ARTICLE 1 Droplet Digital PCR Detects Low-Density Infection in STOMACH a Significant Proportion of Helicobacter Pylori-Negative Gastric Biopsies of Dyspeptic Patients María José Ramírez-Lázaro, PhD1,2, Sergio Lario, PhD1,2, María Elisa Quílez, PhD2, Antònia Montserrat, MD1,2, Maria Rosa Bella, MD, PhD3, Félix Junquera, MD, PhD1,2, Lorena García-Martínez, MSc2, Àlex Casalots, MD3, Tamara Parra, MD3 and Xavier Calvet, MD1,2,4 INTRODUCTION: Helicobacter pylori-infected individuals may present low-density infection, undetectable by conventional tests such as histology, rapid urease test, or urea breath test. Droplet digital polymerase chain reaction (ddPCR) is more sensitive than other polymerase chain reaction methods. We aimed to evaluate the ability of ddPCR to detect H. pylori infection in patients diagnosed as negative by conventional tests. METHODS: Dyspeptic patients (n 5 236) were tested for H. pylori by histology, urea breath test, and rapid urease test. Patients were classified as having 3 positive (n 5 25, control group), 2 positive (n 5 12), one positive (n 5 41), or zero positive (n 5 158) diagnostic tests. DNA was extracted from gastric biopsies. Triplicate ddPCR testing for each of the 16S rDNA, ureA, and vacA(s) genes was performed using a QX200 ddPCR system (Bio-Rad). A gene was considered positive when detected by at least 2 of 3 repeated ddPCRs. H. pylori positivity was defined as having 2 or more positive genes. RESULTS: All the biopsies of the control patients were positive for all 3 16S rDNA, ureA, and vacA(s) genes. H. pylori infection was detected in 57 (36%), 22 (54%), and 9 (75%) patients with zero, 1, and 2 positive diagnostic tests, respectively. The density of infection was 5, 121, 599, and 3,133 copies of H. pylori genome equivalents for patients with zero, 1, and 2 of 3 positive test results and for the control group, respectively. DISCUSSION: ddPCR detected low-density “occult” H. pylori infection in a significant proportion (36%) of patients diagnosed as negative by conventional methods. The number of conventional positive tests was related to the density of infection. SUPPLEMENTARY MATERIAL accompanies this paper at http://links.lww.com/CTG/A290 Clinical and Translational Gastroenterology 2020;11:e00184. https://doi.org/10.14309/ctg.0000000000000184 INTRODUCTION the available tests, urea breath test and histology are considered the H. pylori infection is associated with dyspeptic symptoms, peptic most accurate. The efficacy of all these tests is limited by situations ulcer, and gastric cancer. Infection is present in more than half of or treatments that decrease bacterial density, such as upper gas- the human population (1). Its mortality is high, mostly related to trointestinal bleeding or treatment with antibiotics or proton pump gastric cancer; in fact, it is responsible for 783,000 deaths each year inhibitors. Invasive tests are also limited by the possibility of and is the third cause of cancer death worldwide (2). Adequate sampling error because of the irregular distribution of bacteria diagnosis and treatment of the infection are curative for peptic throughout the stomach (7,8). ulcer and may prevent the development of gastric cancer (3). Many previous studies suggest that subclinical infection with Several conventional methods are available for the detection of H. pylori (i.e., when infection density is below the detection active H. pylori infection. The main noninvasive tests are the urea threshold of conventional diagnostic tests [DT]) may be a frequent breath test and the stool H. pylori antigen test (4). Invasive tests event. One of the first reports of “occult” infection dates from 2006 require endoscopy to obtain tissue biopsies and include rapid when, in a study aimed to describe the bacterial gastric diversity of urease test, culture, and histological examination (5,6). Among all 23 healthy individuals, Bik et al. found that H. pylori was detectable 1 Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Instituto de Salud Carlos III, Madrid, Spain; 2Digestive Diseases Service, Hospital Universitari Parc Taulí, Institut d’Investigació i Innovació Parc Taulí I3PT, Universitat Autònoma de Barcelona, Sabadell, Spain; 3Pathology Service, Hospital Universitari Parc Taulí, Institut d’Investigació i Innovació Parc Taulí I3PT, Universitat Autònoma de Barcelona, Sabadell, Spain; 4Departament de Medicina, UAB, Sabadell, Spain. Correspondence: Xavier Calvet, MD, Prof. E-mail: xcalvet@tauli.cat. María José Ramírez-Lázaro, PhD. E-mail: mramirezl@tauli.cat Received December 10, 2019; accepted April 16, 2020; published online June 4, 2020 © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology American College of Gastroenterology Clinical and Translational Gastroenterology 2 Ramı́rez-Lázaro et al. by molecular methods in 7 of 11 patients identified as negative by H. pylori treatment were excluded. Before the endoscopy, the pa- conventional tests (9). Currently, low-density infection has been tient provided signed informed consent, and urea breath test was described in patients on antisecretory or antibiotic treatment performed. During endoscopy, 4 antral biopsies were obtained: 2 for (10,11), in patients with extensive atrophy of the gastric mucosa histology, one for the urease test and culture, and one for molecular STOMACH and/or intestinal metaplasia (12), in gastric MALT lymphoma analysis. The study was performed in accordance with the Decla- (13,14), and in peptic ulcer bleeding (15). ration of Helsinki and was approved by the ethics committee of the Having accurate DT is fundamental to ensuring quality of care Hospital Universitari Parc Taulı́ (Id. number 2015611). by avoiding unnecessary treatments and/or the underuse of effective therapies. For infectious diseases, molecular DT may help improve Conventional DT the quality of results provided by monoclonal tests, microbiology The DT (histology, rapid urease test, urea breath test, and ddPCR) culture, or histology. Polymerase chain reaction (PCR), for instance, were performed by technicians who were unaware of the results of has a sensitivity and specificity close to 100% for the detection of H. the other assessments. pylori in patients with chronic gastritis or nonpeptic ulcer bleeding The urea breath test was performed with the UBiTest 100 mg (16,17). Moreover, previous studies suggest that PCR is capable of (Otsuka Pharmaceutical Europe, Barcelona, Spain) in accordance detecting low-density infection in a significant number of patients with the manufacturer’s specifications. A basal breath sample was with dyspepsia compared with conventional techniques (18,19). collected by blowing into a specially designed bag. After this, patients More recently, a study of histologically H. pylori-negative gastritis took a pill of 100 mg of 13C-labeled urea in 100 mL of water and filled showed that 49% of patients with chronic mucosal inflammation a second breath bag 20 minutes later. The samples were immediately were positive by PCR (20). Finally, PCR was able to detect active processed by nondispersive infrared spectrophotometry (POCo- infection even in a proportion of healthy individuals diagnosed as neTM Infrared Spectrophotometer, Otsuka Pharmaceutical, Tokyo, negative for H. pylori by conventional tests (21,22). Japan). In accordance with the manufacturer’s specifications, an Furthermore, highly sensitive molecular techniques can help increase in the proportion 13C/12C (13CO2 (%)) of 2.5% or more after detecting H. pylori in clinical settings such as peptic ulcer bleeding, urea intake was considered as indicative of H. pylori infection. gastric cancer, or MALT lymphoma in which diagnosis of H. pylori Rapid urease test was performed after mucosal sampling using the is important but difficult to achieve. In this context, we demon- Jatrox-HP test (CHR Heim Arzneimittel GmbH, Darmstadt, Ger- strated that real-time PCR improves the detection of H. pylori in many) and was read according to the manufacturer’s specifications. paraffin samples obtained during an episode of upper gastroin- For histology, biopsies were collected in formalin, stained with testinal bleeding due to peptic ulcer (23). In addition, there seems to Giemsa, and then evaluated by 2 pathologists specialized in digestive be an association between the density of infection and the virulence disease. The pathologists were blinded to the results of the other DT. of the strain, with virulent strains having the lowest density (24). This latter finding also suggests that low-density infection may be H. pylori detection by ddPCR related to more virulent strains and so may not be innocuous. DNA from the endoscopic biopsies was isolated with MasterPure The latest commercially available refinement in PCR technology is DNA purification kit (Epicentre, Madison, WI). Isolated DNA was digital PCR (dPCR) (25). dPCR is a quantitative technology that quantified with a QuantiFluor-P Fluorometer Quantus NGS shows increased sensitivity when compared with conventional or (Promega Corporation, Madison, WI), and the concentration was real-time PCR methods while maintaining specificity (26,27). dPCR adjusted to 50 ng/mL with TE Buffer Low EDTA Ultrapure, USB has proved to be useful for the detection of infectious agents in a va- (USB Corporation Cleveland, OH). riety