Dr.R.Jayaprada
LYMPHATIC FILARIASIS
Outline of presentation
Lymphatic Filariasis
 Introduction
 History
 Epidemiology
 Morphology
 Mode of transmission
...
Lymphatic Filariasis
 WHO has identified lymphatic filariasis (LF) as the second leading cause of
permanent and long-term...
Lymphatic Filariasis
• Transmitted by the bite of infected mosquito responsible for
considerable sufferings/deformity and ...
History
 Symptoms of bancroftian filariasis had been mentioned as elephantiasis
arabicum in the ancient hindu literature ...
EPIDEMIOLOGY
 W. bancrofti occurs in sub-Saharan Africa, Southeast Asia, the Indian
subcontinent, many of the Pacific isl...
Vectors of Lymphatic filariasis
 The major vectors of W. bancrofti are mosquitoes of the genus Culex (in
urban and semi-u...
EPIDEMIOLOGY
Global Scenario
 Population
at risk : 120 million
 No. of countries : > 80
 Mf carriers : 76 million
 Diseased : 44 mi...
National Scenario
 Total Population : 110 C
 Population at risk : 45.4 C
(in 16 States & 5 UT’s)
 Total infected : 51.7...
Taxonomic classification
 Kingdom : Animalia.
 Phylum : Nemathehelminthes.
 Class : Nematoda.
 Subclass : Phasmidia.
...
Morphology of W . bancrofti
 Adult worm: long, thread like, minute, whitish, transparent nematodes.
 Filariform in shape...
Morphology of Brugia
Brugia malayi
 The adult females of B malayi resembles to W. bancrofti but the
adult males differ .
...
Morphology of microfilaria
 First stage larva is called microfilaria.
 Microfilaria are found in the peripheral blood, h...
Morphology of microfilaria
 Granules are broken at definite places serving as the landmarks for
identification of the spe...
Differences b/n microfilaria of bancrofti, malayi & timori
Microfilariae Periodicity Sheath Size Body &tail
Mf. bancrofti ...
Microfilaria of Brugia
malayi. Figure B:
Microfilaria of B. malayi in
a thick blood smear,
stained with Giemsa and
capture...
Micro filarial periodicity
 Nocturnal periodicity: Mf are present in the peripheral blood between
10 P.M to 4 A.M. Mechan...
Modes of transmission & Incubation Period
 Lymphatic Filariasis is transmitted by the bite of Infected mosquito
which har...
Life cycle
 W. bancrofti passes its life cycle in 2 hosts: Man & Mosquito ( Culex
quinquefasciatus, Anopheles, Aedes).
 ...
Life cycle in mosquito
 Sheathed microfilaria ingested by mosquito during its blood meal, loses
its sheath with in 1 hour...
Life cycle in man
Mosquito with Mf bites a man
Third stage larva (L3) deposited on the skin
Reach lymphatic channels
Migra...
Life cycle
Pathogenesis
 Pathogenic effects seen in wuchereriasis are produced by the adult worm
(living/ dead).
 L3 larva & microf...
Causes of lymphangitis
 1.Mechanical irritation caused by the movement of adult worms inside
the lymphatic system.
 2. L...
Causes for lymphatic obstruction
 1. Mechanical blocking of the lumen by dead worms which act as an
embolus.
 2.Oblitera...
Factors affecting pathogenesis
 Factors affecting pathogenesis of filarial manifestations include
 1.Cumulative exposure...
Different stages of lymphatic vessel remodeling and modulation of lymphatic flow during
progression of filarial infection....
Pathogenesis
Immunopathogenesis
Microfilaria Adult
worm
Wolbachi
a
Dead/Decalcifyin
g
Worm
Secondary
infection
Parasitic secretory/excr...
Immunopathogenesis
Clinical manifestations
 Two distinct groups of clinical entities caused by W.bancrofti are:
 1. Lymphatic filariasis
 ...
Clinical manifestations
 1.Endemic normal/ Asymptomatic amicrofilaremia : In endemic areas
of filariasis, a certain propo...
Clinical manifestations
 3) Acute –
 Fever with chills and rigors, lymphedema with pain, lymphadenopathy
(cervical, axil...
Clinical manifestations
 5) Occult filariasis ( Meyers-Kouwenaar syndrome):
 a)Condition in which there is massive eosin...
Tropical pulmonary eosinophilia (Eosinophilic lung Or Weingarten’s
syndrome)
• First described in 1940 and labelled as “ps...
Laboratory diagnosis of Wuchereriasis
 Samples includes :
Peripheral blood , Chylous urine, Exudate of lymph varix, Hydro...
Methods of examination
 1. Blood Microscopy
 2. DEC provocation test
 3.Quantitative buffy coat examination
 4. Urine ...
Blood Microscopy
 2 or 3 drops of peripheral blood are collected by finger prick.
 Blood is collected as follows:
 Noct...
Blood Microscopy
 Microfilaria can be demonstrated in the blood by microscopy by following
methods:
 1.Direct wet mount:...
