Bancroftian filariasis in a paediatric population: an ultrasonographic study

TRANSACTIONSOFTHEROYALSOCIETYOFTROPICALMEDICINEANDHYGIENE(1999)93,633-636

Bancroftian

filariasis

in a paediatric

population:

an ultrasonographic

study

G. Dreyer’,*, J. No&es *, D. Addiss3, A. Santos’, Z. Medeiros’ and J. Figueredo-Silva4 ‘Departamento de Parasitologia, Centro de Pesquisas Aggeu Magalhaes, Recife, Brazil; ‘Service de Urologia, Hospital das Clinicas, Universidade Federal de Pemambuco, Av. Moraes Rego sin, Cidade Universitaria, Recife, PE, 50670-901, Brazil; 3Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Geors’a, USA; 4Laboratorio de Imunopatologia Keiso Asami, Universidade Federal de Pernambuco, Recife, Brazil Abstract Little is known about lymphatic filariasis or the anatomical location of adult Wuchereria bancrofti in children. Seventy-eight children from Greater Recife, 23 microfilaria-positive and 55 microfilaria-negative in -60 & blood, underwent ultrasound examinations of the major superficial lymphatic vessels of the limbs, scrotal area (boys), and breast area (girls). The characteristic movements of adult worms, known as the filaria dance sign (FDS), were detected in 11 (14.1%) children. In 9 boys, the FDS was detected in lymphatic vessels of the scrotal area (8, ages 14- 16) and the inguinal cord (1, age 11). In girls, the FDS was detected in a crural lymphatic vessel and an axillary lymph node. FDS detection was more common in boys (P = 0.06), older children (P = O.OOl), and children with microfilaraemia (P = 0.05). Diffuse lymphangiectasia was visualized in 4 boys (ages 14-16) and 2 children had clinical signs of filariasis. These ultrasonographic findings associate W. bancrofti with both infection and disease in children. Keywords:

lymphatic filariasis, Wuchereriabancrofti, ultrasound, children, Brazil

Introduction Lymphatic filariasis remains a major public health problem in tropical countries around the world. An estimated 120 million people are infected, more than 90% with Wuchereria bancrofti (MICHAEL et al., 1996). In filariasis-endemic areas, the manifestations of lymphatic filariasis include asymptomatic adult-worm infections without microfilaraemia (DREYER et aZ., 1996b), asymptomatic microfilaraemia, and acute and chronic clinical manifestations (OTTESEN, 1992), but the relationship between the development of symptoms and the course of the infection is not completely understood. As the chronic manifestations of lymphatic filariasis appear most frequently during early adulthood or later, clinical investigations have focused on adult populations. In children, limited information exists on W. bancrofti microfilaraemia (LOWMAN, 1944;RAJASEKARIAH et al., 199 1; BRAGA et al., 1997), antigenaemia (LAMMIE et al., 1998), or immune responsiveness to the parasite (HITCH etal., 1989, 1991a, 1991b; DAY et al., 1991). Living adult W. bancrofti were first visualized by ultrasonography in Brazil (AMARAL et al., 1994). The distinctive pattern of adult worm movement on ultrasound, called the filaria dance sign (FDS), has subsequently been reported from India (SURESH et al., 1997), Egypt (FARIS et aZ., 1998), Haiti (D. Addiss, personal communication), and Tanzania (P. Lammie, personal communication). In men, in whom the lymphatic vessels of the scrotal area are the most common site of adult W. bancrofti, the FDS can be detected in 80% of microfilaria carriers (NOR~ES et al., 1996b); in women, the FDS has been detected in the breast (DREYER et al., 1996d). In men, ultrasound has been successfully used to assess, in the adulticidal efficacy of antifilarial drugs vivo, (DREYER et al., 1995a, 1995b, 1996a; NOR~ES et al., 1997), describe preclinical abnormalities in the lymphatic vessels (NOR~ES et al., 1996a), identify amicrofilaraemic adult-worm carriers (DREYER et al., 1996b), and identify living adult worms in patients with tropical pulmonary eosinophilia (DREYER et aZ., 1996c). As no similar ultrasound studies have been done in children, the present investigation was undertaken to identify by ultrasonography living adult worms in both microfilaria carriers and amicrofilaraemic individuals.