of sample types and, specifically, in the detection and genotyping TaqMan hydrolysis probe assays were used to amplify the of resistance genes in H. pylori infection (28–30) and to determine the fragments of the 16S ribosomal ribonucleic acid (rRNA) and ureA role of CYP219 polymorphisms in triple therapy efficacy (31). genes of H. pylori. The intercalating EvaGreen assay was used to Droplet digital PCR (ddPCR) is a method for performing dPCR amplify the vacA gene (s segment) of H. pylori. Primers and probes that is based on the generation of water-oil emulsion droplets. In used to amplify the target genes are listed in Table 1, Supplemen- ddPCR, the PCR reaction of an individual sample is fractionated into tary Digital Content 1, http://links.lww.com/CTG/A290. thousands of droplets (20,000) and subjected to end point PCR. Am- The ddPCR conditions performed using the QX200 Droplet plification of the template molecules occurs in each individual droplet, Digital PCR System (Bio-Rad, Pleasanton, CA) are summarized in and positive droplets are counted using a fluorescence detector. the supplementary material file, http://links.lww.com/CTG/A290. In this study, our aim was to evaluate the presence of low- The results of ddPCR were generated using QX200 Droplet density H. pylori infection in dyspeptic patients diagnosed as Reader (Bio-Rad) and analyzed using QuantaSoft software version negative by rapid urease test, urea breath test, and/or histology 1.7.4.0917 (Bio-Rad). Each of the 3 16S rRNA, ureA, and vacA using highly sensitive ddPCR. genes were amplified in triplicate by ddPCR. A ddPCR reaction was considered positive if one or more droplets were positive for Eva- PATIENTS AND METHODS Green reactions and/or TaqMan reactions. Study subjects To minimize false positive results, we set the restrictive criteria. Outpatients sent to the endoscopy unit of the Hospital Universitari A biopsy was considered positive for a given gene when at least 2 of Parc Taulı́ for dyspeptic symptoms from 2006 to 2014 were pro- the 3 replicates were positive, and H.pylori infection was considered spectively recruited for our study. Patients were contacted before the present only if 2 or all of the 3 genes tested were positive ddPCRs. endoscopy. Those who agreed to participate in the study were instructed to avoid all antisecretory drugs including proton pump inhibitors in the 2 weeks before the endoscopy. Patients who were Human b-actin amplification unable to stop antisecretory drugs, those who had received anti- The quality of the DNA isolated from the biopsy specimens was biotics in the 4 weeks before the endoscopy, and those with previous confirmed by an initial PCR. We used PCR on each DNA extract Clinical and Translational Gastroenterology VOLUME 11 | JUNE 2020 www.clintranslgastro.com Droplet Digital PCR 3 to amplify a 83 bp fragment from the b-actin gene region (ACTB Validation of PCR assays gene, Prime Time qPCR Assay, IDT, Coralville, IA). All the antral biopsy specimens from the 25 positive controls were positive on all 3 PCR assays. On the other hand, all samples were Bacterial strains positive for human b-actin amplification, indicating that no PCR STOMACH To evaluate the specificity of the technique for vacA (s), ureA, and inhibitors were present. 16S rRNA, 11 bacterial species were used, obtained either from the clinical isolates of Staphylococcus epidermidis, Corynebacterium sp., Species specificity of the PCR assays Proteus sp., Candida albicans, and Neisseria meningitidis or from All non-Helicobacter species were negative for the 3 genes am- the American Type Culture Collection (ATCC) (Rockville, MD) of plified (vacA, ureA, and 16S). The 2 strains of H. pylori were Pseudomonas aeruginosa, Enterococcus faecalis, Escherichia coli, positive for all the 3 genes analyzed. Staphylococcus aureus, Streptococcus pneumoniae, and Haemo- philus influenzae. Strains J99 and 26695 of H. pylori were used as H. pylori detection frequency and copies of genome equivalents a positive control. quantified by ddPCR H. pylori infection was detected by ddPCR in 57 (36%) patients Data and statistical analysis with zero positive DT, in 22 (54%) with one positive DT, and in 9 The mean number of H. pylori copies of genome equivalents were (75%) with two positive DT (Table 2). The distribution frequency compared across all groups with a Kruskal-Wallis test, followed by of the amplified genes is shown in Table 2. Individual results of the Dunn post hoc tests. Statistical analyses were performed using triplicate ddPCR for each are shown in Figure 2 and Tables 2 and 3, GraphPad Prism 6 (La Jolla) and Microsoft Excel 2010 (Microsoft