Recent update in diagnosis and treatment of human pythiosis

Vascular pythiosis

Vascular pythiosis usually affects the arteries of lower extremities (Fig. 2). Skin is likely a portal of entry where the zoospore of P. insidiosum attaches and germinates to invade arteries and surrounding tissue. Typical patients have an underlying hematological disorder and an agriculture-related occupation. Most patients present with chronic non-healing skin lesions, arterial insufficiency syndrome (i.e., intermittent claudication, paresthesia, absence of arterial pulse, and evidence of arteritis or thrombosis), gangrenous ulcer, and pulsatile mass (i.e., a groin mass of the iliac or femoral aneurysm, and an abdominal mass of aortic aneurysm) (Krajaejun et al., 2006b; Permpalung et al., 2015; Reanpang et al., 2015; Sermsathanasawadi et al., 2016). Skin changes (i.e., color and texture), as the sequelae of arterial insufficiency, can be observed in patients with vascular pythiosis. The infection usually ascends to a proximal arterial issue, which leads to thrombosis, arterial occlusion, and aneurysm (ruptured aneurysm results in mortality). Vascular pythiosis of the upper extremities has been occasionally reported (Worasilchai et al., 2018; Khunkhet, Rattanakaemakorn & Rajatanavin, 2015). There are only a few cases of carotid artery involvement, an extremely rare but potentially fatal condition that results in meningitis, cerebral septic emboli, brain abscesses, and death. Patients with an immunosuppressed or neutropenic condition may present with abrupt-onset infection (i.e., within days after the onset of symptoms), with or without a history of exposure to aquatic habitats (Chitasombat et al., 2018a; Hoffman, Cornish & Simonsen, 2011; Hilton et al., 2016), and develop necrotic skin lesions/cellulitis that progress to vascular infections shortly afterward (within a few days or weeks).

Early recognition of pythiosis and the use of a rapid laboratory test leads to prompt and definitive therapy. Laboratory investigation (see the “Laboratory diagnosis” section) is essential for the identification of the P. insidiosum infection. Specimen handling is a critical step to enhance the success rate of the laboratory investigation. Specimens such as blood clot, pus, and vessel tissue (at the site of thrombosed vessels and proximal margin), should be sent, without formalin preservation, to a clinical microbiology laboratory for culture identification, staining, and/or molecular testing. Resected vessel and soft tissue specimen sent for histological examination should be labelled—proximal or distal—in order to identify the disease-free surgical margin by Grocott-Gömöri methenamine silver (GMS) staining (Sermsathanasawadi et al., 2016). Further immunohistochemistry staining for P. insidiosum is helpful to confirm the diagnosis, especially if the standard microbiological culture is not available or fails to identify the pathogen (Inkomlue et al., 2016; Keeratijarut et al., 2009).

Ocular pythiosis

Patients with ocular pythiosis (also known as Pythium keratitis) usually have a history of predisposing factors (see the “Predisposing factors” section), and present with ocular pain, irritation, photophobia, decreased visual acuity, conjunctival redness, and eyelid swelling, similar to another microbial keratitis. Clinical onset of ocular pythiosis could range from two days to over a month. Pythium keratitis is often clinically indistinguishable from fungal keratitis, because the causative agents share some clinical features (such as, the presence of grayish-white stromal infiltrates with feathery margins on slit-lamp biomicroscopy) and microscopic findings (such as, septate linear branching structures in a corneal scraping sample). However, the natural history and clinical clues at the margin or surrounding areas of the main infiltrates would help to differentiate Pythium keratitis from fungal keratitis.

Patients with Pythium keratitis usually present with typical clinical manifestations, including multiple, linear, tentacle-like infiltrates and dot-like or pinhead-shaped infiltrates, involving the subepithelial, anterior stromal, and midstromal layers in surrounding cornea and radiating in a reticular pattern from the central area of the lesion towards the limbus (Fig. 3) (Lekhanont et al., 2009; Lelievre et al., 2015; Bagga et al., 2018; Chatterjee & Agrawal, 2018; Agarwal et al., 2019; He et al., 2016; Thanathanee et al., 2013; Sharma et al., 2015; Agarwal et al., 2018). Radial keratoneuritis has been observed in some cases (Lekhanont et al., 2009; He et al., 2016). Corneal lesions should be closely monitored, as the typical features of Pythium keratitis may develop during the course of the disease (Chatterjee & Agrawal, 2018). In advance disease, these clinical clues may be obscured by dense corneal stromal infiltration (Anutarapongpan et al., 2018; Badenoch et al., 2001). The rapid and progressive nature of ocular pythiosis, despite intensive antimicrobial treatment, distinguishes Pythium keratitis from other fungal infections. An extensive and aggressive infiltration of P . insidiosum could result in corneal perforation, anterior chamber, limbal and scleral invasions, or endophthalmitis in a few days or weeks (Lekhanont et al., 2009; Lelievre et al., 2015; Rathi et al., 2018; Agarwal et al., 2019; Badenoch et al., 2001; He et al., 2016; Maeno et al., 2019; Ros Castellar et al., 2017; Thanathanee et al., 2013; Sharma et al., 2015; Agarwal et al., 2018).

