Loa loa is a human filarial parasite endemic to west

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1 Diagnostic Problems in a Patient With Amicrofilaremic Loa loa 53 Masahide Yoshikawa, MD, * Yukiteru Ouji, PhD, * Noriko Hayashi, PhD, * Kei Moriya, MD, * Mariko Nishiofuku, MD, * Shigeaki Ishizaka, MD, * Makoto Itoh, PhD, Eisaku Kimura, MD, Fukumi Nakamura, MD, and Yukifumi Nawa, MD * Department of Parasitology, Nara Medical University School of Medicine, Kashihara, Japan ; Department of Parasitology, Aichi Medical University School of Medicine, Nagakute, Japan ; Parasitic Diseases Unit, Department of Infectious Diseases, Faculty of Medicine, University of Miyazaki, Kiyotake, Japan DOI: /j x We report a Japanese patient with loiasis who became infected in Cameroon. Despite the clinical history and laboratory data providing adequate evidence for suspecting loiasis, microfilariae were not detected in the blood. It is important to note that most infected travelers whose home countries are in nonendemic regions are amicrofilaremic. Loa loa is a human filarial parasite endemic to west and central African tropical rain forest regions, with an estimated 3 to 13 million people infected. 1 3 L loa endemic countries include Republic of Cameroon (Cameroon), Gabonese Republic (Gabon), Republic of Congo (Congo), Democratic Republic of the Congo, Central African Republic, Federal Republic of Nigeria, and Republic of Equatorial Guinea. Loiasis is not seen in inhabitants of nonendemic countries, except for those who have traveled or immigrated to affected areas. 3 5 We recently treated a Japanese female who contracted loiasis while traveling in the rain forest of southeast Cameroon and living for 1 month in a village among Baka Pygmy people. She developed fugitive swelling (Calabar swelling) 6 months after her return from Cameroon. In the following 16 months, Calabar swelling appeared and disappeared on a few occasions and finally worm migration into the conjunctiva was observed. Blood examinations showed eosinophilia, elevated immunoglobulin (Ig)E level, and a gradual increase in the ratio of antifilarial antibodies (IgG4 fraction). Despite the clinical history and laboratory data providing adequate evidence for suspecting loiasis, microfilariae were not detected in the blood. Herein, we describe Corresponding Author: Masahide Yoshikawa, MD, Department of Parasitology, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara , Japan. myoshika@naramed-u.ac.jp the patient profile and discuss diagnostic problems in patients with amicrofilaremic loiasis. Case Report A 21-year-old Japanese female musician developed nonerythematous and nonpitted swelling on the back of her right hand in November 2004, which slowly migrated to the wrist and disappeared within a few days. She visited a nearby physician after spontaneous disappearance of those symptoms, though no blood examination was conducted at that time. In January 2005, a similar skin edema appeared in the right forearm and disappeared within a few days; however, the patient did not seek medical consultation for that occurrence. The patient revisited the doctor in March 2005 for a regular medical checkup, and a blood examination showed moderate eosinophilia [total white blood cell (WBC) count 7,300/ L, eosinophils 2,190/ L], with normal IgE (50 IU/mL; normal <100) and serum chemistry values. She had no history of allergic diseases. The patient stated that she had visited Cameroon and stayed in a Baka Pygmy village in the rain forest, located in Dimako (coordinates: lat 4 23 N, long E; altitude 680 m), a subdivision district and small town situated slightly south of the East Province capital city of Bertoua, which sees little tourism due to the humidity and has mosquito- and blackfly-infested forests. She joined the Baka Pygmy community to study their 2008 International Society of Travel Medicine, Journal of Travel Medicine, Volume 15, Issue 1, 2008, 53 57

54 music and lived with them in their traditional manner for about 1 month in April and May 2004 and also remembered well being bitten by Chyrops genus flies numerous times during her stay in the

