Case Report: A Series of Three Meningoencephalitis Cases Caused by Acanthamoeba spp. from Eastern India

ABSTRACT.

Acanthamoeba spp. are rare etiological agents of meningoencephalitis with high mortality. We present three cases of Acanthamoeba meningoencephalitis in immunocompetent individuals from Eastern India. The first patient presented with fever and headache; the second with headache, visual disturbance, and squint; and the third presented in a drowsy state. The cases presented on March 3, 18, and 21, 2023 respectively. The first two patients had concomitant tubercular meningitis for which they received antitubercular therapy and steroid. Their cerebrospinal fluid showed slight lymphocytic pleocytosis and increased protein. The diagnosis was done by microscopy, culture, and polymerase chain reaction. They received a combination therapy comprising rifampicin, fluconazole, and trimethoprim-sulfamethoxazole. The first patient additionally received miltefosine. She responded well to therapy and survived, but the other two patients died despite intensive care. Detection of three cases within a period of 1 month from Eastern India is unusual. It is imperative to sensitize healthcare providers about Acanthamoeba meningoencephalitis to facilitate timely diagnosis and treatment of the disease.

INTRODUCTION

Acanthamoeba spp. are aerobic, unicellular, eukaryotic protists that can exist both as a parasite and a free-living organism.¹ Their life cycle includes trophozoite and cyst stages. Central nervous system (CNS) invasion may occur by hematogenous spread after inoculation of respiratory tract or broken skin.²

Acanthamoeba meningoencephalitis may present with headache, neck stiffness, altered mental status, and cognitive decline, nausea, vomiting, low-grade fever, lethargy, gait abnormalities, and disturbances in coordination, visual disturbance, focal motor deficits, seizures, and coma. Definitive diagnosis is made by examination of brain tissue or cerebrospinal fluid (CSF).¹

Acanthamoeba meningoencephalitis may be diagnosed by CSF polymerase chain reaction (PCR). After extraction, DNA is subjected to conventional PCR amplification for detection of Acanthamoeba diagnostic fragments 3 (DF3) of 18SrDNA using genus-specific primer: forward (JDP1) 5′-GGCCCAGATCGTTTACCGTGAA-3′ and reverse (JDP2) 5′-TCTCACAAGCTGCTAGGGAGTCA-3′ amplifying a region of 450–500 bp.³ The amplified DNA products are separated by 1.5% agarose electrophoresis and visualized in a gel documentation system (Figure 1).

Figure 1.

Agarose gel image for Acanthamoeba-specific polymerase chain reaction. Lane 1, negative control; lane 2–5, patient samples amplifying 450- to 500-bp band of Acanthamoeba spp.; lane 6, 100-bp molecular marker; lane 7, positive control.

Currently there is no consensus about the most effective combination therapy against Acanthamoeba meningoencephalitis. Rifampicin, fluconazole, trimethoprim-sulfamethoxazole, miltefosine, flucytosine, pentamidine, and sulfadiazine have been used to treat cases.^1,4

CASE 1

A 23-year-old housewife from Topsia, Kolkata, India, presented with headache, blurred vision, and squint for 55 days on March 3, 2023. She was diagnosed with tuberculous meningitis in a private hospital and treated with four-drug antitubercular therapy (ATT; rifampicin, isoniazid, pyrazinamide, and ethambutol) and prednisolone.

During admission to our institute, she was alert, conscious, cooperative, hemodynamically stable, and afebrile, with SpO₂ 98% at room air. There was no lymphadenopathy. Neck rigidity was present, Kerning’s sign was positive, and she had bilateral sixth cranial nerve palsy (Figure 2). Glasgow Coma Scale (GCS) was 15/15, tone and power of all muscles were normal, and plantar reflex was bilaterally flexor. Fundoscopic findings were normal. Examination of other systems showed no abnormality.

Figure 2.

Bilateral sixth cranial nerve palsy.

