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Parasitology

 

 


Volume 10.  The Blood Nematodes

 

 

Co- Authors:  M. Arcari 1, A. Baxendine 1 and C. E. Bennett2

 

1. Diasys Ltd    2. University of Southampton

  CONTENTS

 

10.1. The Blood Nematodes        

 

1        Wuchereria bancrofti                                                                          2        Brugia malayi 

5        Brugia timori                                                                       

7        Loa loa                                                                                          

 8       Mansonella species                                                                       

10      Mansonella perstans                                                           

10      Mansonella ozzardi                                    

 10.2. Laboratory diagnosis

Detection of microfilariae in blood                                                             14

Staining methods for microfilariae                                                             15

 

10.3.    Microfilaria worms found in tissue and skin

Onchocerca volvulus                                                                                   18

Mansonella streptocerca                                                                                    21

Dracunculus medinensis                                                                                    23

 

 

          References


10. The Blood Nematodes

 

These nematodes are known as filariae and consist 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 do require intermediate hosts for the completion of their life cycle.

 

The morphology of these nematodes comsist of a cylindroid pharynx with an anterior muscular portion and a posterior glandular portion; the males have well-developed alae 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 parasitise man consist of Wuchereria bancrofti, Brugia malayi, Brugia timori and Loa loa, Onchocerca volvulus, Mansonella perstans, Mansonella streptoserca 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 1 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, 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 microfilaraemia 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, while in Papua New Guinea Mansonia 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. (Fig. 1)

 

The blood stages of filariae, mifcrofilariae, 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.

 


 

 


Figure 1. Diagram showing the life cycle of Wuchereria bancrofti, the filarial nematode known to cause the disfiguring disease, elephantiasis, in man.

 

 

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 haematoxylin stain.  (Fig. 2 & Table 1 & 2)


 

 


Figure 2. 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. (Haematoxylin stained x400) (www.dpd.cdc.gov)

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 harbour the adult worms.  Oedema 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. (Fig. 3) 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. 

 


 

Figure 3. Lymphatic filariasis: Elephantiasis is the last consequence of the swelling of limbs and scrotum. 
Elephantiasis of the limbs. (www.cdfound.to.it)

 

 

 

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

 

Laboratory diagnosis

See section below

 

Sheath may or may not stain with Giemsa; does stain with haematoxylin 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 haematoxylin. 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 microfilaraemia 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, 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. Mansonoa bonneae are important vectors in Malaysia, breeding in swamp forest and biting by night, although sometimes by day as well.

 

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

 

Lifecycle

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 lose 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. (Fig. 4)

 


 

Figure 4. Diagram showing the life cycle of Brugia malayi, a nematode which causes lymphatic filariasis.

 

 

 

Morphology

The adult worms of B. malayi are smaller than those of W. bancrofti.  The microfilariae of Brugia malayi are 170 – 230mm in length and have 2 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 haematoxylin stain. (Fig. 5 & Table 1 & 2)


 

 

 


Figure 5. 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. (Haematoxylin stain, x400) (www.dpd.cdc.gov)

 

Clinical disease

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

 

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

 

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

 

Laboratory diagnosis

See section below

 

Kinked microfilaria. Sheath stains deep pink with Giemsa stain. Does stain with haematoxylin 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 310mm 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

 

 


Figure 6. Diagram showing the life cycle 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, where they can be in ingested by the Crysops 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 boduy. 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. (Fig. 6)

 


Morphology

Adult males of Loa loa are 2 – 3.5cm long and the females from 5 – 7cm.  The microfilariae of Loa loa are 250 - 300mm.  They possess a sheath which stains blue-grey with Delafield’s haematoxylin.  The sheath does not stain with Giemsa.  The tail gradually tapers to a rounded end, the densely packed nuclei extending to the tip. (Fig. 7 & Table 1 & 2)

 


 

Figure 7. 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. (haematoxylin stain, x400)

 

 

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 mintues. Hypereosinophilia and increased antibody levels, especially IgE are also noted. Eyeworm episodes are as equally common in man as well as women with common re-ocurrences. 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 localised subcutaneous oedema.  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 localised pain, urticaria and pruritis. There is a higher frequency of calabar swellings in women with common re-occurences.

 

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

 

Laboratory diagnosis

See section below

 

Kinked and sheathed microfilaria. Sheath does not stain with Giemsa stain; does stain with haematoxylin stains. Nuclei crowded extending to tip of tail; tip of tail tapers. Cephalic space as long as it is broad. Innerbody 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


 

Figure 8. General life cycle of Mansonella species of filarial nematodes.

 

 

There is a general life cycle for the Mansonella species of filarial nematodes. The microfilaria are 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. (Fig. 8)

 

 

 

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 200mm in length and the nuclei extend to the tip of the tail which is rounded. (Fig. 9 & Table 1)

 

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 9. 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) (Peters & Gilles, 1995)

 

 

 

Laboratory diagnosis

See section 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 New world and West Indies.  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 - 240mm 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. (Fig. 10 & Table 1 & 2)

 

Clinical disease

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

 


 

Figure 10. 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. (haematoxylin stain, x920) (Peters & Gilles, 1995)

 

Laboratory diagnosis

See section 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 1. Morphology of the blood microfilariae known to infect man.


