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Chapter 10. 
The Blood Nematodes  


10.1 The Blood Nematodes  

These nematodes are known as filariae and consist of a group of nematodes which have successfully invaded the blood stream, connective tissue or serous cavities of vertebrates. They are long thread –like nematodes.  

Many of them are of medical and veterinary importance attacking man and various domestic animals being transported by various vectors, including mosquitoes. The nematodes from this order require intermediate hosts for the completion of their life cycle.  

The morphology of these nematodes consist of a cylindroid pharynx with an anterior muscular portion and a posterior glandular portion; the males have well-developed alae (a flat, wing-like anatomic process or part) and spirally coiled tails.  

Sexually mature female worms release microfilaria, which are pre-larval stages. These are released into the bloodstream. Most species are known to be ovoviviparous and some have ‘sheathed’ microfilaria.  

The filarial nematodes which parasitize man consist of Wuchereria bancrofti, Brugia malayi, Brugia timori and Loa loa, Onchocerca volvulus, Mansonella perstans, Mansonella streptocerca and Dipetalonema streptocerca.  

They inhabit a range of locations within the body; lymph glands, deep connective tissue, subcutaneous tissues or mesenteries. Invasions of these tissues usually result in inflammatory reactions which is a typical symptom of a human filarial infection. In some cases these result in fleshy deformities known as elephantiasis.  

It has been estimated that approximately one billion people in tropical and subtropical countries are exposed to the risk of filarial infections and at least 200 million are infected with filariasis. The species which are primarily responsible for these human filarial infections are Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus.  


Wuchereria bancrofti

Introduction

Wuchereria bancrofti is a nematode causing lymphatic filariasis throughout the tropics and subtropics and is transmitted by the mosquito.  There are two strains of W. bancrofti;

1.     The nocturnal periodic strain which is widely distributed in endemic regions (i.e. Africa, India and the Far East and also parts of China, Korea and Japan) with the microfilariae being in their highest concentrations between the hours of 10pm and 2am.

2.     The sub-periodic strain which is found in the Pacific region, and has a microfilaremia all the time with the highest numbers being detected between noon and 8pm.   

Humans are the only known reservoir host of W. bancrofti. Infection rates in some communities in East Africa exceed 30% of adults causing revolting swellings of the legs or genital system, known as elephantiasis in man.  The adult worm occurs in tightly coiled nodular masses in the major lymphatic ducts.  

The main vector is Culex quinquefasciatus, a mosquito that is particularly common in towns and cities, breeding in organically polluted water, resting in houses and feeding by night on their human occupants. Typical breeding sites include: storm drains blocked with domestic refuse, accumulations of domestic waste water, inadequately covered septic tanks and pit latrines.  

In rural areas throughout Africa, Anopheles gambiae and Anopheles funestus are involved in transmission. Elsewhere other anopheline mosquito species may transmit bancroftian filariasis in rural areas.  In Papua New Guinea, Mansonia species may act as a vector.  

Life Cycle

Microfilariae enter the host during a blood meal when the vector, a mosquito, punctures the skin.  The infective larvae enter through the wound and migrate to the peripheral lymphatics where they grow to mature male and female worms. They can live there for several years.  After mating, the gravid females release sheathed microfilariae into the peripheral blood where they can be detected 8-12 months after the initial infected bite.  

The mosquito acquires the infection by ingestion of the microfilaria in the blood meal.  The microfilariae lose their sheath on arrival in the stomach of the mosquito due to gastric juices.  The larvae migrate to the thoracic muscles and develop into infective larvae over a period of 6-14 days.  The larvae then migrate to the mouthparts of the mosquito which infects the host during a blood meal.

The blood stages of filariae, microfilariae, vary in the times when they are present in the peripheral blood, corresponding with the peak biting time of the vector. Thus, in nocturnally periodic forms the microfilaria are present in the peripheral blood circulation at night; during the day they reside in the deep tissues, particularly the lungs.  

 

PHIL Image 3425


Illustration 10-1.
Diagram showing the life cycle of Wuchereria bancrofti, the filarial nematode known to cause the disfiguring disease, elephantiasis, in man. (SOURCE:  PHIL 3425 - CDC/Alexander J. da Silva, PhD/Melanie Moser)


Morphology

The adult worms are white and threadlike.  The male measures between 2.5–4cm whereas the female is larger, measuring between 8-10cm.  

The microfilariae are 230-275mm in length. The tail of the microfilariae of W. bancrofti tapers to a delicate point and exhibits no terminal nuclei. The sheath the microfilariae of W. bancrofti stains with hematoxylin stain.   

