Thursday, October 15, 2026

Liver Fatty Acids Profile of Rats Fed Ketogenic Diet

Introduction

The ketogenic diet (KD) ‒ characterized by a low carbohydrate content and high-fat levels ‒induces a metabolic shift to the use of ketones as a source of energy for cells. It is also defined as a carbohydrate-restricted diet with a 4:1 ratio of fats to proteins and carbohydrates [1,2]. Several clinical studies have shown that KD a reduction of midlife mortality and a prolongation of health due to its ability to control body weight and its beneficial effects in the prevention of type 2 diabetes, cancer, and neurological diseases [3- 6]. Recently, in a rat model of irritable bowel syndrome (IBS), KD was able to restore intestinal cell damage through the upregulation of endocannabinoids receptors (CB1-R and CB2-R) [7] improving intestinal permeability. Besides, the KD administration positively affected gastrointestinal mucosa in IBS rats and elicited molecular compensatory mechanisms able to reduce inflammation and oxidative stress in the gut, restoring mitochondrial function and baseline autophagy [7,8].

Literature data demonstrate that KD exerts beneficial effects on subjects with obesity [9,10]. Significant weight loss was associated with reduced visceral fat mass in overweight and obese patients treated with KD [11]. Moreover, consumption of a low-carbohydrate KD is involved in a control blood pressure values and the serum levels of total cholesterol, low-density lipoprotein (LDL), and triglycerides [12]. Besides, literature suggested KD as an effective way to treat non-alcoholic fatty liver disease (NAFLD) [13]. During the improvement of human NAFLD induced by a short-term KD (six days), liver redox status significantly changed. It appears that KD, at least as a short-term treatment, can significantly reduce liver fat content and insulin resistance [13]. However, in rodents, different studies have reported that KD can promote the progression of liver steatosis [14,15]. The greater palatability of high-fat diets justifies the greater food consumption by these animals, consequently increasing body weight [16]. High-fat diets seem to favor the fat accumulation in hepatocytes associated with increased lipid peroxidation metabolites in hepatic tissue [3,16]. Notably, the pro-oxidative environment caused by enhanced levels of dietary lipids, promotes the development and progression of steatosis in the liver [17]. The liver has a prominent role in carbohydrate and lipid metabolisms so that excessive amounts of free fatty acids in hepatocytes cause fatty liver, insulin resistance, and lipotoxicity [18-20].

Fatty acids constitute essential components of membranes that influence their physiological functions [21] and their tissue composition is the result of food intake, lifestyle, and environmental factors [22,23]. Based on this evidence and in line with the existing literature background, the purpose of the present study was to investigate whether a high-fat KD with beneficial effects at intestinal levels may affect liver function in Wistar rats. Through a lipidomic approach, the study was aimed to understand the relationship between KD and fatty acid profile of liver tissue and investigate the clinical implications that occur.

Materials and Methods

Animals and Treatment

Twenty-five Wistar rats (5 weeks of age) were used in this study. All the animals received care in compliance with the “Guide for the Care and Use of Laboratory Animals” and the experimental procedures were approved by the Italian Ministry of Health (approval date: 28 November 2018, n. 901/2018-PR). The animals, at weaning, were divided at random into two groups and treated for ten weeks. Particularly, 12 animals were fed a standard diet (control group, CTR group), and 13 were fed a low-carbohydrate, high-fat KD (KD group). Diets (4RF21 standard diet and ketogenic diet) were purchased by Mucedola Srl, Settimo Milanese, Italy, and their composition is shown in Table 1. No sign of suffering, stress, or pain was observed in the animals during the experiment. At sacrifice, by anesthetic overdose, the liver from each animal was collected and stored at -80°C until assayed.

Table 1: Main constituents of Standard Diet and Ketogenic Diet.

Fatty Acids Analysis

Liver tissue fatty acids analysis was carried out using Fatty Acid Extraction Kit Low Standard, according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, U.S.A.). Briefly, 0.15 g of the liver tissue was homogenized in 3 ml of extraction solvent. After homogenization, 5 ml of aqueous buffer were added, and the sample was filtered by a syringe. The eluted fraction containing the lipid extract was trans-esterified by adding boron trifluoride-methanol solution and hexane and incubated at 95° C for 1 hour. After adding hexane, the samples were vortexed and centrifuged at 500 x g for 5 minutes and the fatty acid methyl esters contained in the top hexane layer were evaporated. The dried pellet was reconstituted with 100 μl of hexane and injected into a gas chromatography equipment (Thermo Fisher Scientific, Milan, Italy). The quantification of fatty acids was carried out as previously described [24].

