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. 2023 Nov 8;18(11):e0290394. doi: 10.1371/journal.pone.0290394

Clinical manifestations and outcome of patients with primary amoebic meningoencephalitis in Pakistan. A single-center experience

Shakeel Ur Rehman 1, Salman Farooq 2,*, Muhammad Bilal Tariq 3, Nosheen Nasir 1, Mohammad Wasay 1, Sobia Masood 2, Musa Karim 2
Editor: Kelli L Barr4
PMCID: PMC10631667  PMID: 37939056

Abstract

Primary amoebic meningoencephalitis (PAM) is a rapidly progressing central nervous system (CNS) infection caused by Naegleria fowleri, a free-living amoeba found in warm freshwater. The disease progression is very rapid, and the outcome is nearly always fatal. We aim to describe the disease course in patients admitted with PAM in a tertiary care center in Karachi, Pakistan between the periods of 2010 to 2021. A total of 39 patients were included in the study, 33 males (84.6%). The median age of the patients was 34 years. The most frequent presenting complaint was fever, which was found in 37 patients (94.9%) followed by headache in 28 patients (71.8%), nausea and vomiting in 27 patients (69.2%), and seizures in 10 patients (25.6%). Overall, 39 patients underwent lumbar puncture, 27 patients (69.2%) had a positive motile trophozoites on CSF wet preparation microscopy, 18 patients (46.2%) had a positive culture, and 10 patients had a positive PCR. CSF analysis resembled bacterial meningitis with elevated white blood cell counts with predominantly neutrophils (median, 3000 [range, 1350–7500] cells/μL), low glucose levels median, 14 [range, 1–92] mg/dL), and elevated protein levels (median, 344 [range, 289–405] mg/dL). Imaging results were abnormal in approximately three-fourths of the patients which included cerebral edema (66.7%), hydrocephalus (25.6%), and cerebral infarctions (12.8%). Only one patient survived. PAM is a fatal illness with limited treatment success. Early diagnosis and prompt initiation of treatment can improve the survival of the patients and reduce mortality.

Introduction

Naegleria fowleri is the only pathogenic protozoa in the Genus Naegleria. This amoeba-flagellate was discovered in 1899, but it was not until 1970s that the link between Naegleria Fowleri and primary amoebic meningo-encephalitis (PAM) was identified [1, 2]. Naegleria fowleri has 3 stages in its life cycle: cyst, trophozoite and flagellate, with trophozoite being the only infective stage. The protozoa penetrate the nasal tissue and migrating to the brain via the olfactory nerves causing primary amoebic meningo-encephalitis [1, 3]. Rarely, it can also spread in patient undergoing solid organ transplantation due to the immunosuppressed state of these patients. PAM generally affects children and young adults. In a case series of 142 patients done in the United States between 1937 and 2018 [4], the median age of the patients was 12 years (83% aged ≤18 years); males (76%) were predominately affected.

The acquisition of this infection is linked to very specific risk factors such as engaging in recreational activities involving swimming in freshwater lakes, river, canals, swimming pools, and during ablution as a part of religious practices [1, 5, 6]. Drinking infected water does not lead to the development of PAM.

After an incubation period of 7 days, patients experience fulminant, necrotizing, and hemorrhagic meningo-encephalitis. Initial symptoms mimic that of bacterial meningitis, characterized by severe headaches, stiff neck, fever (38.5°C–41°C), altered mental status, seizures, and coma, leading almost always to death with a fatality rate of over 95% [7, 8].

This study aims at identifying the spectrum of symptoms with which patients present to the hospital, reviewing the clinical course, duration of symptoms and lab parameters, specifically looking at the factors that predict poor outcome and the factors that delay the disease process and ultimately mortality.

Materials and methods

An observational study was conducted on patients hospitalized with clinical features consistent with PAM at Aga Khan University Hospital (AKUH) between the periods of January 2010 to December 2021. In this case series, patients were included if the diagnostic workup was suggestive of PAM and who had received empiric treatment for PAM. Data was collected on clinical signs and symptoms, diagnostic workup including laboratory parameters and brain imaging findings, clinical course, and treatment outcome.

All patients who underwent lumbar puncture were tested for motile trophozoites on CSF wet preparation microscopy, CSF culture and PCR. Brain imaging findings were reported by expert neuroradiologist, and interval imaging comparisons were also made in patients where repeat brain scans were done. The study received approval from the Ethical Review Committee under # 2021-6074-18800, The Aga Khan University, Karachi. All the data will be saved on a computer with a password and access was allowed to only primary and corresponding authors.

Collected data were summarized as mean ± standard deviation (SD), median [IQR] or frequency (%). Patients were stratified based on gender and age and CSF investigations and imaging results were compared with the help of Chi-square test/Fisher’s exact test at 0.05 level of significance. The analysis was done using IBM SPSS version 21.

