Evaluation of antigen detection and polymerase chain reaction for diagnosis of amoebic liver abscess in patients on anti-amoebic treatment

ABSTRACT: BACKGROUND: Diagnosis of amoebic liver abscess (ALA) in patients on anti-amoebic drugs is difficult. There is scanty data on this issue using Entamoeba histolytica (E. histolytica) lectin antigen and polymerase chain reaction (PCR). We studied lectin antigen, PCR, and IgG antibody in liver abscess patients. Liver aspirate of 200 patients, of which 170 had anti-amoebic drug prior to drainage, was tested for E. histolytica lectin antigen by ELISA, PCR, bacterial culture, and serum IgG antibody by ELISA. Classification of abscesses was based on result of anti-amoebic IgG antibody and bacterial culture, E. histolytica PCR and bacterial culture, and E. histolytica lectin antigen and bacterial culture was evaluated. FINDINGS: Using anti-amoebic IgG antibody and bacterial culture, 136/200 (68.0%) were classified as ALA, 12/200 (6.0%) as pyogenic liver abscess (PLA), 29/200 (14.5%) as mixed infection, and 23/200 (11.5%) remained unclassified. Using amoebic PCR and bacterial culture 151/200 (75.5%) were classified as ALA, 25/200 (12.5%) as PLA, 16/200 (8.0%) as mixed infection, and 8/200 (4.0%) remained unclassified. With E. histolytica lectin antigen and bacterial culture, 22/200 (11.0%) patients were classified as ALA, 39/200 (19.5%) as PLA, 2/200 (1.0%) as mixed infection, and 137/200 (68.5%) remained unclassified. CONCLUSIONS: E. histolytica lectin antigen was not suitable for classification of patients who had prior anti-amoebic treatment. However, PCR may be used as alternative test to anti-amoebic antibody in diagnosis of ALA.     

Profiling of the bacteria responsible for pyogenic liver abscess by 16S rRNA gene pyrosequencing

Pyogenic liver abscess (PLA) is a severe disease with considerable mortality and is often polymicrobial. Understanding the pathogens that cause PLA is the basis for PLA treatment. Here, we profiled the bacterial composition in PLA fluid by pyrosequencing the 16S ribosomal RNA (rRNA) gene based on next-generation sequencing (NGS) technology to identify etiological agents of PLA and to provide information of their 16S rRNA sequences for application to DNA-based techniques in the hospital. Twenty patients with PLA who underwent percutaneous catheter drainage, abscess culture, and blood culture for isolates were included. Genomic DNAs from abscess fluids were subjected to polymerase chain reaction and pyrosequencing of the 16S rRNA gene with a 454 GS Junior System. The abscess and blood cultures were positive in nine (45%) and four (20%) patients, respectively. Pyrosequencing of 16S rRNA gene showed that 90% of the PLA fluid samples contained single or multiple genera of known bacteria such as Klebsiella, Fusobacterium, Streptococcus, Bacteroides, Prevotella, Peptostreptococcus, unassigned Enterobacteriaceae, and Dialister. Klebsiella was predominantly found in the PLA fluid samples. All samples that carried unassigned bacteria had 26.8% reads on average. We demonstrated that the occurrence of PLA was associated with eight known bacterial genera as well as unassigned bacteria and that 16S rRNA gene sequencing was more useful than conventional culture methods for accurate identification of bacterial pathogens from PLA.     

Detection of excretory <Emphasis Type="Italic">Entamoeba histolytica</Emphasis> DNA in the urine,and detection of <Emphasis Type="Italic">E. histolytica</Emphasis> DNA and lectin antigen in the liver abscess pus for the diagnosis of amoebic liver abscess

Background  

Amoebic liver abscess (ALA) and pyogenic liver abscesses (PLA) appear identical by ultrasound and other imaging techniques. Collection of blood or liver abscess pus for diagnosis of liver abscesses is an invasive procedure, and the procedure requires technical expertise and disposable syringes. Collection of urine is a noninvasive procedure. Therefore, there has been much interest shown towards the use of urine as an alternative clinical specimen for the diagnosis of some parasitic infections. Here, we report for the first time the detection of E. histolytica DNA excreted in the urine for diagnosis of the cases of ALA.     

