BACKGROUND:Atezolizumab plus bevacizumab (AB) is a standard first-line therapy for unresectable hepatocellular carcinoma (HCC). However, data on the efficacy and safety of AB rechallenge (re-AB) after discontinuation are limited. METHODS:We conducted a retrospective analysis of 13 patients with unresectable HCC who received initial AB treatment and re-AB at the University of Yamanashi and Fujita Health University. Rechallenge was defined as reinitiating AB after prior discontinuation followed by one or more other systemic therapies. Tumor responses were assessed using RECIST v1.1 and modified RECIST (mRECIST), and adverse events (AEs) were evaluated according to CTCAE v5.0. RESULTS:During initial AB, the objective response rate (ORR), disease control rate (DCR), and median progression-free survival (mPFS) were 15%, 69%, and 3.7 months, respectively. AB was discontinued due to disease progression or immune-related AEs. Re-AB was administered after a median of two intervening regimens. In the re-AB phase, ORR, DCR, and mPFS were 15%, 54%, and 4.7 months, respectively. Despite prior progression, two patients achieved partial response and five had stable disease. No worsening of previously observed AEs occurred during re-AB. CONCLUSIONS:AB rechallenge may be a safe and potentially effective treatment option for selected patients with unresectable HCC, even after discontinuation due to disease progression or adverse events.

Abstract Background and Aim Recently, balloon‐occluded retrograde transvenous obliteration (BRTO), performed for spontaneous portosystemic shunts (SPSS), has been receiving attention as a measure to improve liver function in cirrhotic patients with portal hypertension. However, it is unclear whether SPSS diameter is associated with changes in hepatic venous pressure gradient (HVPG) and liver function after BRTO. Methods In 34 cirrhotic patients receiving BRTO for hepatic encephalopathy/gastric varices, the association of SPSS diameter with liver function at baseline and 6 months after BRTO and the accompanying changes in HVPG were investigated. Results Patients had Child–Pugh (CP) scores of A/B/C (7/19/8), SPSS diameters of ≤10 mm/11–20 mm/<20 mm (8/21/5), and an average observation period of 3.2 (0.3–8.5) years. SPSS diameter was significantly associated with male sex, alcohol use, and values of albumin, prothrombin time (PT%), and NH3 at baseline. Moreover, the SPSS diameter was significantly correlated with the changes in HVPG observed upon BRTO (r = 0.55, P = 0.005), and a large shunt diameter was significantly associated with a greater increase in HVPG. At 6 months, significant improvements in albumin, PT%, bilirubin, and NH3 were observed overall, but the improvement was marked in those with larger shunt diameters if they had CP A/B. Conclusion SPSS diameter was strongly associated with liver function at baseline and after BRTO and also with changes in HVPG, indicating that SPSS diameter is an important predictor of BRTO outcome.

The method of analyzing individual resistant hepatitis C virus (HCV) by a combination of haplotyping and resistance-associated substitution (RAS) has not been fully elucidated because conventional sequencing has only yielded short and fragmented viral genomes. We performed haplotype analysis of HCV mutations in 12 asunaprevir/daclatasvir treatment-failure cases using the Oxford Nanopore sequencer. This enabled single-molecule long-read sequencing using rolling circle amplification (RCA) for correction of the sequencing error. RCA of the circularized reverse-transcription polymerase chain reaction products successfully produced DNA longer than 30 kilobase pairs (kb) containing multiple tandem repeats of a target 3 kb HCV genome. The long-read sequencing of these RCA products could determine the original sequence of the target single molecule as the consensus nucleotide sequence of the tandem repeats and revealed the presence of multiple viral haplotypes with the combination of various mutations in each host. In addition to already known signature RASs, such as NS3-D168 and NS5A-L31/Y93, there were various RASs specific to a different haplotype after treatment failure. The distribution of viral haplotype changed over time; some haplotypes disappeared without acquiring resistant mutations, and other haplotypes, which were not observed before treatment, appeared after treatment. Conclusion: The combination of various mutations other than the known signature RAS was suggested to influence the kinetics of individual HCV quasispecies in the direct-acting antiviral treatment. HCV haplotype dynamic analysis will provide novel information on the role of HCV diversity within the host, which will be useful for elucidating the pathological mechanism of HCV-related diseases.