Corneal scrapings should be subjected to clinical microbiology examination (i.e., fungal culture, polymerase chain reaction (PCR), and DNA sequencing; see the “Laboratory diagnosis” section) to establish a definitive diagnosis. An ocular specimen is usually obtained in a minimal amount, which could lead to a false-negative result by such laboratory tests. Serological tests are not useful because the serum anti-P. insidiosum antibody in patients with Pythium keratitis is usually undetectable (Krajaejun et al., 2006b; Permpalung et al., 2019; Krajaejun et al., 2009; Jindayok et al., 2009). Repeated corneal cultures or combining specimens, including corneal scrapings and corneal buttons, could increase the chance of a positive result (Hasika et al., 2019).

In vivo confocal microscopy (IVCM) is an emerging, non-invasive, real-time imaging technique that enables morphological and quantitative analysis of ocular surface microstructure in both health and disease. Clinically, IVCM is a useful diagnostic tool for the early diagnosis of microbial particularly fungal and Acanthamoeba keratitis (Alzubaidi et al., 2016). P. insidiosum hyphae can be identified using IVCM in 95% of culture- or PCR-positive patients (Anutarapongpan et al., 2018). Pythium keratitis manifest as hyperreflective branching structures (vary in size; 1.5–7.5 µm in diameter and 90-400 µm in length), septate linear branching structures resembling fungal filaments, and irregular filaments arranged in a sinuous, various branching pattern, forming “X”,” Y”, and” S” shapes as seen by IVCM (Lelievre et al., 2015; He et al., 2016; Tanhehco et al., 2011; Thanathanee et al., 2013). However, microbial characteristics captured by IVCM are non-specific and overlapped among Pythium, Aspergillus, and Fusarium keratitis (Anutarapongpan et al., 2018). Nevertheless, IVCM can provide rapid visualization of Pythium hyphae in infected cornea and is recommended when Pythium keratitis is suspected (Anutarapongpan et al., 2018).

Unusual features of pythiosis

Patients with pythiosis can present with various skin manifestations, such as vesicle/bulla, skin ulcer, cellulitis, chronic swelling, painful subcutaneous lesion, infiltrative lump and ulcer on the limb (Krajaejun et al., 2006b) and rapidly progressive P. insidiosum infection (i.e., necrotizing cellulitis) (Krajaejun et al., 2006b; Kirzhner et al., 2015). If left untreated, cutaneous/subcutaneous infection could progress to vascular pythiosis, which can lead to amputation of the affected limb (Krajaejun et al., 2006b; Khunkhet, Rattanakaemakorn & Rajatanavin, 2015). Craniofacial infection, such as periorbital/orbital necrotizing cellulitis, is a rapid-onset clinical feature observed in children and young adults with a recent history of exposure to wetland (Krajaejun et al., 2006b; Hilton et al., 2016; Kirzhner et al., 2015; Triscott, Weedon & Cabana, 1993). Rarely, P. insidiosum infection can disseminate to a visceral organ and result in mortality (Krajaejun et al., 2006b), which in a patient with gastrointestinal involvement leads to upper gastrointestinal bleeding, bloody mucous stools, gastric/ileal ulcer, and peritonitis (Krajaejun et al., 2006b).

