Diagnostic determination of Norovirus infection as one of the major causes of infectious diarrhea in HIV patients using a multiplex polymerase chain reaction assay

Abstract

Although infectious diarrhea is one of the most common complications in human immunodeficiency virus (HIV)-infected patients, robust diagnostic methods for determining potential pathogens are still limited in the clinic. Norovirus, a type of calicivirus, has been shown to be the most common cause of gastroenteritis. Here, we used multiplex polymerase chain reaction as a diagnostic tool to verify Norovirus as the diarrhea-related pathogen in HIV-infected patients with unknown etiological information. Stool specimens obtained from 81 HIV-infected patients with diarrhea were analyzed by BioFire FilmArray Gastrointestinal (GI) panel. Among 26 HIV-infected patients with unknown etiological information, we detected Norovirus in 14 stool specimens of these patients with 100% sensitivity and specificity as confirmed by reverse transcription polymerase chain reaction (RT-PCR), and one specimen showed both Norovirus and enterotoxigenic Escherichia coli infection. Among the remaining 55 patients with verified Clostridium difficile infection, nine patients also detected positive for Norovirus infection. In conclusion, using FilmArray GI panel and RT-PCR, we determined that Norovirus infection as one of the main pathogens responsible for diarrhea in HIV patients.

Keywords

HIV/AIDS diarrhea automated nested multiplex polymerase chain reaction system diagnostics

Introduction

Acquired immunodeficiency syndrome (AIDS) is caused by human immunodeficiency virus (HIV) infection which targets and compromises the immune system. Globally, it is estimated that approximately two million new infections and 35 million deaths from HIV occur every year, making HIV/AIDS a huge public health burden.¹ Admittedly, this disease has become a manageable chronic illness in some countries because of the development and promotion of effective treatments such as highly active antiretroviral therapy (HAART).² Nevertheless, epidemiological evidence suggests that opportunistic infections (OIs) are still very common among people living with HIV (PLWH), the spread of which varies among different populations. Among the most infectious OIs, diarrheal pathogens continue to be the most prevalent since up to half of PLWH populations are reported to exhibit pathological diarrhea.^3–5

Generally, PLWH with diarrhea can be identified as infectious or noninfectious according to four different categories of pathogens, including bacteria, fungi, viruses, and protozoa despite other potential opportunistic pathogens.⁶ The incidence of infectious diarrhea in HIV patients has declined significantly since the introduction of HAART in recent years. However, infectious diarrhea still occurs in some individuals, particularly in PLWH exhibiting low CD4⁺ T lymphocyte counts (less than 200 cells/mm³).^7,8 Although the major pathogens that induce diarrhea in HIV patients are bacterial, the full spectrum of other pathogens is still unclear. In this study, using a multiplex PCR panel (FilmArray Gastrointestinal [GI] panel), we identified Norovirus as one of the main non-bacterial causes of diarrhea in HIV patients. This study not only provides us with a therapeutic target for diarrhea in HIV patients, but also showed that the multiplex PCR panel assay, which can detect up to 22 different typical pathogens, can be a rapid and accurate assay with a broad etiological spectrum to support the diagnosis and treatment for PLWH who present with diarrhea.

Materials and methods

Patients and samples selection

This study was carried out as a retrospective study in adult HIV-infected in-patients who suffered from diarrhea between April 2016 and April 2017 at Beijing Ditan Hospital, China. The exclusion criteria included any one of the following: age less than 18 years, no analysed stool specimens, missing HIV viral load data and CD4⁺ T cell counts, pregnancy, or individuals with a GI immune disease. In total 81 HIV/AIDS patients with obvious diarrhea symptoms were selected for this study. If HIV serological status was unclear, rapid HIV testing was performed to confirm eligibility. Stool samples from each patient were collected and stored at −80°C for further analysis (Figure 1). This study was approved by the Ethics Committee of Beijing Ditan Hospital and written informed consent was obtained from every patient included in the study.

Figure 1.

Work flow of this study. GI: gastrointestinal; RT-PCR: reverse transcription polymerase chain reaction.