Objective Owing to advances in direct-acting antiviral (DAA) therapy, a considerable number of patients with hepatitis C virus (HCV)-positive hepatocellular carcinoma (HCC) are now able to achieve a sustained viral response (SVR) after curative treatment of HCC. However, the beneficial effect of a DAA-SVR on the survival remains unclear. Methods A total of 205 patients with HCC who were HCV-positive with Child-Pugh A at the onset from 2008 to 2018 were categorized into 2 groups: 140 patients untreated for HCV throughout the entire course after HCC development (untreated group) and 65 patients treated for HCV with DAAs following HCC treatment who achieved an SVR (SVR group). After propensity score matching, 63 patients from each group were selected. Using these patients, the survival and maintenance of Child-Pugh A after HCC treatment were compared between the untreated group and SVR group. Results There was a significant difference in the overall survival (p<0.001) and the rate of maintaining Child-Pugh A (p<0.001) between the groups. The 5-year survival rates were 96% (SVR group) and 60% (untreated group), and the proportions of patients with Child-Pugh A at 5 years after HCC treatment were 96% (SVR group) and 38% (untreated group). Conclusion In patients with HCV-positive HCC, achieving a DAA-SVR after HCC treatment significantly improved the overall survival rate compared with HCV-untreated patients. The contribution of DAA-SVR during the course of HCC treatment to a longer survival is mainly due to the prevention of the progression of Child-Pugh A to B/C. Further research is needed to determine whether aggressive antiviral therapy is also effective for HCC patients with Child-Pugh B/C.

BACKGROUND:Recently, with the advent of sofosbuvir/velpatasvir therapy, sustained virological response (SVR) can now be achieved even in patients with decompensated cirrhosis (dLC). However, the prognosis after SVR does not always improve in dLC, and appropriate indicators enabling prediction of prognosis is desired.PATIENTS AND METHODS:Serum IP-10/CXCL10 levels were measured in 47 patients (15 chronic hepatitis [CH], 17 compensated cirrhosis [cLC], and 15 dLC) receiving direct acting antiviral (DAA) therapy, and their changes during the therapy were examined.RESULTS:All the patients achieved SVR. In patients with CH, the average IP-10 level was 367, 102, and 68 pg/ml respectively at baseline, at the end of therapy and at 12 weeks after SVR (SVR12), and was decreased upon DAA therapy (P < 0.001). In patients with cLC, IP-10 was respectively 215, 91, and 77 pg/ml, and was decreased upon DAA therapy (P < 0.001) while it was 283, 131, and 182 pg/ml in patients with dLC and there was no evident decrease (P = 0.55). When patients with dLC were further classified depending on the difference in Child-Pugh (CP) score improvement at SVR12, a significant decrease in IP-10 was observed after treatment in those with improvement (P = 0.023) while a significant increase was observed in those without improvement (P = 0.016).CONCLUSION:While serum IP-10 level was decreased in patients with CH/cLC and dLC with post-SVR-CP improvement following SVR, it was increased in patients with dLC without post-SVR CP improvement. The result indicates that IP-10 dynamics may be useful for predicting liver function after DAA therapy.

Aim Recently, serum hepatitis B virus (HBV)‐RNA has been reported to be detectable even when HBV particle production is inhibited by nucleot(s)ide analogues (NAs). However, the dynamics of the HBV‐RNA sequence compared with those of HBV‐DNA during the emergence of antiviral resistance are yet to be elucidated. Methods First, we quantified serum HBV‐RNA in 181 infected patients, and its relationships with clinical characteristics as well as HBV markers were investigated. Next, we undertook simultaneous deep sequencing of HBV‐RNA/HBV‐DNA and their dynamics among four patients receiving NA therapy who were experiencing viral breakthrough. Results Serum HBV‐RNA was detected in 25% (31/123) of cases among patients with HBV without NAs, and the detection rate was significantly high in hepatitis B e antigen‐positive cases with high viral activity. In patients with chronic hepatitis, hepatitis B core‐related antigen was significantly correlated with serum HBV‐RNA irrespective of NA use. In the analysis of the four patients experiencing viral breakthrough, no NA resistance mutation was detected in the serum HBV‐RNA immediately before the breakthrough. However, NA‐resistant sequences appeared at the rates of 0%, 3%, 14%, and 100%, and the NA‐resistant HBV‐RNA sequence rate was correlated with the peak HBV‐DNA titer multiplied by the HBV‐DNA detection duration during the breakthrough (R 2 = 0.978) observed before redisappearance of HBV‐DNA following the addition of new NA. Conclusion Serum HBV‐RNA could reflect the transcriptional activity of covalently closed circular DNA and hepatitis B core‐related antigen. The dynamics of HBV‐RNA could help understanding of the turnover process of HBV covalently closed circular DNA in the liver.

