FormalPara Key Summary Points

Diagnosis of amoebic liver abscess is challenging in a non-endemic setting, because of suboptimal performance of conventional diagnostic methods.

Molecular techniques have emerged as a new diagnostic reference standard.

FilmArray gastrointestinal panel can be useful in detecting several enteric pathogens including Entamoeba histolytica in stool samples.

We report a patient with amoebic liver abscess that was diagnosed thanks to an unconventional application of FilmArray gastrointestinal panel on intraoperative drainage fluid.

Introduction

Amoebiasis is a frequent infectious disease caused by the protozoan parasite Entamoeba histolytica and is one of the most common causes of infectious diarrhea among travelers returning from endemic areas (estimated incidence of 14 patients in every 1000 returning travelers) [1]. Most infections are asymptomatic (luminal amoebiasis) and less than 20% of patients develop symptomatic infection. When E. histolytica disseminates, it can cause more severe infections such as watery or bloody diarrhea, which can ultimately result in massive bowel necrosis with perforation and peritonitis, or toxic megacolon [2,3,4]. Among extraintestinal manifestations (pneumonia, pericarditis, cerebral amoebiasis), liver abscess is the most common manifestation and develops more commonly in men than women, even more than 20 years after the last visit to an endemic area [5,6,7,8].

As a result of the fecal–oral route transmission, amoebiasis is distributed worldwide, especially in areas with poor sanitation such as the tropics; areas with the highest rates of infection include India, Africa, and Central and South America. The prevalence in the Americas between 1990 and 2022 ranged from 0.08% to 82.6%, being more frequent in Venezuela, Colombia, and Mexico [9]. However, the exact burden of amoebiasis is difficult to quantify because of the heterogeneity of studies, and the scarce diagnostic tools and screening programs in endemic countries [4].

Categories at higher risk for exposure are humanitarian aid workers, immigrants, and long-term travelers. It has been estimated that up to 50 million people are affected, mostly in developing countries, causing over 100,000 deaths every year [3, 4].

Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba bangladeshi, Entamoeba coli, Entamoeba hartmanni, and Entamoeba polecki are seven species that infect the human intestinal lumen, although the last two are rare and considered nonpathogenic [3, 10].

After ingestion of mature cysts from contaminated food, water, or sexual contact, motile trophozoites are released in the small intestine and can pass through the colonic epithelium spreading to the peritoneum, liver, lungs, or brain. Symptoms may occur within weeks after ingestion but may develop even years after infection [9]. Amoebic liver abscess is caused by the unbalanced host’s immune response after the invasion of E. histolytica trophozoites in tissues with the recruitment of immune cells such as monocytes, neutrophils, and macrophages forming a capsule. The massive necrosis of hepatocytes causes the typical “anchovy paste” aspect of the fluid, when aspirated [11, 12].

Traditionally, the diagnosis and characterization of Entamoeba spp. have been based mainly on microscopy examination of protozoan morphology. However, this technique is time consuming, heavily affected by the operator’s skills, and is unable to differentiate among protozoa with similar morphological features. More recently, other research tools have become available, such as antigen and antibody detection. Since most people in endemic areas have been exposed to E. histolytica, antibody detection, mainly conducted by ELISA, is unable to distinguish past from current infection, and consequently needs a combination of antigenic test or PCR detection to confirm active disease. Moreover, serology may be falsely negative early in the course of the disease. Antigen detection, mainly performed on stools, has several advantages compared to other methods: it can differentiate between different species, it has a good specificity and sensitivity, and it can be carried out by non-experienced personnel [13]. A limitation of this method, however, is the ease of denaturation, which makes it possible to test only fresh or frozen samples and that an important reduction in sensitivity was observed when the patient had been exposed to antibiotic therapy [13, 14]. Molecular assays such as DNA detection by real-time PCR showed an impressive sensitivity and specificity (around 100%), an optimized turnaround time, and the ability to differentiate between different species. Therefore, PCR should be preferred when available and affordable over antigen detection and microscopy for definitive identification of E. histolytica. In fact, PCR-based assays avoid not only misdiagnosis but also overtreatment [15,16,17].

The FilmArray gastrointestinal panel has recently become available in some hospitals and can detect several gastrointestinal pathogens, including Clostridioides difficile toxin A/B, six diarrheagenic Shigella spp./E. coli, four parasites including E. histolytica, and five viruses. The results are available in 1 h and it has an overall 98.5% sensitivity and 99.2% specificity [18,19,20].

All patients with clinical disease need to be treated with a tissue-active agent (metronidazole or tinidazole) and a luminal cysticidal agent (paromomycin) [2, 4, 7, 17].