Results

Demographics

A total of 39 patients were included in the study, with 33 males (84.6%) and 6 females (15.4%) The median age of the patients was 34 years. The median duration of symptoms was three days while the median length of hospital stay was two days (Table 1). Most cases were reported during May to September with the highest number seen in July (Fig 1).

Table 1. Distribution of demographic characteristics and clinical presentation of patients admitted with primary amoebic meningo-encephalitis.

Characteristics Summary Statistics
Total (N) 39
Gender %
Male 33 (84.6)
Female 6 (15.4)
Median age (years) 34
18 to 35 years 22 (56.4)
>35 years 17 (43.6)
Median length of stay (days) 2 (2–3)
Median duration of symptoms (days) 3 (2–3)
Presenting symptoms and signs
Fever 37 (94.9)
Nausea/vomiting 27 (69.2)
Drowsiness 3 (7.7)
Headache 28 (71.8)
Seizure 10 (25.6)
Decreased consciousness 25 (64.1)
Irritability 18 (46.1)
Median Glasgow Coma Scale (GCS) on arrival 12 (10–13)
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Fig 1. Distribution of admitted cases of primary amoebic meningo-encephalitis by calendar months over 2010 to 2021.

Fig 1

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Clinical presentation

The most frequent presenting complaints included fever found in 37 patients (94.9%) followed by headache in 28 patients (71.8%), nausea and vomiting in 27 patients (69.2%), and seizures in 10 patients (25.6%) (Table 1).

Treatment

The treatment for PAM was initiated in 39 patients which included intravenous amphotericin B 1.5 mg/kg every 12 hours and 1.5 mg intrathecal, intravenous azithromycin 10 mg/kg once daily (OD), Intravenous fluconazole 800mg once daily (OD), rifampin per oral 600mg once daily (OD), chlorpromazine 500mg Q6H (up to 15 to 20 mg per kg), and per oral miltefosine 50mg every eight hourly per day in 27 patients. The remaining 10 patients did not receive miltefosine but received the rest of the treatment regimen. All patients received IV Dexamethasone 0.6mg per kg in divided doses along with the rest of the treatment. Out of these 39 patients only one patient survived.

The patient who survived was a 24 years old gentleman with had no comorbid, presented with fever and vomiting for two days, along with altered sensorium for one day and generalized tonic colonic seizure on arrival in emergency. There was no history of water recreation. On arrival he was hemodynamically stable, GCS was 9/15, neck was rigid, with brisk reflexes and bilateral extensor plantars.

Initial MRI on 6/9/14 showed no acute infarct, intracranial hematoma or abnormal parenchymal enhancement. There was Loss of CSF FLAIR signals with subtle meningeal enhancement, suggestive of leptomeningeal inflammation. The initial CSF DR done on first day showed low Glucose (29 mg/dl), high protein (213 mg/dl), high RBCs 642/cu mm, and raised WBC count (523/cu mm) with 80% Lymphocytes. CSF PCR and culture was positive for Naegleria Fowleri. Patient was started on 1). Miltefosine per oral 50mg Q8H, 2) Intravenous. Amphotericin B 1.5 mg/kg every 12 hours and 1.5 mg intrathecal per day, 3) per oral Azithromycin 750mg once daily (OD), per oral Fluconazole 800mg on day one then followed by 400mg once daily (OD), Rifampin per oral 600mg daily, IV Dexamethasone 0.6mg per kg per day. Patient was also empirically started on meningitic doses of Intravenous Meropenem 200mg Q8H and Intravenous Vancomycin 750mg Q6H. Intensive care unit team was taken on board due to low GCS and respiratory distress but did not require invasive ventilation. A repeat CSF DR done on third day of treatment showed a Glucose of 29 mg/dl, Protein 119 mg/dl, Tlcs 856 /cu mm with 60% Lymphocytes.

The hospital course of the patient got complicated with acute liver injury and kidney injury leading to the complete renal shutdown. Patient was started on hemodialysis which continued from 27/9/2014 till 3/10/2014 while all the antibiotics were also continued. On 28/9/2014 GCS started to improve from 8/15 to 13/15 on 4/10/2014.

Last MRI was done on 21/9/2014 which showed (Abnormal signal intensity area in left frontal lobe not showing diffusion restriction on DWI however showing focal hemorrhage. There was also diffuse abnormal signals in bilateral caudate nuclei showing diffusion restriction on DWI, appearing low on ADC and showing foci of hemorrhage. Findings were most likely due to vasculitic infarction with hemorrhagic conversion.

PAM protocol was stopped on 4/10/2014 after total of 28 days of treatment through combined decision of Infectious Disease team and Neurology team based on improved patient’s condition and risk of additional drug Side effects.

Patient was discharged home on 7/10/2014 with a 31 days long hospital course with a GCS of 15/15 but with mild right sided residual weakness. On follow up clinic visit after one and half month, the patient had marked improvement with independent function of daily living.

