Free-living amoebic encephalitis – Case series

Abstract

Introduction:

Free-living amoeba is ubiquitous in fresh water, mud, and moist soil; although seldom pathogenic to humans, Naegleria fowleri, Acanthamoeba spp., and Balamuthia spp. are known to cause infections of the central nervous system.

Methods:

We report two interesting cases, both of which presented with a rapid-onset and fulminant course. The first case details a 36-year-old male with a history of surgically corrected nasal bone fracture 15 years back, who presented with acute onset of fever, headache and convulsions.

Results:

Direct smears of cerebrospinal fluid (CSF) revealed motile trophozoites resembling N. fowleri and were later confirmed by molecular diagnosis (polymerase chain reaction [PCR]). Subsequently, the source was identified as water used for religious purposes. Unfortunately, the patient succumbed to cardiac arrest. The second case is that of a 4-year-old boy from Palakkad district, Kerala, who presented with sudden onset of nasal discharge, fever, headache, vomiting, seizures and altered sensorium. His CSF smear examination showed motile trophozoites resembling Acanthamoeba spp. While undergoing treatment, he seized thrice before going into cardiac arrest. Despite extensive resuscitation measures, the child could not be revived and passed away. The postmortem CSF sample sent for molecular analysis confirmed infection by Acanthamoeba spp.

Conclusion:

Rapid progression and lack of definite treatment options make this a highly fatal condition. Although clinical presentations of both patients were indicative of pyogenic meningitis, parasitic infection was suspected when the CSF was found turbid with no bacteria, high protein, and low sugar. High index of suspicion helped us to get an early preliminary diagnosis from direct microscopy.

INTRODUCTION

Parasitic infection of the central nervous system (CNS) by free-living amoebae is rarely encountered. These free-living amoebae are ubiquitous and worldwide in distribution. Pathogenic species include Naegleria fowleri and Acanthamoeba spp., which present as primary amoebic meningoencephalitis (PAM) and granulomatous amoebic encephalitis (GAE), respectively.[1] These are well known to inhabit fresh water, tap water, coastal water, sewage, poorly chlorinated swimming pools, artificial lakes, heating and ventilation units.[2]

N. fowleri infection follows the entry of water-containing amoeba into the nose. It is responsible for a rapidly fatal infection involving the CNS, which occurs most often in healthy young individuals with a recent history of swimming in freshwater. It usually results in death within 72 h of the onset of symptoms. N. fowleri induces an intense inflammatory response that results in hemorrhage and lytic necrosis.[3] Most of the cases are misdiagnosed as pyogenic meningitis, with the majority of PAM diagnoses made postmortem.

GAE usually occurs in immunocompromised patients with diabetes mellitus, acquired immune deficiency syndrome, alcoholism and immunosuppressive medication, bone marrow failure, lymphoproliferative and hematoproliferative disorders, renal failure, splenectomy and dysproteinemia.[4,5] GAE can also be seen in otherwise healthy individuals as well.

Rapid progression of the disease with limited awareness among medical professionals makes this disease a diagnostic challenge. Early diagnosis and management may improve the prognosis of the disease.

CASE REPORTS

Case 1

A 36-year-old male who was previously healthy was brought to our hospital casualty with generalized tonic–clonic seizure and altered sensorium. He had a 2-day history of throbbing headache predominantly over frontal areas, associated with nausea, photophobia and high-grade fever not relieved by antipyretics. Probative clinical history included a 15-year-old nasal bone fracture which was corrected surgically.

At initial examination, the patient was drowsy, febrile (39.5°C), with a sluggishly reactive left pupil, and terminal neck stiffness. Examination of all other systems was within normal limits. There was a progressive deterioration of consciousness, following another generalized tonic–clonic seizure, due to which the patient was shifted to the intensive care unit and started on Ceftriaxone, Acyclovir and Steroids (Vancomycin avoided due to a history of patient’s allergy). The laboratory workup found is mentioned below [Table 1].

Table 1.

Laboratory investigations

Test	Result
Total leukocyte count	13,300/mm3
Neutrophils	93%
Lymphocytes	4%
Eosinophils	1%
Hemoglobin	13.7 g/dL
Sodium	133 mmol/L
Potassium	4.4 mmol/L
Liver function test	Within normal limit
Renal function test	Within normal limit

Lumbar puncture was performed which showed a highly turbid cerebrospinal fluid (CSF) sample. Cytological and biochemical analyses were done which showed the following [Table 2].