Western Blotting Analysis

Fifty μg of liver total proteins were subjected to 4-12% SDSPAGE polyacrylamide gel electrophoresis and transferred onto the PVDF membrane (Bio-Rad Laboratories, Milan, Italy). Subsequently, the membrane was incubated overnight at 4°C with the following primary antibodies: NAD-dependent deacetylase sirtuin-1 (SIRT1) (Bio-Rad Laboratories, Milan, Italy), peroxisome proliferatoractivated receptor-γ (PPAR-γ) (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), tumor necrosis factor (TNF-α) (Immunological Science), human neutrophil cytochrome b-light chain (p22/phox) (Abcam, Cambridge, UK), β-Actin (Cell Signaling Technology, Beverly, MA, U.S.A.). Then, the membrane was washed with TBS-T, and the secondary antibody conjugated with horseradish peroxidaseconjugated rabbit or mouse (Bio-Rad Laboratories) was used, where appropriate. The signal of each protein was visualized using the ChemiDoc™ system (Bio-Rad, Milan, Italy) and the Image Lab software. All data were normalized against β-actin expression.

Determination of Enzymatic Antioxidant Activity and Lipid Peroxidation

The levels of [Cu-Zn] Superoxide dismutase (SOD 1) and the Mitochondrial Superoxide dismutase (SOD 2) in the rat liver tissue were measured with Rat SOD 1 and Rat SOD 2 Enzyme-Linked Immunosorbent Assay (ELISA) Kits (Fine Test, Wuhan, China), respectively. Lipid peroxidation was measured in the rat liver by determining malondialdehyde (MDA), using the Rat MDA Assay Kit ELISA (My Bio Source, San Diego, CA, U.S.A.) by following the manufacturer’s instructions.

Statistical Analysis

Sample size calculation has been performed having a fixed power of 80% and a first type error of α of 0.025 (α ½ = 0.025); then, a minimum number of 6 animals for each experimental group was needed. The statistical differences among the two rat groups were evaluated by the student t-test and they were considered significant with a p-value <0.05.

Results

The Effects of the Ketogenic Diet on Body Weight and Liver Tissue Fatty Acids Profile

Figure 1: Total levels of omega-3- and omega-6- PUFAs, and the omega-6/omega-3- PUFAs ratio in rat liver tissue of the CTR and KD group. All data are expressed as mean ± standard deviation (SD) of three different experiments. p-Value indicates significant differences among the groups (p < 0.05, Student t-test); *** p< 0.001.

Table 2: Characteristics of experimental rat groups.

Note: All data are expressed as mean ± standard deviation (SD); p-Value indicates significant differences among the groups (p < 0.05, Student t-test); ns: not significant.

Table 3: Mean percentage of fatty acid composition in liver tissue of the rats treated with the standard diet (CTR group) and KD diet (KD group).

Note: All data are expressed as mean ± standard deviation (SD); p-Value indicates significant differences among the groups (p < 0.05, Student t-test); ns: not significant.

Table 2 shows the characteristics of two experimental rat groups. Compared to the CTR group, a significant increase in body weight was observed in animals after KD treatment (352.7±83.84 vs 454.0±132.2, p=0.033). Interestingly, the administration of KD affected the tissue fatty acids profile in the liver (Table 3). After 10 weeks of KD treatment, the levels of single saturated fatty acids (SFAs), such as palmitic and stearic acid, and single monounsaturated fatty acids (MUFAs) were significantly decreased compared to CTR group. A significant decrease was also observed for the content of arachidonic acid (AA), an omega-6 polyunsaturated fatty acid. In contrast, linoleic acid (LA), an essential omega-6 fatty acid, was significantly increased in liver tissue from KD-fed rats, probably due to the higher oils content in the KD. After KD treatment, all main omega-3 polyunsaturated fatty acids, except for eicosapentaenoic acid (EPA), significantly increased in the liver tissue. Figure 1 summarizes the differences in the levels of total omega-6-, omega-3- Polyunsaturated fatty acids (PUFAs), and the omega-6/omega-3- PUFAs ratio detected between the two groups of rats. In particular, the significant increase in total omega-6- PUFAs justified the increase of omega-6/omega-3- PUFAs ratio observed in the KD group. Saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs). Data are expressed as mean ± standard deviation (SD) of three different experiments; p-Value indicates significant differences among the groups (p < 0.05, Student t-test); *** p<0,0001, ns: not significant.

