In June 2008, a 30-year-old, previously healthy man was referred to the Aga Khan University Hospital with a 2-day history of high-grade fever, severe headache, and seizures. He was comatose with a fixed and dilated left eye pupil. Magnetic resonance imaging showed basal meningeal enhancement. A lumbar puncture found an opening pressure of 44 cm H2O. Cerebrospinal fluid (CSF) analysis showed low glucose (<5 mg/dL), high protein level (1,028 mg/dL), and lymphocytic pleocytosis (900 cells/mm3 with 85% lymphocytes). Gram stain and latex agglutination (LA) test results were negative for bacteria. Wet film of CSF showed motile amebic trophozoites, but CSF volume was insufficient for amebic culture. A preliminary diagnosis of PAM was made, and therapy was begun with intravenous amphotericin B plus oral rifampin and fluconazole. The patient died 4 days after admission.
In September 2008, a previously healthy 25-year-old man was admitted with a 24-hour history of fever, vomiting, and neck rigidity. CSF analysis indicated hypoglycorrhachia, elevated protein level, and neutrophilic pleocytosis. Gram stain and LA test results were negative for bacteria, and wet preparation of CSF showed motile amebic trophozoites. Ptosis of the left eye and a fixed dilated pupil developed on the day of admission, and he was intubated for airway protection. Despite therapy with intravenous amphotericin B, oral fluconazole, and rifampin, his condition deteriorated, and he died 14 days after admission. CSF was cultured on nonnutrient agar on a lawn of Escherichia coli
American Type Culture Collection (Manassas, VA, USA) 29522 in Page ameba saline (1
). Cultured amebae produced flagellates in distilled water at 37°C within 15–30 min.
On the basis of this apparent upsurge of cases, a laboratory policy was instituted at the Aga Khan University Hospital to perform wet mounts of all processed CSF samples that were consistent with bacterial meningitis but had negative Gram stain and LA test results. Although no new cases of PAM were detected in 2008, 11 were identified from April through November 2009. Case-patients were referred from tertiary-care hospitals in Karachi ().
Clinical features and CSF analysis results for 13 patients with Naegleria fowleri meningoencephalitis, Karachi, Pakistan, 2008–2009*
Twelve of 13 patients were male; ages ranged from 16 to 64 years (mean ± SD 31.0 ± 15.33 years). All were residents of Karachi but lived in different districts (). Only 1 patient acknowledged a history of swimming. All patients’ conditions were treated with amphotericin B (1.5 mg/kg/d) and rifampin (600 mg/d). Most patients also received either fluconazole or itraconazole. All required intubation and ventilation within 24 hours of hospital admission, and treatment for PAM was started within 48–72 hours of admission. Nonetheless, only 2 patients survived for >5 days after admission; the mean ± SD time from symptom onset to death was 6.38 ± 3.15 days (range 3–15 days).
CSF samples from all patients (except the first) were positive for amebic culture. Furthermore, CSF samples collected from 3 patients in July 2009 were confirmed by real-time PCR for N. fowleri
DNA at the Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA. Briefly, primers NaeglF192 (3′-GTG CTG AAA CCT AGC TAT TGT AAC TCA GT-5′) and NaeglR344 (5′-CAC TAG AAA AAG CAA ACC TGA AAG G-3′) were used to amplify a 153-bp fragment, detected by the hexachlorofluorescein (HEX)–labeled probe NfowlP (5′-HEX-AT AGC AAT ATA TTC AGG GGA GCT GGG C-BHQ1-3′). PCR was performed in a Mx3000P real-time thermocycler (Stratagene, La Jolla, CA, USA), with 2 initial incubations at 50°C for 2 min (incubation for uracil-DNA-glycosylase activity) and 95°C for 2 min (activation of Platinum Taq
DNA-polymerase), respectively, followed by 40 cycles of 95°C for 15 s and 63°C for 60 s. Fluorescence was measured after each 63°C incubation. Results were analyzed by using Mx3000P version 2.0 software (2
Domestic tap water was obtained for amebic culture from 2 patients’ homes (second and seventh patients in the series). Water samples (100 mL) were passed through a Millipore filter (Millipore Corp., Billerica, MA, USA), which was then inoculated face down on a nonnutrient agar plate with a lawn of E. coli as described above. Amebae were isolated from both water samples. However, only cultured amebae from the seventh patient’s sample were analyzed by real-time PCR, and N. fowleri DNA was detected in the sample.