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Strongyloidiasis in a high risk community of Dhaka, Bangladesh
Transactions of the Royal Society of Tropical Medicine and Hygiene 106 (2012) 756–762
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Strongyloidiasis in a high risk community of Dhaka, Bangladesh Yasmin Sultana a,b,∗ , Gwendolyn L. Gilbert a,b , Be-Nazir Ahmed c , Rogan Lee a,b a b c
Centre for Infectious Diseases and Microbiology Laboratory Services, ICPMR, Westmead Hospital, Westmead, NSW, Australia Discipline of Medicine, Sydney Medical School, University of Sydney, NSW, Australia Institute of Epidemiology Disease Control and Research, Mohakhali, Dhaka, Bangladesh
a r t i c l e
i n f o
Article history: Received 15 May 2012 Received in revised form 29 August 2012 Accepted 29 August 2012 Available online 22 October 2012 Keywords: Strongyloides stercoralis Slum Seroprevalence Copro-prevalence
a b s t r a c t Residents of a slum community of Dhaka city, Bangladesh were tested by serological and faecal examination for evidence of Strongyloides stercoralis infection. In stool specimens from a total of 147 participants Strongyloides larvae were found in 34 (23.1%) by Harada-Mori culture, 15 (10.2%) by agar plate culture (APC) and one (0.7%) by microscopy. Strongyloides IgG, IgG1 and IgG4 antibodies were found in 90 (61.2%), 46 (31.3%) and 53 (36.1%) of participants, respectively. A positive correlation was observed between total IgG levels and the presence of isotypes IgG1 and IgG4 (p<0.001). Six sera (4.0%) reacted to the recombinant filaria antigen Bm 14, three of which were Strongyloides IgG positive. This indicates either there is cross reactivity or some participants are co-infected with lymphatic filariasis. No correlations were found between positive serology and Strongyloides infection in stool, socio- demographic factors or domestic hygienic practices. However, positive stool cultures showed significant associations with irregular nail trimming, walking bare-foot and irregular hand washing after defecation (p<0.05). Other enteric parasites were detected in stools of some participants but their presence showed no correlation with S. stercoralis infection or socio demographic factors. This study confirms that squatters in this slum community in Dhaka have a high prevalence of S. stercoralis infection identified both by serological and coprological methods. © 2012 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction Strongyloides stercoralis is a soil-transmitted intestinal nematode estimated to infect 30–100 million people globally.1 Those individuals co-infected with human lymphotrophic virus-1 (HTLV-1) are more likely to develop severe forms of strongyloidiasis and be less responsive to treatment.2 The mortality rate of disseminated infections is reported to be as high as 87%.3 Strongyloidiasis is now listed as one of the so-called neglected tropical diseases,4 but is poorly described due to lack of epidemiological information.5 Confirmation of Strongyloides infection by coprological examination is difficult because of irregular
∗ Corresponding author. Tel.: +61 2 98457662; fax: +61 2 98938659. E-mail address: [email protected] (Y. Sultana).
excretion of the parasite especially in chronic cases; prevalence of infection is thus underestimated.6,7 Definitive diagnosis of strongyloidiasis is by detection of larvae in stool. Direct smears, formal-ether concentration (FEC), Baermann-Moraes culture, spontaneous sedimentation, Harada-Mori culture and agar plate culture (APC) are common coprological methods used to detect S. stercoralis in stools.8 Agar plate culture has been shown to be more sensitive than direct smear, FEC, Baermann-Moraes technique or filter paper techniques.9,10 The detection of serum antibodies may facilitate the diagnosis and is more sensitive than coprodiagnosis but its specificity can be low. False positive antibody reactions can arise either due to recent past infection or cross-reactions with other helminthic infection such as filariasis.11,12 A previous study has shown that reactive IgG1 and IgG4 to Strongyloides antigen had less cross reaction from other
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helminth infections compared to total IgG, suggesting that these isotypes are more specific than reactive total IgG.13 Specific coproantigen detection and molecular assays have been shown to be promising alternatives with high specificity and sensitivity,14–16 but may not be available in resource poor countries. Countries with poor hygiene and sanitation are found to have high strongyloidiasis prevalence.17,18 Strongyloidiasis is endemic in Africa, the West Indies, Southeast Asia, South America, Pakistan and Bangladesh.6 Coprological and recent serological studies, in slum areas of Dhaka, confirmed its continued existence in Bangladesh.19,20 The aim of the present study, conducted in a slum community of Dhaka, was to ascertain the prevalence of S. stercoralis infection based on faecal examination and serology. Other objectives of this study were to identify risk factors for strongyloidiasis including socio-demographic and household factors and HTLV-I and II co-infection. 2. Materials and methods 2.1. Study group The study was conducted in a slum located at Adabar of Mohammadpur, a western suburb of Dhaka city, Bangladesh. This site was demolished six months after completion of sample collection. During November 2009 to January 2010, a house-to-house survey of the community was performed. A total of 147 individuals who participated in this study provided both serum and stool samples. We also collected socio-demographic information using a questionnaire. As most of the participants were illiterate, consent was obtained verbally from each respondent and the questionnaire was completed during the same visit. At the end of this study participants who were infected with strongyloidiasis were offered albendazole (the only drug registered for treatment of this infection in Bangladesh) 400 mg/day, on two consecutive days. The study was approved by the National Research Ethics Committee, Bangladesh Medical Research Council (BMRC; Dhaka, Bangladesh). 2.2. Stool examinations 2.2.1. Direct smear and formal ether concentration The faecal sample collected from each participant was examined microscopically by direct smear within 6 hours of collection. An FEC using the MINI PARASEP SF (Laboratory Diagnostics, Taren Point, NSW, Australia) was prepared from each stool sample. Iron haematoxylin (Oxoid, Adelaide, Australia) stained smears from these faecal concentrates were examined for the presence of Strongyloides and other enteric parasites. 2.2.2. Agar plate culture and Harada-Mori culture Both APC (single) and Harada-Mori methods (triplicate) were used to culture S. stercoralis.8 Two grams of stool was used for each culture replicate; thus a total of 6 g of stool was used in Harada-Mori methods and only 2 g in APC culture. The plates were sealed with adhesive tape to prevent drying of the agar. Cultures were incubated at