Address for correspondence: Dr Gerusa Dreyer, Hospital das Clinicas, Departamento de Cirurgia, Universidade Federal de Pernambuco, Av. Moraes Rego sin, Cidade Universitaria, Recife, FE, 50670-901, Brazil; fax +55 81 4276455.

Materials and Methods The study population consisted of children and teenagers who participated in a ‘night blood survey’ to detect circulating microfilariae in residents of Olinda, a community in Greater Recife, Brazil. The protocol was approved by the ethics committee of Hospital das Clinicas, Universidade Federal de Pernambuco. A total of 273 children (144 males and 129 females) ranging in age from 5 to 16 years (mean, 10.5 years) were included. Testing for microfilaraemia was initially done by thick smear (-60 + of blood) collected between 23:00 and 0 1:OO.To quantify microfilarial density, all children who were initially microfilaria-positive had 1 mL of venous blood collected at night, filtered, and examined for microfilariae. The first 55 children who were microfilaria-negative and who visited the filariasis clinic at the Aggeu MagalhZes research centre for follow-up had 5 mL of venous blood collected between 23:00 and 01:OO and another 5-mL sample taken 15 days later (Figure). The venous blood was filtered through a 3-p polycarbonate membrane, stained with Carrazzi-haematoxylin, and examined under a microscope for W. bancrofti microfilariae. Children were considered amicrofilaraemic if no microfilariae were detected in 10 mL

Figure. Design of the ultrasonographic study of children in Recife, Brazil, for bancroftian filariasis. FDS, filaria dance sign.

G. DREYER ETAL.

634

of blood. All children who had venous blood examined for microfilariae also were evaluated clinically, and the major superficial lymphatic vessels of the limbs were examined for living adult worms and lymphangiectasia with a portable ultrasound machine using a 75 MHz probe (Pie Medical 200, The Netherlands). Ultrasound examinations were performed with the child in the supine position during three 40-min sessions within a l-week interval. Each child was examined in the upper limbs (arms, forearms, and axillae) and lower limbs (legs, thighs, and inguinal area); in boys, the inguinal cords and scrotal area were also examined, and in girls, the mammary area was examined. A child was considered not to have the FDS only after 3 negative examinations. All children who had a detectable FDS and 24 of those who did not were examined again by an independent ultrasonographer, who was blinded to the results of the previous examinations. To encourage children to remain still during the examination, a cartoon was shown on television. All individuals who were found to be infected with I&‘. bancroft (either microtilariae or adult worms) were treated with diethylcarbamazine (DEC). For continuous variables, differences between groups were tested for statistical significance using the nonparametric Kruskal-Wallis test. For dichotomous variables, the 2-tailed Fisher exact test or the x2 test with Yates’ correction was used.