 

Image 10-1. Microfilaria of Wuchereria bancrofti. The microfilaria is sheathed, its body is gently curved, and the tail is tapered to a point. The nuclear column (the cells that constitute the body of the microfilaria) is loosely packed, the nuclei can be visualized individually and do not extend to the tip of the tail. (Micrograph) (SOURCE:  PHIL 3008 - CDC/Dr. Mae Melvin)


Clinical Disease

Many patients are asymptomatic.  Patients may present with fever, lymphangitis and lymphadenitis. Lymphangitis commonly affects the lower extremities and there may also be genital and breast involvement.  An inflammatory reaction occurs in the lymphatic vessels that harbor the adult worms.  Edema develops which may resolve after the first few attacks.  A late complication resulting in thickening and verrucous changes in the skin known as elephantiasis may occur after recurring lymphangitis. Secondary bacterial and fungal infections may occur in patients with long-standing elephantiasis.   

Obstruction of the genital organs may result in hydrocoele formation and scrotal lymphoedema.  Obstruction of the retroperitoneal lymphatics may cause the renal lymphatics to rupture into the urinary tract producing chyluria. 

Image 10-2. Lymphatic filariasis: Elephantiasis is the last consequence of the swelling of limbs and scrotum. (SOURCE:  PHIL 373 – CDC)


Some patients with filariasis do not exhibit microfilaremia but develop tropical pulmonary eosinophilia which is characterized by peripheral eosinophilia, wheeze and cough.  High eosinophilia, high IgE level and high anti-filarial antibody titers are features of this syndrome.

Laboratory Diagnosis

See section 10.2 below  

Sheath may or may not stain with Giemsa; does stain with hematoxylin stains. Discrete nuclei. Empty space between the nuclei and the body wall. No nuclei in tip of tail. Innerbody is rarely visible in Giemsa. Does not stain with hematoxylin. Cephalic space as long as it is broad. Tip of tail may be bent underneath the body. Found in blood.  


Brugia malayi

Introduction

Brugia malayi is a nematode causing lymphatic filariasis in South East Asia.  There are two strains of B. malayi;

1.   The nocturnal periodic strain which is widely distributed in Asia, the microfilariae being in their highest concentrations between the hours of 10pm and 2am.

2. The sub-periodic strain which is found in Malaysia, Indonesia and the Philippines where humans exhibit a microfilaremia all the time with the highest numbers being detected between noon and 8pm.  

Nocturnally periodic Brugian filariasis is primarily a rural disease, being transmitted by various Anopheles species of mosquitoes and also by Mansonia species, a mosquito that usually bites during the night.  

Nocturnally sub-periodic B. malayi is transmitted almost exclusively by Mansonia species, often different species than those involved in transmitting the periodic form. Mansonia bonneae are important vectors in Malaysia, breeding in swamp forests and biting by night, although sometimes by day as well.  

This species like W. bancrofti also parasitizes the lymph nodes and lymphatics; the adults of the two species are indistinguishable, causing Malayan filariasis.  

Life Cycle

The adult worm inhabits the lymphatics and the female produces sheathed microfilariae which circulate in the peripheral blood.  The mosquito acquires the infection by ingestion of the microfilaria in the blood meal.  The microfilaria loses their sheath on arrival in the stomach of the mosquito.  The larvae migrate to the thoracic muscles and develop into infective larvae over a period of 6-14 days.  The larvae then migrate to the mouth parts of the mosquito and enter the skin of the definitive host through the puncture wound when a blood meal is taken. The infective larvae enter the peripheral lymphatics where they grow to mature male and female worms.   

 PHIL Image 3379

Illustration 10-2. Diagram showing the life cycle of Brugia malayi, a nematode which causes lymphatic filariasis. (SOURCE:  PHIL 3379 - CDC/Alexander J. da Silva, PhD/Melanie Moser)


Morphology

The adult worms of B. malayi are smaller than those of W. bancrofti.  The microfilariae of Brugia malayi are 170–230µm in length and have two terminal nuclei that are distinctly separated from the other nuclei in the tail.  The last terminal nucleus is quite small and is at the tip of the tail.  The sheath stains deep purple with hematoxylin stain.  

Image 10-3. Microfilaria of Brugia malayi. These microfilariae are sheathed measuring approximately 170–230mm in length and have 2 terminal nuclei that are distinctly separated from the other nuclei in the tail.  The microfilariae in this species are tightly coiled, and the nuclear column is more tightly packed, preventing the visualization of individual cells. (Blood Smear) (SOURCE:  PHIL 3003 - CDC/Dr. Mae Melvin)


Clinical Disease

Clinical features of B. malayi are similar to those of W. bancrofti, however in B. malayi, unlike Wuchereria bancrofti, genital involvement, hydrocoele and chyluria are rare.  