Identification of an avirulent Entamoeba histolytica strain with unique tRNA-linked short tandem repeat markers

Highly polymorphic, non-coding short tandem repeats (STR) are scattered between the tRNA genes in Entamoeba histolytica in a unique tandemly arrayed organization. STR markers that correlate with the virulence of individual E. histolytica strains have recently been reported. Here we evaluated the usefulness of tRNA-linked STR loci as genetic markers in identifying virulent and avirulent strains of E. histolytica from 37 Japanese E. histolytica samples (12 diarrheic/dysenteric, 20 amebic liver abscess (ALA), and 5 asymptomatic cases). Twenty three genotypes, assigned by combining the STR sequence types from all 6 STR loci, were identified. One to 8 new STR sequence types per locus were also discovered. Genotypes found in asymptomatic isolates were highly polymorphic (4 out of 5 genotypes were unique to this group), while in symptomatic isolates, almost half of the genotypes were shared between diarrhea/dysentery and ALA. One asymptomatic isolate (KU27) showed unique STR patterns in 4 loci. This strain, though associated with the typical pathogenic zymodeme II, failed to induce amebic liver abscess by animal challenge, which suggests that inherently avirulent E. histolytica strains exist, that are associated with unique genotypes. Furthermore, STR genotyping and in vivo challenge of 2 other asymptomatic isolates (KU14 and KU26) verified the covert virulence of these strains.     

TNFα-Mediated Liver Destruction by Kupffer Cells and Ly6Chi Monocytes during Entamoeba histolytica Infection

Amebic liver abscess (ALA) is a focal destruction of liver tissue due to infection by the protozoan parasite Entamoeba histolytica (E. histolytica). Host tissue damage is attributed mainly to parasite pathogenicity factors, but massive early accumulation of mononuclear cells, including neutrophils, inflammatory monocytes and macrophages, at the site of infection raises the question of whether these cells also contribute to tissue damage. Using highly selective depletion strategies and cell-specific knockout mice, the relative contribution of innate immune cell populations to liver destruction during amebic infection was investigated. Neutrophils were not required for amebic infection nor did they appear to be substantially involved in tissue damage. In contrast, Kupffer cells and inflammatory monocytes contributed substantially to liver destruction during ALA, and tissue damage was mediated primarily by TNFα. These data indicate that besides direct antiparasitic drugs, modulating innate immune responses may potentially be beneficial in limiting ALA pathogenesis.     

Precision-cut hamster liver slices as an ex vivo model to study amoebic liver abscess

Entamoeba histolytica is the etiological agent of amoebiasis, the second cause of global morbidity and mortality due to parasitic diseases in humans. In approximately 1% of the cases, amoebas penetrate the intestinal mucosa and spread to other organs, producing extra-intestinal lesions, among which amoebic liver abscess (ALA) is the most common. To study ALA, in vivo and in vitro models are used. However, animal models may pose ethical issues, and are time-consuming and costly; and cell cultures represent isolated cellular lineages. The present study reports the infection of precision-cut hamster liver slices with Entamoeba histolytica trophozoites. The infection time-course, including tissue damage, parallels findings previously reported in the animal model. At the same time amoebic virulence factors were detected in the infected slices. This new model to study ALA is simple and reproducible, and employs less than 1/3 of the hamsters required for in vivo analyses.     

Imported Amoebic Liver Abscess in France

Background

Worldwide, amoebic liver abscess (ALA) can be found in individuals in non-endemic areas, especially in foreign-born travelers.

Methods

We performed a retrospective analysis of ALA in patients admitted to French hospitals between 2002 and 2006. We compared imported ALA cases in European and foreign-born patients and assessed the factors associated with abscess size using a logistic regression model.

Results

We investigated 90 ALA cases. Patient median age was 41. The male:female ratio was 3.5∶1. We were able to determine the origin for 75 patients: 38 were European-born and 37 foreign-born. With respect to clinical characteristics, no significant difference was observed between European and foreign-born patients except a longer lag time between the return to France after traveling abroad and the onset of symptoms for foreign-born. Factors associated with an abscess size of more than 69 mm were being male (OR = 11.25, p<0.01), aged more than 41 years old (OR = 3.63, p = 0.02) and being an immigrant (OR = 11.56, p = 0.03). Percutaneous aspiration was not based on initial abscess size but was carried out significantly more often on patients who were admitted to surgical units (OR = 10, p<0.01). The median time to abscess disappearance for 24 ALA was 7.5 months.

