Our target population comprised patients from different parts of Kolkata admitted to the Infectious Diseases (I. D.) Hospital between November 2007 and October 2008 with diarrhoeal complaints. Non-diarrhoea cases were not included in this study. A total of 1103 samples were screened; they were enrolled throughout the year by systematic sampling by our surveillance system. An epidemiological survey of the study population was also conducted, including stratification by age and sex, nature of parasitic infestations, month-wise prevalence of different parasites and GIS mapping of the areas where parasites are prevalent. The aim of this systematic study was to reveal the current burden of common enteric parasitic infestations in and around Kolkata.
Study design
A laboratory-based surveillance system was introduced. The I. D. Hospital in Kolkata provides treatment to about 20,000 to 25,000 acute diarrhoea patients annually. Patients admitted to the hospital with diarrhoeal complaints were included in the study using a systematic sampling process: on two randomly selected days each week, every fifth patient with diarrhoea or dysentery but no associated complaints was enrolled. The system remained unbiased for sex and age of the patient at the time of selection. Faecal samples from all the enrolled patients were collected and analysed. A total of 19,500 patients were admitted to the I.D. Hospital during our study period; 1107 (5.6%) of these were enrolled by the system and 1103 fecal samples were processed for the study. The parasites were detected firstly by conventional microscopy, which is still considered the gold standard for any epidemiological study, but as this method has comparatively low sensitivity and has limitations in distinguishing among different parasites (e.g. Entamoeba dispar from Emtamoeba histolytica), we also used molecular detection techniques, ELISA and PCR, to increase the sensitivity and specificity of detection.
Sample preparation
For microscopic analysis the samples were concentrated using the method of Ridley [3], mixed with 2.5% potassium dichromate for preservation [4] and stored at 4°C. Three separate aliquots from each sample were stored with no preservative at -80°C pending ELISA and PCR studies.
Microscopic screening
Immediately they were received, the unpreserved samples were examined by microscopy. Three separate techniques were used to identify the parasites in the faecal samples: iodine wet mount staining for trophozoites and parasite cysts; modified Kinyoun's Acid fast staining for Cryptosporidium sp. [5]; and Trichrome staining for Giardia sp. and Entamoeba sp. [6].
Molecular Screening
Antigen-capture Enzyme Linked Immunosorbent Assay (ELISA) was performed on all the samples to detect the above-mentioned parasites using commercially available kits, i.e. GIARDIA II, E. HISTOLYTICA II and CRYPTOSPORIDIUM II (TECHLAB, USA). Parasite DNA was isolated using a Stool DNA Isolation Kit (QIAGEN) according to the manufacturer's protocol, then PCR was performed on all samples using the following parasite-specific primers:-
(a) Giardia lamblia
A beta-giardin gene fragment-specific primer (MAH433F 5'-CATAACGACGCCATCGCGGCTCTCAGGAA-3' as forward primer and MAH592R 5'-TTTGTGAGCGCTTCTGTCGTGGCAGCGCTAA-3' as reverse primer) with a PCR amplicon size of 218 bp was used [7].
(b) Entamoeba histolytica
The target for PCR amplification was SSU rRNA (EH1 5'-GTACAAAATGGCCAATTCATTCAATG-3' as forward primer and EH2 5'-ACTACCAACTGATTGATAGATCAG-3' as reverse primer); the amplicon size is 135 bp [8]. The primers used are highly specific for Entamoeba histolytica and differentiate it from the non-pathogenic Entamoeba dispar.
(c) Cryptosporidium sp
The primers used were those described by Xiao et al. (1999) with an amplicon size of 1325 bp (18 SF: 5'-TTCTAGAGCTAATACATGCG-3' as forward primer and 18 SR: 5'-CCCTAATCCTTCGAAACAGGA-3' as reverse primer); then nested PCR was performed with an amplicon size of 825 bp (5'-GAAGGGTTGTATTTATTAGATAAAG-3' as forward primer and 5'-AAGGAGTAAGGAACAACCTCCA-3' as reverse primer) [9].
Statistical analysis
The data from the above identification procedures and the epidemiological survey were entered into a pre-designed pro forma in the SQL server with an inbuilt entry validation checking facilitated program. Data were randomly matched and checked to establish consistency and validity. SPSS.14.0 was used for statistical analysis. In this study, positive cases in the inferential age group were explored for parasites (Giardia lamblia, Cryptosporidium sp and Entamoeba histolytica) by Multinomial Logistic Regression (MLR) [10, 11].
GIS analysis
A GIS map was constructed using the Choropleth Mapping Analysis System [12] for all patients enrolled by the surveillance system in order to establish the catchment areas of the parasites by evaluating their spatial distributions in Kolkata.