Although the usefulness of liquid biopsy as a biomarker in the treatment of hepatocellular carcinoma (HCC) has been suggested, its usefulness in transcatheter arterial chemoembolization (TACE) or tyrosine kinase inhibitor (TKI) therapies has not been reported in detail. In this study, we investigated the clinical value of a cell‐free (cf)DNA quantification system targeting the human telomerase reverse transcriptase (hTERT) promoter mutation in advanced HCC treatment. Plasma from 67 patients with advanced HCC, treated with TACE and TKI, was used for extraction of cfDNA. We defined cfDNA with the hTERT promoter C228T mutation as circulating mutant DNA (mutant DNA) and without the mutation as circulating wild‐type DNA (wild‐type DNA). We analyzed the changes in mutant and wild‐type DNA levels during HCC treatment and examined the relationship between changes in the cfDNA level and the clinical course. Mutant DNA was detected in 73.1% (49/67) of the patients during HCC treatment. In univariate analysis, factors associated with detection of mutant DNA before treatment were the intrahepatic maximum tumor diameter (P = 0.015) and protein induced by vitamin K absence (PIVKAII) (P = 0.006). The degree of mutant DNA change after TACE was significantly correlated with tumor volume (P < 0.001), reflecting the treated tumor volume. Responders with peak cfDNA levels within 1 week of TKI initiation had significantly better progression‐free survival than nonresponders (P = 0.004). Conclusion: Changes in blood hTERT promoter mutant DNA levels during TACE or TKI treatment indirectly reflect the amount of HCCs and are useful for predicting long‐term treatment responses.

Abstract Background and Aim There are no globally approved, distinguishing criteria enabling the classification of gastric adenomas and intramucosal carcinomas for differential diagnosis of noninvasive neoplasia (NIN). Methods Next‐generation sequencing of 50 cancer‐related genes was undertaken on 68 pathologically diagnosed microdissected gastric neoplasms (25 adenomas, 27 intramucosal carcinomas, and 16 submucosal carcinomas) obtained during endoscopic submucosal dissection. Findings from magnifying endoscopy with narrow‐band imaging (M‐NBI) of 52 NINs (the 25 adenomas and 27 intramucosal carcinomas) were compared with these data. Results Among all 68 neoplasms, the most frequently mutated genes were APC (76% in adenoma, 11.1% in intramucosal carcinoma, and 0% in submucosal carcinoma; P < 0.001) and TP53 in intramucosal and submucosal carcinomas (8% in adenoma, 48.1% in intramucosal carcinoma, and 75% in submucosal carcinoma; P < 0.001). Dividing the NIN neoplasms into five groups according to their mutational status (A1: APC mutation, A2: APC + α mutation, B: APC + TP53 mutation, C: TP53 mutation, D: no mutation in either APC or TP53) resulted in almost identical diagnoses by pathology and M‐NBI for groups A1 (12/13, 92%), C (12/13, 92%), and D (16/17, 94%) but not for groups A2 (3/7, 43%) or B (0/2, 0%). This finding implies that NINs with the APC + α mutation have carcinoma‐like endoscopic features despite most being judged as adenomas by pathology. Conclusion A diagnosis of NINs by pathology or M‐NBI in the subset of gastric tumors classified by cancer‐related mutations is not completely identical, suggesting the possible additional role of M‐NBI in diagnosing NINs. Further studies are needed to confirm this.