Investigations and outcomes

Of the 39 patients that underwent lumbar puncture, 27 patients (69.2%) had positive motile trophozoites on CSF wet preparation microscopy, 18 patients (46.2%) had a positive culture, and 10 patients (25.6%) had a positive PCR. Cerebrospinal fluid (CSF) analysis resembled bacterial meningitis with elevated white blood cell counts with predominantly neutrophils (median, 3000 cells/μL [range, 1350–7500 cells/μL]), low glucose levels median, 14 mg/dL [range, 1–92 mg/dL]), and elevated protein levels (median, 344 mg/dL [range, 289–405 mg/dL]) (Table 2). Imaging results were abnormal in approximately three-fourths of the patients.

Table 2. Distribution of CSF and lab investigations, and imaging results of patients admitted with primary amoebic meningo-encephalitis.

Characteristics Summary Statistics (%)
Total (N) 39
CSF Investigations
Positive Wet Mount 27 (69.2)
Positive Culture 18 (46.2)
Positive PCR 10 (25.6)
Median WBC (units) 3000 (1350–7500)
Median LYMP (units) 20 (10–25)
Median POLY (units) 80 (75–90)
Median Protein (units) 344 (289–405)
Median Glucose (units) 14 (5–45)
Lab Investigations
Median HB (units) 13.7 (11.7–15.2)
Median PLT (units) 207 (178–236)
Median CREAT (units) 1.1 (0.9–1.4)
Median BICARB (units) 17.1 (15.5–18.3)
Median SODIUM (units) 136 (132–140)
Median SGPT (units) 27 (21–44)
Imaging
1st CT Performed 34 (87.2)
Basal Enhancement 3 (8.8)
Infarction 3 (8.8)
Cerebritis 1 (2.9)
Lepto Enhance 5 (14.7)
Uncus Herniation 0 (0)
Hydroceph 6 (17.6)
Cereb Edema 13 (38.2)
Ventriculitis 1 (2.9)
Midline Shift 0 (0)
Tonsil Herniation 2 (5.91)
2nd CT Performed 17 (43.6)
Basal Enhancement 0 (0)
Infarction 1 (5.9)
Cerebritis 0 (0)
Lepto Enhance 0 (0)
Uncus Herniation 0 (0)
Hydroceph 3 (17.6)
Cereb Edema 15 (88.2)
Ventriculitis 0 (0)
Midline Shift 0 (0)
Tonsil Herniation 6 (35.3)
MRI performed 9 (23.0)
Basal Enhancement 7 (77.8)
Infarction 1 (11.1)
Cerebritis 0 (0)
Uncus Herniation 0 (0)
Hydroceph 1 (11.1)
Cereb Edema 2 (22.2)
Ventriculitis 0 (0)
Midline Shift 0 (0)
Tonsil Herniation 2 (22.2)
Outcome
Died 38 (97.4)
Alive 1 (2.6)
Collective Imaging results
Basal Enhancement 9 (23.0)
Infarction 5 (12.8)
Cerebritis 1 (2.6)
Lepto Enhance 5 (12.8)
Uncus Herniation 0 (0)
Hydrocephalus 10 (25.6)
Cerebral Edema 26 (66.6)
Ventriculitis 1 (2.6)
Midline Shift 0 (0)
Tonsil Herniation 9 (23.1)
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Cerebral edema (66.6%), hydrocephalus (25.6%) and cerebral infarction (12.8%) were the most common findings. Only one patient managed to survive (Table 2).

Role of gender

There were no statistically significant differences between male and female patients in terms of CSF investigations and imaging results (Table 3). However, the disease has a predilection towards male gender. There was a tendency of higher positive wet mount among male patients with frequency of 72.7% vs. 50.0%; p = 0.348 for male and female patients, respectively.

Table 3. Comparison of CSF investigations and imaging results of patients admitted with primary amoebic meningo-encephalitis by gender.

Male Female P-value
CSF Investigations
Positive Wet Mount 24 (72.7) 3 (50) 0.348
Positive Culture 17 (51.5) 1 (16.7) 0.19
Positive PCR 8 (24.2) 2 (33.3) 0.636
Combine Imaging results
Basal Enhancement 7 (21.2) 2 (33.3) 0.607
Infarction 4 (12.1) 1 (16.7) >0.999
Cerebritis 1 (3) 0 (0) >0.999
Lepto Enhance 5 (15.2) 0 (0) 0.574
Uncus Herniation 0 (0) 0 (0) -
Hydroceph 8 (24.2) 2 (33.3) 0.636
Cereb Edema 20 (60.6) 6 (100) 0.081
Ventriculitis 1 (3) 0 (0) >0.999
Midline Shift 0 (0) 0 (0) -
Tonsil Herniation 7 (21.2) 2 (33.3) 0.607
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While imaging results showed higher tendency of cerebral edema among female patients with frequency of 60.6% vs. 100.0%; p = 0.081 for female and male patients, respectively (Table 3).