tions, 28 who were microtilaria-positive and 50 who were microfilaria-negative (in 10 mL of blood). Of these, 11 (14.1%) children, 9 boys and 2 girls, had living adult worms visible on ultrasound examination. The 12 nests (1 boy had 2 nests) were detected on each of the 3 ultrasound examinations, and were confirmed by the independent ultrasonographer. The ultrasound image of the FDS, both in the B and M modes, was identical to that reported in adults (AURAL et al., 1994; DREYER et al., 1996~; NOR~ES et al, 1996b). The length of time required to detect the adult worms ranged from 4 to 20 min, being shorter for children with greater lymphatic vessel diameter. The FDS was detected in 7 (25%) microfilaria-positive children and 4 (8%) children who were micro’rilaria-negative (P = 0,048, Fisher exact test). In girls, living adult worms were detected in a lvmphatic vessel in the&Ural area and in the efferent vessel of an axillarv lvmnh node. In bovs. the FDS was detected in lymphatic iessels in the scrotal area (8 boys) and the inguinal cord (1 boy, age 11 years). Among boys aged 14-16 years, the FDS was detected only in the scrotal area; 4 nests were supratesticular, 3 were paratesticular, and 2 were infratesticular (1 boy had 2 nests). Mean lymphatic vessel diameter at the site of the adult worm nest was 2.1 mm (range, 1.4-3.5 mm). Vessel diameter tended to increase with age; in 3 children aged G 11 years, mean vessel diameter was 1.6 mm, compared to 2.2 mm in the 8 children who were aged > 11 years (P = 0.18). Diffuse lymphangiectasia (not just at the site of the adult worm) was detected in 4 bovs, ages 14- 16 vears (Table 1). Only 2 children had clinical &idence of W. b&crofti disease: a ‘i-year-old girl had painless adenopathy (3 cm X 4 cm on palpation) in the right axilla, and a 15-year-old boy had a hydrocoele and lymphangiectasia that was palpable on physical examination. Detection of the FDS was associated with age and sex of the child. Comuared to the 67 children who had no detectable FDS, those with ultrasonographic evidence of adult worm infection were significantlv older (mean, 13.7 years compared to 9.5 years, P = 6.001, I(r;lskalWallis test) and more likelv to be male (82% comnared to 46%, P =‘0.06, x”) (Table 2). The 9‘boys with detectable FDS were older than the 2 girls (mean ages 15.0 and 8.0 years, respectively, P = 0.025); in contrast, the ages of all 78 children who underwent ultrasound examination did not differ significantly by sex (10.8 and 9.5 years, respectively, P = 0.14). Among 55 children who were microfilaria-negative in 60 r.ls, of blood, 9 (16.4%) were found to be infected: 5

Results Among 273 children who submitted blood for examination by thick smear, 23 (8.4%; 10 males and 13 females) were found to be microfilaria-positive. Mean age did not differ significantly between children who were microfilaria-positive (10.8 years) and those who were microtilaria-negative (10.1 years, P = 0.44), and the _ proportion of females was similar for both groups _ - _ (57% and 46%, P = 0.48). A total of 250 children were microfilaria-negative in 60 & of capillary blood; the 55 who were se&ted for further study were similar to the remaining 195 in age (mean 9.9 and 10.2 years, respectively, P- 0.52) and sex (46% and 47% female. resuectivelv. P = 0.991. Five (9.1%) of the 55 ‘micro&aria-negat&e’ children were found to have microfilariae on filtration of 10 mL of venous blood (range, l-6 per 10 mL). The mean age of these 5 children. all males. did not differ sismiticantlv from that ofthe 5b who remained microfilaria-Negative in 10 mL of blood (11.8 and 9.7 years, respectively, P = 0.38). A total of 78 children underwent ultrasound examina-

Table 1. Characteristics Recife, Brazil

Sex

Age (years)

of 11 children

who had adult

Mf/mL

FDS site

12 130 O/10 mL

Crural (left) Efferent vessel, right axillary lymph node Inguinal cord (left) Paratesticular (right), supratesticular (left)

;

9 7

E

11 15

198 1498

E M M M

16 16 14 16 16 16 15

1880 630 168 O/10 mL O/10 mL O/10 mL l/10 mL

FDS, filaria dance sign.

Supratesticular Supratesticular Infratesticular Infratesticular Paratesticular Supratesticular Paratesticular

(right) (right) (right) (left) (left) (right) (right)

W. bancrofti

Vessel diameter (at FDS) (mm) 1.7 1.6

detected

on ultrasound

examination,

Diffuse lymphangiectasia

Clinical signs

No No

None Painless adenopathy

1.5 3.1 3.0

No Yes (severe) Yes (severe)

1.9 2.4 3.5 2.7 1.5 2.0 1.4

Yes (mild) Yes (mild) Yes (mild) No No No No

None Bight hydrocoele, lymphangiectasia on physical examination None None None None None None None

