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Improved agar plate culture conditions for diagnosis of Strongyloides stercoralis
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Improved agar plate culture conditions for diagnosis of Strongyloides stercoralis Wannee Kaewrat , Chatchawan Sengthong , Manachai Yingklang , Kitti Intuyod , Ornuma Haonon , Sudarat Onsurathum , Rungthiwa Dangtakot , Phitsamai Saisud , Arunnee Sangka , Sirirat Anutarakulchai , Somchai Pinlaor , Ubon Cha’on , Porntip Pinlaor PII: DOI: Reference:
S0001-706X(19)30537-6 https://doi.org/10.1016/j.actatropica.2019.105291 ACTROP 105291
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Acta Tropica
Received date: Revised date: Accepted date:
20 April 2019 9 October 2019 4 December 2019
Please cite this article as: Wannee Kaewrat , Chatchawan Sengthong , Manachai Yingklang , Kitti Intuyod , Ornuma Haonon , Sudarat Onsurathum , Rungthiwa Dangtakot , Phitsamai Saisud , Arunnee Sangka , Sirirat Anutarakulchai , Somchai Pinlaor , Ubon Cha’on , Porntip Pinlaor , Improved agar plate culture conditions for diagnosis of Strongyloides stercoralis, Acta Tropica (2019), doi: https://doi.org/10.1016/j.actatropica.2019.105291
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Highlights
We optimized agar plate culture (APC) for detection of Strongyloides stercoralis.
A pH of 6.0, 0.5% NaCl and temperature of 29-30 °C were found to be optimum.
Testing in an endemic community confirmed this.
Improved agar plate culture conditions for diagnosis of Strongyloides stercoralis
Wannee Kaewrat1,7, Chatchawan Sengthong2,7 , Manachai Yingklang2,7, Kitti Intuyod2,7 Ornuma Haonon2,7, Sudarat Onsurathum2,7, Rungthiwa Dangtakot 3,7, Phitsamai Saisud4, Arunnee Sangka4,7, Sirirat Anutarakulchai5,7, Somchai Pinlaor2,7, Ubon Cha’on6,7, Porntip Pinlaor4,7*
1
Science Program in Clinical Pathology and Management, Faculty of Associated Medical
Sciences, Khon Kaen University, Khon Kaen 40002, Thailand 2
Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002,
Thailand 3
Science Program in Biomedical Science, Khon Kaen University, Khon Kaen 40002, Thailand
4
Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of
Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand 5
Department of Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002,
Thailand 6
Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002,
Thailand 7
Chronic Kidney Disease Prevention in The Northeastern Thailand, Faculty of Medicine, Khon
Kaen University, Khon Kaen 40002, Thailand
*Correspondence: [email protected]
ABSTRACT Strongyloides stercoralis infection causes gastrointestinal symptoms and can lead to severe disease in immunocompromised hosts. Live larvae are passed in feces, encouraging the common use of diagnosis by cultivation methods including agar plate culture (APC), the gold-standard technique. Nevertheless, APC has limitations, especially since there can be considerable day-today fluctuations in numbers of larvae produced. Herein, we collected stool samples from heavily infected subjects with strongyloidiasis in Khon Kaen Province, Thailand, to evaluate modifications (temperature, pH, nutrition source and salinity) to APC conditions to maximize the number of S. stercoralis worms counted. Best results were obtained using a modified APC with the following conditions: pH 6.0, 0.5% of NaCl, addition of yeast extract for nutrition and incubation at 29-30 °C. This modified APC was more sensitive for detection of S. stercoralis than was standard APC or the formalin-ethyl acetate concentration technique. In brief, this finding suggests that a modification of standard APC conditions increases the counts of S. stercoralis.
Key words: Strongyloides stercoralis, physicochemical factors, agar plate culture, worm development.
