Filarisis

Concise discussion on Fialrial worms including Morphology, Life cycle, pathogenesis, clinical manifestations and laboratory diagnosis including newer techniques for UG and PG students.

1. Filariasis Dr. Suprakash Das Assist. Prof.

2. Lecture Objectives Introduction of filariasis Parasitological description –Morphology and Life cycle History of filariasis in ancient and modern times. Epidemiology of filariasis and the burden of the disease. Pathogenesis Clinical spectrum of filarial diseases. Laboratory diagnosis- Conventional and newer methods Treatment of filariasis in short.

3. Introduction Nematodes belonging to the superfamily Filarioidea are slender thread-like worms (Latin, filum and thread), which are transmitted by the bite of blood-sucking insects. The filarial worms reside in the subcutaneous tissues, lymphatic system, or body cavities of humans. The adult worm generally measures 80-100 mm in length and 0.25-0.30 mm in breadth ; the female worm being longer than the males. The tail of the male worm has perianal papillae and unequal spicules but no caudal bursa. The female worms are viviparous and give birth to larvae known as microfilariae . The microfilariae released by the female worm, can be detected in the peripheral blood or cutaneous tissues, depending on the species. In some species, the microfilariae retain their egg membranes which envelop them as sheath. They are known as sheathed microfilariae . In some other species of filarial nematodes, the egg membrane is ruptured and is known as unsheathed microfilariae .

4. Introduction Once the microfilariae are classified on the basis of sheath as "sheathed" or "unsheathed", their further differentiation can be done on the characteristic arrangement of nuclei. Periodicity  Depending on when the largest number of microfilariae occur in blood, filarial worms can exhibit nocturnal, diurnal periodicity or no periodicity at all. The basis of periodicity is unknown but it may be an adaptation to the biting habits of the vector. The life cycle of filarial nematodes is passed in two hosts: (1) definitive host is man and (2) intermediate host are the blood-sucking arthropods. The microfilariae complete their development in the arthropod host to produce the infective larval stages. These are transmitted to humans by arthropod, which are their vectors also during the next feed. Adult worms live for many years whereas microfilariae survive for 3-36 months.

5. Introduction Eight species of filarial worms infect humans, of them six are pathogenic- (1) Wuchereria bancrofti , (2) Brugia malayi and (3) B. timori cause lymphatic filariasis; (4) Loa loa causes malabar swellings and allergic lesions; (5) Onchocerca volvulus causes eye lesions and dermatitis; (6) Mansonella streptocerca leads to skin diseases; and two of them, (7) M. ozzardi and (8) M. perstans are virtually nonpathogenic. Infection with any of the filarial worms may be called filariasis, but Traditionally, the term filariasis refers to lymphatic filariasis caused by Wuchereria or Brugia species.

6. LYMPHATIC FILARIASIS- Introduction   Lymphatic filariasis (LF) is the second most common mosquito-borne disease globally . LF infection occurs by exposure to mosquito bites. There are three parasites, which cause human LF  Wuchereria bancrofti , Brugia malayi , and Brugia timori all of them are transmitted by Anopheles , Aedes , and Culex . W. bancrofti is responsible for more than 90% of infections globally, B. malayi is mostly contributed to the transmission of the remainder.

7. LYMPHATIC FILARIASIS- Introduction   The third parasite, B. timori , is common in a few countries in Southeast Asia. The most important filarial diseases for humans are lymphatic filariases, in which the adult worms are found in the lymphatic system. The lymphatic form of filariasis will be the focus of the site. Lymphatic is also referred to sometimes as “elephantiasis.” Elephantiasis is actually an extreme clinical feature of filariasis. 

8. LYMPHATIC FILARIASIS- History   Ancient Time  It is known that lymphatic filariasis occurred in the Nile region, and ancient artifacts suggest that the disease may have been found as early as 2000BC. A statue of Pharaoh Mentuhotep depicts swollen limbs, a characteristic of elephantiasis. Artifacts from the Nok civilization in West Africa also show scrotal swelling, another characteristic of elephantiasis. The first reliable documentation of lymphatic filariasis symptoms is from an exploration of Goa between 1588 and 1592 ( by writings of  Jan Huygen Linschoten ) Recent Times  In 1863, French surgeon Jean-Nicolas Demarquay  M icrofilariae in fluid extracted from a hydrocoele (another common symptom of lymphatic filariasis) firstly. Three years later, Otto Henry Wucherer  M icrofilariae in urine.

