Free-living amoebae (FLA) are unicellular eukaryotes that can develop as free-living organisms in the environment or as parasites in hosts. N. fowleri (Naegleria fowleri), Acanthamoeba spp., and B. mandrillaris (Balamuthia mandrillaris) represent amphizoic amoebas that exhibit pathogenic and free-living species (1). They can reach the central nervous system (CNS) by hematogenous spread from a primary site in wounded skin or upper respiratory tract via nasal passage, which can cause serious cerebral infections, named primary amoebic meningoencephalitis (PAM), granulomatous amoebic encephalitis (GAE), and balamuthia amoebic encephalitis (BAE) respectively (2).

It is not entirely clear why different FLAs cause different types of encephalitis. From a phylogenetic point of view, Acanthamoeba spp. and B. mandrillaris are more closely related. Both of these pathogens have two developmental stages: the trophozoite as a nutrition feeding form and the cyst as a resting form. N. fowleri presents an additional flagellated stage. In the presence of water, the trophozoite transitions to this temporary motile stage and allows the amoebae to find other favorable environmental niches. Likewise, recent studies (3, 4) have shown that the immune response to the three FLAs is different. Acanthamoeba spp. and B. mandrillaris primarily involve macrophages and T cells, and induce the formation of granulomas. In contrast, N. fowleri elicits an acute inflammatory response that primarily involves neutrophils and macrophages, the production of pro-inflammatory cytokines, and severe tissue damage (5).

The clinical symptoms of these three cerebral infections are similar, but brain inflammation and disease course are different. In the early stage, general symptoms such as headache and fever may appear. And according to the distribution and severity of the lesion, neurological symptoms such as confusion, hemiparesis and seizures may appear. Currently, pathogenic FLA encephalitis is an infectious disease with a high mortality rate due to the small number of cases and difficulty of diagnosis and treatment. Consensus treatment has not yet been established. Even with treatment, these pathological changes result in a high mortality rate of over 90%. In China, the first case of PAM caused by N. fowleri was reported in Henan in 1979, and several cases of encephalitis caused by FLA were subsequently reported nationwide. There were an increasing number of cases reported in the last decade. There are great differences in the description and analysis of clinical data reported by different research reports.

Encephalitis by FLA can cause severe clinical problems. Three kinds of infections, despite low morbidity, have relatively high mortality rates, which is a considerable challenge for early diagnosis and efficient therapy. This article comprehensively analyzes the literature reports in China and the medical records in our hospital. Combining these clinical data, the article aimed to determine the clinical characteristics and differences among PAM, GAE, and BAE in the Chinese population to develop strategies to manage the three FLA encephalitis.

This systematic review was performed according to “Preferred Reporting Items for Systematic Reviews and Meta-Analyses” (PRISMA) statement (6).

To our knowledge, this article is the largest clinical case series of patients from China with three pathogenic FLA encephalitis, including PAM, GAE, and BAE. There are obvious differences and similarities between three FLA encephalitis. We analyzed the exposure location, clinical symptoms, diagnosis, treatment, and outcomes, and hope it will be helpful to early differential diagnosis of different meningoencephalitis and initiate amoebic-specific therapy more rapidly.

In our study, BAE has almost 3 times more cases than PAM or GAE, but this may not indicate a greater susceptibility to B. mandrillaris. In general, the sample size on free-living amoebic encephalitis is limited in China. BAE has a slower disease course than PAM. And because of the manifestation of skin lesions before the appearance of encephalitis symptoms in most of BAM patients, BAE may be more likely to be identified and reported. Besides, in the last 5 years, BAE has been reported more frequently than PAM and GAE, and medical personnel are more aware of BAE. There is also a case series on B. mandrillaris infection (including 16 patients with BAE) in China (37). The above may be the reason for the number of cases of BAE was higher than that of other PAM and GAE.