Role of age

The CSF investigations showed significantly higher proportion of positive CSF cultures among patients aged > 35 years with frequency of 70.6% vs. 27.3%; p = 0.007 for patients with >35 years and 18 to 35 years of age, respectively. Similarly, imaging investigations showed higher proportion of tonsillar herniation among patients aged 18 to 35 years with frequency of 36.4% vs. 5.9%; p = 0.052 for patients with 18 to 35 years and >35 years of age, respectively (Table 4).

Table 4. Comparison of CSF investigations and imaging results of patients admitted with primary amoebic meningo-encephalitis by age.

18 to 35 years >35 years P-value
CSF Investigations
Positive Wet Mount 14 (63.6) 13 (76.5) 0.389
Positive Culture 6 (27.3) 12 (70.6) 0.007
Positive PCR 6 (27.3) 4 (23.5) >0.999
Combine Imaging results
Basal Enhancement 6 (27.3) 3 (17.6) 0.704
Infarction 4 (18.2) 1 (5.9) 0.636
Cerebritis 0 (0) 1 (5.9) 0.436
Lepto Enhance 1 (4.5) 4 (23.5) 0.147
Uncus Herniation 0 (0) 0 (0) -
Hydroceph 5 (22.7) 5 (29.4) 0.721
Cereb Edema 16 (72.7) 10 (58.8) 0.497
Ventriculitis 0 (0) 1 (5.9) 0.436
Midline Shift 0 (0) 0 (0) -
Tonsil Herniation 8 (36.4) 1 (5.9) 0.052
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Discussion

The progression of PAM disease is very rapid, with extremely high mortality rates. In this study our aim was to be identifying the spectrum of symptoms with which patients present to the hospital, reviewing the clinical course, duration of symptoms and lab parameters, specifically looking at the factors that predict poor outcome and the factors that delay the disease process and ultimately mortality. Our data showed that almost all features of Naegleria Fowleri induced PAM are like the cases found in other parts of the world. Common presenting features include fever, headache, vomiting and at late presentation, seizures with or without altered neurological status [9].

In our study, peak incidence was reported during the months of May to September which coincides with a hotter temperature [10]. Similar to earlier reports, only a small proportion of patients had confirmed history of swimming or water recreational activity which is strongly linked with the pathogenesis of Naegleria PAM [11], while the other portion of patients had no such history. Virtually all patients were Muslims, as a result the infection probably transpired through ablution in conjunction with tap water.

The highest number of PAM cases in the USA were reported in children younger than 14 years, but in Pakistan, most cases are reported in adults aged 26–45 years, which may possibly be due to a genetically unique strain in Pakistan. More research is needed to look at the genome sequence for a likely emerging resistant strain in Pakistan [12].

The male predominance seen in our sample and overall reported cases may be related to activities of water exposure from different sources compared to females with limited exposure or may be predisposed due to sex-linked hormones as has been hypothesized for other infections such as Entamoeba histolytica liver abscesses [13].

Poor healthcare system, and unavailability of potent drugs to manage this disease is also a huge health risk for our country [14, 15]. Unfortunately, our data also revealed that Naegleria Meningitis has high mortality rate (97.4%) which was attributed to late presentation, delay in accurate diagnosis, and short incubation period leading to death in 72 hours of first symptoms appearance [16–18].

It is a single center audit for the duration of more than 10 years of 39 Naegleria cases. Although almost all the findings are consistent with earlier similar work, the source of spread is somewhat different in our part of the world as compared to west.

Besides working on traditional mode of spread of Naegleria which includes swimming/recreational activities in contaminated water, water reservoirs for domestic/daily usage in underground tanks should also be considered a potential source. Like other seasonal pandemics such as dengue, COVID, malaria, flu, hemorrhagic fever, we should also prepare ourselves for Naegleria surge and use social, electronic, and print media to educate our community to efficiently combat these fatal diseases. Efforts should be made at government level to improve living infrastructure in provision of clean and safe chlorinated water to all and to improve healthcare facilities. Chlorination is the most extensively used disinfectant for water treatments due to its low cost, ease to produce, store, transport and use as well as its high oxidizing potential. Usually used as chlorine gas, sodium or calcium hypochlorite, it provides a minimum level of residual disinfectant able to prevent microbial recontamination [19]. Chlorine dioxide have been as well reported by Dupuy et al., (2014) for its efficacy to inhibit three different FLA strains [20]. The Karachi Water and Sewerage Board (KWSB) is the principal agency in Karachi, Pakistan which oversees issues with water distribution and chlorination in the city, has been ordered by the health authorities to ensure that the municipal water supply is adequately chlorinated with the WHO recommendations, and preventive actions have also been developed to stop the spread of this sickness [21].

Limitations

Small sample size as well as single-center study.

Conclusions

PAM is a fatal illness with limited treatment success. Treating Physicians should have a high suspicion for PAM in the setting of acute meningo-encephalitis with rapidly progressive symptoms with negative CSF results for bacteria and common viruses. Early diagnosis and prompt initiation of treatment may improve the survival of the patients and reduce mortality.