Many patients are asymptomatic. Patients may present fever. Lymphaginitis and lymphadenitis develop in the lower extremities. An inflammatory reaction occurs in the lymphatic vessels that harbor the adult worms. Edema develops which may resolve after the first few attacks. However, in long standing disease after several episodes of lymphaginitis, thickening and verrucous changes in the skin known as elephantiasis occurs.  

Some patients with lymphatic filariasis do not exhibit microfilaremia. However, they do have high eosinophilia, high IgE level and high anti-filarial antibody titers.  


Laboratory Diagnosis

See section 10.2 below  

Kinked microfilaria. Sheath stains deep pink with Giemsa stain. Does stain with hematoxylin stains. Nuclei crowded and fill the whole body. Empty space between nuclei and body wall. Cephalic space twice as long as it is broad. Innerbody may or may not stain; when it does, it is prominent. Found in blood.


Brugia Timori

Brugia timori is found in the islands of Indonesia and exhibits a strictly nocturnal periodicity.  The lifecycle and disease closely resembles that of Brugia malayi.  However, the microfilariae can be distinguished from those of B. malayi in that they are about 310µm in length.  The sheath satins pink with Giemsa and the nuclei at the tip of the tail are similar to those of B. malayi

Loa loa

Introduction

Loa loa, also known as the African eye worm, is a filarial nematode endemic in the rain forests of West and Central Africa.  It is transmitted by Chrysops species, also known as mango flies or horse flies and humans are the only known reservoir. It is estimated that 2-13 million humans are infected with the larvae.  

Adults migrate in the subcutaneous tissues of man and monkeys, with them eventually migrating across the eyeball under the conjunctiva.

Life Cycle

PHIL Image 3399

Figure 10-3. Diagram showing the life cycle of Loa loa, the African eye worm. (SOURCE:  PHIL 3399 - CDC/Alexander J. da Silva, PhD/Melanie Moser)

The adult worms live in the subcutaneous and deep connective tissues and the microfilariae are found in the peripheral blood, where they can be in ingested by the Chrysops fly (day biting fly) The adults can live in the tissues for up to 17 years.  

Once the microfilariae have been taken up by the Chrysops during a blood meal they develop within the fat body. They develop through to L3 within 10–12 days. The microfilariae, L3 re-enter the hosts blood stream when the fly takes another blood meal. They reach adult worms within 4- 6 months living in the subcutaneous and deep connective tissues. 

The microfilariae exhibit diurnal periodicity, the highest numbers being detected in blood between 10am and 2pm.  

Morphology

Adult males of Loa loa are 2–3.5cm long and the females from 5-7cm.  The microfilariae of Loa loa are 250-300µm.  They possess a sheath which stains blue-grey with Delafield’s hematoxylin.  The sheath does not stain with Giemsa.  The tail gradually tapers to a rounded end, the densely packed nuclei extending to the tip.  

   

Image 10-4. Microfilaria of Loa loa, the African eye worm. The adult worms live in the subcutaneous and deep connective tissues and the microfilariae are found in the peripheral blood, The microfilaria are kinked and sheathed. Nuclei crowded extending to tip of tail; tip of tail tapers. (hematoxylin stain, x400) (SOURCE: Unknown)


Clinical Disease

Many patients infected with Loa loa appear to be asymptomatic and the migration of the adult worm through the subcutaneous tissues often goes unnoticed, unless passing beneath the conjunctiva of the eye. They can be seen crossing the eye, but it is a rapid process taking approximately 15–20 minutes. Hyperesinophilia and increased antibody levels, especially IgE are also noted. Eye-worm episodes are as equally common in man as well as women with common reoccurrences. There is an increased incidence with age.  

The most common pathology associated with Loa loa infections are Calabar swellings, which are inflammatory swellings resulting in a localized subcutaneous edema.  These swellings are due the host’s response to the worm or its metabolic products and can be found anywhere in the body but most commonly in the extremities.  These swellings last from 1–3 days. They develop rapidly and last one to three days, usually accompanied by localized pain, urticaria and pruritis. There is a higher frequency of Calabar swellings in women with common reoccurrences.  

Serious complications such as cardiomyopathy, encephalopathy, nephropathy and pleural effusion have been recorded.

Laboratory Diagnosis

See section 10.2 below  

Kinked and sheathed microfilaria. Sheath does not stain with Giemsa stain; does stain with hematoxylin stains. Nuclei crowded extending to tip of tail; tip of tail tapers. Cephalic space as long as it is broad. Inner body does not usually stain. Found in blood.


