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Identification of Volatile Biomarkers of Giardia Duodenalis Infection in Children with Persistent Diarrhoea

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Identification of Volatile Biomarkers of Giardia Duodenalis Infection in Children with Persistent Diarrhoea Parasitology Research (2019) 118:3139–3147 https://doi.org/10.1007/s00436-019-06433-4 PROTOZOOLOGY - ORIGINAL PAPER Identification of volatile biomarkers of Giardia duodenalis infection in children with persistent diarrhoea C. Ubeda1 & E. Lepe-Balsalobre 2 & C. Ariza-Astolfi2 & J. M. Ubeda-Ontiveros2 Received: 28 March 2019 /Accepted: 21 August 2019 /Published online: 12 October 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract Currently, chronic diarrhoea syndrome in children is a very common pathology whose aetiology is sometimes difficult to identify. Methodologies for the diagnosis of infections have diversified, and gas chromatography/mass spectrometry (GC/MS) is a very useful tool. The aim of this study was to identify volatile biomarkers of the presence of Giardia duodenalis in the faeces of patients with chronic diarrhoea (with and without giardiasis) using static headspace extraction followed by GC/MS. The analysis of the volatiles extracted from the headspace had enough sensitivity to detect differences in the volatile profiles in the faeces of the patients with and without Giardia duodenalis infection and discriminate between them. Dimethyl disulphide and trisulphide were found in the faeces of patients without giardiasis but not in the faeces of patients with G. duodenalis. Finally, three possible biomarkers, acetic acid, 1,4-dimethoxy-2,3-butanediol and 1,3-dimethoxy-2-propanol, were proposed to identify patients with giardiasis; these compounds were not present in the patients without the parasite. Multivariate analysis revealed that principal component 1 separated the stool samples according to the presence of infection by G. duodenalis despite the inter-individual variability in biological specimens such as faeces. Keywords Biomarkers . Volatile compounds . Giardia duodenalis . Gas chromatography . Chronic diarrhoea . Persistent diarrhoea Introduction 14 days and is not exclusively associated with an infectious cause (Lee et al. 2012). Therefore, persistent diarrhoea is in- In children, prolonged episodes of diarrhoea are characterised cluded in a large overall category called chronic diarrhoea by an increased frequency, a reduced consistency and an in- syndrome. The characteristics of the stools and their accom- creased volume of bowel movements that do not improve and panying symptoms vary according to the pathophysiology of that last more than 2 weeks; these episodes are considered as the diarrhoea (osmotic, secretory, steatotic), its impact on the chronic and/or persistent diarrhoea (Keating 2005; Baran nutritional status of the child and, sometimes, its aetiology 2014). The concepts of “persistent diarrhoea” and “chronic (Baran 2014). In this context, one of the most common proto- diarrhoea” are different. Persistent diarrhoea is an episode of zoans associated with persistent diarrhoea is Giardia diarrhoea with an infectious aetiology that develops acutely duodenalis, which has been reported in some studies to have but lasts longer than 14 days (Mathai et al. 2011), whereas a prevalence of 66.4% in patients presenting with this symp- chronic diarrhoea is defined as diarrhoea that lasts more than tom (McHardy et al. 2014). The detection of G. duodenalis by direct immunofluores- cence has a sensitivity that ranges between 99 and 100%, with Section Editor: Lihua Xiao a specificity of 100% compared with conventional techniques (66% for conventional microscopy and 93% for conventional * J. M. Ubeda-Ontiveros trichrome staining) (García et al. 1992). Current methodolo- [email protected] gies for the diagnosis of diseases have become diversified, and 1 Instituto de Ciencias Biomédicas, Facultad de Ciencias, Universidad the use of immunological-based assays and molecular Autónoma de Chile, Providencia, Región Metropolitana, Chile methods has increased (Parčina et al. 2018;Hooshyaretal. 2 Department of Microbiology and Parasitology, Faculty of Pharmacy, 2019). The search for specific biomarkers is the current ten- University of Seville, 41012 Seville, Spain dency, and in this scientific field, gas chromatography (GC) 3140 Parasitol Res (2019) 118:3139–3147 has been demonstrated to be a reliable tool to determine char- point of view, the HS extraction method is cheaper than the acteristic volatile organic compounds for a large range of dis- SPME technique and prolongs the durability of the equipment eases (Shirasu and Touhara 2011). Furthermore, the detection because it introduces less interference. of volatiles at low levels allows the diagnosis of a disease in the early stages of development. Alterations in the volatile profile of various samples (faeces, urine, sweat, breath) reflect Material and methods the physiological and physiopathological metabolic processes that may take place in the human body. The employment of Samples scents for the detection of diseases has been used from the time of Hippocrates through today; scent is currently