The Effects of the Ketogenic Diet on Liver Protein Levels

The effects of KD administration on the rat liver levels of SIRT1, TNF-α, PPAR-γ, NFκB, and p22/phox are shown in Figure 2. The group receiving the KD treatment presented higher levels of TNF-α, PPAR-γ, NFκB, and p22/phox protein than the control group. In contrast, a statistically significant reduction in SIRT1 protein levels was observed in rats fed KD. The significant increase of the PPAR-γ and p22/phox protein expression demonstrated that KD caused an inflammatory environment in the liver, which was also justified by a significant reduction in SIRT1 protein.

The Effects of the Ketogenic Diet on Enzymatic Antioxidant Activity and Lipid Peroxidation

The analysis of enzymatic antioxidant activity based on the determination of SOD 1 and SOD 2 demonstrated that the rats fed KD showed higher value than the controls (Figure 3). No difference was observed in the hepatic MDA levels between the two experimental rat groups (Figure 4), suggesting that the higher degree of inflammation in the liver tissue occurred without significant changes in the oxidative status.

Figure 2: Western blotting analysis of SIRT1 (a), TNF-α (b), PPAR-γ (c), NFκB (d) and p22/phox (e) protein expression, normalized with β-Actin protein, in CTR and KD group. Panel f shows representative blots evaluated in two experimental groups. All data are expressed as mean ± standard deviation (SD) of three different experiments. p-Value indicates significant differences among the groups (p < 0.05, Student t-test); ** p<0.001 and *** p< 0.0001.

Figure 3: SOD 1 (panel a) and SOD 2 (panel b) levels in the CTR and KD groups. All data are expressed as mean ± standard deviation (SD) of three different experiments. ** p<0.001 and *** p< 0.0001.

Figure 4: MDA levels in CTR and KD groups. All data are expressed as mean ± standard deviation (SD) of three different experiments.

Discussion

The effects of KD on liver metabolism and function can be mixed. The present study shows that KD, on the one hand, promotes the increase of tissue inflammation in the liver, on the other, it activates defense mechanisms to protect the liver environment. Significant changes in liver tissue fatty acids profile have been observed after KD intake in rats. Most of these changes can be considered as environmental factors that lead to improving or counteracting tissue inflammation. The anti-inflammatory effects of KD are confirmed by the decrease of the AA content in liver tissue. While the increase of total omega-6-PUFAs detected in rats fed KD is probably due exclusively to the levels of LA derived by the diet. Several studies showed that the high-LA diet could cause no significant increase in AA [25,26]; the LA derived from the diet could be less exposed to enzymatic turnover and, therefore, AA production starting from its precursor is reduced. SIRT1 is known to regulate several downstream pathways eliciting antiinflammatory effects [27,28]. Higher SIRT1 protein levels have been observed during calorie restriction in tissues, as the brain, muscles, liver, and kidneys [29-31]. SIRT1 seems to regulate both liver and adipose tissue inflammation, and its protective function in high-fatinduced liver pathology has been demonstrated in SIRT1+/- mice [32]. Different studies have reported that the expression of SIRT1 is downregulated in most metabolic disorders, including obesity [29,33], demonstrating that the reduction of SIRT1 expression is often linked to an upregulation of pro-inflammatory cytokines, as TNF-α.