Supplementary Digital Content 1, http://links.lww.com/CTG/ Corp). Significance was set at P , 0.05. A290. All the biopsies of the three positive DT control patients were positive for 16S rDNA, ureA, and vacA(s) genes. RESULTS The number of conventional positive tests was related to the Study subjects density of the infection (Figure 3). The density of the infection in A total of 634 dyspeptic patients were initially recruited. Sixty-one the three positive DT control group was 1,88 3 103, 1,11 3 103, and patients with some DT not performed were excluded. Of the 6,40 3 103 copies of H. pylori genome equivalents for vacA(s), remaining 573 patients with 3 DT performed, 322 were negative for ureA, and 16S rRNA, respectively. The infection density then de- some or all tests. In this group, 111 patients lacking clinical data (n creased progressively by 3 orders of magnitude down to ;10 copies 5 35) or insufficient biopsy sample (n 5 34) were excluded as were in the zero positive DT group (Figure 3 and Table 4, Supplementary patients diagnosed with intestinal metaplasia, lymphocytic gastri- Digital Content 1, http://links.lww.com/CTG/A290). These dif- tis, neuroendocrine tumor, or hyperplastic polyps (n 5 42). Of the ferences in infection density were significant for each of the groups 251 patients with all positive DT, 25 were randomly selected as when compared with the zero positive DT samples. a positive control group. Finally, 236 patients with dyspeptic symptoms who had valid Role of the histological findings for detecting “occult” results of 3 conventional DT were selected for the molecular H. pylori infection analysis. Patients were classified as having 3 positive (n 5 25, Chronic active gastritis (mucosal neutrophil infiltration) was control group), 0 positive (n 5 158), 1 positive (n 5 41), or 2 found in 24 of 25 patients with three positive DT (96%) and in positive results (n 5 12) by conventional DT (Figure 1). Patient only 4 of 158 patients with zero positive DT. As expected, characteristics, including endoscopic and histological diagnoses, chronic active gastritis increased proportionally with the are shown in Table 1. number of positive DT: 9 of 41 patients with one positive DT Figure 1. Study patients’ flow diagram. DT, diagnostic tests. American College of Gastroenterology Clinical and Translational Gastroenterology 4 Ramı́rez-Lázaro et al. Table 1. Patient characteristics 0 DT Hp(1) (n 5 158) 1 DT Hp(1) (n 5 41) 2 DT Hp(1) (n 5 12) 3 DT Hp(1) (n 5 25) STOMACH Gender (male/female) (n) 55/103 17/24 3/9 14/11 Age (mean 6 SD) 47 6 14 49 6 14 50 6 18 47 6 14 Endoscopic diagnosis (n): Normal 128 39 8 15 Duodenal ulcer/erosive duodenitis 21 2 2 6 Gastric ulcer 1 0 1 0 Esophagitis (LA A or B/LA C or D) 2/1 0 0 2/2 Other 5 0 1 0 Histological diagnosis (n): Gastritis (no/yes/Nd) 11/144/3 3/38/0 0/12/0 0/25/0 Gastritis activity (no/yes/Nd) 150/4/4 32/9/0 3/9/0 1/24/0 Gastritis severity (0/1/2/Nd) 12/141/2/3 3/35/3/0 2/7/3/0 0/15/10/0 Lymphoid follicle (no/yes/Nd) 120/29/9 24/17/0 4/6/2 8/15/2 Helicobacter pylori 0 4 8 25 Metaplasia/atrophy 0/1 0/3 0/0 0/0 DT, diagnostic tests; LA, Los Angeles esophagitis classification; Nd, no data; mild (A or B) or severe (C or D). (22%) and 9 of 12 patients with two positive DT (75%) (Table 1). DISCUSSION Neutrophil infiltrate was, however, not useful for diagnosing In this study, we found that ddPCR detects “occult” H. pylori in occult infection. Chronic active gastritis was present in only 2 of a significant proportion (36%) of dyspeptic patients diagnosed as 57 patients with zero negative DT and Hp positive by ddPCR negative by 3 conventional methods. The prevalence of infection in (4%) and in 2 of 101 patients with negative ddPCR (2%) (see patients with 1 or 2 positive tests was even higher, at 49% and 75%, Table 4, Supplementary Digital Content 1, http://links.lww. respectively. Thus, ddPCR seems to be more sensitive than conven- com/CTG/A290). tional tests. In addition, because it is a quantitative method, we were Among patients with positive ddPCR, lymphoid follicles were able to quantify the density of the infection. Quantification indicated present in 11 (19%), 12 (55%), and 6 (67%) patients with zero, that H. pylori escapes conventional DT when the bacterial load is low; one, or two positive DT. Corresponding figures in patients neg- in fact, we found a clear linear correlation between the bacterial ative by ddPCR were 18 (18%), 5 (26%), and 0, respectively (see density and the number of positive conventional tests (Figure 2). Tables 5 and 6, Supplementary Digital Content 1, http://links. Several authors suggest that PCR-based diagnosis can be