Routine examinations

Demographical and etiological information for each patient was collected. Clinical information was obtained and validated by the Laboratory of Infectious Diseases Centre of Beijing Ditan Hospital. The HIV viral load was detected by PCR, and CD4⁺ and CD8⁺ T cell counts were measured using a flow cytometer. The routine examinations for stool specimens included different smear and dyeing detection methods, such as direct smear method for detecting Giardia lamblia, acid fast stain (Ziehl–Neelsen) for Mycobacterium, improved acid-fast stain for Cryptosporidium, and Gram stain for fungi and mold. Isolation cultures with stool were performed to identify Salmonella, Shigella dysenteriae, enteroinvasive Escherichia coli and fungi; Gold-labeled antibody staining and aerobic culture for the detection of Clostridium difficile and Campylobacter jejuni, respectively, were also used.

FilmArray multiplex PCR array

FilmArray multiplex PCR array was performed using BIOFIRE® FILMARRAY^® system (FilmArray GI panel, BioFire Diagnostics, LLC, Salt Lake City, Utah) according to manufacturer’s protocol in our hospital. Briefly, 3–5 g stool samples were cryopreserved at −80°C in Cary Blair Transport medium. Next, 200 µl of transport medium containing the stool specimen was used for FilmArray GI panel analysis. This assay includes automated nested multiplex PCR system, sample preparation, reverse transcription, multiplex nested PCR, and fluorescence detection, which can be accomplished in one step by loading the sample and hydration solution into the panel as described by the manufacturer (Figure 2). Each sample was analyzed in triplicates and the experiment was repeated three times to exclude false positive/negative results. In total 22 known pathogens, 13 bacterial, 5 viral, and 4 parasitic, were included in this panel.⁹

Figure 2.

Brief description of the FilmArray multiplex PCR system with GI panel. This panel includes two major steps, pouch preparation and machine processing. The red injection port was loaded with stool sample and sample mix while the blue injection port was injected with hydration solution. An automated nested multiplex PCR is carried out within the platform, in which 22 pathogens are targeted, and the result is generated using endpoint melting curve assays.

RT-PCR verification

To validate the findings by the FilmArray GI panel, we performed established RT-PCR as the laboratory-developed test (LDT), which is approved by China’s Food and Drug Administration. RNA was extracted from 0.25 g of stool specimen with RNeasy Power Microbiome Kit (Qiagen, Cat No. 26000-50) and then diluted ten times for the RT-PCR reaction. Multiplex real-time PCR detection kits for Norovirus (group I and II) were purchased from Jiangsu Uninovo Biological Technology Co. Ltd. The TaqMan probes provided in the kit could be used for the simultaneous amplification and detection of Norovirus groups I and II in which Norovirus RNA and RNAase-free water served as the positive and negative controls, respectively. The 20.0 µl PCR mix system consisted of 17.2 µl of RT-PCR reaction mix, 2 µl of extracted RNA template, and 0.8 µl of enzyme mixed according to the manufacturer’s description. The RT-PCR started with reverse transcription at 42°C for 10 min; denaturation at 94°C for 10 s; 5 preamplification cycles of 94°C for 5 s, 50°C for 20 s, 72°C for 20 s; and finally, 45 amplification cycles of 94°C for 5 s, 56°C for 50 s, and 72°C for 15 s.

Statistical analysis

All data in this study were analyzed using IBM SPSS Statistics software 22.0. Demographic characteristics of HIV/AIDS and the numbers of antiviral therapy were shown in percentages. CD4⁺ T lymphocyte counts and history of diarrhea were presented as median ± standard deviation, and differences were calculated by t-test or Fisher’s exact test. P values less than 0.05 were considered statistically significant.

Results

Verification of pathogens in PLWH with routine methods

Among the 81 patients, 55 patients were identified as having different pathogenic infections using routine examination methods, while in the remaining 26 patients, pathogen identification via routine methods was unsuccessful. Among these 26 PLWH with unknown pathogens, 20 were male (76.9%) and 6 were female, with a median age of 41 years (IQR, 29–48) and median CD4⁺ T cells count of 22 cells/mm3 (IQR, 7–128). Fifty percent (13/26) of patients had been treated with HAART, for a median period of 12 months (IQR, 1.5–36); 59.7% (15/26) had a history of diarrhea with a median duration of 30 days (IQR, 9–37). The detailed information is summarized in Table 1.