1. Introduction Human strongyloidiasis is mainly caused by Strongyloides stercoralis infection and is still a major health problem worldwide, especially in tropical and subtropical countries including Thailand (Schar et al., 2013), where the reported prevalence of S. stercoralis infection ranges from 0.05 to 57.0% (Jongsuksuntigul et al., 2003; Laoraksawong et al., 2018; Sithithaworn et al.,
2003). Infection with S. stercoralis in healthy people generally does not induce severe gastrointestinal symptoms. In contrast, in immunocompromised patients, such as those with chronic kidney disease, diabetes mellitus or HIV, the parasites can rapidly multiply leading to hyperinfection (De Souza et al., 2018; Krolewiecki and Nutman, 2019; Qu et al., 2016; Winnicki et al., 2018). Moreover, patients receiving immunosuppressive drugs or corticosteroid treatment are prone to autoinfection and disseminated infection, the latter being the most important lifethreatening complication of strongyloidiasis (Abanyie et al., 2018; Camargo et al., 2019; Mileo Bacelar Guerreiro et al., 2018; Paula et al., 2018). Although stool examination techniques, such as simple smear, the Kato-Katz technique and formalin-ether concentration are commonly used in prevalence studies of intestinal parasites, they are inadequate for S. stercoralis detection (Buonfrate et al., 2015; Campo Polanco et al., 2014). The Baermann technique and Koga agar plate culture have better sensitivity (Campo Polanco et al., 2014) but are still unsatisfactory (Buonfrate et al., 2018; Krolewiecki and Nutman, 2019). Currently, the agar plate culture (APC) method is considered the gold-standard for diagnosis of S. stercoralis infection. It is used in standard laboratory screening in endemic countries, including Thailand (Formenti et al., 2019; Koga et al., 1991; Kristanti et al., 2018). This technique maintains the parasite development by simulation of natural climatic and physicochemical requirements such as nutrition, humidity and temperature (Viney and Lok, 2015). Nevertheless, APC has some limitations, including those caused by fluctuation in numbers of larvae released in feces each day (Uparanukraw et al., 1999). Also, the APC method has low sensitivity in the case of light infections (Anamnart et al., 2010; Intapan et al., 2006; Ketzis, 2017). Shiwaku et al. (1988) modified some physicochemical parameters in cultures, such as temperature and fecal dilution, to try to increase development of free-living generations
of S. stercoralis under cultivation. Other conditions such as pH, nutrition and salinity in the APC remain to be optimized. Here, we have investigated the influence of temperature, pH, nutrition and salinity on the numbers of S. stercoralis recovered. This provides a better understanding of the optimal culture conditions for the APC, the most widely used diagnostic tool for S. stercoralis infection.
2. Materials and Methods 2.1 Study population, study design and ethics statement The human ethical review committee of Khon Kaen University (HE611068) approved the protocol of study. This was a part of larger study, the Chronic Kidney Disease Northeastern Thailand (CKDNET) study. Informed consent forms and plastic containers were provided to each volunteer before stool collection. The study was conducted from January 2017 to May 2018 at Donchang Sub-District, Muang District, Khon Kaen Province, northeastern Thailand, where a high prevalence of 23% of S. stercoralis infection was previously reported in 2003 (Sithithaworn et al., 2003). The sample size was calculated as described elsewhere (Sithithaworn et al., 2003): based on a prevalence of 23%, with 95% confidence interval (z = 1.96) and 0.5 of an error, we calculated that 273 individuals were required for the study. However, 793 individuals (>35 years of age) volunteered and were enrolled for the study. All were screened for S. stercoralis infection using standard APC (Intapan et al., 2005). At the end of the work, individuals infected with S. stercoralis were treated with a single dose of 200 µg/kg of ivermectin. 2.2 Initial screening for S. stercoralis infection by standard agar plate culture Two grams of fecal material from each subject (793 subjects) were immediately placed on standard APCs individually (APC type A – see below) and covered with parafilm during
fieldwork in the community. All plates were transferred to the Department of Parasitology, Faculty of Medicine, Khon Kaen University, Thailand where they were incubated at room temperature (29-30 °C) for three to five days. On days 3, 4 and 5, each plate was washed with 10 ml of sterile normal saline solution, which was decanted and centrifuged at 3000 rpm for 10 min to collect the worms. The worm species was identified under a light microscope. 2.3 Sources of fecal material for evaluation of modified APC tests Following the initial screening, 20 heavily infected individuals were asked to provide additional stool samples. From these, two sets of pooled stool samples were made, material from ten subjects for each set. The first pooled sample was used for testing effects of agar plate types (which reflected the differences of nutrition sources), pH and temperature, and the second was used for testing salinity. Each pooled sample was mixed thoroughly to ensure homogeneous distribution of larvae before experiments were started. The number of larvae per gram (LPG) was determined in each pooled sample using the formalin-ethyl acetate concentration technique (FECT), performed as described elsewhere (Sithithaworn et al., 2003). In brief, two grams of fecal sample were fixed with 10% formalin solution and shaken vigorously. After centrifugation, the supernatant was discarded and the pellet was resuspended in 0.85% sodium chloride solution (normal saline solution; NSS), filtered through gauze and centrifuged at 2,500 rpm for 5 min. After removal of supernatant, the pellet was resuspended in ethyl acetate, the mixture was recentrifuged at 2,500 rpm for 5 min. Finally, the top three layers were discarded and the numbers of S. stercoralis larvae in the sediment were counted under a light microscope. 2.4 Preparation and evaluation of modified APC conditions Two types of APC (types A and B) were prepared according to the manufacturer's instructions, each type with a series of different pH, salinity and nutrient conditions and
incubated at various temperatures. The unmodified APC type A was the standard APC as used for many years in our laboratory and elsewhere. This contained 3 g beef extract (as nutrient), 5 g of peptone and 15 g of Difco™ nutrient agar, BD (BD Bioscience, MD, USA) in 1L of distilled water. The APC type B was prepared based on Oxoid™ nutrient agar (Cat.# LP0029B, Oxoid Ltd., Hampshire, UK), which included 1 g of Lab-Lemco powder, 2 g of yeast extract (as nutrient), 15 g of agar powder, 5 g of peptone and 5 g of NaCl in 1L of distilled water. Several different pH conditions (pH 4, pH 5, pH 6, pH 6.5, pH 7, pH 8 and pH 9) were used in plates made with both agar types. The pH was adjusted to desired pH levels by adding either 0.1 M hydrochloric acid solution or 0.1 M sodium hydroxide solution to the agar solution. Then, the agar solution was autoclaved at 121 °C for 15 min. Finally, two grams of feces from the first pooled sample were placed on the APC plates types A and B and incubated for three to five days at two temperature conditions (29 to 30 °C: room temperature and 27 °C: control temperature). The second set of pooled fecal samples was used to evaluate the effects of salinity. Plates were made up with various concentrations of NaCl as follows; 0.5%, 1%, 1.5% and 2% in agar type A (containing beef-extract nutrition) and agar type B (containing yeast-extract nutrition). The optimal pH and temperature conditions from the first experiment (pH 6 and incubation at 29-30 °C) were used. Worm counts, including all developmental stages present, were done on days 3, 4, and 5. Each plate was washed with 10 ml of distilled water and collected on each of those days. The washings were centrifuged at 3000 rpm for 10 minutes and the worm pellet mixed with 2 ml of 10% formalin. After thorough mixing, 10 µl of the suspended worms were counted under a light microscope by two independent investigators. The data were derived from the mean of worm count on days 3, 4 and 5. All experiments were performed in duplicate.
