The Problem of Filarial Disease

The term “filariasis” may seem remote to some – but for others in the medical field, it is far too close to home. Filariasis refers to a group of neglected tropical diseases caused by nematodes of the superfamily Filarioidea, transmitted through arthropod vectors. These diseases are classified as lymphatic or cutaneous/ocular filariasis based on which tissues are the primary home of adult worms (1). It is estimated that over 120 million people are infected worldwide, 40 million of whom are disfigured or incapacitated by the disease. The social, psychological, and economic burden of filariasis – amounting to a loss of 2.8 million Disability Adjusted Life Years annually (2) – is clear. The World Health Organization has committed to eliminating lymphatic filariasis as a public health problem by 2020 – and river blindness, a cutaneous/ocular form of the disease by 2025 – by i) mass drug administration in endemic regions and ii) targeting the vectors to halt transmission (3,4). However, even regions that have eliminated filariasis may see its re-emergence due to travel in and out of the area (5).

How and where
Parasitic nematodes responsible for human lymphatic filariasis include Wuchereria bancrofti (which causes over 90 percent of lymphatic filariasis), Brugia malayi, and Brugia timori. Nematodes causing cutaneous and ocular filariasis include Onchocerca volvulus, Loa loa, Mansonella perstans, Mansonella ozzardi, and Mansonella streptocerca. Transmission of lymphatic filariasis occurs through the Culex (in urban and suburban areas), Anopheles (in rural areas), and Aedes (Pacific islands) mosquitoes. Black flies are vectors for M. ozzardi and O. volvulus, and deer flies for L. loa. Biting midges transmit M. ozzardi, M. perstans, and M. streptocerca (1,2,6).

To make an accurate diagnosis, the geographic distributions of these parasites are key.

• W. bancrofti has widespread distribution, mainly in the tropics and subtropics including Asia, Pacific islands, Africa, South America, and the Caribbean basin.
• B. malayi is concentrated in southeast Asia, including India, China, Philippines, Malaysia, Indonesia, Korea, and Vietnam.
• B. timori is limited to some islands of eastern Indonesia, including Timor (2).
• O. volvulus is endemic to west and central Africa, as well as parts of eastern Africa and Yemen.
• L. loa is found only in the tropical regions of west-central Africa; exercise caution in diagnosing loiasis outside Africa. Dirofilaria causes ocular infections clinically similar to loiasis in Europe and Asia (7,8).
• M. perstans is distinctive to Africa south of the Sahara, Central and South America, and some Caribbean islands.
• M. ozzardi is distributed from Central to South America and in the Caribbean.
• M. streptocerca is native to tropical areas of western and central Africa (9).

The circle of life
Filarial nematodes need two hosts to complete their life cycle: mosquitoes or flies as intermediate hosts for development and maturation, followed by humans as definitive hosts for reproduction (10,11). Humans have adult worms localized to lymphatics (W. bancrofti, B. malayi, B. timori), subcutaneous tissues or eyes (L. loa, O. volvulus, M. streptocerca, M. ozzardi),and body cavities (M. perstans). Adult worms can survive for years in human hosts. When they undergo sexual reproduction, female worms release millions of larvae (microfilariae) into the skin or blood. These motile microfilariae stay in pulmonary vessels during the day and migrate to peripheral blood at night, reaching peak peripheral blood concentration – and thus ideal sample collection time – around midnight in parasites with nocturnal periodicity (W. bancrofti and Brugia spp.).

How does it work? A vector picks up microfilariae circulating in blood or cutaneous tissue during a feed. The development stages, which occur in the vector and take 10–14 days, are L1 (inactive), L2 (pre-infective), and L3 (infective). Finally, the vector bites a human host and releases L3 larvae that migrate to their respective localization site.

Studies have demonstrated the presence of Wolbachia bacteria as endosymbionts in some filarial nematodes. This association is implicated in disease pathogenesis, and host immune response, and adult worm viability and development. However, when the nematode dies, these bacteria are released and can initiate an immunologic reaction (12) – which is why it may be effective to accompany antifilarial treatment with antibiotics targeting Wolbachia.