Mansonella species

Introduction

Members of the genus Mansonella are filarial nematodes which rarely cause serious disease.  However, they can be found in geographical areas where Wuchereria bancrofti, Loa loa and Onchocerca volvulus also occur and therefore must be differentiated from these pathogenic microfilariae.  Unlike the pathogenic blood filariae, they do not exhibit periodicity.  

Life Cycle

PHIL Image 3404

Image 10-4. General life cycle of Mansonella species of filarial nematodes.  (SOURCE:  PHIL 3404 - CDC/Alexander J. da Silva, PhD/Melanie Moser)


There is a general life cycle for the Mansonella species of filarial nematodes. The microfilaria is picked up by the vector Culicoides sp. (biting midges) during a blood meal. The larvae develop within the body of the Culicoides sp. and are re-introduced into the human host when the vector takes another blood meal. They are found in various sites around the human host body.


Mansonella perstans

The microfilariae of M. perstans have been found in Africa and South America. This is a mildly pathogenic species in man and apes. They are found in the deep connective tissue and serous cavities.

Morphology

The adult worms live in the peritoneal, pleural and pericardial cavities and their size is comparable to the pathogenic species already discussed. The microfilariae are unsheathed are about 200µm in length and the nuclei extend to the tip of the tail which is rounded.

Clinical Disease

It is difficult to assess the disease associated with M. perstans, however pruritis, fever and subcutaneous swellings have been associated with infection of M. perstans.  The adult worm appears to cause little or no host reaction. Eosinophilia is common.  

 

Figure 10-5. Microfilariae of Mansonella perstans, a mildly pathogenic nematode to man. The microfilariae are unsheathed are about 200mm in length and the nuclei extend to the tip of the tail which is rounded. (Giemsa stained x920) (SOURCE: Unknown)


Laboratory Diagnosis

See section 10.2 below 

Small, thin microfilaria. Does not have a sheath. Nuclei extend to end of tail; last nucleus bigger; tip of tail is blunt. Nuclei stain deeply and “run together”. Found in blood.


Mansonella ozzardi

Mansonella ozzardi nematodes are confined to the westerm hemisphere. These are non-pathogenic filarial nematodes. The parasites cause nodules in the skin of the vertebrate hosts.  

Morphology

The adult worms are located in the mesenteric tissues and their size is comparable to the pathogenic species already discussed (0.6m long).  The microfilariae are found in the peripheral blood and range between 173-240µm in length.  The nuclei do not extend to the tip of the tail which has a pointed end. The male adult worm is almost unknown.

Clinical Disease

Infections caused by M. ozzardi are generally symptomless, however lymphadenopathy, arthralgia, fever and eosinophilia have been reported.

 

Image 10-6. Microfilariae of Mansonella ozzardi, Non-pathogenic filarial nematode to man. The microfilariae are found in the peripheral blood and range between 173 - 240mm in length.  The nuclei do not extend to the tip of the tail which has a pointed end. (SOURCE:  PHIL 382 - CDC/Dr. Mae Melvin)


Laboratory Diagnosis

See section 10.2 below  

Small thin microfilaria. Does not have a sheath. Nuclei do not extend to end of tail; tip of tail tapers. Stains very lightly; tip of tail difficult to see. Found in blood and skin.

Species

Size of Microfilariae

Morphology of microfilariae

Wuchereria bancrofti

210 – 320mm by 8 - 10mm

Sheathed. Tail pointed and clear

Brugia malayi

170 – 260mm by 5 - 6mm

Sheathed. Tail pointed with 2 nuclei

Loa loa

230 – 300mm by 6 - 8mm

Sheathed. Tail blunt with nuclei

Mansonella perstans

200mm by 6mm

Unsheathed. Tail blunt with nuclei

Mansonella ozzardi

250mm by 6 - 7mm

Unsheathed. Tail pointed and clear

Table 10-1. Morphology of the blood microfilariae known to infect man. (SOURCE: CDC)

 

Species

Geographic distribution

Pathogenicity

Adults (site of infection)

Microfilariae (characteristics)

Vector

Wuchereria bancrofti

Asia, Pacific, Tropical Africa, Americas

Lymphangitis, fever, elephantiasis hydrocoele, chyluria

Lymphatics

Found in blood, sheathed, periodicity variable

Culicidae (mosquitoes)

Brugia malayi

South and East Asia

Lymphagitis, fever. Elephantiasis

Lymphatics

Found in blood, sheathed, nocturnally periodic or subperiodic

Culicidae (mosquitoes)

Dipetalonema perstans

Africa and South America

No definite pathogenicity

Peritoneal & pleural cavity

Found in blood, unsheathed, nocturnally subperiodic

Culicoides (biting midges)

Dipetalonema streptocerca

Africa (Ghana and Congo)