2.5 Data analysis Analysis of effects of physicochemical factors (pH, temperature, salinity and nutrition) on the means of worm counts was done to compare between groups using independent t-tests. Comparisons of all conditions between each APC type were performed using ANOVA. Differences were considered as statistically significant if the P-value was less than 0.05. All analyses were performed using SPSS software version 19.
3. Results Strongyloides stercoralis infection screening and effect of the modification APC on the worm counts According to our screening using the standard APC (APC type A: unmodified) technique, the overall prevalence of S. stercoralis infection in Donchang District, Khon Kaen Province, Thailand was 16.28%. Fecal samples from heavily infected individuals (as determined by FECT) were pooled into two sets (N = 10 each) for further study. The intensities of S. stercoralis infection (LPG, determined by FECT) were 136 and 160 in the first and second pooled samples, respectively. The first pooled sample was used to determine the efficacy of different types of APC (type A vs type B) and the effect of pH and temperature of both APC types on the worm number. When cultivated at 27 °C, the highest worm count for APC type A was observed at pH 6 whereas that of APC type B was observed at pH 6.5 (Figure 1). Notably, APC type B yielded higher worm counts than did type A at all pH levels, except at pH 7 and pH 9. Considering the effects of temperature, cultivation at room temperature (29-30 °C) led to a significantly higher worm count in APC Type A than did cultivation at 27 °C at all pH conditions tested, except pH
4, pH 7 and pH 9 (p<0.0001) (Figure 1a). A similar finding was also observed in APC type B, except at pH 7 and pH 9 (p<0.0001) (Figure 1b). Interestingly, at pH 4, pH 6 and pH 6.5, significantly more worms were obtained from APC type B than from APC type A at room temperature (p<0.0001) (Figure 2). APC type B at pH 6 and room temperature provided optimal conditions for S. stercoralis detection. Curiously, at pH 7, standard APC (Type A) yielded more number of worms at 27°C (510 worms) than at 29-30 °C (110 worms), but the difference was not statistically significant (P>0.05). A similar finding was also observed in the modified APC (Type B), which yielded worm counts of 350 and 260 worms for temperatures 27 °C and 29-30 °C, respectively. Since APC type B had NaCl as one of its principle components, we assumed that salinity might affect the worm count of S. stercoralis. We therefore investigated this possibility using the second pooled sample. APC type A and B were prepared at pH 6 but with at different NaCl concentrations (0-2%). On APC type A plates, when cultivated at room temperature, the worm counts were highest at the concentration of 0.5% NaCl followed by 0%, 1 %, 2% and 1.5%, respectively (p < 0.05; Figure 3a). APC type B exhibited the highest worm counts with 0.5% NaCl. Counts were significantly lower at 1 %, 1.5% and 2% NaCl (p < 0.05; Figure 3b). An improved agar plate culture method for diagnosis of Strongyloides stercoralis We compared the ability of standard APC (type A: unmodified), modified APC (type B using optimized conditions, mAPC) and FECT methods to detect S. stercoralis in 20 subjects randomly selected from a community at Khok Samran Sub-District, Khon Kaen Province, Thailand. Modified APC detected infection in 16 cases, standard APC in 9 cases and FECT was positive in only 2 cases. Modified APC found infection in 6 cases which were negative according to both standard APC and FECT. Only four cases were negative according to all three methods.