Here, KD treatment exerted a reduction in liver SIRT1 protein levels and a concomitant induction of TNF-α, NFκB, and p22/phox protein expression. In a normal condition, NFκB has been observed in the cytoplasm of hepatocytes in a latent form but, when exposed to pro-inflammatory stimuli, it participates in the activation of tissue inflammation [31]. In addition, the upregulation of NFκB was reported to be closely connected with the overexpression of the p22/phox protein [32]. Experimental studies have also shown that all these proteins are linked together, actively intervening in inflammatory processes [34-36]. The significant induction of PPAR-γ, detected in liver tissue of rats fed KD agreed with other studies showing increased PPAR-γ expression following excessive fat intake [37,38]. PPAR-γ is a transcription factor that controls hepatic fatty acids metabolism and, therefore, it results in enhanced hepatic steatosis and abnormal conditions of liver function [39].

Simultaneously, the diet-induced pro-inflammatory state in the rat liver was somewhat offset by increased antioxidant activity, increased total PUFA levels, and a significant reduction in SFA and MUFA. In the present study, the beneficial effect of KD is confirmed by the significant increase in SOD 1 and SOD 2 (Figure 3), in rats fed KD compared to the control. SODs are present in all aerobic living cells since O2 is a common product of oxygen metabolic reactions. SOD 1 is the main intracellular form of SOD and accounts for 80% of the total SOD protein. Previously, some studies declared SOD 1 to be predominantly cytosolic [40]. Nevertheless, later studies have found it in the whole cell, including in the mitochondrial intermembrane space and nucleus [41]. On the other hand, SOD 2 is located exclusively in the mitochondrial matrix (MM) [41]. Together, SODs provide to remove the damaging O2- free radicals from cells and tissues.

In addition to their conventional function as O2- detoxification enzymes, emerging evidence suggests that SOD 1 and SOD 2 are actively implicated in the modulation of several cellular processes [42,43]. One well-recognized mechanism is related to H2O2˗the dismutating product of O2- by SOD˗, can act as a second messenger in growth and metabolic processes regulation [44]. Thus, by increasing SODs, KD can induce antioxidant enzyme activity in liver tissue, probably in response to pro-inflammatory dietary stimuli. The tissue fatty acids profile observed in the present study after KD treatment agrees with experimental evidence underlying the role of SFAs and MUFAs in regulating cell membrane fluidity [45]. A low content of SFAs and MUFAs seems to improve the fluidity of the cell membrane and allows to counteract a possible cell damage, rapidly [45].

Moreover, the therapeutic potential of PUFAs, mainly omega-3 PUFAs, on metabolic liver diseases is well known [46]. EPA and Docosahexaenoic acid (DHA) also seem to exert protective effects against bile acids-induced cell death in human hepatocytes [47,48]. In addition, diet rich in omega-3 PUFAs are recommended for its benefits in reducing platelet adherence [49], lowing cholesterol, and reducing pro-inflammation and pro-oxidation mediators [50]. Several investigations described that the first line of defense of the cell against environmental stresses is the membrane and its content in SFAs, MUFAs, and PUFAs is important for the correct physiological functions [23]. The current study shows the possibility that the KD, through the lipidome, could improve the diet-induced inflammatory state of liver tissue. Herein, this finding implicates a significant and novel role for the tissue lipids profile in regulating complex metabolic pathways in the liver.

Conclusion

The positive effects of KD in rat liver tissue support the role of diet in modulating tissue fatty acids composition and in regulating molecular pathways related to counteracting inflammation. The clinical relevance of the study consists in suggesting a controlled use of KD in the subjects with metabolic disorders, likely for a short period, to avoid a diet-induced inflammatory effect in the liver.

BJSTRAngelaRoy

Wednesday, October 14, 2026

Rare Case of Polymicrobial Sepsis with Cupravidus Pauculus in a Sclerodermic Patient

