skin of man https://t.me/docinmaykingFig. 36.2: Life cycle of Leishmania donovani. *RES-Reticuloendothelial system. Characters Old World VL New World VL Indian VL (kala-azar) Infantile VL African VL Mediterranean VL Agent Leishmania donovaniL. infantum L. donovani L. chagasi Vector Phlebotomus argentipesP. perniciosus P. orientalis, P. martiniLutzomyia longipalpis EpidemiologyIndia Middle East, Central Asia, China and Mediterranean basin Sudan, Ethiopia, Kenya and Uganda Central and South America Age a"ectedYoung adults Infants and children <5 years of ageAdults Children Reservoir Anthroponotic (human)Zoonotic (canine) Anthroponotic, Rarely-Zoonotic (rodents) Zoonotic (canine) PKDL Common Less common Common Less common Lymph node invovlement Less common More common, Aggravated by poor nutrition Less common Less common Abbreviations: VL, visceral leishmaniasis; PKDL, post-kala-azar dermal leishmaniasis. 362 SECTION 4 Bloodstream and Cardiovascular System Infections In humans: Promastigotes are phagocytosed by the skin macrophages, where they transform into amastigote forms The amastigote forms multiply inside the macrophages, causing cell rupture and are released into the circulation Amastigotes are carried out in the circulation to various organs like liver, spleen and bone marrow and invade the reticuloendothelial cells like macrophages, endothelial cells, etc. In sand"y: During the blood meal, the amastigotes are ingested and transformed into promastigote forms in the insect gut, which multiply and then migrate to their foregut. !e cycle continues when this sand"y bites a new host. Pathogenicity Various factors contribute to the pathogenesis of VL. The phagocytosis of the promastigotes is facilitated by binding of its surface antigens such as 63 kDa glycoprotein (gp-63) and lipophosphoglycan (LPG) to speci$c receptors on macrophages LPG is the principle virulence factor, exhibits variety of functions. It prevents phagosome maturation and protects the parasite against hydrolytic enzymes secreted from the phagolysosome GPI (glycosyl-phosphatidyl-inositol) is a major surface protein on amastigotes, helps in protecting from phagolysosomal attack inside the macrophage. Host Immune Response Depending on the host immune response (TH1 or TH2) the amastigotes are either killed or allowed to multiply inside the macrophages. T helper-1 Response If TH1 cells are activated, leads to a cell-mediated immune response which contain the disease spread by secreting interferon γ (IFN- γ) to activate macrophages, which in turn kill the amastigotes. TH1response is observed in cutaneous leishmaniasis and patients after recovery/treated for VL !ese individuals exhibit a delayed-type hypersensitivity (DTH) to leishmanial antigens (positive leishmanin skin test). T helper-2 Response If TH2 cells are activated, leads to a humoral immune response due to increased production of IL-10 and IL-4; which in turn causes polyclonal B cell activation leading to hypergammaglobulinemia. It is observed in patients developing active VL and in di#use CL Patients do not show positive leishmanin skin test !e parasites use the macrophage much like a Trojan horse. Amastigotes are released periodically by rupture of the macrophages !ey disseminate through the regional lymphatics and the vascular system to infect the reticuloendothelial cells of various organs !is results in remarkable enlargement of the spleen, liver and bone marrow dysfunction. Clinical Features (VL) VL is also called as kala-azar (a hindi term meaning “black fever”). Incubation period ranges from 2–6 months. !e hallmark of VL is a pentad of fever, progressive weight loss, hepatosplenomegaly, pancytopenia and hypergammaglobulinemia. Fever: !e most common symptom of VL; abrupt in onset, moderate to high-grade and associated with chills and rigors. Typically, it is described as double or triple rise of fever in 24 hours Splenomegaly: It is the most consistent sign. !e spleen may become hugely enlarged (soft nontender and friable) and palpable below the umbilicus (Fig. 36.3A) Hepatomegaly (non-tender, moderate degree) soon follows splenomegaly Lymphadenopathy: Common in most of the African endemic regions (rare in Indian subcontinent). Femoral and inguinal nodes are a#ected commonly Hyperpigmentation is observed on face, hands, feet, and abdomen; hence the name kala-azar or black fever. !is is a characteristic feature of Indian VL Pedal edema and ascites: Occur due to hypoalbuminemia, may be seen in advanced illness Mucosal lesions in mouth and nasopharynx—seen in Sudan, rare in India Hematological abnormalities: Occurs due to bone marrow dysfunction Pancytopenia: Anemia, leukopenia and thrombocytopenia Hypergammaglobulinemia (due to polyclonal B cell activation). Leishmanoma: Nodular skin lesions seen in African cases only Secondary infections: Such as measles, pneumonia, tuberculosis, bacillary or amoebic dysentery and gastroenteritis are common Other !ndings include weight loss and hair changes (thinning, hypopigmentation, etc.) Death occurs due to superimposed infection, severe anemia and hemorrhages. Post-kala-azar Dermal Leishmaniasis (PKDL) PKDL is a nonulcerative lesion of skin occurs in 2–50% of patients of VL following treatment with antimonials. It is aggravated by exposure to sunlight. Mainly seen in India and East African countries It develops as hypopigmented macule near mouth which spreads to face, arms and trunk and $nally becomes nodules resembling leprosy (Figs 36.3B to D) https://t.me/docinmaykingCHAPTER 36 Visceral Leishmaniasis and Trypanosomiasis 363 A B C D Figs 36.3A to D: Clinical features of leishmaniasis: A. Splenomegaly seen in visceral leishmaniasis; B. Hypopigmented skin changes in early PKDL; C and D. Extensive facial nodular lesions in late PKDL. Source: World Health Organization “Manual on visceral leishmaniasis control” Slide1/Desjeux; Slide 4 and 5/ El Hassan; Slide 6/ Bryceson (with permission). Ocular lesions like conjunctivitis and uveitis are associated in some patients Sometimes, PKDL may directly occur in subclinical patients without a history of VL Diagnosis: By detection of amastigotes in the nodular lesions and by serological tests such as direct agglutination test (DAT) and antibodies to rK39 antigen; positive in most of the cases PKDL in Indian subcontinent: Occurs in 2–20% of VL cases, usually after 2–10 years and persists for long periods (20 years). It can a#ect any age group. Amphotericin B is the drug of choice, given for 4 months. Leishmaniasis with HIV Co-infection Co-infection of HIV with VL has been reported from many countries. Southern Europe (France, Italy, Spain and Portugal) accounts for majority of co-infections; where 50–75% of VL are HIV positive and 7–17% of HIV-infected people with fever have amastigotes It is also reported from other places like Africa (Ethiopia, Sudan), Brazil and India In India, it is reported from Bihar, sub-Himalayan region and other North Indian states Both HIV and Leishmania a#ect each other’s pathogenesis Effect on HIV: Leishmania appears to cause activation of latent HIV. It expresses high level of chemokine receptors (CCR5) on macrophages E#ect on Leishmania: HIV causes activation of TH2 cells response leading to disease progression. Clinical feature: In HIV co-infected patients, apart from the classical features of VL, other forms such as CL, MCL, PKDL, and atypical presentation such as chronic diarrhea and pleural e#usion may be observed Diagnosis: Antibody detection tests are usually negative. Amastigotes are demonstrated from unusual sites such as bronchoalveolar lavage "uid and bu#y coat region of blood. L ABORATORY DIAGNOSIS Visceral leishmaniasis Microscopy—Giemsa staining, detects LD bodies (i.e. macrophage #lled with amastigote forms) ¾ Splenic aspiration: Most sensitive ¾ Bone marrow aspiration: Most common specimen ¾ Lymph node aspiration ¾ Liver biopsy ¾ Peripheral blood smear (in HIV-infected people) ¾ Biopsy of various organs (in HIV-infected people) Culture (detects promastigotes)—useful for species identi#cation and drug sensitivity testing ¾ NNN medium ¾ Schneider’s liquid medium Antibody detection in serum ¾ ELISA, IFA and direct agglutination test ¾ ICT using rk39 or rKE16 antigens Antigen detection—carbohydrate antigen in the urine (latex agglutination test) Molecular method—PCR, real-time PCR detecting speci#c kinetoplast (mitochondrial) DNA Leishmanin test (montenegro test)—indicates good CMI (DTH reaction); positive in all stages, except active VL and di"use CL Animal inoculation—golden hamster Nonspeci!c tests to detect: ¾ Hypergammaglobulinemia—by Napier’s aldehyde test and Chopra’s antimony test ¾ Pancytopenia—by complete blood count Laboratory Diagnosis In an endemic area, any case presented with fever >2 weeks, splenomegaly and/or weight loss is suspected of having VL and should be subjected to laboratory con$rmation. https://t.me/docinmayking364 SECTION 4 Bloodstream and Cardiovascular System Infections A B Figs 36.4A and B: L. donovani amastigotes: showing a macrophage containing multiple Leishmania amastigotes: A. Schematic; B. In bone marrow smear stained with Giemsa. Note that each amastigote has a nucleus (red arrow) and a rod-shaped kinetoplast (black arrow). Source: B. DPDx Image Library, Centers for Disease Control and Prevention (CDC), Atlanta (with permission). Microscopy Demonstration of amastigotes inside the macrophages (also known as Leishman-Donovan bodies or LD bodies) is the gold standard method for the diagnosis of VL (Figs 36.4A and B). Smears should be stained with Leishman, Giemsa or Wright stains. !e various samples include: Splenic aspiration: !e sensitivity of splenic smear examination is excellent (>98%) but less preferred because of the risk of splenic puncture. Grading of LD bodies from splenic smear is useful in determining the parasitic load and monitoring the response to