Seroepidemiology and serological follow up of anti leptospiral igg in children in southern vietnam

de Vriesa aDivision of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Center, Amsterdam, The Netherlands bDepartment of Microbiology, Cho Ray Hospital, 201 B Nguyen Ch

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Acta Tropica

j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / a c t a t r o p i c a

Seroepidemiology and serological follow-up of anti-leptospiral

IgG in children in Southern Vietnam

Khoa T.D Thaia,∗, Tran Thi Thanh Ngaa,b, Hoang Lan Phuonga,d, Phan Trong Giaoa,d, Le Quoc Hunga,d, Tran Quang Binhd, Nguyen Van Namc, Rudy A Hartskeerle, Peter J de Vriesa

aDivision of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Center, Amsterdam, The Netherlands

bDepartment of Microbiology, Cho Ray Hospital, 201 B Nguyen Chi Thanh, District 5, Ho Chi Minh City, Viet Nam

cBinh Thuan Provincial Malaria Center, 133 A Hai Thuong Lan Ong, Phan Thiet City, Viet Nam

dTropical Diseases Clinical Research Center, Cho Ray Hospital, 201 B Nguyen Chi Thanh, District 5, Ho Chi Minh City, Viet Nam

eDepartment of Biomedical Research, Leptospirosis Reference Centre, Koninklijk Instituut voor de Tropen, Amsterdam, The Netherlands

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

per-sistence 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

infec-tions, 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 2 years Data from this study indicate that IgG antibody responses against Leptospira may persist at least for 2 years in children without

manifesta-tions 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

1 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

investiga-tions 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).

al., 1973) MAT is more specific than ELISA and offers identifica-tion 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 pre-ferred technique in resource poor settings

The interpretation of serology is complicated by cross-reactivity between different serovars and the variable persistence of leptospi-ral antibodies (Levett, 2001) The time course of rising and declining antibodies varies between individuals and serovars and antibod-ies 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

Lep-tospira 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 0001-706X/$ – see front matter © 2008 Elsevier B.V All rights reserved.

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

chil-dren (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

haem-orrhagic 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 2 years

2 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, 5 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

pri-mary 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

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

labo-ratory 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

par-tially purified extract of Leptospira biflexa, which contains genus

specific epitopes for all Leptospira subtypes IgG-ELISA was

per-formed 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

manufac-turer 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 <5 U/ml were considered negative,

ODR > 5 U/ml and <9 U/ml were interpreted as probable previous

exposure to Leptospira and ODR IgG values ≥9 U/ml as certain past-exposure 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 2 years, the true annual incidence of infection was cal-culated as the proportion of seroconversion among sero-negative children in 2003, using the following formula:

dy

This can be rewritten as y(t) = y(0) exp(−t), in which y is the

preva-lence of sero-negative subjects and  is a measure of the force of infection

The annual incidence rate is thus, when t = 1 year,

3 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 9 years: 73; 10 years: 80; 11 years: 89; 12 years: 13; 13 years: 7) were tested 2 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).

3.3 Seroconversion of Leptospira IgG antibodies and annual incidence

Among the 211 children who tested negative in 2003, 22 (10.4%,

95% CI: 7.0–15.3) tested positive for IgG antibodies against

Lep-tospira in 2005 (Fig 1, left) Eighteen of these (8.5%) had an

intermediate ODR and four (1.9%) had a clearly positive ODR result,

classified as certainly being exposed to Leptospira over the last 23

months The decline rate of sero-negatives of 10.4% between 2003

and 2005 corresponds to an annual incidence of Leptospira

infec-tions of 5.6% (95% CI: 3.7–8.3) If only the four children with high

ODR values were taken into account, the incidence rate of Leptospira

infections would be 0.99% (95% CI: 0.39–2.52)

4 Discussion

This study shows a waxing and waning pattern of Leptospira IgG antibodies in healthy children in Southern Vietnam Leptospira IgG

persist for 2 years in 61% of the children; 10% of previously sero-negative children show seroconversion in 2 years

While the microscopic agglutination test (MAT) is the gold stan-dard, commercially available ELISAs are easier to perform, cheaper,

K.T.D Thai et al / Acta Tropica 106 (2008) 128–131

Fig 1 Leptospira IgG antibody responses of 262 children at follow-up evaluation, as determined by ELISA Antibody Leptospira IgG responses of 211 children who were

sero-negative (left) and 51 who were sero-positive in 2003 (right) The dotted line indicates the cut-off (≥5 U/ml, sero-positive; <5 U/ml, sero-sero-negative); horizontal bar, geometric

mean.