2 54 music and lived with them in their traditional manner for about 1 month in April and May 2004 and also remembered well being bitten by Chyrops genus flies numerous times during her stay in the village. Although a blood smear examination was not performed, serological tests were strongly positive against Dirofilaria immitis and Brugia phangi. At that point, the records of the patient were referred to one of the coauthors (F. N.), and loiasis was suspected to be the most likely cause. Under suspicion of being infected with L loa, the patient visited the department of infection in a medical center in May 2005, though no symptoms had been seen since January According to the information letter from that hospital, the patient showed eosinophilia (WBC 7,200/ L, eosinophils 2,950/ L), a normal IgE value (82 IU/mL), and no microfilariae in peripheral blood drawn at noon. Thereafter, the patient was hospitalized for 2 days for blood sampling at midnight and a provocative test with a single dose of diethylcarbamazine (DEC). Fifty milligrams of DEC was given and blood drawn just before and 1 hour after administration. However, microfilariae were not detected in any of the blood samples and no symptoms appeared after the administration of DEC. We were not informed regarding the method used for examining microfilariae, and no further treatment was given at the medical center. Following a 10-month asymptomatic period, edema again began to develop on her left forearm in November 2005, which disappeared within 1 week. Two months later in February 2006, the patient came to another one of the authors (M. Y.) at Nara Medical University. A blood examination at that time demonstrated severe eosinophilia (WBC 9,200/ L, eosinophils 4,700/ L) and elevated IgE (659 IU/mL). Further, a gradual increase in the anti- B phangi antibody (IgG4 fraction) titer was disclosed (January 2005, 43 IU; May 2005, 66 IU; February 2006, 225 IU). Chest x-ray and electrocardiogram results showed no abnormalities, while neither hematuria nor proteinuria was found, and a smear examination of peripheral blood failed to reveal microfilariae. We also performed a concentration test using the Knott technique, 6,7 in which 1 ml of blood was premixed with 10 ml of 2% formalin, and the sediment was examined. Repeated examinations using the Knott technique of blood samples drawn on different days during daytime hours between noon and 2:00 pm resulted in an unsuccessful demonstration of microfilaremia. Although the patient was amicrofilaremic, we made a working diagnosis of loiasis on the basis of her Yoshikawa et al. recent history of travel to an area where the parasitic nematode L loa is endemic as well as the clinical presentation, including repeated symptoms and laboratory data. We considered that the patient would benefit from therapy because of the occasional appearance of Calabar swelling and hypereosinophilia that nearly reached 5,000/ L. Treatment was scheduled for the end of March 2006, when her upcoming musical tour abroad would finish. In the middle of March while on tour, swelling appeared on the right forehead of the patient ( Figure 1 ) and a thin threadlike worm began to wiggle in the right conjunctiva after several days, though the parasite could not be felt a few days later. This episode of worm migration to the conjunctiva convinced us that the patient was infected with L loa. When she returned home at the end of March, no abnormalities were found in an ophthalmologic examination, and microfilariae were still undetectable in blood drawn at noon, as well as in smear and Knott method examinations. However, severe eosinophilia with eosinophils exceeding 5,000/ L (WBC 9,300/ L, eosinophils 5,200/ L) and a high level of IgE (885 IU/mL) remained. We decided to introduce treatment and prescribed 200 g/kg of ivermectin on March 30. No adverse reactions were observed, and microfilariae remained undetectable in blood samples collected at 1 and 24 hours after administration. Thereafter, a second administration of ivermectin at the same dose was given on April 17. On that day, the eosinophil count in peripheral blood had begun Figure 1 Nonerythematous and nonpitted swelling in the right forehead. The picture was taken using an instant camera by a friend of the patient. After several days, the patient noticed a thin thread-like worm that began to wiggle in the right conjunctiva.