Blood Microscopy
 3. DEC provocation test:
 In this test, 2-8 mg/ kg of Diethyl carbamazepine (DEC) is given
orally to s...
Blood Microscopy
 4.Quantitative buffy coat examination:
 Quantitative buffy coat system (which requires commercial
equi...
Concentration of blood
 Various concentration methods are available for recovery of Mf.
 a. Knott’s method of concentrat...
Knott’s method of concentration by sedimentation.
 Principle : This technique is used to recover low number of microfilar...
Knott’s method of concentration by sedimentation
 Procedure: 1)1 ml of fresh whole blood or anticoagulated blood ( 9ml
of...
Membrane filtration concentration
 Principle: Membrane filtration method have been developed for
recovering from patients...
Membrane filtration concentration
 Procedure:
 1. 1 ml of fresh whole blood or anticoagulated blood into a 15 ml syringe...
Membrane filtration concentration
 6. Without disturbing the filter, Swiney adapter is removed from the
syringe, and appr...
Microscopy
 5.Urine Microscopy: Mf are demonstrated in the chylous urine.
 Usually 10-20 ml of early morning sample is c...
Immunodiagnosis
 Immunodiagnostic methods include:
 1.Serological tests & 2. Cellular assays.
 Serology-based diagnosis...
Antibody-detection assays
 These include:
 1.IHA, 2.IFA, 3.ELISA, 4.RIA,5.Luminescence immunoassay.
 Native antigen is ...
Antibody-detection assays
 Antibody-based diagnostic assays using four recombinant antigens,
WbSXP are commercially avail...
Antigen-detection assays
 These assays are useful in distinguishing recent & past infection.
 Two monoclonal antibody ba...
Antigen-detection assays
 ICT card has a sensitivity of 96-100% & specificity is 100 %.
 Main advantage of antigen based...
Serodiagnosis Contd
 ‘Seva Filachek’ is a dipstick based ELISA system which has been permitted
by government of India (Si...
Cellular assays
• These include:
• 1.Filarial skin test (Intradermal test ): It is an immediate hypersensitivity
test. Fil...
Other
 Molecular methods
 PCR: It has been developed which can detect as low as 1 pg of filarial
DNA.
 PCR is positive ...
Treatment
 Diethyl carbamazepine is the drug of choice for Lymphatic filariasis.
 It is filaricidal in action.
 1st day...
Prevention & Control
 Prevention & Control depends upon:
 1.Mosquito control 2. Chemotherapeutic control.
 Mosquito con...
Brugia. malayi
 Adult Brugia is distinctly dilated at the anterior end & has a
complete spicule.
 Mf is sheathed & has n...
Brugia. malayi
 Brugia. malayi causes malayan filariasis.
 It has both nocturnal periodicity– 10 P.M to 4 A.M & sub-peri...
 Laboratory diagnosis is similar to bancroftian filariasis.
 Antibody-based diagnostic assays using four recombinant ant...
B. timori
 Human infection with B.timori is found in Timor island at the eastern end of the
Indonesian archipelago.
 No ...
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Lymphatic Filariasis jp

  1. 1. Dr.R.Jayaprada LYMPHATIC FILARIASIS
  2. 2. Outline of presentation Lymphatic Filariasis  Introduction  History  Epidemiology  Morphology  Mode of transmission  Life cycle  Pathogenesis  Signs and symptoms  Laboratory diagnosis  Prevention and Control
  3. 3. Lymphatic Filariasis  WHO has identified lymphatic filariasis (LF) as the second leading cause of permanent and long-term disability after leprosy(1-3).  Filariasis is the pathological condition caused by infection of filarial nematodes transmitted by different vectors. Infection with 3 closely related Nematodes  Wuchereria bancrofti (90%)  Brugia malayi  Brugia timori (10%) 1.Centers for Disease Control and Prevention (CDC). Morbidity & Mortality Weekly Report. 2011; 60(24):814-18. 2. Addiss DG, Brady MA. Morbidity Management in the Global Programme to Eliminate Lymphatic Filariasis: A Review of the Scientific Literature. Filaria J 2007;6:2. 3.National Vector Borne Disease Control Programme, Directorate General of Health Services, Ministry of Health and Family Welfare, Government of India. Website: Available from: http://nvbdcp. gov.in/filariasis-new.html. [Assessed on 2011 Sept 23].
  4. 4. Lymphatic Filariasis • Transmitted by the bite of infected mosquito responsible for considerable sufferings/deformity and disability • Infection occurs in blood vessels, lymphatic system , connective tissues and serous cavities of man * All the parasites have similar life cycle in man * Adults are seen in Lymphatic vessels * Off springs seen in peripheral blood during night
  5. 5. History  Symptoms of bancroftian filariasis had been mentioned as elephantiasis arabicum in the ancient hindu literature ( Susruta, 600 BC).  Demarquay (1863) first demonstrated the microfilariae in hydrocele fluid.  Subsequently, Wucherer in brazil described the microfilariae in chylous urine in 1866.  Lewis (1872) in India demonstrated the microfilariae in the peripheral blood.  Bancroft (1876-77) first demonstrated adult females & Sibthorpe (1888) first found males.  Manson(1878) first demonstrated the culex mosquitoes as the intermediate host & also described the nocturnal periodicity of microfilariae in the peripheral blood.