Data Availability

The data will be available on following contact: The Contact information of Institutional Research Ethics Committee are: Tel: +92 21 3493 0051 EXT:4988/2445 Email: erc.pakistan@aku.edu.

Funding Statement

The authors received no specific funding for this work.

References

  • 1.Schuster FL, Visvesvara GS. Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals. Int J Parasitol. 34(9):1001–27. doi: 10.1016/j.ijpara.2004.06.004 [DOI] [PubMed] [Google Scholar]
  • 2.Yoder JS, Eddy BA, Visvesvara GS, Capewell L, Beach MJ. The epidemiology of primary amoebic meningoencephalitis in the USA, 1962–2008. Epidemiol Infect. 2010;138(7):968–75. doi: 10.1017/S0950268809991014 [DOI] [PubMed] [Google Scholar]
  • 3.Hannafin B, LoVecchio F, Rosenberg D. Update on emerging infections: News from the Centers for Disease Control and Prevention. Primary amebic meningoencephalitis-Arizona, Florida, and Texas, 2007. Ann Emerg Med. 2009;54(3):469–72. doi: 10.1016/j.annemergmed.2009.07.007 [DOI] [PubMed] [Google Scholar]
  • 4.Capewell LG, Harris AM, Yoder JS, Cope JR, Eddy BA, Roy SL, et al. Diagnosis, clinical course, and treatment of primary amoebic meningoencephalitis in the United States, 1937–2013. J Pediatric Infect Dis Soc. 2015;4(4):e68–75. doi: 10.1093/jpids/piu103 [DOI] [PubMed] [Google Scholar]
  • 5.Cope JR, Murphy J, Kahler A, Gorbett DG, Ali I, Taylor B, et al. Primary amebic meningoencephalitis associated with rafting on an artificial whitewater river: case report and environmental investigation. Clin Infect Dis. 2018;66(4):548–53. doi: 10.1093/cid/cix810 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Shakoor S, Beg MA, Mahmood SF, Bandea R, Sriram R, Noman F, et al. Primary amebic meningoencephalitis caused by Naegleria fowleri, Karachi, Pakistan. Emerg Infect Dis. 2011;17(2):258–61. doi: 10.3201/eid1702.100442 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Johnson RO, Cope JR, Moskowitz M, Kahler A, Hill V, Behrendt K, et al. Notes from the Field: Primary Amebic Meningoencephalitis Associated with Exposure to Swimming Pool Water Supplied by an Overland Pipe-Inyo County, California, 2015. MMWR Morb Mortal Wkly Rep. 2016;65(16):424. doi: 10.15585/mmwr.mm6516a4 [DOI] [PubMed] [Google Scholar]
  • 8.Visvesvara GS, Moura H, Schuster FL. Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea. FEMS Immunol Med Microbiol. 2007;50(1):1–26. doi: 10.1111/j.1574-695X.2007.00232.x [DOI] [PubMed] [Google Scholar]
  • 9.Ghanchi NK, Jamil B, Khan E, Ansar Z, Samreen A, Zafar A, et al. Case series of Naegleria fowleri primary ameobic meningoencephalitis from Karachi, Pakistan. Am J Trop Med Hyg. 2017;97(5):1600–2. doi: 10.4269/ajtmh.17-0110 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Bellini NK, Santos TM, da Silva MT, Thiemann OH. The therapeutic strategies against Naegleria fowleri. Exp Parasitol. 2018;187:1–11. doi: 10.1016/j.exppara.2018.02.010 [DOI] [PubMed] [Google Scholar]
  • 11.Ghanchi N. Increasing cases of Naegleria fowleri infections from Karachi Pakistan. Int J Infect Dis. 2018;73:185. [Google Scholar]
  • 12.Naqvi AA, Yazdani N, Ahmad R, Zehra F, Ahmad N. Epidemiology of primary amoebic meningoencephalitis-related deaths due to Naegleria fowleri infections from freshwater in Pakistan: An analysis of 8-year dataset. Arch Pharm Pract. 2016;7(4):119–29. [Google Scholar]
  • 13.Klein SL. Hormonal and immunological mechanisms mediating sex differences in parasite infection. Parasite Immunol. 2004;26(6‐7):247–64. doi: 10.1111/j.0141-9838.2004.00710.x [DOI] [PubMed] [Google Scholar]
  • 14.Mungroo MR, Khan NA, Siddiqui R. Naegleria fowleri: diagnosis, treatment options and pathogenesis. Expert Opin Orphan Drugs. 2019;7(2):67–80. [Google Scholar]
  • 15.Siddiqui R, Khan NA. Primary amoebic meningoencephalitis caused by Naegleria fowleri: an old enemy presenting new challenges. PLoS Negl Trop Dis. 2014;8(8):e3017. doi: 10.1371/journal.pntd.0003017 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Jahangeer M, Mahmood Z, Munir N, Tahir IM, Akram M, Shah SM, et al. Naegleria fowleri: Sources of infection, pathophysiology, diagnosis, and management; a review. Clin Exp Pharmacol Physiol. 2020;47(2):199–212. doi: 10.1111/1440-1681.13192 [DOI] [PubMed] [Google Scholar]
  • 17.Chow FC, Glaser CA. Emerging and reemerging neurologic infections. Neurohospitalist. 2014;4(4):173–84. doi: 10.1177/1941874414540685 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.De Jonckheere JF. Origin and evolution of the worldwide distributed pathogenic amoeboflagellate Naegleria fowleri. Infect Genet Evol. 2011;11(7):1520–8. doi: 10.1016/j.meegid.2011.07.023 [DOI] [PubMed] [Google Scholar]
  • 19.Gopal K, Tripathy SS, Bersillon JL, Dubey SP. Chlorination byproducts, their toxicodynamics and removal from drinking water. J Hazard Mater. 2007;140(1–2):1–6. doi: 10.1016/j.jhazmat.2006.10.063 [DOI] [PubMed] [Google Scholar]
  • 20.Dupuy M, Berne F, Herbelin P, Binet M, Berthelot N, Rodier MH, et al. Sensitivity of free-living amoeba trophozoites and cysts to water disinfectants. Int J Hyg Environ Health. 2014;217(2–3):335–9. doi: 10.1016/j.ijheh.2013.07.007 [DOI] [PubMed] [Google Scholar]
  • 21.Tabassum S, Naeem A, Gill S, Mumtaz N, Khan MZ, Tabassum S, et al. Increasing cases of Naegleria fowleri during the time of COVID 19; an emerging concern of Pakistan. Int J Surg. 2022;105:106881. doi: 10.1016/j.ijsu.2022.106881 [DOI] [PMC free article] [PubMed] [Google Scholar]

