Abstract
Sapovirus are important agents of acute gastroenteritis (AGE) and they are associated with outbreaks and sporadic cases worldwide. They infect people of all ages, but mainly children, the elderly and immunocompromised individuals are affected. The aim of this study was investigate sapovirus and to determine viral loads in fecal samples from patient undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Fecal samples were submitted to extraction of the genetic material using a commercial kit, and RT-qPCR TaqMan was used for sapovirus screening and determination of viral loads, using a standard curve with serial dilutions of a recombinant plasmid. Positive samples were sequence by Sanger method. Sapovirus was detected in one patient, 5.3% (1/19). Viral excretion lasted for 16 days. Viral load varied from 1.73 × 106 to 8.97 × 106 GC/g. One of the positive samples was characterized as GI.1 genotype. This is the first study to determine sapovirus loads in samples from allo-HSCT and to identify GI.1 genotype in immunocompromised patients.
Keywords: Sapovirus, Immunocompromised patients, GI.I sapovirus, Allo-HSCT, RT-qPCR
Human caliciviruses (sapovirus and norovirus) are important agents of acute gastroenteritis (AGE) and are associated with outbreaks and sporadic cases worldwide [1, 8]. They are classified in the Caliciviridae family, genera Sapovirus and Norovirus, respectively. The genus Sapovirus is further subdivided into genogroups and genotypes. The genogroups (G) I–V have been recognized to date, but only the GI, GII, GIV and GV genogroups infect humans [7].
Sapoviruses are transmitted by fecal–oral route, through ingestion of contaminated water and food and by person-to-person contact. The human caliciviruses infections are usually self-limited and the main associated symptoms are diarrhea and vomiting. They can affect people of all ages, but mainly children, the elderly and immunocompromised individuals [5, 8]. Noroviruses have been detected more frequently in immunocompromised individuals, whereas sapoviruses reports among this population are still scarce [3, 5, 10]. Although the clinical impact of norovirus infection in immunocompromised patients remains to be fully understood, there is report of a worse prognosis for patients that were submitted to renal transplant [12]. Norovirus prolonged shedding by hematopoietic stem cell transplant (HSCT) patients, has also been reported [5].
During the transplant conditioning period, these patients are submitted to immunosuppression, which is maintained for some time after the procedure [15]. Diarrhea is a frequently reported by these patients, and graft-versus-host disease (GVHD) is the main complication presented, that may also present diarrhea as a symptom [11, 12]. In addition, the patient becomes more susceptible to opportunistic infections [15]. Therefore, monitoring HSCT patients for various pathogens is important for a better prognosis and optimal treatment of the patient. The aim of this study was to investigate and to determine sapovirus loads in serially collected fecal samples of patients undergoing allogeneic HSCT (allo-HSCT) at a reference center for bone marrow transplantation in Mid-West Brazil.
This was a longitudinal retrospective study that included 19 patients submitted to allo-HSCT at a reference bone marrow transplant unit center (Hospital Araújo Jorge) in Goiás, Brazil. Fecal samples were collected, from October 2012 to July 2014. Patients' follow-up varied from four to 586 days. One fecal sample was also obtained from each patient before the transplant. After transplantation, fecal samples were collected weekly; however, when the patient presented diarrhea, samples were obtained more frequently. After hospital discharge, samples were collected during outpatient consultations. Information on patients’ symptoms during the period of the study was obtained from patients’ medical records. This study was approved by Ethics Review Committee of Universidade Federal de Goiás (CAAE: 67782917.3.0000.5083) and samples were collected after a signed informed consent form.
Fecal samples were processed (20% fecal suspensions in PBS pH 7.4) and stored at − 80 °C until further testing. Samples were extracted using a commercial kit QIAamp® RNA Mini Kit (Qiagen—Hilden, Germany) following the manufacturer's instructions. Viral loads were determined by RT-qPCR TaqMan using with primers (SaV124F, SaV1F, SaV1245R) and probe (SaV124TP), according to Oka et al. 2006 [6], specific for genogroups I, II and IV, targeting the polymerase/capsid viral genome region, using a standard curve with serial dilutions of recombinant plasmid containing the polymerase/capsid partial region.
Positive samples were submitted to amplification with SLV5317 and SLV5749 primer pairs targeting the ORF1 (polymerase/capsid junction), generating a 434 bp product [16]. Products were purified by using 65% isopropanol and 70% ethanol, and then submitted to a sequencing reaction [Big Dye Terminatorv3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA)] in an automatic sequencer (DNA ABI PRISM 3130, Applied Biosystems). The sequence quality and consensus sequence were obtained using BioEdit 7.0.5.3 [4]. The genotype was characterized using an online platform (Norovirus Typing Tool 2.0), which also allows sapovirus characterization. The nucleotide sequence was deposited in the NCBI under the accession number: MN097151.