4. Discussion Since APC is the most sensitive technique to detect S. stercoralis infection (Marchi Blatt and Cantos, 2003; Pocaterra et al., 2017), we used it for community screening. We found a lower overall prevalence (16.28%) than did two previous reports from the same locality and using the same method; 28.9% in 2003 (Sithithaworn et al. in 2003) and 23.0% in 2018 (Laoraksawong et al., 2018). These differences might be due to fluctuations in climate, land cover, soil texture, soil nutrition and soil pH; all of which have an effect on prevalence of S. stercoralis infection (Chaiyos et al., 2018). Agar plate culture (APC) recipes and culture conditions have been partially evaluated in the past, e.g. temperature (Shiwaku et al., 1988). Previously, temperatures ranging from 25-28 °C have been considered as optimum for S. stercoralis development (Anamnart et al., 2015) and have been used for APC incubation in standard laboratory practice, particularly in strongyloidiasis-endemic areas including the Laboratory Service of the Department of Parasitology, Faculty of Medicine, Khon Kaen, Thailand (Intapan et al., 2006). However, our results indicated that a temperature of 29-30 °C (ambient temperature in Thailand during the hot season in March and April) was optimal for worm development. We have also found mature worms on days 4 and 5 in mAPC, indicating that worms undergo development and growth. An increase in mean worm count at 29-30 °C rather than 27°C in both agar types indicates the need for temperature optimization. Similarly, enhanced worm development of S. ratti was found at temperatures >25 °C in cultivation studies (Minato et al., 2008; Sakamoto and Uga, 2013). Soil pH has rarely been reported from S. stercoralis-endemic areas. A pH of 8 was found in Egypt (Etewa et al., 2016). In the present study, we have demonstrated for the first time that a
pH of 6 and incubation at room temperature leads to significantly increased worm counts. In contrast, the lowest worm count was obtained at pH 7 in both types of APC and at both temperatures, implying that this neutral pH might not be suitable for S. stercoralis survival. Some previous studies have used 0.5% NaCl in agar plates for maintenance of S. stercoralis (Pocaterra et al., 2017), but a range of concentrations has not been explored. In this study, we varied the salinity and found that worm development was greatest when NaCl was adjusted to 0.5%. Overall, we found that using 0.5% NaCl, pH 6 and yeast extract as the main nutrient, as in APC type B, could significantly increase the number of worms counted compared to APC type A at the same saline concentration, pH and temperature (29-30 °C). Use of yeast extract as the main nutrient will yield a far higher worm count than the “traditional” APC, which uses meat extract. In addition, our modified APC (type B) could detect a higher worm count than APC type A on day 1 and a significantly higher count on day 3 onward (data not shown). Assuming that nutrient in APC type B might serve as nutritional factor to enhance of worm growth and development and enhance the worm count. Alternatively, nutrient in APC type B may promote more bacterial growth, which in turn, serve as a food source for worms, leading to the enhanced worm development and worm count. We also investigated the efficacy of modified APC (agar type B) in another community (Khok Samran Sub-District) also in Khon Kaen Province, Thailand, compared to standard agar (type A) and FECT. Modified APC of a single stool sample detected S. stercoralis infection more frequently than did standard APC and FECT. In agreement, standard APC was far better for detection of S. stercoralis infection than was FECT (Anamnart et al., 2013). Therefore, our modified APC could increase the sensitivity of screening in a community survey, providing more
accurate diagnosis in immunocompromised patients, such as some kidney disease patients who treating with steroid drugs and hence, at greater risk for disseminated strongyloidiasis even a light infection. Based on our findings, enhanced worm counts and increased sensitivity were achieved using APC with pH 6, 0.5% NaCl, yeast-extract nutrition and incubation temperatures of 29-30 °C. Use of these conditions in a modified APC will improve standard laboratory diagnosis of human strongyloidiasis in endemic areas, and in broad-scale community surveys.
Conflict of interest The authors declare no conflicts of interest exists.
Acknowledgments This study was supported by CKDNET (grant no. CKDNET2559007). The authors would like to thank all of people whom voluntarily participated in this study. KI thanks the scholarship under the Post-Doctoral Training Program from Research Affairs and Graduate School, Khon Kaen University, Thailand. (Grant no. 60163). We would like to acknowledge Prof. David Blair from Publication Clinic KKU, Thailand, for his comments and editing the manuscript.
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Figure legends
Fig 1. Effects of temperature and pH on the number of worms counted after cultivation in APC types A and B: White bar is temperature of this study (29-30 °C) and black bar is the temperature of control (27 °C). All developmental stages of S. stercoralis were counted on days 3, 4 and 5 according to standard practice. Error bar is mean of three-day counts from duplicate experiments. RT = room temperature (29-30 °C); N.S. = no significant difference; **** = significant difference at p < 0.0001 using independent t-tests.
Fig 2. Comparison the number of worm counts between APC type A and type B cultivation: White bar is agar plate culture type A and black bar is agar plate type B under various conditions of pH at the same room temperature (29-30 °C). All developmental stages of S. stercoralis were counted on days 3, 4 and 5 according to standard practice. Error bar is mean of three-day counts from duplicate experiments. N.S. = no significant difference; **** = significant difference at p < 0.0001; *** = significant difference at p < 0.001 using independent t-tests.