Introduction

Cupriavidus pauculus (C. pauculus), formerly named Wautersia paucula, is a Gram-negative, aerobic, non-fermentative bacterium, part of the Cupriavidus species [1]. This germ is commonly found in the environment, such as plants, soil and water, including bottled mineral water [2]. Only few worldwide case reports are currently available in literature [2-6]. We report the case of a polymicrobial severe infection produced by different multi-drug resistant (MDR) microorganisms, including C. pauculus in an immunosuppressed patient. A 41-year-old female patient was admitted in January 2019 into our clinic for persistent fever, dry cough, chest pain and progressive dyspnoea, with onset within 6 days before presentation. The patient was non-smoker, with medical history of scleroderma and CREST syndrome (Raynaud phenomenon) and recently returned from Thailand. On examination, she was subfebrile (37.6°C), and pulmonary auscultation revealed bilateral diminished vesicular murmur and inspiratory crackling in both lungs, with high variations in oxygen saturation between 86 - 97% at room air), hypotension (92/70 mmHg) and tachycardia (132 beats per minute). Laboratory tests identified leukocytosis (11040/mmc) and neutrophilia (92.8%) (Figure 1A), severe inflammation (CRP>324 mg/dL) (Figure 1B), hypochromic anaemia, nitrogen retention syndrome, and significant alteration of arterial gas levels (PaO2/FiO2 = 102.083). Legionella urinary antigen test was negative. The day of the hospital admission, 3 peripheral blood cultures were performed and were positive 72 hours later for penicillin susceptible Streptococcus pneumoniae (Table 1).

Table 1: Susceptibility results.

Note: AK, amikacin; AMP, ampicillin; AMX, amoxicillin; AMC, amoxicillin-clavulanate; AZM, azithromycin; FEP, cefepime; CTX, cefotaxime; CPD, cefpodoxime; CPT, ceftaroline; CAZ, ceftazidime; CRO, ceftriaxone; CXM, cefuroxime; CHL, chloramphenicol; CIP, ciprofloxacin; CLR, clarithromycin; CLI, clindamycin; ERY, erythromycin; GEN, gentamicin; IPM, imipenem; LEV, levofloxacin; MEM, meropenem; MXF, moxifloxacin; PEN, penicillin; PRL, piperacillin; TPZ, piperacillin/tazobactam; TEC, teicoplanin; TIC, ticarcillin; TOB, tobramycin; SXT, sulphamethoxazole/trimethoprim; VA, vancomycin

Figure 1: Laboratory markers.

Figure 2: Imagistic results

a) Alveolar condensation and opacification with ascending upper limit.

b) Condensation syndrome with aeric bronchogram and fluid in left pleura.

c) And regression of lesions.

Chest X-ray (Figure 2A) revealed alveolar condensation in the right upper lobe and bilateral pleural effusion. Antibiotic treatment initiated with cefotaxime 6 g/day and doxycycline at admission, was subsequently modified according to susceptibility results to vancomycin 2 g/day, and imipenem 2g/day. Sepsis was improved during the next days of treatment, until a sudden aggravation was manifested by high fever, severe productive cough, and vomica in the sixth day of hospitalization. Sputum examination identified inflammatory reaction and in culture were identified multidrug resistant (MDR) microorganisms, namely Pseudomonas aeruginosa and Stenotrophomonas maltophilia (Table 1). Pleural ultrasound revealed bilateral pleural fluid and subjacent alveolar condensation. Fluid obtained by left thoracentesis was cloudy, xantochrome, with high cellularity (5.787 cells/mmc, 90% neutrophils), and bacteriologically negative. Antibiotic therapy was deescalated to ampicillin 10 g/day, TMP/SMX 4 tablets/day and levofloxacin 750 mg/day. After 14 days of treatment, thoracic CT (Figure 2B) revealed an aspect of bronchopneumonia with abscess formation and medium left pleural effusion with passive atelectasis. Examination of pleural fluid objectified a significant improvement, with decrease of cellularity to 560 cells/field. On the 27th day of hospitalization, bacteriological sputum examination revealed absence of MDR pathogens. By contrast, the non-fermentative gram-negative bacteria (C. pauculus) has been isolated from the patient’s sputum on blood agar. The bacterium has been identified using ID32GN strips (bioMérieux, France). Clinical significance of the isolated bacterium was supported by the presence of gramnegative bacilli associated with the inflammatory cells in the sputum Gram-stained smear and the isolation in high amount in culture. Based to susceptibility test (Table 1), patient continued antibiotic treatment with levofloxacin and TMP/SMX. The 7 – day course of therapy was effective, and symptoms were ameliorated, biological markers (Figures 1A & 1B) and chest X-ray image (Figure 2C) regressed. Entire duration of the hospitalization was 36 days. Within 7 days after discharge, patient presented rare episodes of residual cough, a medium-intensity opacity in the right upper lobe on X-ray and inflammatory syndrome. The inflammatory syndrome was also identified at 2 – month follow up and was expected due to systemic immunological disorder.