9. LYMPHATIC FILARIASIS- History   Timothy Lewis  M icrofilariae in both blood and urine. The adult worm was discovered by Joseph Bancroft . Patrick Manson in 1877  P inpoint the microfilariae in mosquitoes. In 1900, George Carmichael Low discovered microfilariae in the proboscis of mosquitoes, and finally pinpointed the true mechanism of transmission, which is attributed to infective bite from a mosquito vector.

10. Jean-Nicolas Demarquay Otto Henry Wucherer Joseph Bancroft Patrick Manson

11. LYMPHATIC FILARIASIS- Epidemiology One of the most important infectious diseases worldwide  Lymphatic filariasis most common. 120M people in at least eighty countries are infected with the parasites associated with lymphatic filariasis. 90% of this infection  W. bancrofti . 1B people are estimated to be at risk for Infection. 25M men have the genital disease (hydrocele) and almost 15M, mostly women, have lymphoedema or elephantiasis of the leg. Many kinds of mosquitoes can transmit the parasite, depending on the geographic area. For example, in Africa  Anopheles Americas  Culex , Aedes and Mansonia  Pacific and Asia.  

12. LYMPHATIC FILARIASIS- Life Cycle The extrinsic life cycle starts when the microfilariae are ingested with the human blood by a bite of a mosquito. The microfilariae migrate through the gut wall of the mosquito to thoracic muscles where they become shorter and thicker, later develop into the first-stage larvae (L1). After 5–7 days, the L1 grow and develop to become the second stage (L2), which is more active and finally by 10–11 days, they develop to become the infective stage larvae (L3). After maturity, most of the infective larvae (L3) move to the mosquito’s proboscis, where they become ready to infect another human.

13. LYMPHATIC FILARIASIS- Life Cycle When the mosquito bites the host, L3 is put on the skin surface and after pulling the proboscis; they get into the wound and travel to the lymphatics. After about 9–10 days of entering, the L3 molt to become the fourth stage larvae (L4). The L4 stage needs several days to few months before it develops and becomes an adult. In the human body, adult worms (male and female) live in lymph vessels and lymph nodes. After mating, the females produce numerous microfilariae, which migrate into the lymphatic system and spread through the bloodstream 

14. LYMPHATIC FILARIASIS- Parasite Morphology Adult worm  The adults are whitish, translucent, thread-like worms with smooth cuticle and tapering ends. The female is larger (70-100 × 0.25 mm) than the male (25-40 x 0.1 mm). The posterior end of the female worm is straight, while that of the male is curved vertically and contains two spicules of unequal length. Males and females remain coiled together usually in the abdominal and inguinal lymphatics and in the testicular tissues. The female worm is viviparous and directly liberates sheathed microfilariae into lymph. The adult worms live for many years, probably 10-15 years or more.

15. LYMPHATIC FILARIASIS- Parasite Morphology Microfilariae:  The microfilaria has a colorless, translucent body with a blunt head, and pointed tail. It measures 250-300 µm in length and 6-10 µm in thickness. It can move forwards and backwards within the sheath which is much longer than the embryo. It is covered by a hyaline sheath, within which it can actively move forwards and backwards as sheath is much longer than the embryo. When stained with Leishman or other Romanowsky stains, structural details can be made out. Along the central axis of the microfilaria, a column of granules can be seen, which are called somatic cells or nuclei.

16. LYMPHATIC FILARIASIS- Parasite Morphology The granules are absent at certain specific locations- a feature which helps in the identification of the species. The specific locations are as following  At the head end is a clear Space devoid of granules, called the cephalic space . In Microfilaria bancrofti , the cephalic space is as long as it is broad, while in Microfilaria malayi , it is longer than its breadth. With vital a stains, a stylet can be demonstrated projecting from the cephalic space. The anterior half of the microfilaria, is an oblique area devoid of granules called the nerve ring . Approximately midway along the length of the microfilaria is the anterior V-spot, which represents the rudimentary excretory system. The posterior V-spot (tail spot) represents the cloaca or anal pore.

17. W. Bancfrofi microfilariae in Thick blood smear

18. LYMPHATIC FILARIASIS- Pathogenesis Infection caused by W. bancrofti is termed as wuchereriasis or bancroftian filariasis. The disease can present as:  Classical filariasis Occult filariasis. Classical filariasis : It occurs due to blockage of lymph vessels and lymph nodes by the adult worms. The blockage could be due to mechanical factors or allergic inflammatory reaction to worm antigens and secretions. The affected lymph nodes and vessels are infiltrated with macrophages, eosinophils, lymphocytes and plasma cells. The vessel walls get thickened and the lumen narrowed or occluded, leading to lymph stasis and dilatation of lymph vessels.

