Acta Tropica 106 (2008) 128–131 Contents lists available at ScienceDirect Acta Tropica journal homepage: www.elsevier.com/locate/actatropica Seroepidemiology and serological follow-up of anti-leptospiral IgG in children in Southern Vietnam Khoa T.D Thai a,∗ , Tran Thi Thanh Nga a,b , Hoang Lan Phuong a,d , Phan Trong Giao a,d , Le Quoc Hung a,d , Tran Quang Binh d , Nguyen Van Nam c , Rudy A Hartskeerl e , Peter J de Vries a a Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Center, Amsterdam, The Netherlands Department of Microbiology, Cho Ray Hospital, 201 B Nguyen Chi Thanh, District 5, Ho Chi Minh City, Viet Nam Binh Thuan Provincial Malaria Center, 133 A Hai Thuong Lan Ong, Phan Thiet City, Viet Nam d Tropical Diseases Clinical Research Center, Cho Ray Hospital, 201 B Nguyen Chi Thanh, District 5, Ho Chi Minh City, Viet Nam e Department of Biomedical Research, Leptospirosis Reference Centre, Koninklijk Instituut voor de Tropen, Amsterdam, The Netherlands b c a r t i c l e i n f o Article history: Received 12 July 2007 Received in revised form 18 February 2008 Accepted 25 February 2008 Available online 29 February 2008 Keywords: Leptospirosis Seroepidemiologic studies Follow-up studies IgG ELISA (enzyme-linked immunosorbent assay) Vietnam a b s t r a c t A follow-up study was conducted with 23 months interval to investigate the seroepidemiology and persistence of Leptospira IgG antibodies among healthy children in Binh Thuan province, Southern Vietnam Sera from 262 children (7–13 years of age) were collected and analysed with a commercially available enzyme-linked immunosorbent assay (ELISA) for Leptospira IgG Seroconversion was observed in 10.4% (22 of 211, 95% CI: 5.6–26.7) of the children, of whom 18 (8.5%) had probably and four (1.9%) had certainly been exposed to Leptospira Based on the reduction of sero-negatives of 1.9% among children who have been certainly exposed, the annual seroconversion rate, a measure of the incidence rate of Leptospira infections, corresponds to 0.99% (95% CI: 0.39–2.52) In 61% (31 of 51, 95% CI: 47.1–73.0) of the children with past-infection, Leptospira IgG antibodies remain detectable after years Data from this study indicate that IgG antibody responses against Leptospira may persist at least for years in children without manifestations of leptospirosis Results of study uncover the true incidence of leptospirosis infection, the dynamics of waxing and waning antibody concentrations and points at a larger burden of clinically non-significant Leptospira infections in Southern Vietnam This also indicates background reactivity for serological testing and thus serological result of a single serum sample must be carefully interpreted © 2008 Elsevier B.V All rights reserved Introduction Human leptospirosis has a worldwide distribution and in the tropical areas it is probably the most common bacterial zoonotic disease (Vinetz, 2001) Leptospirosis infection results from direct or indirect exposure to urine of animals infected with the spirochetes of the pathogenic serovars of the genus Leptospira The spectrum of clinical presentation of leptospirosis in humans is wide The clinical diagnosis is therefore difficult, particularly in tropical countries where other febrile diseases such as malaria, dengue and influenza may mimic leptospirosis (Levett, 2001) The diagnosis of leptospirosis is confirmed by laboratory investigations Enzyme-linked immunosorbent assay (ELISA) is commonly used for diagnosis, while the microscopic agglutination test (MAT) remains the reference method for serological diagnosis (Cole et ∗ Corresponding author at: Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Center, F4-217, PO Box 22700, 1100 DE Amsterdam, The Netherlands Tel.: +31 20 5664380; fax: +31 20 6972286 E-mail address: t.d.thai@amc.uva.nl (K.T.D Thai) 0001-706X/$ – see front matter © 2008 Elsevier B.V All rights reserved doi:10.1016/j.actatropica.2008.02.005 al., 1973) MAT is more specific than ELISA and offers identification of infecting serovars, however, MAT is a time-consuming and expensive technique, which is usually available only in dedicated reference laboratories Therefore, ELISA has become a much preferred technique in resource poor settings The interpretation of serology is complicated by cross-reactivity between different serovars and the variable persistence of leptospiral antibodies (Levett, 2001) The time course of rising and declining antibodies varies between individuals and serovars and antibodies remain detectable for variable length of time after recovery (Cumberland et al., 2001; Everard and Bennett, 1990; Lupidi et al., 1991; Romero et al., 2003; Silva et al., 1995) Most studies on this aspect followed Leptospira antibodies at multiple-time points in adults with acute leptospirosis However, the persistence of Leptospira antibodies in healthy individuals or children has never been quantified Vietnam is known to be endemic for leptospirosis since the 1930s (Berman et al., 1973b; de Lajudie and Brygoo, 1953; Allen et al., 1968) The southern province Binh Thuan seems ideal for the transmission of Leptospira because of its hot and humid climate, dense