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25–28 ◦ C for a week and examined daily. Any specimen, in which at least one larva was identified by either direct microscopy or culture, was regarded as positive. Positive samples were confirmed by microscopic examination of Strongyloides larva for morphological structures. 2.3. Strongyloides ELISA Sera were separated from freshly collected blood and stored at −20 ◦ C prior to testing. IgG against Strongyloides antigen was detected by an indirect ELISA using soluble antigen from Strongyloides ratti third stage larvae (L3) as previously described.20 All the sera with detectable Strongyloides IgG were screened for isotypes IgG1 and IgG4. 2.4. Other serological studies 2.4.1. Detection of filarial antibody All sera were tested for antibody to Wuchereria bancrofti using an indirect ELISA for the detection of Bm14 specific IgG4 antibody (Cellabs Pty Ltd, Brookvale, NSW, Australia). Results were interpreted according to the manufacturer’s instructions. 2.4.2. Detection of HTLV-I and II antibody Presence of HTLV-I and II antibody in sera was measured by the Murex HTLV I+II assay (Abbott Australasia Pty Ltd, Botany, NSW, Australia) and results were interpreted according to the manufacturer’s instructions. 2.5. Risk factors associated with strongyloidiasis Participants who provided blood and stool samples also completed a questionnaire. Data collected included: gender, age, level of education, occupation, average monthly income, number of family members and type of toilet used. Other data collected were aspects of personal hygiene such as wearing shoes, trimming nails regularly, washing hands with soap after defecation and history of diarrhoea in the past six months. These factors were then evaluated against positive serology and positive culture of each specimen. 2.6. Statistical analysis Statistical package, SPSS version 17 (SPSS Inc., Chicago, IL, USA) was used: to compare the observed prevalence of infection as determined by serology and stool examination; to determine significant risk factors from the questionnaire; to show associations, if any, of each with positive serology and stool examination; and to identify association of strongyloidiasis with other enteric parasitic infestations using 2 statistics. Levels (as shown by OD values) of IgG, IgG1 and IgG4 antibodies to S. ratti were compared by Pearson’s correlation statistics. Logistic regression was used to compute the risk of infection with age, entered as a continuous variable. Values of p<0.05 were considered statistically significant.
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3. Results 3.1. Stool examination (direct smear, formalin ether concentration and culture) Strongyloides stercoralis larvae were detected by microscopy in only one specimen which failed to show larvae in culture. No larvae were found by microscopic examination of FEC. Larvae were detected in 34 (23.1%) stool samples using the Harada Mori culture method but in only 15 (10.2%) of these by APC. There was no association between detection of S. stercoralis larvae in stool with either age or sex. Other enteric parasites found in formal-ether concentrations of stool sample are listed in Table 1. Ascaris lumbricoides and Trichuris trichiura each were found in more than one third of samples and were both present in 28 (19%) stools. No association was observed between the presence of Strongyloides larvae and the other enteric parasites including Ascaris and Trichuris. However, the presence of A. lumbricoides and T. trichiura together in each stool sample was statistically significant (p<0.05). 3.2. Strongyloides serology (ELISA) Total Strongyloides IgG was found in 90 of 147 (61.2%) sera in the studied group. IgG1, IgG4 and both subclasses were detected in 46/90 (51.1%), 53/90 (58.8%) and 34/90 (37.8%) of the total IgG positive sera respectively. There was a significant positive correlation between the level of total IgG and both isotypes by Pearson correlation statistics (p<0.001). More females than males were serologically positive, but the difference was not statistically significant. In addition, age showed no association with seropositive cases. Table 1 Other enteric parasites identified in stool samples of 147 participants examined for the presence of Strongyloides stercoralis Organism
Number detected (%) n=147
Number co-infected with S. stercoralis (%) n=34
Ascaris lumbricoides Trichuris trichura Entamoeba coli Entamoeba histolytica Idomoeba butchilli Enterobius vermicularis Endolimax nana Blastocystis hominis Giardia lamblia
57 (38.8) 52 (35.4) 8 (5.4) 4 (2.7) 11 (7.5) 2 (1.4) 2 (1.4) 2 (1.4) 1 (0.7)
16 (47.1) 15 (44.1) 3 (8.8) 2 (5.9) 4 (11.8) 0 1 (2.9) 1 (2.9) 0
Table 2 Comparison of IgG reactivity to Strongyloides antigen and detection of larvae in stools Serology, IgG
Culture, APC/Harada-Mori Positive (%)
Negative (%)
Total
Positive Negative Total
24 (16.3) 10 (6.8) 34
66 (44.9) 47 (32.0) 113
90 57 147
APC: agar plate culture.
Of the 147 participants, 24 (16.3%) had both Strongyloides antibodies and Strongyloides larvae in their stool (Table 2). Sixty six (44.9%) had Strongyloides antibody but no larvae detected in their stools and 10 (6.8%) had larvae in their stools but no detectable antibody. No significant correlation was found between participants with reactive total IgG and presence of larvae in their stools. Of the 34 copropositive respondents, 17 (50%), 14 (41%) and 12 (35%) had detectable IgG1, IgG4 and both isotypes, respectively (Table 3). A significant association was observed between positive culture and IgG1 (p<0.05). Positive Strongyloides IgG had no correlation to presences of other faecal helminths found in their respective faecal specimen. 3.3. Risk factors associated with positive serology and Strongyloides stercoralis positive stools The socio-demographic information collected from each participant regarding education level, occupation and other household factors showed no correlation with positive serology (Table 4). However, a number of personal hygiene practices (namely being bare-foot, not washing hands after defecation and irregular nail trimming) were associated with a greater risk of being infected by S. stercoralis identified by stool culture (Table 5). 3.4. Other serological results Six (4.1%) of 147 sera showed positive results for IgG4 antibody against recombinant antigen (Bm 14) for lymphatic filariasis; of these, three had low positive reactions and were also seropositive for Strongyloides total IgG and IgG isotypes. All six individuals with reactive IgG4 to lymphatic filariasis had no Strongyloides larvae detected in their faecal specimens. Two individuals from the studied group had detectable antibodies to HTLV-I and II. No larvae were found in their respective faecal specimens. They had low Strongyloides IgG antibodies.