Discussion

The pathology of scleroderma is complex and is not fully understood yet. Cell-cell and cell-matrix interactions are responsible for the activation of the immune system and hyperproduction of growth factors and cytokines with pathogenic potential (IL-1, IL-4, IL-6, IL-8, IL-13, and IL-17), as well as vascular damage and excessive synthesis of extracellular matrix resulting in a significant increase in the amount and deposition of collagen [7]. Systemic scleroderma is a systemic disease with multiorgan damage, directly affects the lungs where it causes interstitial damage, pulmonary hypertension or pleural involvement (pleurisy has been identified in 7% cases). However, opportunistic respiratory infections remain an important cause of mortality and morbidity in scleroderma, with a mortality rate of up to 33% [8]. Pneumonia can be triggered by pathogens with tropism for the respiratory system, including Enterobacteriaceae, Pseudomonas aeruginosa, and Staphylococcus aureus [8]. Our patient considered herself otherwise healthy and did not investigated her underlying condition appropriately. Although she was aware of her underlying autoimmune disorder, she did not have any vaccine administered (e.g. pneumococcal vaccine), even when travelling to a country with endemic potential [9]. All these factors provided the breeding ground for the severe polymicrobial infection produced by Streptococcus pneumoniae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and subsequently C. pauculus.

Cupriavidus spp. are ubiquitous environmental organisms, mainly in water and plants. C. pauculus is the most important member of this family responsible for opportunistic infections and which can sometimes cause outbreaks [2,4]. The germ was initially classified as CDC group IV c-2 bacteria and later named Ralstonia paucula or Wautersia paucula by different authors. Based on the similarity of the genomic sequence, both were renamed as C. pauculus [1,6]. C. pauculus is responsible for different infections, such as community or nosocomial respiratory infections (including ventilation – associated pneumonia), meningitis, tenosynovitis, cellulitis, septic arthritis, peritonitis, sepsis [2,3]. The first cases of nosocomial infections produced by C. pauculus were described in hospitalized children [4]. The infection has been identified in all age groups, but has a high severity in newborns and immunocompromised patients (e.g. neoplasia, transplantation, AIDS or inflammatory bowel disease) [6,8,10]. Respiratory infections affect mainly immunocompromised patients but preexisting of metastatic foci of infection in lungs could increase the risk of infection with C. pauculus even in immunocompetent patients [11]. In our case, pneumococcus and the MDR germs could be predisposing factors for the C. pauculus infection. We were unable to identify the source of the C. pauculus infection. We do not know if the pathogen was present since admission, but C. pauculus was not isolated in samples drawn during early hospitalization. However, the patient underwent many invasive procedures (including thoracentesis) and required prolonged oxygen therapy.

We may even consider this as a nosocomial infection through a possible contamination of water from the bubble humidifier bottles used for oxygen therapy. Contaminated water from hydrotherapy pools, nebulization solution or even thermos-regulator reservoir water in the exracorporeal membrane oxygenation circuit (ECMO) has been identified as the source of infection with this microorganism in various forms of infection [4]. In addition, the use of catheters, parenteral nutrition or systems for hemodialysis or have also been identified as the source of nosocomial infection caused by C. pauculus [12,13], but such devices were not used in our case. However, including a possible contamination of bottled water consumed by the patient [14] or even the recent international travel history could lead to a supposition of a diagnosis of community – acquired infection in our patient. Current clinical experience regarding an optimal antibiotic treatment of C. pauculus infection is limited. The sensitivity of this germ is quite variable. Data from the literature have shown that C. pauculus is susceptible to broad spectrum beta-lactams, fluoroquinolones and TMP/SMX, most strains are resistant to aminoglycosides and inconstantly sensitive to colistin [3,12,15]. In our case, the susceptibility testing was performed by disk-diffusion method; colistin susceptibility was assessed by broth microdilution (MIC = 0.5 mg/L). Interpretation of the susceptibility tests was done using the EUCAST criteria for Acinetobacter spp. (version 9.0). The strain was susceptible to standard dosing regimen of aminoglycosides, carbapenems, and levofloxacin, and susceptible to ciprofloxacin in increased exposure circumstances. Our results confirm what other authors have mentioned previously, namely that empirical treatment with imipenem followed by TMP/SMX and levofloxacin, offered favourable evolution in C. pauculus infection [12,15]. The peculiarity of the case derives from identification of polymicrobial infection produced by MDR germs, including C. pauculus which has not been reported previously in our region.