Decision Letter 0

Kelli L Barr

Transfer Alert

This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.

3 May 2023

PONE-D-23-06572Clinical Manifestations and Outcome of Patients with Primary Amoebic Meningoencephalitis in Pakistan. A Single Center ExperiencePLOS ONE

Dear Dr. Farooq,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================The reviewers have made comments that will improve this manuscript. I agree that adding information on the patient that survived would be wonderful since there are so few reports of PAM survivors.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 1. The scientific name of Naegleria fowleri should be correctly addressed as italics through the manuscript.

2. IRB approval number should be clearly provided.

3. Table 1: GCS means Glasgow Coma Scale? If yes, please describe it clearly.

4. In Treatment: Why only 37 patients, not 39? Detailed description for one survivor would be interesting.

5. Were there any specific reasons to classify the patients into two groups, 18-35 and over 35 years old, by age?

6. Tables 3 and 4: The numbers of patients based on the classification could be included.

7. Table 4: Few values such as infarction, lepto enhance, and tonsil herniation in combined imaging results showed differences between the two groups, even though the number of patients were not high. 8. Discussion on the differences in the discussion section would be appreciated.

9. Line 155: Proper references should be added.

10. Considering the main points of this manuscript were the clinical features in PAM patients, discussion could be improved further by comparing the previously reported clinical features in Pakistan and other global areas.

11. English could be improved. English editing is recommended.

Reviewer #2: According to my expertise, this article fulfills all the requirements mentioned in the PLOSOne guidelines.

The researchers of the article have done a rigorous analysis on their sample population and provided with the results in written and table forms as well which makes it easier to comprehend.

The method and material portion upheld the ethical code and was approved by IRB.

However, there are few changes that I would like to suggest.

1.Out of these 39 patients only one patient who survived received all

105 these treatment for total of 24 days and was stopped by infectious disease team.

Suggestion a: Please mention the clinical criteria used by the infectious disease team to decide when to stop treatment for this patient. This information can provide further insight into prognostic factors and recovery criteria that can be used by doctors managing this disease in future.

Suggestion b: Kindly also mention any demographic or clinical presentation factors that differ between this patient and the other deceased patients, as the mortality rate of this disease is high. Elaborating on why this particular patient may have survived could help the medical community identify potential factors that contribute to better outcomes. This information could inform further studies that could hypothesize about the causal relationship between these factors and disease severity.

2.Efforts should be made at government level to improve living infrastructure in provision

of clean and safe chlorinated water to all and to improve healthcare facilities.

Suggestion: kindly provide a reference for the claim that chlorinated water can reduce the spread of Naegleria, and if so, how? This information could provide guidelines for the prevention of this infection. Alternatively, could you mention any already-published guidelines used by developed countries to prevent the spread of this disease?"

3.

Reviewer #3: First of all, I want to thank to all of the authors of the manuscript. This study needs to be revised with more detailed and proper written language.