Clinical manifestations
The spectrum of disease differs between patients living in endemic regions and those who have traveled or recently migrated to those regions. In general, filarial disease has a severe, acute presentation in recent travelers or newly exposed individuals, whereas people native to endemic areas experience a more chronic and debilitating disease course.

Lymphatic filariasis
In lymphatic filariasis, asymptomatic or subclinical microfilaremia is the most common presentation. In endemic areas, this begins in early childhood (13). Patients may exhibit lymphangiectasis or dilated lymphatics (including scrotal lymphatics), microscopic hematuria, and proteinuria (1).

In acute filarial lymphangitis, patients present with high-grade fever, retrograde lymphatic inflammation, lymph node enlargement, and transient edema. Genital lymphatic involvement in W. bancrofti filariasis leads to funiculitis, epididymitis, and scrotal pain. Acute dermatolymphangioadenitis is characterized by fever, chills, myalgia, red tender edematous inflammatory plaques, and vesicles or ulcers. Ongoing lymphatic inflammation and subsequent obstruction leads to elephantiasis: brawny edema, thickening of subcutaneous tissue, and hyperkeratosis. Hydrocele, scrotal lymphedema and chyluria may be noted, and obstructive lymphedema may involve other organs. Recently exposed individuals present with short-lived lymphadenitis, followed by retrograde lymphangitis (1,5,14).

Tropical pulmonary eosinophilia is an occult filariasis characterized by marked eosinophilia, paroxysmal cough and wheeze (mostly at night), low-grade fever, and weight loss. Microfilariae are not usually identified in peripheral blood, but may be rarely observed in lung biopsies. A rapid initial response to diethylcarbamazine is hallmark of this disease (15).

Onchocerciasis
Cutaneous manifestations include intense pruritus, generalized papular eruptions, eczematous dermatitis, lichenification, and hypo- or hyperpigmentation (“leopard skin”). Longstanding cases may exhibit lax skin with atrophy. Onchocercomata – firm, non-tender, mobile subcutaneous nodules composed of adult worms – are present over bony prominences. Ocular symptoms may include conjunctivitis, keratitis, corneal opacities, uveitis, and optical atrophy. Patients also frequently experience hanging groin with enlarged inguinal and femoral lymph nodes covered by atrophied loose skin (14,16,17).

Streptocerciasis
The clinical features of this disease resemble those of onchocerciasis. They include pruritus, skin rash, and lymphadenopathy, but lack the subcutaneous nodules (16).

Loiasis
Loiasis is mostly asymptomatic. Episodic Calabar swellings (transient, localized subcutaneous angioedema) over extremities may be observed, as may subconjunctival presence of microfilariae. These are transitory and resolve with complete recovery (1,16). Atypical manifestations include arthralgia or arthritis, heart failure due to endomyocardial fibrosis, respiratory symptoms due to pleural effusion or pulmonary fibrosis, and rarely gastrointestinal and renal symptoms. Encephalopathy, either spontaneous or more often after initiation of ivermectin, may occur in patients with high parasite load (>30,000 microfilaria/mL). Some patients experience partial or complete vision loss.

Mansonellosis
Most patients infected with M. perstans are asymptomatic – but some may present with transient subcutaneous swellings, rash, pruritus, effusions, or systemic symptoms. Individuals with M. streptocerca infection present with papular dermatitis and pruritus, most commonly on the upper parts of the body (18).

Making the diagnosis
Diagnosis of filariasis can be made by direct visualization of adult worms in biopsy; by observing microfilariae in peripheral blood, aspirate smears, and skin snips; and by serologic and molecular testing.

Histopathology
The lymphatics of the lymph nodes, breast, epididymis, and legs are commonly affected. In subclinical lymphangiectasia, intact adult worms are found lying freely in dilated lymphatics lined by thickened endothelium. The worms are 30–75 µm wide and have a thin cuticle with fine transverse striations (19). There is paucity of inflammatory response. Fibrosis alternates with atrophied or hypertrophied smooth muscle. Nonspecific reactive lymphoid hyperplasia occurs in the lymph node nearest to the dilated lymphatics harboring the parasite (20).