19. LYMPHATIC FILARIASIS- Pathogenesis The worms inside lymph nodes and vessels may cause granuloma formation, with subsequent scarring and even calcification. Inflammatory changes damage the valves in lymph vessels, further aggravating lymph stasis. Increased permeability of lymph vessel walls lead to leakage of protein-rich lymph into the tissues. This produces the typical hard pitting or brawny edema of filariasis . Fibroblasts invade the edematous tissues, laying down fibrous tissue, producing the nonpitting gross edema of elephantiasis. Recurrent secondary bacterial infections cause further damage. Occult fllariasis :  It occurs as a result of hypersensitivity reaction to microfilarial antigens, not directly due to lymphatic involvement. Microfilariae are not found in blood, as they are destroyed by the allergic inflammation in the tissues.

20. LYMPHATIC FILARIASIS- Clinical Manifestations The most common presentations of lymphatic filariasis are  Symptomatic (subclinical) microfilaremia, Acute adenolymphangitis (ADL) and Chronic lymphatic disease. Most of the patients appear clinically asymptomatic but virtually all of them have subclinical disease including  Microscopic hematuria or proteinuria, Dilated lymphatics (visualized by imaging) and in men with W. bancrofti infection, Scrotal lymphangiectasia [ Lymphangiectases represent superficial lymphatic dilatation caused by a wide range of scarring processes. Lymphangiectasia occurs as a consequence of lymphatic damage by an external cause, leading to obstruction of local lymphatic drainage.] (detected by ultrasound).

21. LYMPHATIC FILARIASIS- Clinical Manifestations Acute adenolymphangitis is characterized by  High fever, Lymphatic inflammation (lymphangitis and lymphadenitis) and Transient local edema. Fever is of high grade, sudden in onset, associated with rigors and last for 2 or 3 days. Lymphangitis is inflamed lymph vessels seen as red streaks underneath the skin. Lymphatics of the testes and spermatic cord are frequently involved, with epididymo -orchitis and funiculitis. Acute lymphangitis is usually caused by allergic or inflammatory reaction to filarial infection, but may often be associated with streptococcal infection also.

22. LYMPHATIC FILARIASIS- Clinical Manifestations Lymphadenitis Inflammation of lymph nodes. Most common affected lymph nodes being inguinal nodes followed by axillary nodes. The lymph nodes become enlarged, painful and tender. Lymphedema This follows successive attacks of lymphangitis and usually starts as swelling around the ankle, spreading to the back of the foot and leg. It may also affect the arms, breast, scrotum, vulva, or any other part of body. Initially, the edema is pitting in nature , but in course of time, becomes hard and nonpitting.

23. LYMPHATIC FILARIASIS- Clinical Manifestations Lymphangiovarix Dilatation of lymph vessels commonly occurs in the inguinal, scrotal, testicular and abdominal sites. The lymphangitis and lymphadenitis can involve both the upper and lower extremities but involvement of genital lymphatic occurs exclusively with W. bancrofti infection. The genital involvement  Funiculitis, Epididymitis and Hydrocele formation. Hydrocele:  very common manifestation of filariasis. Accumulation of fluid occurs due to obstruction of lymph vessels of the spermatic cord and also by exudation from the inflamed testes and epididymis. The fluid is usually clear and straw colored but may sometimes be cloudy, milky, or hemorrhagic. The hydrocele may be unilateral or bilateral and is generally small in size in the early stage, but may occasionally assume enormous proportions in association with elephantiasis of the scrotum. The largest reported hydrocele weighed over 100 kilograms.

24. LYMPHATIC FILARIASIS- Clinical Manifestations Lymphorrhagia  Rupture of lymph varices leading to release of lymph or chyle and resulting in Chyluria, Chylous diarrhea, Chylous ascites and Chylothorax , depending on the involved site. Elephantiasis  Repeated leakage of lymph into tissues first results in lymphedema, then to elephantiasis, in which there is Nonpitting brawny edema Growth of new adventitious tissue Thickened skin, cracks, and fissures Secondary bacterial and fungal infections, commonly seen in leg but may also involve other parts of body.