used to Samplesoffaecesfromchildrenbetween2and6yearsold detect the presence of diseases such as cancer, Alzheimer’s suffering from chronic diarrhoea symptoms (20–30 days with disease and Parkinson’s disease (Khatib et al. 2014; Wang diarrhoea) collected in May 2018 in the Hospital Universitario et al. 2014;Mazzatentaetal.2015). Virgen de Valme, Seville (Spain) were analysed. The micro- Among the diseases able to be diagnosed with gas chroma- scopic diagnosis of Giardia was carried out for all the sam- tography, infections are very interesting because the presence ples. Ten samples were from patients with diarrhoea syndrome of a specific microorganism is commonly associated with a with a non-parasitic aetiology (NPD), and seven samples were characteristic smell in various organic samples due to micro- from patients with diarrhoea syndrome with Giardia presence bial metabolism (Ho and Reddy 2011; Bos et al. 2013). For (GD). Among them, eight samples were from males and 9 example, faeces from Clostridium difficile patients produces were from females. The samples were collected in standard 5-methyl-2-furancarboxyaldehyde (Probert et al. 2004) and plastic faeces sample receptacles and frozen on the same day isocaproic acid (Tait et al. 2014), faeces from patients with of collection for 1 week at − 20 °C until analysis. Campylobacter jejuni present 1-butoxy-2-propanol and 3- methyl furan (Garner et al. 2007), and faeces from patients Reagents and standards with Vibrio cholerae produces extremely large quantities of dimethyl disulphide and p-menth-1-en-8-ol (Garner et al. For the semi-quantification of volatile compounds, 4-methyl- 2009). As mentioned above, G. duodenalis is one of the most 2-pentanol was used as an internal standard supplied by prevalent protozoans causing persistent diarrhoea in children. Merck (Darmstadt, Germany) and sodium chloride (NaCl) In general, patients with giardiasis are reported to expel a foul purchased from Sigma-Aldrich (Madrid, Spain). The Kovats gas with a distinctive foul smell through eructations or flatuses index was calculated using a solution of n-alkanes (C6-C30) (Mørch et al. 2009). This characteristic smell has been from Merck ((Darmstadt, Germany). analysed by Bond et al. (2015)infaecesfrompatientswith G. duodenalis; results suggested that 2,2,4,4- Extraction and analysis of volatile compounds tetramethyloctane, acetic acid and 2,2,4,6,6- pentamethylheptane were potential biomarkers of giardiasis. The extraction method employed was static headspace (HS). In this study, volatile compounds from faeces were extracted This method was carried out placing 3.5 g of faeces in a by solid phase microextraction (SPME), a sensible technique 20-mL vial to which 3.5 mL of a NaCl-distilled water mixture employed to extract major and minor volatile compounds (20%) was added to force the migration of the volatile com- from samples. The aim of this study was to detect volatile pounds to the headspace. Next, 10 μLof4-methyl-2-pentanol biomarkers of the presence of G. duodenalis from the faeces (0.75 mg L−1) was added as an internal standard. Finally, the of patients with chronic diarrhoea. The authors would like to vial was closed with an aluminium screw cap with a silicone note that the aim was not to propose a direct detection method septum/PETF (VWR International Eurolab S.L., Barcelona, for G. duodenalis with gas chromatography, but rather to de- Spain) and placed in the autosampler. tect molecules that are markers of the presence of Giardia in Analyses were carried out in a Thermo Scientific Gas faeces to employ these molecules as targets to develop simple Chromatograph coupled to a triple quadrupole mass spec- diagnostic methods, i.e. colorimetric methods, in the future. trometer (TSQ8000-Thermo Scientific) equipped with an For this purpose, we employed the following extraction tech- autosampler and automatic injector. The sample in the vial nique, which is used for the extraction of volatile compounds was incubated for 30 min at a temperature of 50 °C with that are found in samples in large quantities: static headspace continuous stirring while heating. At the end of this time, extraction followed by gas chromatography coupled to mass 800 μL of gas was taken from the headspace and injected into spectrometry (HS/GC/MS). The detection of biomarkers the gas chromatograph using a 1-mL syringe of heated capac- found in high amounts in faeces would indicate them as pos- ity (50 °C) in split mode 10:1. The injection temperature was sible targets for the development of more cost effective detec- 250 °C, and the capillary column employed was a ZB- tion methods for this parasite. Additionally, from a technical WAXplus column (30 m × 0.25 mm, 0.25-μm thickness, Parasitol Res (2019) 118:3139–3147 3141 Zebron). The carrier gas was helium at a constant flow of et al. (2015) found significant differences in the amounts of 1 mL/min. The temperature of the oven started at 40 °C, was propyl propanoate and ethyl butanoate between patients with maintained for 1 min and then rose to 100 °C at a rate of giardiasis and the control group, finding more of both in the 2.5 °C/min and then up to 200 °C at a rate of 15 °C/min.
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