Abstract Background and Aim Liquid biopsy is a method that can efficiently detect tumor genetic abnormalities from body fluids such as blood and urine. Detection sensitivity and the available number of mutations in cell‐free DNA (cfDNA) are limited. In this study, we develop a highly sensitive and comprehensive method to detect mutations from cfDNA by concentrating tumor fractions of small cfDNA in advanced colorectal cancers. Methods Biopsied specimens and 37 serum samples were collected from 27 patients with advanced colorectal carcinoma. A serum‐extracted cfDNA was divided into enriched fractionated small cfDNA and unfractionated cfDNA. Both cfDNAs were subjected to digital polymerase chain reaction (PCR) to evaluate their KRAS, BRAF, CDKN2A, and TP53 status. Consequently, their mutant allele frequencies (MAFs) were compared and analyzed by next‐generation sequencing (NGS) in conjunction with tissue‐derived DNA. Results NGS analyses revealed mutations in TP53 (63%), KRAS (63%), APC (30%), and PIK3CA (22%). Digital PCR could detect mutations in 25 of 27 samples (93%) of unfractionated cfDNA, a rate that increased to 100% when samples were enriched with fractionated small cfDNA (6.8 vs 10.7%, P < 0.001). NGS also showed increased MAFs in fractionated small cfDNA compared to unfractionated cfDNA (16.3 vs 18.8%, P = 0.012) and a tendency to detect a greater number of cancer‐related genes in fractionated cfDNA. Conclusions Fractionated small cfDNA increased MAFs of gene mutations and increases the possibilities to detect cancer‐related genes even in advanced cancer patients from whom it is difficult to obtain tissue samples.

AIM:The landscape of cancer-related genetic aberrations in hepatocellular carcinoma (HCC) has gradually become clear through recent next-generation sequencing studies. However, it remains unclear how genetic aberrations correlate with imaging and histological findings.METHODS:Using 117 formalin-fixed paraffin-embedded specimens of primary liver tumors, we undertook targeted next-generation sequencing of 50 cancer-related genes and digital polymerase chain reaction of hTERT. After classifying tumors into several imaging groups by hierarchal clustering with the information from gadoxetic acid enhanced magnetic resonance imaging, contrast-enhanced computed tomography, contrast-enhanced ultrasound, and diffusion-weighted imaging magnetic resonance imaging, the correlation between genetic aberrations and imaging and histology were investigated.RESULTS:Most frequent mutations were hTERT (61.5%), followed by TP53 (42.7%), RB1 (24.8%), and CTNNB1 (18.8%). Liver tumors were classified into six imaging groups/grades, and the prevalence of hTERT mutations tended to increase with the advancement of imaging/histological grades (P = 0.026 and 0.13, respectively), whereas no such tendency was evident for TP53 mutation (P = 0.78 and 1.00, respectively). Focusing on the mutations in each tumor, although the variant frequency (VF) of hTERT did not change (P = 0.36 and 0.14, respectively) in association with imaging/histological grades, TP53 VF increased significantly (P = 0.004 and <0.001, respectively). In multivariate analysis, stage III or IV (hazard ratio, 3.64; P = 0.003), TP53 VF ≥ 50% (hazard ratio, 3.79; P = 0.020) was extracted as an independent risk for recurrence in primary HCC patients.CONCLUSIONS:Increased prevalence of hTERT mutation and increased TP53 mutation VF are characteristic features of HCC progression, diagnosed with imaging/histological studies.