Host (intrinsic) factors

Vascular pythiosis has been strongly associated with thalassemia (Krajaejun et al., 2006a; Permpalung et al., 2015; Worasilchai et al., 2018; Reanpang et al., 2015; Sermsathanasawadi et al., 2016; Thitithanyanont et al., 1998). When stimulate with P. insidiosum zoospore, peripheral blood mononuclear cells collected from thalassemic patients showed lower level of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon gamma (IFN-γ) production, compared with the non-thalassemic controls (Ud-Naen et al., 2019). Other hematologic disorders that predispose individuals to vascular pythiosis include paroxysmal nocturnal hemoglobinuria, aplastic anemia, myelodysplasia, idiopathic thrombocytopenic purpura, and leukemia (Krajaejun et al., 2006b), and to a lesser extent, young age, alcoholism, malnourishment, immunosuppression, HIV infection, cancer, and neutropenia (Krajaejun et al., 2006b; Pan et al., 2014; Shenep et al., 1998; Chitasombat et al., 2018a; Hoffman, Cornish & Simonsen, 2011; Hilton et al., 2016; Kirzhner et al., 2015; Franco et al., 2010; Chitasombat et al., 2018b).

In contrast to vascular pythiosis, ocular pythiosis commonly affects healthy individuals (Krajaejun et al., 2006b; Lekhanont et al., 2009; Krajaejun et al., 2004). However, in a series reported from Northeastern Thailand, 50% of patients with Pythium keratitis had thalassemia hemoglobinopathy (Thanathanee et al., 2013), though the prevalence, and the heterogeneity, of thalassemia hemoglobinopathies is high among Thai people living especially in Northeastern Thailand. Thus, whether thalassemia is a predisposing factor in ocular pythiosis is not known, warranting the need for additional studies to understand the relationship between ocular pythiosis and thalassemia hemoglobinopathies. Additionally, a fulminant form of Pythium keratitis can be observed in immunocompromised patients (such as those with diabetes or Crohn’s disease treated with immunotherapy), which resulted in poor clinical outcomes (i.e., enucleation) (Rathi et al., 2018; Neufeld et al., 2018).

Environmental (extrinsic) factors

P. insidiosum inhabits stagnant water and soil, and colonizes aquatic vegetation (Supabandhu et al., 2008; Vanittanakom et al., 2014; Mendoza, Hernandez & Ajello, 1993). Potential risk factors for pythiosis include farming exposure, direct contact with a water resource (i.e., lake, river, lagoon, swamp, or even swimming pool), and a history of travel to endemic countries, such as Thailand (Krajaejun et al., 2006b; Lelievre et al., 2015; Hasika et al., 2019; Appavu, Prajna & Rajapandian, 2019). Inappropriate hygiene of contact lens can pose a risk (Lekhanont et al., 2009; Lelievre et al., 2015; Badenoch et al., 2001; Maeno et al., 2019; Bernheim et al., 2019; Tanhehco et al., 2011; Barequet, Lavinsky & Rosner, 2013; Neufeld et al., 2018; Raghavan et al., 2018), and exposure to dust or some foreign body in their eyes (Bagga et al., 2018; Hasika et al., 2019) predisposes individuals with no agricultural activity to this infection. The incidence of pythiosis follows a seasonal trend (Hasika et al., 2019; Thanathanee et al., 2013) with outbreaks of Pythium keratitis occurring during the rainy season in Thailand (Thanathanee et al., 2013) and around June to September during monsoon season in South India (Hasika et al., 2019).

Direct examination

Clinical specimens (i.e., purulent discharge, corneal scraping, blood clot, and pathologic tissue) obtained from a lesion are subjected to direct wet mount examination, using 10% KOH preparation (Krajaejun et al., 2006b; Mendoza, Ajello & McGinnis, 1996). Although the results obtained may not be specific, microscopic observation of broad and occasionally sparsely septate filamentous elements alerts to a further identification of the causative organism (i.e., P. insidiosum and some other pathogenic fungi).

Sample handling and culture identification

Transportation of an infected patient tissue with suspected pythiosis to a clinical microbiology laboratory is a critical step for successful isolation of P. insidiosum. Temperature control is essential to ensure viability, and failure to isolate P. insidiosum is linked to storage of clinical specimen in low-temperature containers, such as icebox, refrigerator, or freezer (Brown & Roberts, 1988; Bentinck-Smith et al., 1989). Optimal growth of P. insidiosum occurs between 28 °C and 37 °C and extreme temperature as low as 8 °C or as high as 42 °C can completely inhibit or even kill the organism (Krajaejun et al., 2010). When immediate transportation of a culture specimen is not possible, it is recommended to store the sample in sterile distilled water while transferring to the clinical microbiology laboratory (Mendoza, Ajello & McGinnis, 1996).