treatment Bone marrow aspiration: It is the most common specimen. !e sensitivity is around 80–85% Lymph node aspiration: It is useful only in African cases of VL and has a low sensitivity (53–65%) Liver biopsy: Less sensitive and carries the risk of hemorrhage Peripheral blood: Smears made from bu#y coat area (after blood is centrifuged) are particularly useful in HIV patients. Amastigotes are found within mononuclear cells and neutrophils Biopsy specimens of various organs: Like oropharynx, stomach, or intestine; useful in patients with AIDS. Culture Useful specimens are aspirations from spleen, bone marrow and bu#y coat. NNN medium: Novy-MacNeal-Nicolle (NNN) medium is the culture medium of choice (Fig. 36.5B) Schneider’s Drosophila insect medium can also be used alternatively Inoculated specimens are incubated at ambient temperature (24–26°C) and examined weekly for 4 weeks before declared as negative Amastigotes transform into promastigotes in the culture "uid which are detected by staining with Giemsa stain (Fig. 36.5A) A B Figs 36.5A and B: A. Smear made from culture !uid shows promastigote forms (Giemsa stain); B. NNN medium. Source: A. DPDx Image Library, Centers for Disease Control and Prevention (CDC), Atlanta (with permission); B. World Health Organization, “Manual on visceral leishmaniasis control” (Slide22/Alvar) (with permission). Culture is found to be positive in 75% of cases Culture is useful for species identification and drug sensitivity testing. Antibody Detection in Serum In general, the serological tests are sensitive, but less specific. False-positive results may occur due to crossreacting antibodies in patients with leprosy, Chagas’ disease, CL, and other infections. Antibodies cannot di#erentiate current and past infection. More so, antibodies maybe absent or present in low titer in patients with AIDS Direct agglutination test (DAT): Serial dilution of patient serum is added with stained extract of L. donovani amastigote on microtiter plate and incubated for 18 hours If speci$c antibodies are present, agglutination (matt formation) is visible by naked eyes. Button formation indicates absence of antibodies (Fig. 36.6) Fig. 36.6: Direct agglutination test. Source: “Manual on visceral leishmaniasis control”, World Health Organization (Slide32/Alvar) (with permission). https://t.me/docinmaykingCHAPTER 36 Visceral Leishmaniasis and Trypanosomiasis 365 It is found to be 100% sensitive and specific. It is simple, rapid and does not need any instrument Disadvantage: However, antibodies persist up to 5 years after the treatment. Immunochromatographic test (ICT): ICT detects leishmanial antibody by using L. chagasi recombinant kinesin antigen (rK39) It claims 100% sensitivity and 98% specificity, however, the sensitivity is low in East Africa and in HIV patients Like DAT, it is also simple, rapid and does not need any instrument (useful in $eld studies) Recently, ICT based on another novel antigen rKE16 (from L. donovani) has been developed. Other tests available are ELISA and indirect "uorescent antibody (IFA) test. Antigen Detection Recently, latex agglutination test has been available detecting a heat-stable, low-molecular-weight carbohydrate antigen in the urine of VL patients. It has good speci$city but variable sensitivity (40–80%) Antigen detection is more useful (i) in HIV-VL coinfection, (ii) as a prognostic marker, (iii) indicating active infection. Molecular Methods Various formats such as PCR, nested PCR and quantitative detection by real-time PCR are available targeting Leishmania specific kinetoplast (mitochondrial) DNA. It is mostly con$ned to the reference laboratories with sensitivity varying from 70% to 93%. Leishmanin Test (Montenegro Test) It is a skin test to detect delayed hypersensitivity to a suspension of killed L. donovani promastigote injected intradermally. Positive test (induration of >5 mm in 72 hours) indicates prior exposure to Leishmania antigens. !erefore, it is useful for epidemiological survey to estimate the burden of the disease It is positive in people with good CMI: Asymptomatic individuals, cutaneous leishmaniasis, after recovery from VL and leishmaniasis recidivans However, this test is negative when CMI is low: Such as in case of active VL and di#use CL. Animal Inoculation Intranasal inoculation of specimens to golden hamsters yields amastigotes after several months. It is not in use nowadays. Nonspeci!c Tests Complete blood count—to detect pancytopenia Elevated liver enzymes Reversal of albumin globulin ratio (reflects hypergammaglobulinemia) Other Non-speci$c tests such as Napier’s Aldehyde test and Chopra’s antimony test were in use in the past to detect hypergammaglobulinemia; now not in use. T REATMENT Visceral leishmaniasis The various drugs used in the treatment of VL are: (i) pentavalent antimonials, (ii) liposomal amphotericin B, (iii) miltefosine, (iv) paromomycin. The WHO recommended regimens used for treatment