Here we present a clinical case of a patient with amoebic liver abscess promptly diagnosed by FilmArray gastrointestinal panel performed on liver drainage fluid.

Case Presentation

A 63-year-old Italian man with unremarkable medical history required medical attention because of abdominal pain and fever in July 2023. He resides in the UK and had lived in Venezuela for 3 months 10 years earlier. He denied any documented exposure to contaminated water and any sexual behavior at risk. He was initially hospitalized in another hospital, where a CT scan of the abdomen was performed showing the presence of a voluminous liver abscess of 11 cm involving the S6 and S7 segments. A drainage was positioned into the abscess but culture of the fluid was negative. Therefore, an empirically broad-spectrum antimicrobial therapy with piperacillin/tazobactam was started with benefit on symptoms, and size reduction of liver lesion at a follow-up CT scan. The patient was finally discharged after hepatic drainage removal.

One month after discharge, he experienced new onset of daily fever up to 38 °C and abdominal pain, so he presented to medical attention (day 0), at the Policlinic San Martino Hospital in Genoa. New abdominal CT scan was performed with evidence of a dimensional increase of the known liver lesion (14 × 16 cm) involving the S4, S5, S7, and S8 segments with compression of the intrahepatic tract of the inferior vena cava, portal system, and suprahepatic veins, and leading to dilatation of the intrahepatic biliary tract (Fig. 1).

Fig. 1
figure 1

CT scan of amoebic liver abscess before surgery

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An empirical broad-spectrum antimicrobial therapy with piperacillin/tazobactam was started at admission and escalated to meropenem and tigecycline after infectious disease consultation because of persistence of symptoms and failure of the previous antibiotic.

Among the microbiological investigations performed, serology for E. histolytica test was positive, using an ELISA method performed with an E. histolytica IgG DRG kit (DRG Instruments GmbH, Germany), whereas detection of amoebic antigen in stools and blood was negative using an immunochromatographic method performed with an ImmunoCardSTAT CGE kit (Meridian Bioscence, Milan, Italy).

On day 3, a percutaneous liver drainage was positioned, with evacuation of purulent, brown-colored material (with the presence of 92,000/mm3 leukocytes and 88,000/mm3 lymphocytes on cytometry). From drainage culture a contaminant coagulase-negative Staphylococcus spp. was isolated, and the amoebic antigen test was negative.

On day 12 after admission, the patient needed laparotomy, with resection of a voluminous septate liver lesion (Fig. 2). Intraoperative drainage fluid presented as brown purulent material, resembling the classic amoebic “anchovy paste” appearance. At this point, Biofire FilmArray gastrointestinal multiplex PCR panel (BioMérieux, France) was performed on the sterile intraoperative drainage fluid, with rapid molecular detection of the E. histolytica gene, despite the same test performed on stool being negative.

Fig. 2
figure 2

Intraoperative picture of amoebic liver abscess resection

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Therefore, targeted antimicrobial therapy with metronidazole 750 mg every 8 h was started on the same day (day 12). Since the majority of gram-negative pathogens responsible for bacterial liver abscess are not detected by the FilmArray panel, empirical antibiotic therapy previously started was discontinued only when superinfection of the lesion was excluded by negative traditional culture on drainage fluid (day 17). A 7-day course of intraluminal therapy with paromomycin was also performed after completion of a well-tolerated 10-day course of metronidazole therapy, to promote intestinal decolonization of the parasite.

The subsequent histological examination results confirmed the presence of hepatic fibrino-necrotic areas in which red blood cells and leukocytes were observed, in association with very focal cellular elements of histiocytoid morphology, PAS (periodic acid Schiff) positive, consistent with organisms of Entamoeba spp. The adjacent parenchyma showed chronic inflammation and hepatocyte necrosis without significant fibrosis (Fig. 3).

Fig. 3
figure 3

Histological detail of liver abscess showing cellular elements of histiocytoid morphology (hematoxylin and eosin, on the left), PAS positive (on the right), consistent with organisms of Entamoeba spp. associated with  fibro-necrotic debris and inflammatory cells

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The patient experienced a postoperative complication with the occurrence of post-surgical bilioma with consistent pleural effusion and concomitant lung thickening, for which surgical reintervention with pleural drainage positioning was necessary. An infectious origin of this complication was ruled out by performing amoebic antigen, FilmArray gastrointestinal panel, and culture examination was negative; moreover, the patient improved without further antimicrobial therapy. After a regular postoperative course, the patient was transferred from intensive care unit to surgical department in good general clinical condition.