P. insidiosum grows well on standard agar types, such as Sabouraud dextrose agar, potato dextrose agar, corn meal agar, and blood agar (Chaiprasert et al., 1990; Grooters et al., 2002). After inoculation of a viable P. insidiosum-containing tissue on agar of choice, a yellow-to-white or colorless-to-white flat colony can be observed within a few days (the color may depend on the culture medium used and the age of the colony), with a radial growth rate of ∼5 mm/day (De Cock et al., 1987; Krajaejun et al., 2010). Under the microscope, P. insidiosum appears as a right-angle branching, sparsely septate, broad (up to 10 µm in diameter) hyphae (Mendoza, Ajello & McGinnis, 1996). Zoosporangium (a sac formed at the hyphal tip that contains multiple mobile biflagellate zoospores) is a unique characteristic that differentiates P. insidiosum from fungi. Zoosporogenesis can be induced in an experienced laboratory by using sterile grass leaves and the induction solution, as described in details elsewhere (Chaiprasert et al., 1990; Mendoza & Prendas, 1988). Although P. insidiosum is a prominent pathogenic oomycete of humans, other oomycetes, such as Lagenidium spp. and Pythium aphanidermatum have also been reported, to a much lesser extent, as a human pathogen (Calvano et al., 2011; Reinprayoon et al., 2013; Farmer et al., 2015). Of late, the accurate identification of P. insidiosum requires immunodiagnostic, molecular, or proteomic assay (Badenoch et al., 2001; Vanittanakom et al., 2004; Intaramat et al., 2016; Krajaejun et al., 2018).

Serological tests and biomarkers

Detection of anti-P. insidiosum antibody can facilitate the diagnosis of pythiosis. Several methods have been developed to detect serum P. insidiosum-specific antibodies. Two types of crude proteins, (i) culture filtrate antigen (CFA) and (ii) soluble antigen from broken hyphae (SABH) (Krajaejun et al., 2006a), extracted from P. insidiosum serve as antigens for serological tests. Immunodiffusion (ID) is a simple, cost-effective, and specific assay that is designed to observe in-gel immunoprecipitation lines generated by anti-P. insidiosum antibodies in patient serum and the P. insidiosum crude protein extract (Imwidthaya & Srimuang, 1989; Pracharktam et al., 1991). Major drawbacks of ID include poor detection sensitivity leading to a false-negative result, and longer turnaround time (∼24 h) (Krajaejun et al., 2009; Jindayok et al., 2009; Krajaejun et al., 2002; Chareonsirisuthigul et al., 2013). An enzyme-linked immunosorbent assay (ELISA) has circumvented the limitation of ID, as it shows higher sensitivity and shorter turnaround time (∼3–4 h) (Intaramat et al., 2016; Krajaejun et al., 2002; Chareonsirisuthigul et al., 2013). Western blot (WB) for immunodetection of P. insidiosum (Krajaejun et al., 2006a; Supabandhu et al., 2009) has been developed though its diagnostic application is limited due to the multi-step procedures involved and complexity. Hemagglutination (HA) and immunochromatographic test (ICT) are rapid and user-friendly assays that detect anti-P. insidiosum antibodies within 30–60 min (Krajaejun et al., 2009; Jindayok et al., 2009; Intaramat et al., 2016). However, implementation of a serodiagnostic test in a non-endemic region is challenging. Transportation of the specimen could impact the assay turnaround time.

Comparison of the above-mentioned serological assays, using a set of 37 pythiosis patients and 248 control sera, shows that ELISA (sensitivity, 100%; specificity, 100%) and ICT (sensitivity, 100%; specificity, 100%) outperform ID (sensitivity, 68%; specificity, 100%) and HA (sensitivity, 84%; specificity, 82%) (Chareonsirisuthigul et al., 2013). Evaluation of ELISA and ICT using a different set of 28 pythiosis and 80 control sera, demonstrates the greater sensitivity of ELISA compared with ICT (96% vs. 86%) (Intaramat et al., 2016). Each serological assay has its own advantages and disadvantages. Selection of an appropriate test for serodiagnosis of pythiosis depends on assay availability, experience of the laboratory personnel, preferred test performance and turnaround time. Most importantly, the serological assays described here often fail to detect anti-P. insidiosum antibodies in patients with ocular pythiosis (Krajaejun et al., 2009; Jindayok et al., 2009; Intaramat et al., 2016). Therefore, one should be aware of false-negative results when performing the assays on samples from an ocular patient.