Fig 3. Effects of various salinities on the number of worms counted in agar plates type A and B at pH 6 and room temperature (29-30 °C). a: the number of worms counted in agar type A (containing beef-extract nutrition). * = significant difference at p < 0.05 compared to control (no NaCl); # = significant difference at p < 0.001. b: the number of worms counted in agar type B (containing yeast-extract nutrition). * = significant difference at p < 0.05 compared to control (0.5% NaCl) using ANOVA.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Improved agar plate culture conditions for diagnosis of Strongyloides stercoralis Wannee Kaewrat , Chatchawan Sengthong , Manachai Yingklang , Kitti Intuyod , Ornuma Haonon , Sudarat Onsurathum , Rungthiwa Dangtakot , Phitsamai Saisud , Arunnee Sangka , Sirirat Anutarakulchai , Somchai Pinlaor , Ubon Cha’on , Porntip Pinlaor PII: DOI: Reference:
S0001-706X(19)30537-6 https://doi.org/10.1016/j.actatropica.2019.105291 ACTROP 105291
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
20 April 2019 9 October 2019 4 December 2019
Please cite this article as: Wannee Kaewrat , Chatchawan Sengthong , Manachai Yingklang , Kitti Intuyod , Ornuma Haonon , Sudarat Onsurathum , Rungthiwa Dangtakot , Phitsamai Saisud , Arunnee Sangka , Sirirat Anutarakulchai , Somchai Pinlaor , Ubon Cha’on , Porntip Pinlaor , Improved agar plate culture conditions for diagnosis of Strongyloides stercoralis, Acta Tropica (2019), doi: https://doi.org/10.1016/j.actatropica.2019.105291
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Highlights
We optimized agar plate culture (APC) for detection of Strongyloides stercoralis.
A pH of 6.0, 0.5% NaCl and temperature of 29-30 °C were found to be optimum.
Testing in an endemic community confirmed this.
Improved agar plate culture conditions for diagnosis of Strongyloides stercoralis
Wannee Kaewrat1,7, Chatchawan Sengthong2,7 , Manachai Yingklang2,7, Kitti Intuyod2,7 Ornuma Haonon2,7, Sudarat Onsurathum2,7, Rungthiwa Dangtakot 3,7, Phitsamai Saisud4, Arunnee Sangka4,7, Sirirat Anutarakulchai5,7, Somchai Pinlaor2,7, Ubon Cha’on6,7, Porntip Pinlaor4,7*
1
Science Program in Clinical Pathology and Management, Faculty of Associated Medical
Sciences, Khon Kaen University, Khon Kaen 40002, Thailand 2
Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002,
Thailand 3
Science Program in Biomedical Science, Khon Kaen University, Khon Kaen 40002, Thailand
4
Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of
Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand 5
Department of Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002,
Thailand 6
Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002,
Thailand 7
Chronic Kidney Disease Prevention in The Northeastern Thailand, Faculty of Medicine, Khon
Kaen University, Khon Kaen 40002, Thailand
*Correspondence: [email protected]
ABSTRACT Strongyloides stercoralis infection causes gastrointestinal symptoms and can lead to severe disease in immunocompromised hosts. Live larvae are passed in feces, encouraging the common use of diagnosis by cultivation methods including agar plate culture (APC), the gold-standard technique. Nevertheless, APC has limitations, especially since there can be considerable day-today fluctuations in numbers of larvae produced. Herein, we collected stool samples from heavily infected subjects with strongyloidiasis in Khon Kaen Province, Thailand, to evaluate modifications (temperature, pH, nutrition source and salinity) to APC conditions to maximize the number of S. stercoralis worms counted. Best results were obtained using a modified APC with the following conditions: pH 6.0, 0.5% of NaCl, addition of yeast extract for nutrition and incubation at 29-30 °C. This modified APC was more sensitive for detection of S. stercoralis than was standard APC or the formalin-ethyl acetate concentration technique. In brief, this finding suggests that a modification of standard APC conditions increases the counts of S. stercoralis.
Key words: Strongyloides stercoralis, physicochemical factors, agar plate culture, worm development.
1. Introduction Human strongyloidiasis is mainly caused by Strongyloides stercoralis infection and is still a major health problem worldwide, especially in tropical and subtropical countries including Thailand (Schar et al., 2013), where the reported prevalence of S. stercoralis infection ranges from 0.05 to 57.0% (Jongsuksuntigul et al., 2003; Laoraksawong et al., 2018; Sithithaworn et al.,
2003). Infection with S. stercoralis in healthy people generally does not induce severe gastrointestinal symptoms. In contrast, in immunocompromised patients, such as those with chronic kidney disease, diabetes mellitus or HIV, the parasites can rapidly multiply leading to hyperinfection (De Souza et al., 2018; Krolewiecki and Nutman, 2019; Qu et al., 2016; Winnicki et al., 2018). Moreover, patients receiving immunosuppressive drugs or corticosteroid treatment are prone to autoinfection and disseminated infection, the latter being the most important lifethreatening complication of strongyloidiasis (Abanyie et al., 2018; Camargo et al., 2019; Mileo Bacelar Guerreiro et al., 2018; Paula et al., 2018). Although stool examination techniques, such as simple smear, the Kato-Katz technique and formalin-ether concentration are commonly used in prevalence studies of intestinal parasites, they are inadequate for S. stercoralis detection (Buonfrate et al., 2015; Campo Polanco et al., 2014). The Baermann technique and Koga agar plate culture have better sensitivity (Campo Polanco et al., 2014) but are still unsatisfactory (Buonfrate et al., 2018; Krolewiecki and Nutman, 2019). Currently, the agar plate culture (APC) method is considered the gold-standard for diagnosis of S. stercoralis infection. It is used in standard laboratory screening in endemic countries, including Thailand (Formenti et al., 2019; Koga et al., 1991; Kristanti et al., 2018). This technique maintains the parasite development by simulation of natural climatic and physicochemical requirements such as nutrition, humidity and temperature (Viney and Lok, 2015). Nevertheless, APC has some limitations, including those caused by fluctuation in numbers of larvae released in feces each day (Uparanukraw et al., 1999). Also, the APC method has low sensitivity in the case of light infections (Anamnart et al., 2010; Intapan et al., 2006; Ketzis, 2017). Shiwaku et al. (1988) modified some physicochemical parameters in cultures, such as temperature and fecal dilution, to try to increase development of free-living generations
of S. stercoralis under cultivation. Other conditions such as pH, nutrition and salinity in the APC remain to be optimized. Here, we have investigated the influence of temperature, pH, nutrition and salinity on the numbers of S. stercoralis recovered. This provides a better understanding of the optimal culture conditions for the APC, the most widely used diagnostic tool for S. stercoralis infection.