Table 3 Comparison of participant’s reactivity IgG1 and IgG4 to Strongyloides antigen and detection of larvae in stools Serological Results
Copropositive, n=34 (% of IgG positive sera)
OR (95% CI)
p value
IgG positive IgG1 positivea IgG4 positive Both IgG1 and IgG4 positivea Either IgG1 or IgG4 positive
24 (70.6) 17 (50.0) 14 (41.2) 12 (35.3) 19 (55.9)
1.71 (.75–3.91) 2.90 (1.31–6.41) 1.33 (0.61–2.91) 2.26 (0.97–5.24) 1.84 (0.85–4.00)
0.231 (NS) 0.031 0.613 (NS) 0.055 0.172 (NS)
NS: Not significant. a p<0.05 Significant association with positive culture.
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Table 4 Life style factors associated with total IgG reactive to Strongyloides antigen Household factor Educational level Illiterate Non formal Standard 1–5 and above Occupation Service Labor Student Housewife Other Type of toilet Sanitary Non sanitary Washes hands Regularly Irregularly Trims nails Regularly Irregularly Shoes Wears shoes Bare footed Diarrhoea in past 6 months Absent Present
Percentage within the total IgG positive group, n=90
Percentage within total sample group, n=147
44/82 (53.7) 33/50 (66.0) 13/15 (86.7)
48.0 36.0 14.0
30.0 22.4 8.8
03/09 (33.3) 23/40 (57.5) 12/18 (66.7) 50/76 (65.8) 02/04 (50.0)
3.3 25.0 13.3 55.0 2.2
2.0 15.6 8.1 34 1.3
36/46 (78.3) 54/101 (53.5)
40.0 60.0
24.0 36.0
11/20 (55.0) 79/127 (62.2)
12.0 87.0
7.4 53.7
35/51 (68.6) 55/96 (57.3)
38.0 61.0
23.0 37.4
35/58 (60.3) 55/89 (61.8)
38.0 61.0
23.0 37.4
10/15 (66.7) 80/131 (61.1)
11.0 88.0
6.8 54.4
No. of IgG reactive sera in each category (%)
Table 5 Life style factors associated with Strongyloides stercoralis detected in stools Household factor Educational level Illiterate Non formal Standard 1–5 and above Occupation Service Labor Student Housewife Other Type of toilet Sanitary Non sanitary Washes handsa Regularly Irregularly Trims nailsa Regularly Irregularly Shoesa Wears shoes Bare footed Diarrhoea in past 6 months Absent Present a
No. in each category (%)
Percentage within positive culture, n=34
Percentage within total sample group, n=146
19/82 (23.2) 13/50 (26.0) 02/15 (13.3)
55.8 38.2 5.8
13.0 8.9 1.3
02/09 (22.2) 12/40 (30.0) 05/18 (27.8) 13/76 (17.1) 02/04 (50.0)
5.8 35.2 14.7 38.2 5.8
1.3 8.2 3.4 8.9 1.3
10/46 (21.7) 24/101 (23.8)
29.4 70.5
6.8 16.4
0/20 (0.00) 34/127 (26.8)
0 100
0 23.1
06/46 (13.0) 28/101 (27.7)
17.6 82.3
4.1 19.0
08/58 (13.8) 26/89 (29.2)
23.5 76.4
5.4 17.8
06/15 (40.0) 28/131 (21.4)
17.6 82.3
4.1 19.0
p<0.05 Significant association with positive culture.
4. Discussion One hundred and forty seven squatters in an urban slum of Dhaka were screened for infection with S. stercoralis using both coprological and serological methods. The prevalence of S. stercoralis determined by parasitological
diagnosis was 23% using direct microscopy and culture (APC and Harada-Mori) methods. Seroprevalence, based on total IgG response was 61%, which is almost three times higher than by copro examination but the difference was not statistically significant. The observed disparity between copro prevalence and seroprevalence in this study has also
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been reported previously in Thailand, Argentina, Uganda and among refugees in Canada in whom serology was more likely to be positive than fecal detection.12,21–24 The discrepancy in copro-prevalence and seroprevalence in our study could be explained firstly by the presence of cross reactions between Strongyloides and other helminthic infections, especially filariasis; secondly by the relatively low sensitivity of culture methods; and thirdly, by larvae being passed intermittently in stools of immunocompetent individuals. Serological cross-reaction of Strongyloides with filaria using S. stercoralis as an antigen and with Schistosoma using S. ratti antigen are reported.11,25 In our study all sera (n=147) were tested with a recombinant filarial antigen Bm14. A serological study using this antigen showed sensitivities of 91% and 96% for detections of Wuchereria bancrofti and Brugia infections respectively; specificity was checked with non filarial helminth infections and no cross reactions were observed with either Strongyloides or Echinococcus species.26 Only six sera from our study were positive for filarial IgG4 and three of those also had reactive total IgG to Strongyloides antigen. Thus 4% of participants in this study group had serological evidence of lymphatic filariasis. Detection of IgG1 and IgG4 has been shown to be more specific than total IgG response for detection of strongyloidiasis in humans.13,27 Sera from patients with intestinal parasites other than S. stercoralis showed positive IgG1 (1/3; 3%) and IgG4 (7/30; 23%) to Strongyloides antigen indicating serological cross reactivity.13 In our study, IgG1 and IgG1 plus IgG4 positive sera were significantly correlated to positive stool culture (p<0.05) while the total IgG was not. This suggests that the isotypes are more specific in detecting strongyloidiasis. However, we found that three sera reacted to both Strongyloides IgG and Bm14 also had positive IgG1 and IgG4 reactivity to Strongyloides antigen. We can not be sure whether these are either co-infected with filariasis and S. stercoralis or have a filarial infection that is causing a false positive Strongyloides IgG reaction, as larvae were not found in the respective stool sample. Even with improved specificity by detecting reactive IgG isotypes cross reactivity from other helminth infections remains a problem in serological diagnosis of strongyloidiasis. Previous studies show that APC was more sensitive than other coprological methods even in low worm burdens.9,10,28,29 However, in our study only 15 (43%) of all copro positive samples were detected by the APC method. Lower detection rates of only 15% were reported in a study from Paraguay7 while another study in Argentina found no difference between Harada and APC methods in detecting S. stercoralis in stool.23 The low detection rate in the APC method in our study was most likely due to fungal overgrowth. The studied group lived in a temporarily occupied area where residents might have defecated on the ground allowing contamination with soil fungi. A low detection rate in the APC method due to soil fungal contamination has been reported from a previous study in Peru.12 Another possible reason for a higher detection rate in the HaradaMori method in our study was that we used triplicate preparations compared to only one for APC, thus increasing