Conclusions/Significance

In this study on imported ALA was one of the largest worldwide in terms of the number of cases included males, older patients and foreign-born patients presented with larger abscesses, suggesting that hormonal and immunological factors may be involved in ALA physiopathology. The long lag time before developing ALA after returning to a non-endemic area must be highlighted to clinicians so that they will consider Entamoeba histolytica as a possible pathogen of liver abscesses more often.     

Natural Killer T Cells Activated by a Lipopeptidophosphoglycan from Entamoeba histolytica Are Critically Important To Control Amebic Liver Abscess

The innate immune response is supposed to play an essential role in the control of amebic liver abscess (ALA), a severe form of invasive amoebiasis due to infection with the protozoan parasite Entamoeba histolytica. In a mouse model for the disease, we previously demonstrated that Jα18^-/- mice, lacking invariant natural killer T (iNKT) cells, suffer from more severe abscess development. Here we show that the specific activation of iNKT cells using α-galactosylceramide (α-GalCer) induces a significant reduction in the sizes of ALA lesions, whereas CD1d^−/− mice develop more severe abscesses. We identified a lipopeptidophosphoglycan from E. histolytica membranes (EhLPPG) as a possible natural NKT cell ligand and show that the purified phosphoinositol (PI) moiety of this molecule induces protective IFN-γ but not IL-4 production in NKT cells. The main component of EhLPPG responsible for NKT cell activation is a diacylated PI, (1-O-[(28∶0)-lyso-glycero-3-phosphatidyl-]2-O-(C16:0)-Ins). IFN-γ production by NKT cells requires the presence of CD1d and simultaneously TLR receptor signalling through MyD88 and secretion of IL-12. Similar to α-GalCer application, EhLPPG treatment significantly reduces the severity of ALA in ameba-infected mice. Our results suggest that EhLPPG is an amebic molecule that is important for the limitation of ALA development and may explain why the majority of E. histolytica-infected individuals do not develop amebic liver abscess.     

Prevalent HLA Class II Alleles in Mexico City Appear to Confer Resistance to the Development of Amebic Liver Abscess

Amebiasis is an endemic disease and a public health problem throughout Mexico, although the incidence rates of amebic liver abscess (ALA) vary among the geographic regions of the country. Notably, incidence rates are high in the northwestern states (especially Sonora with a rate of 12.57/100,000 inhabitants) compared with the central region (Mexico City with a rate of 0.69/100,000 inhabitants). These data may be related to host genetic factors that are partially responsible for resistance or susceptibility. Therefore, we studied the association of the HLA-DRB1 and HLA-DQB1 alleles with resistance or susceptibility to ALA in two Mexican populations, one each from Mexico City and Sonora. Ninety ALA patients were clinically diagnosed by serology and sonography. Genomic DNA was extracted from peripheral blood mononuclear cells. To establish the genetic identity of both populations, 15 short tandem repeats (STRs) were analyzed with multiplexed PCR, and the allelic frequencies of HLA were studied by PCR-SSO using LUMINEX technology. The allele frequencies obtained were compared to an ethnically matched healthy control group (146 individuals). We observed that both affected populations differed genetically from the control group. We also found interesting trends in the population from Mexico City. HLA-DQB1*02 allele frequencies were higher in ALA patients compared to the control group (0.127 vs 0.047; p= 0.01; pc= NS; OR= 2.9, 95% CI= 1.09-8.3). The less frequent alleles in ALA patients were HLA-DRB1*08 (0.118 vs 0.238 in controls; p= 0.01; pc= NS; OR= 0.42, 95% CI= 0.19-0.87) and HLA-DQB1*04 (0.109 vs 0.214; p= 0.02; pc= NS; OR= 0.40, 95% CI= 0.20-0.94). The haplotype HLA-DRB1*08/-DQB1*04 also demonstrated a protective trend against the development of this disease (0.081 vs. 0.178; p=0.02; pc=NS; OR= 0.40, 95% CI= 0.16-0.93). These trends suggest that the prevalent alleles in the population of Mexico City may be associated with protection against the development of ALA.     