ULTFCASONOGRAPHY OFFILAFUASIS

635

Table 2. Factors associated with detection of the filaria dance sign (FDS) on ultrasound examination of 28 microfilaria-positive and 50 microfilarianegative (in 10 mL venous blood) children, Recife, Brazil Characteristic Microfilaria-positive Microfilaria-negative Mean age in years (range) Gender Male Female

FDS-positive 7 (25%) 4 (8%) 13.7 (7-16) 9 2

FDS-negative 21 94:

Discussion Little is known about the spectrum of clinical manifestations or the natural history of lymphatic filariasis in children. In areas where transmission of lymphatic tilariasis is intense, the prevalence of micro’iilaraemia is usually very low in young children and increases rapidly between 5 and 15 years of age (LOWMAN, 1944; PANI uZ.,1991; RAJASEI&FUAHet al., 1991; HIGHTOWERetaL, 1993: BRAGA et aZ.. 1997). The recent use of assavs to detect circulating adult-worm antigen has shown that W bancrofii infection may be much more common in children than previously recognized, and that infection occurs several years earlier. For example, in a cohort of children in Leogane, Haiti, the prevalence of microtilaraemia in 20 pL of blood increased from zero to 1.3% between 2 and 4 years of age, while the prevalence of antigenaemia increased from 6% to 30% (LAMMIE et al., 1998). Our ultrasonographic findings in this study also provide evidence for subclinical filarial disease in children. The ultrasound images of living adult worms (in 11 children) and diffusely dilated lymphatic vessels (in 4 boys, aged 14-16 years) were similar to those seen previously in men with W. bancrofti infection (AMARAL et aZ., 1994; NOR~ES et aZ., 1996a, 1996b). Similarly, although clinical disease associated with W. bancrofti infection is rarely reported in children, histopathological studies suggest that it occurs but is likely to be overlooked. TUNGMAhWet al. (1991, 1992) reviewed the cases of”75 patients who underwent diagnostic lymphnode or lymphatic nodule biopsy in Recife, Brazil. Of & these, 41 (55%) were aged <20 years (JUNGMANN et

FIGUEREDO-SILVA,1989). The diagnosis of lymphatic filariasis, which was unexpected prior to biopsy, was based on the presence of adult worms, which were

associated with a wide range of alterations in the lymph nodes and lymphatic vessels (JUNGMANN et al., 1991, 1992). In the current study, 2 (18%) of 11 children with a detectable FDS also had clinical manifestations of lymphatic ‘tilariasis (painless adenopathy and hydrocoele with palgable lvrnphangiectasia). This study is the firs to document ultrasonographic detection of livine adult W. bancrofti in children. Adult worms were detected more frequently in boys and in

P

28 50

0.05

(5-17) 31 36

(9.1%) were microfilaria-positive by filtration of 10 mL blood (i.e., they had ultra-low microfilarial density) and 5 (9.1%) had a detectable FDS (1 child had both detectable microfilaraemia and a FDS). Although these 55 children had not been randomly selected from among the 250 who were microfilaria-negative in 60 III- of blood, they were very similar in age and sex, and all 250 children lived in filariasis-endemic areas of Greater Recife. If one assumes their risk of occult W. bancrofti infection to be similar to that of the entire group, then about 41 (16.4%) of these 250 children would be infected. Only 23 of the original 273 children in this study were microfilarianositive in 60 LIL of blood. Thus. the number of infected children in this population was’about 2.8 times higher (n - 64) than were identified by a 6O+L fingerstick (n = 23).