Routine blood investigations (complete hemogram, urea, creatinine, liver function tests) were normal. Routine microbiological workup (HIV I and II, hepatitis B surface antigen, anti–hepatitis C virus antibody, serum dengue IgM, serum Leptospira IgM, and serum scrub typhus IgM) was negative. Contrast-enhanced computed tomogram (CECT) and magnetic resonance imaging (MRI) of the brain showed no detectable abnormality in brain morphology but suggested bilateral optic nerve edema/neuritis. Cerebrospinal fluid study showed normal opening pressure, cell count 200/mm³ with lymphocyte 94%, neutrophils 6%, sugar 30 mg/dL, protein 96 mg/dL, Gram stain, bacterial culture, acid-fast bacilli (AFB) stain, cryptococcal antigen (CRAG), fungal stain, fungal culture, acute encephalitis syndrome (AES) panel (herpes simplex virus DNA PCR, scrub typhus PCR, Japanese encephalitis PCR, chikungunya PCR) were negative. GeneXpert^® (Cepheid, Sunnyvale, CA) Mycobacterium tuberculosis that was rifampicin sensitive was detected in CSF. A CSF wet mount showed a few motile, free-living amoeba morphologically resembling Acanthamoeba spp. Growth of Acanthamoeba spp. was also observed in culture. For the nonnutrient agar (NNA) culture, the CSF samples were inoculated on the NNA plates with a lawn of Escherichia coli and incubated at 37°C for 7 days to monitor the growth of trophozoites and cysts. Subsequently, Acanthamoeba PCR from the CSF sample was positive.

We started her on fluconazole 400 mg/day, trimethoprim-sulfamethoxazole (160 mg/800 mg) twice daily, and miltefosine 50 mg twice daily. Rifampicin was continued in a dose of 600 mg/day as part of ATT. Steroid was started as dexamethasone 0.4 mg/kg intravenously and gradually tapered. After 10 days of treatment, she developed hepatotoxicity with transaminitis. Four-drug ATT and fluconazole were stopped, and she was put on modified liver-friendly ATT including amikacin, levofloxacin, and ethambutol. After 10 days, liver enzymes normalized. Rifampicin, fluconazole, and isoniazid were gradually reintroduced, and amikacin was withdrawn. The patient improved significantly with resolution of headache, recovery of sixth cranial nerve palsy, and absence of meningeal signs. On repeat CSF study Acanthamoeba PCR was negative.

CASE 2

A 15-year-old female student from Sundargarh, Orissa, India, presented with fever and headache for 25 days on March 18, 2023. She visited the Park Circus area of Kolkata for 7 days 1 month before this illness.

During admission, she was alert, conscious, cooperative, hemodynamically stable, and afebrile, and her SpO₂ was 98% in room air. She had no lymphadenopathy. Neck rigidity and Kerning’s sign were present. Her GCS was 15/15. There was no cranial nerve palsy, normal tone and power of all muscles, and plantar reflex was bilateral flexor. Fundoscopy revealed no abnormality. Examination of all other systems was normal.

Routine blood investigations were normal, and routine microbiological workup was negative. Brain CECT and MRI revealed effaced cortical sulci with slightly prominent lateral ventricles (Figure 3). Her CSF study showed normal opening pressure, cell count 400 cells/mm³ with lymphocyte 98%, neutrophils 2%, sugar 26 mg/dL, protein 111 mg/dL, Gram stain and culture, AFB stain, fungal stain, fungal culture, GeneXpert for M. tuberculosis, CRAG, and AES panel were negative. A CSF wet mount showed a fair number of motile free-living amoeba with acanthopodia morphologically resembling Acanthamoeba spp. (Figure 4). Subsequently Acanthamoeba PCR was positive from the CSF sample.

Figure 3.

Magnetic resonance imaging scan of brain showing effaced cortical sulci with slightly prominent lateral ventricles.

Figure 4.

Cerebrospinal fluid wet mount showing Acanthamoeba trophozoites.

We started her on rifampicin 600 mg once a day, fluconazole 400 mg/day, and trimethoprim-sulfamethoxazole (160 mg/800 mg) twice daily. After 1 month, repeat CSF wet mount and PCR for Acanthamoeba was negative. Repeat GeneXpert^® for M. tuberculosis from CSF sample was positive. Treatment of tubercular meningitis with four-drug ATT and dexamethasone was initiated. The patient improved with resolution of headache and meningeal signs.

However, she developed severe headache, vomiting, and altered sensorium within a few days after discharge. On readmission, her GCS was 13/15 (E₄V₄M₅) with neck rigidity and one episode of seizure. Repeat MRI brain showed hydrocephalus and infarction in the right posterior cerebral artery territory. She had repeated seizures and was transferred to the intensive care unit. Despite intensive care support, her condition deteriorated, requiring mechanical ventilation. She died 3 months after her diagnosis of Acanthamoeba meningoencephalitis.