  6. 6. EPIDEMIOLOGY  W. bancrofti occurs in sub-Saharan Africa, Southeast Asia, the Indian subcontinent, many of the Pacific islands, and focal areas of Latin America and the Caribbean (including Haiti).  B. malayi occurs mainly in China, India, Malaysia, the Philippines, Indonesia, and various Pacific islands.  B. timori occurs on the Timor Island of Indonesia.  Overall, approximately two-thirds of individuals infected with lymphatic filariasis are in Asia.
  7. 7. Vectors of Lymphatic filariasis  The major vectors of W. bancrofti are mosquitoes of the genus Culex (in urban and semi-urban areas), Anopheles (in rural areas of Africa and elsewhere) and Aedes(in islands of the Pacific).  The parasites of B. malayi are transmitted by various species of the genus Mansonia; in some areas, anopheline mosquitoes are responsible for transmitting infection.  Brugian parasites are confined to areas of east and south Asia, notably India, Indonesia, Malaysia and the Philippines.  An estimated 120 million people in tropical and subtropical areas of the world are infected with lymphatic filariasis; of these, almost 25 million men have genital disease (most commonly hydrocele) and almost 15 million, mostly women, have lymphoedema or elephantiasis of the leg.  Approximately 66% of those at risk of infection live in the South-East Asia Region and 33% in the African Region.
  8. 8. EPIDEMIOLOGY
  9. 9. Global Scenario  Population at risk : 120 million  No. of countries : > 80  Mf carriers : 76 million  Diseased : 44 million  Hydrocele : 27 million  Lymphoedema : 16 million  TPE : 1 million
  10. 10. National Scenario  Total Population : 110 C  Population at risk : 45.4 C (in 16 States & 5 UT’s)  Total infected : 51.7 million (Wb - 99.4 % and Bm - 0.6 %)  No. of diseased : 22.5 million  Mf carriers : 29.2 million  Hydrocele : 12.9 million
  11. 11. Taxonomic classification  Kingdom : Animalia.  Phylum : Nemathehelminthes.  Class : Nematoda.  Subclass : Phasmidia.  Super family : Filariodea.  Family : Acanthocheilonematidae.  Genus : Wucheraria, Brugia.  Species : bancrofti, malayi, timori.
  12. 12. Morphology of W . bancrofti  Adult worm: long, thread like, minute, whitish, transparent nematodes.  Filariform in shape.  Anterior end is surrounded by 2 rows of ten sessile papillae.  Posterior end contains anus at its terminal end.  Male measures 2.5 to 4 cm in length by 0.1 mm in thickness and female measures 8 to 10 cm in length and 0.3 mm in thickness  Male: Tail end is sharply curved ventrally with 2 spicules of unequal length.  Female: Tail end is narrow & abruptly pointed.  Females are ovoviviparous ( Laying eggs with well developed embroyos).  Life span is usually 5-10 years.
  13. 13. Morphology of Brugia Brugia malayi  The adult females of B malayi resembles to W. bancrofti but the adult males differ . Brugia timori  Similar to B. malayi .
  14. 14. Morphology of microfilaria  First stage larva is called microfilaria.  Microfilaria are found in the peripheral blood, hydrocele fluid and chylous urine.  It has a  A . Hyaline sheath  B . Cuticle lined by subcuticular cells & is seen only with vital stains.  C. Somatic cells or nuclei : These appear as granules in central axis of the body & extend from head to the tail end. Granules do not extend upto the tip of the nuclei & serve as distinguishing feature of Mf. bancrofti.
  15. 15. Morphology of microfilaria  Granules are broken at definite places serving as the landmarks for identification of the species. They include the following:  A. Nerve ring, an oblique space.  B. Anterior ‘V’ spot, represents rudimentary excretory system.  C. Posterior ‘V’ spot, represents the terminal part of the alimentary canal (anus/cloaca).  D.G-cells, also called genital cells.  E.Innenkorper of fulleborn or Central body of manson: Represents the rudimentary alimentary canal.
  16. 16. Differences b/n microfilaria of bancrofti, malayi & timori Microfilariae Periodicity Sheath Size Body &tail Mf. bancrofti Nocturnal Sheathed Large & thick Body curves are graceful & sweeping. Tail is tapering &pointed. No nuclei are in the tail end. Mf. malayi Nocturnal Sheathed Large & thick Body is coiled & kinked. Nuclei are large& dense. Tail is tapering. Two nuclei are present in the tail end. Mf.timori Sub-periodic Sheathed Large & thick Body stains lightly. Tail is irregularly tapering. Two nuclei are present at the tail end.