con- lww.com/CTG/A290). sidered a gold standard, provided that specific H. pylori primers are Finally, H. pylori detection by ddPCR vs individual DT is used to target more than one conserved gene (32–34). In our case, to summarized in Table 7, Supplementary Digital Content 1, http:// avoid false positive results, ddPCR positivity was determined on the links.lww.com/CTG/A290. amplification of 3 specific H. pylori genes, each of them in triplicate. Table 2. Distribution frequency of the amplified genes by ddPCR ddPCR positive for Hp (n(%)) No. of Hp(1) genes Hp gene 0 DT Hp(1) (158 patients) 1 DT Hp(1) (41 patients) 2 DT Hp(1) (12 patients) 1 vacA (s) 54 (34%) 30 (73%) 9 (75%) ureA 76 (48%) 23 (56%) 9 (75%) 16S 59 (37%) 22 (54%) 11 (92%) 2 vacA(s)-ureA 38 (24%) 20 (49%) 9 (75%) vacA(s)-16S 38 (24%) 20 (49%) 9 (75%) ureA-16S 47 (30%) 20 (49%) 9 (75%) 3 vacA-ureA-16S 33 (21%) 19 (46%) 9 (75%) 2 or 3 2 or 3 ddPCR Hp (1) 57 (36%) 22 (49%) 9 (75%) ddPCR, droplet digital polymerase chain reaction; DT, diagnostic tests. Clinical and Translational Gastroenterology VOLUME 11 | JUNE 2020 www.clintranslgastro.com Droplet Digital PCR 5 STOMACH Figure 3. H. pylori copies of genome equivalents detected by ddPCR in dyspeptic patients. vacA s (blue), ureA (red), and 16S (green) bars. The results are expressed as means 6 SEM: *P , 0.05/**P , 0.01/***P , 0.005/****P , 0.001 vs. 0 DT Hp(1). DT, diagnostic tests; ddPCR, droplet digital polymerase chain reaction. the disease after H. pylori treatment; the authors attributed this finding to “occult” infection. In addition, Guell et al. (15) reported that 79% of the patients with peptic ulcer bleeding who tested neg- ative for H. pylori during the bleeding episode had active infection, which was only detected when the tests were repeated a few weeks after the episode. Our group previously reported that real-time PCR detects H. pylori in more than two-thirds of histology-negative samples from biopsies obtained during an episode of bleeding (23) and in 27% of histology-negative samples from dyspeptic patients with a “false positive” urea breath test (36). The prevalence and relevance of “occult” H. pylori infection in dyspeptic patients or in those with ulcer are currently unknown. It is possible, for example, that “occult” infection may account for a proportion of idiopathic peptic ulcers, an entity that seems to be increasingly frequent worldwide (37). It may be worth investigating the prevalence of “occult” infection in these cases. ddPCR examinations in the long- term studies after eradication treatment would also be of interest. Apart from MALT lymphoma, peptic ulcer bleeding, and Figure 2. Individual results of triplicate ddPCR for each amplified gene. gastric atrophy, a growing body of evidence indicates that occult Each line in the figure represents a patient. Each gene amplification was infection may pass undetected after one or more of the con- performed in triplicate. Color was assigned to number of positive ddPCR ventional tests in dyspeptic patients without these conditions, replicates for Helicobacter: Dark green color (3 positive replicates), light and even in healthy volunteers. In a study of the gastric green color (2 positive replicates), yellow color (one positive replicate), and microbiota of 23 healthy individuals, Bik et al. (21) analyzed red color (zero positive replicates). DT, diagnostic tests; ddPCR, droplet H. pylori infection by rapid urease test, histology, culture, se- digital polymerase chain reaction. rology, and 16S rRNA sequencing and detected H. pylori in- fection in 12 individuals by conventional tests (52%) and in 19 of In addition, we selected primers that showed a very high specificity in the 23 by molecular tests (83%). Kiss et al. (20) showed that previous publications (23,35). Therefore, our study suggests that approximately 50% of histological gastritis and H. pylori-neg- ddPCR may be a useful alternative for detecting H. pylori infection, ative are PCR positive. Some authors suggest that patients with especially for patients with very low-degree, “occult” infection. In chronic active gastritis in the absence of visible H. pylori should fact, the results suggest that persistent low-density infection may be considered as infected individuals in whom H. pylori has remain undetected in a proportion of patients submitted to endos- temporarily disappeared from the mucosa because of PPI or copy for dyspepsia. The potential causal role of the infection in these antibiotic use (38). This topic remains, however, unclear. Ana- symptoms, however, remains to be determined. lyzing epidemiological data, the same authors also suggested Standard