**4. Diagnosis**

Identification of *Abiotrophia* and *Granulicatella* spp. in blood cultures is extremely difficult. NVS do not grow well in subcultures and may be regarded as contaminant bacteria. Their extreme pleomorphism may lead to misidentification of these bacteria as other bacteria, even fungi [36, 37]. Christensen and Facklam [38] studied 101 NVS isolates and reported that isolates were gram variable and pleomorphic, forms varied from bacilli with spore like swellings to cocci predominantly in pairs and chains when gram stain preparations were made from agar plates. NVS can demonstrate bulbous swelling and filament formation and they can form rough colony morphology on chocolate agar that can be suggestive of other microorganisms such as *Streptobacillus moniliformis* or *Erysipelothrix rhusiopathiae* [36].

NVS show morphologic variations depending on the pyridoxal concentrations in the growth medium [39]. Due to the difficulties in identification of these bacteria, it is crucial for microbiology staff to be vigilant and be aware of the pleomorphic nature of the NVS to prevent misidentification.

NVS should be suspected when gram stain shows microbial cells but cultures are negative [2]. Once their nutritional growth requirements are supplemented in media, NVS convert to streptococci-like cells [39] and gram positivity making them easier to identify, although it was also shown that correcting nutritional deficiency may not convert all abnormalities [40]. For *G. elegans*, addition of cysteine to growth media would have the effect of reversal of pleomorphic morphology but addition of pyridoxal HCl does not [29]. The difficulty in identifying these organisms leads to delays in diagnosis and thus timely initiation of appropriate antimicrobial treatment [41].

Contemporary blood culture methods enable *Abiotrophia* and *Granulicatella* spp. to grow routinely, visible colonies of these organisms appear in 48 h on subculture from positive blood culture media supplemented with *Brucella* and chocolate subculture media [3].

Cargill et al. noted that anaerobic blood culture bottles became positive sooner than aerobic blood cultures bottles; (3.56 h, standard deviation 8.49 h) although they noted that this was not significant or reliable [31].

Specific phenotypic characteristics of NVS can be identified by examining their patterns of production of α-galactosidase, β-galactosidase, β-glucosidase, N-acetyl-β-glucosaminidase and β-glucuronidase, and fermentation of trehalose, pullulan, tagatose and sucrose [32, 38].

*A. defectiva* produces α-galactosidase, β-galactosidase and produces acid from trehalose, sucrose and pullulan. Its acid production from tagatose is variable [38].

*G. adiacens* produces β-glucuronidase and produces acid from sucrose and tagatose. *G. elegans* hydrolyses arginine. Its hippurate hydrolysis is variable. It produces acid from sucrose*. G. balaenopterae* hydrolyses arginine and produces acid from trehalose and pullulan. [38]

*G. para-adiacens* produces β-glucosidase, does not produce α- or β-galactosidase or arginine and does not ferment trehalose, pullulan or tagatose [32].

Molecular diagnostic techniques can be used for rapid and accurate diagnosis of NVS in blood or tissue samples. PCR amplification of 16S rRNA and restriction fragment length polymorphism (RFLP) for routine detection of NVS was developed by Ohara-Nemoto et al. in 1997 [42]. For culture negative infective endocarditis, molecular techniques appear to be more sensitive in resected valvular tissue compared to blood samples [43].

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a fast, reliable and cost-effective technique used to identify microorganisms by utilizing MALDI-TOF MS devices in the clinical microbiology labs [44]. These devices carry the potential to complement or replace the phenotypic identification of various microorganisms including bacteria [44]. MALDI-TOF MS is a rapid and accurate diagnostic tool that has been used to identify and timely diagnose NVS [45].

In culture negative endocarditis, *Abiotrophia* and *Granulicatella* spp. should be suspected and supplemented media should be performed for the organisms to grow. Once the growth is achieved, PCR amplification of 16S rRNA or MALDI-TOF mass spectrometry can be utilized for rapid and accurate diagnosis [46].
