**4.1 Clinical diagnosis**

Calabar swelling commonly appears on the arm, elbow, face, or chest, and is often accompanied by localized pruritus and discomfort. Another diagnostic sign is ocular passage (Figure 4) of the adult worm (eye worm). These two signs are the most frequent and specific among autochtones and visitors. However, eye worm is more frequent in autochtones than in visitors, while calabar swelling is more common among visitors (82%) (Churchill et al., 1996). Visitors may developed a syndrome of immunological hyperresponsiveness (Nutman et al., 1986) characterized by high titers of antifilarial antibodies, elevated IgE levels (Klion et al., 1991), hypereosinophilia, frequent pruritus around the angiodema, and complications such as endomyocardial fibrosis, renal disease, lymphoma (Gerd et al., 1996), and subcutaneous nodules. Calabar swelling and eye worm may appear alone or simultaneously. Retinal hemorrhage can occur in case of high microfilaremia (Toussaint & Danis 1965), especially after treatment with DEC or ivermectin. Examination of the fundus is therefore necessary, even in the absence of antifilarial treatment. Onset of calabar swelling and eye worm may be followed by cardiac (Andi et al., 1981), renal (Pilay et al., 1973; Bariety et al., 1967) or neurological disorders. Non specific symptoms include pruritus, fever, urticaria, rash, myalgia and arthralgia.

Fig. 4. Ocular passage of *Loa loa* adult worm

#### **4.2 Biological diagnosis**

#### *Direct specific diagnosis*

Direct specific diagnosis is based on detection of *Loa loa* microfilaria in peripheral blood or of the migrating adult worm. In wet films of blood samples taken around noon, *Loa loa* microfilaria are highly mobile, with a snake-like movement among red blood cells, enveloped in a translucid membrane (sheath). Body size is 231-300 µm by 5-7 µm. A concentration technique on fresh blood can be used: 1 ml of blood is diluted 10X in PBS (Akue et al., 1996), then red cells are lyzed with 2% saponin and the solution is centrifuge at 2000 rpm for 10 minutes. The pellet is smeared on a slide and examined under a microscope. Microfilariae appear as described above. Microfilariae can also be stained with Giemsa in a

Encephalitis Due to *Loa loa* 347

Fig. 6. Amplification of *Loa loa* 15 kDa gene by PCR for diagnosis

thick blood film, allowing *Loa loa* to be distinguished from other filariae by their size (253- 300 um long), their unstained sheath, and the presence of several nuclei inside the body, reaching the tail but not the cephalic extremity. Direct examination of cerebrospinal fluid (CSF) after centrifugation may be positive for microfilariae in case of neurological signs. The migrating adult worm is most readily detected during its ocular passage. The adult is 2-7 cm long, the female being longer than the male.

Fig. 5. Stained *Loa loa* microfilaria

thick blood film, allowing *Loa loa* to be distinguished from other filariae by their size (253- 300 um long), their unstained sheath, and the presence of several nuclei inside the body, reaching the tail but not the cephalic extremity. Direct examination of cerebrospinal fluid (CSF) after centrifugation may be positive for microfilariae in case of neurological signs. The migrating adult worm is most readily detected during its ocular passage. The adult is 2-7 cm

long, the female being longer than the male.

Fig. 5. Stained *Loa loa* microfilaria

Fig. 6. Amplification of *Loa loa* 15 kDa gene by PCR for diagnosis

Encephalitis Due to *Loa loa* 349

and death after a few days. Laboratory tests show numerous *Loa loa* microfilaria in peripheral blood, cerebrospinal fluid (CSF) and urine. Loaisis encephalopathy is classified according to the neurological manifestations, their time of onset, and biological findings, in three categories: definite, probable and possible (Scientific working Group on SEA, 2003). **6.1.1** Definite *Loa loa* encephalopathy: microscopic examination of brain tissue obtained by autopsy or needle biopsy is consistent with *Loa loa* encephalopathy (vasculopathy with evidence of *Loa loa* microfilariae), and onset of central nervous system (CNS) disorders

