This study aims to evaluate of olfactory and gustatory functions of COVID-19 patients and possible risk factors for olfactory and gustatory dysfunctions. The cross-sectional study included adult patients who were diagnosed with COVID-19 in Gazi University Hospital between April 2020 and June 2020. Volunteered patients participated in a survey in which olfactory and gustatory functions and various clinical information were questioned. Sinonasal Outcome Test-22 was also administrated to all patients.A hundred and seventy-one patients participated in this study. Olfactory and gustatory dysfunctions rates were 10.5% (n: 18) and 10.5% (n: 18), respectively. Patients without any symptom other than smell and taste dysfunctions were clustered as group 1 and patients who are clinically symptomatic were clustered as group 2. Olfactory dysfunction occurred in 8% of group 1 and 17.4% of group 2 (p = 0.072). Gustatory dysfunction rate of smokers was 19.7% and significantly higher than gustatory dysfunction rate of nonsmokers (5.5%) (p = 0.007). Twenty-seven-point-eight percent of the patients with olfactory dysfunction (n = 5) were male and 72.2% (n: 13) were female. Sex did not show significant effect on rate of olfactory dysfunction. Twenty-five patients participated in psychophysical olfactory function test. No participant reported olfactory dysfunction at the time of test. Of the participants, 64% (n: 16) were normosmic and 36% (n: 9) were hyposmic according to Sniffin’ Stick test. Olfactory and gustatory dysfunctions are more common in patients who are clinically symptomatic than those diagnosed during contact tracing. Objective tests may show that frequency of olfactory dysfunction is greater than frequency of self-reported olfactory dysfunction. Corona virus disease 2019 (COVID-19) is a pandemic that emerged from East Asia in late 2019 and rapidly spread to the rest of the world and caused by severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) [1]. The disease is held responsible for more than 2 million deaths worldwide and still poses a threat to the public health in most of the countries. The most common manifestations of the disease are fever, coughing, sore throat, and dyspnea . Smell and taste dysfunctions have been frequently reported since the onset of the disease. In one of the very first reports of the neurologic manifestation of the disease, Mao et al. reported anosmia and ageusia in 5.1% and 5.6% of the cases, respectively [3]. Although incidences of taste and smell dysfunctions vary among the reports, Lechien et al. reported that 85.6% of the patients in Europe suffered from olfactory dysfunction and 88.8% had gustatory dysfunction [4]. Early reports claimed chemosensory disorders may be the initial signs of the disease, especially in the asymptomatic patients [4–7]. Sudden onset of anosmia and ageusia within 24–48 h is reported to be highly predictive for the disease and these symptoms often occur within 5 days from the onset of disease [8].. Epithelial damage of nasal and oral mucosa may have a role in olfactory and gustatory dysfunctions . SARS-CoV-2 is shown to be neuro-invasive and invasion of the olfactory nerve and trigeminal nerve may cause olfactory and gustatory dysfunctions [17]. Another hypothesis claims that central nervous system involvement with focal encephalitis in olfactory and gustatory cortex may be the cause of olfactory and gustatory dysfunctions. The detection of viral RNA in cerebrospinal fluid of the patients may support this hypothesis [3].Alternation of chemosensory functions due to a viral infection is not a new phenomenon for otolaryngologists. In adults, 40% of anosmia cases are caused by a viral upper respiratory tract infection [9]. Common pathogens of upper respiratory tract, rhinovirus, parainfluenza, Epstein-Barr virus, and coronavirus are known to cause olfactory and gustatory dysfunctions . On the other hand, olfactory dysfunction pathogenesis in COVID-19 seems to be on a different aspect since anosmia may occur without rhinorrhea or any other findings related to upper respiratory tract infection . Lechien et al. reported the occurrence of anosmia or hyposmia in the absence of rhinorrhea and nasal congestion in 79.7% of the patients [4]. To explain mechanism of chemosensory disorders in COVID-19 several hypotheses were raised. SARS-Cov-2 binds directly the angiotensin converting enzyme 2 (ACE 2) cell receptors. ACE2 receptors take place frequently on olfactory epithelium and on oral cavity mucosa particularly on the tongue but may also be detected on glial cells and neurons of central nervous system as well [12–14]Even though the mechanism of olfactory and gustatory dysfunctions in COVID-19 patients is not fully discovered, these symptoms keep importance for suspecting of disease and early diagnosis. In this study, we aim to evaluate olfactory and gustatory dysfunctions of COVID-19 patients with a subjective self-reported questionnaire. We also investigated the long-term effects of COVID-19 on olfaction with an objective psychophysical test. This cross-sectional study was approved by Gazi University Ethical committee of clinical research. The study included adult patients who diagnosed with COVID-19 in Gazi University Hospital between April 2020 and June 2020. The diagnosis was made with a positive SARS-Cov-2 PCR test. The patients were invited to this study by phone. All participants provided informed consent. In the survey, each patient was interviewed about basic demographic info, the time of diagnosis, hospitalization time, presence of olfactory and gustatory dysfunctions, onset of these symptoms (days before or days after diagnosis), recovery of olfactory and gustatory dysfunctions, history of any rhinologic surgery. Sinonasal Outcome Test-22 (SNOT-22) was also administrated to all patients. Patients with the history of smell or taste dysfunctions prior to COVID-19 and history of previous rhinologic surgery were excluded from the study. Two hundred and ninety-eight patients had been diagnosed with COVID-19 between April 2020 and June 2020. Ninety-six patients refused to participate in the study. Five patients with olfactory dysfunction prior to COVID-19 and 26 patients with a history of rhinologic surgery were excluded from the study. A hundred and seventy-one patients met the inclusion criteria.Information on disease severity and treatment modality was obtained from the patients’ files. The patients were divided into groups according to disease severity. Patients without any symptom other than smell and taste dysfunction were clustered as group 1 and patients who are clinically symptomatic were clustered as group 2. Group 2 was further divided in two groups as the patients with pneumonia were clustered as group 2b and the others were clustered as group 2a.Patients whose recovery of the disease was shown with two consecutive negative SARS-CoV2 tests were invited to the clinic for objective assessment of olfaction with Sniffin’ stick test battery . Twenty-five patients volunteered to participate. The test was performed within 45 days and at least 30 days after the diagnosis of COVID-19. No participant had a history of head trauma or another episode of upper respiratory tract infection after the diagnosis of COVID-19. The tests were performed as previously described by Rumeau et al. [18]. All the tests were performed in a well-ventilated, odor-free room. The investigator who performed the test used personal protective equipment during the procedure. An interval of at least 8 h took place between tests to prevent spread of the disease. Threshold (T), Discrimination (D), Identification (I), and global scores (TDI) were recorded for each patient. Patients with a TDI score lower than 15 are considered anosmic, patients with a score between 15 and 30 are considered as hyposmic, and patients with a score equal to or higher than 30 are considered normosmic. Statistical analysis was performed with IBM SPSS v 22.0 . The Shapiro–Wilk test was used for assessing normality. To display demographic information, mean ± standard deviation (SD) was used for normally distributed variables and median (min–max) for nonnormally distributed variables. Chi-square was used for categorical data. The Student t-test and the Mann–Whitney U test were used to compare the normal and nonnormally distributed data between two groups, respectively. Spearman’s rank correlation coefficient was used for correlation of nonnormally distributed data. The level of statistical significance was set at p ≤ 0.05 with a 95% confidence interval.A hundred and seventy-one patients participated in this study. Of the participants, 58.5% (n: 100) were female and 41.5% (n: 71) were male. The median age of the participants was 36 (min–max: 18–71). Of the patients, 35.7% (n: 61) were smokers and 12.9% (n: 22) were previously diagnosed with allergic rhinitis. Participants’ demographic and clinic information is summarized on Table 1. The patients were clustered in three groups according to disease severity, 73.1% of the patients (n: 125) were in group 1 (asymptomatic other than smell or taste dysfunction), 20.5% (n: 35) were in group 2a (symptomatic disease without pneumonia), and 6.4% (n: 11) were in group 2b (with pneumonia). In total, 26.9% of the patients (n: 46) were clinically symptomatic (group 2). Patients who suffered from olfactory dysfunction and gustatory dysfunction comprised 10.5% (n: 18) and 10.5 (n: 18) of all the patients, respectively. Olfactory and gustatory dysfunctions were present together in 6.4% of the patients (n: 11). Clinical information of patients with and without olfactory dysfunction is summarized in Table 2, and clinical information of patients with and without gustatory dysfunction is summarized in Table 3. Olfactory dysfunction occurred before diagnosis in 8 patients. The median time between onset of olfactory dysfunction and diagnosis was 2.5 days (min–max: 1–5). In 10 of the patients, olfactory dysfunction occurred after diagnosis with a median interval of 1 day (min–max: 1–7). All but one patient recovered from olfactory dysfunction in a median time of 7 days (min–max: 0–30). One patient was still suffering from olfactory dysfunction 45 days after diagnosis. Gustatory dysfunction occurred before diagnosis in 8 participants within a median time of 2 days (min–max: 1–5) and after diagnosis in 10 participants within a median time of 1 day (min–max: 1–3). Gustatory dysfunction resolved in 16 of the patients within a median time of 8.5 days (min–max: 1–30). . Olfactory dysfunction rate in group 1 was lower than that in group 2 but that was not statistically significant (p = 0.072). Olfactory dysfunction rates in groups 2a and 2b were 17.1% and 18.2%, respectively. Gustatory dysfunction occurred in 8% of group 1 and in 17.4% of group 2 and that