the sensitivity.16 Furthermore, ideal laboratory conditions for culture could not be maintained in our study. Temperature control in the laboratory was not consistent allowing increases of ambient temperature above 28 ◦ C coupled with high humidity making it ideal for fungal overgrowth in APC. The Harada-Mori culture method may be more effective in a resource poor setting but may not reach the sensitivity levels found with APC when optimum culture conditions are achieved. Twenty-four (16.3%) of the respondents in the current study had confirmed strongyloidiasis, with both positive serology and positive stool culture. Ten (6.8%) respondents had Strongyloides larvae in stool but no detectable antibody in sera. Apart from the lower sensitivities of coproanalysis, acute S. stercoralis infection does not necessarily display positive serology; delayed seroconversion thus leads to false negative results.30 Those participants in our study who were either recently infected or possibly immunosuppressed with S. stercoralis may be not be detected by serological methods. Difficulties in collecting field samples from a shifting population limited us to a single collection. Detection rates of Strongyloides in a single stool examination are estimated to be below 30% due to low densities and irregular output of parasites; testing three to five samples improves the sensitivity up to 85%.10,11,31 In our study, the parasitic load in the stool at the time of sample collection was less than five larvae detected by Harada-Mori cultures, indicating low larval output in the majority of positive specimens. A high antibody level and eosinophilia might hamper worm maturation leading to false negative results in stool examination.21 Other enteric parasites found in stool samples had no statistical association with the presence of Strongyloides IgG and isotypes. However, there was a significant correlation between infections with A. lumbricoides and T. trichiura (p<0.05). Surprisingly, there was no correlation between these common helminths and any associated risks identified in our questionnaire. Factors such as education, occupation and personal hygiene (e.g., type of toilet, use of shoes and type of dwelling) did not correlate with seroprevalence. This is consistent with our previous study in Bangladesh.20 However, copro analysis in an earlier study in Bangladesh showed higher risks of S. stercoralis infection among people who either used the community latrine, lived in a house with an earth floor or were Pakistani refugees who settled in Dhaka in 1971.19 In our study, those participants who were not trimming their nails regularly, not washing their hands after defecation and not wearing shoes were more likely to be infected with S. stercoralis (p<0.05) by coproanalysis. Another study in Jamaica showed that over crowded families were predisposed to S. stercoralis infection.32 Risk factors which enhance the chance of exposure to infective larval stages depend both on the level of disease endemicity in the community and survival of the parasite in the environment. Individuals co-infected with S. stercoralis and HTLV1 are more refractory to treatment2,33,34 and might require repeated dosing with ivermectin. We found two participants who were seropositive both for HTLV I and II and
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Strongyloides antibodies. However, no larvae were cultured from their respective faecal samples. This retro viral infection shows low seroprevalence in Dhaka, which is consistent with a previous study.20 The lack of correlation between coproanalysis and serology for detection of S. stercoralis infection in our study population may indicate that the two methods are detecting different groups of infected individuals. Those who are either recently infected or are immunosuppressed are more likely to have detectable larvae in their stools, especially when a single stool specimen is collected. These individuals, however, are less likely to have detectable antibodies because they have not seroconverted or have depressed antibody responses. Serology will identify immunocompetent individuals who are chronic intermittent excreters of low larval numbers. These cases are therefore likely to be copronegative with a single sample. Unfortunately, this hypothesis can not be supported by the current information in our study and further investigation in this community could not be achieved because the slum site was demolished. Nevertheless, our study shows that S. stercoralis infection remains prevalent in Bangladesh particularly in squatters found in these temporary shelters. Authors’ contributions: RL, B-N A and YS participated in the conception and design of the study; B-N A Ahmed supervised the data collection; YS carried out the entire tests and interpreted the data; GLG and RL revised the article critically for intellectual contents; all authors confirmed the final approval of the version of the manuscript to be published. RL and YS are the guarantors of the paper. Acknowledgements: The study was conducted in collaboration with the Institute of Epidemiology, Disease Control and Research (IEDCR), Dhaka, Bangladesh. We gratefully acknowledge the assistance of Dr. Brian O’Toole for data analysis and the technical assistance of John Clancy, ICPMR, Westmead Hospital, NSW, Australia. Funding: This research was supported by an Australian Leadership Scholarship (ALA) Award funded by The Australian Agency for International Development (Aus AID) and part by Centre for Infectious Diseases and Microbiology-Public Health. Competing interests: None declared. Ethical Approval: The study was approved by the National Research Ethics Committee, Bangladesh Medical Research Council (BMRC; Dhaka, Bangladesh; Ref: BMRC/NREC/2007-2010/1256; Dated 20-01-2009). References 1. Bethony J, Brooker S, Albonico M, et al. Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm. Lancet 2006;367: 1521–32. 2. Terashima A, Alvarez H, Tello R, Infante R, Freedman DO, Gotuzzo E. Treatment failure in intestinal strongyloidiasis: an indicator of HTLV-I infection. Int J Infect Dis 2002;6:28–30. 3. Siddiqui AA, Berk SL. Diagnosis of Strongyloides stercoralis infection. Clin Infect Dis 2001;33:1040–7.
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29. Ines Ede J, Souza JN, Santos RC, et al. Efficacy of parasitological methods for the diagnosis of Strongyloides stercoralis and hookworm in faecal specimens. Acta Trop 2011;120:206–10. 30. Baaten GG, Sonder GJ, van Gool T, Kint JA, van den Hoek A. Travelrelated schistosomiasis, strongyloidiasis, filariasis, and toxocariasis: the risk of infection and the diagnostic relevance of blood eosinophilia. BMC Infect Dis 2011;11:84. 31. Winsberg GR, Sonnenschein E, Dyer AR, Schnadig V, Bonilla E. Prevalence of intestinal parasites in Latino residents of Chicago. Am J Epidemiol 1975;102:526–32.