The susceptible population of three FLA encephalitis are mostly healthy young people and children. Patients were predominantly male, especially PAM. And BAE is more likely to occur in younger age groups. The reason for this pattern of sex and age distribution is unclear. The increased number of infections in young males may be related to differences in exposure to outdoor activities. Some behaviors, hobbies, or works could be putting these people at a higher risk. Such as engage in agricultural activities, or dive, swim, jump into water in rivers or ponds, or soak in a hot spring, increasing the risk of skin breakdown or water forcing its way into the nasal cavity. These activities have been documented in some patients. However, these activities also do not fully explain the increased risk of infection. These are similar to the susceptible populations in the United States (39, 40), India (41), Thailand (42), and Peru (43). But our group seems to find that the median age of PAM in China is higher than in the rest of the globe, for example, the United States (39). This may be due to the fact that some high-risk behaviors, hobbies, or works described above are more common among adults in China. Among the GAE and BAE patients reported in the United States (44) and Peru (43, 45, 46), it is more likely to occur in immunocompromised patients. Similarly, China did show similar epidemiologic patterns. Immunocompromised youngers due to tumors or chronic diseases (e.g., breast cancer, AIDS, long-term glucocorticoids use, and skin lesions) may increase the risk of contracting GAE or BAE or accelerating the course of GAE or BAE. And immunocompromised patients have relatively poorer outcomes in later treatment. The southern regions of China reported cases more frequently. Many FLA cases also been reported in tropical regions of the world, which may be associated with the dry and hot climate environment. Besides, southern China generally has a higher economic and medical level than northern China, with a greater ability to identify and report cases. A variety of factors may have contributed to this regional trend. And there were an increasing number of cases reported in southern China over time. It is also likely due to improvements in awareness and diagnostic capacity. Broader case reporting is needed to validate this epidemiologic pattern. Besides, we also found that PAM cases with data on a clear season of onset were mostly in the warm, hot summer months, which is consistent with the thermophilic nature of N. fowleri. All three FLA encephalitis exposure to amoeba-contaminated water or soil sources was possible prior to the onset of symptoms. But unfortunately, in only one case (23) did the research group collect both a sample of the patient's CSF and an aqueous sample of the patient's inhaled muddy water. The results indicated that the Acanthamoeba spp. indeed, existed in the rice field.

The onset of PAM is mostly acute or subacute. The median incubation period for PAM cases in China was 4 (1–6) days, similar to other countries. We found that time from onset of symptoms to death of PAM patients in China take longer than the rest of the world (47). It has been reported that different geographic distribution of each genotype isolated from environmental and clinical specimens (48). But there is no conclusive evidence to prove different types of N. fowleri have virulence differences. Besides, we found that although Chinese PAM patients had a long disease duration, their disease progressed quickly. Most of the several patients with longer disease duration had motile amoeba or trophozoite-like organisms found in the CSF and were anti-amoebic treated accordingly. However, by the time the diagnosis was clear, the patients were in a coma and anti-amoebic treatment did not save their lives. Timely anti-amoebic treatment may prolong the disease course. Most patients with GAE and BAE have an insidious onset and a chronic course.

The clinical manifestations of the three encephalitis are similar, but there are some significant differences. Neurological symptoms of PAM present as acute and fulminant hemorrhagic meningoencephalitis. GAE and BAE have a slower disease progression than PAM. Two patients (19, 20) with GAE had ocular symptoms as the onset symptom, which is noteworthy. The genus Acanthamoeba is currently classified into 23 genotypes (T1–T23), and of these some are known to be capable of causing GAE mainly in immunocompromised patients while other genotypes cause Acanthamoeba keratitis (AK) mainly in otherwise healthy patients. There are partial genotypes known to cause both AK and GAE (T2, T4, T5, T10, and T12) (49). Whether ocular symptoms can be indicative of GAE is a question that needs to be further explored in future studies. In BAE, 77.8% of patients initially had skin lesions, the most common lesion seen is a painless fuchsia plaque. And develop encephalitis several months or years later. The most common location is on the central face, over the nose, or on one extremity, which is similar to cases reported in Peru (43, 45, 46). The patients may develop encephalitis several months or years after skin lesions. However, cases from the US often progress directly to encephalitis without skin lesions (40). It is not known why some patients initially develop skin lesions while others develop encephalitis directly. Different genotypic differences in geographically distributed B. mandrillaris may lead to difference in pathobiology. But B. mandrillaris is the only pathogenic species without genotypic classification. It has been reported the variable length of the rps3 type II intron (region of the mitochondrial genome) between the different strains of B. mandrillaris suggesting that it may be promising for the development of molecular genotyping (49). More B. mandrillaris from different geographical locations need to be sequenced for molecular genotyping. Besides, we hypothesized that it also may be related to the invasion pathways. A total of 11 patients had a clear history of trauma. China has a large rural population with a high risk of skin trauma and skin infection compared to the urban population.