The MAK16 Gene of Entamoeba histolytica and Its Identification in Isolates from Patients

To identify sequences of Entamoeba histolytica associated with the development of amebic liver abscess (ALA) in hamsters, subtractive hybridization of cDNA from E. histolytica HM-1:IMSS under 2 growth conditions was performed: 1) cultured in axenic medium and 2) isolated from experimental ALA in hamsters. For this procedure, 6 sequences were obtained. Of these sequences, the mak16 gene was selected for amplification in 29 cultures of E. histolytica isolated from the feces of 10 patients with intestinal symptoms and 19 asymptomatic patients. Only 5 of the 10 isolates obtained from symptomatic patients developed ALA and amplified the mak16 gene, whereas the 19 isolates from asymptomatic patients did not amplify the mak16 gene nor did they develop ALA. Based on the results of Fisher''s exact test (P<0.001), an association was inferred between the presence of the mak16 gene of E. histolytica and the ability to develop ALA in hamsters and with the patient''s symptoms (P=0.02). The amplification of the mak16 gene suggests that it is an important gene in E. histolytica because it was present in the isolates from hamsters that developed liver damage.     

Effects of ALA,EPA and DHA in high-carbohydrate,high-fat diet-induced metabolic syndrome in rats

We compared the cardiovascular, hepatic and metabolic responses to individual dietary n-3 fatty acids (α-linolenic acid, ALA; eicosapentaenoic acid, EPA; and docosahexaenoic acid, DHA) in a high-carbohydrate, high-fat diet-induced model of metabolic syndrome in rats. Additionally, we measured fatty acid composition of plasma, adipose tissue, liver, heart and skeletal muscle in these rats. The same dosages of ALA and EPA/DHA produced different physiological responses to decrease the risk factors for metabolic syndrome. ALA did not reduce total body fat but induced lipid redistribution away from the abdominal area and favorably improved glucose tolerance, insulin sensitivity, dyslipidemia, hypertension and left ventricular dimensions, contractility, volumes and stiffness. EPA and DHA increased sympathetic activation, reduced the abdominal adiposity and total body fat and attenuated insulin sensitivity, dyslipidemia, hypertension and left ventricular stiffness but not glucose tolerance. However, ALA, EPA and DHA all reduced inflammation in both the heart and the liver, cardiac fibrosis and hepatic steatosis. These effects were associated with complete suppression of stearoyl-CoA desaturase 1 activity. Since the physiological responses to EPA and DHA were similar, it is likely that the effects are mediated by DHA with EPA serving as a precursor. Also, ALA supplementation increased DHA concentrations but induced different physiological responses to EPA and DHA. This result strongly suggests that ALA has independent effects in metabolic syndrome, not relying on its metabolism to DHA.     

IL-6 KO mice develop experimental amoebic liver infection with eosinophilia

Interleukin 6 (IL-6) is a multifunctional cytokine that regulates various aspects of the immune response, such as acute phase reaction and hematopoiesis, and is an important signal that coordinates activities of liver cells, macrophages, and lymphocytes. Amoebic liver lesions have been studied, usually in hamsters, due to the problem of abscess development in mice. We report here the development of an experimental amoebic liver abscess (ALA) model in mice deficient in IL-6. Axenically grown amoebae were injected directly into the livers of C57BL/6 wild type (WT) and IL-6 KO -/- mice; the abscesses produced were counted and the inflammatory process was examined on 5, 10, and 20 days postinfection. Our results showed that IL-6 KO -/- mice develop ALA, in contrast to the WT strain, which usually do not have signs of abscess or infection. Histological analysis of the abscesses showed extended inflammatory response, mainly mediated by eosinophils, which strongly infiltrate the abscess in IL-6 K -/- mice. The present results suggest that in mice, IL-6 could play a role in the resistance against ALA.     