Total

0.001 40 38

0.063

older children. Because the number of children studied was relatively small and because the 9 boys with detectable worms were older than the 2 girls, it was not possible to assess whether age or sex is the more important factor, or whether age differentially influences adult worm location or ease of detection in boys and girls. An equivalent number of boys and girls (40 and 38, respectively) was examined by ultrasound, and the mean age of those examined was similar for both groups. Further, both in this study and in population-based studies in Greater Recife (BRAGA et al., 1997), microtilaraemia prevalence was similar for boys and girls. This contrasts with the general finding of an increased prevalence of microfilaraemia in men compared to women (BRABIN,

1990). Taken together, these data suggest that the mevalence of W. bancrohi infection is similar amona boys and girls, but thatdadult worms are more easily detected in boys, particularly once they reach puberty. In addition, the ultrasonographic findings are consistent with the hypothesis that lymphatic vessel dilatation, and subsequent clinical disease, are triggered or enhanced at the time of puberty, at least in males. Additional work is needed to evaluate this hypothesis. Among boys aged > 14 years, the worms were detected only in the scrotal area. This is also the most common site of adult-worm detection (NOR~ESet al., 1996b; DREYER et al., 1999) and clinical disease (hydrocoele) in men. These data suggest that, in boys, puberty influences the ‘preferred’ location of living adult W. bancrofti. In the 3 youngest children, ages 7- 11 years, adult worms were detected in the axillary, crural, and inguinal areas. This was the first reported demonstration of living adult W. bancrofti in an axillary lymph node. The worms were detected in the efferent lymphatic vessel of this node, and were associated with painless adenopathy. Although dead or degenerating adult worms can be detected within the lymph node parenchyma in biopsy specimens, in our experience, apparently intact adult worms in biopsy specimens are found only in association with lymphatic vessel endothelium. The ultrasonographic image in this r/-year old girl corresponded exactly with the histological findings described in lymph node biopsies from patients living in the filariasis-endemic area of Greater Recife: in 10% of those cases, both afferent and efferent dilated vessels contained intact parasites associated with lymphoid hyperplasia (JUNGMANNet al., 1991; FIGUEREDOSILVA et aZ., 1994). As previously documented in adults (DREYER et aZ., 1996b), the use of ultrasound in this study made it possible to identify children who were microfilaria-negative adult-worm carriers. Among the 55 children who were microfilaria-negative in 60 Ils, of blood, 5 (9.1%) had a detectable FDS on ultrasound. Four of these children were microfilaria-negative in 10 mL of blood. Thus. in this arouv of 55 children. 4 (7.3%) were microfilaria-negutive adult-worm carriers. . ’ The ultrasonographic findings, in combination with filtration of larger amounts of venous blood to detect circulating microfilariae, indicate that the W. bancrofti infection rate in school-age children may be about 3 times greater than that estimated by sampling 60 &