was not statistically significant (p = 0.072). Gustatory dysfunction rates in group 2a and 2b were 17.1% and 18.2%, respectively. Olfactory dysfunction occurred in 8% of group 1 and 17.4% of group 2The median ages of the patients who suffered from olfactory dysfunction and who did not were 39.5 years (min–max: 18–55) and 36 years (min–max: 19–71), respectively (p > 0.005).Patients with a previous diagnosis of allergic rhinitis had a higher olfactory dysfunction rate compared to the other patients  but that was not statistically significant (p > 0.05). Gustatory dysfunction rates were also higher in the patients with allergic rhinitis (22.7% vs 8.7%) (p > 0.05). Olfactory dysfunction rates in smokers and nonsmokers were 13.1% and 9.1%, respectively. Gustatory dysfunction rate of smokers was 19.7% and significantly higher than gustatory dysfunction rate of nonsmokers (5.5%) (p = 0.007).Olfactory dysfunction occurred in 7.0% of the male patients and 13.0% of the female patients but the difference was not significant (p = 0.16).Mean recovery time of olfactory dysfunction was 17.3 ± 12.3 days in group 2 and 9.2 ± 8.2 days in group 1. The difference was insignificant (p > 0.05). The mean recovery time of olfactory dysfunction of smokers was 12.6 ± 11.5 days and was similar with the mean recovery time of nonsmokers (12.4 ± 10.3 days). The mean SNOT-22 score of all the patients was 11.6 ± 13.2 (min–max: 0–59). In groups 1, 2a, and 2b, the mean scores were 10.3 ± 11.7, 17.9 ± 17.2, 6.73 ± 9.9, respectively. There was not statistically significant difference between SNOT-22 scores of groups 1 and 2 (p = 0.19). The mean SNOT-22 score of patients who suffered from olfactory dysfunction was 20 ± 13.4 and the mean score of patients who did not was 10.6 ± 12.9. The difference was statistically significant. (p = 0.001) (Table 4). The mean SNOT 22 scores of patients who had gustatory dysfunction and who did not were 27.2 ± 14.9 and 9.8 ± 11.8, respectively. The difference was also statistically significant p < 0.001). Analysis of the correlation between SNOT-22 scores and recovery times (days) of olfactory and gustatory dysfunctions did not show significance (p > 0.05 R: 0.315). Objective olfactory evaluation with Sniffin’ sticks test battery was performed to 25 volunteered patients, 52% of whom (n: 13) were female and 48% of whom (n: 12) were male. The median age of the patients was 38 years (min–max: 23–52). Twenty-two patients were in group 1 and three patients were in group 2a. Twenty-four percent of the patients had a previous diagnosis of allergic rhinitis and 48% were smokers. None of the participants had self-reported olfactory dysfunction at the time of Sniffin’ sticks test. Only 2 patients reported olfactory dysfunction due to COVID-19 and both claimed to be totally recovered. The median SNOT-22 score of these 25 patients was 9 (min–max: 0–59). The median T, D, I, and TDI scores were 10.33 (min–max: 1–16), 10 (min–max: 6–16), 11 (min–max: 8–15), and 32 (min–max: 19.33–43), respectively. Sixty-four percent of the patients (n: 16) were normosmic and 36% (n: 9) were hyposmic. Eleven-point-one percent (n: 1) of the hyposmic patients had self-reported olfactory dysfunction due to COVID-19. Eighty-one-point-three percent of normosmic patients were in group 1. All the hyposmic patients were in group 1. Sixty-three-point-six percent of the hyposmic patients and 42.1% of tne normosmic patients were smokers. Twenty-five percent of normosmic patients and 22.2% of normosmic patients were diagnosed with allergic rhinitis. Smoking habit and previous diagnosis of allergic rhinitis did not differ significantly between hyposmic and normosmic patients. Demographic and clinical information of the patients participated in Sniffin’ Stick Test was summarized in Table 5.Anosmia is a well-known symptom of viral upper respiratory tract infections. A viral infection is the reason of anosmia in 40% of the cases in adult patients [9]. Many viruses such as rhinovirus, Epstein-Barr virus, and parainfluenza may cause mechanical obstruction with mucosal inflammation and rhinorrhea, resulting in olfactory dysfunction . However, the smell disorder associated with COVID-19 has a different pathogenesis that can occur without rhinorrhea and nasal obstruction . Lechien et al. claimed that 79.7% of COVID-19 patients with anosmia or hyposmia did not complain about rhinorrhea and nasal obstruction [4]. On the other hand, in our study, the mean score of “nasal obstruction” (question-2 of SNOT-22) of the patients with self-reported olfactory dysfunction was significantly higher than the patients without. In the literature, rates of olfactory dysfunction vary among studies between 3.2% and 98.3%  and gustatory dysfunction varies between 5.6% and 88% . In a metaanalysis reported by Agyeman et al., olfactory dysfunction and gustatory dysfunction rates were found to be 41.0%  and 38.2%  respectively [21]. In our study we found that rates of olfactory and gustatory dysfunctions were both 10.5%. We attribute our low rates of olfactory and gustatory dysfunctions to the fact that most of the participants in the study were asymptomatic patients diagnosed during contact tracing.COVID-19 may present in a wide clinical spectrum, from asymptomatic cases to severe illness, with or without pneumonia [22]. In our study, the patients were clustered in three groups according to disease severity, 73.1% of the patients were in group 1 (asymptomatic other than smell or taste dysfunction), 20.5% were in group 2a (symptomatic disease without pneumonia) and 6.4% were in group 2b (presented with pneumonia). The patients in group 1 had a lower rate of olfactory dysfunction compared to group 2 but that was not statistically significant. Several authors reported lower olfactory disorder rates in severe COVID-19 [23–26]. However, in the studies that used psychophysical olfactory tests, no relationship between disease severity and olfactory dysfunction was found .Some studies reported significantly higher olfactory dysfunction rates in females than in males . In our study, the rate of self-reported olfactory dysfunction was higher in females (13% vs. 7%) but that was not statistically significant. The relation between smoking and self-reported olfactory dysfunction varies among studies . In our study, the rate of olfactory dysfunction of smokers was similar to that of nonsmokers. The mean ages of the patients with and without self-reported olfactory dysfunction were 39.5 (min–max: 18–55) and 36 (min–max: 19–71) respectively. Vaira et al. evaluated a large group of patients with psychophysical olfactory tests and did not find a relationship between age, sex, smoking, and olfactory dysfunction [31].In our study, gustatory dysfunction rate of smokers was significantly higher than that of nonsmokers. No information was found in the literature on how smoking affects the susceptibility to gustatory dysfunction in COVID-19 patients. On the other hand, smoking is known to cause alternations of gustatory function [32]. All but one patient recovered from olfactory dysfunction in median time of 7 days (min–max: 0–30). One patient was still suffering from olfactory dysfunction 45 days after diagnosis. Gustatory dysfunction resolved in 16 of the patients within a median time of 8.5 days (min–max: 1–30). These findings were in line with the literature. In the study by Lechien et al. 96.7% of the patients recovered in two weeks [4]. Klepfenstein et al. pointed out that the average duration of anosmia was 8.9 days and ≥14 days for 20% of individuals [33]. Lee et al. reported that patients with olfactory or gustatory dysfunctions recovered within 3 weeks; with the average recovery time of 7 days [29].SNOT-22 score of group 2a was higher than the others. Lechien et al. also reported higher SNOT-22 scores in the patients with moderate disease than patients with mild or severe disease [34]. Since the first 12 questions of SNOT-22 mostly cover the symptoms of upper respiratory tract infection, it was not surprising to have higher SNOT-22 scores in patients with moderate symptoms without pulmonary disease. Samaranayake et al. pointed out that “nasal blockage” (question 2) and “runny nose” (question 4) were more prevalent in patients with mild or moderate disease than patients with severe disease [35]. In the second step of our study, we aimed to evaluate olfactory function of recovered patients with sniffin’ sticks test battery. All the tests were performed at least 30 days after the diagnosis. No participant reported olfactory dysfunction at the time of test. According to TDI scores, 64% of the patients were normosmic and 36% were hyposmic. Moein et al. reported that nearly 65% of the patients were unaware of their olfactory dysfunction [20]. Vaira et al. reported that 14.5% of the patients without self-reported olfactory dysfunction were actually hyposmic [31]. On the other hand, being unaware of olfactory dysfunction is not rare with a prevalence of %22 in normal population [36]. According to this information, it may be wrong to say that hyposmia in our patients is caused by COVID-19, but it could be speculated that olfactory dysfunction is more common in COVID patients than patients’ self-report. There was not any significant difference of age, sex, severity of disease, and diagnosis of allergic rhinitis between normosmic and hyposmic patients. The median SNOT-22 score was higher in hyposmic patients. This study has several limitations. Firstly, both the survey and psychophysical olfactory tests were performed after the patients’ recovery. The survey was based on patients’ self-reported data. This posed a risk for recall bias. Secondly, almost all the patients who participated in psychophysical olfactory tests were those who did not report olfactory dysfunction during COVID-19. Therefore, no comment could be made on the permanence of the olfactory dysfunction caused by COVID-19. A third limitation was that the olfactory functions of the patients who underwent psychophysical olfactory tests were not objectively known prior to disease. Therefore, it could not be clarified whether the hyposmia in our patients was due to COVID or not.Olfactory and gustatory dysfunctions have been a remarkable issue for physicians since the beginning of the COVID-19 outbreak. These symptoms occur in the early period of the disease. Olfactory and gustatory dysfunctions are more common in patients who are clinically symptomatic than those diagnosed during contact tracing. Olfactory dysfunction is not related to the severity of the disease. Objective tests may show that frequency of olfactory dysfunction is greater than frequency of self-reported olfactory dysfunction.The study was approved by the local ethical committee of Gazi University. All the participants provided informed consent.