  17. 17. Microfilaria of Brugia malayi. Figure B: Microfilaria of B. malayi in a thick blood smear, stained with Giemsa and captured at 500x oil magnification Microfilaria of Wuchereria bancrofti. Figure A: Microfilaria of W. bancrofti in a thick blood smear stained with Giemsa and captured at 500x oil magnification Microfilaria of B. timori in a thick blood smear, stained with Giemsa and captured at 500x oil magnification. Courtesy: CDC
  18. 18. Micro filarial periodicity  Nocturnal periodicity: Mf are present in the peripheral blood between 10 P.M to 4 A.M. Mechanism is presumed to be related with night feeding habit of culex pipens fatigans.  Nocturnal periodicity is shown by Mf.bancrofti, B.malayi & B.timori  Diurnal periodicity: Mf are present in large number in peripheral blood during day hours. Eg: Loa loa.  Nocturnal sub-periodicity & diurnal sub-periodicity: Mf are present in the peripheral blood continuously throughout day & night with a slight increase in number during afternoon & evening. Eg: Sub-periodic W. bancrofti & B.malayi.
  19. 19. Modes of transmission & Incubation Period  Lymphatic Filariasis is transmitted by the bite of Infected mosquito which harbours L3 larva.  L1: 1-3 hours  L2: 3-4 days  L3: 5-6 days  Pre-patent period: (L3 to Mf) Not known  Clinical Incubation period: 8-16 months
  20. 20. Life cycle  W. bancrofti passes its life cycle in 2 hosts: Man & Mosquito ( Culex quinquefasciatus, Anopheles, Aedes).  Mode of transmission: Bite of infected female mosquito.  Infective form : Third stage larva.  In India & china: Culex pipens fatigans.  In pacific islands: Anopheles punctulatus.  In polynesian islands: Aedes polysienensis.
  21. 21. Life cycle in mosquito  Sheathed microfilaria ingested by mosquito during its blood meal, loses its sheath with in 1 hour or 2 hour Migrates to thoracic muscles of the mosquito next 48 hrs First stage larva (L1) ( Thick, short, sausage shaped forms with short spicy tail ) Moults once/ twice in 3-7 days time Second stage larva (L2) ( Larger sausage shaped larva on 10th or 11th day Third stage larva (L3)Infective stage. Enters the proboscis of the mosquito on 14th day. Development in mosquito is completed within 10-20 days. In mosquitoes, Mf do not multiply to increase in number, each microfilara develops into one L3 larva.
  22. 22. Life cycle in man Mosquito with Mf bites a man Third stage larva (L3) deposited on the skin Reach lymphatic channels Migrates to the inguinal lymph nodes Develop into adult worm & sexually mature Adult male & female worms are coiled up in the regional lymphatics &lymph nodes Fertilization Gravid female gives birth to Mf Reach the thoracic/R. lymphatic duct Venous system Peripheral blood
  23. 23. Life cycle
  24. 24. Pathogenesis  Pathogenic effects seen in wuchereriasis are produced by the adult worm (living/ dead).  L3 larva & microfilaria are not pathogenic.  Following stages occur sequentially in the pathogenesis of lymphatic filariasis:  1.Dilatation of lymphatic vessels.  2.Infecion of the lymphatics ( Lymphangitis).  3. Obstruction of the lymph nodes.
  25. 25. Causes of lymphangitis  1.Mechanical irritation caused by the movement of adult worms inside the lymphatic system.  2. Liberation of toxic metabolites by the growing larvae & secretion of some toxic fluid by fertilized females Allergic manifestations like urticaria, fugitive swellings & lymphedema.  3. Absorption of toxic products liberated from dead worms undergoing disintegration Allergic manifestations.  4.Bacterial infection---secondary invaders.
  26. 26. Causes for lymphatic obstruction  1. Mechanical blocking of the lumen by dead worms which act as an embolus.  2.Obliterative endolymphangitis: Endothelial proliferation & inflammatory thickening of the walls of the lymphatic vessels.  3.Excessive fibrosis of the lymphatic vessels: caused by recurrent & repeated attacks of lymphangitis.  4.Fibrosis of the afferent lymph nodes draining the particular area. Effects of lymphatic obstruction: Two types of conditions are produced:  A. Lymph varix —varicosity of lymphatic vessels.  B. Elephantiasis ---Hypertrophy of the affected part.