human population and rural settlement with agricultural K.T.D Thai et al / Acta Tropica 106 (2008) 128–131 activities In previous studies, we demonstrated a high prevalence of antibodies against leptospires in healthy adults as well as in children (Thai et al., 2006; Wagenaar et al., 2004) The seroprevalence of Leptospira IgG among children was 12.8% (Thai et al., 2006) A striking discrepancy is, however, that leptospirosis is diagnosed seldomly and that severe leptospirosis, Weil’s syndrome or haemorrhagic pneumonitis is very rare in this region In order to study the dynamics of serum Leptospira IgG antibodies and to study if there is an increase of sero-positivity rate, we followed up healthy children over a period of years Material and methods 2.1 Study site and population The study site was described in details previously (Thai et al., 2005) Briefly, Binh Thuan province (1,140,429 inhabitants in 2004), located on the south-eastern coast of Vietnam, 150 km northeast of Ho Chi Minh City, has a tropical monsoon climate with a average temperature of 27 ◦ C The rainy season lasts from May to October and the annual rainfall is 1152 mm The mean economic activity consists of agriculture, such as cultivation of rice and fruit and small-scale cattle breeding We conducted two serosurveys in two communes, Ham Kiem and Ham Hiep, km west and 15 km south of the provincial capital Phan Thiet The first survey in 2003 included all children >7 years of age at two primary schools in the two communes (Thai et al., 2006) A commercially available ELISA was performed for the presence of Leptospira IgG serum antibodies All children who tested positive for Leptospira IgG serum antibodies and randomly selected sample of children who tested negative in 2003, were retested in 2005 2.2 Ethical consideration In cooperation with the People’s Committee of the village, the local health post staff and schoolteachers, all pupils of the primary school and their parents were informed about the study and consent was obtained from all The study was carried out under a protocol that was approved by the provincial Health services and the community health centers of Ham Kiem and Ham Hiep and the Scientific Committee of Cho Ray Hospital, Ho Chi Minh City 129 exposure to Leptospira and ODR IgG values ≥9 U/ml as certain pastexposure to Leptospira 2.4 Statistical analysis Statistical analysis was performed using statistical software (SPSS 11.5, SPSS Inc., Chicago, IL, USA) With the assumption that leptospirosis is endemic in Southern Vietnam, with a constant force of infection and seroconversion rate, and that antibodies persist for longer than years, the true annual incidence of infection was calculated as the proportion of seroconversion among sero-negative children in 2003, using the following formula: dy = −y dt (1) This can be rewritten as y(t) = y(0) exp(−t), in which y is the prevalence of sero-negative subjects and is a measure of the force of infection The annual incidence rate is thus, when t = year, − exp(−) (2) Results 3.1 Sample population In 2003, 123 children (123/961; 12.8%) were found to be positive for Leptospira IgG, indicating previous exposure to Leptospira In 51 of these children (57% boys) who were still at school, sera were tested again in 2005 Among 838 children who tested negative in 2003, 494 were still at primary school and 211 of these children (53.1% boys) were randomly selected and also re-tested in 2005 In total, 262 children (age groups years: 73; 10 years: 80; 11 years: 89; 12 years: 13; 13 years: 7) were tested times at different time-points 3.2 Persistence of Leptospira IgG antibodies At follow-up, 31 of the 51 (61%) children who tested positive in 2003 still had IgG reactivity with Leptospira The geometric mean of IgG response was 7.6 U/ml in 2003; 23 months later this had declined 1.3-fold, to 5.7 U/ml (Fig 1, right) 2.3 Serum handling and ELISA Serum was collected with finger puncture as described before (Thai et al., 2006) Serum samples were stored at −20 ◦ C locally All samples were transported on ice in a cooling box to the laboratory for virology in Cho Ray Hospital, Ho Chi Minh City, where they were stored at −70 ◦ C IgG antibodies against Leptospira were measured using a commercially available monowell ELISA testkit for IgG (Virion\Serion GmbH, Wurzburg, Germany) This test is based on crude antigens from an isolated, concentrated and partially purified extract of Leptospira biflexa, which contains genus specific epitopes for all Leptospira subtypes IgG-ELISA was performed exactly according to the manufacturer’s instructions as described before (Thai et al., 2006) A selection of 10 samples was also tested for validation of quality at the laboratory of the manufacturer Optical density (OD) values were measured at 405 nm, with 630 nm as a reference, with a conventional automatic absorbance reader (EvolisTM , Bio-Rad Laboratories, Inc., Hercules, CA, USA) Results were expressed as the ratio between the sample OD-value and the OD-value of a calibration sample, enclosed in the ELISA kit (ODR), both after subtraction of the OD-value of an enclosed blank specimen ODR IgG values U/ml and