Tuberculosis lymphadenitis (TBLN) diagnosis is often challenging in most resource poor settings. Often cytopathologic diagnosis of TBLN suspected patients is inconclusive impeding timely clinical management of TBLN suspected patients, further exposing suspected patients either for unnecessary use of antibiotics or empirical treatment. This may lead to inappropriate treatment outcome or more suffering of suspected patients from the disease. In this study, an integrated diagnostic approach has been evaluated to elucidate its utility in the identification of TBLN suspected patients.A cross-sectional study was conducted on 96 clinically diagnosed TBLN suspected patients, where fine needle aspirate (FNA) samples were collected at the time of diagnosis. FNA cytology, Ziehl-Neelsen (ZN), Auramine O (AO) staining, GeneXpert MTB/RIF and Real time PCR (RT-PCR) were performed on concentrated FNA samples. Considering culture as a gold standard, the sensitivity, specificity, positive and negative predictive values were calculated. Cohen’s Kappa value was used to measure interrater variability and level of agreement and a P-value of <0.05 was considered as statistically significant.Mycobacterium tuberculosis (Mtb) using ZN staining, 27 (28.1%) using AO staining, 51 (53.2%) using FNAC, 43 (44.7%) using GeneXpert MTB/RIF, 51 (53.1%) using Real time PCR (RT-PCR) and 36 (37.5%) using Lowenstein-Jensen (LJ) culture. Compared to LJ culture, the sensitivities of GeneXpert MTB/RIF, RT-PCR, and FNAC were 91.7%, 97.2%, and 97.2%, respectively and the specificities were 83.3%, 73.3%, and 68.3%, respectively. GeneXpert MTB/RIF and RT-PCR when combined with FNAC detected 61 (63.5%) cases as having Mtb, and the sensitivity and specificity was 100% and 58.3%, respectively.Out of the 96 FNA sample, 12 (12.5%) were identified to have Mtb detection tools and the detection sensitivity even improved when FNA cytology was combined with GeneXpert MTB/RIF, performed on concentrated FNA sample, suggesting the combined tests as an alternative approach for improved diagnosis of TBLN.FNA cytology and RT-PCR detected more TBLN cases compared to other  Mycobacterium tuberculosis (Mtb) complex and remains to be the major cause of death globally 10uM IS1081_Probe [6FAM]-Real time to detect MTB complex in DNA extracted from FNA sample.Master Mix for 1X sample was prepared by mixing 2.5 μl of IS1081f and IS1081r, 0.5 μl Probe FAM labeled, 12.5 μl hot start master mix, and 2 μl molecular grade water in a biosafety cabinet, an aliquot of 20ul of the master mix was added into each Real time-PCR reaction tube. DNA templates i.e. (controls and unknown samples) were added to each tube containing the master mix.The tubes were placed in a Rotor Gene 3000 Real Time PCR machine  and run with thermal conditions of initial denaturation at 95°C for 15 mins, and 40 cycles of 95°C for 15 sec, 58°C for 1 min. Samples were considered positive if Ct value was less than 36.Mtb infection. Of these 51 positive samples, 44.8% of the FNA samples were collected from cervical site and the 1% was that collected from cervical and auxiliary areas Of all FNA samples processed, 51 (53.1%) were aspirates mixed with blood, 36 (37.5%) purulent and the remaining 9 (9.4%) were aspirates with cheesy-like material. Depending on the site of swollen lymph node, the majority of the lymph nodes were from cervical region (83.3%), followed by auxiliary (9.4%) and inguinal (6.3%). A small proportion of the lymph nodes (1.04%) were also from cervical and auxiliary regions. Based on cytological examinations, 53.1% (51) of the FNA samples suggested A Higher number of FNAC positive smear was observed in group 1, whereas the least number was observed in last group Mtb growth on LJ culture media out of the 96 FNA samples collected, where the colony characteristics ranged from single colony to multiple colonies. Four samples got contaminated and were excluded from the analysis. The average growth period for LJ culture ranged between 3 and 6 weeks. Fine needle aspirate cytology, RT-PCR and GeneXpert MTB/RIF detected more Mtb bacilli  compared to the microscopic staining techniques (Zn (12) and AO (27)) ) ; however.According to an earlier report , a posit.Previous history of anti-TB treatment didn’t show significant association with GeneXpert MTB/RIF resistance. None of the participants showed resistance to rifampicin as detected by GeneXpert.The detection rate of all parameters included in this study as compared with FNAC was summarized as shown in ZN and AO staining have detected 1 additional positive case which was missed by FNAC. From all FNAC positives cases, 11 of them were also positive with ZN. While AO detected 26 positive cases from all FNAC positive results.Of the 42 cytology negative results, 4 were positive with GeneXpert MTB/RIF assay and 13 cases were negative by GeneXpert MTB/RIF from all FNAC positive cases. RT PCR detected additional 6 positive cases which were FNAC negative. Among the 3 inconclusive results, 1/3 was positive with both RT PCR and GeneXpert MTB/RIF assay while 2/3 were negative.Positivity rate of molecular methods were compared with that of FNAC. RT PCR detected 6 positive cases which were FNAC negative and 9 GeneXpert negative cases as positive. GeneXpert also detected 4 FNAC negative cases as positive and 1 RT PCR negative case as positive. FNAC in turn detected 7 positive cases missed by RT PCR and 13 negative cases by GeneXpert were positive by FNAC.The Sensitivity, specificity, positive predictive value, negative predictive value, and positive and negative likelihood ratio were calculated with 95% confidence interval with culture being gold standard. The sensitivity of GeneXpert MTB/RIF assay, 91.7% [95%CI (91.8%– 100%)], was slightly lower than RT PCR, the specificity, 83.3% [95%CI (73.9%– 92.8%)], was higher when compared with that of RT PCR. The sensitivity, specificity, positive predictive value, and negative predictive value of cytology were 97.2% [95%CI (91.8%– 100%)], 68.3% [95%CI (60%– 82.5%)], 68.6% [95%CI (55.8%– 81.3%)], and 97.6% [95%CI (93%– 100%)] respectively. The molecular methods (GeneXpert and RT PCR) were combined together with FNAC to see if the laboratory diagnosis of TBLN can be improved. As shown in the table, the three methods in combination detected 61 positive cases. The level of agreement with FNAC  was 0.772. To see the combination effect of the molecular methods separately, GeneXpert MTB/RIF assay with FNAC, and RT PCR with FNAC were compared with the result from FNAC. Kappa value GeneXpert assay with FNAC was 0.856 with P value of <0.001. Similarly, the kappa value was 0.815 with p value of <0.001 for combined effect of RT PCR with FNAC. The detection rate of combined methods (Molecular methods + FNAC), GeneXpert + FNAC, and RT PCR + FNAC were calculated in comparison with culture. The detection rate when GeneXpert+RT PCR+FNAC combined together was higher prevailing 61 positive cases. A substantial agreement was shown when GeneXpert was combined with FNAC. The sensitivity, specificity, PPV, NPV, level of agreement, and P value of combined method (FNAC+RT PCR+GeneXpert), GeneXpert + FNAC, and RT PCR + FNAC were calculated in comparison with culture (gold standard). The sensitivity of GeneXpert+RT PCR+FNAC, GeneXpert+FNAC, and RT PCR+ FNAC was 100% while the specificity of Xpert+FNAC, 66.7%, was relatively higher. Diagnosis of TBLN in Ethiopia mainly relies on FNAC. The method was proven to be simple, sensitive, and inexpensive but often times, it is based mainly on suggestive features of tuberculosis such as epithelioid granuloma and caseous necrosis rather than depending on the direct detection of bacteria which might occur due to factors other than Tuberculosis and may produces inconclusive results in some instances . Some reet al [et al has shown the detection rate of culture from all GeneXpert positive cases was 44.3% [et al [The time-consuming nature of culture was also noted well with the current study. The earliest growth recorded in this study was three weeks and the longest period being six weeks. In this study, Culture has shown a positive detection rate of 37.5% of the total participants. Among FNAC positive cases 70.5% of them were also positive with culture. The remaining positive cases missed by culture from all FNAC positive cases might reflect the non-specific nature of FNAC. Same finding has been shown by Kant et al . Among tas 44.3% . Based o% [et al : a kappaCulture method also detected positive cases of 28.6% from all ZN negative cases. Zewdie et al observed an inconsistent finding with 8.7% positivity of culture method from all ZN negative cases . In bothet al showed increased positivity of GeneXpert, that is 60.1%, in comparison with the present study [This study aimed at integrating the molecular methods: GeneXpert MTB/RIF assay and RT PCR with FNAC to improve the laboratory diagnosis of TBLN patients. GeneXpert MTB/RIF assay is a simple, sensitive, and automated method endorsed by the WHO. The method relied on the identification of rpoB gene associated with RIF drug resistance. RT PCR assay in this study targets IS1081 gene present in all MTBC and in a stable copy number of 5–7 repeats per genome , 18. Thent study . In thiset al who reported 38 (19.6%) additional positive cases by GeneXpert from culture negative cases [When compared with culture, GeneXpert has detected an additional 10 (16.67%) positive cases which were culture negative. This finding showed a slight similarity with Samreen ve cases . The kapet al reported 15.6% additional positive cases by GeneXpert which did not have suggestive feature of TBLN by FNAC (19). Among all FNAC positive cases, 74.5% were also positive with GeneXpert MTB/RIF assay and 86.2% were also positive with RT PCR assay. Regarding level of agreement with FNAC, both molecular methods showed an agreement with a kappa value of 0.6 and 0.675 for GeneXpert and RT PCR respectively.GeneXpert and RT PCR have detected 4 (7.8%) and 6 (11.7%) additional positive cases respectively which were negative with FNAC. Tadesse In contrast to the finding from Mengistu et al, which reported a sensitivity and specificity of 78% and 74% for GeneXpert MTB/RIF assay , the preet al, which showed a sensitivity of 70% and higher specificity of 99% [et al [The sensitivity and specificity of RT PCR in this study was found to be 97.2% and 73.3%. This finding is not consistent with Babafemi y of 99% . A very % [et al  which haFNAC has a sensitivity of 97.2% and a specificity of 68.3%. Tamana E-Nur also reported the same pattern of FNAC having a higher sensitivity and lower specificity. The finding from Tamana E-Nur showed the sensitivity of 79.7% and specificity of 48.1% . This seet al has shown that the combined use of PCR with FNAC was helpful in improving the diagnosis of TBLN. They have shown how the sensitivity and specificity of FNAC can be improved from 48% and 87.5% to 84% and 100% respectively [A study by Manitchotpisist ectively . The molWe concluded that the combination of molecular methods, performed on concentrated FNA sample, with FNAC observed in this study clearly showed how the diagnostic value could be enhanced to an improved state than that observed in FNAC alone. Especially combining FNAC with GeneXpert MTB/RIF assay generated a very useful result in delivering affirmative result hence initiating early and appropriate treatment, and in providing better health care service.We recommend that easily accessible, simple and rapid diagnostic methods such as GeneXpert MTB/RIF assay and RT PCR, performed on concentrated FNA sample, can be used to improve the diagnostic challenge of tubercular lymphadenitis, and we also recommend country wide studies to be performed which would give a better picture regarding the application of molecular tests in combination with FNAC, and the challenges seen in the diagnosis of Tubercular lymphadenitis.S1 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