Cutaneous edema, elephantiasis

Subcutaneous tissues

Found in skin, unsheathed, nonperiodic

Culicoides (biting midges)

Mansonella ozzardi

Central and South America

No definite pathogenitis

Peritoneal cavity

Found in blood, unsheathed, nonperiodic

Culicoides (biting midges)

Loa loa

Tropical Africa

Skin swellings, allergic reactions

Subcutaneous tissues

Found in blood, sheathed, diurnally periodic

Chrysops (Tabanidae or Horse fly)

Onchocerca volvulus

Africa, Central and South America

Skin nodules, ocular complications (blindness)

Subcutaneous tissues

Found in skin, unsheathed, nonperiodic

Simulium (Black fly)

  Table 10-2. Comparison of the main human filarial nematodes. (SOURCE: CDC)

 


10.2. Laboratory Diagnosis

Detection of Microfilariae in Blood

Collection of specimens

The specimen collection times should be selected in accordance with the patient’s clinical symptoms and travel history.  

Species

Geographic location

Periodicity

Collection time


Wuchereria bancrofti

Tropics / Subtropics

Nocturnal

2400 hours

Wuchereria bancrofti
 

Pacific

Diurnal subperiodic

1600 hours


Brugia malayi

SE Asia and SW India

Nocturnal

2400 hours

Brugia malayi
 

Indonesia

Nocturnal subperiodic

2100 hours

Brugia timori
 

Indonesia

Nocturnal

2400 hours


Loa loa

West / Central Africa

Diurnal

1300 hours


Mansonella perstans

Africa / South America

Non periodic

Any time

Mansonella ozzardi
 

Central & South America

Non periodic

Any time

Table 10-3. Periodicity and the advised collection times of the human filarial nematodes. (SOURCE: CDC)

Detection Methods

(i)  Polycarbonate membrane filtration.

This technique is very sensitive, enabling very low parasitemias to be detected. It is now the most widely used technique for separating microfilariae from blood.

Nucleopore polycarbonate membranes, 25µm diameter, 5µm pore size, are held in a Millipore Swinnex filter holder, using a rubber gasket to secure the membrane.  

Method

a)      Place the membrane on the holder with a drop of water.

b)      Draw up 10-20 ml of 1:1 saline diluted blood into a 20ml syringe

c)      Connect the syringe to the filter and gently push the blood through the filter membrane.

d)      Repeat until all of the blood has been filtered.

e)      Draw up 20 ml of saline into the syringe, flush through the filter, repeat using air.

f)        Unscrew the top of the filter and discard the gasket into chloros; use forceps to transfer the membrane to a slide.

g)      Add a drop of saline to the membrane and cover with a coverslip.

h)      Examine the membrane under the microscope, using a x10 objective. Examine any microfilariae found using a x40 objective to note the presence of a sheath.

 (ii)  Saline/saponin method.

Reagent

One percent saponin in normal saline.

Method

a)      Deliver 2ml of blood (fresh or anticoagulated) into a centrifuge tube and add 8ml of 1% saponin in saline.

b)      Mix the blood by inversion, then allow it to stand at room temperature for 15 minutes to allow the blood to hemolyze.

c)      Centrifuge at 2,000rpm for 15 minutes to deposit the microfilariae.

d)      Discard the supernatant and use the deposit to make a wet preparation.

e)      Examine the slide using the x10 objective. Active microfilariae can be seen and produce a snake-like movement as they disturb the cell suspension.

If it is not easy to inspect the microfilariae due to excess “wriggling” a little 10% formalin can be run under the coverslip to immobilize them.   

Confirmation of species can be made by using appropriate staining methods to demonstrate nuclear morphology.  


Staining Methods for Microfilariae 

When filariasis is suspected, a geographical and clinical history helps to determine the most appropriate collection time.  Thick and thin blood films can be examined. However this is an insensitive method due to the low microfilaremia, and larger volumes of blood need to be examined.   

There are 4 characteristics that are generally used in diagnosing microfilaria:

    1.     The presence of absence of a sheath.

    2.     The presence or absence of nuclei in the tip of the tail.

    3.     The innerbody – can or cannot be demonstrated.

    4.     The size of the microfilaria.


The two methods commonly used are:  

(i)  Supravital Staining

Reagent

0.75% cresyl blue in saline or 1.0% methylene blue in saline.  

These reagents can be used to stain live microfilariae by allowing the stain to flow under the coverslip on to a polycarbonate membrane preparation or a centrifuged preparation. The dye will stain the nuclei of the microfilariae and also provide a contrasting background to look for a sheath. It may take several minutes for the dye to penetrate the organisms and the slide should be kept in a moist chamber to prevent the preparation from drying out.  