The precise diagnosis of the disease and the causal agent promptly is the critical step to deliver the appropriate treatment. It is challenging to diagnose and differentiate PAM, GAE, and BAE by the laboratory or imaging examination, and the results were similar to those observed in the United States (44). But the specificity of neuroimaging for the diagnosis has yet to be evaluated. PAM in neuroimaging mostly had only one infected lesion with diffuse cerebral edema and signs of increased intracranial pressure. Besides, one case of PAM showed normal neuroimaging findings, but the patient had already been experiencing acute symptoms such as headache, fever, and confusion for 2 days. Normal imaging findings were also observed early in a patient with GAE who presented with only headache symptoms. This false-negative result was also seen in a patient in the United States (50), which is worrisome. Patients may not receive early intervention if medical professionals are not aware of the disease. Neuroimaging of GAE and BAE typically shows multiple, well-defined, focal, ring-enhancing, space-occupying lesions. Brain lesions in the thalamus and parietal lobe were much more frequent for GAE. The frontal lobe, parietal lobe, temporal lobe, and occipital lobe were affected most in cases of BAE.

Moreover, among patients diagnosed premortem, 4 of PAM, 2 of GAE, and 10 of BAE patients were diagnosed by NGS. NGS constitutes a rapid and accurate method for pathogen identification, particularly in diagnosing unexplained diseases. Its turnover time is < 48 h, and is less affected by previous antibiotic exposure (51). And the samples for NGS analysis are generally flexible, including CSF, blood, tissue, etc. This unbiased NGS provides an operable diagnosis for rare pathogenic infections, which is helpful for clinicians to timely diagnose and find targeted and effective treatment. Although the cost of NGS testing is significantly lower compared to Sanger sequencing, it is still very expensive compared to other traditional laboratory tests. Currently, a single NGS test can cost up to six hundred dollars. The high cost is one of the factors that limit the widespread use of NGS in clinical settings. But with the further maturation of the technology, NGS should be further improved in the future in terms of cost reduction, diagnostic standard improvement, quality control improvement, etc. Therefore, we propose that the NGS method should be applied more widely in the clinical practices to help physicians make diagnosis. Suspicion of FLA encephalitis should be raised when the presence of motile amoeba is found in CSF wet-mount smear or trophozoite-like organisms is found in CSF by Giemsa stains. N. fowleri was found in the CSF more often than the other two types of amoebae, most likely because of its characteristic motile flagellated form. It is worth noting that negative CSF samples findings cannot exclude the possibility of infection in suspected patients. Inadequate atypical morphological features are difficult to distinguish different amoeba. If an amoeba is detected in CSF, or there are associated risk factors, such as the history of freshwater exposure, skin lesions, or ineffective antibiotic therapy, confirmatory testing by immunohistochemical staining, PCR, or NGS are required for the diagnosis of FLA encephalitis. For BAE, if the patients present first with a skin lesion, they may expect to be more easily diagnosed by skin biopsy, immunohistochemical staining, PCR or NGS. Therefore, it is important to correctly identify the cutaneous manifestation of the disease.

Life expectancy very poor, with only 6 successful treatment cases reported in China. No single agent can be proposed as the ideal therapy by itself. Amphotericin B is an antifungal drug, and has been used in the treatment of PAM since 1970. Although amphotericin B is still considered the drug of choice for treating PAM, it has limited efficacy and some side effects, such as anemia, fever, nausea, and dose-related nephrotoxicity. It has been reported that amphotericin B was administered to most confirmed PAM patients, but only 7 patients survived and also received other treatments (47). The Centers for Disease Control and Prevention (CDC) in the United States based on case reports or in vitro studies recommends the standard treatment for PAM, which is a combinational therapeutic cocktail that includes amphotericin B, an azole (ketoconazole, fluconazole, miconazole), azithromycin, rifampin, and miltefosine (52, 53). However, treatment may be successful only at early stages. The onset of PAM is urgent and progresses rapidly in a short period of time, and there is often not receiving targeted and effective treatment in time. The only surviving PAM patient in China had a less acute presentation. And he had received amphotericin B, in combination with rifampicin, chloramphenicol, and other non-specific treatments. Including decompressive occipital lobectomy, insertion of a ventricular catheter and removal of the bone flap to reduce intracranial pressure actively. This may be the key to the survival of the patient. Whether chloramphenicol has any anti-amoebic effect remains unproven. Miltefosine is originally developed as an anticancer drug, and currently used as an effective antileishmanial therapy. Because it was used to treat the two survivors in the United States in 2013 (39). Recent reports showed good in-vitro activity of miltefosine against B. mandrillaris, Acanthamoeba spp., and N. fowleri species (54–57). But it's not yet available in China.