Table 1.

The demographic information for all the patients involved in this study.

Patient ID	Gender	Age	HIV therapy	Diarrhea period	Diarrhea times/day	CD4⁺ counts	CD8⁺ counts	CD4/CD8	WBC count (10⁹/ml）	Neutrophil percentage	Lymphocyte count (10⁹/ml）	Lymphocyte percentage	CRP^# (mg/l)	Film array result
1	M	39	0	7 d	4	96	573	0.17	7.85	77.7	1.27	16.2	31.4	Norovirus
2	M	38	3 y	10 d	5–6	0	13	0	1.09	64.34	0.13	11.94	110.8	Norovirus
3	M	41	2 w	2 m	4–8	27	369	0.07	1.39	54.6	0.5	36	4.5	Norovirus+ Enterotoxigenic E. coli
4	M	42	1 m	1 m	4–7	142	761	0.19	2.18	49	0.44	20.2	181.4	Norovirus
5	M	32	2 m	7 d	2–3	4	14	0.31	0.72	70.84	0.06	8.34	151	Norovirus
6	F	46	4 y	6 m	3–5	19	65	0.29	5.54	89.61	0.41	7.42	15.9	Norovirus
7	M	26	0	1 m	2–3	5	193	0.03	14.08	83.74	1.47	10.44	80.2	Norovirus
8	F	62	0	1 m	3–4	229	333	0.69	7.26	71.1	0.94	12.9	19.5	Norovirus
9	F	46	0	1 m	2	16	119	0.13	2.4	72.9	0.32	13.3	204.8	Norovirus
10	M	26	0	3 d	6–7	3	178	0.02	5.12	78.02	1.04	20.33	3	Norovirus
11	M	32	0	15 d	3	11	574	0.02	7.76	79.61	0.9	11.62	20.6	Norovirus
12	M	70	0	18 d	10–20	131	456	0.29	1.11	4.54	0.99	89.24	264.4	Entamoeba histolytica
13	M	21	1	2 d	10	28	752	0.04	5.55	72.8	0.99	17.8	91.7	Negative
14	M	30	4 m	4 m	2	19	388	0.05	18.15	91.61	0.62	3.42	125.6	Negative
15	M	77	2 y	7 d	1–2	79	406	0.19	6.44	85.51	0.54	8.42	319.3	Negative
16	M	54	1 y	2 m	10	458	505	0.91	3.6	52.3	1.2	34.3	0.9	Negative
17	M	27	0	1 m	2	3	163	0.02	9.86	92.84	0.34	3.42	382.1	Negative
18	M	36	6	1 m	2	58	181	0.32	5.41	87.2	0.28	5.2	170	Negative
19	F	46	1 m	1 m	3	179	333	0.54	3.77	49.14	1.08	28.64	9.1	Norovirus
20	M	41	0	4 m	5–6	14	567	0.03	5.77	84.61	0.55	9.52	46.3	Norovirus
21	M	47	0	21 d	3–4	8	296	0.03	3.11	71.74	0.6	19.34	29.2	Entamoeba histolytica
22	F	45	3 y	1 m	2–3	632	552	1.14	6.42	56.3	2.18	34	9	Negative
23	M	21	0	1 y	3–5	12	276	0.04	2.6	60	0.52	20	0.5	Norovirus
24	M	55	3 y	1 m	3–4	127	16	7.7	14.91	71.6	2.46	16.5	15.5	Negative
25	M	24	0	3 m	2	7	135	0.05	3.8	67	0.5	12.2	1.1	Negative
26	F	60	0	1 m	4	26	85	0.31	0.57	3.34	0.25	43.94	17.2	Negative

CRP: C-reactive protein; WBC: white blood cell.