Host inflammatory response occurs in response to the death of an adult worm and intensifies as the filarial worm degenerates. The dead worm lies within fibrinous material adherent to the vessel wall. Eosinophil-rich inflammation, initially mild, becomes florid with time, obliterating the lumen. The degenerated worms and fragmented cuticles form their own inflammatory centers, consisting of lymphocytes, plasma cells, histiocytes, and eosinophils; granulomatous reactions (20,21) and central neutrophilic micro-abscess formations may be present. Finally, the dead parasite calcifies, surrounded by fibrous healing. Granulation tissue forms in the surrounding zone and obliterated vessels may undergo recanalization (20). Differential diagnosis includes:

• cat scratch disease-related lymphadenitis, which shows similar central necrosis surrounded by micro-abscesses and granulomas
• other granulomatous lymphadenitis
• Hodgkin lymphoma (19)

The subcutaneous nodules of onchocerciasis reveal coiled adult worms surrounded by a fibrous capsule (22). Skin biopsy shows the presence of microfilariae in all layers of the dermis, along with chronic inflammation comprising a variable number of lymphocytes, plasma cells, and histiocytes, with a few eosinophils. After treatment initiation, plenty of eosinophils are seen around degenerating microfilariae. Granulomas may be present in some cases. All patients show dermal scarring with loss of elastic fibers and fibrinoid necrosis of collagen. The cornea in punctate keratitis reveals edema and lymphocyte and eosinophil infiltration. Inflammation is extensive in heavy parasitemia, and the accompanying neovascularization and fibrous scarring leads to sclerosing keratitis (22,23).

Lymph nodes from patients with onchocerciasis show thickening and fibrosis of the capsule, obliteration of the subcapsular sinuses dilated in the early phase, and replacement fibrosis of lymphoid tissue. The characteristic perivascular symmetrical fibrosis with fibrinoid material deposition is probably due to immune complex deposition. The medulla displays plenty of eosinophils, plasma cells, and histiocytes. Microfilariae can be identified in fibrous capsule, interstitial tissue, and rarely in the medulla (24).

Skin biopsies from cutaneous loiasis lesions reveal adult worms with randomly arranged cuticular nodules. Dirofilaria is an important differential; these species have taller, more numerous muscle cells and possess longitudinal cuticular ridges on skin biopsy (7). Loiasis lymphadenitis shows unique features: atrophied lymphoid follicles and sinusoidal dilatation containing histiocytes and eosinophils. Other features include capsular and trabecular fibrosis and dilatation of capsule and medullary lymphatics (25).

Streptocercal lymphadenitis reveals depletion of lymphoid tissue due to inhibitory factors released by microfilariae (24).

Cytologic findings
Parasites may be identified in cytological specimens, such as fine needle aspirates, effusion cytology, and gynecologic smears. Several case reports have been published on the cytological diagnosis of filarial disease from various sites. Aspirate or fluid may appear milky, turbid, or hemorrhagic. Microfilariae are detected on a background of inflammatory cells rich in eosinophils (see Figure 1). At times, giant cells and epithelioid cell granulomas are part of the inflammatory reaction. Rarely, cytology reveals adult worms or fragments (26, 27). 

Erroneous diagnoses can present a challenge; extraneous material like cotton fibers, vegetable matter, or airborne contamination with fungal conidia may be mistaken for microfilariae (28). Accurate diagnosis relies on recognizing the characteristic morphologic pattern of the microfilariae.

Peripheral blood smear
Adult worms residing in lymphatics are usually inaccessible; the traditional method of diagnosing lymphatic filariasis is to detect microfilariae in the peripheral blood, particularly thick smears (see Figure 2). Circulating microfilariae can be identified in peripheral blood smears and occasionally bone marrow aspirate (29). Associated eosinophilia is a frequent hematologic finding in filariasis; in particular, counts are strikingly high in tropical pulmonary eosinophilia, a complication of lymphatic filariasis.