Table 2.

Cytological and biochemical investigtation reports

Test	Result
Total leukocyte count	300/mm3
Neutrophils	6%
Lymphocytes	39%
Protein	1100 g/dL
Sugar	5 mg/dL

Gram staining showed numerous pus cells, with no obvious organisms. Fortunately, wet film examination showed motile trophozoites resembling Naegleria spp. Figure 1.

Figure 1.

Motile trophozoites resembling Naegleria spp

CSF sediment culture on nonnutrient agar with a lawn culture of Escherichia coli ATCC 25922 yielded motile trophozoite forms of Naegleria spp. after 24 h of incubation (Trail sign positive) Figure 2.

Figure 2.

Positive Trail sign

The critically ill patient desaturated and went into ventricular tachycardia, further developed a myocardial infarction from which he could not be revived.

Immediate to releasing the preliminary laboratory report suggesting Naegleria infection, a detailed investigation into the source of infection indicated a suspicious exposure incident with well water through the nasal route for religious purposes. Subsequently, six water samples from the patient’s locality (received through FHC Desamangalam), along with his CSF sample was sent to PGIMER, Chandigarh, for confirmation of diagnosis Further, studies of water showed that chlorine levels of the water supply were inadequate.

Case 2

A 4-year-old boy from Palakkad, who had no known comorbidities, presented with altered sensorium and episodes of seizures. He had fever for 4 days associated with nasal discharge and headache, which was followed by vomiting for 3 days. The child destabilized later becoming increasingly drowsy and irritable with excessive crying. Physical examination revealed signs of meningeal irritation. Although empirical therapy with Intravenous Ceftriaxone and Vancomycin along with Dexamethasone was started, within 2 days of admission, seizures recurred and escalated to right hemiparesis.

Laboratory reports gave a picture suggestive of pyogenic infection [Table 3].

Table 3.

Biochemical investigations

Test	Result
Total leukocyte count	33,500/mm3
Neutrophils	96%
Lymphocytes	3%
Eosinophils	1%
Hemoglobin	10.5 g/dL
Sodium	131 mmol/L
Potassium	4.4 mmol/L
Liver function test	Within normal limit
Renal function test	Within normal limit

The CSF sample received in our microbiology laboratory was visibly turbid and was processed as per protocol. Gram stain showed plenty of pus cells without any bacteria. However, the unstained wet film revealed the motile trophozoites resembling Acanthamoeba spp. Figure 3 Although the boy exhibited mild improvement on Intravenous Amphotericin B, Fluconazole, Cotrimoxazole and Rifampicin subsequent to the initial report, signs of meningeal irritation worsened within the next week. He had an episode of generalized tonic–clonic seizure, following which he passed away due to cardiac arrest. The CSF sediment culture on nonnutrient agar with a lawn culture of E. coli yielded the motile trophozoite forms of Acanthamoeba spp. after 24 h of incubation (positive Trail sign). The laboratory diagnosis of Acanthamoeba spp. was confirmed from CSF samples collected postmortem, by PGIMER, Chandigarh.

Figure 3.

Motile trophozoites resembling Acanthamoeba spp

The amoebae were identified by the method standardized at PGIMER, Chandigarh.[6] Briefly, samples were subjected to pan-FLA PCR that targets the 18 S rRNA gene sequence. The samples found to be positive for Acanthamoeba spp. and N. fowleri by pan-FLA PCR were then confirmed by parasite-specific PCR targeting of Acanthamoeba spp. and N. fowleri. For Acanthamoeba spp., the JDP primers targeting the 18 S rRNA gene were used. N. fowleri was identified using the genus- and species-specific primers targeting the ITS1 and ITS2 regions. The amplified DNA products were separated on a 1.5% agarose gel and viewed in a gel documentation system (ProteinSimple AlphaImager HP System Automated Gel Imaging, Santa Clara, CA, USA).