To observe and analyze the visual outcomes of cataract surgery of ten years at a tertiary eye care hospital, Karachi.An observational study with retrospective data search was conducted in Al Ibrahim Eye Hospital (AIEH), Karachi. Data of all adults (above 16 years) who underwent cataract surgery from 2010-2019 was retrieved from HIMS. Presence of opacity in the lens was labelled as cataract. Surgery was advised when patient’s BCVA was found to be ≤ 6/18. Visual assessments of the patients were done on day 01, one week and six weeks postoperatively. Postoperatively, 6/6 – 6/12 was considered as good, 6/18 as mild visual impairment, < 6/18 to 6/60 as moderate visual impairment and < 6/60 as severe visual impairment.A total of 1,027,840 patients visited AIEH with different eye diseases. Among 1027840 individuals, cataract was identified in 88443 (8.6%). Surgery was advised to 58371 and performed in 38616. Records of operated cases (38616) were retrieved. Mean age of patients was 54.96±14.22 years. There were 20578 (53.29%) males and 18038 (46.71%) females who underwent surgery . At the end of sixth week, 3561 (18.4%), patients were found to have “good vision”, 12242 (63.43%) had mild visual impairment, 2676 (13.86%) had moderate visual impairment and 822 (4.26%) had severe visual impairment. Corneal Complications was the commonest cause (33.56%) at sixth week.The institution achieved WHO recommended criteria of “good visual outcome” (6/6 to 6/18) of 81.83% which is near to recommended ≥ 90% and severe visual impairment of 4.26%. Cataract is the gradual cloudiness of the transparent ocular lens. It is regarded as a single largest cause of reversible blindness worldwide. Nearly 45% of global blindness is attributed to cataract, accounting 15 million out of 33.6 million cases of the global blindness.2The burden of vision loss in Pakistan had been on the rise since 1990 and is estimated to increase at a steady rate by 2025. Pakistan ranked third, among south Asian countries, after India and Bangladesh in the prevalence of blindness and vision impairment. Presbyopia, refractory disorder and cataract contributed >90% cases of blindness and severe vision loss. Nearly >50% of these cases in the country were attributed to cataract.2Cataract is primarily an aging phenomenon that cannot be prevented. However, vision impairment by cataract can be restored by surgery. Though cataract surgery is the commonest procedure performed by the ophthalmic surgeons, substantial variations in cataract surgery rate (CSR) between countries were observed. Cataract surgery rate is used as a proxy indicator of access to cataract services in a country.4Present study has followed the new recommendationsIt was an observational study with retrospective data retrieved from HIMS for the year 2010-2019 carried out at Al Ibrahim Eye Hospital, a tertiary eye care center, Karachi. Data of all adults (above 16 years) undergoing cataract surgery by Extra-capsular cataract extraction (ECCE), phacoemulsification with IOL (Phaco + IOL) implant and Intra capsular cataract extraction (ICCE) with or without IOL were included in the study. Missing records, dropped follow-ups, cataract surgery combined with other ocular surgery like trabeculectomy, vitrectomy, keratoplasty, complicated and secondary cataract were excluded.st week and 6th week after surgery. BCVA was recorded at all visits. Visual acuity (BCVA) was graded according to new recommendations where visual outcome is categorized into 4 grades: good 6/6 to 6/12, minimal visual impairment 6/18, moderate visual impairment < 6/18 to 6/60 and severe visual impairment < 6/60.All patients visiting the out patient’s department of Al Ibrahim Eye Hospital for ophthalmic consultation, underwent detail ophthalmic examination like Best corrected visual acuity (BCVA), Slit lamp examination, IOP and Fundus examination. Presence of opacity in the lens was labelled as cataract. B-scan was performed in patients whose fundus was not visible with 90 D fundus examination. Surgery was advised when patient’s BCVA found to be < 6/18 or patient was not satisfied with his present vision. Pre-operative investigation included Hepatitis B, C, HIV and Random Blood Sugar level. Keratometry and biometry was done to determine IOL number. The choice of procedure was based on type and grade of cataract. Extracapsular extraction was done in mature and hyper mature cataracts where phacoemulsification was problematic. IOL was implanted in both procedures. Post-operatively topical antibiotics and steroids were prescribed to all patients. First follow-up was scheduled the next day of surgery. Subsequent follow-up was at 1WHO recommends following categories and levels to be achieved:6Almost all national studiesData was retrieved from Hospital Information Management System (HIMS) and exported to SPSS version 23.0. Mean and Standard deviation was calculated for continuous variable like age. Frequencies and percentages were reported for categorical variables like age groups, gender, type of surgery, categories for visual acuity and causes of reduced BCVA. Chi-square test was applied to see the significance. P-value ≤ 0.05 considered to be statistically significant.During the year 2010-2019, total of 1,027,840 patients visited Al- Ibrahim Eye Hospital with different eye problems. Among them 88443 (8.6%) individuals were labelled as cataract, Age of patients ranged from 16 to more than 60 years. Mean age of patients operated was 54.96±14.22 years. An increasing frequency of age group was seen from 46 to 60 years. Gender distribution was observed as male 20578 (53.29%) and female 18038 (46.71%). Phacoemulsification with PC IOL (Phaco + PC IOL) was done in 33990 (88.02%) and Extracapsular Cataract extraction with Posterior Chamber Intraocular Lens (ECCE + PC IOL) was done in 3684 (9.54%) patients. Remaining individuals 942 (2.4%) had Intra capsular cataract extraction (ICCE) with or without IOL .Total number completing follow up of 6 weeks were 19301 (50%) Those with “good vision” were 3561 (18.4%), with mild visual impairment were 12242 (63.43%), with moderate visual impairment were 13.86% and those with sever vision impairment were 4.26% .st postoperative day, 27.41% (10588) on 2nd follow up at one week and 50% (19315) at 6th week. At 6 week follow up, 18.27 % of the Phaco IOL had “ good vision”, 65.04% had mild visual impairment, 13.03% had moderate and only 3.64% had severe visual impairment. Respective results with ECCE IOL were 19.2%, 55.7%, 17.78% and 7.18%. Severe visual impairment with Phaco is 3.64%, which is well within recommended level of < 5%; but 7.18% with severe visual impairment in ECCE IOL is more than desirable (Drop outs were 9.84% (3801) patients on 1esirable .th week follow-up is shown in  undergone cataract surgery, which is 12.92% lower in women. Various studies from Pakistan reported Cataract surgical coverage (CSC) of 69.9 – 72.2% among men while reported CSC for women was 39.6 – 60.2 %. Studies also reported gross gender inequity in terms of CSC from other countries of South Asia. Like in India CSC was found to be 27% higher in men than women. Nearly 2/3Reporting the outcome of cataract surgery is important in order to establish the bench mark and to audit the performance of an institute. Nearly 19755 (33.9%) individuals advised for surgery did not avail intervention. It remains to be explored why 33.9% people with cataract did not avail surgery although being non-profit organization, surgery is offered at very subsidized rate. These patients availed the free consultation; but did not avail the intervention. Second observation in this study was patient dropout rate in follow ups after cataract surgery. Our findings indicated that people seek and receive cataract surgery but miss out on regular follow-up consultations. Most likely reason of both these occurrences is financial constraint. Though being charitable institute services in this center is offered at very subsidize rate, for the very poor segment of people it remains out of pocket. People were too poor to afford even subsidized cost and come for follow up. There exists strong relationship of poverty with cataract surgical rate (CSR), and postoperative visual acuity.19Over the period of ten years, change in the operation procedure was observed from ECCE (17.38%) to Phacoemulsification (82.6%). ECCE has been replaced by Phaco surgery except in very hard cataracts. Our main worry is moderate vision impairment (<6/18 to 6/60) of 13.03% with Phaco surgery.Our results are comparable with national and afro Asian countries. Most of these studies followed the old recommendations where “good vision” includes 6/6 to 6/18, border line 6/24-6/60 and poor vision <6/60. In our study if 6/18 is considered as “good vision”, the number will be 81.88% with Phaco and 74.9% with ECCE IOL. Report from university college hospital in IBADAN showed “Good vision” in 78.8%, borderline vision in 17.4%, and severe visual impairment in 3.8% after cataract surgery.21Rapid assessment of cataract surgical services in Malakand district showed good, borderline and poor visual acuity of 40.5%, 22% and 14.3% respectively.Like any surgical procedure, cataract surgery is also liable to complications. These complications result in poor visual outcomes and are dependent on many preoperative, intraoperative and postoperative factors. Corneal damage during surgery is the most common cause of postoperative moderate as well as severe vision impairment. This is common in the learning phase of Phaco emulsification. The second common reason for unfavorable visual outcome was due to refractive errors resulting from the surgery 13th week. We are completely unaware of the visual status of these patients. Another important aspect of this study is variability of the operating surgeon from trainee ophthalmologist to senior surgeon over 10 years.Though present study is the largest study reporting post-operative outcomes of cataract, study demands carful interpretation. Deposition of data in HMIS is not real time. Data entry operator transferred data from hard copy to the data bank at a convenient time. This may result in erroneous entry which is rectified by randomly cross checking and matching the hard file entry with data bank. Nearly 33.9% people attended the hospital and diagnosed with cataract did not avail surgery in our center. Moreover, postoperative patient’s dropouts reached to 50% (19315) at 6The institution achieved WHO recommended criteria of “good visual outcome” (6/6 to 6/18) of 81.83% which is near to recommended ≥ 90% and severe visual impairment of 4.26%.MSM manuscript writing & final review.SA manuscript writing, critical review & literature search.SB did final review of the article.TM did data collection, summarizing of data & editing of manuscriptMFF did Statistical analysis and result write up.UH review from clinical point of view as an ophthalmologistMA design of study and comments on review article.