(ii)  Permanent Staining

Permanent stains should show up the nuclei, including the pattern of nuclei in the tail region and stain the sheath if necessary.

The stains of choice are;

1.   Hematoxylin

2.   Giemsa

3.   Rapid Field’s

1.  Hematoxylin

Delafield’s hematoxylin will stain the nuclei and the sheath well and unlike Ehrlich’s hematoxylin does not require heating

Reagents

·         Delafield’s hematoxylin (BDH)

·         1% acid alcohol

·         Methanol

Method

a)    Make thin films, allow to air dry then fix in methanol for five minutes

b)    Stain with Delafield’s hematoxylin for 20 minutes

c)     “Blue” the nuclei by placing the slide in a coplin jar and allow a stream of running water to flow into the jar for 20 minutes.

d)    Decolorize with 1% acid alcohol for 5-10 seconds before “blueing” in tap water again. Control this process by examination under the microscope until the nuclei are clear and distinct.

e)    Allow the slide to dry before mounting in DPX.

f)      The nuclei should stain blue and the sheath grey.

2.  Giemsa

Reagents

  • Methanol
  • Giemsa stain
  • Immerson Oil

 Method

a)    Make a thin film and allow to air dry.

b)    Fix in methanol for one minute.

c)     Tip off the methanol and flood the slide with Giemsa stain diluted 1:6 with buffered distilled water pH 6.8. The diluted stain must be freshly prepared each time.

d)    Stain for 20 – 25 minutes.

e)    Run buffered water on to the slide to float off stain and to prevent deposition of precipitate on to the film. Allow the slide to drain dry.

f)      Examine the film using the oil immersion objective. Nuclei should stain red


3.  Rapid Field’s

Reagents

  • Methanol
  • Field’s Stain A solution
  • Field’s Stain B solution
  • Immersion Oil

Method

a)    Make thin film and allow to air dry for five minutes.

b)    Fix the smear in methanol for one minute.

c)     Flood the slide with 1ml of Field’s stain B (diluted 1:4 with distilled water)

d)    Immediately add an equal volume of Field’s stain A, mix well on slide and allow to stand for one minute.

e)    Rinse well in tap water and drain dry.

f)      Examine the film using the oil immersion objective and immersion oil.

g)  The nuclei should stain red. 


10.3. Microfilaria Worms Found in Tissue and Skin  

The main species of microfilariae found in the skin and tissue are Onchocerca volvulus and Mansonella streptocerca.  Microfilariae of Onchocerca volvulus and less often, Mansonella streptocerca migrate through the dermis causing itching and skin texture changes and occasionally arrive in the eye where they cause blindness.  Detection of these microfilariae is from skin snips or nodule biopsies.  When high numbers of microfilariae are present, they can occasionally be found in the blood and urine.


Onchocerca volvulus

Introduction

Onchocerca volvulus is mainly found in West Africa and Central and South America.  Onchocerciasis, also known as river blindness, is a major public health problem, especially in West Africa despite the fact that an eradication program has been established.  It is one of the world’s most distressing diseases of helminth origin, often resulting in blindness. Onchocerca volvulus is transmitted by the species Simulium or black fly whose breeding habitat is by fast flowing rivers or streams, therefore there is a patchy distribution of the disease as it is specified to where water courses are. The adult worms are found in nodules or onchodermata in superficial sites, but may invade other tissues.   It is estimated that there are 18 million cases worldwide with 17.5 million being found in Africa. Nigeria is the most infected region. The rate of morbidity is high in relation to those with an infection.

Life Cycle

PHIL Image 3413

Illustration 10-5. Diagram showing the life cycle of Onchocerca volvulus, a filarial nematode which causes onchocerciasis, or River blindness. It is known as river blindness due to the vector, Simulium damnosum, breeding in fast flowing rivers. (SOURCE:  PHIL 3413 - CDC/Alexander J. da Silva, PhD/Melanie Moser)


The life cycle is similar to W. bancrofti, except that the intermediate hosts are various species from the genus Simulium (Black flies), the most important species is Simulium damnosum.

The microfilariae are ingested by a Black fly during a blood meal, from where they are carried to the midgut where they penetrate the epithelium and migrate, via the hemocoele, to the indirect flight muscles. Here they undergo two molts, L1–L3 and develop into infective L3 larvae which move to the mouth parts. Development is completed in 6–9 days.  

When the infected fly takes another blood meal the infective larvae are once again transmitted into another host (definitive host). The microfilariae are released from the mouth parts and transmitted directly into the hosts bloodstream. Molting takes place form L3 - L4 within 2-5 days and the larvae then migrate widely through the body under the skin and between muscles, ligaments and tendons. The final molt to L5 occurs at 1.5–2.5 months after transmission. Male worms are known to mature in about four months later. Female worms initiate the formation of the nodules and the males may join later. The sexually mature female worms release microfilariae which migrate out from the nodules into the skin and other tissues, most significantly into the eye.  