Conclusion

Patient with scleroderma must be aware of their disease and ensure a proper medical monitoring since the autoimmune disorder is a risk factor for infections caused by multidrugresistant or rare germs, such as C. pauculus. This case illustrates the need to address and treat infection produced by C. pauculus in immunocompromised individuals as an infection with possible severe evolution. Our results are in line with previous data, namely that empirical treatment with imipenem may offered favourable evolution in C. pauculus infection. Informed Consent Statement: Informed Consent Statement: Written informed consent was obtained from the patient for publication of this case report and accompanying images.

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Thursday, October 8, 2026

Investigation of the Diagnostic Accuracy of Rapid Influenza Diagnostic Kit Method in Comparison to the Real Time Reverse Transcription Polymerase Chain Reaction

Introduction

Influenza is a highly contagious viral respiratory infection caused by influenza viruses who’s epidemic and pandemic has resulted in significant mortality and morbidity. It has been reported that annual epidemic of influenza results in an estimated 3 – 5 million cases of severe illness and about 290000 – 650000 deaths globally. This study was aimed to investigate the rapid influenza diagnostic tests that detect influenza viral antigens are used to screen patients with suspected influenza and offer the advantage of providing a timely result, compared with real time PCR for influenza, that can influence clinical decision making with in short time [1]. This test can help to decrease unwanted diagnostic testing, to advice antiviral treatment, to decrease inappropriate use of antibiotics and to reduce the duration of treatment in the emergency department or hospitalization [2-8].

Rapid influenza tests have also been used for public health purposes to investigate suspected influenza outbreaks. Identification of influenza virus infection by rapid tests can facilitate prompt implementation of control measures before confirmatory test results are available from shell vial culture, tissue cell viral culture, or RT-PCR. Rapid influenza tests have also been used for public health purposes to investigate suspected influenza outbreaks. Identification of influenza virus infection by rapid tests can facilitate prompt implementation of control measures before confirmatory test results are available from shell vial culture, tissue cell viral culture, or RT-PCR. A wide range of sensitivities of the rapid influenza tests have been reported, whereas specificities have been reported to be high, compared with reference tests [1,8-12]. In the context of enrolling participants in studies to knowledge of transmission of influenza virus, a rapid influenza diagnostic test was used to screen persons with influenza-like illness in the Nepal during the 2016 influenza seasons. Here, we report our findings that compared the accuracy of the rapid test with that of RT-PCR an investigation of these results.

Methods

Study Design

This study is cross-sectional and consecutive study carried out to find out the sensitivity and specificity of Quick Vue rapid influenza diagnostic kit test with reference to real time RT-PCR. This study was conducted at National Influenza Center, Reference Laboratory, National Public Health Laboratory, Teku, Kathmandu. This study was undertaken in individuals having Influenza like illness visiting National Public Health Laboratory. The numbers of the suspected clinical samples included in the study were sixteen hundred eighty-three. This study was performed in 2016.

Collection of Sample and Transportation

After obtaining Ethical approval, informed consent, patient information, demographic characteristics, clinical history, two throat swabs were collected. According to the WHO (2011), throat swab is a suitable specimen for upper respiratory tract infection, which can easily be collected. Out of two throat swabs collected, one was used for the rapid detection of influenza at the site of sample collection while another was transported to laboratory for viral RNA extraction by using WHO manual for the collection and transportation of clinical sample. Clinical samples which could not be processed on the collection day were refrigerated at 4oC no longer than 4 days and for longer storage than that storage at -70oC were done. The clinical specimens collected were processed according to the standard protocols for detection of influenza virus.