  27. 27. Factors affecting pathogenesis  Factors affecting pathogenesis of filarial manifestations include  1.Cumulative exposure to bites,  2.Quantity of accumulating adults,  3. Number of secondary infections,  4.Degree and type of host immune response (Th1/Th2) and  5.Genetic predisposition (4-6).  Hydrocele is less common in microfilaria carriers than endemic normals.  Lymphatic filarial parasites also harbor an endosymbiont – Wolbachia - that contributes to inflammation (7). 4.Adjobimey T, Hoerauf A. Induction of Immunoglobulin G4 in Human Filariasis: An Indicator of Immunoregulation. Ann Trop Med arasitol 2010; 104:55-64. 5.Ravichandran M, Reghunathan J, Narayanan RB, Jayaram K, Kaliraj P. Modulation of Cellular Immune Responses by Cytokines in Bancroftian Filariasis. Ind J Clin Biochem 1997; 12(1):27-31. 6.Lammie PJ, Cuenco KT, Punkosdy GA. The Pathogenesis of Filarial Lymphedema: Is it the Worm or is it the Host? Ann NY Acad Sci 2002; 979:131-42. 7.Debrah AY, Mand S, Specht S, Marfo-Debrekyei Y, Batsa L, Pfarr K et al. Doxycycline Reduces Plasma VEGF- C/sVEGFR-3 and Improves Pathology in Lymphatic Filariasis. PLoS Pathogens 2006;
  28. 28. Different stages of lymphatic vessel remodeling and modulation of lymphatic flow during progression of filarial infection. (A) Normal lymphatic collecting vessel showing normal flow patterns and lymphatic drainage regulated by the unidirectional valves in the absence of filarial parasitic infection. (B) Onset and progression of acute filarial infection with microfilariae and adult worms lodged within the vessel. Normal host immune response is initiated. Slight hypertrophy of the lymphatic muscle cell layers is observed with a partial impairment of lymph flow. (C) Chronic filarial infection results in a major host immune response due to toxins released by dead or live parasites. Various immune cells are observed at the site of infection leading to a strong inflammatory reaction. Secondary infections with bacteria harboring Wolbachia exacerbates the condition leading to a chronic infection state or elephantiasis. The lymphatic vessels exhibit largely dysfunctional valves, vessel dilation, impaired lymphatic muscle contractility and insufficient drainage. The resultant fluid accumulation and retrograde lymph flow associated with severe lymphedema. Activation and remodeling of lymphatic endothelial cells during this chronic stage could potentially result in either endothelial dysfunction or promote lymphangiogenesis.
  29. 29. Pathogenesis
  30. 30. Immunopathogenesis Microfilaria Adult worm Wolbachi a Dead/Decalcifyin g Worm Secondary infection Parasitic secretory/excretory Products (over 850 identified) Immunomodulatory Gly p (ES- 62), Circulating microbial products Early activation of immune response Th2 response Antiinflammatory cytokine production IL-4, IL-10, TGF-ß Wolbachia ligands(>90 identi) SP, HSP, LPS Inflammatory Cytokine prodution IL1,6,IFNγ,TNFα,N O Upregulation of pro inflammatory Cyto IL6,12,17,22, IFNγ,TNFα, GM CSF Desensitisation to TLR & CD 40 pathways Activation of inflammatory mediators Lymphangiogenic & Angiogenic factors, VEGF- A,C, D, Ang 1,2. Upregulated acute phase proteins CRP, Haptoglobin.. Early activation of immune response Th1 & Th2Depressed & Desensitized host immune response Acute inflammatory response Inflammatory host immune response Asymptomatic or Acute pathology Chronic pathology Lymphatic endothelial cells Lymphatic muscle cells Proliferation morphological changes..< permeability Fibrosis Cellular hyperplasia, impaired contractility
  31. 31. Immunopathogenesis
  32. 32. Clinical manifestations  Two distinct groups of clinical entities caused by W.bancrofti are:  1. Lymphatic filariasis  2. Occult filariasis.  Clinical manifestations depend on stages of the disease lymphatic filariasis as follows:  1.Endemic normal  2.Asymptomatic stage  3. Acute filariasis  4.Chronic filariasis  5.Occult filariasis  6.Less frequent lesions: granuloma of spleen & other organs, presence of adult W. bancrofti in the anterior chamber of the eye.
  33. 33. Clinical manifestations  1.Endemic normal/ Asymptomatic amicrofilaremia : In endemic areas of filariasis, a certain proportion of population living in these areas do not develop overt clinical features or any microfilaria in the peripheral blood after an exposure to L 3 larva.  2. Asymptomatic stage:  Persons in this stage have microfilaraemia in their blood & do not show any clinical manifestations.  It is suggested that in these people, TH1 component of inflammatory response is down regulated & TH2 component is stimulatory. Cytokine IFNα is depressed & IL-4 is elevated.  After many years later, Hyporesponsiveness breaks down Inflammation begins
  34. 34. Clinical manifestations  3) Acute –  Fever with chills and rigors, lymphedema with pain, lymphadenopathy (cervical, axillary, inguinal and generalised – Acute Filarial Lymphangitis/Acute Dermatolymphangioadenitis), chyluria, hematuria, inflammatory granuloma or abscesses, pain in testes, funiculitis, epididymoorchitis.  4) Chronic - funiculitis, epididymoorchitis, hydrocele, lymph varices, chyluria, elephantiasis, breast edema(1-3,8-10). 8.Sabesan S, Palaniyandi M, Das PK, Michael E. Mapping of lymphatic filariasis in India. Ann Trop Med Parasitol 2000; 94:591-606. 9.Hotez, PJ., Molyneux, DH., Fenwick A., Kumaresan, J, Sachs SE, Sachs JD et al. Control of Neglected Tropical Diseases. N Engl J Med 2007; 357:1018-27. 10.Lahariya C, Tomar SS. How Endemic Countries can Accelerate Lymphatic Filariasis Elimination? An Analytic Review to Identify Strategic and Programmatic Interventions. J Vector Borne Dis 2011; 48:1-6.