of VL is given in Table 36.2. Pentavalent antimonials It has been the drug of choice and widely used in the past for several decades. However as the resistance has been emerged, currently, its use is restricted to regions where resistance has not been developed. Dosage: It is given as 20 mg/kg per day IM or IV for 30 days Mechanism of action: It is converted to its active trivalent form in body, induces oxidative stress in the parasite Resistance: Resistance to antimonials has been reported mostly from Bihar, India. Liposomal amphotericin B It has been the current drug of choice of leishmaniasis, especially in areas where resistance to antimonials have been reported. Dosage: It is given as 3–5 mg/kg per daily dose by IV infusion for 3–5 days up to a total dose of 15 mg/kg Mechanism of action: It acts by disrupting the cell membrane by forming pores. Miltefosine Dosage: It is given as 150 mg/day; orally for 28 days Mechanism of action: by interacting with lipids, inhibiting cytochrome C oxidase Resistance: Resistance to miltefosine is rare, mainly reported from India and Nepal. Paromomycin It is an aminoglycoside antibiotic with anti-leishmanial activity. It is given IM at a dose of 15 mg per kg per day for 21 days. Immunotherapy Interferon-γ has been used in antimonial resistant and in selected relapse cases from Kenya, Brazil, and India. Prevention of VL National Vector-borne Disease Control Programme National Vector-borne Disease Control Programme (NVBDCP) is a national program in India which works for the control of six common vector-borne diseases in India. It has launched the accelerated plan for kala-azar elimination in 2017. The target for elimination is to reduce the annual incidence of kala-azar to less than one per 10,000 populations at block PHC level The blocks in endemic area of India are classified into four categories based on annual incidence of kalaazar; each category has a specific action plan aiming towards kala-azar elimination, as described in Table 36.3. https://t.me/docinmayking366 SECTION 4 Bloodstream and Cardiovascular System Infections Table 36.2: WHO recommendation for treatment of VL in di!erent regions of the world. TRYPANOSOMIASIS Region Indian subcontinent (ranked by preference) Drug regimen(s) • Liposomal amphotericin B • Combinations ¾ Liposomal amphotericin B plus miltefosine ¾ Liposomal amphotericin B plus paromomycin ¾ Miltefosine plus paromomycin • Amphotericin B deoxycholate • Miltefosine • Paromomycin • Pentavalent antimonials Trypanosomes are hemo"agellates that reside in peripheral blood and tissues of their host. Based on their geographical distribution, they can be classi$ed into two types: African trypanosomes: Trypanosoma brucei complex. !ey are transmitted by the vector tsetse "y. !ey cause African sleeping sickness American trypanosomes: Trypanosoma cruzi; which is the causative agent of Chagas’ disease and is transmitted by the insect vector reduviid bug (also called triatomine bug). East Africa and YemenMediterranean Basin, Middle East, Central Asia and in America (due to L. infantum) Pentavalent antimonial plus paromomycin Liposomal amphotericin B CHAGAS’DISEASE (TRYPANOSOMA CRUZI) Trypanosoma cruzi is the causative agent of South American trypanosomiasis or Chagas’ disease, named after its discoverer Carlos Chagas, in 1909. Abbreviation: WHO, World Health Organization. Table 36.3: Categorization of blocks in endemic area* of India based on annual incidence of kala-azar, 2014. Categories De!nition Measures to be taken Category I (94 blocks) Blocks above elimination threshold (high transmission areas) Annual incidence >1 case per, 10,000 population Intensive case detection Vector control activities Case based surveillance Category II (23 blocks) Blocks with borderline endemicity with Annual incidence 0.8 to <1 per10,0000 population (low transmission area) Requires enhanced surveillance Category III (290 blocks) Fluctuation blocks with increasing or decreasing trend either year to year or with a gap of few years Operation factors to be looked in Category IV (207 blocks) Silent blocks, i.e. areas reporting nil cases annually for at least two consecutive years Robust surveillance for validation of nil status Preparedness for detection if cases occur *Endemic region include Bihar, Jharkhand, Uttar Pradesh and West Bengal. Vaccine Trials Currently, no vaccine is available for the prevention of leishmaniasis. However, several trials are on-going. Control Measures Vector control measures should be followed such as: Personal prophylaxis by using insect repellents or bed nets (as discussed in Chapter 35) Control of canine or rodent reservoir Phlebotomus does not "y high above the ground level and it is nocturnal in habitat. So, sleeping at top "oors also can prevent transmission Early treatment of all cases (mainly anthroponotic V Can you give a notes of it without missing any point
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