A 36-year-old man from Kolkata, in West Bengal, India, sought care in July 2024 for a 6-day history of intermittent fever, characterized by 1–2 spikes per day and a recorded high temperature of 102°F. The patient reported fever with malaise but no chills or rigors, along with a 5-day history of dull, continuous left upper quadrant abdominal pain that worsened with deep inspiration, sneezing, or when lying in the left lateral decubitus position. A slow-developing heavy feeling in the left upper abdomen, along with nausea, a bad taste in the mouth, and 1 episode of nonbilious vomiting, also occurred. The patient reported weight loss from 53 kg to 47 kg over 4 weeks, despite preserved appetite. In addition, he had a history of occasional nonproductive cough without wheezing, hemoptysis, or shortness of breath. The patient had a left-lobe hepatic abscess diagnosed in March 2023 in Kolkata and was treated empirically with intravenous injection and oral metronidazole. The causative agent was not identified. The bad taste in the patient’s mouth was likely not related to the previous metronidazole treatment, because he had not been on the medication in the previous 3 weeks.

The patient was a cigarette smoker but had abstained from alcohol for 4 years. On examination, his heart rate was 104 beats/min and blood pressure 146/74 mmHg; oxygen saturation (98% room air) and respiratory rate (16 breaths/min) were unremarkable. Physical examination revealed pallor, hepatomegaly (2 cm below right costal margin), splenomegaly (6 cm below left costal margin), and left upper quadrant tenderness. The patient had absent breath sounds over the left intercostal and subaxillary areas but showed no signs of cyanosis, clubbing, or lymphadenopathy. Neurologic examination results were unremarkable. Iron deficiency anemia was noted. Serologic testing revealed negative results for HIV, hepatitis B surface antigen, hepatitis C virus antibody, malarial parasite detection antigen, and Brucella IgM and IgG. The patient also had a nonreactive Rose Bengal plate test and standard tube agglutination test for Brucella. A chest radiograph revealed an elevated left hemidiaphragm and left pleural effusion. An abdominal ultrasound revealed hepatomegaly and a large splenic abscess (18 cm x 14 cm × 17 cm, 2000 mL) with mild subdiaphragmatic fluid. Contrast-enhanced computed tomography of the chest and abdomen confirmed a collapsed left lower lobe and a splenic abscess. The patient was managed with drainage and supportive care.

Figure 1

Figure 1. Microscopic view of Entamoeba moshkovskiitrophozoites (arrowhead) observed in a pleural fluid sample recovered from a patient with an extraintestinal infection, eastern India. Slide used a wet-mount preparation,. Scale...

Pleural fluid analysis with Gram and Ziehl-Neelsen stains showed no microorganisms, and Mycobacterium tuberculosis was not detected. The wet mount showed predominantly macrophages, erythrocytes, epithelial cells, and many protozoal structures resembling E. histolytica with erythrophagia (Figure 1). However, splenic abscess aspirate did not reveal amoebae or amoeba-like structures. The direct smear revealed degenerated blood elements and necrosis, with no granuloma or malignant cells. We conducted additional characterization of the E. histolytica–like organism by using molecular methods.

Figure 2

Figure 2. PCR amplification of DNA from an extraintestinal Entamoeba moshkovskii infection in a patient from eastern India. A) PCR amplification of the 18S rRNA locus of E. moshkovskii...

We extracted DNA by using the QIAamp DNA Mini Kit (QIAGEN, https://www.qiagen.com). We performed conventional PCR targeting the 18S rRNA gene on splenic aspirate and pleural fluid by using previously published species-specific primers for E. histolytica and E. moshkovskii (5,12). Both the splenic abscess aspirate and pleural fluid DNA tested negative for E. histolytica but positive for E. moshkovskii (Figure 2, panel A). To confirm the presence of E. moshkovskii, we targeted the chitinase gene for testing. We designed primer sequences to amplify the upstream and downstream regions of the chitinase open reading frame (Appendix Table). Both splenic abscess aspirate and pleural fluid samples again tested positive for E. moshkovskii when targeting the chitinase locus (Figure 2 panel B). We then purified the PCR products by using the Roche PCR Gel Extraction kit (Roche, https://www.roche.com) and sequenced bidirectionally with the BigDye Terminator v3.1 kit (Thermo Fisher Scientific, https://www.thermofisher.com).

Figure 3

Figure 3. Phylogenetic trees constructed from the 18S rRNA and chitinase gene sequences obtained from Entamoeba moshkovskiiisolates from a patient in eastern India (red circles) and reference sequences. A) Phylogenetic...

BLAST analysis (https://blast.ncbi.nlm.nih.gov) confirmed the organism as E. moshkovskii, showing 100% identity to the reference Laredo strain (18S rRNA; GenBank accession no. AF149906.1). However, the chitinase gene corresponding to AmoebaDB (https://amoebadb.org/amoeba/app) entry EMO_056190 exhibited 3 mutations at positions 36 T/C, 127 G/A, and +2 T/G. The mutation at position 36 was synonymous, whereas the substitution at position 127 resulted in a nonsynonymous change, replacing glutamic acid with lysine. We constructed phylogenetic trees generated from the obtained 18S rRNA and chitinase gene sequences to confirm species identity and evaluate evolutionary relationships (Figure 3, panels A, B).

Of note, PCR analysis of DNA isolated from the patient’s stool samples by using the 18S rRNA targeted assay detected the presence of E. moshkovskii and not E. histolytica (Figure 2, panel C). In addition, results of ELISA testing for E. histolytica–specific IgG in serum were negative. Taken together, those findings indicate that neither the hepatic abscess nor the splenic abscess was associated with E. histolytica infection.

The patient was initially started on intravenous (IV) meropenem empirically. However, after confirmation of E. moshkovskii infection, we changed the treatment to IV metronidazole (750 mg IV/8 h for 1 d), followed by oral metronidazole (400 mg 3×/d after food for 7 d). The patient was also prescribed ferrous sulfate 200 mg (1 dose by mouth), B-complex tablets, and other medications as needed. Pigtail drainage continued for the first week. The patient recovered after 3 weeks.

E. moshkovskii DNA alongside E. histolytica in 5 of 115 liver abscess cases was previously reported; however, extraintestinal E. moshkovskii infection, particularly in pleural fluid, is extremely rare (13). In the case we report, E. moshkovskii might have reached the pleural cavity through direct extension or rupture of a splenic abscess.

Metronidazole is the primary drug for treating amoebiasis and was effective in this patient, showing empirical activity against E. moshkovskii (14). However, the optimal dosage for this species remains undefined. Drug sensitivity differs among Entamoeba spp.; for instance, E. moshkovskii is resistant to emetine, which is effective against E. histolytica (15). Diagnosis is also challenging. Molecular tests often target only E. histolytica, leaving E. moshkovskii infections unrecognized. In addition, E. moshkovskii produces fewer cysts than E. histolytica, reducing the reliability of microscopic detection and increasing the risk for false negatives (5).

Because of the high regional prevalence of E. moshkovskii, extraintestinal infections caused by this species need attention. Our case underscores the need to better understand transmission and to develop improved diagnostic methods to ensure effective management and prevent drug resistance in E. moshkovskii.