AimAlthough the viral markers hepatitis B surface antigen (HBsAg) and hepatitis B core‐related antigen (HbcrAg) could reflect intrahepatic hepatitis B virus (HBV) replication activity and constitute important biomarkers for hepatocellular carcinoma (HCC), the value of using these two markers in combination for assessing HCC risk has not been clarified in detail.MethodsFour hundred and forty‐nine consecutive patients with chronic HBV infection were included in the study and the association of HBsAg and HBcrAg with HCC risk was investigated cross‐sectionally, as well as longitudinally.ResultsWhen the high value cut‐offs of HBsAg and HBcrAg were defined as 3.0 log IU/mL and 3.0 log U/mL, respectively, patients with a history of HCC were found frequently in the low HBsAg group (P = 0.002) and high HBcrAg group (P < 0.001). When HBsAg and HBcrAg were combined, an HCC history was most frequent in the subset with low HBsAg and high HBcrAg, among the HBeAg‐negative patients (odds ratio [OR], 7.83; P < 0.001), irrespective of nucleos(t) ide analogue (NA) therapy (NA: OR, 4.76; P < 0.001; non‐NA: OR, 9.60; P < 0.001). In a longitudinal analysis of the subsequent development of HCC, carried out on the 338 patients without an HCC history at enrollment, HCC developed significantly more frequently in the low HBsAg/high HBcrAg group (P = 0.005).ConclusionsPatients with low HBsAg/high HBcrAg values are at high risk of developing HBV‐related HCC, according to this cross‐sectional and longitudinal analysis, indicating that the combination of HBsAg and HBcrAg values is an excellent biomarker for assessing HCC risk.

AIM:Deletions are observed frequently in the preS1/S2 region of hepatitis B virus (HBV) genome, in association with liver disease advancement. However, the most significant preS1/S2 region and its influences on viral markers are unclear.METHODS:The preS1/S2 HBV regions of 90 patients without antiviral therapy were subjected to deep sequencing and deleted regions influencing viral markers were investigated.RESULTS:From the deletion frequency analysis in each patient, deletions were observed most frequently in the preS2 codon 132-141 region. When the patients were divided into three groups (0-0.1%: n = 27, 0.1%-10%: n = 34, 10-100%: n = 29), based on the deletion frequency, FIB-4 (p < 0.01), HBV DNA (p < 0.01), HBcrAg (p < 0.01) and preS1/S2 start codon mutations (p < 0.01, both) were significantly associated with the deletion. When clinical and viral markers were investigated by multivariate analysis for their association with the deletion, FIB-4 (p < 0.05), HBcrAg (p < 0.05), and preS1 start codon mutation (p < 0.01) were extracted as independent variables. When the influence of the preS codon 132-141deletions on HBsAg and HBcrAg, relative to HBV DNA, was investigated, the HBsAg/HBV DNA ratio was lower (0-10% vs. 10%-100%, p<0.05), while the HBcrAg/HBV DNA rati o was higher (0-0.1% vs. 10%-100%, p<0.05) in the presence of the preS codon 132-141deletions.CONCLUSION:The preS codon.132-141 deletions have a significant influence on the clinical characteristics and viral markers, even when present as a minor population. Importantly, the preS codon 132-141 deletions have a clear influence on the viral life cycle and pathogenesis.

Mycobacterium triplex, a slowly growing, non-pigmented mycobacterium, i.e. a Runyon group III organism, was named after the triple-peak cluster pattern of mycolic acid obtained by HPLC in 1996 [[1]Floyd M.M. Guthertz L.S. Silcox V.A. Duffey P.S. Jang Y. Desmond E.P. et al.Characterization of an SAV organism and proposal of Mycobacterium triplex sp. nov.J Clin Microbiol. 1996; 34: 2963-2967Crossref PubMed Scopus (69) Google Scholar]. Mycobacterium triplex resembles Mycobacterium simiae or Mycobacterium avium-intracellulare complex (MAC) in biochemical tests. The American Thoracic Society and the Infectious Disease Society of America state that M. triplex was previously identified as MAC by using conventional biochemical identification methods and that the organism might have minimal pathogenicity [[2]Griffith D.E. Aksamit T. Brown-Elliott B.A. Catanzaro A. Daley C. Gordin F. et al.An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases.Am J Respir Crit Care Med. 2007; 175: 367-416Crossref PubMed Scopus (4303) Google Scholar]. Documentation of the clinical characteristics of uncommon non-tuberculous mycobacterial infection is crucial for better