During the clinical course of pythiosis, levels of anti-P . insidiosum antibodies can be monitored using one of the serological tests. A gradual decrease in anti-P. insidiosum antibody levels over the course of months, in association with clinical improvement, indicate favorable prognosis (Jindayok et al., 2009; Pracharktam et al., 1991; Krajaejun et al., 2002). In contrast, increased or sustained antibody levels may be observed after administration of the P. insidiosum immunotherapeutic antigen (PIA) vaccine, prepared from crude protein extracts of the pathogen, in clinically-cured pythiosis patients (Worasilchai et al., 2018; Krajaejun et al., 2002). Recently, Worasilchai et al. (2018) reported the use of serum β-d-glucan as a surrogate marker for monitoring 50 vascular pythiosis patients after a combination of medical and surgical treatment. A decrease in β-d-glucan levels was observed over a few months in 45 patients who survived, in contrast to the high level of β-d-glucan in five patients who died. However, the serum β-d-glucan was not specific to P. insidiosum and therefore, all possible causes that affect β-d-glucan level should be ruled out to better understand the role of β-d-glucan.

Histological examination

Standard histological stains, such as GMS and Periodic acid-Schiff (PAS) demonstrate the presence and extent of P. insidiosum in infected tissues (Krajaejun et al., 2006b; Keeratijarut et al., 2009; Mendoza, Prasla & Ajello, 2004). However, such stains may not differentiate P. insidiosum from other fungi (i.e., Aspergillus spp., Fusarium spp., Mucorales). Hematoxylin and eosin (H&E) staining shows tissue reactions, such as infiltration of eosinophils, purulent granuloma, and giant cells at the P. insidiosum infection site (Krajaejun et al., 2006b).

As opposed to the standard stains (i.e., GMS, PAS and H&E), several immunohistological staining assays have been developed to facilitate microscopic detection of P. insidiosum (Inkomlue et al., 2016; Keeratijarut et al., 2009; Triscott, Weedon & Cabana, 1993; Mendoza, Ajello & McGinnis, 1996; Mendoza, Kaufman & Standard, 1987; Brown et al., 1988). These assays rely on the use of rabbit antiserum generated against crude protein extract of P. insidiosum. For example, Mendoza et al. implemented a direct immunofluorescent assay using the rabbit anti-P. insidiosum antibodies conjugated with fluorescein isothiocyanate (Mendoza, Ajello & McGinnis, 1996; Mendoza, Kaufman & Standard, 1987). Brown et al. (1988), Triscott, Weedon & Cabana (1993), and Keeratijarut et al. (2009) introduced indirect immunochemical assays that require sequential staining reactions with rabbit anti-P. insidiosum antibodies (primary antibody), mouse or bovine anti-rabbit IgG (secondary antibody) tagged with peroxidase, and enzymatic substrates. These immunostaining assays require careful examination under a light microscope to confirm the presence of P. insidiosum in infected tissues. Interpretation of results may be complicated by false-positive staining of other fungi, such as Conidiobolus and Fusarium species (Keeratijarut et al., 2009; Grooters & Gee, 2002). Thus, confirmation of the P. insidiosum infection requires additional laboratory investigations. When fungal elements are histologically detected by GMS or PAS, a negative immunostaining result could exclude the possibility of P. insidiosum, due to the high sensitivity and negative predictive value of the tests (Keeratijarut et al., 2009).

A novel immunohistological method targeting elicitins that are only present in oomycetes, including P. insidiosum (Jiang et al., 2006; Lerksuthirat et al., 2015), has been reported to be highly specific. Recently, Inkomlue et al. used a rabbit antiserum against recombinant elicitin (ELI025) of P. insidiosum for the development of an immunohistochemical assay (Inkomlue et al., 2016), and found that all 38 P. insidiosum samples were detected, but not 49 control samples of various fungi, including Fusarium species and Mucorales.

Molecular analysis

Conventional molecular techniques used to detect P. insidiosum are sequence homology analysis and PCR. The most popular target sequence is rDNA (also known as ribosomal RNA gene repeat). The rDNA contains 18S ribosomal RNA, internal transcribed spacer 1, 5.8S ribosomal RNA, internal transcribed spacer 2, and 28S ribosomal RNA (Grooters & Gee, 2002). These molecular techniques require a combination of universal fungal- or P. insidiosum-specific primers, template DNA extracted from pure culture or infected tissue, PCR reagents and equipment, and DNA sequencing facility (Badenoch et al., 2001; Vanittanakom et al., 2004; Znajda, Grooters & Marsella, 2002). Use of molecular techniques can substitute microbiological and immunological methods, especially when the latter fail to detect P. insidiosum. Badenoch et al. used universal fungal primers to amplify and sequence a DNA fragment from an organism isolated from a patient with an unknown ocular infection (Badenoch et al., 2001). Homology analysis of the obtained sequence (by BLAST searching against the GenBank database) identified the organism as P. insidiosum. Although the sequence homology analysis is a time-consuming and multi-step procedure, it is a standard method for the identification of many microorganisms, including P. insidiosum. One reason is that most reagents and tools required for this test are readily available in many clinical microbiology laboratories.