Methane measurements, and sample collections for rumen metabolite and microbial community composition were carried out at the end of weeks 2, 4, 6, 8, 10, 14, 24 and 49. Animal growth and dry matter intake (DMI) were regularly monitored over the duration of the experiment. Methane emissions decreased up to 90% whilst hydrogen emissions increased in treated compared to control calves, but only for up to 2 weeks after treatment cessation. The near complete methane inhibition did not affect calves’ DMI and growth. The acetate:propionate ratio decreased in treated compared to control calves during the first 14 weeks but was similar at weeks 24 and 49. The proportions of Methanobrevibacter and Methanosphaera decreased in treated compared to control calves during the first 14 weeks; however, at week 24 and 49 the archaea community was similar between groups. Bacterial proportions at the phylum level and the abundant bacterial genera were similar between treatment groups. In summary, methane inhibition increased hydrogen emissions, altered the methanogen community and changed the rumen metabolite profile without major effects on the bacterial community composition. This indicated that the main response of the bacterial community was not a change in composition but rather a change in metabolic pathways. Furthermore, once methane inhibition ceased the methanogen community, rumen metabolites and hydrogen emissions became similar between treatment groups, indicating that perhaps using the treatments tested in this study, it is not possible to imprint a low methane microbiota into the rumen in the solid feed of pre-weaned calves.The present study aimed to determine whether dietary supplementation with methanogen inhibitors during early life may lead to an imprint on the rumen microbial community and change the rumen function and performance of calves to 49-weeks of rearing. Twenty-four 4-day-old Friesian x Jersey cross calves were randomly assigned into a control and a treatment group. Treated calves were fed a combination of chloroform (CF) and 9,10-anthraquinone (AQ) in the solid diets during the first 12 weeks of rearing. Afterward, calves were grouped by treatments until week 14, and then managed as a single group on pasture. Solid diets and water were offered  Methano) to CH4 , but the removal . The usehe rumen , change  species . Howeverin vitro and in vivo have shown that chloroform (CF) and 9,10-anthraquinone (AQ) are potent methanogen inhibitors  during early and adult life on rumen microbial establishment, rumen function and performance in calves. The use of CF and AQ was to provide a methane inhibition effect that would last over the treatment period even if the rumen community adapts to one of the inhibitors. The objective of the present study was to determine whether feeding CF and AQ in the solid feed diet during early life may lead to an imprint on the rumen microbial community, change the fermentation pathways and alter growth performance of calves to 49 weeks of age.Studies hibitors . The mechibitors . In contormation . The useormation  with theTwenty-four female dairy calves (Friesian x Jersey cross) were randomly allocated, following a simple randomization procedure, to a control and a treatment group. The treatment group had the methane inhibitors CF and AQ mixed into their starter concentrate and partial mixed ration (PMR) diets. The control diet did not contain methane inhibitors. The treatment period lasted for 12 weeks followed by a 37-week period in which both groups were fed similar diets. Animal manipulations were reviewed and approved (AE13132) by the Grasslands Animal Ethics Committee and complied with the institutional Codes of Ethical Conduct for the Use of Animals in Research, Testing and Teaching, as prescribed in the New Zealand Animal Welfare Act of 1999 and its amendments.Calves were sourced from a single commercial farm at 4 days of age and an average weight of 33 ± 3.7 kg (mean ± S.D.). On arrival to the animal facility, calves were weighed and assigned to one of the experimental groups. Calves were housed in individual pens (1.5 m × 3 m) bedded with wood shavings. Experimental groups were housed in separate temperature-controlled rooms to avoid cross contamination of ruminal microbes between treatments. At week 10, after weaning off milk, calves were moved from individual to a single group pen within each treatment group. At week 14, calves from both groups were transferred outdoors and managed as one mob on pasture. LW was determined weekly during the first 10 weeks of life, fortnightly until week 24 of age, and monthly thereafter. ADG was calculated at the end of the experiment.ad libitum from day 8. From week 4, calves were offered ad libitum access to starter concentrate plus a PMR. Calves were weaned off starter concentrate from weeks 12 to 14. At week 14, calves were moved outdoors and managed as a single mob on a ryegrass and red clover mixed sward, with continue free access to the PMR. Calves were weaned off PMR diet from week 15 to 17, reducing intake by 10% per day. Fresh water was available ad libitum throughout the study.All calves were fed 4 L/d  of colostrum during the first 4 days of life prior to enrollment into the trial. From day 5 onward, calves were fed 4 L of reconstituted milk replacer  and 49.5% lactose; Milligans, Oamaru, New Zealand) split into two equal feeds of 2 L fed at 0800 and 1600 h using individual feeders. At week 4, calves were transitioned to once per day milk feeding, where 4 L was offered in the morning only. At week 10, calves were fully weaned from MR over a 10-day period reducing individual milk intake by 10% per day. Starter concentrate  was offered The ingredients used in the starter concentrate and PMR diets are given in ®, TBCDF-F, Cyclodextrin Technologies Development Ltd., Gainesville, FL, United States) to stop evaporation from the feed and to render it odorless. 50 ml of CF were added to β-CD dissolved at 10% (w/v) in 10 L of water. The mix was kept at 4°C and shaken every hour during an 8 h period, after which it settled over night. The supernatant liquid was decanted and the sediment filtered using Whatman no. 54 filter paper, dried in the fridge at 4°C and transferred to a jar. Concentrations of 9,10-AQ and CF fed to the calves in the starter concentrate and PMR diets were 500 and 50 mg/kg of feed, respectively. The choice and dose of the CF/AQ mix was based on a dose response test run in vitro . Both AQ and CF were pre-mixed into approximately 5 kg of starter concentrate using a food processor and then mixed into the total batch amount using a concrete mixer. The same process was used for the PMR diet, where the inhibitors were first mixed into 5 kg of soybean meal, then into the total soybean meal using a concrete mixer and, finally, the rest of the ingredients were incorporated in a large mixer. The final mixes were prepared twice a week and stored at 4°C until used.The inhibitors used in this study were 9,10-AQ  and CF . The CF was complexed with β-cyclodextrin , when solid diets, i.e., the starter concentrate and/or PMR diets (week 2–14), or fresh grass (week 24 and 29), were offered. During the milk feeding period, MR was offered before entering to the chambers in the morning (0800 h) and on weeks 2 and 4 also before the afternoon feed allocation (1600 h). For the last two measurements, the same type of pasture that the animals were consuming in their allocated paddocks was cut daily  and transported to the Animal Facility at Grasslands. For measurements at week 24 and 49, calves were moved to indoor yards and adapted to eat fresh cut pasture in pens for five to 7 days prior to entering to the chambers. Animals in the chambers were offered solid diets ad libitum and refusals were collected to determine DMI from the difference between feed allowance and refusals.Emissions of CHchambers  for 24 hg, 10 min, 4°C) and an aliquot of 0.9 ml of the supernatant was collected into 0.1 ml of internal standard (19.8 mM ethylbutyrate in 20% v/v phosphoric acid) and stored at −20°C until analysis. Rumen samples for subsequent DNA extraction and microbial community analysis were snap-frozen and stored at −20°C.Rumen samples were taken via stomach tubing after removing the calves from the respiration chambers . Each sample was subsampled for SCFA analysis (1.8 ml) and DNA extraction (0.9 ml). Samples for SCFA analysis were centrifuged  and 0.8 ml of the supernatant was collected into a crimp cap glass vial. Gas chromatography was used to analyze SCFA composition  method . A totalo of 5:1 .PRJEB37781.Before sequencing, pooled libraries were checked for quality control (QC) with the Labchip GX Touch HT Instrument  using the DNA High Sensitivity assay. Amplicons were sequenced at the Massey Genome Service/New Zealand Genomics Limited using Illumina MiSeq system . The pooled library was run on one Illumina MiSeq; 2 × 250 base PE run version 2 chemistry . An Illumina prepared PhiX control library for the run was loaded onto the Illumina MiSeq run at 20% volume. Sequence reads were provided in fastq format. Raw sequence reads were deposited in the European Nucleotide Archive under the accession number Sequencing reads were quality-filtered using the DynamicTrim function of SolexaQA . Reads wThe effects of including CF and AQ (methane inhibitors) in the solid feed diet of calves from 1 to 12 weeks of age on DMI, ADG and LW, rumen function (SCFA and enteric emissions) and microbial community composition (bacteria and archaea) were evaluated during and after treatment.NLME package in R  model via the restricted maximum likelihood (REML) framework as implemented in the age in R . DMI, LWijk is response at time j for the kth animal in the ith treatment, μ is the general mean, βk is the covariate for the animals within treatments , αi is the effect of the ith treatment, γj is the effect of the jth time, (αγ)ij is the interaction between treatment and time, uik is the normally distributed random experimental error for the experimental units  with constant variance σu2, and eijk is the normally distributed random experimental error on repeated measures with variance σe2. The resulting models were analyzed by repeated-measures ANOVA and Tukey’s post-test to determine the longitudinal effects of the intake of methanogen inhibitors during early life. Predicted means from the model, together with estimates of the standard error of the mean (SEM), permutation F-test of the model , and pairwise comparisons (Tukey test) were obtained using the PREDICTMEANS package in R  of the geometric mean, permutation