management of patients. Pulmonary M. triplex disease is rare, though several cases have been reported in the last two decades. We report two cases of pulmonary M. triplex infection in immunocompetent women with acquired clarithromycin resistance after cessation of ethambutol, resulting in disease progression. In 2005, Patient 1, a woman in her fifties, presented to Fukujuji Hospital complaining of 1 week of chest discomfort. She did not have any history of smoking or immunosuppressive drug use but had had pulmonary TB at the age of 21 years. Chest X-ray showed infiltrations in the left middle and lower lung fields. Chest CT scans revealed nodular opacities and bronchiectatic changes in the lingular segment and the left lower lobe (see Supplementary material, Fig. S1). Although her three consecutive sputum smears were negative for acid-fast bacilli, the cultures became positive thereafter. The isolates were identified as M. triplex by 16S rRNA sequencing. Because her symptoms resolved spontaneously before non-tuberculous mycobacteria identification, she was followed up once every 3 months without treatment. One year later, she presented with productive cough and haemoptysis, and her bronchial washing specimen culture again became positive for M. triplex. Her chest X-ray findings also worsened, so combination chemotherapy with clarithromycin (600 mg), rifampicin (300 mg) and ethambutol (750 mg) was initiated. After 1 year of treatment, she complained of visual impairment, although her respiratory symptoms were relieved, and the chest X-ray findings improved slightly. Considering the optical neuropathy, the physician replaced ethambutol with sitafloxacin. The MIC of clarithromycin against the isolate at that time was 1 mg/L. After changing the regimen, her chest X-ray findings gradually deteriorated. Follow-up CT scans showed multiple cavitary lesions in the lingular segment and left lower lobe of the lung while she was treated with the same regimen for 5 years. Sputum cultures were consecutively positive for M. triplex, and no other non-tuberculous mycobacterium was isolated. Five years after the cessation of ethambutol, the MIC increased to >32 mg/L, and the case was considered to have acquired clarithromycin resistance (Table 1).Table 1rrl sequence analysis of stock strains of the two Mycobacterium triplex cases as well as MICsStrain16S rRNArrl (23S rRNA)Antibiotics MIC (mg/L)CLRMFXAMKPatient 1, (2006)M. triplex (100%)wild-type1216Patient 1, (2017)M. triplex (100%)A 2058 C>32>816Patient 2, (2014)M. triplex (100%)A 2058 T>32432M. triplex (DSM44626)10.516Abbreviations: CLR, clarithromycin; MFX, moxifloxacin; AMK, amikacin.Note: We analysed the stock strains, which were collected before initiating treatment (in 2006) and after the development of macrolide resistance (in 2017).The strain at the time of diagnosis in patient 2 was not collected. DSM44626 is the type strain. Open table in a new tab Abbreviations: CLR, clarithromycin; MFX, moxifloxacin; AMK, amikacin. Note: We analysed the stock strains, which were collected before initiating treatment (in 2006) and after the development of macrolide resistance (in 2017). The strain at the time of diagnosis in patient 2 was not collected. DSM44626 is the type strain. In 2004, Patient 2, a woman in her fifties, was diagnosed with pulmonary MAC disease and treated with a standard three-drug combination regimen. She discontinued the treatment after 18 months due to optical neuropathy. Six years later, her sputum culture became positive again, and the isolates were identified as M. triplex; the MIC of clarithromycin was 8 mg/L. Treatment with clarithromycin, rifampicin and levofloxacin without ethambutol was initiated 1 year after diagnosis. Her symptoms persisted for 34 months as CT findings showed progression even after treatment initiation, and the MIC of clarithromycin against M. triplex increased to >32 mg/L (Table 1). To confirm the observed acquired resistance to clarithromycin, we performed sequence analysis of rrl using three isolates [[3]Meier A. Kirschner P. Springer B. Steingrube V.A. Brown B.A. Wallace Jr., R.J. et al.Identification of mutations in 23S rRNA gene of clarithromycin-resistant Mycobacterium intracellulare.Antimicrob Agents Chemother. 1994; 38: 381-384Crossref PubMed Scopus (166) Google Scholar]. The isolate from patient 1 at diagnosis had no mutation in rrl, whereas the isolate in 2017 harboured a mutation (A2058C). The isolate from patient 2 in 2014 also had a mutation in rrl (A 2058 T) (Table 1). The present cases show that M. triplex can cause chronic pulmonary disease in immunocompetent hosts, with a refractory clinical course possibly due to acquired clarithromycin resistance. Clarithromycin resistance was acquired due to the regimen without ethambutol, which is one of the major causes of macrolide resistance in MAC [[2]Griffith D.E. Aksamit T. Brown-Elliott B.A. Catanzaro A. Daley C. Gordin F. et al.An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases.Am J Respir Crit Care Med. 2007; 175: 367-416Crossref PubMed Scopus (4303) Google Scholar]. Nine cases of M. triplex infections have been reported to date; five extra-pulmonary, two paediatric cervical lymphadenitis, one polyserositis in liver transplantation, and two disseminated infections in severe immunosuppression due to human immunodeficiency virus infection. Among four cases of pulmonary diseases [4Piersimoni C. Zitti P. Mazzarelli G. Mariottini A. Nista D. Zallocco D. Mycobacterium triplex pulmonary disease in immunocompetent host.Emerg Infect Dis. 2004; 10: 1859-1862Crossref PubMed Scopus (14) Google Scholar, 5Campos C.E. Dias C.F. Unis G. de Souza Caldas P.C. Redner P. de Carvalho L.D. et al.First detection of Mycobacterium triplex in Latin America.Int J Mycobacteriol. 2016; 5: 89-91Crossref PubMed Scopus (6) Google Scholar], three patients were immunocompetent. The other patient was infected with human immunodeficiency virus but had retained CD4 cells (514 cells/μL) on antiretroviral therapy. The described cases suggest that M. triplex can cause a pulmonary infection, even in an immunocompetent host. Additionally, all the individuals reported with pulmonary M. triplex disease have particular characteristics similar to those of pulmonary MAC disease; such as female gender, 50 years of age or older, and nodular bronchiectasis (see Supplementary material, Table S1) [4Piersimoni C. Zitti P. Mazzarelli G. Mariottini A. Nista D. Zallocco D. Mycobacterium triplex pulmonary disease in immunocompetent host.Emerg Infect Dis. 2004; 10: 1859-1862Crossref PubMed Scopus (14) Google Scholar, 5Campos C.E. Dias C.F. Unis G. de Souza Caldas P.C. Redner P. de Carvalho L.D. et al.First detection of Mycobacterium triplex in Latin America.Int J Mycobacteriol. 2016; 5: 89-91Crossref PubMed Scopus (6) Google Scholar]. No standard treatment regimen for M. triplex infection has been established. Two of four previously reported pulmonary infections were susceptible to clarithromycin, and treatment with clarithromycin and ethambutol resulted in good clinical outcomes [4Piersimoni C. Zitti P. Mazzarelli G. Mariottini A. Nista D. Zallocco D. Mycobacterium triplex pulmonary disease in immunocompetent host.Emerg Infect Dis. 2004; 10: 1859-1862Crossref PubMed Scopus (14) Google Scholar, 5Campos C.E. Dias C.F. Unis G. de Souza Caldas P.C. Redner P. de Carvalho L.D. et al.First detection of Mycobacterium triplex in Latin America.Int J Mycobacteriol. 2016; 5: 89-91Crossref PubMed Scopus (6) Google Scholar]. In this report, patient 1 had been successfully treated until ethambutol cessation. However, acquisition of clarithromycin resistance, probably due to ethambutol cessation, led to the poor outcome. The present cases suggest that pulmonary M. triplex disease may respond poorly to the combination of clarithromycin, rifampicin and fluoroquinolone, similar to pulmonary MAC disease. The treatment of macrolide-resistant MAC disease with surgical therapy along with aminoglycosides recommended by American Thoracic Society and the Infectious Disease Society of America could also be suitable for macrolide-resistant M. triplex disease [[2]Griffith D.E. Aksamit T. Brown-Elliott B.A. Catanzaro A. Daley C. Gordin F. et al.An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases.Am J Respir Crit Care Med. 2007; 175: 367-416Crossref PubMed Scopus (4303) Google Scholar]. In conclusion, treatment of M. triplex infection should be conducted using a multidrug regimen based on clarithromycin and ethambutol at the recommended doses. Moreover, the physician must understand that the modified regimen without ethambutol for any reason may lead to the development of CLR resistance. All authors report no conflicts. This work was supported by the Japan Agency for Medical Research and Development, AMED (project number 17fk0108116h0401).