25. LYMPHATIC FILARIASIS- Clinical Manifestations Occult filariasis:  Massive eosinophilia (30-80%) Hepatosplenomegaly Pulmonary symptoms like dry nocturnal cough, dyspnea and asthmatic wheezing. Arthritis, Glomerulonephritis, Thrombophlebitis, Tenosynovitis, etc. Classical features of lymphatic filariasis are absent. Meyers Kouwenaar syndrome is a synonym for occult filariasis.

26. LYMPHATIC FILARIASIS- Clinical Manifestations Tropical pulmonary eosinophilia  This is a manifestation of occult filariasis which Presents with Low-grade fever, Loss of weight, and Pulmonary symptoms such as dry nocturnal cough, dyspnea and asthmatic wheezing. Children and Young adults are more commonly affected in areas of endemic filariasis including the Indian subcontinent. There is a marked increase in eosinophil count (>3000 µm - ≥ 50,000 µm) . Chest X-ray shows mottled shadows similar to miliary tuberculosis . It is associated with a high level of serum immunoglobulin E ( IgE ) and filaria antibodies . Serological tests with filarial antigen are usually strongly positive . The condition responds to treatment with diethylcarbamazine (DEC) , which acts on microfilariae.

27. LYMPHATIC FILARIASIS- Laboratory Diagnosis The diagnosis of filariasis depends on  Clinical features, History of exposure in endemic areas and Laboratory findings. Demonstration of microfilaria  Microfilaria can be demonstrated in blood, chylous urine, exudate of lymph varix and hydrocele fluid. Peripheral blood is the specimen of choice. The method has the advantage that the species of the infecting filaria can be identified from the morphology of the microfilaria seen. It is also the method used for carrier surveys.

28. LYMPHATIC FILARIASIS- Laboratory Diagnosis In India and other areas, where the prevalent filarial species is nocturnally periodic, it is best to collect "night blood" samples between 10 pm and 4 am. Microfilaria can be demonstrated in unstained as well as stained preparations and in thick as well as thin smears. Unstained film  Examination under the low power microscope shows the actively motile microfilariae lashing the blood cells around. The timing of blood collection is critical and should be based on the periodicity of the microfilariae. The examination may be conveniently made the next morning as microfilariae retain their viability and motility a for a day or 2 at room temperature.

29. Microfilaria in unstained Blood film

30. LYMPHATIC FILARIASIS- Laboratory Diagnosis Stained film  A "thick and thin" blood smear is prepared on a clean glass slide and dried. The thick part of the smear is dehemoglobinized by applying distilled water. The smear is fixed in methanol and stained with Giemsa, Leishman, or polychrome methylene blue stains. Microfilariae may be seen under the low power microscope in the thick film. The morphology of microfilariae can be studied in thin film. The microfilaria of W. bancrofti are sheathed and appear smooth curves in stained smear and are 298 µm long and 7.5-10 µm in diameter. By using a micropipette for taking a known quantity of blood (20-60 mm") for preparing the smear and counting the number of microfilariae in the entire stained smear, microfilaria counts can be obtained.

31. LYMPHATIC FILARIASIS- Laboratory Diagnosis Concentration techniques  Knot's concentration technique:  Anticoagulated blood (1 mL) is placed in 9 mL of 2% formalin and centrifuged 500 x g for 1 minute. The sediment is spread on a slide to dry thoroughly. The slide is stained with Wright or Giemsa stain and examined microscopically for microfilariae. Nucleopore filtration:  In the filtration methods used at present, larger volumes of blood, up to 5 mL, can be filtered through millipore or nucleopore membranes (3 µm diameter). The membranes may be examined as such or after staining, for microfilariae. The filter membrane technique is much more sensitive, so that blood can be collected even during day time for screening.

32. LYMPHATIC FILARIASIS- Laboratory Diagnosis The disadvantages of the technique are the cost and the need for venipuncture. Diethylcarbamazine provocation test  A small dose of DEC (2 mg per kg body weight) induces microfilariae to appear in peripheral blood even during day time. For surveys, blood samples can be collected 20-50 minutes after the administration of one 100 mg tablet of DEC to adults. Other specimens: Microfilaria may be demonstrated in centrifuged deposits of  Lymph, Hydrocele fluid, Chylous urine or Other appropriate specimens. Usually 10-20 mL of the first early morning urine is collected for examination and demonstration.