The absence of microfilariae in the peripheral blood does not exclude a diagnosis of filariasis. Even in patients with overt disease, microfilaria may be absent; adult worms may still be sexually immature, have passed reproductive age, or died (20). The time of sample collection should be appropriate for expected peak concentration; blood samples should be collected at midnight for filarial forms with nocturnal periodicity and midday for diurnal periodicity. Microfilariae survive in venous EDTA blood for 48 hours at room temperature. Often surrounded by empty space, they are found at the edges and tail of smears – so it’s vital to screen the entire smear at 4X magnification, or else microfilariae may be missed. Wet-mount preparation may reveal motile microfilariae.

When microfilariae cannot be detected in smears, concentration techniques are indicated. The filtration method is the most sensitive, but risks potential infection. The recommended approach is the lysed capillary blood method, which can reveal moving microfilariae through a partially closed condenser iris or dark-field microscopy. Field’s stain A or 1% methylene blue can facilitate species identification. Where the lysed capillary blood cannot be applied, buffy coat preparation also concentrates microfilariae (30).

Species identification is done on stained smears by morphologic assessment of sheath (present or absent), distribution pattern of nuclei, and size of microfilaria. Sheath identification is an important feature, but interpretation should be in context of size. W. bancrofti, B. malayi, and L. loa are sheathed filariae; Mansonella spp. are unsheathed. However, some features can complicate sheath identification:

• Sheathed microfilariae may lose their sheaths if there is a delay in sample processing.
• The sheath may be retracted.
• Sheath color is dependent on pH and type of stain.
• The sheath cannot be identified in skin snip preparations.

For size context, microfilaria can be compared with red blood cells. W. bancrofti and Brugia spp. are equal to or larger than red blood cells, whereas Mansonella spp. are half the size.

Nuclear pattern is the most important diagnostic parameter (see Table 1).

Skin snips
Diagnosis of O. volvulus and M. streptocerca microfilariae in skin snips – a specialized bloodless biopsy – is standard. Skin snips are collected from multiple sites. The skin tissue is immersed in isotonic saline, covered with a coverslip, and the slide is examined for motile microfilariae after 15 to 20 minutes. Prolonged incubation is required in low-density infections.

Serologic and molecular testing
Serologic testing and detection of antigen or antibody are convenient diagnostic modalities compared with demonstration of microfilaria in smears. These testing methods are unaffected by periodicity and thus do not require nocturnal sampling. Sensitivity is high, meaning that these assays can identify the patients with low-density microfilariae as well as amicrofilaremic patients.

• W. bancrofti filariasis can be diagnosed by detecting circulating filarial antigen by enzyme-linked immunosorbent assay (ELISA) and rapid immunochromatographic card test. These are extremely sensitive and specific methods. Filarial antigen tests can yield positive results for at least three years following mass treatment in endemic regions and are thus not suitable for determining the efficacy of control programs. Filariasis test strips are more sensitive at low antigen levels and have longer shelf life (5,31,32).
• Brugia can be detected by a rapid test for IgG4 antibodies with high sensitivity and specificity. Rapid antibody-based assays are available to diagnose onchocerciasis. The main drawback of serologic testing is cross-reactivity with W. bancrofti and other protozoal parasites, making it insufficient for establishing the diagnosis independently (32,33).
• Polymerase chain reaction (PCR)-based assays are available for diagnosing W. bancrofti, Brugian spp., L. loa, and O. volvulus (13,33).
• Finally,hypergammaglobulinemia may be present and serum IgE levels are high in all filarial diseases.

In essence, a combination of characteristic geographic or travel history, clinical presentation, blood eosinophilia, demonstration of microfilariae, and/or serologic testing allows a correct diagnosis of filarial nematodes. No single feature is enough for diagnosis – and this is a particular problem in resource-limited regions where diagnosticians may lack the resources for a multimodal approach and patients may lack the ability to reach a clinic once, let alone attend repeatedly for diagnosis and treatment. The reasons behind regions with endemic filariasis are manifold and complex, but for those of us tasked with identifying and treating the disease, this combination of methods may allow us to modify our approach to suit our resources and our patients’ needs.