2. Materials and Methods 2.1 Study population, study design and ethics statement The human ethical review committee of Khon Kaen University (HE611068) approved the protocol of study. This was a part of larger study, the Chronic Kidney Disease Northeastern Thailand (CKDNET) study. Informed consent forms and plastic containers were provided to each volunteer before stool collection. The study was conducted from January 2017 to May 2018 at Donchang Sub-District, Muang District, Khon Kaen Province, northeastern Thailand, where a high prevalence of 23% of S. stercoralis infection was previously reported in 2003 (Sithithaworn et al., 2003). The sample size was calculated as described elsewhere (Sithithaworn et al., 2003): based on a prevalence of 23%, with 95% confidence interval (z = 1.96) and 0.5 of an error, we calculated that 273 individuals were required for the study. However, 793 individuals (>35 years of age) volunteered and were enrolled for the study. All were screened for S. stercoralis infection using standard APC (Intapan et al., 2005). At the end of the work, individuals infected with S. stercoralis were treated with a single dose of 200 µg/kg of ivermectin. 2.2 Initial screening for S. stercoralis infection by standard agar plate culture Two grams of fecal material from each subject (793 subjects) were immediately placed on standard APCs individually (APC type A – see below) and covered with parafilm during
fieldwork in the community. All plates were transferred to the Department of Parasitology, Faculty of Medicine, Khon Kaen University, Thailand where they were incubated at room temperature (29-30 °C) for three to five days. On days 3, 4 and 5, each plate was washed with 10 ml of sterile normal saline solution, which was decanted and centrifuged at 3000 rpm for 10 min to collect the worms. The worm species was identified under a light microscope. 2.3 Sources of fecal material for evaluation of modified APC tests Following the initial screening, 20 heavily infected individuals were asked to provide additional stool samples. From these, two sets of pooled stool samples were made, material from ten subjects for each set. The first pooled sample was used for testing effects of agar plate types (which reflected the differences of nutrition sources), pH and temperature, and the second was used for testing salinity. Each pooled sample was mixed thoroughly to ensure homogeneous distribution of larvae before experiments were started. The number of larvae per gram (LPG) was determined in each pooled sample using the formalin-ethyl acetate concentration technique (FECT), performed as described elsewhere (Sithithaworn et al., 2003). In brief, two grams of fecal sample were fixed with 10% formalin solution and shaken vigorously. After centrifugation, the supernatant was discarded and the pellet was resuspended in 0.85% sodium chloride solution (normal saline solution; NSS), filtered through gauze and centrifuged at 2,500 rpm for 5 min. After removal of supernatant, the pellet was resuspended in ethyl acetate, the mixture was recentrifuged at 2,500 rpm for 5 min. Finally, the top three layers were discarded and the numbers of S. stercoralis larvae in the sediment were counted under a light microscope. 2.4 Preparation and evaluation of modified APC conditions Two types of APC (types A and B) were prepared according to the manufacturer's instructions, each type with a series of different pH, salinity and nutrient conditions and
incubated at various temperatures. The unmodified APC type A was the standard APC as used for many years in our laboratory and elsewhere. This contained 3 g beef extract (as nutrient), 5 g of peptone and 15 g of Difco™ nutrient agar, BD (BD Bioscience, MD, USA) in 1L of distilled water. The APC type B was prepared based on Oxoid™ nutrient agar (Cat.# LP0029B, Oxoid Ltd., Hampshire, UK), which included 1 g of Lab-Lemco powder, 2 g of yeast extract (as nutrient), 15 g of agar powder, 5 g of peptone and 5 g of NaCl in 1L of distilled water. Several different pH conditions (pH 4, pH 5, pH 6, pH 6.5, pH 7, pH 8 and pH 9) were used in plates made with both agar types. The pH was adjusted to desired pH levels by adding either 0.1 M hydrochloric acid solution or 0.1 M sodium hydroxide solution to the agar solution. Then, the agar solution was autoclaved at 121 °C for 15 min. Finally, two grams of feces from the first pooled sample were placed on the APC plates types A and B and incubated for three to five days at two temperature conditions (29 to 30 °C: room temperature and 27 °C: control temperature). The second set of pooled fecal samples was used to evaluate the effects of salinity. Plates were made up with various concentrations of NaCl as follows; 0.5%, 1%, 1.5% and 2% in agar type A (containing beef-extract nutrition) and agar type B (containing yeast-extract nutrition). The optimal pH and temperature conditions from the first experiment (pH 6 and incubation at 29-30 °C) were used. Worm counts, including all developmental stages present, were done on days 3, 4, and 5. Each plate was washed with 10 ml of distilled water and collected on each of those days. The washings were centrifuged at 3000 rpm for 10 minutes and the worm pellet mixed with 2 ml of 10% formalin. After thorough mixing, 10 µl of the suspended worms were counted under a light microscope by two independent investigators. The data were derived from the mean of worm count on days 3, 4 and 5. All experiments were performed in duplicate.
