Leptospirosis has been recognized as an emerging global public health problem because of its increasing incidence mainly in developing countries; it is partly due to natural calamities such as cyclone and floods (P. Vijayachari, 2008). It remains a major endemic environmental disease in most countries in the Asia Pacific Region in which annual incidence is estimated at 10-100 per 100,000 pop and a neglected public health problem for most of the countries.
Most of infections are mild or subclinical ranging from undifferentiated fever to influenza like symptoms with or without jaundice or hemorrhage. Only a small proportion develops various complications which include one or multiple organ failures. Of these, the case-fatality ratio is estimated at ~40% or more. The wide spectrum of clinical signs and symptoms associated with probable, acute, leptospiral infections contributes to the potential of significant underreporting.
The ability of all countries in the region to accurately report and monitor leptospirosis hinges strongly on their respective capacity to provide accurate and reliable diagnostic and reporting services. Whereas effective surveillance systems with appropriate laboratory support exist in developed countries in the Asia Pacific region, they are generally lacking in the disease-endemic, developing countries. Even when present, the systems may not meet acceptable international quality assurance standards. Despite the existence of the WHO case definition for leptospirosis, the infrequent use of confirmatory laboratory testing and the inability to link clinical, epidemiologic, and laboratory data have hindered system utility
Cambodia and neighboring countries have been considered endemic for leptospirosis (Laras 2002) although surveillance does not exist and incidence data are lacking. Recent seroprevalence studies conducted in flood-prone rural areas in Laos (Laras 2002) and in southern Vietnamese children (Kawaguchi 2008) showed an overall seroprevalence of 23.9% and 12.8%, respectively.
To make a robust estimate of the actual incidence of leptospirosis, seasonality and the prevalence of antibodies against Leptospira sp by age group in persons< 20 years of age living in rural and urban areas of Cambodia during 2006 – 2009. We will use archived anonymous, unlinked serum specimens collected for a dengue surveillance study.
The protocol has been approved by National Ethic Committee for Health Research on April 8th 2011.
The study conducted anonymous, unlinked testing for antibodies to Leptospira spp and PCR in on paired sera of Cambodian patients with fever illnesses. These patients were part of a cohort of villagers <20 years of age residing in urban and rural areas of Kampong Cham province, Cambodia. Kampong Cham is the most populated province of Cambodia with ~1.7 million people and a capital city of ~90,000 people.
Serum samples derived from population-based active surveillance of fever illnesses. During 2006 – 2009, active, community-based surveillance for febrile illnesses was conducted in a convenience sample of 32 villages and 10 urban areas in four districts of Kampong Cham province. Not all study villages/urban areas were included for the entire period; 5 were included for 3 years, 13 for 2 years and the remainder for only one year.
Active surveillance was mainly conducted during the rainy season. A total 10,086, 7,673 and 7169 (study midpoints) individuals <20 years of age were followed-up during June 1st-December 31st, 2007; April 1st to December 31st, 2008, and Jan. 1st to Dec. 31st, 2009 respectively. As described previously (Vong PLoS ntd 2010), a census was updated in each village to identify eligible families, a village team (VT) visited families once a week to identify persons with fever (axillary temperature >37.5°C) or history of fever in the previous seven days. Febrile persons were visited by an investigation team (IT) to obtain informed consent, and assent where indicated, conduct an interview with a standardized case report form (CRF) and collect blood samples. Febrile persons were also visited 14 – 21 days after disease onset to collect information related to type of care sought and collect a convalescent blood specimen. Serum specimens were tested for infection by various pathogens using molecular, serological and virological methods as described previously (Vong PLoS Negl Trop Dis 2010).
The number of refusals to participate in the study was 0.6% per year on average (range 0.4-0.9%). Few participants (0.9% on average; range 0.8-1.1%) moved outside the surveillance area.
Selecting the sample for Leptospira testing
We considered that to maximize success, urban villages would be excluded from the study. Only rural villages – which represent the majority of the population – were targeted by the serological survey.
In order to get as accurate a representation of the epidemiological situation as possible, the two aspects of the study were conducted on different sample frames from which samples were drawn using random sampling. In both cases, the sample tested was structured to match the 0 – 19 age structure of the population in Kampong Cham documented for 2008 (see Annex 1).
