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Loa loa is a human fi larial 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 Cam-eroon (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 nonen-demic 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 Cam-eroon and living for 1 month in a village among Baka Pygmy people. She developed fugitive swell-ing (Calabar swelling) 6 months after her return from Cameroon. In the following 16 months, Cala-bar swelling appeared and disappeared on a few occasions and fi nally worm migration into the conjunctiva was observed. Blood examinations showed eosinophilia, elevated immunoglobulin (Ig)E level, and a gradual increase in the ratio of antifi larial antibodies (IgG4 fraction). Despite the clinical history and laboratory data providing ade-quate evidence for suspecting loiasis, microfi lariae were not detected in the blood. Herein, we describe

the patient profi le and discuss diagnostic problems in patients with amicrofi laremic 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 spon-taneous 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 consulta-tion for that occurrence.

The patient revisited the doctor in March 2005 for a regular medical checkup, and a blood examina-tion 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 his-tory 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 (coordi-nates: lat 4°23 ′ N, long 13°34 ′ 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 blackfl y-infested forests. She joined the Baka Pygmy community to study their

Diagnostic Problems in a Patient With Amicrofi laremic Loa loa

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: 10.1111/j.1708-8305.2007.00167.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, microfi lariae were not detected in the blood. It is important to note that most infected travelers whose home countries are in nonendemic regions are amicrofi laremic.

Corresponding Author: Masahide Yoshikawa, MD, Department of Parasitology, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan. E-mail: myoshika@naramed-u.ac.jp

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music and lived with them in their traditional man-ner for about 1 month in April and May 2004 and also remembered well being bitten by Chyrops genus fl ies numerous times during her stay in the village. Although a blood smear examination was not per-formed, serological tests were strongly positive against Dirofi laria immitis and Brugia phangi. At that point, the records of the patient were referred to one of the coauthors (F. N.), and loiasis was sus-pected to be the most likely cause.

Under suspicion of being infected with L loa , the patient visited the department of infection in a med-ical center in May 2005, though no symptoms had been seen since January 2005. According to the in-formation 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 microfi lariae 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. How-ever, microfi lariae 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 microfi -lariae, 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 electro-cardiogram results showed no abnormalities, while neither hematuria nor proteinuria was found, and a smear examination of peripheral blood failed to re-veal microfi lariae. We also performed a concentra-tion test using the Knott technique, 6,7 in which 1 mL of blood was premixed with 10 mL of 2% for-malin, 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 microfi laremia. Al-though the patient was amicrofi laremic, we made a working diagnosis of loiasis on the basis of her

recent history of travel to an area where the parasitic nematode L loa is endemic as well as the clinical pre-sentation, including repeated symptoms and labo-ratory data.

We considered that the patient would benefi t 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 mu-sical tour abroad would fi nish. In the middle of March while on tour, swelling appeared on the right forehead of the patient ( Figure 1 ) and a thin thread-like 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 migra-tion 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 microfi lariae were still undetectable in blood drawn at noon, as well as in smear and Knott method examinations. However, severe eosinophilia with eosinophils ex-ceeding 5,000/ � L (WBC 9,300/ � L, eosinophils 5,200/ � L) and a high level of IgE (885 IU/mL) re-mained. We decided to introduce treatment and prescribed 200 � g/kg of ivermectin on March 30. No adverse reactions were observed, and microfi -lariae remained undetectable in blood samples col-lected 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.

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to decrease to 1,060/ � L, while it declined further to 420/ � L on May 8.

The patient is currently under long-term obser-vation. 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 anti-body has not been examined recently, we expect a gradual decrease.

Discussion

Loiasis is caused by inoculation of infective third-stage larvae (L3) of the fi larial nematode L loa by Chrysops genus fl ies and is endemic to the forests of west and central African countries. 1 – 3 Once bitten by infected fl ies, 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 microfi lariae are found in blood and ingested by fl ies.

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, de-spite high levels of microfi laremia, though episodic Calabar swellings and pruritis are occasionally seen. Those residents generally have moderate eosino-philia and variable antibody levels. However, the clinical features of L loa infection in temporary resi-dents of endemic regions such as travelers are known to be different from those in native resi-dents. 4,8 The features of patients from nonendemic areas are often characterized by predominant aller-gic symptoms, such as frequent recurrent episodes of angioedema and a state of marked immunologi-cal hyperresponsiveness showing profound hyper-eosinophilia, increased levels of serum IgE, and high titers of the antibody to fi lariae. 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 labora-tory data providing adequate evidence for suspect-ing loiasis, microfi lariae were not detected in the blood.