2.5 Data analysis Analysis of effects of physicochemical factors (pH, temperature, salinity and nutrition) on the means of worm counts was done to compare between groups using independent t-tests. Comparisons of all conditions between each APC type were performed using ANOVA. Differences were considered as statistically significant if the P-value was less than 0.05. All analyses were performed using SPSS software version 19.
3. Results Strongyloides stercoralis infection screening and effect of the modification APC on the worm counts According to our screening using the standard APC (APC type A: unmodified) technique, the overall prevalence of S. stercoralis infection in Donchang District, Khon Kaen Province, Thailand was 16.28%. Fecal samples from heavily infected individuals (as determined by FECT) were pooled into two sets (N = 10 each) for further study. The intensities of S. stercoralis infection (LPG, determined by FECT) were 136 and 160 in the first and second pooled samples, respectively. The first pooled sample was used to determine the efficacy of different types of APC (type A vs type B) and the effect of pH and temperature of both APC types on the worm number. When cultivated at 27 °C, the highest worm count for APC type A was observed at pH 6 whereas that of APC type B was observed at pH 6.5 (Figure 1). Notably, APC type B yielded higher worm counts than did type A at all pH levels, except at pH 7 and pH 9. Considering the effects of temperature, cultivation at room temperature (29-30 °C) led to a significantly higher worm count in APC Type A than did cultivation at 27 °C at all pH conditions tested, except pH
4, pH 7 and pH 9 (p<0.0001) (Figure 1a). A similar finding was also observed in APC type B, except at pH 7 and pH 9 (p<0.0001) (Figure 1b). Interestingly, at pH 4, pH 6 and pH 6.5, significantly more worms were obtained from APC type B than from APC type A at room temperature (p<0.0001) (Figure 2). APC type B at pH 6 and room temperature provided optimal conditions for S. stercoralis detection. Curiously, at pH 7, standard APC (Type A) yielded more number of worms at 27°C (510 worms) than at 29-30 °C (110 worms), but the difference was not statistically significant (P>0.05). A similar finding was also observed in the modified APC (Type B), which yielded worm counts of 350 and 260 worms for temperatures 27 °C and 29-30 °C, respectively. Since APC type B had NaCl as one of its principle components, we assumed that salinity might affect the worm count of S. stercoralis. We therefore investigated this possibility using the second pooled sample. APC type A and B were prepared at pH 6 but with at different NaCl concentrations (0-2%). On APC type A plates, when cultivated at room temperature, the worm counts were highest at the concentration of 0.5% NaCl followed by 0%, 1 %, 2% and 1.5%, respectively (p < 0.05; Figure 3a). APC type B exhibited the highest worm counts with 0.5% NaCl. Counts were significantly lower at 1 %, 1.5% and 2% NaCl (p < 0.05; Figure 3b). An improved agar plate culture method for diagnosis of Strongyloides stercoralis We compared the ability of standard APC (type A: unmodified), modified APC (type B using optimized conditions, mAPC) and FECT methods to detect S. stercoralis in 20 subjects randomly selected from a community at Khok Samran Sub-District, Khon Kaen Province, Thailand. Modified APC detected infection in 16 cases, standard APC in 9 cases and FECT was positive in only 2 cases. Modified APC found infection in 6 cases which were negative according to both standard APC and FECT. Only four cases were negative according to all three methods.