To our knowledge, this is the first study to determine sapovirus loads in samples from allo-HSCT and to identify GI.1 genotype in immunocompromised patients. Sapovirus was detected in 5.3% (1/19) of the patients. The positive patient was diagnosed with acute lymphocytic leukemia (ALL), and was 21 years old at the time of the sample collection. The follow-up period lasted for 104 days. The patient was admitted to the hospital on 06/14/2013 and received the transplant 12 days after. Higher positivity rate was observed in our study (5.3%) when compared with other studies that have identified sapovirus in samples from immunocompromised patients, such as patients submitted to transplants [3, 10, 12] although, recently, another study reported a higher rate of sapovirus positivity (9.7%) in children submitted to hematopoietic cell transplant, being the only calicivirus identified [14]. The identification of this calicivirus in samples from such patients demonstrates the importance of including sapovirus monitoring, for a better understanding of the impact of this virus in the population.
The first fecal sample obtained in this period was negative for sapovirus (Table 1). After that, the patient excreted sapovirus for 16 days, with the second, third and fourth samples being positive for sapovirus, with viral loads of 8.97 × 106 GC/g, 1.73 × 106 GC/g and 3.57 × 106 GC/g respectively.
Table 1.
Timeline of sapovirus monitoring in fecal samples from the allo-HSCT sapovirus-positive patient
| Sample number | Sample collection date | Presence of diarrhea | Results of SaV by RT-qPCR | Viral load (GC/g) |
|---|---|---|---|---|
| 01 | 06/20/2013 | No | Negative | – |
| 02 | 06/29/2013 | Yes | Positive | 8.97 × 106 |
| 03 | 07/02/2013 | Yes | Positive | 1.73 × 106 |
| 04 | 07/12/2013 | Yes | Positive | 3.57 × 106 |
| 05 | 07/16/2013 | Yes | Negative | – |
| 06 | 07/22/2013 | Yes | Negative | – |
| 07 | 07/27/2013 | Yes | Negative | – |
| 08 | 08/31/2013 | No | Negative | – |
| 09 | 08/06/2013 | No | Negative | – |
| 10 | 08/17/2013 | Yes | Negative | – |
| 11 | 08/18/2013 | Yes | Positive | 2.57 × 106 |
| 12 | 09/30/2013 | Yes | Negative | – |
Gastrointestinal symptoms (diarrhea) were reported starting from the date of the third sample collection (07/02/2013), lasting for approximately 25 days. Even though diarrhea persisted, the fifth, sixth and seventh samples obtained were all negative for sapovirus. The patient was discharged on 07/29/2013, and during the outpatient visits, the eighth and ninth samples were obtained, being negative for sapovirus. At this time patient did not present diarrhea; however, when the 10th sample was obtained the patient reported the occurrence of diarrhea, and the sample was negative for sapovirus. The 11th sample was positive for sapovirus, with a viral load of 2.57 × 106 GC/g and diarrhea was also reported. The last sample (12th) was obtained after 43 days and it was negative for sapovirus; however, the patient still reported diarrhea.
Persistent excretion of sapovirus (14 to 30 days) detected in samples from this patient, was similar to that reported in a renal transplanted patient [12], and chronic excretion (> 30 days) of sapovirus has also been documented in a bone marrow transplant patient study [10]. Human adenovirus were also detected in this patient from our study, in a serum sample which was obtained between the 11th and 12th fecal samples collection, in a study previously conducted by Santos et al. 2014 in this cohort of patients [13]. In addition, this patient was also positive for human bocavirus in the 8th and 11th fecal samples [2]; which characterized co-infection, since the the 11th fecal sample was also positive for sapovírus in our study.
One of the positive samples by RT-qPCR had good quality to be sequenced, and was characterized as GI.1 genotype. This GI.1 sequence had high nucleotide identity (99%) to a sequence obtained from a fecal sample of a day-care child, in previous study [9], indicating that this genotype had been circulating in this region since 2009.
In conclusion, this data reinforces the need of monitoring sapovirus among other gastroenteric virus in transplanted patients, since infection by these agents can result in symptoms that mimic GVHD. The misdiagnosis may lead to an increase in immunosuppression which could result in complications. Furthermore, the identification of more than one gastrointestinal agent in the same samples from the same patient should be better investigated, because this could contribute to a worse prognosis for these immunocompromised patients.
Acknowledgments
We thank all the staff members from the bone marrow transplant unit of Hospital Araujo Jorge who collected the patients' samples. This study was supported by Grant of Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (MCTI/CNPq/Universal 2014). The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare no conflicts of interest.
Footnotes
Publisher's Note
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Thairiny Neres Silva and Nathânia Dábilla have contributed equally to this work.
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