test BH; . TransfoP < 0.001) in both treatment groups. Total mixed ratio intake was 11% lower (P = 0.022) in treated compared to control calves. Concentrate intake had a treatment by time interaction effect tendency (P > 0.090), but the post hoc (Tukey test) analysis did not show differences (P ≥ 0.468) between treated and control calves. No other treatment by time effects were observed for PMR, pasture and total DMI of all solid feed diets. LW and ADG of treated and control calves is presented in P < 0.001) for overall LW gain throughout the study, nevertheless this was primarily influenced by a 5.6% difference in LW between treated and control calves at week 44  of an effect of treatment on ADG (Trt = 0.613 kg/d [C.I. = 0.590 – 0.637] vs. Ctrl = 0.642 kg/d [C.I. = 0.619 – 0.666]). ADG declined from week 14 to 18 following removal of the starter concentrate with recovery of growth rates observed by week 20  were evident during the first 14 weeks of rearing for yield and production of CH4 and H2, and the percentage of acetate, propionate, caproate, valerate and isovalerate. Treated calves had average CH4 yield decreases (P < 0.001) of 7.9 ± 1.90-fold (mean ± SEM), and H2 yield (P < 0.001) increases of 88.7 ± 48.78-fold compared to control calves, but similar values for CH4 yield and H2 yield in both groups at 24 and 49 weeks. The average proportion of acetate decreased 1.2 ± 0.02-fold (P < 0.001) and those of propionate increased 1.3 ± 0.05-fold (P = 0.001) in treated compared to control calves, but the proportions of butyrate were similar (P = 0.246) in both groups. When CH4 was inhibited, treated calves had valerate 2.1 ± 0.17-fold greater (P < 0.001), caproate 2.8 ± 0.08-fold greater (P < 0.001) and isovalerate (P < 0.001) 2.4 ± 0.19-fold greater average proportions than control calves. No interaction effects were observed for isobutyrate (P = 0.462) or total SCFA concentrations (P = 0.723). Similar SCFA profiles were observed in both groups at weeks 24 and 49.The intake of methane inhibitors on the rumen function is shown in A total of 9,527,448 reads were obtained from 190 rumen samples, using the Illumina MiSeq platform, with an average of 42,445 bacteria and 7,699 archaea sequences per sample. The number of OTUs was 1,500 and 40 for bacteria and archaea, respectively. A total of 245 bacteria and 17 archaea taxa were analyzed after using a minimal sample cut-off of 200 reads.P < 0.001) over time, but it was similar for the effects of treatment (P = 0.713) and treatment by time interaction (P = 0.907). The archaeal Shannon index (P < 0.001), where treated calves had a greater diversity at weeks 2 (P < 0.001) and 4 (P = 0.002) compared to control calves. In treated calves (0.48 ± 0.024), the archaea community showed a 24.5% greater (P = 0.002) Shannon index than in control calves (0.36 ± 0.024). Time effects (P < 0.001) were evident in calves at weeks 6 (0.33 ± 0.038) and 14 (0.30 ± 0.037) and had a lower (P < 0.001) archaeal diversity than calves at week 49 (0.56 ± 0.038).on index  showed aThe beta diversity of the bacteria dataset is shown in Firmicutes  and Bacteroidetes (43.1 ± 7.22%) were the most abundant phyla. Whilst Proteobacteria, Spirochaetae, Tenericutes, Fibrobacteres, Actinobacteria, and Cyanobacteria together represented only 12.8 ± 4.71% of the bacteria phyla in the rumen. The effect of methane inhibitors on the abundant bacterial phyla at different sampling times is shown in Fibrobacteres (P = 0.048) and Cyanobacteria (P = 0.003). Pairwise analysis (P = 0.137) did not show differences for Fibrobacteres at any of the sampling times between treatments. The relative abundance of Cyanobacteria decreased in treated calves at weeks 10 and 14  with respect to control calves. Time effects were observed (P < 0.001) in the abundant bacterial phyla composition, for all except the Actinobacteria (P = 0.125). The proportions of Bacteroidetes and Firmicutes were constant until week 49, when Bacteroidetes dominated the ruminal community. Other changes over time were the general decline in Proteobacteria and at the same time an increase in Spirochetes until weaning (week 10) that decreased thereafter.The bacterial community, from the selected cut offs of OTUs, showed 18 different phyla. These phyla corresponded to 32 classes, 46 orders, 81 families and 244 genera –6. PhylaPrevotella 1 was the most abundant genus (24.7 ± 11.23%), followed by Christensenellaceae R-7 group (5.2 ± 5.54%), Rikenellaceae RC9 gut group (4.3 ± 3.87%), Ruminococcus 2 (4.1 ± 5.61%) and Sharpea (3.2 ± 6.54%). The abundant bacteria genera for treated and control calves during different sampling times are shown in P ≤ 0.048) were observed in 15 abundant bacteria genera; however, pairwise comparison adjusted to Benjamini–Hochberg (BH) showed significance (P ≤ 0.040) in only 11 abundant bacteria genera for treated when compared to control calves at random weeks during and 2 weeks after treatment. Treatment effects (P < 0.031) were observed in 9 of the 41 abundant bacteria genera. Samples from inhibitor-treated calves indicated proportional increases of Rikenellaceae RC9 gut group, Succiniclasticum, Lachnospiraceae NK3A20 group, Ruminococcaceae UCG 002 and p-2534-18B5 gut group, whilst decreases of Ruminococcus 1, Ruminococcaceae NK4A214 group, Ruminoclostridium 5 and Ruminococcaceae UCG 005 when compared to control calves. Most of the abundant bacterial genera were affected by time (P ≤ 0.029), except for Succinivibrionaceae UCG 002, Ruminiclostridium 5 and Eubacterium ventriosum group (P ≥ 0.261).At the genus level, 41 bacteria genera had a relative abundance ≥0.50% across sampling times. These 41 genera accounted for 85.7 ± 5.38% (mean ± S.D.) of the community across sampling times. On average, P ≤ 0.011) were observed during and 2 weeks after the intake of methane inhibitors. Post hoc analyses of Methanomassiliicoccales (Mcc.) Group 12 sp. ISO4-H5 showed greater  relative abundance from 2 to 10 weeks, and Methanosphaera (Msp.) sp. ISO3-F5 showed lower (P < 0.001) abundance at weeks 6 and 14 , in treated when compared to control calves. Methanobrevibacter (Mbb.) ruminantium and Mmc. Group 4 sp. MpT1 had greater (P < 0.001) proportions in treated calves in week 2 ; however, Mmc. Group 4 sp. MpT1 showed significant decreases (P < 0.001) in treated calves at weeks 6, 8 and 14 (between 9 and 57-fold reductions) when compared to control calves. Treatment effects showed greater proportions (P < 0.001) of Mmc. Group 12 sp. ISO4-H5 (2.9% [C.I. = 1.71 – 4.87] vs. 0.4% [C.I. = 0.25 – 0.72]), but lower proportions of Mbb. gottschalkii  and Msp. sp. ISO3-F5  in treated when compared to control calves. At weeks 24 and 49, the archaeal groups did not differ between treatments, and Mmc. Group 12 sp. ISO4-H5 was almost imperceptible in the rumen of both groups of calves. Time effects (P ≤ 0.016) were observed across all the abundant archaea species, except for Mbb. gottschalkii whose relative abundance did not change (P = 0.061) with time.The archaeal community was represented by 17 species at a cut-off of ≥200 amplicons across sampling times. The archaeal community of treated calves had a reduced number of amplicons during and 2 weeks after administration of methane inhibitors compared to control calves . The eff4 inhibitors that were incorporated into the solid diet of calves up to 12 weeks of age to investigate the impact on the rumen microbial composition, fermentation profiles, enteric emissions and animal feed intake and growth. In the present study, the inclusion of a CF/AQ mix in the PMR reduced the intake of this diet, but it did not affect the total DMI of solid feeds (concentrate and PMR) of treated calves, resulting in similar growth to control calves during the treatment interventions. Observed results from the current study supported those reported in adult ruminants that showed no effects on feed intake and animal production when receiving comparable doses of halogenated or synthetic compounds to reduce CH4 emissions  spp. are obligatory hydrogen-dependent methylotrophic methanogens and require compounds like methanol, methylamine, dimethylamine, and trimethylamine as their major energy and carbon source  were altered by the CF/AQ mix fed to the calves. The results in the present study agree with those reported in lambs and calves also receiving methane inhibitors from birth until weaning off milk and 3 weeks after weaning off milk, respectively, in which alterations of the fermentation pathways did not affect the bacterial diversity in the rumen when compared to controls . Bacteri2 concentrations observed between treatment groups in the present study, the bacterial community at the phylum level was similar between studied groups. Our results differed from those in cannulated steers where increases in relative abundance of Bacteroidetes and decreases in Firmicutes in response to CF were described   in the diet  may be impaired by the increased H2 pressure in the rumen.Cellulolytic microbes such as g of LW)  and in sg of LW) . These rmulation  because mulation . In contthe diet . Inhibitncreased . The claactivity . The inc ethanol . TherefoPrevotella capable of degrading a broad spectrum of polysaccharides and peptides in the diet. They also have the ability to use different pathways in response to H2 pressure which makes them very flexible under high H2 conditions (2 pressure, produced by the inhibition of methanogen microbes with CF/AQ in the diet, and to explore whether imprinting of the rumen microbiota is possible.Methane inhibition did not have any effect on the most abundant genus nditions  as induc4 inhibition/H2 pressure has been removed. Metabolic flexibility of the rumen microbiota, however, may be overcome if the treatments begin earlier in the rumen and more work is needed to establish the start and the duration of such early life interventions.Collectively, this study showed that CF/AQ mix inclusion in the starter diets during the first 12 weeks of rearing did not affect the DMI and growth of dairy calves. Methane inhibition changed the composition of the target community, the methanogens, but had only minor effects on the bacterial community indicating the importance of the metabolic flexibility of many rumen microorganisms. This metabolic flexibility, however, may account for the observation that there was no lasting effect of the microbial community as it returns to the energetically more favorable pathways once CHPRJEB37781.The data presented in the study are deposited in the European Nucleotide Archive, accession number The animal study was