Naegleria fowleri, the causative agent of primary amoebic meningoencephalitis (PAM), is a free-living amoeba. It is a water-borne infection usually detected in children and young people with healthy immune systems who swim, dive and perform activities in fresh and hot springs. It is an important study in terms of addressing the Clinical Symptoms and Outcomes of Patients with Primary Amoebic Meningoencephalitis in Pakistan. The points mentioned below need to be considered in more detail.

- "Naegleria fowleri" or "Naegleria" needs to be corrected in italics throughout the manuscript.

- -In line 56, it is stated that the transmission route of N. fowleri can not only be through contaminated water, such as swimming in freshwater lakes, rivers, canals, and swimming pools, but also "dry infection" with dust in the air. It would be better to mention this transmission route here.

- In line 93, it should also mention the decrease in consciousness about the clinical symptoms that are the backbone of the study.

- The term "GCS" is not mentioned anywhere in the manuscript.

- In line 102, the spelling of "2 gm/day" should be corrected.

- In line 104, explaining the drug treatment complex administered for a surviving patient in more detail would be more beneficial.

- It is interesting t that 27 patients were positive by microscopy, while only ten were positive by PCR. Besides, the PCR method is more sensitive and specific, and PCR results are essential in confirming the microscopy. It would be better if you could explain how you evaluate this situation.

- Cerebral edema is 66.7% in line 114 and 66.6% in the table; whichever is correct should be corrected.

- Looking at the table, tonsil herniation (23.1%) is among the most common ones.

- "While imaging results showed higher tendency of cerebral edema among female patients with frequency of 60.6% vs. 100.0%; p=0.081 for female and male patients, respectively" in line 122 This sentence should be reviewed. Because while 100% cerebral edema is seen in women in table 3, it is expressed as 60.6% in men.

- In the discussion section, the clinical symptoms of the patients and the treatment options given should be discussed in more detail. For example, it should be discussed compared to clinical symptoms and treatments in a single case (https://doi.org/10.1007/s11686-021-00514-0).

**********

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Reviewer #1: No

Reviewer #2: Yes: Syeda Maria Hassan

Reviewer #3: Yes: Mehmet AYKUR

**********

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Attachment

Submitted filename: Reviewer Comments to Author_02.05.2023.docx

Click here for additional data file. (17.3KB, docx)
PLoS One. 2023 Nov 8;18(11):e0290394. doi: 10.1371/journal.pone.0290394.r002