Entamoeba histolytica: rapid detection of indian isolates by cysteine proteinase gene-specific polymerase chain reaction

Amoebiasis, caused by Entamoeba histolytica, is still one of the major problems for developing countries like India. Early detection of the parasite is a must for its prevention and control. In this study, PCR analysis of the cysteine proteinase gene from clinical isolates of symptomatic intestinal and amoebic liver abscess (ALA) cases has been compared with the stool microscopy, serology, and ultrasonography methods. The clinical isolates negative for E. histolytica by stool microscopy demonstrated the presence of the cysteine proteinase gene by PCR amplification. Also the gene copy number was increased in ALA samples compared with intestinal cases. Hence an accurate, early, and easier detection was possible by cysteine proteinase gene amplification directly from the clinical samples.     

Futile cycle of β-oxidation and de novo lipogenesis are associated with essential fatty acids depletion in lipoatrophy

Total absence of adipose tissue (lipoatrophy) is associated with the development of severe metabolic disorders including hepatomegaly and fatty liver. Here, we sought to investigate the impact of severe lipoatrophy induced by deletion of peroxisome proliferator-activated receptor gamma (PPARγ) exclusively in adipocytes on lipid metabolism in mice. Untargeted lipidomics of plasma, gastrocnemius and liver uncovered a systemic depletion of the essential linoleic (LA) and α-linolenic (ALA) fatty acids from several lipid classes (storage lipids, glycerophospholipids, free fatty acids) in lipoatrophic mice. Our data revealed that such essential fatty acid depletion was linked to increased: 1) capacity for liver mitochondrial fatty acid β-oxidation (FAO), 2) citrate synthase activity and coenzyme Q content in the liver, 3) whole-body oxygen consumption and reduced respiratory exchange rate in the dark period, and 4) de novo lipogenesis and carbon flux in the TCA cycle. The key role of de novo lipogenesis in hepatic steatosis was evidenced by an accumulation of stearic, oleic, sapienic and mead acids in liver. Our results thus indicate that the simultaneous activation of the antagonic processes FAO and de novo lipogenesis in liver may create a futile metabolic cycle leading to a preferential depletion of LA and ALA. Noteworthy, this previously unrecognized cycle may also explain the increased energy expenditure displayed by lipoatrophic mice, adding a new piece to the metabolic regulation puzzle in lipoatrophies.     

Quantitative detection of Proteus species by real-time polymerase chain reaction using SYBR Green I

A SYBR Green real-time polymerase chain reaction (PCR) method for rapid detection of Proteus species was developed and evaluated. Of 322 clinical and food samples tested, 75 samples were positive for Proteus species by using conventional PCR and real-time PCR assays. The results were consistent with standard culture methods and the Vitek auto-microbe system, indicating a 100 % specificity obtained by both PCR assays. For the real-time PCR method, the minimum detectable level was 10 colony forming units (CFU) /ml, which was a 10³ multiple higher than the conventional PCR method. Correlation coefficients of standard curves which were constructed using the threshold cycle (Ct) versus copy numbers of Proteus showed good linearity (R ²?=?0.997). In conclusion, several significant advantages such as higher sensitivity and rapidness were observed by using the SYBR Green real-time PCR method for identifying Proteus species.     

Testosterone Increases Susceptibility to Amebic Liver Abscess in Mice and Mediates Inhibition of IFNγ Secretion in Natural Killer T Cells

Amebic liver abscess (ALA), a parasitic disease due to infection with the protozoan Entamoeba histolytica, occurs age and gender dependent with strong preferences for adult males. Using a mouse model for ALA with a similar male bias for the disease, we have investigated the role of female and male sexual hormones and provide evidence for a strong contribution of testosterone. Removal of testosterone by orchiectomy significantly reduced sizes of abscesses in male mice, while substitution of testosterone increased development of ALA in female mice. Activation of natural killer T (NKT) cells, which are known to be important for the control of ALA, is influenced by testosterone. Specifically activated NKT cells isolated from female mice produce more IFNγ compared to NKT cells derived from male mice. This high level production of IFNγ in female derived NKT cells was inhibited by testosterone substitution, while the IFNγ production in male derived NKT cells was increased by orchiectomy. Gender dependent differences were not a result of differences in the total number of NKT cells, but a result of a higher activation potential for the CD4⁻ NKT cell subpopulation in female mice. Taken together, we conclude that the hormone status of the host, in particular the testosterone level, determines susceptibility to ALA at least in a mouse model of the disease.     