Rapid Antigen Diagnosis of Influenza Virus

The Quick Vue Influenza A+B test involves the extraction of influenza A and B viral antigens. The patient specimen is placed in the extraction reagent tube, during which time the virus particles in the specimen are disrupted, exposing internal viral nucleoproteins. After extraction, the test strip is placed in the extraction reagent tube where nucleoproteins in the specimen will react with the reagents in the test strip. If the extracted specimen contains influenza antigens, a pink-to-red test line along with a blue procedural control line will appear on the test strip indicating a positive result. The test line for Influenza A or B will develop at separate specified locations on the same test strip. If influenza type A or type B antigens are not present, only a blue procedural control line will appear. In case of presence of very low levels of influenza type A or type B antigens, the test kit cannot detect viral antigen and gives result as in negative, a blue control line.

RNA Extraction

The RNA was extracted manually using the QAIamp® Viral RNA mini kit (QIAGEN GmbH, Hilden, Germany) as per the manufacturer’s instructions.

Nucleic Acid Detection of the Influenza Virus

The CDC Real-time RT-PCR protocol for detection and characterization of Influenza includes a panel of oligonucleotide primers and dual-labeled hydrolysis (taqman®) probes to be used in real time RT-PCR assays for the in-vitro qualitative detection and characterization of human Influenza Virus in respiratory specimens and viral cultures. The Influenza A and B primer and probe set was designated for universal detection of type A and type B influenza viruses. Influenza A sub typing primer and probe sets were designed to specially detect contemporary human A/H1, human A/H3, and A/H1N1 pdm09 influenza viruses.

Data Collection and Analysis

Data were collected after the valid results were obtained for each specimen. The data were entered in Microsoft Excel 2013 as well as IBM SPSS Statistics Version 20.

Laboratory Safety and Infection Control

Influenza is air-borne disease. It is very contagious disease and transmitted to the person through the air borne droplet up to 6 feet away. If proper safety method is not used, then it can cause serious health hazard to the laboratory workers. It was mandatory to wear PPE (personal protective equipment) before handling the clinical samples. No laboratory staff handling clinical samples could touch pen, copy/files, computers, door locks, hair, eyes etc. Triple layers packing of the contagious samples of influenza virus was mandatory to prevent the possible spread of virus. Every clinical sample was processed in the safety cabinet. There was a different room for allocation, extraction, master mix and PCR. All the waste materials were discarded only after autoclaving. All the diagnosis was carried out in BSL II laboratory.

Results

A total 1683 samples were processed for influenza testing by RDT Quick Vue Influenza A + B and PCR simultaneously. Among 1683 samples 491 were positive by rapid testing and PCR as well. Whereas 122 were positive only by PCR (Tables 1 & 2). And out of 1520 negative samples, 1508 were negative by rapid testing & PCR as well and 12 were negative only by the PCR (Figure 1).

Figure 1: Comparison of Rapid Test with PCR for Influenza Testing.

Table 1: Comparison of Results of Influenza Antigen-Based RDT with PCR (n = 1683).

Table 2.

Discussion

Influenza can be a major public health burden. Both influenza A and influenza B have caused estimated 3 – 5 million cases of severe illness and about 290000 – 650000 deaths globally. The diagnosis of influenza infections includes viral isolation in cell culture, immunofluorescence assays, nucleic acid amplification tests, immunochromatography‐based rapid diagnostic tests, etc. The rapid diagnosis which is relatively simple and easy to perform test than other test and take shorten period to diagnose that help to manage the patient treatment and reduce the overall cost of the treatment. Here we have evaluated rapid influenza test through Quick Vue Influenza A+B test kit in comparison to RT-PCR. The Performance of the rapid test was moderately good, with a sensitivity (80%) and specificity (98%) in comparison with a real time RT-PCR assay. In Janice K. Louie et al study on the Quick Vue test for influenza have found comparable specificities (98%) but lower sensitivities (26%) R. However other previous studies show related to this show comparable sensitivity and specificity R. The discrepancies in the result might be due to the study design and sample size, across the range of Ct value.

Conclusion

In our study it shows overall good positive predictive and negative predictive value help to provide about influenza in quickly as point of care testing that help in the clinical management of the patient in the outbreak with less trained manpower and costeffective kits. However more data and test need to be done to evaluate the clinical performance of RDT for influenza diagnosis on extrapolated to other age group & clinical information for specific symptoms and severity is require [13-15].

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