Using species-specific primers, PCR amplification can identify P. insidiosum from pure culture or infected tissue without DNA sequencing of the amplicon (Vanittanakom et al., 2004; Grooters & Gee, 2002; Znajda, Grooters & Marsella, 2002). Compared to sequence homology analysis, the PCR assay significantly reduces turnaround time from days to hours. Grooters et al. and Znajda et al. have co-developed a nested PCR assay, using the universal fungal primers (ITS1 and ITS2 or ITS2P) for the first-round reaction and the species-specific primers (PI-1 and PI-2) for the second-round reaction to specifically amplify P. insidiosum DNA (Grooters & Gee, 2002; Znajda, Grooters & Marsella, 2002). Vanittanakom et al. showed that the primers PI-1 and PI-2 (Grooters & Gee, 2002) failed to detect a Thai P. insidiosum strain. Therefore, a new set of P. insidiosum-specific primers (ITSpy1 and ITSpy2) was used in a simplified one round-PCR reaction (Vanittanakom et al., 2004) that was able to detect all four Thai strains tested. Thongsri et al. reported a single-tube nested PCR using a different set of primers (CPL6, CPR8, YTL1, and YTR1) for specific detection of P. insidiosum (Thongsri et al., 2013). More recently, Rujirawat et al. developed a multiplex PCR assay, using four primers (ITS1, Ra, R2, and R3) to target several single nucleotide polymorphisms of rDNA (Rujirawat et al., 2017). The multiplex PCR assay can simultaneously identify and genotype P. insidiosum.

Keeratijarut et al. showed that the rDNA-targeting primers, ITSpy1 and ITSpy2, were unable to PCR amplify certain P. insidiosum strains (Keeratijarut et al., 2014). They proposed the exo-1,3- β-glucanase-encoding gene (exo 1) of P. insidiosum as an alternative PCR target, and designed exo 1-specific primers, Dx3 and Dx4, for a conventional PCR assay. Based on a head-to-head comparison for PCR-based detection of 35 Thai P. insidiosum strains, the primers, Dx3 and Dx4, showed higher detection sensitivity than with ITSpy1 and ITSpy2 (100% vs. 91%) (Keeratijarut et al., 2014). Besides, the primers, Dx3 and Dx4, did not amplify non-specific target DNA from a variety of fungal species tested, showing 100% detection specificity. Keeratijarut et al. introduced an exo 1-targeting real-time PCR assay using the primers, Pr77 and Pr78, for detection of P. insidiosum (Keeratijarut et al., 2015). The detection sensitivity (100%) and specificity (100%) of the real-time PCR was high (Keeratijarut et al., 2015), but shortened the assay turnaround time by eliminating laborious and toxic steps (i.e., gel electrophoresis and ethidium bromide staining).

Proteomic analysis

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a powerful diagnostic tool (Singhal et al., 2015; Sanguinetti & Posteraro, 2017; Jang & Kim, 2018) that can be applied for accurate identification of clinically-important microorganisms at low cost and short turnaround time. Unlike serological and molecular assays, MALDI-TOF MS does not require pathogen-specific reagents, such as primer, antigen, and antibody. Crude proteins of an unknown microorganism are extracted using an optimized protocol and subjected to the generation of main spectral profile (MSP) by MALDI-TOF MS analysis. The obtained MSP is searched against a reference database containing MSPs of many typed microorganisms. The matched organism with a significant score is, therefore, the identity of the unknown sample. Large mass spectrometric databases of a wide variety of microorganisms are currently available and accessible to assist the MALDI-TOF MS-based microbial identification (Singhal et al., 2015; Lau et al., 2013; Becker et al., 2014). Several mass spectrometric databases are now supplemented with the P. insidiosum MSPs aiding the identification of P. insidiosum (Bernheim et al., 2019; Krajaejun et al., 2018; Mani et al., 2019). The microorganism in question is reported as P. insidiosum if the MSP generated significantly matched its corresponding mass spectrum deposited in the reference database.

Vascular pythiosis

Ocular pythiosis

Clinical outcomes