  35. 35. Clinical manifestations  5) Occult filariasis ( Meyers-Kouwenaar syndrome):  a)Condition in which there is massive eosinophilia (30-80%),  b)Absolute eosinophilic count >3000/ mm3,  c) Generalized lymphadenopathy  d) Hepatosplenomegaly  e) Pulmonary symptoms  f) Absence of microfilaraemia.  Adult worm produces Mf continuously, but they do not reach the peripheral blood because they are destroyed in the tissues.
  36. 36. Tropical pulmonary eosinophilia (Eosinophilic lung Or Weingarten’s syndrome) • First described in 1940 and labelled as “pseudotuberculosis with eosinophilia”(11). • Term tropical pulmonary eosinophilia (TPE) was first coined by Weingarten in 1932 to a syndrome of wheezing, fever, eosinophilia, cough with scanty sputum (blood tinged), spleenomegaly and bilateral mottling of the lungs. • Mf may be demonstrated in the lung biopsy specimens. • TPE is caused by a type 1 hypersensitivity reaction to filarial antigens (W. bancrofti or B. malayi). It presents as an eosinophilic alveolitis with an airway component. • Total serum IgE levels (10,000 to 100,000 ng/mL) and antifilarial antibody titers are characteristically elevated. 11.Frimod1. t-Moller C, Barton RM. A pseudo-tuberculosis condition associated with eosinophilia. Indian Med Gaz 1940; 75 : 607-13.
  37. 37. Laboratory diagnosis of Wuchereriasis  Samples includes : Peripheral blood , Chylous urine, Exudate of lymph varix, Hydrocele fluid, Lymph node biopsy, skin specimen  Diagnosis of Bancroftian filariasis  Direct evidence /Specific tests Indirect evidence/ Non specific test  Microfilariae in Peripheral blood, Adult worms Chylous urine, Hydrocele fluid in biopsied L.node Allergic tests Serological tests 1. Blood for eosinophilia 2. Intradermal test for immediate hypersensitivity
  38. 38. Methods of examination  1. Blood Microscopy  2. DEC provocation test  3.Quantitative buffy coat examination  4. Urine microscopy  5.Microscopy of hydrocele fluid & lymph node aspiration.
  39. 39. Blood Microscopy  2 or 3 drops of peripheral blood are collected by finger prick.  Blood is collected as follows:  Nocturnal periodic W.bancrofti: Between 10 P.M & 4 A.M in the night.  Sub-periodic Nocturnal W.bancrofti : Between 8 P.M & 4 A.M during night.  Sub-periodic diurnal W.bancrofti : Between 2 P.M & 6 P.M in the after noon.
  40. 40. Blood Microscopy  Microfilaria can be demonstrated in the blood by microscopy by following methods:  1.Direct wet mount: 2-3 drops of blood is collected on a clean glass slide & examined after placing a cover slip on it. Live microfilariae are identified by characteristic serpentine movement in the blood plasma.  2.Stained thick blood smear: Thick blood smear stained with giemsa or leishman or wright or delafield’s hematoxylin stain is most commonly used method for demonstration of Mf.  Sheathed Mf with absence of nuclei in the tail tip is diagnostic of W. bancrofti Mf.  Delafield’s hematoxylin stain is used widely to demonstrate the greater structural detail of Mf. This stain enhances the nuclei & sheath.
  41. 41. Blood Microscopy  3. DEC provocation test:  In this test, 2-8 mg/ kg of Diethyl carbamazepine (DEC) is given orally to stimulate nocturnal periodic Mf to circulate in the peripheral blood during the day time.  After 30 minutes, capillary blood is collected by finger prick for demonstration of Mf by direct wet mount or by staining the blood smear.  This test is contraindicated in the areas where Loa loa or Onchocerca volvulus infections are also found because of Mazzotti reaction & blindness in onchocerciasis.
  42. 42. Blood Microscopy  4.Quantitative buffy coat examination:  Quantitative buffy coat system (which requires commercial equipment and fluorescence microscopy) may be used to enhance sensitivity.  Additional thin smears would be required to determine identification of any microfilaria present.
  43. 43. Concentration of blood  Various concentration methods are available for recovery of Mf.  a. Knott’s method of concentration by sedimentation.  b. Membrane filtration concentration methods using nuclepore membrane filter or millipore membrane filter Most sensitive method for recovery of Mf.
  44. 44. Knott’s method of concentration by sedimentation.  Principle : This technique is used to recover low number of microfilariae from blood.  Formalin solution is used to lyse the RBC’s in a large blood sample, and the organisms are concentrated in sediment by centrifugation.  Disadvantage of this technique is that the Mf are killed & immobilized and are therefore not readily revealed by any motility.  Limitations:  A. Motility will not be visible after formalin fixation.  B. Identification to the species may be difficult without additional staining.