32. Lindo JF, Robinson RD, Terry SI, et al. Age-prevalence and household clustering of Strongyloides stercoralis infection in Jamaica. Parasitol 1995;110:97–102. 33. Hirata T, Uchima N, Kishimoto K, et al. Impairment of host immune response against Strongyloides stercoralis by human T cell lymphotropic virus type 1 infection. Am J Trop Med Hyg 2006;74:246–9. 34. Zaha O, Hirata T, Uchima N, Kinjo F, Saito A. Comparison of anthelmintic effects of two doses of ivermectin on intestinal strongyloidiasis in patients negative or positive for anti-HTLV-1 antibody. J Infect Chemother 2004;10:348–51.
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Strongyloidiasis in a high risk community of Dhaka, Bangladesh Yasmin Sultana a,b,∗ , Gwendolyn L. Gilbert a,b , Be-Nazir Ahmed c , Rogan Lee a,b a b c
Centre for Infectious Diseases and Microbiology Laboratory Services, ICPMR, Westmead Hospital, Westmead, NSW, Australia Discipline of Medicine, Sydney Medical School, University of Sydney, NSW, Australia Institute of Epidemiology Disease Control and Research, Mohakhali, Dhaka, Bangladesh
a r t i c l e
i n f o
Article history: Received 15 May 2012 Received in revised form 29 August 2012 Accepted 29 August 2012 Available online 22 October 2012 Keywords: Strongyloides stercoralis Slum Seroprevalence Copro-prevalence
a b s t r a c t Residents of a slum community of Dhaka city, Bangladesh were tested by serological and faecal examination for evidence of Strongyloides stercoralis infection. In stool specimens from a total of 147 participants Strongyloides larvae were found in 34 (23.1%) by Harada-Mori culture, 15 (10.2%) by agar plate culture (APC) and one (0.7%) by microscopy. Strongyloides IgG, IgG1 and IgG4 antibodies were found in 90 (61.2%), 46 (31.3%) and 53 (36.1%) of participants, respectively. A positive correlation was observed between total IgG levels and the presence of isotypes IgG1 and IgG4 (p<0.001). Six sera (4.0%) reacted to the recombinant filaria antigen Bm 14, three of which were Strongyloides IgG positive. This indicates either there is cross reactivity or some participants are co-infected with lymphatic filariasis. No correlations were found between positive serology and Strongyloides infection in stool, socio- demographic factors or domestic hygienic practices. However, positive stool cultures showed significant associations with irregular nail trimming, walking bare-foot and irregular hand washing after defecation (p<0.05). Other enteric parasites were detected in stools of some participants but their presence showed no correlation with S. stercoralis infection or socio demographic factors. This study confirms that squatters in this slum community in Dhaka have a high prevalence of S. stercoralis infection identified both by serological and coprological methods. © 2012 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction Strongyloides stercoralis is a soil-transmitted intestinal nematode estimated to infect 30–100 million people globally.1 Those individuals co-infected with human lymphotrophic virus-1 (HTLV-1) are more likely to develop severe forms of strongyloidiasis and be less responsive to treatment.2 The mortality rate of disseminated infections is reported to be as high as 87%.3 Strongyloidiasis is now listed as one of the so-called neglected tropical diseases,4 but is poorly described due to lack of epidemiological information.5 Confirmation of Strongyloides infection by coprological examination is difficult because of irregular
∗ Corresponding author. Tel.: +61 2 98457662; fax: +61 2 98938659. E-mail address: [email protected] (Y. Sultana).
excretion of the parasite especially in chronic cases; prevalence of infection is thus underestimated.6,7 Definitive diagnosis of strongyloidiasis is by detection of larvae in stool. Direct smears, formal-ether concentration (FEC), Baermann-Moraes culture, spontaneous sedimentation, Harada-Mori culture and agar plate culture (APC) are common coprological methods used to detect S. stercoralis in stools.8 Agar plate culture has been shown to be more sensitive than direct smear, FEC, Baermann-Moraes technique or filter paper techniques.9,10 The detection of serum antibodies may facilitate the diagnosis and is more sensitive than coprodiagnosis but its specificity can be low. False positive antibody reactions can arise either due to recent past infection or cross-reactions with other helminthic infection such as filariasis.11,12 A previous study has shown that reactive IgG1 and IgG4 to Strongyloides antigen had less cross reaction from other
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helminth infections compared to total IgG, suggesting that these isotypes are more specific than reactive total IgG.13 Specific coproantigen detection and molecular assays have been shown to be promising alternatives with high specificity and sensitivity,14–16 but may not be available in resource poor countries. Countries with poor hygiene and sanitation are found to have high strongyloidiasis prevalence.17,18 Strongyloidiasis is endemic in Africa, the West Indies, Southeast Asia, South America, Pakistan and Bangladesh.6 Coprological and recent serological studies, in slum areas of Dhaka, confirmed its continued existence in Bangladesh.19,20 The aim of the present study, conducted in a slum community of Dhaka, was to ascertain the prevalence of S. stercoralis infection based on faecal examination and serology. Other objectives of this study were to identify risk factors for strongyloidiasis including socio-demographic and household factors and HTLV-I and II co-infection. 2. Materials and methods 2.1. Study group The study was conducted in a slum located at Adabar of Mohammadpur, a western suburb of Dhaka city, Bangladesh. This site was demolished six months after completion of sample collection. During November 2009 to January 2010, a house-to-house survey of the community was performed. A total of 147 individuals who participated in this study provided both serum and stool samples. We also collected socio-demographic information using a questionnaire. As most of the participants were illiterate, consent was obtained verbally from each respondent and the questionnaire was completed during the same visit. At the end of this study participants who were infected with strongyloidiasis were offered albendazole (the only drug registered for treatment of this infection in Bangladesh) 400 mg/day, on two consecutive days. The study was approved by the National Research Ethics Committee, Bangladesh Medical Research Council (BMRC; Dhaka, Bangladesh). 2.2. Stool examinations 2.2.1. Direct smear and formal ether concentration The faecal sample collected from each participant was examined microscopically by direct smear within 6 hours of collection. An FEC using the MINI PARASEP SF (Laboratory Diagnostics, Taren Point, NSW, Australia) was prepared from each stool sample. Iron haematoxylin (Oxoid, Adelaide, Australia) stained smears from these faecal concentrates were examined for the presence of Strongyloides and other enteric parasites. 2.2.2. Agar plate culture and Harada-Mori culture Both APC (single) and Harada-Mori methods (triplicate) were used to culture S. stercoralis.8 Two grams of stool was used for each culture replicate; thus a total of 6 g of stool was used in Harada-Mori methods and only 2 g in APC culture. The plates were sealed with adhesive tape to prevent drying of the agar. Cultures were incubated at