3 Amicrofilaremic Loiasis to decrease to 1,060/ L, while it declined further to 420/ L on May 8. The patient is currently under long-term observation. After the treatment with ivermectin, she has been asymptomatic for more than 7 months, and a laboratory examination performed on November, 6 months after the second administration, showed a normal eosinophil count in blood (WBC 4,100/ L, eosinophils 160/ L) and declining IgE (260 IU/ ml). Although the titer of anti- B phangi IgG4 antibody has not been examined recently, we expect a gradual decrease. Discussion Loiasis is caused by inoculation of infective thirdstage larvae (L3) of the filarial nematode L loa by Chrysops genus flies and is endemic to the forests of west and central African countries. 1 3 Once bitten by infected flies, development into adult worms takes about 6 to 12 months, and they can survive for up to 17 years, during which the adult male and female worms migrate into subcutaneous tissues. The microfilariae are found in blood and ingested by flies. The patient in our study spent about 1 month in the rain forest in Cameroon. Most of the infected residents in endemic areas are asymptomatic, despite high levels of microfilaremia, though episodic Calabar swellings and pruritis are occasionally seen. Those residents generally have moderate eosinophilia and variable antibody levels. However, the clinical features of L loa infection in temporary residents of endemic regions such as travelers are known to be different from those in native residents. 4,8 The features of patients from nonendemic areas are often characterized by predominant allergic symptoms, such as frequent recurrent episodes of angioedema and a state of marked immunological hyperresponsiveness showing profound hypereosinophilia, increased levels of serum IgE, and high titers of the antibody to filariae. We consider that the patient in our study was a representative case of loiasis that developed in a resident of a nonendemic region. Despite the clinical symptoms and laboratory data providing adequate evidence for suspecting loiasis, microfilariae were not detected in the blood. It has been reported that microfilariae are not detected in most patients from nonendemic areas, 3,4 as well as in a significant percentage of those from endemic areas. 9,10 Many among those amicrofilaremic individuals are known to have been infected with L loa by episode of subconjunctival migration 55 of at least one adult worm. The precise mechanisms underlying amicrofilaremia despite the presence of distinct symptoms causing suspicion of loiasis are unknown, though the probability of becoming microfilaraemic seems to increase with age, suggesting the importance of chronic exposure to bites from infected Chrysops or the total number of L loa L3 inoculations. In contrast, another report speculated that the main deciding factor related to microfilaremia is the level of exposure to vectors within the past few years. 14 The number of inoculations with L loa L3 is considered to be considerably fewer with patients from nonendemic regions than with those from endemic regions because of the short duration or limited exposure to the endemic environment, which may be the reason for the absence of microfilaremia. Further, it is conceivable that the immunological conditions of infected individuals against filarial infections may regulate the development of microfilaremia, as clinical and immunological differences between patients from endemic and nonendemic areas have been reported. 4,8 Distinctly provoked immune responses, often observed in patients from nonendemic regions, may interfere with the production of microfilariae from female adult worms. In contrast, the hyporesponsiveness against filarial infection seen in those from endemic regions may permit microfilaremia. Therefore, one problem often faced is difficulty in demonstrating microfilariae in blood in patients from nonendemic areas. To raise the efficiency of microfilariae detection, we paid attention to the following two points. First, the microfilariae of L loa exhibit diurnal periodicity. Therefore, we collected blood samples during the daytime, at noon and 2:00 pm. Second, in addition to blood smears, we used 1 ml of peripheral blood with a Knott technique, which allowed for an increased volume of blood tested. Instead of the Knott technique, a filter-trap method using a polycarbonate membrane with 3 to 5 m pores is another choice. 