It has been reported that microfi lariae are not detected in most patients from nonendemic areas, 3,4 as well as in a signifi cant percentage of those from endemic areas. 9,10 Many among those amicrofi lare-mic individuals are known to have been infected with L loa by episode of subconjunctival migration

of at least one adult worm. The precise mechanisms underlying amicrofi laremia despite the presence of distinct symptoms causing suspicion of loiasis are unknown, though the probability of becoming mi-crofi laraemic seems to increase with age, 11 – 13 sug-gesting the importance of chronic exposure to bites from infected Chrysops or the total number of L loa L3 inoculations. In contrast, another report specu-lated that the main deciding factor related to micro-fi laremia 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 envi-ronment, which may be the reason for the absence of microfi laremia. Further, it is conceivable that the immunological conditions of infected individuals against fi larial infections may regulate the develop-ment of microfi laremia, as clinical and immunolog-ical differences between patients from endemic and nonendemic areas have been reported. 4,8 Distinctly provoked immune responses, often observed in pa-tients from nonendemic regions, may interfere with the production of microfi lariae from female adult worms. In contrast, the hyporesponsiveness against fi larial infection seen in those from endemic regions may permit microfi laremia.

Therefore, one problem often faced is diffi culty in demonstrating microfi lariae in blood in patients from nonendemic areas. To raise the effi ciency of microfi lariae detection, we paid attention to the fol-lowing two points. First, the microfi lariae 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 fi lter-trap method using a polycarbonate membrane with 3 to 5 � m pores is another choice. 6,15 Nevertheless, microfi lariae could not be detected in our patient. Further, microfi lariae were undetectable after a provocative administration of DEC and therapeu-tic administration of ivermectin.

Detection of antifi larial antibodies is useful to diagnose fi larial infection in patients from nonen-demic areas, and IgG4 antibodies are known to be detected even in individuals without evidence of microfi laremia. 16 In the patient in our study, enzyme-linked immunosorbent assay using B phangi as the target antigen revealed a gradual increase in the titer of IgG4 antibodies, indicating an active fi larial infection. However, the infected fi larial

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species could not be identifi ed because of extensive antigenic cross-reactivity. Although there are sev-eral reports documenting antigen detection using antisera, 17,18 the problem of cross-reactivity still ex-ists. A certain fraction of circulating antigens was reported be a possible diagnostic marker in loiasis for amicrofi laremic patients, 19 but gel fi ltration is required. Recently, the usefulness of a polymerase chain reaction (PCR), followed by a nested PCR using primer sets specifi c for the repeat three se-quence of the gene encoding the L loa 15 kD pro-tein, was reported for amicrofi laremic 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 mi-crofi lariae 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 tech-nique used in the present case.

Although there are no defi nite criteria concern-ing indications for treatment of amicrofi laremic pa-tients 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 treat-ment. There are three kinds of drugs available at present for the treatment of loiasis: DEC, 21 iver-mectin, 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 microfi laria, prophylactic use of steroids is required to prevent severe adverse reac-tions that have been reported following treatments with DEC and ivermectin. 25,26 DEC and albenda-zole each require consecutive daily administrations for a few weeks. In contrast, ivermectin is generally given only once or a few times. For the present ami-crofi laremic 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 ivermec-tin and became normal, which we considered to be a good indicator for evaluating treatment effi cacy as well as a long-lasting asymptomatic period.

In conclusion, we treated a case of loiasis that de-veloped in a Japanese traveler after returning from an endemic area. Despite clinical symptoms and laboratory data providing adequate evidence for suspecting loiasis, microfi lariae were not detected in the blood. It is important to be aware that most infected patients from nonendemic regions are ami-crofi laremic, even after repeated examinations by

concentration testing methods. For amicrofi lare-mic loiasis, a nested PCR assay may be the most use-ful and should provide defi nitive information.

Declaration of Interests

The authors state that they have no confl icts of interest.

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A sign discovered in a tertiary referral hospital in Inner Mongolia. The correct translation would have been “Abdominal Ultrasound Room” (Submitted by Jane Youngs).