636

blood for microfilariae. Of the 2 techniques, the noninvasive nature of ultrasound makes it the preferable tool for diagnosis of W. bancroft infection in individual children who are microfilaria-negative by thick smear. The discrepancy between the number of infected children detected by thick smear and the number of children who are actually infected appears to be even greater in pre-school children. In Leogane, Haiti, the prevalence of circulating Maria1 antigen was more than 15 times greater than the prevalence of microlilaraemia (in 20 & of et aZ., 1998). blood) in children aged 2-4 years (LAMMIE These data support the strategy of mass treatment with adulticidal drugs for lymphatic filariasis, and suggest that the proportion of children who will benefit from such therapy will be considerably greater than those who are known to be infected on the basis of a 60-$ thick smear. However, little is known about the natural history of lymphatic filariasis, the nature of lymphatic damage caused by W. bancrofti infection, or the macrofilaricidal efficacy of DEC in children. Longitudinal paediatric studies are therefore crucial for a better understanding of these issues. In this context, the non-invasive, widely available and relatively inexpensive technique of ultrasonography represents a remarkable advance for understanding the natural history of bancroftian filariasis. Acknowledgements The study was funded by the Amaury Coutinho NonGovernmental Organization. The authors thank the Oswald0 Cruz Foundation and the University Federal de Pernambuco, where the study was conducted. The authors are grateful for the trust of the parents of children who participated in the study and for the children themselves, who patiently endured the ultrasound examinations and bravely cooperated with the collection of blood. References Amaral, F., Dreyer, G., Figueredo-Silva, J., Noroes, J., Cavalcanti. A.. Samico. S. C.. Santos. A. & Coutinho. A. (1994). Live ‘adult worms detected by-ultrasonography in human bancroftian filariasis. American Journal of Tropical Medicine andHygiene, 48, 178-185. Brabin, L. (1990). Sex differentials in susceptibility to lymphatic tilariasis and implications for maternal child immunity. Epidemiology and Infection, 105, 335-353. Braga, C., de Albuquerque, M. F. M., Schindler, H., Rezende, A., Maciel, A., Silva, M. C. M., Furtado, A., Carvalho, A. B., Lapa, T. & Ximenes, R. A. A. (1997). Per81 epidemiologico da filariose linfatica em criancas residentes em areas endemicas. Jomal de Pediatia, 73, 95-100. Day, K. I’., Gregory, W. F. & Maizels, R. M. (1991). Agesnecific acouisition of immunitv to infective larvae in a bancroftian-filariasis endemic arka of Papua New Guinea. Parasite Immunology, 13, 277-290. Dreyer, G., Amaral, F., Noroes, J., Medeiros, Z. & Addiss, D. (1995a). A new tool to assess the adulticidal efficacy in vivo of anti’tilarial drugs for bancroftian fllariasis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 89, 225-226. Dreyer, G., Noroes, J., Amaral, F., Nen, A., Medeiros, Z., Coutinho, A. & Addiss, D. (1995b). Direct assessment ofthe adulticidal efficacy of a single-dose of ivermectin for bancroftian filariasis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 89,44 l-443. Dreyer, G., Addiss, D., Noroes, J., Amaral, F., Rocha, A. & Coutinho, A. (1996a). Ultrasonographic assessment of the adulticidal efficacy of repeated high-dose ivermectin in bancroftianfilariasis. TroaicalMedicineandInternationalHealth. _ 1._ 427-432. Dreyer, G., Santos, A., Noroes, J., Amaral, F., Rocha, A. & Addiss, D. (1996b). Amicrofilaraemic carriers of adult Wuchereria bancrofti. Transactions of the Roval Societv of Tronical Medicine and Hygiene, 90,288-289. ” - ” Dreyer, G., Noroes, J., Rocha, A. & Addiss, D. (1996~). Detection of living adult Wuchereria bancrofti in a patient with tropical pulmonary eosinophilia. BrazilianJournal of Medical and Biological Research, 29, 100% 1008. Dreyer, G., Brandlo,A. C., Medeiros, Z. & Addiss, D. (1996d). Detection of living adult Wuchereria bancrofn’ in the female breast. Memorias do Instituto Oswald0 Cruz, 91, 95-96. Dreyer, G., Santos, A., Noroes, J. & Addiss, D. (1999). Proposed panel of diagnostic criteria, including the use of ultrasound, to refine the concept of ‘endemic normals’ in