The patient was aware of the conduct and publication of this case study and provided verbal and written informed consent. The study was carried out in accordance with the principles of the Helsinki Declaration of 1964 and its later amendments.

Discussion

Here we present a clinical case of amoebic liver abscess that was diagnosed thanks to an unconventional application of FilmArray gastrointestinal panel on the intraoperative drainage with rapid detection of the pathogen. This choice led to a fast diagnosis and let us prompt initiation of targeted therapy, despite the negative result of amoebic antigen and the unavailability of the gold standard diagnostic technique in our hospital (PCR methods).

Amoebiasis represents a significant public health concern, particularly in endemic regions, whereas in Italy, in the absence of clear risk factors, it is more difficult to observe and diagnose this condition. However, careful analysis of predisposing factors such as a relevant travel history, even years after the last visit to an endemic region, should raise the clinical suspicion in cases with suggestive clinical presentations, like a single hepatic abscess unresponsive to previous antibacterial treatment.

This clinical case shows the challenging diagnostic process of amoebic liver abscess, which requires high clinical suspicion to conduct specific diagnostic investigations such as serology, antigenic testing, or molecular techniques. In addition, many of these tests are not routinely available, especially in smaller hospitals, thereby significantly delaying diagnosis. In this case, although a careful anamnesis revealed a past journey to an endemic region many years earlier and positive serology result was available, the diagnosis remained difficult because antigenic test was negative on stools, serum, and drainage fluid, preventing confirmation of the diagnosis. This highlights the suboptimal sensitivity of antigenic tests, especially for amoebic liver abscess, which is a critical issue and still represents one of the main barriers to early treatment [4]. The most relevant aspect about our case is the decisive role of FilmArray gastrointestinal panel as a diagnostic method that allowed the confirmation of clinical suspicion by revealing the presence of E. histolytica gene in the drainage fluid.

Rapid molecular techniques are an extraordinarily useful tool, especially in cases where antigenic test is negative, since the direct visualization of the microorganism in histological specimens is uncommon and requires a highly experienced anatomopathologist [21, 22]. Other clinical cases recently suggested this unconventional application where multiplex PCR testing allowed a rapid and robust diagnosis of amoebiasis in patients with cystic focal liver lesions, overcoming the diagnostic limitations of traditional diagnostic tools [23,24,25]. The advantages of this method include the speed with which the results are provided but also the possibility of confirming the diagnosis if classical PCR techniques are not available, and when antigenic detection is negative. Another aspect to consider is that the application of FilmArray gastrointestinal panel could also lead to a rapid differential diagnosis with other pathogens which commonly cause liver abscess, as the panel includes a wide range of bacterial and parasitic pathogens. However, since the panel do not cover all the potential pathogens that can cause bacterial liver abscess, it is mandatory to send biological fluid for traditional cultures, and only after obtaining a negative result can a bacterial etiology be excluded. Another limit of this application of the FilmArray gastrointestinal panel is the lack of standardization, so other studies are needed to determine its effective sensitivity and specificity in this diagnostic setting.

Unexpectedly, our patient had subsequent diagnostic confirmation by direct evidence of the pathogen on histology, which is rare given the low sensitivity of direct microscopic analysis. In fact, examination of liver abscess pus only gives a microscopic diagnosis in less than 20% of cases, as amoebae are typically found in the periphery of abscesses rather than in the central aspirated pus, as occurred in this case [21, 22]. However, it should be emphasized that the sample analysis was time-consuming as the microorganism was not easily detectable despite being carried out by highly qualified personnel. For this reason, although the role of histology was crucial to make further confirmation of the diagnosis, it was not sufficient to allow rapid initiation of targeted antimicrobial therapy since the definitive results were available many days later.

In this framework, our case showed the essential role of the FilmArray gastrointestinal panel for the diagnosis of amoebic liver abscess, so it can be supposed that the sensitivity and specificity of this test could be greater than standard diagnostic tools. However, its use on liver abscess pus remains unvalidated and its real diagnostic value remains unknown.

Our patient finally received targeted antimicrobial therapy with metronidazole, together with source control by hepatic resection of the abscess, as recommended by international guidelines, and thus completely recovered, highlighting how a prompt diagnosis and appropriate therapy can overcome a potentially life-threatening condition.

Conclusion

This case report describes the usefulness of the application of Biofire FilmArray gastrointestinal multiplex PCR panel (BioMérieux, France) on intraoperative samples to rapidly diagnose amoebic liver abscess. Further research and awareness are needed to improve the diagnosis and management of amoebiasis, especially in non-endemic regions, considering the potential benefit of applying this innovative rapid molecular technique.