Effect of bovine lactoferrin in a therapeutic hamster model of hepatic amoebiasis

Entamoeba histolytica is the causative agent of amoebiasis, a disease that produces dysentery as a result of the perforation of the large intestine. This parasite often invades other organs, primarily the liver, leading to an amoebic liver abscess (ALA), which can cause death. Metronidazole is the drug of choice for the treatment of ALA; however, it produces toxic side effects in patients. Lactoferrin (Lf) is a glycoprotein of the innate immune response that sequesters iron in the mucosae. Lf possesses immune-regulatory properties, such as antiinflammatory and antioxidant activities. Moreover, the microbicidal activity of apoLf, which lacks bound iron, has been shown. In this study, we evaluated the therapeutic effect of bovine Lf (bLf) against ALA in a model of hepatic amoebiasis in hamsters. Interestingly, hamsters treated intragastrically with Lf (2.5 mg/100 g mass) over a period of 8 days showed no clinical signs of disease and ALA was effectively decreased, with only 0.63% detectable lesion, compared with 63% in untreated animals. Furthermore, liver function and blood cells approached normal levels among those receiving bLf treatment. These results suggest that bLf may aid in the therapy of amoebiasis, likely without producing undesirable effects in patients.     

Autoimmunity in amoebiasis.

Amoebic liver abscess (ALA) and symptomatic intestinal amoebiasis cases were assessed by indirect haemagglutination assay for auto-reactive IgG and IgA class of antibodies in response to healthy human serum IgG and IgA. The present results indicated the presence of autoreactive IgG and IgA class of antibodies in ALA and intestinal amoebiasis respectively.     

On-line process monitoring and chemometric modeling with 2D fluorescence spectra obtained in recombinant E. coli fermentations

2D spectrofluorometry produces a large volume of spectral data during fermentation processes with recombinant E. coli, which can be analyzed using chemometric methods such as principal component analysis (PCA), principal component regression (PCR) and partial least square regression (PLS). An analysis of the spectral data by PCA results in scores and loadings that are not only visualized in the score-loading plots but are also used to monitor the fermentation processes on-line. The score plots provided useful qualitative information on four fermentation processes for the production of extracellular 5-aminolevulinic acid (ALA). Two chemometric models (PCR and PLS) were used to examine the correlation between the 2D fluorescence spectra and a few parameters of the fermentation processes. The results showed that PLS had slightly better calibration and prediction performance than PCR.     

细菌性肝脓肿临床特点的回顾性分析

目的:总结细菌性肝脓肿(pyogenic liver abscesses,PLA)的临床特点、诊断及治疗经验,提高诊疗水平。方法:回顾性分析哈尔滨医科大学第二临床医学院2013年1月-2014年12月收治的75例PLA患者的临床资料。结果:75例患者的平均年龄57岁,最大78岁,最小34岁。其中,男45例,女30例,男:女为1.5:1。75例患者中表现为发热者69例(92.0%)、寒战28例(37.3%)、右上腹痛66例(88.0%),伴恶心呕吐者28例(37.3%),黄疸15例(20.0%),肝肿大8例(10.7%)。有糖尿病病史患者28例(37.3%)。胆系疾病(78.7%)是本组资料主要的易患因素,其中53例患有胆囊炎(70.7%)。外周血白细胞增高67例(89.3%),血红蛋白降低41例(54.7%),白蛋白降低46例(61.3%);75例患者转氨酶升高58例(77.3%)。肝右叶单发脓肿多见(75例患者中36例,占48%)。51例B超确诊,24例行B超+CT或CT检查。48例行穿刺液细菌培养及25例血培养,其中肺炎克雷伯氏菌为主要病原菌。平均住院天数为19天,多数患者经皮下肝脓肿穿刺或置管引流而好转,1例漏诊为肺炎,2例患者反复入院,1例因菌血症死亡。结论:PLA的临床表现多样,以发热最为常见;多为肝右叶单发,部分患者血红蛋白及血清白蛋白降低。糖尿病为PLA最常见基础疾病,胆系疾病是主要的易患因素。肺炎克雷伯杆菌是本组PLA患者的主要致病菌。