reviewed and approved by Grasslands Animal Ethics Committee.SM designed the study, secured funding, and generated the respiration chamber data. OC-C generated the microbial community data and wrote the initial draft manuscript. SG and OC-C undertook the statistical analysis. All authors contributed to the data interpretation and manuscript editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Escherichia coli and isolates 54% (39/72) of Salmonella enteritidis isolates, which were all clinically pathogenic multidrug-resistant strains. Morphological observation showed that phage swi3 belonged to the Myoviridae family with an icosahedral head (80 nm in diameter) and a contractile sheathed tail (120 nm in length). At the optimal multiplicity of infection of 1, the one-step growth analysis of swi3 showed a 25-min latent period with a burst size of 25-plaque-forming units (PFU)/infected cell. Phage swi3 remained stable both at pH 6.0–8.0 and at less than 50°C for at least 1 h. Genomic sequencing and bioinformatics analysis based on genomic sequences and the terminase large subunit showed that phage swi3 was a novel member that was most closely related to Salmonella phages and belonged to the Rosemountvirus genus. Phage swi3 harbored a 52-kb double-stranded DNA genome with 46.02% GC content. Seventy-two potential open reading frames were identified and annotated, only 15 of which had been assigned to functional genes. No gene associated with pathogenicity and virulence was identified. The effects of phage swi3 in treating pathologic E. coli infections in vivo were evaluated using a mouse model. The administration of a single intraperitoneal injection of swi3 (106 PFU) at 2 h after challenge with the E. coli strain (serotype K88) (108 colony-forming units) sufficiently protected all mice without toxic side effects. This finding highlighted that phage swi3 might be used as an effective antibacterial agent to prevent E. coli and S. enteritidis infection.A novel virulent bacteriophage vB_EcoM_swi3 (swi3), isolated from swine feces, lyzed 9% (6/65) of  Escherichia coli and Salmonella enteritidis are the major opportunistic pathogens in animals and humans and are frequently reported worldwide  are viruses that exclusively infect bacteria and are widely distributed in the environment . In the oviridae . Moreoved phages .E. coli strain and 54% (39/72) of S. enteritidis strain, and it showed a good protective effect in a mouse model challenged with pathogenic E. coli. These data provide valuable information to assess the potential of phage swi3 as a biocontrol agent against pathogenic bacteria.In this study, a novel polyvalent bacteriophage vB_EcoM_swi3 was isolated and characterized. Phage swi3 showed a wide host range; it could lyze 9% (6/65) of E. coli and 72 pathogenic S. enteritidis clinical isolates were used in this study . The bacterial strains were cultured in Luria–Bertani culture (LB) at 37°C and stored in 30% glycerol at −80°C.Sixty-five pathogenic Female BALB/c mice, 5 weeks of age (20–25 g in weight), were purchased from the Experimental Animal Center of Shandong, China.All animal procedures were performed in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals, approved through the State Council of the People’s Republic of China (1988.11.1), and with the approval of the Animal Welfare and Research Ethics Committee at Qingdao Agricultural University.E. coli (serotype K88), hereafter named E. coli K88, was used in this study for phage isolation. Feces and seawater samples were collected from a pig farm in Shandong, China. Phage isolation was performed using the standard enrichment method as described before but with slight modifications . Then, 100 μl of the filtered supernatant was added to 100 μl of log-phase E. coli K88 and mixed with 3 ml of prewarmed NA top agar (0.7% agar), spread on an NA plate, and cultured for 4 h at 37°C. A single plaque was selected and picked from the plate. Then, the plaque was leached overnight with 0.9% normal saline. This procedure was repeated three times to obtain purified phages. Electron micrographs of purified phage particles were obtained according to a standard method  was spotted on each bacterial lawn on agar plates and incubated at 37°C for 4 h. The presence of plaques was examined. Additionally, the efficiency of plating was tested as described before . Phage swi3 was cocultured with E. coli K88 in 10 ml of LB broth at different MOIs   of phage swi3,  and 10) . A cultu6 PFU) was mixed with 200 ml of bacterial culture (5 × 106 CFU/ml), incubated at 37°C for 5 min, and then centrifuged, and the cell pellet was resuspended in 500 μl of LB broth. Aliquots were taken, and the phage titers were immediately determined by the double-layered agar method. This assay was performed in triplicate.The burst size and the latent period of phage swi3 were determined by one-step growth analysis as previously described . In brie8 PFU/ml) at 40, 50, 60, 70, and 80°C, and aliquots (100 μl) were taken at 20, 40, and 60 min. To evaluate the stability of phage swi3 at various pH values , phage suspensions were incubated in LB broth with different pH values at 37°C for 1, 2, and 3 h and then assayed by the double-layered agar method. All assays were performed in triplicate.The stability of phage swi3 was tested as previously described . Briefly1. Contigs were assembled using the de novo assembly algorithm Newbler, version 3.0, with default parameters , Salmonella phage LSE7621 (MK568062.1), Salmonella phage UPF_BP2 (NC_048649.1), and Salmonella phage vB_SenM_PA13076 (MF740800.1) were compared and performed using Mauve software with a default parameter  according to the manufacturer’s instructions. Then, the extracted genomic DNA was sent to Biozeron Company  for high-throughput sequencing on an Illumina HiSeq platform and assembled with ABySS, v2.0.2rameters . PotentieneMark3 . Additioarameter .E. coli K88 was first determined. In brief, 28 5-week-old female BALB/c mice were randomly divided into four groups. Each group was treated orally with different doses of E. coli K88 . The LD50 was determined based on the mortality of mice and used in subsequent challenge tests. To test the protective effect of phage swi3 on mice, 42 5-week-old female BALB/c mice were randomly divided into six groups (a–f). Groups a–d were inoculated orally with 108 CFU of E. coli K88. Then, groups a–c were inoculated intraperitoneally with a single dose  of phage swi3 at 2 h after the bacterial challenge. Control group d was inoculated with an equivalent volume of LB instead of phage swi3, control group e was treated with 107 PFU of phage swi3 without E. coli K88 challenge, and control group f only took an equal volume of LB orally. Then, the mice were observed daily for 5 days to record the clinical signs and cumulative mortality. At the same time, blood and feces were collected every 2 h on the first day and every 24 h after the first day to detect the metabolic dynamics of bacteria and phage swi3 in the mice.To test the antibacterial activity of phage swi3, the 50% lethal dose (LD50) of MT768059.The sequence data of swi3 were deposited at GenBank under accession number E. coli K88 and observed by transmission electron microscopy. Phage swi3 has a regular icosahedral head (80 nm in diameter) and a contractile sheathed tail 120 nm in length . According to the novel universal system of bacteriophage naming, the suggested full name of phage swi3 will be vB_EcoM_swi3 , seven genes associated with transcription and replication , and two lysis-related genes (orf 44 and orf 45). A detailed phage swi3 genome annotation showed that the orfs related to transcription and replication were mainly concentrated in the downstream part of the whole genome, while the structurally related orfs were mainly concentrated in the upstream part of the whole genome sequence  sequence, the tail-associated protein (encoded by orf 27), and the tail tape measure protein (encoded by orf 33), using the neighbor-joining method with the default parameters in MEGAX software.The phylogenetic tree of phage swi3 was constructed based on the genomic sequence, the terminase large subunit  was included in ICTV, with 98% coverage rate and 97.73% identity. The phylogenetic tree showed that 11 phages belonged to two groups, phage swi3 and eight more phages belonged to the Rosemountvirus genus, and the other two phages belonged to the Loughboroughvirus genus , and all of them belonged to us genus . Based oa phages . The gena phages .orfs: the tail-associated protein (encoded by orf 27) and the tail tape measure protein (encoded by orf 33). Their phylogenetic trees were constructed. Based on the tail-associated protein, only four comparable phages were found in the NCBI database with a high homology (>82.7%), and all of them were Salmonella phages. Other phages in the database showed less than 45% homology . After the challenge with E. coli K88, all mice in control group d died within 1 day; their lungs, liver, spleen, and kidneys showed varying degrees of bleeding and swelling, and E. coli K88 was isolated from these diseased organs. All mice in control groups f and e had good health until the end of the experiment, and no organ lesions were found  at 2 h after oral challenge with E. coli K88 could sufficiently protect all mice without toxic side effects.The LD50 of re found . In groure found . The resE. coli K88 entered the blood and reached its peak (108 CFU/ml) 1.5 h after inoculation. Phage swi3 was intraperitoneally injected into the mice, detected in the blood in a short time, and then cleared E. coli K88 within 8.5–51 h in the blood; no bacteria or phage could be detected after 51 h  on the tail fiber so that the phage adsorbed to the surface of the host bacteria and then initiated the process of phage infection  can be found below: The animal study was reviewed and approved by the Animal Welfare and Research Ethics Committee at Qingdao Agriculture University.BS performed the experiments, analyzed the data, and wrote this manuscript. LH, HR, and WL performed the experiments. CZ designed the experiments and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The erratum corrects an error in Equation 1 of the published article. Neurophotonics8(1), 015001 (2021) doi: https://doi.org/10.1117/1.NPh.8.1.015001] was originally published on 11 July 2021 with an error in Equation 1. The equation should have had negatives in both of the exponent expressions .This article [The negatives were added to the exponent expressions to correct the equation and the article was republished on 4 February 2022.