  45. 45. Knott’s method of concentration by sedimentation  Procedure: 1)1 ml of fresh whole blood or anticoagulated blood ( 9ml of blood + 1 ml of 5% sodium citrate) in a centrifuged tube containing 10 ml of 2% formalin is mixed thoroughly.  2. Centrifuge for 5 min at 300 Χ g.  3. Pour off the supernant fluid without disturbing the sediment.  4. A portion of sediment is placed on the slide & a cover slip is applied and examined microscopically under low power (10X) & high power (40X).  5. If Mf are present, a thick film is prepared from the remainder sediment, air dried, fixed in absolute methanol for 5 min, air dried again & stained with giemsa/ Delafield haematoxylin.
  46. 46. Membrane filtration concentration  Principle: Membrane filtration method have been developed for recovering from patients with light infections  It is one of the most efficient for the clinical laboratory when other procedures used to recover Mf are unsatisfactory.  Limitations:  A. Giemsa or hematoxylin staining may be necessary to identify the organisms to species level.  B. Identification of Mf on filters to the species level may be difficult.
  47. 47. Membrane filtration concentration  Procedure:  1. 1 ml of fresh whole blood or anticoagulated blood into a 15 ml syringe containing 10 ml of distiiled water.  2. Mixture is shaken for 2-3 min to ensure that all blood cells are lysed  3. A 25 mm Nuclepore filter (5µm porosity ) is placed over a moist 25 mm filter paper pad, & placed in a swiney filter adapter.  4. Swiney filter adapter is attached to the syringe containing the lysed blood.  5. Lysed blood is pushed through the filter with gentle steady pressure on the piston.
  48. 48. Membrane filtration concentration  6. Without disturbing the filter, Swiney adapter is removed from the syringe, and approx 10 ml of distilled water is drawn into the syringe. Adapter is replaced & gently water is pushed through the filter to wash the debris from the filter.  7. Filter is removed from the adapter, placed it on the slide & allowed it air dry.  8.Then stained with giemsa / Delafield hematoxylin.
  49. 49. Microscopy  5.Urine Microscopy: Mf are demonstrated in the chylous urine.  Usually 10-20 ml of early morning sample is collected, centrifuged & sediment is examined under microscope for Mf.  6.Microscopy of hydrocele fluid/ L.node aspiration:  Ether/ Chloroform /xylol is used dissolve the fat globules & same method as urine is employed.
  50. 50. Immunodiagnosis  Immunodiagnostic methods include:  1.Serological tests & 2. Cellular assays.  Serology-based diagnosis tools can be divided into two categories:  a) Antigen-detection assays and b) Antibody-detection assays.  These include the enzyme-linked immunosorbent assay (ELISA), also called enzyme immunoassay (EIA), and all its derived tests such as the Falcon assay screening test ELISA (FAST-ELISA) and the dot-ELISA.  Other assays include the hemagglutination (HA) test, indirect or direct immunofluorescent antibody (IFA or DFA) tests, complement fixation (CF) test, and immunoblotting and rapid diagnostic tests (RDTs)(12). 12.Ndao M. Diagnosis of parasitic diseases: old and new approaches. Interdiscip Perspect Infect Dis. 2009
  51. 51. Antibody-detection assays  These include:  1.IHA, 2.IFA, 3.ELISA, 4.RIA,5.Luminescence immunoassay.  Native antigen is W.bancrofti Mf obtained from microfilaraemic cases or heterologous filarial worms ( Brugia.malayi, Dirofilaria immitis, etc).  Most of the newer tests are using recombinant filarial antigen.  Falcon assay screening test ELISA (FAST-ELISA) consists of using synthetic and recombinant peptides to evaluate antibody responses to an antigen
  52. 52. Antibody-detection assays  Antibody-based diagnostic assays using four recombinant antigens, WbSXP are commercially available.  They are based on the detection of antifilarial IgG4 antibodies.  Disadvantages: These assays show cross reactivity with sera from other filarial & helminthic infections.  These cannot discriminate between past & current infections as filarial antibodies persist longer even after clinical cure.
  53. 53. Antigen-detection assays  These assays are useful in distinguishing recent & past infection.  Two monoclonal antibody based ELISA’s that detect circulating filarial antigen in the serum are available.  A) ELISA employing mab AD12 detects a 200 KD antigen of adult W.bancrofti in the serum.  B) ELISA using the mab Og43C that detects adult W.bancrofti as well as Mf antigen in the serum.  Immunochromatography test (ICT) filariasis card test is a new & rapid filarial antigen test that detects soluble W.bancrofti antigens in the serum of the infected patients.  This test is developed by ICT diagnostics (Balgowlah, New South Wales, Australia).  It uses mab AD12 to detect the antigen.
  54. 54. Antigen-detection assays  ICT card has a sensitivity of 96-100% & specificity is 100 %.  Main advantage of antigen based assays are:  1.Blood can be collected during daytime for demonstration of the antigen.  2. Demonstration of antigen in the urine is a most recent approach for the diagnosis of bancroftian filariasis.
  55. 55. Serodiagnosis Contd  ‘Seva Filachek’ is a dipstick based ELISA system which has been permitted by government of India (Signal MF) for microfilarial antigen(IC-Ag) as well as filarial antibodies (IgG4) in diagnosis of filarial infection in different clinical groups.  The detection of IgG4 antibody titre of 1:300 and above against specific microfilarial antigen was found to be useful.