6,15 Nevertheless, microfilariae could not be detected in our patient. Further, microfilariae were undetectable after a provocative administration of DEC and therapeutic administration of ivermectin. Detection of antifilarial antibodies is useful to diagnose filarial infection in patients from nonendemic areas, and IgG4 antibodies are known to be detected even in individuals without evidence of microfilaremia. 16 In the patient in our study, enzymelinked immunosorbent assay using B phangi as the target antigen revealed a gradual increase in the titer of IgG4 antibodies, indicating an active filarial infection. However, the infected filarial

4 56 species could not be identified because of extensive antigenic cross-reactivity. Although there are several reports documenting antigen detection using antisera, 17,18 the problem of cross-reactivity still exists. A certain fraction of circulating antigens was reported be a possible diagnostic marker in loiasis for amicrofilaremic patients, 19 but gel filtration is required. Recently, the usefulness of a polymerase chain reaction (PCR), followed by a nested PCR using primer sets specific for the repeat three sequence of the gene encoding the L loa 15 kd protein, was reported for amicrofilaremic individuals infected with L loa. 20 According to that report, 19 of 20 occult-infected individuals, who experienced an ocular passage of an adult L loa but showed no microfilariae in examinations of 2 ml of peripheral blood, were found to be positive by the nested PCR technique. Thus, it is highly possible that a positive result could be obtained by the nested PCR technique used in the present case. Although there are no definite criteria concerning indications for treatment of amicrofilaremic patients from nonendemic areas, we think that those displaying symptoms are indicated for treatment, especially those with hypereosinophilia. However, there is not an urgent need for immediate treatment. There are three kinds of drugs available at present for the treatment of loiasis: DEC, 21 ivermectin, 22 and albendazole, 23,24 of which DEC has been used for about 50 years. The dosage of DEC should be increased in a step-by-step manner. In patients with heavy microfilaria, prophylactic use of steroids is required to prevent severe adverse reactions that have been reported following treatments with DEC and ivermectin. 25,26 DEC and albendazole each require consecutive daily administrations for a few weeks. In contrast, ivermectin is generally given only once or a few times. For the present amicrofilaremic case, any of those treatments could have been chosen, though we treated our patient with ivermectin, using two administrations at 200 g/kg with a 17-day interval. The eosinophil count promptly decreased after treatment with ivermectin and became normal, which we considered to be a good indicator for evaluating treatment efficacy as well as a long-lasting asymptomatic period. In conclusion, we treated a case of loiasis that developed in a Japanese traveler after returning from an endemic area. Despite clinical symptoms and laboratory data providing adequate evidence for suspecting loiasis, microfilariae were not detected in the blood. It is important to be aware that most infected patients from nonendemic regions are amicrofilaremic, even after repeated examinations by Yoshikawa et al. concentration testing methods. For amicrofilaremic loiasis, a nested PCR assay may be the most useful and should provide definitive information. Declaration of Interests The authors state that they have no conflicts of interest. References 1. Stoll NR. This wormy world. J Parasitol 1947 ; 33 : Thomson MC, Obsomer V, Dunne M, et al. Satellite mapping of Loa loa prevalence in relation to ivermectin use in west and central Africa. Lancet 2000 ; 356 : Garcia LS. Loa loa. In: Garcia LS, ed. Diagnostic medical parasitology. 