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25–28 ◦ C for a week and examined daily. Any specimen, in which at least one larva was identified by either direct microscopy or culture, was regarded as positive. Positive samples were confirmed by microscopic examination of Strongyloides larva for morphological structures. 2.3. Strongyloides ELISA Sera were separated from freshly collected blood and stored at −20 ◦ C prior to testing. IgG against Strongyloides antigen was detected by an indirect ELISA using soluble antigen from Strongyloides ratti third stage larvae (L3) as previously described.20 All the sera with detectable Strongyloides IgG were screened for isotypes IgG1 and IgG4. 2.4. Other serological studies 2.4.1. Detection of filarial antibody All sera were tested for antibody to Wuchereria bancrofti using an indirect ELISA for the detection of Bm14 specific IgG4 antibody (Cellabs Pty Ltd, Brookvale, NSW, Australia). Results were interpreted according to the manufacturer’s instructions. 2.4.2. Detection of HTLV-I and II antibody Presence of HTLV-I and II antibody in sera was measured by the Murex HTLV I+II assay (Abbott Australasia Pty Ltd, Botany, NSW, Australia) and results were interpreted according to the manufacturer’s instructions. 2.5. Risk factors associated with strongyloidiasis Participants who provided blood and stool samples also completed a questionnaire. Data collected included: gender, age, level of education, occupation, average monthly income, number of family members and type of toilet used. Other data collected were aspects of personal hygiene such as wearing shoes, trimming nails regularly, washing hands with soap after defecation and history of diarrhoea in the past six months. These factors were then evaluated against positive serology and positive culture of each specimen. 2.6. Statistical analysis Statistical package, SPSS version 17 (SPSS Inc., Chicago, IL, USA) was used: to compare the observed prevalence of infection as determined by serology and stool examination; to determine significant risk factors from the questionnaire; to show associations, if any, of each with positive serology and stool examination; and to identify association of strongyloidiasis with other enteric parasitic infestations using 2 statistics. Levels (as shown by OD values) of IgG, IgG1 and IgG4 antibodies to S. ratti were compared by Pearson’s correlation statistics. Logistic regression was used to compute the risk of infection with age, entered as a continuous variable. Values of p<0.05 were considered statistically significant.
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3. Results 3.1. Stool examination (direct smear, formalin ether concentration and culture) Strongyloides stercoralis larvae were detected by microscopy in only one specimen which failed to show larvae in culture. No larvae were found by microscopic examination of FEC. Larvae were detected in 34 (23.1%) stool samples using the Harada Mori culture method but in only 15 (10.2%) of these by APC. There was no association between detection of S. stercoralis larvae in stool with either age or sex. Other enteric parasites found in formal-ether concentrations of stool sample are listed in Table 1. Ascaris lumbricoides and Trichuris trichiura each were found in more than one third of samples and were both present in 28 (19%) stools. No association was observed between the presence of Strongyloides larvae and the other enteric parasites including Ascaris and Trichuris. However, the presence of A. lumbricoides and T. trichiura together in each stool sample was statistically significant (p<0.05). 3.2. Strongyloides serology (ELISA) Total Strongyloides IgG was found in 90 of 147 (61.2%) sera in the studied group. IgG1, IgG4 and both subclasses were detected in 46/90 (51.1%), 53/90 (58.8%) and 34/90 (37.8%) of the total IgG positive sera respectively. There was a significant positive correlation between the level of total IgG and both isotypes by Pearson correlation statistics (p<0.001). More females than males were serologically positive, but the difference was not statistically significant. In addition, age showed no association with seropositive cases. Table 1 Other enteric parasites identified in stool samples of 147 participants examined for the presence of Strongyloides stercoralis Organism
Number detected (%) n=147
Number co-infected with S. stercoralis (%) n=34
Ascaris lumbricoides Trichuris trichura Entamoeba coli Entamoeba histolytica Idomoeba butchilli Enterobius vermicularis Endolimax nana Blastocystis hominis Giardia lamblia
57 (38.8) 52 (35.4) 8 (5.4) 4 (2.7) 11 (7.5) 2 (1.4) 2 (1.4) 2 (1.4) 1 (0.7)
16 (47.1) 15 (44.1) 3 (8.8) 2 (5.9) 4 (11.8) 0 1 (2.9) 1 (2.9) 0
Table 2 Comparison of IgG reactivity to Strongyloides antigen and detection of larvae in stools Serology, IgG
Culture, APC/Harada-Mori Positive (%)
Negative (%)
Total
Positive Negative Total
24 (16.3) 10 (6.8) 34
66 (44.9) 47 (32.0) 113
90 57 147
APC: agar plate culture.
Of the 147 participants, 24 (16.3%) had both Strongyloides antibodies and Strongyloides larvae in their stool (Table 2). Sixty six (44.9%) had Strongyloides antibody but no larvae detected in their stools and 10 (6.8%) had larvae in their stools but no detectable antibody. No significant correlation was found between participants with reactive total IgG and presence of larvae in their stools. Of the 34 copropositive respondents, 17 (50%), 14 (41%) and 12 (35%) had detectable IgG1, IgG4 and both isotypes, respectively (Table 3). A significant association was observed between positive culture and IgG1 (p<0.05). Positive Strongyloides IgG had no correlation to presences of other faecal helminths found in their respective faecal specimen. 3.3. Risk factors associated with positive serology and Strongyloides stercoralis positive stools The socio-demographic information collected from each participant regarding education level, occupation and other household factors showed no correlation with positive serology (Table 4). However, a number of personal hygiene practices (namely being bare-foot, not washing hands after defecation and irregular nail trimming) were associated with a greater risk of being infected by S. stercoralis identified by stool culture (Table 5). 3.4. Other serological results Six (4.1%) of 147 sera showed positive results for IgG4 antibody against recombinant antigen (Bm 14) for lymphatic filariasis; of these, three had low positive reactions and were also seropositive for Strongyloides total IgG and IgG isotypes. All six individuals with reactive IgG4 to lymphatic filariasis had no Strongyloides larvae detected in their faecal specimens. Two individuals from the studied group had detectable antibodies to HTLV-I and II. No larvae were found in their respective faecal specimens. They had low Strongyloides IgG antibodies.