Step 1: determine structure of general population in Kampong Cham, 2008
Step 2: determine age structure of population for the 2007 – 2009 studies (N = 8295)
Step 3: Apply general population structure to randomized sample, overrepresenting higher age groups
1. Incidence study
This study of IgM seroconversion, complemented by PCR, aimed at documenting incidence in the less than 20 year olds. Samples were drawn from those taken at the end of each rainy season in girls and boys of rural villages and aged less than 20 years (samples available from the Dengue study). Also to maximize chances of leptospira positivity, tested samples were those which were negative for dengue, JEV, influenza A and B, RSV and HMPV as the fevers observed were highly likely to be due to those infections in samples positive for those viruses.
Among the 24 928 persons aged less than 20 followed up between 2007 and 2009, 3001, 2539 and 2755 were sampled for fever in the years 2007, 2008 and 2009 respectively, totaling 8,295 samples held at the Institut Pasteur in Cambodia. A total of 1133 (13.65%) of these samples were positive for dengue, JEV, influenza, RSV or HMPV and were excluded from the sampling frame for the incidence survey. The serostudy bore on a randomly selected subset of 2359 (33%) among the remaining 7162 subjects. The percentage of positive results in the representative subset were calculated as well as their 95% probability confidence intervals using the binomial exact method (Epi-info). Percentages of positivity were extrapolated to the total number of samples and the study period converted to person-months to compute incidence rates per 1000 persons-months and confidence intervals under a Poisson distribution. Confidence intervals were computed for alpha = 0.05 using an Excel worksheet.
2. Prevalence study
This study of IgG aims at documenting prevalence in the < 20 year olds. Samples were drawn from those taken at any time during the study to smooth out the effect of any single year with particular meteorological phenomena such as drought or floods. They included girls and boys of rural villages of all ages but <20 yo (samples available from dengue study), whatever their status for incident dengue, JEV, HMPV, VRS or influenza. Based on sample size calculations, a randomized sample of 398 subjects was generated from the 8295 samples held at the Institut Pasteur du Cambodge, notwithstanding the results for other incident infections.
For the incidence study, a case of acute leptospirosis infection was defined as a patient with febrile illness who had serological evidence of seroconversion on paired sera. Seroconversion is defined by negative IgM antibodies on the first sample and positive IgM antibodies on the second sample. For the prevalence study, a case of past leptospirosis (symptomatic or otherwise) was defined as a patient with detectable specific IgG.
Tests and testing algorithm
Firstly, for the incidence study sera collected from clinical patients with febrile non-dengue clinical symptoms were screened for anti-Leptospira IgM antibodies by enzyme-linked immunosorbent assay (ELISA) using PanBio Leptospira IgM ELISA kit (Panbio Ltd, Brisbane, Australia) at the IPC on the second sample (D-14-21 days of fever onset). Secondly, to save costs, we only tested patients with positive IgM on the second sample for IgM antibodies on the first (acute phase) sample (D0-7 days of fever onset). All cases for which seroconversion was observed between the first and second sample were tested by real-time polymerase chain reaction (PCR) assay using a TaqMan probe targeting lipL32, which is present only in pathogenic Leptospira spp (Stoddard, Diagn Microbiol Infect Dis 2009) to detect the bacteria in blood.
For the prevalence study, sera samples were tested for anti-Leptospira IgG antibodies by enzyme-linked immunosorbent assay (ELISA) using Serion ELISA classic Leptospira IgG (Virion/Serion GmbH, Wurzburg, Germany).
Of the 2,359 persons randomly selected for IgM testing on the second sample, a total of 99 (4.20%) returned positive while they were found seronegative on an early sample. There are therefore 99 seroconversions documented using the IgM assay.
The denominator, however, should be the total number of persons susceptible at the start of the study. Therefore it should be 7150 plus the 1133 cases who were found positive for other pathogens but who were theoretically susceptible to leptospirosis at baseline. That number is 8,295. If we extrapolate the 99 (4.25%) cases found among the 2,359 randomized subjects to the total sample frame of 8295 that means 0.0420 x 8295 ≈ 349 positive cases in 8,295 persons tested for febrile illness during the three successive studies.
As these 8295 persons tested were the symptomatic cases among a larger group of persons followed, the global estimated attack rate for symptomatic infection by pathogenic leptospires among all persons aged <20 years and followed up is therefore around 349/24928 = 1.40% [CI95%: 1.26% – 1.55%] for the cumulated study (exposure) periods totaling 28 months.
Table 1: Preliminary IgM results (testing and extrapolated) for leptospira IgM seroconversion in symptomatic <20 year olds, 2007-2009 IPC studies, rural Kampong Cham villages, Cambodia.