**6.1.2** Probable *Loa loa* encephalopathy: encephalopathy (without seizures, usually febrile) in a previously healthy person with no other cause of encephalopathy, and onset of CNS symptoms and signs within 7 days of treatment with metizan, progressing to coma without remission; and >10 000 mf/ml of peripheral blood pre-treatment, or >1000 mf/ml within 6 months post-treatment, or >2700 mf/ml within 6 months of treatment, and /or L. loa

**6.1.3** Possible L. loa encephalopathy: encephalopathy (without seizures, usually febrile) in a previously healthy person with no other underlying cause of encephalopathy, and onset of CNS symptoms and signs within 7 days of treatment with mectizan, progressing to coma without remission, and semi-quantitatively or qualitatively positive (+, ++ or +++) for L. loa

Spontaneous cases are rare but may be under-estimated. A number of apparently cases of spontaneous encephalitis have been been reported ( Bonet ,1943; Gallais et al., 1954; Carayon et al., 1959; Same Ekobo et al., 1981; Tuna Lukiana et al., 2006). In some case described by Kivit (1952), patients might have taken antifilarial drugs. The symptomatology is variable and may start with calabar swelling accompanied by itching, asthenia, facial edema, abdominal pain, diarrhea, violent headache, renal failure, hemiplegia or double hemiplegia, with mental disorders, functional impairment, altered consciousness including coma, usually terminating in sudden death. The process can last between 1 and 3 months, with hyperthermia in some case. The electroencephalogram may be abnormal (Bogaert et al., 1955). Microfilaria will be present in CSF and usually in peripheral blood, with albuminuria,

Treatment of *L. loa* encephalitis is based on nursing, nutritional support and re-hydration. According to Serious Adverse events (SEA) Experts in *Loa loa* endemic areas (Scientific working Group on SAE, 2003) Corticosteroids and antihistamines should be avoided. The reasons for avoidance of corticosteroids are the lack of evidence of efficacy for this condition and potential harmful effect; while the antihistaminic treatment should be avoid because of the lack of efficacy and they sedate patient with a neurologic condition, interfering with diagnosis and neurologic assessment. The protocol suggested here is based on that described by Gardon et al., 1999. It is based on vital monitoring (pulse, arterial pressure, temperature, consciousness (Glasgow score), hydration, complete neurological and clinical examination every hour then every three hours. When the patient is dehydrated and systolic pressure is below 9 cmHg: perfuse 500 ml of Ringer lactate solution over 30 min; if no improvement, continue to perfuse until systolic pressure reaches 10 cmHg and

within 7 days of treatment with mectizan, progressing to coma without remission.

microfilariae in CSF.

microfilariae in peripheral blood or CSF.

red blood cells and leukocytes in urine (Lukiana et al., 1996).

**7. Treatment of loiasis encephalopathy** 

**6.2 Spontaneous encephalopathy** 

#### *Indirect (presumptive) diagnosis*

Indirect diagnosis is based on hypereosinophilia (25%) in general among non indigenous population from endemic zone, and elevated total IgE. As most infected people are amicrofilaremic, indirect methods based on antibody or gene detection are valuable. One of the first such methods was the immunofluorescent antibody test (IFAT) using fixed microfilaria. Others include ELISA detection of specific IgG4 against a crude extract of *Loa loa* worm, a method that appears to be specific and sensitive for both microfilaremic and amicrofilaremic forms (Akue et al., 1994). Its sensitivity and specificity (relative to *Mansonnella perstans*) are reported to be better than 90% for parasitologically proven loiasis in a co-endemic area. However, crude extracts are in limited supply. A luciferase immunoprecipitation system (LIPS) based on detection of IgG to *Loa loa* recombinant antigen L1SXP-1 has been recently developed and shows high specificity but limited sensitivity. A rapid LIPS format improves the specificity by limiting cross-reactivity with *O. volvulus* (Burbelo et al., 2008). The same L1XP-1 antigen was used to develop an ELISA method for the detection of specific IgG4 antibodies, but sensitivity was poor (56%) (Klion et al., 2003). Molecular diagnosis may consist of detecting the ladder R3 gene (Ajuh et al., 1995) of *Loa loa* in DNA extracted from whole blood (Touré et al., 1997). However, although highly specific, the test is impractical in rural areas and non specialized laboratories. In general, these methods, although specific, are not sensitive enough to detect all cases of loiasis and are not available at many points of care.