The treatment of GAE usually involves combination therapy also. The available drugs include liposomal amphotericin B, rifampin, azoles (voriconazole, itraconazole, fluconazole, ketoconazole), caspofungin, sulfadiazine, pyrimethamine, cotrimoxazole (trimethoprim–sulfamethoxazole), meropenem, linezolid and moxifloxacin, pentamidine isethionate and miltefosine (3, 56, 58). The 2007 survivor was aggressively managed for his elevated intracranial pressure, including anti-infective therapy and lesion excision surgery. The 2009 survivor was given amphotericin B, Rifampicin, and other nonspecific. The 2021 survivor was given cotrimoxazole and metronidazole. However, one GAE patient in 2021 was already in critical condition when she received targeted medication after the diagnosis was clear. And this patient's disease progressed extremely rapidly. He passed away despite receiving aggressive treatment with most of the recommended medications. These cases proved that treatment effects may be related to the severity and duration of the disease, and early diagnosis.

The treatment of one BAE survivor in China consisted of amphotericin B, macrolides (azithromycin), sulfonamides, azoles (fluconazole), and flucytosine. Because there are so few surviving cases, this treatment option requires further research. We observed one patient who was treated with essentially a similar regimen, but unfortunately passed away. This may be related to this patient progressing very rapidly after the onset of brain symptoms. This drug combination, may still represent the best choice for current treating BAE until new drugs are developed in further. The second survivor was treated with lincomycin and interferon-γ mainly for skin lesion. The brain lesion was detected by imaging before the onset of brain symptoms, and the medicine for encephalitis is not reported. Thereafter, it was surgically excised and cured. Based on a limited number of successful clinical cases and previously identified in vitro drug susceptibility, CDC recommends the therapeutic drug for BAE consists of pentamidine, 5-flucytosine, fluconazole, and a macrolide (clarithromycin or azithromycin) with one of the following: sulfadiazine, miltefosine, thioridazine or liposomal amphotericin B (53, 59). Recently, Laurie et al. (60) screened more than 2000 drugs and identified in vitro efficacy of quinoline nitroxoline is better than that of other antiamebic medications; it also is safe and well tolerated. A patient in the United States survived after receiving treatment with a regimen that included the repurposed drug nitroxoline (61). We believe that quinoline nitroxoline maybe a promising treatment for B. mandrillaris infection.

We suggest clinicians should ideally investigate for less common potential pathogens, in patients with bacterial meningitis who are unresponsive to first-line antibiotic treatments. And treatment strategies usually combine various classes of drugs with different action mechanisms for individualized therapies. In addition to anti-amoebic medications, they are managed with physical procedures such as CSF drainage, hyperosmolar therapy, moderate hyperventilation, and hypothermia. Cerebral hernia is a common cause of death. Therefore, management of high intracranial pressure should be promptly instituted. External ventricular draining is the preferred procedure (62). Diagnostic biopsy is previously applied in three FLA encephalitis. It is now suggested that early surgical resection of intracranial lesions combined with drug therapy may be an important method for the treatment of three FLA encephalitis. In China, one survivor in PAM, one survivor in GAE, and two survivors in BAE were treated by excision of the infected brain tissue. However, due to the limited number of surgical survivors, prognostic factors and effective treatment options need to be further studied.

Some inherent selection bias may limit this article due to differences in medical experience, and the ability to identify and report cases in different provinces and cities. And FLA encephalitis is a rare case, the sample size is small, and the conclusions are limited. Patients present with fever, headache and vomiting, which are very similar to bacterial or viral encephalitis and are very prone to misdiagnosis. Misdiagnosis is more likely to occur when FLA is coinfected with bacteria and viruses. This misdiagnosis can easily result in patients not receiving timely and effective treatment, since FLA is the main responsible for encephalitis. Due to the common misdiagnosis of bacterial meningitis, numerous cases may remain unreported.

FLA encephalitis is a rare but pathogenic infection that currently lacks specificity in clinical, laboratory, and imaging manifestations, despite advances in clinical understanding, diagnostic methods, and treatment. Increasing awareness of FLA encephalitis could improve the detection and reporting of cases, thereby increasing our knowledge of the disease. Rapid clinical diagnosis relies primarily on the vigilance of clinical physicians and rapid pathogenic detection methods. NGS of the CSF is an unbiased and rapid method, and it can lead to earlier diagnosis and intervention. Therefore, we emphasize the early identification of encephalitis caused by this less common pathogen to enable early diagnosis and subsequent treatment, improving patient outcomes.

X-TC, QZ, and C-QZ participated in the study concept and design. X-TC, S-YW, and F-FC participated in the acquisition, statistical analysis, and interpretation of the results. X-TC, QZ, S-YW, F-FC, and C-QZ prepared the manuscript. X-TC and C-QZ revised the draft paper for content. All authors contributed to the article and approved the submitted version.