G. DREYER ETAL. lymphatic filariasis. 3oumal of Tropical Medicine and International Health, in press. Faris, R., Hussain, O., El Setouhy, M., Ramzy, R. M. R. & Weil, G. J. (1998). Bancroftian tilariasis in Egypt: visualization of adult worms and subclinical lymphatic pathology by scrotal ultrasound. American Journal of Tropical Medicine and Hygiene, 59, 864-867. Figueredo-Silva, J., Dreyer, G., Guimarles, K., Brandt, C. & Medeiros, Z. (1994). Bancroftian lymphadenopathy: absence of eosinophils in tissues despite peripheral blood hypereosinophilia. Journal of Tropical Medicine and Hygiene, 97,55-59. Hightower, A. W., Lammie, P. J. & Eberhard, M. L. (1993). Maternal filarial infection-a persistent risk factor for infection in offspring? Parasitology Today, 9,4 18-42 1. Hitch, W. L., Lammie, P. J. & Eberhard, M. L. (1989). Heightened anti-filarial immune resuonsiveness in a Haitian ped&ic population. American Jouktal of Tropical Medicine and Hygiene, 41,657-663. Hitch, W. L., Lammie, P. J. & Eberhard, M. L. (1991a). Antitilarial cellular resnonses detected in a Haitian aediatric population by use of a microblastogenesis assay. journal of Infectious Diseases, 164, 8 1 l-8 13. Hitch, W. L., Hightower, A. L., Eberhard, M. L. & Lammie, P. J. (1991b). Analysis of isotype-specific antitilarial antibody levels in a Haitian pediatric population. American Journal of Tropical Medicine and Hygiene, 44, 16 1- 167. Jungmann, P. & Figueredo-Silva, J. (1989). Bancroftian filariasis in the metropolitan area of Recife (Pernambuco State, Brazil): clinical aspects in histologically diagnosed cases. Brazilian Journal of Medical and Biological Research, 22, 687-690. Jungmann, P., Figueredo-Silva, J. & Dreyer, G. (1991). Bancroftian lymphadenopathy: a histopathologic study of fiftyeight cases from northeastern Brazil. American Journal of Tropical Medicine and Hygiene, 45,325-33 1. Jungmann, P., Figueredo-Silva, J. & Dreyer, G. (1992). Bancroftian lymphangitis in northeastern Brazil: a histopathological study of 17 cases. Journal of Tropical Medicine and Hygiene, 95,114-118. Lammie, P. J., Reiss, M. D., Dimock, K. A., Streit, T. G., Roberts, J. M. & Eberhard, M. L. (1998). Longitudinal analysis of the development of filarial infection and antifilarial immunity in a cohort of Haitian children. AmericanJournal of Tropical Medicine and Hygiene, 59, 2 17-22 1. Lowman, E. W. (1944). Incidence of filariasis in children. US Naval Medical Bulletin, 42, 34 1-343. Michael, E., Bundy, D. A. P. & Grenfell, B. T. (1996). Reassessing the global prevalence and distribution of lymphatic filariasis. Parasitology, 112,409-428. Noroes, J., Addiss, D., Santos, A., Medeiros, Z., Coutinho, A. & Dreyer, G. (1996a). Ultrasonography evidence of abnormal lymphatic vessels in young men with adult Wuchereria bancrofti infections in the scrotal area. Journal of Urology, 166, 409-412. Noroes, J., Addiss, D., Amaral, F., Coutinho, A., Medeiros, Z. & Dreyer, G. (1996b). Occurrence of living adult Wuchereria bancrofti in the scrotal area of men with microfilaraemia. Transactions of the Royal Society of Tropical Medicine and Hygiene, 90, 55-56. Nordes, J., Dreyer, G., Santos,A., Mendes,V. G., Medeiros, Z. & Addiss, D. (1997). Assessment of the efficacv of diethvlcarbamazine on adult Wuchereria bancrofti in v&o. Transactions of the Royal Society of Tropical Medicine and Hygiene, 91, 78-81. Ottesen, E. A. (1992). Infection and disease in lymphatic filariasis: an immunological perspective. Parasitology, 104, s71-s79. Pani, S. I’., Balakrishan, N., Srividya, A., Bundy, D. A. P. & Grenfell, B. T. (199 1). Clinical epidemiology of Bancroftian filariasis: effect of age and gender. Transactions of the Royal Society of Tropical Medicine and Hygiene, 85, 260-264. Rajasekariah, G. R., Parab, P. B., Chandrashekar, R., Deshpande, L. & Subrahmanyam, D. (199 1). Pattern of Wuchereria bancrofcimicrofilaraemia in young and adolescent school children in Bassein, India, an endemic area for lymphatic filariasis. Annals of Tropical Medicine and Parasitology, 8.5, 663-665. Suresh, S., Kumaraswami, V., Sureah, I., Rajesh, K., Suguna, G., Vijayasekaran, V., Ruckmani, A. & Rajamanickam, M. G. (1997). Ultrasonographic diagnosis of subclinical filariasis. Journal of Ultrasound Medicine, 16, 45-49. Received 26 May 1999

1999; accepted for publication

4 August