  56. 56. Cellular assays • These include: • 1.Filarial skin test (Intradermal test ): It is an immediate hypersensitivity test. Filarial antigen is injected on skin. After 30 minutes a wheal over 2 cm appears. • 2. Invitro lymphocyte responses to filarial antigens. • Disadvantage of cellular assay being neither of the two are specific.
  57. 57. Other  Molecular methods  PCR: It has been developed which can detect as low as 1 pg of filarial DNA.  PCR is positive only when circulating Mf are found in the peripheral blood.  So PCR is negative in cases of chronic infections.  Xenodiagnosis:  Demonstration of Mf in the stomach-blood of the specific mosquito vector which was allowed to bite an infected individual.  Imaging methods : Chest X-ray, Ultrasound.  Haemogram shows eosinophilia.
  58. 58. Treatment  Diethyl carbamazepine is the drug of choice for Lymphatic filariasis.  It is filaricidal in action.  1st day—50 mg after food.  2nd day---50 mg tid daily.  3rd day--- 100 mg tid daily.  4th day to 21st day– 5mg/kg /day in 3 divided doses.  Use of tetracycline to kill the adult worm & to kill wolbachia bacteria which is having a symbiotic relation with W.bancrofti.  Other drugs include: Ivermectin-single oral dose-150µg/kg is used to destroy the Mf, but not the adult worm.  Recently evaluated drugs include Levamisole, mebendazole and centprazine ( CDRI, Lucknow).
  59. 59. Prevention & Control  Prevention & Control depends upon:  1.Mosquito control 2. Chemotherapeutic control.  Mosquito control:  a) Clinical control– By spraying insecticides like DDT, Malathion etc.  b) Biological control– By the use of carnivorous bacteria ( Bacillus sphaericus, strain 1593), carnivorous fish (Poecilia reticulata molliensis) & spore forming bacterium ( Bacillus thuringenesis serotype 11-14). c) Environmental control– By efficient drainage & sewage system to eliminate the mosquito breeding places. d) Reduction of non vector contact by use of mosquito nets & house screens.  Chemotherapeutic control:  It is based on the mass or selective treatment of the cases by administering DEC or use of DEC medicated salt in the populations exposed to filarial infections.
  60. 60. Brugia. malayi  Adult Brugia is distinctly dilated at the anterior end & has a complete spicule.  Mf is sheathed & has nuclei at the end of the tail. One nucleus is present at the external end of the tail.  Adult always inhabits the lymphatics. Mf are present in the peripheral blood.  Brugia has 9 species.  Species pathogenic to man are:  B. malayi, B.timori, B. pahangi, B.beaveri.
  61. 61. Brugia. malayi  Brugia. malayi causes malayan filariasis.  It has both nocturnal periodicity– 10 P.M to 4 A.M & sub-periodic diurnal periodicity.  Life cycle is same as that of W.bancrofti.  It is transmitted by mosquito vectors– Mansonia, A.barbirostris, but not by Culex.  Man is the definitive host, apart from humans leaf monkeys ( Presbytis spp) are also definitive hosts.  Infective form : Third stage larva (L3) similar to W.bancrofti.  Pathogenesis & pathology:  Like W.bancrofti, it causes lymphangitis & elephantiasis.  Malayan filariasis is characterized by absence of chyluria and genital involvement.
  62. 62.  Laboratory diagnosis is similar to bancroftian filariasis.  Antibody-based diagnostic assays using four recombinant antigens, Bm14,BmSXP, and BmR1 have become commercially available.  They are based on the detection of antifilarial IgG4 antibodies.  The BmR1 ELISA as well as dipstick (Brugia Rapid immunochromatography based) antibody tests have very high sensitivity for Brugia malayi (~100%), Bm14 ELISA is sensitive for both Wuchereria bancrofti and Brugia malayi (~91%-96%).  A sandwich ELISA detecting antibodies to recombinant antigen Bm-SXP-1 has been found useful to detect B. malayi infection.  Treatment: same as bancroftian filariasis.  Prophylaxis: In India, removal of water plant, Pistia stratiodes, Water hyacinth reduced the breeding of Mansonioides annulifera, mosquito vector.
  63. 63. B. timori  Human infection with B.timori is found in Timor island at the eastern end of the Indonesian archipelago.  No animal reservoir host has yet been discovered.  Natural vector mosquito is A. barbirostris.  Adults are found in lymphatic system.  Clinical manifestations are milder than other lymphatic filariasis.  Lymphangitis, lymphadenitis, lymphedema (confined below knee) & abscess along the lymph trunk or nodes are common clinical features.  Diagnosis depends on the detection of Mf in the peripheral blood collected during night.  Mf.timori has a length: width ratio of cephalic space of 3:1 (Mf.malayi 2:1), 5 -7 terminal nuclei, sheath not stained with giemsa stain.  Laboratory diagnosis, treatment & prophylaxis are similar to those of B.malayi.
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