This study aims to assess the association between positron emission tomography-computed tomography (PET-CT) parameters and the response to immune checkpoint inhibitors in unresectable head and neck squamous cell carcinoma (HNSCC).A total of 105 patients receiving immunotherapy (pembrolizumab or sintilimab with/without cisplatin) were retrospectively enrolled in this study; pretreatment data regarding metabolic tumor volume (MTV) and maximum standardized uptake value (SUVmax) were collected. The primary interest of the study was objective response rate (ORR), and the secondary was progression−free survival (PFS).3 (range: 8.5–100.3), ORRs in tumors with total MTV of ≥40.6 and <40.6 cm3 were 43.1% and 23.1%, respectively; the difference was statistically significant (p = 0.018). Survival analysis indicated similar PFS rates in the two groups (p = 0.057). The mean total SUVmax was 12.5, ORRs in tumors with total SUVmax ≥12.5 and <12.5 were 40.0% and 26.0%, respectively; the difference was not significant (p = 0.092). Survival analysis reported patients with total SUVmax of ≥12.5 had significantly worse PFS (p = 0.001) than patients with total SUVmax of <12.5.The mean total MTV was 40.6 cm3 translated into improved clinical response but not into better PFS; total SUVmax had no effect on clinical response, but total SUVmax ≥12.5 was associated with worse PFS.In HNSCC, total MTV ≥40.6 cm Recently, the Food and Drug Administration (FDA) approved the use of PD-1 inhibitor (pembrolizumab) with or without cisplatin as the first-line therapy for recurrent or metastatic HNSCC (Head and neck squamous cell carcinoma (HNSCC) is the seventh commonest malignancy in the world , even tric HNSCC .However, the overall response rate to PD-1 inhibitor monotherapy is about 20%; thus, there is a need to identify biomarkers that will help with patient selection . Some rePET-CT is widely used for detecting disease metastasis in clinical practice, and its parameters including maximum standard uptake value (SUVmax) and metabolic tumor volume (MTV), are significantly associated with patient survival in HNSCC , 11.Therefore, this study aims to assess the association between PET-CT parameters and response to immune checkpoint inhibitors in unresectable HNSCC.Our hospital institutional research committee approved our study, and all participants signed an informed consent agreement. All methods were performed in accordance with the relevant guidelines and regulations. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.2 and cisplatin 100 mg/m2, on day 1; fluorouracil by continuous infusion 1,000 mg/m2, on days 1 to 5, TPF) as palliative treatment after multidisciplinary consultation, based on Chinese Society of Clinical Oncology guidelines for recurrent and metastatic HNSCC; those who had a PET-CT scan performed within 1 week prior to treatment. Patients were excluded if they had insufficient follow-up data or had received other treatment for the primary or recurrent disease. Information regarding demographic and pathologic data, Eastern Cooperative Oncology Group (ECOG) performance, PET-CT parameters, prior treatment, and follow-up was extracted and analyzed.Between January 2018 and January 2021, medical records of HNSCC patients were retrospectively reviewed; inclusion criteria were as follows: those with disease defined as unresectable; those who received pembrolizumab or sintilimab with/without cisplatin  or sintilimab  was used solely or concurrently with TPF chemotherapy based on comprehensive consideration of the patient’s economic status and willingness, health condition, PD-L1 expression, and so on for HNSCC patients from January 2018. Pembrolizumab or sintilimab was given intravenously at a dose of 200 mg every 3 weeks until intolerable toxicity or disease progression occurred. Six cycles of TPF chemotherapy was given.From July 2013, immunohistochemical analysis of p16  was performed for every patient with HNSCC. The level of positivity of p16 overexpression was consistent with that in previous studies : 0–+, dePD-L1 expression was assessed using the 22C3 pharmDx assay  and calculated by CPS, it was defined as the number of PD-L1-positive cells divided by the total number of tumor cells ×100; a minimum of 100 viable tumor cells must have been present for the specimen to be considered evaluable  was performed by several scanners. Before the scan, patients were required to fast for at least 6 h. If glucose levels were >200 mg/dl, PET-CT scan was postponed. Every patient received 10–20 mCi of [The patient was usually examined by palpation and image assessment after every two to four cycles of treatment; timely evaluation was performed if there was any new sign of disease progression or other complications.The primary study interest was objective response rate (ORR); the secondary interest included progression−free survival (PFS) and OS. Responses were formulated as complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD) according to RECIST, version 1 , and werp < 0.05 was considered to be significant.The Chi-square test  was used to analyze the association between clinicopathologic variables, PET-CT parameters, and ORR, and the factors which were significant in univariate analyses were then analyzed in multivariate analysis to detect the independent predictors. The Kaplan-Meier method was used to compare the PFS and OS. Cox model was used to determine the independent prognostic factors. All statistical analyses were performed using SPSS 20.0, and a 3 (range: 8.5–100.3). Fourteen (13.3%) patients had a CPS of less than 1, 66 (62.9%) had a CPS greater than 1 but less than 20, and 25 (23.8%) had a CPS no less than 20. Forty-six (43.8%) patients received immunotherapy alone, and 59 (56.2%) received both immunotherapy and TPF chemotherapy  were male and 30 (28.6%) were female with a mean age of 56.8 years (range: 38–79). Twenty-nine (27.6%) of them had an ECOG score of 0, 61 (58.1%) had a score of 1, and 15 (14.3%) had a score of 2. Twenty (19.0%) patients were current smokers, 61 (58.1%) were former smokers, and 24 (22.9%) were never smokers. Thirty-five (33.3%) patients had a primary disease, 70 (66.7%) had recurrent/metastatic disease, and had all received prior treatment in form of surgery or radiotherapy; 35 of them had also received chemotherapy. Primary tumors were located in the oral cavity in 20 (19.0%) patients, in the oropharynx in 31 (29.5%), in the larynx in 32 (30.5%), and in the hypopharynx in 22 (21.0%). Positivity of p16 occurred in 11 (10.5%) patients. The mean total SUVmax was 12.5 (range: 4.7–48.6), while the mean total MTV was 40.6 cmotherapy Table 1.A total of 35 patients showed positive clinical response; there were four cases of CR and 31 cases of PR, the overall ORR was 33.3%, and SD and PD occurred in 15 (14.3%) and 55 (52.4%) patients, respectively.p = 0.006). The ORRs in tumors with total MTV ≥40.6 cm3 and<40.6 cm3 were 43.1% and 23.1%, respectively; the difference was significant (p = 0.018). In tumors with CPS ≥20, the ORR was 52.0%, it was apparently higher than those of the other two subgroups (p = 0.044). Compared with immunotherapy alone, the addition of TPF yielded a significantly increased ORR of 42.4% (p = 0.026). The ORRs in tumors with total SUVmax ≥12.5 and<12.5 were 40.0% and 26.0%, respectively; the difference was not significant (p = 0.092). Moreover, the treatment response had no association with age, sex, ECOG score, or smoking status   Table 2.Adverse events occurred in all patients, with 268 grades 1–2 events and 12 grades 3–4 events. The most common grades 1–2 and grades 3–4 events were anorexia and neutropenia, respectively. Two patients died of serious adverse reaction Table 4.3, and 21% in those with total MTV ≥40.6 cm3; the difference was not significant , the 2-year PFS rate was 31% in patients with total MTV <40.6 cm3, and 42% in those with total MTV ≥40.6 cm3, the difference was significant  had similar estimated OS rates at 18, 24, and 30 months. However, in a study by Burtness et al.  was likely to decrease the treatment response rate. As for metastatic melanoma treated with anti-PD-1 therapy, Nakamoto et al. (MTV was a commonly analyzed parameter of PET-CT; it referred to the volume of tumor tissue with high metabolic activity. Our previous studies have confirmed its negative effect on disease prognosis in HNSCC , but wheo et al.  did not SUVmax was another important parameter of PET-CT, which refers to the ability of tumor tissues to take up tracers; in general, the higher the SUVmax, the higher the possibility and degree of malignancy. A number of researchers have described that patients with high SUVmax were more likely to have larger tumors, advanced stage disease, and poor prognosis , 23, 24.Limitations in this study must be acknowledged: first, its retrospective nature had inherent bias; second, our sample size was small, which decreased our statistic power; third, our follow-up time was limited, there might be more interesting findings in the future; fourth, PD-1 inhibitors consisted of two different drugs in this study, both of which had been confirmed to have good ability of binding to receptor PD-L1 , 25.3 was related to improved clinical response but did not translate into better PFS; total SUVmax had no effect on clinical response but total SUVmax ≥12.5 was associated with worse PFS.In summary, in HNSCC, total MTV of ≥40.6 cmThe original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.Henan Cancer hospital institutional research committee approved our study, and all participants signed an informed consent agreement. The patients/participants provided their written informed consent to participate in this study.All authors have contributed to the concept design, data collection, data analysis, manuscript writing, and manuscript revision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.