0-20 y.o. exposed (followed-up) during study (N)
Fever cases sampled during study
Samples randomized and tested for leptospirosis
Positive for lepto.
(% of C)
to total sampled (D% x B)
Months exposed that year or total
p. 100 [95%CI] in A for each study period and total
Incidence rate p.1000 person-months in A [95%CI] for each study and total
[0.98 – 1.48%]
[1.10 – 1.65]
[1.29 – 1.88%]
[1.08 – 1.57]
[1.26 – 1.55%]
[1.39 – 1.72]
The estimated attack rate for symptomatic leptospirosis in Kampong Cham villages during 2007-2009 was 1.4% (varying from 1.21% to 1.56%, depending on the year). The incidence rate for symptomatic leptospirosis was 1.55 per 1000 persons-months exposure, varying from 1.30° p-m to 2.04 ° p-m.
Of the 99 sera samples tested for PCR, 2 cases were positive. This low number can be explained by the possible pre-treatment with antibiotics prior to the blood sample, self-medication being common in Cambodia. Serum is not also the best sample to use for PCR: plasma would have been more suitable.
A previous study with Cambodian patients hospitalized presenting clinical symptoms compatible with leptospirosis tested for serology using the Panbio IgM Elisa kit and the micro-agglutination reference test (MAT) showed that 50% of the samples IgM positive had agglutinins against pathogenic serovars of Leptospira (Berlioz-Arthaud, Bull. Soc. Pathol. Exot 2010). We can therefore conclude that there may in fact be not 99 but approximately 50 true positives for symptomatic, incident leptospirosis. Attack and incidence rates may be halved. This will be confirmed during subsequent testing using the reference technique.
The number of IgG carriers is unexpectedly low. The prevalence study in a sample of 398 persons drawn from the 24,928 found 9 IgG seropositives for a seroprevalence of 2.3% [CI95%: 1.1 – 4.4%]. Among these, 8 were samples taken in 2007 (4 were aged 15 or above and 3 were aged 0 – 4).
Preliminary elements for discussion
The estimated figures for the attack rate and the incidence per 1000 person-months of symptomatic leptospirosis in Kampong Cham villages, 2007-2009 remain in the same order of magnitude.
There was, on the other hand, a very marked observed discrepancy between the IgG seroprevalence and the estimated attack and incidence rates. There are several possible explanations to this
1/ the nature of the anti-Leptospira antibody response : detection of IgG antibodies is variable. Anti-Leptospira IgG may sometimes not be detected at all, or be detectable for only relatively short periods of time, but may sometimes persist for several years;
2/ the technique used to detect IgGs lacked of sensitivity and thus underestimated the true positives;
3/ the sample was not optimally randomly chosen and overrepresented seronegatives;
4/ the sample and seroprevalence are correct but the seroprevalence was low due to outbreaks being geographically very limited;
5/ the sample and seroprevalence are correct, but there are actually only 2-4 years of their lifetime during which people get infected by Leptospira;
6/ the sample and seroprevalence are correct, but several infections with different serovars occur in a limited number of persons belonging to a high-risk group;
7/ the sample and seroprevalence are correct, but an unusually high number of cases occurred in 2007-2009.
Published sources will be sought in the literature to determine the seroprevalence, attack and/or incidence rates documented in the region in recent years. Further testing will also be performed in subjects with IgM already present at fever onset to determine the prevalence in IgG. Detailed biostatistical analysis will be undertaken linking serological data with socio-demographic data from these studies to compare years and age groups and geolocalize seroconversions. Preliminary incidence estimates will be further refined in subsequent analysis to compute incidence rates for comparable age groups and month of each year, taking into account rainfall data as shown in Annex.
Our incidence data emphasize the importance of leptospirosis among patients with fever residing in Kampong Cham province during the years 2007-2009. There is a need for Cambodia to have a reference laboratory capable of performing the gold standard serological test (microscopic agglutination test, MAT) which has high specificity and can give important information about which serogroups are present within a population.
Figure 1: Schematic representation of study populations and randomized samples standardized for sex and age on which the leptospirosis study was conducted.
Figure 2: Monthly average for rainfall measured in several points of Kampong Cham province, 2007-2009 (Source: Agus Rachmat, Namru-2).
Figure 3: Laboratory Results for incidence study
Figure 4: Laboratory Results for prevalence study