tests are suboptimal in certain clinical situations such as that chronic active gastritis in the absence of H. pylori may be MALT lymphoma, peptic ulcer bleeding, or extensive atrophy. a distinct entity with an as yet unknown etiologic agent (39). As Several reports have indicated the presence of H. pylori at low levels an alternative, occult H. pylori infection may help explain this in these clinical settings. For example, Raderer et al. (14) reported 6 situation. Conversely, even in the absence of active gastritis, patients with gastric MALT lymphoma with negative results for all H. pylori has been detected by immunohistochemistry (40) or H. pylori conventional tests who presented complete resolution of histology (41). Our study reports similar findings using a far American College of Gastroenterology Clinical and Translational Gastroenterology 6 Ramı́rez-Lázaro et al. more sensitive test such as ddPCR and adds support to the Study Highlights various reports in the literature suggesting that undetectable infection is not exceptional. For example, it is well-known that WHAT IS KNOWN antisecretory drugs or antibiotics may reduce the density of the STOMACH infection to levels undetectable by conventional tests; for this 3 Several conventional methods are available for the detection reason, it is recommended that these drugs should be stopped in of active H. pylori infection; the efficacy of all these tests, the weeks before the DT are carried out (11). However, the use of however, is limited by specific situations or treatments that PPIs does not seem to explain the occurrence of “occult” in- decrease bacterial density. fection in this study. Patients were interviewed by telephone 3 Subclinical infection with H. pylori may be a frequent event in before inclusion in the study and instructed to avoid PPIs and dyspeptic patients, and low Helicobacter density may be associated with virulent strains. antibiotics. On the day of endoscopy, patients were asked again about PPI and antibiotic use before the urea breath test and the 3 Highly sensitive molecular techniques can help to detect H. pylori in clinical settings in which diagnosis of H. pylori is endoscopy, and those who had not stopped the drugs were important but difficult to achieve. excluded. A limitation of our study is that the molecular tests were 3 ddPCR is a quantitative technology that shows increased sensitivity compared with conventional or real-time PCR performed on a single antral biopsy. It is well-known that methods while maintaining the levels of specificity. H. pylori can show a patchy distribution in the stomach, and the more biopsies are performed, the higher the diagnostic accuracy WHAT IS NEW HERE (42). However, the current cost of ddPCR limited the number of biopsies that could be analyzed for each patient. Another limi- 3 We found that ddPCR detected low-density “occult” H. pylori infection in a significant proportion of dyspeptic patients tation is that because the samples we analyzed were negative by diagnosed as negative by three conventional methods. conventional tests, we were unable to create a control group of patients who were unequivocally negative. 3 Quantification indicated that H. pylori escapes conventional diagnostic tests when the bacterial load is low. We found At present, we still do not know the relevance of the “occult” a clear linear correlation between the bacterial density and H. pylori infection. Conceivably, the low levels of infection may the number of positive conventional tests. be an advantage for these patients, protecting them from disease by modulating the adjacent microbiome or the immune system. TRANSLATIONAL IMPACT Furthermore, the absence of active gastritis may suggest that occult infection is associated with a lower risk of severe clinical 3 ddPCR may be a useful alternative for detecting H. pylori infection, especially for patients with very low-degree, manifestations. However, other data suggest that patients with “occult” infection. occult H. pylori infection may present especially virulent or resistant strains different from those found in high-density 3 It is important to detect and treat this occult infection to prevent transmission, peptic ulcer, and gastric cancer. infections (24). If so, it would be important to detect and treat this unnoticed occult infection to prevent transmission, peptic ulcer, and gastric cancer. Further studies are needed to char- acterize low-density infection and to determine the factors that REFERENCES modulate the bacterial load to prevent antibiotic resistances and 1. Hooi JKY, Lai WY, Ng WK, et al. 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