CASE 3

A 40-year-old hypothyroid housewife, resident of Belur, Bally, West Bengal, India, presented with fever for 37 days and behavioral abnormality for 6 days on March 21, 2023. Thus all three cases reported here presented within the month of March 2023. She was on multidrug therapy for leprosy (rifampicin monthly, clofazimine daily, dapsone daily) with 7 days of the course duration left.

On admission, she was drowsy, irritable, GCS 10/15 (E₂V₃M₅), hemodynamically stable, and afebrile, with SpO₂ 98% at room air. She had multiple episodes of seizures immediately after admission. She had no lymphadenopathy. Neck rigidity and Kernig’s sign were present but no cranial nerve palsy. Tone of all muscles was normal with bilaterally extensor plantar reflex. Fundoscopy revealed no abnormality. Abdominal examination showed nontender hepatomegaly 2 cm below costal margin. Examination of other systems including respiratory and cardiovascular systems showed no abnormalities.

Her routine blood investigations were normal. Routine microbiological workup was negative. Brain imaging including CT and MRI suggested bilateral meningeal enhancement with bilateral periventricular hyperintensities (Figure 5). Her CSF study showed normal opening pressure, cell count 250 cells/mm³, lymphocyte 100%, sugar 45 mg/dL, and protein 144 mg/dL, Gram stain and culture, AFB stain, fungal stain, fungal culture, GeneXpert for M. tuberculosis, CRAG, and AES panel were negative. The CSF wet mount showed a few motile free-living amoebae morphologically resembling Acanthamoeba spp. The diagnosis was confirmed by CSF PCR for Acanthamoeba spp.

Figure 5.

Magnetic resonance imaging scan of brain showing bilateral meningeal enhancement with bilateral periventricular hyperintensities.

She was initially started on empiric ATT and amphotericin B deoxycholate. After diagnosis of Acanthamoeba spp., we started her on fluconazole 400 mg/day, trimethoprim-sulfamethoxazole (160 mg/800 mg) twice daily, and rifampicin 600 mg once daily. However, she developed severe metabolic acidosis requiring mechanical ventilation. She succumbed to death 2 weeks after her diagnosis of Acanthamoeba meningoencephalitis.

DISCUSSION

Among these three cases detected in March 2023, one had favorable outcome. All received rifampicin, trimethoprim-sulphamethoxazole, and fluconazole. The surviving patient additionally received miltefosine. The second case worsened after initial improvement, and the third case deteriorated quickly. All cases were diagnosed by microscopy and confirmed by CSF culture and PCR for Acanthamoeba spp.

A review of Acanthamoeba meningoencephalitis from the United States included 122 cases from 1956 to 2020. The most commonly used regimen for the treatment of Acanthamoeba spp. included miltefosine, fluconazole, flucytosine, pentamidine, and sulfadiazine.⁴ However, we have used an alternative regimen including rifampicin, trimethoprim-sulphamethoxazole, and fluconazole as suggested by the Infectious Disease Society of America guidelines.⁵

A review of 10 granulomatous amoebic encephalitis cases from China caused by Acanthamoeba spp. showed that fever, headache, and hemiplegia were the most common symptoms (each found in 40% cases).⁶ However, none of the patients in our study presented with hemiplegia.

A systematic review from Brazil identified 19 cases of human infection by Acanthamoeba spp. from 1977 to 2008 including a fatal case of meningoencephalitis.⁷ In our study, two of the three cases died.

Miltefosine is effective against Acanthamoeba spp. As a phospholipid analogue, miltefosine may act against Acanthamoeba spp. by disrupting the cell membrane. Miltefosine may provide mortality benefit in Acanthamoeba meningoencephalitis.¹

Acanthamoeba spp. may act as a Trojan horse for other pathogens, including M. tuberculosis.⁸ This may explain the coinfection with tuberculosis in our cases.

We have observed three cases of Acanthamoeba meningoencephalitis in eastern India within 1 month. They were immunocompetent without any apparent common exposure. Clinicians should remain vigilant for diagnosis and treatment of Acanthamoeba meningoencephalitis cases.

Tuberculosis may sometimes be present as coinfection. A regimen including rifampicin, fluconazole, trimethoprim-sulfamethoxazole, and miltefosine may be effective. Awareness, vigilance, and proper diagnostic facilities are essential to identify and treat the disease.