Pathogen determination in stool specimens

All 26 specimens with unknown pathogens were tested with the FilmArray GI panel. There was no failure within the internal controls for the automated nested multiplex PCR, thus all assays were confirmed to be valid. In contrast to the negative results determined by the routine examination methods, the FilmArray GI panel detected three distinct pathogens within stool specimens obtained from these patients. Surprisingly, Norovirus genogroup II (GII) was the most prevalent pathogen (50.0%, 13/26) detected, Entamoeba histolytica was found in two patients’ stool samples and there was one specimen which tested positive for both enterotoxigenic E. coli and Norovirus which was obtained from a patient who had suffered from diarrhea for two weeks. The distribution of the various pathogens is presented in Figure 3(a) and representative images for different pathogens’ signals are shown (Supplementary Figure 1). This result suggested that Norovirus could be one of the major causes of diarrhea in PLWH. To check whether Norovirus also contributed to the diarrhea in the 55 HIV/AIDS patients in which routine methods detected verified pathogenic infection, we also performed the FilmArray multiplex PCR array. Results showed that 16.4% (9/55) patients with verified pathogens also had coinfections with Norovirus (Figure 3(b)). These observations further indicated that Norovirus is one of the major pathogens detectable in PLWH with diarrhea.

Figure 3.

Distribution of pathogens detected in 26 stool specimens. (a) Norovirus was the most common pathogen detected in this study (13/26), followed by E. histolytica only (2/26), and one sample which showed both enterotoxigenic E. coli and Norovirus infection. Note: No pathogens were detected in the remaining ten samples (negative) by FilmArray GI panel. (b) Norovirus was also detected in many patients with verified pathogens. Five patients (5/55) tested positive for Norovirus and C. difficile and four (4/55) patients tested positive for Norovirus and fungi coinfection. EAEC: Enteroaggregative Escherichia coli.

Validation of the diagnostic performance of the FilmArray GI panel

To validate the diagnostic performance of the FilmArray GI panel, we performed RT-PCR designed specifically for detecting Norovirus GI and GII using the same stool samples in a blinded manner. The RT-PCR results revealed 14 positive Norovirus GII samples and 12 negative samples; importantly, there was no positive Norovirus GI detection in the 26 samples. Indeed, we found that the RT-PCR (LDT) result perfectly matched the results generated by FilmArray GI panel. According to the standard reference using the LDT, we concluded both the diagnostic sensitivity and specificity of the FilmArray GI panel in detecting Norovirus GII RNA were 100% (95%CI from 76.84 to 100.0% for sensitivity and 95%CI from 73.54 to 100.0% for specificity). Moreover, the positive predictive value was 100.0% as well as the negative predictive value (Table 2). Comparison of both methods in the determination of Norovirus GII showed a kappa coefficient score of 1.00 We have double confirmed that the CI range is correct on all 26 specimens tested, suggesting the excellent concordance between the established LDT and FilmArray GI panel (Table 2).

Table 2.

Performance evaluation of the FilmArray GI panel using LDT as the reference method.

Methods	Positive	Negative	Sensitivity (95%CI)	Specificity (95%CI)	PPV (95%CI)	NPV (95%CI)
LDTFilmArrayGI panel	14	0	100% (76.84–100.0%)	100% (73.54–100.0%)	100% (75–100%)	100% (75–100%)
LDTFilmArrayGI panel	14	0	100% (76.84–100.0%)	100% (73.54–100.0%)	100% (75–100%)	100% (75–100%)

GI: gastrointestinal; LDT: laboratory-developed test; NPV: negative predictive value; PPV: positive predictive value.

Discussion

Recently, the treatment for PLWH has been significantly improved after the establishment of new therapies such as HAART. However, OIs are still very common among PLWH. Thus, it is very important to investigate how OIs take place, specific types of OIs, and how to control such events. Epidemiological evidence suggests that diarrhea-related infection is a common cause of OI as many PLWH have shown continuous diarrhea symptoms. In China, it is still unknown which pathogens are the major culprits for OIs in PLWH.^10,11 Previous etiological studies of diarrhea in HIV-infected patients mainly focused on detection of bacteria, parasites, or fungi,^2,12 which failed to identify the responsible pathogen in many scenarios. Therefore, identifying the diarrhea-causing pathogen for PLWH would greatly improve our public health strategies and, importantly, guide individual treatment.