Author response to Decision Letter 0

19 Jul 2023

Reviewer #1: 1. The scientific name of Naegleria fowleri should be correctly addressed as italics through the manuscript. Thank you for this feedback. We have corrected the name Naegleria fowleri in italics through the manuscript. 2. IRB approval number should be clearly provided. Thank you for this comment. The IRB file is attached to the submission. For clarification, we have also added the IRB # ( 2021-6074-18800) to the manuscript.For Information: Tel: +92 21 3493 0051 Ext: 4988|2445 Email: erc.pakistan@aku.edu 3. Table 1: GCS means Glasgow Coma Scale? If yes, please describe it clearly. Thank you for your comment. We have added Glasgow Coma Scale to describe GCS clearly in the table. 4. In Treatment: Why only 37 patients, not 39? Detailed description for one survivor would be interesting. We thank the reviewer for this comment. We have corrected the number of patients to 39. We have also added additional detail on the patient that survived. 5. Were there any specific reasons to classify the patients into two groups, 18-35 and over 35 years old, by age? The patient groups were divided into two age groups to see if any difference in incidence, diagnostic,radio-logical and clinical features in younger patients compared to older patients. The age cut off was arbitrarily decided. 6. Tables 3 and 4: The numbers of patients based on the classification could be included.Thank you for this comment. We have added the total number of patients with the qualifications. 7. Table 4: Few values such as infarction, lepto enhance, and tonsil herniation in combined imaging results showed differences between the two groups, even though the number of patients were not high.Thank you for your comment. The differences were not statistically significant at the prespecified level of significance set at 0.05.8. Discussion on the differences in the discussion section would be appreciated. 9. Line 155: Proper references should be added. 10. Considering the main points of this manuscript were the clinical features in PAM patients, discussion could be improved further by comparing the previously reported clinical features in Pakistan and other global areas. 11. English could be improved. English editing is recommended. Thank you for the comment. We have rectified. Reviewer #2: According to my expertise, this article fulfills all the requirements mentioned in the PLOSOne guidelines.The researchers of the article have done a rigorous analysis on their sample population and provided with the results in written and table forms as well which makes it easier to comprehend. The method and material portion upheld the ethical code and was approved by IRB. However, there are few changes that I would like to suggest. 1.Out of these 39 patients only one patient who survived received all 105 these treatment for total of 24 days and was stopped by infectious disease team. Suggestion a: Please mention the clinical criteria used by the infectious disease team to decide when to stop treatment for this patient.This information can provide further insight into prognostic factors and recovery criteria that can be used by doctors managing this disease in future. Suggestion b: Kindly also mention any demographic or clinical presentation factors that differ between this patient and the other deceased patients, as the mortality rate of this disease is high. Elaborating on why this particular patient may have survived could help the medical community identify potential factors that contribute to better outcomes. This information could inform further studies that could hypothesize about the causal relationship between these factors and disease severity. Thank you for your comment. We have added the clinical course of the patient to better convey their treatment. It is difficult to analyze any major differences between this patient and the patients that did not survive. We have added some of our thoughts. 2.Efforts should be made at government level to improve living infrastructure in provision of clean and safe chlorinated water to all and to improve healthcare facilities. Suggestion: kindly provide a reference for the claim that chlorinated water can reduce the spread of Naegleria, and if so, how? This information could provide guidelines for the prevention of this infection. Alternatively, could you mention any already-published guidelines used by developed countries to prevent the spread of this disease? Thank you for this comment. We have added references and a paragraph in the discussion to support this statement. Reviewer #3: First of all, I want to thank to all of the authors of the manuscript. This study needs to be revised with more detailed and proper written language. Naegleria fowleri, the causative agent of primary amoebic meningoencephalitis (PAM), is a freeliving amoeba. It is a water-borne infection usually detected in children and young people with healthy immune systems who swim, dive and perform activities in fresh and hot springs. It is an important study in terms of addressing the Clinical Symptoms and Outcomes of Patients with Primary Amoebic Meningoencephalitis in Pakistan. The points mentioned below need to be considered in more detail. - "Naegleria fowleri" or "Naegleria" needs to be corrected in italics throughout the manuscript. Thank you for your suggestion. We have corrected it in the manuscript. - -In line 56, it is stated that the transmission route of N. fowleri can not only be through contaminated water, such as swimming in freshwater lakes, rivers, canals, and swimming pools, but also "dry infection" with dust in the air. It would be better to mention this transmission route here.Thank you for this comment. We have added this as a statement. - In line 93, it should also mention the decrease in consciousness about the clinical symptoms that are the backbone of the study. Thank you for the comment. We have rectified. - The term "GCS" is not mentioned anywhere in the manuscript.The full form of Glasgow Coma Scale has been added on first mention, and abbreviation as well. Thank you for the comment. We have added the full form. - In line 102, the spelling of "2 gm/day" should be corrected.Thank you for pointing this out. We have corrected this. - In line 104, explaining the drug treatment complex administered for a surviving patient in more detail would be more beneficial.Thank you for your comment. We have added the clinical course of the patient to better convey their treatment. - It is interesting t that 27 patients were positive by microscopy, while only ten were positive by PCR. Besides, the PCR method is more sensitive and specific, and PCR results are essential in confirming the microscopy. It would be better if you could explain how you evaluate this situation. PCR method was introduced at our hospital at 2014 and before this it was diagnosed on wet mount. Also when it was diagnosed on Wet mount PCR was not done in that case. In highly suspected case like short history and early deterioration PCR was done. This has been clarified in the manuscript. - Cerebral edema is 66.7% in line 114 and 66.6% in the table; whichever is correct should be corrected.The correct value is 66.6 and has been corrected.- Looking at the table, tonsil herniation (23.1%) is among the most common ones.- "While imaging results showed higher tendency of cerebral edema among female patients with frequency of 60.6% vs. 100.0%; p=0.081 for female and male patients, respectively" in line 122 This sentence should be reviewed. Because while 100% cerebral edema is seen in women in table 3, it is expressed as 60.6% in men. This was miswritten and has been corrected. - In the discussion section, the clinical symptoms of the patients and the treatment options given should be discussed in more detail. For example, it should be discussed compared to clinical symptoms and treatments in a single case (https://doi.org/10.1007/s11686-021-00514-0). Thank you for the comment. We have rectified.

Attachment

Submitted filename: Response to Reviewers.pdf

Click here for additional data file. (155.2KB, pdf)

Decision Letter 1

Kelli L Barr

8 Aug 2023

Clinical Manifestations and Outcome of Patients with Primary Amoebic Meningoencephalitis in Pakistan. A Single Center Experience

PONE-D-23-06572R1

Dear Dr. Farooq,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Kelli L. Barr, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Syeda Maria Hassan

Reviewer #3: Yes: Mehmet Aykur

**********

Acceptance letter

Kelli L Barr

11 Aug 2023

PONE-D-23-06572R1

Clinical Manifestations and Outcome of Patients with Primary Amoebic Meningoencephalitis in Pakistan. A Single-Center Experience

Dear Dr. Farooq:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Kelli L. Barr

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    Attachment

    Submitted filename: Reviewer Comments to Author_02.05.2023.docx

    Click here for additional data file. (17.3KB, docx)
    Attachment

    Submitted filename: Response to Reviewers.pdf

    Click here for additional data file. (155.2KB, pdf)

    Data Availability Statement

    The data will be available on following contact: The Contact information of Institutional Research Ethics Committee are: Tel: +92 21 3493 0051 EXT:4988/2445 Email: erc.pakistan@aku.edu.

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