Morphology

The whitish adult worm lies coiled within capsules in the fibrous tissue.  The female can measure up to 50cm while the males are shorter measuring up to 5 cm.  The microfilariae of O. volvulus are unsheathed and are usually found in the dermis.  They measure between 221-287µm long.

 

 

Image 10-7. Onchocerca volvulus microfilariae after being released by the adult female worm. They escape to the subcutaneous tissues and the eye and can be recovered with blood-free skin snips. (SOURCE: CDC)


Clinical Disease

Clinical manifestations are due to microfilariae in the epidermis. 

Light infections may be asymptomatic or cause pruritis. This leads to scratching which can result in infection.  Lyphadenopathy may also be a feature of early infection.  After months or years, onchodermatitis results in secondary stage of thickening due to intradermal edema and pachydermis.  There is a loss of elastic fibers resulting in hanging groin, hernias and elephantiasis of the scrotum.  There is finally atrophy of the skin resulting in loss of elasticity.  There is mottled depigmentation of the skin.  

Ocular lesions are related to the intensity of the microfilariae in the skin.  Ocular lesions include sclerosing keratitis, secondary glaucoma and cataract, coroidoretinitis and fluffy corneal opacities.  The major complication of onchocerciasis is the development of lesions in the eye which may result in blindness or other distressing ocular diseases.

Laboratory Diagnosis

    1.  Analysis of Skin Snips 

Small amounts of skin are collected by using a needle to raise the skin and then to slice about 1 mg of skin to a depth of 0.5µm.  Snips are collected from several sites, usually the shoulders or the buttocks and sometimes the chest and calves.  The snips are placed immediately in 0.5ml normal saline in a microtiter plate and left for 4 hours to allow the microfilariae to migrate out of the tissues.  After four hours, the wells are examined using an inversion microscope.  The microfilariae should still be moving and can be identified from the table below. The microfilariae can also be collected by filtration or centrifugation and the deposit containing microfilariae can be stained with Giemsa at pH 6.8.  

    2.  Analysis of Biopsies

Biopsies of tissue nodules can be dabbed on to a slide to produce impression smears and then stained with Giemsa stain at pH 6.8 for the presence of microfilariae.  

Recent advances in diagnostic methods includes and ELISA-based antibody detection assay which utilizes a cocktail of recombinant antigens. The advantages of using this test is that it is highly sensitive (almost 100% in onchocerciasis foci). It is also highly specific (100%), it also uses finger prick blood. Therefore, reducing the painful procedure of gaining a skin snip.  

The disadvantages is that it requires advanced ELISA apparatus and reagents and cannot distinguish between past and present infections due to it detecting antibodies which stay present in the body for a long time after the infection. Another modern detection method is for Parasite DNA detection, which is based on the amplification of specific DNA sequences form microfilariae using molecular biology technology. The advantages of this technique is its exquisite sensitivity and detects active infections only. The disadvantages are that it requires specialized equipment and expensive reagents. Also it still requires a skin snip but a urine assay is a possibility for the future.  

Thick microfilaria. Does not have a sheath. Head often spatulate. Nuclei do not extend to tip of tail. Found only in skin.


Mansonella streptocerca  

Introduction

Microfilaria of M. streptocerca were first reported in the skin of a West African patient in 1922.  These microfilaria are primarily found in the skin but have been also reported in the blood. This species occurs in Ghana, Cameroon and Zaire. The adults are poorly known, and occur in the cutaneous tissue of man and chimpanzee.  

The microfilariae do not exhibit periodicity with the intermediate hosts being Culicoides grahamii and possibly other Culicoides species.   

Life Cycle

The life cycle is the same as that of the blood Mansonella species.

 

Image 10-8. Microfilaria of Mansonella streptocerca. From a skin snip, after a concentration procedure and hematoxylin stained. The microfilaria is typically unsheathed, and its body has a straight attitude. The tail is typically coiled ("shepherd's crook"), and nuclei extend to the end of the tail, as a single-cell row. (SOURCE: CDC)


Clinical Disease

Infection is characterized by pruritic dermatitis and hypopigmented macules.  