Table 3 Comparison of participant’s reactivity IgG1 and IgG4 to Strongyloides antigen and detection of larvae in stools Serological Results
Copropositive, n=34 (% of IgG positive sera)
OR (95% CI)
p value
IgG positive IgG1 positivea IgG4 positive Both IgG1 and IgG4 positivea Either IgG1 or IgG4 positive
24 (70.6) 17 (50.0) 14 (41.2) 12 (35.3) 19 (55.9)
1.71 (.75–3.91) 2.90 (1.31–6.41) 1.33 (0.61–2.91) 2.26 (0.97–5.24) 1.84 (0.85–4.00)
0.231 (NS) 0.031 0.613 (NS) 0.055 0.172 (NS)
NS: Not significant. a p<0.05 Significant association with positive culture.
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Table 4 Life style factors associated with total IgG reactive to Strongyloides antigen Household factor Educational level Illiterate Non formal Standard 1–5 and above Occupation Service Labor Student Housewife Other Type of toilet Sanitary Non sanitary Washes hands Regularly Irregularly Trims nails Regularly Irregularly Shoes Wears shoes Bare footed Diarrhoea in past 6 months Absent Present
Percentage within the total IgG positive group, n=90
Percentage within total sample group, n=147
44/82 (53.7) 33/50 (66.0) 13/15 (86.7)
48.0 36.0 14.0
30.0 22.4 8.8
03/09 (33.3) 23/40 (57.5) 12/18 (66.7) 50/76 (65.8) 02/04 (50.0)
3.3 25.0 13.3 55.0 2.2
2.0 15.6 8.1 34 1.3
36/46 (78.3) 54/101 (53.5)
40.0 60.0
24.0 36.0
11/20 (55.0) 79/127 (62.2)
12.0 87.0
7.4 53.7
35/51 (68.6) 55/96 (57.3)
38.0 61.0
23.0 37.4
35/58 (60.3) 55/89 (61.8)
38.0 61.0
23.0 37.4
10/15 (66.7) 80/131 (61.1)
11.0 88.0
6.8 54.4
No. of IgG reactive sera in each category (%)
Table 5 Life style factors associated with Strongyloides stercoralis detected in stools Household factor Educational level Illiterate Non formal Standard 1–5 and above Occupation Service Labor Student Housewife Other Type of toilet Sanitary Non sanitary Washes handsa Regularly Irregularly Trims nailsa Regularly Irregularly Shoesa Wears shoes Bare footed Diarrhoea in past 6 months Absent Present a
No. in each category (%)
Percentage within positive culture, n=34
Percentage within total sample group, n=146
19/82 (23.2) 13/50 (26.0) 02/15 (13.3)
55.8 38.2 5.8
13.0 8.9 1.3
02/09 (22.2) 12/40 (30.0) 05/18 (27.8) 13/76 (17.1) 02/04 (50.0)
5.8 35.2 14.7 38.2 5.8
1.3 8.2 3.4 8.9 1.3
10/46 (21.7) 24/101 (23.8)
29.4 70.5
6.8 16.4
0/20 (0.00) 34/127 (26.8)
0 100
0 23.1
06/46 (13.0) 28/101 (27.7)
17.6 82.3
4.1 19.0
08/58 (13.8) 26/89 (29.2)
23.5 76.4
5.4 17.8
06/15 (40.0) 28/131 (21.4)
17.6 82.3
4.1 19.0
p<0.05 Significant association with positive culture.
4. Discussion One hundred and forty seven squatters in an urban slum of Dhaka were screened for infection with S. stercoralis using both coprological and serological methods. The prevalence of S. stercoralis determined by parasitological
diagnosis was 23% using direct microscopy and culture (APC and Harada-Mori) methods. Seroprevalence, based on total IgG response was 61%, which is almost three times higher than by copro examination but the difference was not statistically significant. The observed disparity between copro prevalence and seroprevalence in this study has also
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been reported previously in Thailand, Argentina, Uganda and among refugees in Canada in whom serology was more likely to be positive than fecal detection.12,21–24 The discrepancy in copro-prevalence and seroprevalence in our study could be explained firstly by the presence of cross reactions between Strongyloides and other helminthic infections, especially filariasis; secondly by the relatively low sensitivity of culture methods; and thirdly, by larvae being passed intermittently in stools of immunocompetent individuals. Serological cross-reaction of Strongyloides with filaria using S. stercoralis as an antigen and with Schistosoma using S. ratti antigen are reported.11,25 In our study all sera (n=147) were tested with a recombinant filarial antigen Bm14. A serological study using this antigen showed sensitivities of 91% and 96% for detections of Wuchereria bancrofti and Brugia infections respectively; specificity was checked with non filarial helminth infections and no cross reactions were observed with either Strongyloides or Echinococcus species.26 Only six sera from our study were positive for filarial IgG4 and three of those also had reactive total IgG to Strongyloides antigen. Thus 4% of participants in this study group had serological evidence of lymphatic filariasis. Detection of IgG1 and IgG4 has been shown to be more specific than total IgG response for detection of strongyloidiasis in humans.13,27 Sera from patients with intestinal parasites other than S. stercoralis showed positive IgG1 (1/3; 3%) and IgG4 (7/30; 23%) to Strongyloides antigen indicating serological cross reactivity.13 In our study, IgG1 and IgG1 plus IgG4 positive sera were significantly correlated to positive stool culture (p<0.05) while the total IgG was not. This suggests that the isotypes are more specific in detecting strongyloidiasis. However, we found that three sera reacted to both Strongyloides IgG and Bm14 also had positive IgG1 and IgG4 reactivity to Strongyloides antigen. We can not be sure whether these are either co-infected with filariasis and S. stercoralis or have a filarial infection that is causing a false positive Strongyloides IgG reaction, as larvae were not found in the respective stool sample. Even with improved specificity by detecting reactive IgG isotypes cross reactivity from other helminth infections remains a problem in serological diagnosis of strongyloidiasis. Previous studies show that APC was more sensitive than other coprological methods even in low worm burdens.9,10,28,29 However, in our study only 15 (43%) of all copro positive samples were detected by the APC method. Lower detection rates of only 15% were reported in a study from Paraguay7 while another study in Argentina found no difference between Harada and APC methods in detecting S. stercoralis in stool.23 The low detection rate in the APC method in our study was most likely due to fungal overgrowth. The studied group lived in a temporarily occupied area where residents might have defecated on the ground allowing contamination with soil fungi. A low detection rate in the APC method due to soil fungal contamination has been reported from a previous study in Peru.12 Another possible reason for a higher detection rate in the HaradaMori method in our study was that we used triplicate preparations compared to only one for APC, thus increasing