DISCUSSION

Amoebic meningitis is a rare, but fatal CNS infection. PAM can mimic acute pyogenic infections and GAE is a chronic progressive disease. Here, we report two cases in previously healthy individuals. Both of them surrendered to the disease within a week, despite combination treatment with anti-fungal agents and Rifampicin. The majority of the reported cases gave a history of swimming in contaminated waters, but nasal irrigation with tap water for therapeutic and religious purposes has also been reported in the literature.[7,8]

Less than four cases of PAM are reported per year worldwide, 79% of which were males. Most of them were from Asia and Africa during the warmer seasons, as well as in North America during spring and summer.[9,10,11] It is uncertain whether nasal surgery done 15 years prior could have had an impact on disease progression in the first case. Although diagnostic confounders such as neutrophilic pleocytosis cytology with low sugar levels indicated a pyogenic cause, high index of suspicion helped us to get a preliminary diagnosis from direct microscopy. We were also able to culture the parasite on non nutrient agar with a lawn of E. coli and confirm the diagnosis.[12,13]

Reports of survivors (11 cases) consumed a myriad of antimicrobial combinations of Intravenous and Intrathecal Amphotericin B, Ketoconazole, Miltefosine, Rifampicin and Dexamethasone for a minimum of 3–4 weeks as recommended by the Centers for Disease Control and Prevention (CDC).[13,14,15]

GAE commonly presents as a subacute or chronic infection and seldom in immunocompetent patients. Khurana et al. reported a CNS infection in a 3-year-old malnourished child who recovered without any residual symptoms, contrary to our second case.[16] Of about 30 cases of pediatric GAE reported over the past 40 years, 46% were from India, of whom 87% were previously healthy.[17] History of fresh water swimming may be elicited rarely (20% cases). Definitive diagnosis requires the presence of motile trophozoites in CSF, which may be misidentified as macrophages. Serological tests have a minute advantage in GAE diagnosis over PAE due to the indolent progression of the disease, though molecular confirmation is the currently accepted gold standard for both infections.[6,18] Out of the 20 genotypes of Acanthamoeba found in India, T4 is the most common genotype.[19]

Treatment guidelines for both PAM and GAE are poorly elucidated, and therapy is decided based on CDC recommendations, which include Pentamidine, Sulfadiazine, Flucytosine and Fluconazole/Itraconazole, as early as possible. Less than 15% overcome the disease, with the survival rate highest among those who took triple-drug regimen for more than 3 months. However, a quarter of survivors reported long-term neurological sequelae.[20,21]

Environmental surveillance led to the confirmation of local tap water as a source in the first case which directed a rapid response from the public health center to enhance chlorination of all surrounding water supply. The incidence of amoebic meningitis in immunocompetent hosts indicates a critical need for improving water quality and safety to eliminate these water-borne parasites. Periodical and appropriate surveillance for water contamination and implementation of preventive hygienic measures, following the identification of a case and its infective source, is vital to alleviate the incidence of this lethal infection.

Contamination of water with organic material and warm temperatures are ideal for the proliferation of N. fowleri.[15] Acanthamoebae spp. is distributed in soil, air, water, chlorinated water in pools, sewage, dialysis units, air conditioning units and contaminated contact lens solutions. Free-living amoebae have been isolated from tap water samples, ventilators, air conditioners, hemodialysis units and dental irrigation systems of hospitals.[22] Chlorinated water and salt water significantly decrease the risk of N. fowleri infection. Chlorine at 2 ppm is effective against N. fowleri but not for Acanthamoeba spp. It is necessary that wells be properly maintained with adequate chlorine for N. fowleri.[23] Water that has been distilled or purified should be used for sinus rinsing, according to the CDC.[24]

CONCLUSIONS

PAM and GAE are rare fatal diseases involving the CNS, usually found in tropical and subtropical regions. Both are serious public health problems. Prompt identification with timely intervention with an appropriate antimicrobial therapy in the correct dosage is the key.

Public health measures have to be taken for the prevention and control of free-living amoeba including N. fowleri and Acanthamoeba spp. Better awareness of the disease within the medical community and public health awareness through health education are desirable. Clinical microbiologists should be more aware of the importance of careful examination of CSF wet preparation for diagnosis of N. fowleri and Acanthamoeba spp. as early treatment may markedly improve the prognosis of the disease.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.