Laboratory Diagnosis

Mansonella streptocerca can be diagnosed by demonstrating the microfilaria in a skin snip.   Snips are collected from several sites, usually the shoulders and buttocks and sometimes the chest and calves.  The snips are placed immediately in 0.5ml of 0.9% sodium chloride in a microtiter plate and left for four hours to allow the microfilaria to migrate out of the tissues.  After four hours, the wells are examined using an inversion microscope.  The microfilaria should still be moving and can be identified by staining with Giemsa at pH 6.8  

Small, thin, microfilaria. Does not have a sheath. Nuclei extend to end of tail. Tail is hooked; its tip is rounded or forked. Found only skin.

Species

Onchocerca volvulus

Mansonella streptocerca

  Distribution

Tropical Africa, Central and South America

West Africa

Vector

Simulium spp.

Culicoides spp.

Adult location

Subcutaneous nodules

Cutaneous connective tissue

Microfilariae location

Skin

Skin

Microfilariae size

280 - 330µm

180 - 240µm

Morphology

Broad spatulate head;
No sheath, pointed tail

Curled tail;
No sheath

Tail nuclei

Tail free from nuclei

Nuclei extend to tail tip

Table 10-4. Differential features of Onchocerca volvulus and Mansonella streptocerca. (SOURCE: CDC)


Dracunculus medinensis

Introduction

Dracunculus medinensis is a non-filarial parasite as it only has one uterus whereas filaria have two. It is usually associated with places where there is a lack of clean drinking water e.g. step wells in India, covered cisterns in Iran, and ponds in Ghana. The life cycle usually involves copepod intermediate host. They are parasitic in the connective tissue or coelom of vertebrates. The disease associated with this parasite is known as Dracunculiasis.

Life Cycle  

PHIL Image 3391

Illustration 10-6. Diagram showing the life cycle of Dracunculus medinensis, Guinea worm. (SOURCE:  PHIL 3391 - CDC/Alexander J. da Silva, PhD/Melanie Moser)


Mature female worms which are gravid with microfilariae migrate to the superficial layers of skin of humans, especially those regions which are most likely to come in contact with water, such as the ankle, foot, arms and shoulders. Here the worms secrete a substance (substance is unknown) which causes a blister to rise over its anterior end where it has pierced the lower layers. The blister eventually forms into an ulcer which on contact with water, the uterus is projected out of the ulcer cavity, and a cloud of milky white secretion, containing hundred of active larvae, is released. Once out of the water again the uterus dries and shrivels preventing the release of further larvae.  

If the microfilariae are ingested by an appropriate species of Cyclops, they break though the soft mid-intestine wall and come to lie in the body cavity. The larvae undergo two molts and become infective in approximately three weeks. Humans become infected by accidentally ingesting through drinking water the infective Cyclops. Upon ingestion the larvae are activated to penetrate through the gut wall, and migrate through the tissues, molting twice and finally becoming lodges in the viscera or subcutaneous tissues. Maturation of the worms is slow taking about one year to reach sexual maturity before the females are ready to migrate to the skin to release their larvae.  

Morphology

The adult female worm measure up to one meter in length whereas the male measures about 2cm. 

Clinical Disease

After ingestion of the Cyclops, there is no specific pathology associate with the mucosal penetration and larval maturation in the deep connective tissues. Erythema and tenderness can be associated with blister formation. The patient can also exhibit vomiting, diarrhea, asthmatic attacks. Symptoms usually subside when the lesion erupts. If the worm is removed, healing usually occurs without any problems. If the worm is damaged or broken during removal, there may be intense inflammatory reaction with possible cellulitis along the worms migratory tract. This can result in arthritis and synovitis.

 

Figure 10-9. Female Dracunculus medinensis worm (Guinea worm) emerging out of a typical ulcer. Adult worms emerge from these ulcers on contact with water to release their microfilaria. The most effective method for removing these worms are to slowly wind them around a piece of stick, being careful not to break the worm in two.  (SOURCE:  PHIL 1342 – CDC)

Laboratory Diagnosis

The best remedy for removing the adult worm is a slow process of daily gently rolling the worm around a small stick and slowly pulling it out of the skin. With this method you must be careful not to pull apart the worm as it will recoil back into the skin and cause secondary infections.  

This parasite is currently being approached with a strict control program. The program includes stopping people from drinking infected water, putting muslin over water collection jars, educating the communities about the parasite, and adding temphos to the water to kill it off.

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Ch 1. The Ameba
Ch 2. The Ciliates, Coccidia, and Microsporidia
Ch 3. The Flagellates
Ch 4. The Cestodes
Ch 5. The Nematodes
Ch 6. The Trematodes
Ch 7. Tissue Dwelling Nematodes
Ch 8. Larval Cestodes and Nematodes
Ch 9. Malaria
Ch 10. The Blood Nematodes
Ch 11. Babesia, Trypanosomes, and Leishmania
Ch 12. Arthropod Vectors
Ch 13. Artifacts and Confounders