the sensitivity.16 Furthermore, ideal laboratory conditions for culture could not be maintained in our study. Temperature control in the laboratory was not consistent allowing increases of ambient temperature above 28 ◦ C coupled with high humidity making it ideal for fungal overgrowth in APC. The Harada-Mori culture method may be more effective in a resource poor setting but may not reach the sensitivity levels found with APC when optimum culture conditions are achieved. Twenty-four (16.3%) of the respondents in the current study had confirmed strongyloidiasis, with both positive serology and positive stool culture. Ten (6.8%) respondents had Strongyloides larvae in stool but no detectable antibody in sera. Apart from the lower sensitivities of coproanalysis, acute S. stercoralis infection does not necessarily display positive serology; delayed seroconversion thus leads to false negative results.30 Those participants in our study who were either recently infected or possibly immunosuppressed with S. stercoralis may be not be detected by serological methods. Difficulties in collecting field samples from a shifting population limited us to a single collection. Detection rates of Strongyloides in a single stool examination are estimated to be below 30% due to low densities and irregular output of parasites; testing three to five samples improves the sensitivity up to 85%.10,11,31 In our study, the parasitic load in the stool at the time of sample collection was less than five larvae detected by Harada-Mori cultures, indicating low larval output in the majority of positive specimens. A high antibody level and eosinophilia might hamper worm maturation leading to false negative results in stool examination.21 Other enteric parasites found in stool samples had no statistical association with the presence of Strongyloides IgG and isotypes. However, there was a significant correlation between infections with A. lumbricoides and T. trichiura (p<0.05). Surprisingly, there was no correlation between these common helminths and any associated risks identified in our questionnaire. Factors such as education, occupation and personal hygiene (e.g., type of toilet, use of shoes and type of dwelling) did not correlate with seroprevalence. This is consistent with our previous study in Bangladesh.20 However, copro analysis in an earlier study in Bangladesh showed higher risks of S. stercoralis infection among people who either used the community latrine, lived in a house with an earth floor or were Pakistani refugees who settled in Dhaka in 1971.19 In our study, those participants who were not trimming their nails regularly, not washing their hands after defecation and not wearing shoes were more likely to be infected with S. stercoralis (p<0.05) by coproanalysis. Another study in Jamaica showed that over crowded families were predisposed to S. stercoralis infection.32 Risk factors which enhance the chance of exposure to infective larval stages depend both on the level of disease endemicity in the community and survival of the parasite in the environment. Individuals co-infected with S. stercoralis and HTLV1 are more refractory to treatment2,33,34 and might require repeated dosing with ivermectin. We found two participants who were seropositive both for HTLV I and II and
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Strongyloides antibodies. However, no larvae were cultured from their respective faecal samples. This retro viral infection shows low seroprevalence in Dhaka, which is consistent with a previous study.20 The lack of correlation between coproanalysis and serology for detection of S. stercoralis infection in our study population may indicate that the two methods are detecting different groups of infected individuals. Those who are either recently infected or are immunosuppressed are more likely to have detectable larvae in their stools, especially when a single stool specimen is collected. These individuals, however, are less likely to have detectable antibodies because they have not seroconverted or have depressed antibody responses. Serology will identify immunocompetent individuals who are chronic intermittent excreters of low larval numbers. These cases are therefore likely to be copronegative with a single sample. Unfortunately, this hypothesis can not be supported by the current information in our study and further investigation in this community could not be achieved because the slum site was demolished. Nevertheless, our study shows that S. stercoralis infection remains prevalent in Bangladesh particularly in squatters found in these temporary shelters. Authors’ contributions: RL, B-N A and YS participated in the conception and design of the study; B-N A Ahmed supervised the data collection; YS carried out the entire tests and interpreted the data; GLG and RL revised the article critically for intellectual contents; all authors confirmed the final approval of the version of the manuscript to be published. RL and YS are the guarantors of the paper. Acknowledgements: The study was conducted in collaboration with the Institute of Epidemiology, Disease Control and Research (IEDCR), Dhaka, Bangladesh. We gratefully acknowledge the assistance of Dr. Brian O’Toole for data analysis and the technical assistance of John Clancy, ICPMR, Westmead Hospital, NSW, Australia. Funding: This research was supported by an Australian Leadership Scholarship (ALA) Award funded by The Australian Agency for International Development (Aus AID) and part by Centre for Infectious Diseases and Microbiology-Public Health. Competing interests: None declared. Ethical Approval: The study was approved by the National Research Ethics Committee, Bangladesh Medical Research Council (BMRC; Dhaka, Bangladesh; Ref: BMRC/NREC/2007-2010/1256; Dated 20-01-2009). References 1. Bethony J, Brooker S, Albonico M, et al. Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm. Lancet 2006;367: 1521–32. 2. Terashima A, Alvarez H, Tello R, Infante R, Freedman DO, Gotuzzo E. Treatment failure in intestinal strongyloidiasis: an indicator of HTLV-I infection. Int J Infect Dis 2002;6:28–30. 3. Siddiqui AA, Berk SL. Diagnosis of Strongyloides stercoralis infection. Clin Infect Dis 2001;33:1040–7.
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