require less time and can detect antibodies against Leptospira

serovars that are not available for testing with MAT Commercially

available ELISA kits are thus very sensitive but may have

difficul-ties to discriminate between the different infecting strains in highly

endemic areas and may pick up antibodies against low pathogenic

serovars (Cole et al., 1973; Cumberland et al., 1999) This probably

also applies to Vietnam, where since long very virulent Leptospira

interrogans serovars were isolated in different places and among

different population groups (de Lajudie and Brygoo, 1953; Van et

al., 1998) In a previous study, we detected Leptospira IgM

antibod-ies in 17% of serum samples of patients with acute undifferentiated

fever in Binh Thuan province, Vietnam, but specific serovars could

not be confirmed with the MAT (unpublished data) The ELISA

prob-ably picks up antibodies against serovars that are not applied in the

MAT, for which known pathogenic serovars are used Based on these

findings, it is therefore very reasonable to presume that in Binh

Thuan, multiple Leptospira strains prevail, with a predominance of

less pathogenic strains, which cause mild disease

The time course of antibody production after the first infection

by Leptospira is variable, but in general, Leptospira IgG serum

anti-bodies become detectable by the end of the first week after onset

of disease and reach maximum titers by the third or fourth week

(Levett, 2001) Thereafter, patients commonly remain sero-positive

to IgG for variable length of time, depending on the immune status

and serovar (Cumberland et al., 2001; Everard and Bennett, 1990;

Lupidi et al., 1991; Plank and Dean, 2000; Romero et al., 2003; Silva

et al., 1995)

In serosurveys, declining antibodies cause underestimation of

the incidence of first infections The persistence of antibodies

frustrates the serological confirmation of acute leptospirosis in

individual patients Antibodies persist longer when antibody

con-centrations reach high levels during the acute infection However,

repeated exposure to leptospires may boost the immune response

without causing symptomatic infection and this may explain why

the population in areas with favourable conditions for

transmis-sion of leptospirosis, or subjects with a high occupational risk for contracting leptospirosis, maintain high titers of antibodies with-out suffering from acute disease (Johnson et al., 2004; Van et al.,

1998)

Persisting antibodies may protect against repeated infections or may even attenuate the severity (Sonrier et al., 2000; Branger et al.,

2001) In this study, the increase of Leptospira IgG concentrations in

those children who were already positive was relatively small This may be compatible with some boosting of the immune response by sub-clinical re-infections Persisting IgG antibodies may offer cross-reactive protection to different serovars (Adler and Faine, 1977; Bharti et al., 2003)

Seroconversion for Leptospira IgG was observed in 10.4% (22/211)

of previously sero-negative children of whom 1.9% (4/211) showed

a large increase of antibody concentrations, classified as certain

exposure to Leptospira These observations confirm earlier findings

that primary school children in developing countries are frequently exposed to leptospires (Berman et al., 1973a; Johnson et al., 2004; Laras et al., 2002) Interestingly, also in this group of seroconvert-ers, clinical manifestations of leptospirosis in the previous 2 years

were not reported We calculated the total annual incidence of

Lep-tospira infections (0.99%) on the basis the observed seroconversion

rate, probably the result of sub-clinical or asymptomatic infections This is in concordance to the estimated annual incidence of our first serosurvey, which was 1.5% (Thai et al., 2006) Similarly, in the gen-eral population of Binh Thuan, severe leptospirosis is extremely rare despite a high seroprevalence of antibodies (Wagenaar et al., 2004) This also points at frequent sub-clinical and asymptomatic infec-tions, even though the mechanisms of such infections are poorly understood (Ashford et al., 2000) With this repeated seropreva-lence study among the same children over a period of 2 years, we uncovered the dynamics of waxing and waning antibody concen-trations, which is an indication of the true incidence of mainly

sub-clinical or asymptomatic Leptospira infections in the general

population

K.T.D Thai et al / Acta Tropica 106 (2008) 128–131

This study did not study adults and thus a higher incidence of

symptomatic leptospirosis in adults was not excluded Age

depen-dent immunological responses with protection by boosting by

repeated infections, or inversely, with immune enhancement by

repeated infections, similar to the concept of antibody dependant

immune enhancement in dengue, are not inconceivable (Halstead,

1982; Sonrier et al., 2000)

In conclusion, repeated cross-sectional school surveys

uncov-ered the true incidence of Leptospira infections and a waxing and

waning pattern of IgG antibody concentrations in healthy children

High titers of Leptospira IgG may be caused by repeated exposure

to non- or less-pathogenic Leptospira serovars and may persist for

more than 23 months in healthy children As a consequence the

positive predictive value of a single positive ELISA result in subject

from an endemic area is very limited

Acknowledgements

This study was carried out with the support of the Netherlands

Foundation for the Advancement of Tropical Research (WOTRO)

Authors acknowledge Marga Goris and Adrian van de Ven for their

laboratory assistance and technical advises with the ELISA We

thank the Health Posts workers in Ham Kiem and Ham Hiep for

their contribution to this study Thanks also go to co-workers of the

Binh Thuan Provincial Malaria Center

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