Patients with HIV often experience OIs which could be induced by various pathogens, such as Norovirus.¹³ Unfortunately, the current available routine examinations have many limitations in defining the pathogens for HIV patients due to low efficiency and high cost. Therefore, development of a rapid, accurate, and cost-effective assay to identify the responsible pathogens which cause diarrhea in PLWH has immense clinical significance. The FilmArray multiplex PCR system, which provides different panels such as the respiratory panel, meningitis/encephalitis panel, and the GI panel, is one of the most advanced molecular diagnostic approaches for syndromic infectious diseases. The FilmArray multiplex PCR panel, which has been approved by the U.S. FDA and the European Medicines Agency, can detect up to 22 different typical pathogens, including 13 common bacterial GI pathogens, 5 viral and 4 parasitic GI pathogens with a single reaction in 75 min. Compared to common approaches such as bacterial culture and bidirectional sequencing, FilmArray assay exhibits high sensitivity and specificity according to the BioFire Diagnostics instruction booklet based on a study of a total of 1560 specimens (https://filmarray.files.wordpress.com/2017/05/manual-panel-me.pdf). Such multiplex PCR strategies have also been demonstrated to be highly sensitive and specific in the clinic, particularly the respiratory panel which is designed for determination of respiratory pathogens.^14–16 However, diagnostic performance of the GI panel has rarely been reported or evaluated in PLWHA with a compromised immune system.

In this study, we obtained stool specimens from 26 HIV-infected patients with diarrhea of which the etiological cause was unknown by routine examinations. These samples were reanalyzed by the FilmArray GI panel which took in total about 75 min. Surprisingly, this automated nested multiple PCR system rapidly detected Norovirus GII infection in 14 samples. Norovirus GII has been previously reported to be a common diarrhea-causing pathogen in the Chinese population^17–19 and cannot be detected by routine examinations. Importantly, by using established RT-PCR protocol targeting Norovirus, we validated the findings of the FilmArray GI panel with 100% sensitivity and specificity. Furthermore, this technology also recognized two patients infected with E. histolytica, a rare pathogen which usually escapes identification by routine examinations. Moreover, one sample contained both enterotoxigenic E. coli and Norovirus infection, suggesting that FilmArray GI panel can detect multiple types of pathogens in one reaction, which might not be achieved by any other routine examination technologies. Of note, the current FilmArray GI panel only targets 22 pathogens; thus, it does not cover the entire spectrum of diarrhea-related pathogens in HIV patients. This may explain why we still observed 12 negative reactions in this multiplex assay despite diarrhea in those patients. It could also be plausible that the diarrhea was caused by the antiretroviral therapy or other reasons. Nevertheless, further investigation is required to evaluate the performance of the FilmArray GI panel in determining diarrhea-associated pathogens in a larger cohort of PLWH.

Conclusion

Taken together, our study suggests that the FilmArray GI panel is a robust and rapid method for identifying diarrhea-associated pathogens in the context of HIV/AIDS patients with high sensitivity and specificity. Compared to other general screening protocols which are currently widely used, this system has multiple advantages in terms of time, cost, efficiency, and sensitivity. We therefore advocate that the FilmArray GI panel should be promoted for use in PLWH.

Supplemental Material

Supplemental material for Diagnostic determination of Norovirus infection as one of the major causes of infectious diarrhea in HIV patients using a multiplex polymerase chain reaction assay

Supplemental Material for Diagnostic determination of Norovirus infection as one of the major causes of infectious diarrhea in HIV patients using a multiplex polymerase chain reaction assay by Siyuan Yang, Min Li, Jingwei Cheng, Gang Wan, Yunao Zhou, Hongyu Jia, Hongshan Wei, Rui Song, Linjun Sheng, Huizhu Wang, Linghang Wang and Wenhao Hua in International Journal of STD & AIDS

Footnotes

Acknowledgments

We thank all the colleagues of the Clinical Laboratory of Beijing Ditan Hospital, Capital Medical University for their kind help and discussion.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article: The FilmArray GI panel tested in this study was provided by BioFire Diagnostics, LLC, Salt Lake City, Utah.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by Beijing Municipal Administration of Hospitals Clinical Incubation Program Funding (Grant No. PX2016025).

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