4. Discussion Since APC is the most sensitive technique to detect S. stercoralis infection (Marchi Blatt and Cantos, 2003; Pocaterra et al., 2017), we used it for community screening. We found a lower overall prevalence (16.28%) than did two previous reports from the same locality and using the same method; 28.9% in 2003 (Sithithaworn et al. in 2003) and 23.0% in 2018 (Laoraksawong et al., 2018). These differences might be due to fluctuations in climate, land cover, soil texture, soil nutrition and soil pH; all of which have an effect on prevalence of S. stercoralis infection (Chaiyos et al., 2018). Agar plate culture (APC) recipes and culture conditions have been partially evaluated in the past, e.g. temperature (Shiwaku et al., 1988). Previously, temperatures ranging from 25-28 °C have been considered as optimum for S. stercoralis development (Anamnart et al., 2015) and have been used for APC incubation in standard laboratory practice, particularly in strongyloidiasis-endemic areas including the Laboratory Service of the Department of Parasitology, Faculty of Medicine, Khon Kaen, Thailand (Intapan et al., 2006). However, our results indicated that a temperature of 29-30 °C (ambient temperature in Thailand during the hot season in March and April) was optimal for worm development. We have also found mature worms on days 4 and 5 in mAPC, indicating that worms undergo development and growth. An increase in mean worm count at 29-30 °C rather than 27°C in both agar types indicates the need for temperature optimization. Similarly, enhanced worm development of S. ratti was found at temperatures >25 °C in cultivation studies (Minato et al., 2008; Sakamoto and Uga, 2013). Soil pH has rarely been reported from S. stercoralis-endemic areas. A pH of 8 was found in Egypt (Etewa et al., 2016). In the present study, we have demonstrated for the first time that a
pH of 6 and incubation at room temperature leads to significantly increased worm counts. In contrast, the lowest worm count was obtained at pH 7 in both types of APC and at both temperatures, implying that this neutral pH might not be suitable for S. stercoralis survival. Some previous studies have used 0.5% NaCl in agar plates for maintenance of S. stercoralis (Pocaterra et al., 2017), but a range of concentrations has not been explored. In this study, we varied the salinity and found that worm development was greatest when NaCl was adjusted to 0.5%. Overall, we found that using 0.5% NaCl, pH 6 and yeast extract as the main nutrient, as in APC type B, could significantly increase the number of worms counted compared to APC type A at the same saline concentration, pH and temperature (29-30 °C). Use of yeast extract as the main nutrient will yield a far higher worm count than the “traditional” APC, which uses meat extract. In addition, our modified APC (type B) could detect a higher worm count than APC type A on day 1 and a significantly higher count on day 3 onward (data not shown). Assuming that nutrient in APC type B might serve as nutritional factor to enhance of worm growth and development and enhance the worm count. Alternatively, nutrient in APC type B may promote more bacterial growth, which in turn, serve as a food source for worms, leading to the enhanced worm development and worm count. We also investigated the efficacy of modified APC (agar type B) in another community (Khok Samran Sub-District) also in Khon Kaen Province, Thailand, compared to standard agar (type A) and FECT. Modified APC of a single stool sample detected S. stercoralis infection more frequently than did standard APC and FECT. In agreement, standard APC was far better for detection of S. stercoralis infection than was FECT (Anamnart et al., 2013). Therefore, our modified APC could increase the sensitivity of screening in a community survey, providing more
accurate diagnosis in immunocompromised patients, such as some kidney disease patients who treating with steroid drugs and hence, at greater risk for disseminated strongyloidiasis even a light infection. Based on our findings, enhanced worm counts and increased sensitivity were achieved using APC with pH 6, 0.5% NaCl, yeast-extract nutrition and incubation temperatures of 29-30 °C. Use of these conditions in a modified APC will improve standard laboratory diagnosis of human strongyloidiasis in endemic areas, and in broad-scale community surveys.
Conflict of interest The authors declare no conflicts of interest exists.
Acknowledgments This study was supported by CKDNET (grant no. CKDNET2559007). The authors would like to thank all of people whom voluntarily participated in this study. KI thanks the scholarship under the Post-Doctoral Training Program from Research Affairs and Graduate School, Khon Kaen University, Thailand. (Grant no. 60163). We would like to acknowledge Prof. David Blair from Publication Clinic KKU, Thailand, for his comments and editing the manuscript.
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Figure legends
Fig 1. Effects of temperature and pH on the number of worms counted after cultivation in APC types A and B: White bar is temperature of this study (29-30 °C) and black bar is the temperature of control (27 °C). All developmental stages of S. stercoralis were counted on days 3, 4 and 5 according to standard practice. Error bar is mean of three-day counts from duplicate experiments. RT = room temperature (29-30 °C); N.S. = no significant difference; **** = significant difference at p < 0.0001 using independent t-tests.
Fig 2. Comparison the number of worm counts between APC type A and type B cultivation: White bar is agar plate culture type A and black bar is agar plate type B under various conditions of pH at the same room temperature (29-30 °C). All developmental stages of S. stercoralis were counted on days 3, 4 and 5 according to standard practice. Error bar is mean of three-day counts from duplicate experiments. N.S. = no significant difference; **** = significant difference at p < 0.0001; *** = significant difference at p < 0.001 using independent t-tests.
Fig 3. Effects of various salinities on the number of worms counted in agar plates type A and B at pH 6 and room temperature (29-30 °C). a: the number of worms counted in agar type A (containing beef-extract nutrition). * = significant difference at p < 0.05 compared to control (no NaCl); # = significant difference at p < 0.001. b: the number of worms counted in agar type B (containing yeast-extract nutrition). * = significant difference at p < 0.05 compared to control (0.5% NaCl) using ANOVA.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: