key: cord-0964301-eniznrmu authors: Kawamura, Masaru; Hoshina, Takayuki; Ogawa, Masato; Yamamoto, Noboru; Haro, Kaoru; Kumadaki, Tokiko; Fukuda, Kazumasa; Kusuhara, Koichi title: The optimal duration of antimicrobial therapy for lower respiratory tract infection in patients with neuromuscular disorders based on a clone library analysis of the bacterial 16S rRNA gene sequence date: 2020-09-17 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2020.09.035 sha: c07295032f6d616662db0ceb4c768780bfb60783 doc_id: 964301 cord_uid: eniznrmu OBJECTIVES: The aim of this study is to determine the optimal duration of antimicrobial therapy for lower respiratory tract infection (LRTI) in neuromuscular disorder (NMD) patients. METHODS: This prospective study included 13 episodes from 9 NMD patients hospitalized for bacterial LRTI. Sputum samples were collected from these patients during the three consecutive days after their admission. Bacterial cell counts and the proportion of the most predominant bacterium identified by a clone library analysis of the bacterial 16S rRNA gene sequence in the samples obtained before antimicrobial therapy were serially investigated. RESULTS: All episodes were initially treated with ampicillin/sulbactam. In 12 episodes with a therapeutic effect, the bacterial cell counts in the samples obtained on the third day of therapy were significantly lower than those before therapy (P = 0.0013). In most of these episodes, the most predominant bacterium in the sample obtained before therapy was undetectable by the third day of therapy. In the one patient without a therapeutic effect, neither the bacterial cell counts nor the proportion of the most predominant bacterium in the sample obtained before therapy decrease after therapy. CONCLUSION: Short-term antimicrobial therapy is sufficiently effective for LRTI in NMD patients if the initial therapy is effective. ). However, our previous study using a clone library analysis of the bacterial 16S rRNA gene sequence indicated that the frequency of LRTI caused by these bacteria was not as high as previously thought, and that the clinical symptoms in most NMD patients improved following the administration of antimicrobial agents without activity against P. aeruginosa (Ogawa et al., 2019) . Despite these promising findings regarding which agents are most effective, the optimal duration of antimicrobial therapy for LRTI in NMD patients remains unclear. To determine the optimal duration of antimicrobial therapy for LRTI in NMD patients, we investigated the serial changes in the microbiome of sputum samples obtained from NMD patients with bacterial LRTI using a clone library analysis. J o u r n a l P r e -p r o o f This prospective study included NMD patients with a permanent tracheostomy, who were admitted to the Department of Pediatrics at the Hospital of University of Occupational and Environmental Health, Japan from March 1, 2016 to April 30, 2017, under suspicion of bacterial LRTI. All patients were bedridden and had an IQ or DQ of ≤20. The clinical information of the patients was collected using standardized case report form. We diagnosed LRTI when patients had cough and sputum production as clinical symptoms, accompanied by abnormalities on chest auscultation (Gadsby et al., 2016) . Bacterial etiology of LRTI were suspected when a patient showed an increased white blood cell counts (>15,000/L) and/or elevated serum C-reactive protein (CRP) levels (>20 mg/L). In patients with leukocytosis and/or elevated serum CRP levels, bacterial LRTI was diagnosed when phagocytized bacterial cells were seen on the Gram stain smear of the sputum sample. The presence of consolidation on chest X-ray was assessed by two of the authors (T.H., K.K.). Informed consent was obtained from all patients' parents. Our study was approved by the Institutional Review Board of the University of J o u r n a l P r e -p r o o f Occupational and Environmental Health, Japan. In our hospital, ampicillin/sulbactam (120-150mg/kg/day, maximal dose: 6 g/day) was initially administrated intravenously for NMD patients because Moraxella catarrhalis was major causative pathogen of LRTI in these patients (Hoshina et al., 2010) . If improvement in the clinical symptoms was achieved promptly, the initial therapy was discontinued after roughly 5 days. If no therapeutic effect of the initial antimicrobial agent was shown after 2-3 days, we re-evaluated the diagnosis of the patient and investigated the results of microbiological examinations. Changes to the treatment were considered after the re-evaluation and further investigation. Sputum samples were collected from NMD patients with LRTI during the three consecutive days after admission. The samples were obtained by inserting a collection tube into patient's trachea. Part of each sample was processed for Gram staining and The 16S rRNA gene was amplified using a Veriti thermocycler (Applied The PCR products were cloned with a TOPO TA cloning kit (Invitrogen, Carlsbad, Genetic Analyzer (Applied Biosystems). DNASIS Pro software (Hitachi Software Engineering, Tokyo, Japan) was used to check the quality and trimming of the sequences. Highly accurate sequences selected by KB basecaller v1.2 (Applied Biosystems) were compared with the 16S rRNA gene sequences of the type strains using the basic local alignment search tool algorithm. A phylotype sharing 97% or higher homology with the sequence of the type strain using the basic local alignment search tool algorithm was considered to be a presumptive species in this study. The SPSS statistics software program (version 21; SPSS Inc.,Chicago, IL, USA, and IBM, Armonk, NY, USA) was used for the analysis. Wilcoxon signed-rank test was used to compare the differences between the quantitative values in the bacteriological analysis. P-values <0.05 were considered to be statistically significant. During the investigation period, sputum samples were obtained in 13 episodes from 9 NMD patients. The patients' characteristics are shown in Table 1 . In just one episode (No. 8-3), ampicillin/clavulanate was given orally for one day before admission. After admission, ampicillin/sulbactam was initially administrated to all patients. All episodes but one improved after starting antimicrobial therapy. In one episode (No. 4-1), ampicillin/sulbactam was changed to piperacillin/tazobactam because of no therapeutic effect. DNA was extracted from sputum samples to perform the clone library analysis of the bacterial 16S rRNA gene sequence. The cell lysis efficiency was ≥80% in all samples (data not shown). The top three bacteria detected by the method were not always identified by culturing. The proportion of concordance between the two methods was 69.2% (Table 2) The numbers of bacteria stained by ethidium bromide were counted in each sputum sample (before DNA extraction was performed). In patients who showed a therapeutic effect, the total bacterial cell counts gradually decreased after starting the initial antimicrobial therapy (Figure 1 ). These counts on the third day of therapy (median, In two of the episodes that showed a therapeutic effect, the total bacterial cell counts did not decrease after therapy. In one of these episodes (No. 8-3) , the proportion of the most predominant bacterium detected in sputum samples obtained before therapy decreased after therapy (Table 3 ). In the other episode (No. 1-1), neither the total bacterial cell counts nor the proportion of the most predominant bacterium detected in the sputum samples obtained before therapy decreased after therapy. In one episode (No. 3-1), the total bacterial cell counts decreased after therapy, but the proportion of the most predominant bacterium detected in the sputum samples obtained before therapy increased after therapy (Table 3 ). In a patient (No. 4-1, Table 1 ) who was refractory to the initial antimicrobial therapy (ampicillin/sulbactam), the total bacterial cell counts (10 8.18 cells/mL) on the third day of the therapy were higher than those before therapy (10 7 cells/mL), and the proportion of the most predominant bacterium (H. influenzae) in sputum samples obtained before therapy did not decrease after the initial antimicrobial therapy (Figure 2 ). Both total bacterial cell counts and the proportion of the bacterium immediately decreased after switching to a second antimicrobial agent (piperacillin/tazobactam). β-lactamaseproducing ampicillin-clavulanate-resistant H. influenzae was the presumptive causative pathogen of LRTI in this patient ( Table 2 ). In 4 of 13 episodes, respiratory viruses were detected from the sputum samples In this study, the bacterial cell counts in the sputum sample from NMD patients with LRTI serially decreased after starting antimicrobial treatment if the initial therapy was effective. In most patients who saw a therapeutic effect, the most predominant bacterium detected by a clone library analysis in the sputum samples obtained before Given the results of that previous and the present studies, the number of patients with treatment failure may have increased if we had stopped antimicrobial therapy immediately after the disappearance of the most predominant bacterium in the clone library analysis. However, the duration of antimicrobial therapy sufficient for LRTI in NMD patients may be at least the same as that in previous-healthy patients (5-day treatment) , although the optimal duration of the therapy may be difficult to determine based on our results alone. Our previous study indicated that around five days of penicillin therapy was also clinically effective for bacterial LRTI in most NMD patients (Kawamura et al., 2018) . The results of this study indicated for the first time from a microbiological perspective that short-term penicillin therapy, such as that with ampicillin/sulbactam, recommended in the guideline for LRTI in previously-healthy children in Japan was sufficiently effective, even for LRTI in NMD patients. In a patient who developed acute pneumonia due to β-lactamase-producing ampicillin-clavulanate-resistant H. influenzae, the clinical symptoms did not improve, and the total bacterial cell counts did not decrease by the third day of treatment with ampicillin/sulbactam ( Figure 2 ). The efficacy of antimicrobial therapy is generally This study has some limitations. First, it was impossible to completely amplify all of J o u r n a l P r e -p r o o f the bacterial 16S rRNA genes with the universal primers that were used in this study. The sensitivity of the primers for the bacterial species registered in the Ribosomal Database Project II database was approximately 92%. However, the bacteria that were not detectable using these primers do not include pathogenic bacteria. Second, there was no method to evaluate whether the bacteria that were predominantly identified by a clone library analysis were the real causative pathogens of LRTI. In general, an increase in the bacterial load in the sputum is associated with the development of LRTIs (Gadsby et al., 2016; Johansson et al., 2010) . Our hypothesis that predominant bacteria would be causative pathogens may be justified. However, it is also necessary to consider that multiple bacteria may cause LRTI especially in episodes with a low proportion of the most predominant bacterium. Third, the clone library analysis method has more technical limitations than the metagenome sequencing analysis. The clone library analysis has an advantage in that it can precisely identify bacteria at the species level because of the relatively long length of sequences compared to next-generation sequencing (Kawanami et al., 2011; Pérez-Losada et al., 2018) . We performed a clone library analysis in this study because it was the most clinically efficient method of identifying the causative bacteria at the species level. Finally, study population was relatively small; this could have affected the accuracy of the statistical analysis. In conclusion, from a microbiological perspective, it was suggested that the optimal duration of antimicrobial therapy for LRTI in NMD patients was the same as that in previously-healthy children if the initial therapy was shown to be effective. The appropriate antimicrobial therapy is needed in order to prevent an increase in multidrug-resistant bacteria-associated LRTI (Elias et al., 2017; WHO, 2015) . Further largescale studies using this method should be performed in order to determine the optimal antimicrobial therapy for NMD patients with LRTI. Pneumonia ABPC/SBT 6 *1 Clinical symptoms and laboratory data improve not following four days initial treatment (ABPC/SBT) but after the change of antimicrobial therapy (PIPC/TAZ). PIPC/TAZ had been administrated for six days. *2 Influenza virus was also isolated at the acute phase of the disease. 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