Species

Geographic distribution

Pathogenicity

Adults (site of infection)

Microfilariae (characteristics

Vector

Wuchereria bancrofti

Asia, Pacific, Tropical Africa, Americas

Lymphagitis, 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 oedema, 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, occular complications (blindness)

Subcutaneous tissues

Found in skin, unsheathed, nonperiodic

Simulium (Black fly)

 

 

Table 2. Comparison of the main human filarial nematodes

 

 

 

 


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

12.midnight.

Wuchereria bancrofti

Pacific

Diurnal subperiodic

16.00 hours

Brugia malayi

SE Asia and SW India

Nocturnal

12.midnight.

Brugia malayi

Indonesia

Nocturnal subperiodic

21.00 hours

Brugia timori

Indonesia

Nocturnal

12.midnight

Loa loa

West / Central Africa

Diurnal

13.00 hours

Mansonella perstans

Africa / S. America

Non periodic

Any time

Mansonella ozzardi

Central & S America

Non periodic

Any time

 

Table 3. Periodicity and the advised collection times of the human filarial nematodes

 

Detection Methods

(i)  Polycarbonate membrane filtration.

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

Nucleopore polycarbonate membranes, 25 mm diameter, 5 µm 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 20 ml 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.

1% saponin in normal saline.

 

Method.

a)      Deliver 2 ml of blood (fresh or anticoagulated) into a centrifuge tube and add 8 ml 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 haemolyse.

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 immobilise 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 microfilaraemia, 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 2 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.   Haematoxylin

2.   Giemsa

3.   Rapid Field’s

 

1.  Haematoxylin.

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

 

Reagents.

Delafield’s haematoxylin (BDH)

1% acid alcohol

Methanol

 

Method.

a)    Make thin films, allow to air dry then fix in methanol for 5 minutes

b)    Stain with Delafield’s haematoxylin 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)    Decolourise with 1% acid alcohol for 5-10 seconds before “blueing” in tapwater 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 1 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 5 minutes.

b)    Fix the smear in methanol for 1 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 1 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, there 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

 

 

Figure 11. 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.

 

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. (Fig. 11)

 

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 haemocoele, to the indirect flight muscles. Here they undergo two moults, 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. Moulting 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 moult to L5 occurs at 1.5 – 2.5 months after transmission. Male worms are known to mature in about 4 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 - 287mm long. (Fig. 12 & Table 2)


 

 


Figure 12. 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. (Wet mount preparation) (www.cdfound.to.it)

 

 

Clinical Disease

Clinical manifestations are due to microfilarie in the epidermis. (Table 4)

 

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 oedema and pachydermis.  There is a loss of elastic fibres 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 0.5mm.  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 microtitre plate and left for 4 hours to allow the microfilariae to migrate out of the tissues.  After 4 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 utilises 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 specialised equipment and expensive reagents. Also it still require 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

 

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. (Fig. 13)

 

Life cycle

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

 


 

Figure 13. Microfilaria of Mansonella streptocerca. From a skin snip, after a concentration procedure, and  haematoxylin 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.

 

 

Clinical Disease

Infection is characterised by pruritic dermatitis and hypopigmented macules. (Table 4)

 

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 microtitre plate and left for 4 hours to allow the microfilaria to migrate out of the tissues.  After 4 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.



 

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 - 330 um

180 - 240 um

Morphology

Broad spatulate head

No sheath, pointed tail

Curled tail

No sheath

Tail nuclei

Tail free from nuclei

Nuclei extend to tail tip

 

Table 4. Differential features of Onchocerca volvulus and Mansonella streptocerca

 

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

 Figure 14. Diagram showing the life cycle of Dracunculus medinensis, Guinea worm.

 

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. (Fig. 14)

 

If the microfilariae is 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 moults and become infective in approximately 3 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, moulting twice and finally becoming lodges in the viscera or subcutaneous tissues. Maturation of the worms is slow taking about 1 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 1 metre in length whereas the male measures about 2cm. (Fig. 15)

 

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, diarrhoea, 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 15. 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. (Peters & G/illes, 1995)

 

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.

 

At this moment in time this parasite is being effectively controlled due to a strict control programme. The programme includes stopping people from drinking infected water, putting muslin over the water jars, which they use to collect the water in, thus preventing the cyclops from being collected in the water. Educating the communities about the parasite and adding temphos to the water thus killing off any microfilariae in the water.


 

References

 

 

Murray, PR, Drew, WL, Koyayashi, GS & Thomson, JH: Medical Microbiology. Mosby Books Inc., New York (1990)

 

Peters, W & Gilles, HM: Tropical Medicine & Parasitology. Wolfe Medical Publications Ltd.

 

Jeffrey & Leach: Atlas of Medical Helminthology and Protozoology. E & S Livingstone Ltd.

 

Ash, LR & Orihel, TC: Atlas of Human Parasitology. ASCP Press, Chicago.

 

World Health Organisation: Basic Laboratory Methods in Medical Parasitology. ISBN 92 4 154410 4. (1991)

 

Garcia, LS & Bruckner, DA: Diagnostic Medical Parasitology. Elsevior Science Publishing Co. Inc.

 

Muller, R & Baker, JR: Medical Parasitology. Gower Medical Publishing.

 

Smyth, J.D: Introduction to Animal Parasitology. Cambridge University Press (1994)

 

Snell, JJS, Farrell, ID & Roberts, C: Quality Control, Principles and Practice in the Microbiology Laboratory. Public Health Laboratory Service. ISBN 0 901 144 312.

 

I would like the thank the authors from the following web sites:

www.dpd.cdc.gov

www.cdfound.to.it