33. LYMPHATIC FILARIASIS- Laboratory Diagnosis Biopsy  Adult filarial worms can be seen in sections of biopsied lymph nodes, but this is not employed in routine diagnosis. Skin test  Intradermal injection of filarial antigens (extracts of microfilariae, adult worms and third-stage larvae of B. malayi or of the dog filaria Dirofilaria immitis ) induce an immediate hypersensitivity reaction. But, the diagnostic value of the skin test is very limited due to the high rate of false-positive and negative reactions. Imaging techniques Ultrasonography  High frequency ultrasonography (USG) of scrotum and female breast coupled with Doppler imaging may result in identification of motile adult worm (filaria dance sign) within the dilated lymphatics. Adult worm may be visualized in the lymphatics of the spermatic cord in up to 80% of the infected men with microfilaria associated with W. bancrofti .

34. LYMPHATIC FILARIASIS- Laboratory Diagnosis Radiology  Dead and calcified worms can be detected occasionally by X-ray. In tropical pulmonary eosinophilia (TPE), chest X-ray shows mottled appearance resembling military tuberculosis. Intravenous urography, retrograde pyelography, lymphangiography and lymphoscintigraphy may be used to demonstrate abnormal lymphatic urinary fistula. Serodiagnosis ( Demonstration of antibody) Complement fixation, Indirect hemagglutination (IHA), Indirect fluorescent antibody (IFA), Immunodiffusion and Immunoenzyme tests have been described. Indirect immunofluorescence an ELISA detect antibodies in over 95% of active cases and 70% of established elephantiasis. Disadvantages: Cannot differentiate between current and past infections.

35. Calcified Filarial Worm

36. LYMPHATIC FILARIASIS- Laboratory Diagnosis Demonstration of circulating antigen  Highly sensitive and specific test for detection of specific circulating filarial antigen (CFA) have been developed for detection of recent bancroftian filariasis . Trop-bio test  semiquantitative sandwich ELISA for detection of CFA in serum or plasma specimen. Immunochromatographic test (ICT)  is a new and rapid filarial antigen test that detects soluble W. bancrofti antigens using monoclonal antibody (ADI2) in the serum of infected humans. Both assay have sensitivities of 93-100% and specificities approaching 100%. Specific IgG4 antibody  against W. bancrofti antigen WbSXP-1 have been used to develop ELISA for detecting circulating filarial antigen in sera of patients with filariasis. There is however, extensive cross-reactivity between filarial antigens and antigens of other helminths, including intestinal roundworm, thus interpretation of serological findings can be difficult. Advantages: Antigen detection tests are more sensitive than microscopy and can differentiate between current and past infections.

37. LYMPHATIC FILARIASIS- Laboratory Diagnosis Molecular diagnostic technique  Polymerase chain reaction (PCR) can detect filarial deoxyribonucleic acid (DNA) from patient's blood, only when circulating microfilaria are present in peripheral blood but not in chronic carrier state. Usually the test provides sensitivities that are up to tenfold greater than parasitic detection by direct examination and is 100% specific. Recently LAMP technique has also been used. Indirect evidences  Eosinophilia (5-15%) is a common finding in filariasis. Elevated serum IgE levels can also be seen.

38. LYMPHATIC FILARIASIS- Treatment Diethylcarbamazine is the drug of choice. It is given orally in a dose of 6 mg/kg body weight daily for a period of 12 days amounting to a total of 72 mg of DEC per kg of body weight. Following treatment with DEC severe allergic reaction ( Mazzotti reaction) may occur due to death of microfilariae. It kills both microfilaria and adult worm. Antihistamines or corticosteroids may require to control the allergic phenomenon. The administration of DEC can be carried out in three ways: l. Mass therapy  In this approach, DEC is given to almost everyone in community irrespective of whether they have microfilaremia disease manifestation or no signs of infection except those under 2 years of age, pregnant women and seriously-ill patients.

39. LYMPHATIC FILARIASIS- Treatment The dose recommended is 6 mg/kg body weight. In some countries it is used alone and in some, with albendazole or ivermectin. Mass therapy is indicated in highly endemic areas. 2. Selective treatment  Diethylcarbamazine is given only to those who are microfilaria-positive. In India, the current strategy is based on detection and treatment of human carriers and filarial cases. The recommended dose in the Indian program is DEC 6 mg/kg of body weight daily for 12 doses, to be completed in 2 Weeks. In endemic areas, treatment must be repeated every 2 years. 3. Diethylcarbamazine medicated salts  Common salt medicated with 1-4 gram of DEC per kg has been used for filariasis control in Lakshadweep island, after an initial reduction in prevalence had been achieved by mass or selective treatment of microfilaria carriers.

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