A 47-year-old male was hospitalized on February 4, 2022, presenting with recurrent diarrhea for more than 7 months and with a fever for 5 days (highest temperature of 38.3°C). The patient reported about five yellow watery stools per day, accompanied by nausea, abdominal distension, and poor appetite. The patient had a weight loss of more than 10 kilograms in 6 months. The source of this patient’s microsporidium infection was unclear. For the past year, his travel had been limited to Guangdong Province in China. He was diagnosed with B-ALL in July 2019. He had previously received more than 10 antileukemia combination chemotherapies following the modified Berlin-Frankfurt-Munster 90 (BFM-90) protocol (Rajendra et al., 2021) for acute lymphoblastic leukemia, beginning in March 2020 with long-term oral methotrexate (MTX, 20 mg/m², once a week) and 6-mercaptopurine (6-MP, 50 mg/m², once a day) as maintenance chemotherapy. Approximately 1 month and 6 months after the initial symptoms of diarrhea, the patient underwent colonoscopy and gastroscopy, respectively, which showed no obvious abnormal lesions. During the period of diarrhea, repeated culture and routine detection of stool, several bone marrows punctures and a positron emission tomography/computed tomography (PET-CT) examination were performed, and no evidence of tumor recurrence was found.

At admission, the patient was dehydrated and had abdominal distention and pain; taking MTX and 6-MP at the time. Laboratory analyses showed lymphocytopenia (0.103×10⁹/L), with macrocytic and moderate anemia (hemoglobin concentration was 86 g/L, the mean erythrocyte volume was 113 fl), C-reactive protein level was 148 mg/L, procalcitonin was 0.272 ng/ml. His CD4 and CD19 counts were 31 cells/µl (24.3%) and 5 cells/µl (4%) in the peripheral blood, respectively. Stool routine, urine routine, stool culture, urine culture, blood culture, Widal test, waffian reaction, anti-nuclear antibody, anticardiolipin antibody, antineutrophil cytoplasmic antibody, G and GM test, T-spot TB test, EB virus, and cytomegalovirus DNA were all negative. The smear of the stool followed by Wright’s stain had also been performed, and a small number of spores were observed under a light microscope, but it could not confirm the species of spores. The liver and kidney functions were normal and the albumin level was 19.1 g/L. Bone marrow smear and flow cytometry examination showed no abnormalities.

Physical examination revealed that the patient had an unwell and anemic appearance, unpalpable superficial lymph nodes, abdominal distention, abdominal muscle tension, tenderness, shifting dullness, and slight edema of the lower limbs. The cardiopulmonary examination was unremarkable.

Based on these tests above, the diagnosis of local or systemic bacterial, viral, fungal and tuberculosis infection was not supported, nor was the evidence of tumor recurrence.

The patient immediately stopped taking oral chemotherapy for leukemia, was first administered piperacillin/tazobactam, amikacin and caspofungin, and was treated symptomatically with intravenous fluids, antidiarrheal, probiotics, folic acid and vitamin B12. Fever still occurred 5 days after treatment, the highest temperature was 38.4°C, and his diarrhea remained uncontrolled (approximately 5 liquid bowel movements per day). Abdominal distension, abdominal pain, and other related symptoms did not improve. Routine blood and biochemical examinations showed no apparent changes.

Due to a lack of response from the previous antibiotic treatment, the patient underwent another full-body PET-CT, which revealed elevated glucose metabolism throughout the peritoneum (standardized uptake value (SUV) max=12.57) and part of the penis (SUVmax=13.41) and massive peritoneal effusion with peritonitis, suggesting tumor or microbial infiltration ( Figure 1A ). Then,

We performed peritoneal puncture drainage on the patient, peritoneal fluid was obtained from the patient for mNGS testing. E. bieneusi genomic sequences were detected, suggesting a likelihood of infectious disease. The sequencing results were obtained within 24 hours, which revealed E. bieneusi DNA sequence reads was 85, the RNA sequence reads was 3495,the DNA sequences covered 0.20% of the genome ( Figure 2A ), the RNA sequences covered 2.21% of the genome ( Figure 2B ). Meanwhile, target gene sequencing and mutational analysis showed no tumor-specific or germline mutations.

The patients were evaluated using 475 Lymphoma-related NGS panel. DNA extraction, sequencing library preparation, and targeted capture enrichment were carried out following the methods as previously described with modifications (Tong et al., 2019). Target enriched libraries were sequenced on the HiSeq4000 platform (Illumina). Single-nucleotide variants, indels, structural variants, and copy number changes were identified by validated bioinformatics process from paraffin-embedded tissues (DePristo et al., 2011; Amarasinghe et al., 2013; Newman et al., 2014; Shen and Seshan, 2016). Mutant allele frequency (MAF) cutoff for single-nucleotide variants and indels was defined as 1%. The log2 ratio cut-off for copy number gain was defined as 2.0 for tissue samples. A log2 ratio cut-off of 0.6 was used for copy number loss detection.

The DNA of ascitic fluid extracted using the TIANamp Magnetic DNA Kit (Tiangen) according to the manufacturer’s protocols. DNA libraries were prepared using the KAPA Hyper Prep kit (KAPA Biosystems) according to the manufacturer’s protocols and were 75bp single-end sequenced on Illumina NextSeq 550Dx (Illumina). We use in-house developed bioinformatics pipeline for pathogen identification. Our microorganism genome database contained bacteria, fungi, virus and parasite genomic sequences (download from https://www.ncbi.nlm.nih.gov/).

We used the following criteria for positive results of mNGS:

For Mycobacterium, Nocardia and Legionella pneumophila, the result was considered positive if a species detected by mNGS had a species-specific read number≥1. For bacteria (excluding Mycobacterium, Nocardia and Legionella pneumophila), fungi, virus and parasites, the result was considered positive if a species detected by mNGS had at least 3 non-overlapping reads. Pathogens detected in the negative ‘no-template’ control (NTC) were excluded but only if the detected reads was ≥10-fold than that in the NTC.

We attempted to treat the patient with albendazole at 400 mg twice daily for 7 days (14 February to 20 February 2022). The patient became weaker and continued to have severe diarrhea with worsening abdominal distension and pain. Bedside abdominal ultrasound indicated that there was still a large amount of ascites, and more separation bands were visible, which resulted in no peritoneal fluid draining from the abdominal drainage tube. Albendazole tablets probably failed to produce a clinical reaction.

After a review of extensive literature, alternatively, the patient took nitazoxanide (500 mg twice a day for 14 consecutive days) with reported successful treatment in case reports with AIDS and solid tumors after organ transplantation. About three days after the above treatment, his stool began to form and the frequency decreased; he presented no nitazoxanide-related side effects and was discharged home. The patient completed fourteen days of nitazoxanide. A full-body PET-CT was performed more than one month later, which showed a decline in glucose metabolism throughout the peritoneal and penile regions; meanwhile, the ascites had completely resolved ( Figure 1B ). His CD4 count increased to 297 cells/µl (26.3%) in peripheral blood, and his hemoglobin level was normal. He was successfully treated with nitazoxanide, and the clinical symptoms disappeared.

At the recent follow-up, the patient had no anemia, no fever, no diarrhea, no abdominal pain, no abdominal distension, a normal diet, and sleep. A whole-body contrast-enhanced CT was also obtained on July 11, 2022(approximately 5 months after treatment for Microsporidia), which also showed no abnormalities. Considering that the maintenance treatment of the patient’s leukemia was almost over, the patient was instructed to review the signs and examinations related to leukemia regularly.