4th Ed. Washington, DC : ASM Press, 2001 : Nutman TB, Miller KD, Mulligan M, Ottesen EA. Loa loa infection in temporary residents of endemic regions: recognition of a hyperresponsive syndrome with characteristic clinical manifestations. J Infect Dis 1986 ; 154 : Rakita RM, White AC, Kielhofner MA. Loa loa infection as a cause of migratory angioedema: report of three cases from the Texas Medical Center. Clin Infect Dis 1993 ; 17 : Knott J. A method for making microfilarial surveys on day blood. Trans R Soc Trop Med Hyg 1939 ; 33 : Garcia LS. Procedures for detecting blood parasites. In: Garcia LS, ed. Diagnostic medical Parasitology. 4th Ed. Washington, DC : ASM Press, 2001 : Klion AD, Massougbodji A, Sadeler BC, et al. Loiasis in endemic and nonendemic populations: immunologically mediated differences in clinical presentation. J Infect Dis 1991 ; 163 : Dupont A, Zue-N dong J, Pinder M. Common occurrence of amicrofilaraemic Loa loa filariasis within the endemic region. Trans R Soc Trop Med Hyg 1988 ; 82 : Pinder M. Loa loa a neglected filaria. Parasitol Today 1988 ; 4 : Kershaw WE, Keay RW, Nicholas WL, Zahraa A. Studies on the epidemiology of filariasis in West Africa, with special reference to the British Cameroons and the Niger delta. IV. The incidence of loa loa and Acanthocheilonema perstans in the rain-forest, the forest fringe and the mountain grasslands of the British Cameroons, with observations on the species of chrysops and culicoides found. Ann Trop Med Parasitol 1953 ; 47 : Ripert C, Ambroise-Thomas P, Riedel D, et al. Epidemiology of filariasis: L. loa and D. perstans in 7 villages of the southern center of Cameroon [in French]. Bull Soc Pathol Exot Filiales 1977 ; 70 :

5 Amicrofilaremic Loiasis 13. Pion DS, Gardon J, Kamgno J, et al. Structure of the microfilarial reservoir of Loa loa in the human host and its implications for monitoring the programmes of community-directed treatment with Ivermectin carried out in Africa. Parasitol 2004 ; 129 : Pion SD, Demanou M, Oudin B, Boussinesq M. Loiasis: the individual factors associated with the presence of microfilaraemia. Ann Trop Med Parasitol 2005 ; 99 : Dennis DT, Kean BH. Isolation of microfilariae: report of a new method. J Parasitol 1971 ; 57 : Akue JP, Egwang TG, Devaney E. High levels of parasite-specific IgG4 in the absence of microfilaremia in Loa loa infection. Trop Med Parasitol 1994 ; 45 : Jaoko WG. Loa loa antigen detection by ELISA: a new approach to diagnosis. East Afr Med J 1995 ; 72 : Walker-Deemin A, Kombila M, Mouray H, et al. Detection of circulating antigens in Gabonese patients with Loa loa filariasis. Trop Med Int Health 1996 ; 1 : Walker-Deemin A, Ferrer A, Gauthier F, et al. Identification and specificity of a 38 kda Loa loa antigenic fraction in sera from high-microfilaraemic Gabonese patients. Parasitol Res 2004 ; 92 : Toure FS, Mavoungou E, Kassambara L, et al. Human occult loiasis: field evaluation of a nested 57 polymerase chain reaction assay for the detection of occult infection. Trop Med Int Health 1998 ; 3 : Klion AD, Ottesen EA, Nutman TB. Effectiveness of diethylcarbamazine in treating loiasis acquired by expatriate visitors to endemic regions: long-term follow-up. J Infect Dis 1994 ; 169 : Richard-Lenoble D, Kombila M, Rupp EA, et al. Ivermectin in loiasis and concomitant O. volvulus and M. perstans infections. Am J Trop Med Hyg 1988 ; 39 : Klion AD, Massougbodji A, Horton J, et al. Albendazole in human loiasis: results of a double-blind, placebo-controlled trial. J Infect Dis 1993 ; 168 : Tabi TE, Befidi-Mengue R, Nutman TB, et al. Human loiasis in a Cameroonian village: a double-blind, placebo-controlled, crossover clinical trial of a threeday albendazole regimen. Am J Trop Med Hyg 2004 ; 71 : Carme B, Boulesteix J, Boutes H, Puruehnce MF. Five cases of encephalitis during treatment of loiasis with diethylcarbamazine. Am J Trop Med Hyg 1991 ; 44 : Gardon J, Gardon-Wendel N, Demanga-Ngangue, et al. Serious reactions after mass treatment of onchocerciasis with ivermectin in an area endemic for Loa loa infection. Lancet 1997 ; 350 : A sign discovered in a tertiary referral hospital in Inner Mongolia. The correct translation would have been Abdominal Ultrasound Room (Submitted by Jane Youngs).

Lecture 5: Dr. Jabar Etaby

Lecture 5: Dr. Jabar Etaby 1 2 Onchocerca volvulus (Blinding filariasis; river blindness) Microfilaria of Onchocerca volvulus, from skin snip from a patient seen in Guatemala. Wet preparation 3 Some important