|Year : 2015 | Volume
| Issue : 4 | Page : 538-546
Ocular infections caused by Candida species: Type of species, in vitro susceptibility and treatment outcome
SR Motukupally1, VR Nanapur1, KN Chathoth1, SI Murthy2, RR Pappuru3, A Mallick4, S Sharma1
1 Jhaveri Microbiology Centre, Lakshmi Vara Prasad Eye Institute, Hyderabad, Telangana, India
2 Cornea and Anterior Segment Service, Lakshmi Vara Prasad Eye Institute, Hyderabad, Telangana, India
3 Smt. Kanuri Santhamma Retina-Vitreous Service, Lakshmi Vara Prasad Eye Institute, Hyderabad, Telangana, India
4 Ocular Microbiology Service, Lakshmi Vara Prasad Eye Institute, Bhubaneswar, Odisha, India
|Date of Submission||06-Jun-2014|
|Date of Acceptance||15-Jan-2015|
|Date of Web Publication||16-Oct-2015|
Jhaveri Microbiology Centre, Lakshmi Vara Prasad Eye Institute, Hyderabad, Telangana
Source of Support: None, Conflict of Interest: None
Purpose: To report clinical and microbiological profile of patients with ocular candidiasis. Materials and Methods: Patients with ocular candidiasis were retrospectively identified from microbiology records. Significant isolates of Candida species were identified by Vitek 2 compact system. Minimum inhibitory concentration (MIC) of antifungal agents such as amphotericin B, itraconazole, voriconazole, fluconazole and caspofungin was determined by E test and of natamycin by microbroth dilution assay. Data on treatment and outcome were collected from medical records. Results: A total of 42 isolates of Candida were isolated from patients with keratitis-29, endophthalmitis-12 and orbital cellulitis-1. The most common species isolated was Candida albicans (12-keratitis, 4-endophthalmitis, 1-orbital cellulitis). All except one isolate were susceptible to amphotericin B. MIC of caspofungin was in the susceptible range in 28 (96.5%) corneal isolates while 12 out of 29 (41.3%) corneal isolates were sensitive to fluconazole. Resistance to voriconazole was seen in four corneal isolates. All isolates were susceptible to natamycin and all except two isolates were resistant or susceptible dose-dependent to itraconazole. Outcome of healed ulcer was achieved in 12/18 (66.6%) patients treated medically, while surgical intervention was required in 11 patients. Among the isolates from endophthalmitis patients, 11/12 were susceptible to amphotericin B, 6/12 to voriconazole and all to natamycin. Ten out of 11 patients (one patient required evisceration) with endophthalmitis were given intravitreal amphotericin B injection with variable outcome. Conclusions: Ocular candidiasis needs early and specific treatment for optimal results. Candida species continue to be susceptible to most commonly available antifungals including amphotericin B, voriconazole and natamycin.
Keywords: Candida, E test, minimum inhibitory concentration, ocular infections, outcome
|How to cite this article:|
Motukupally S R, Nanapur V R, Chathoth K N, Murthy S I, Pappuru R R, Mallick A, Sharma S. Ocular infections caused by Candida species: Type of species, in vitro susceptibility and treatment outcome. Indian J Med Microbiol 2015;33:538-46
|How to cite this URL:|
Motukupally S R, Nanapur V R, Chathoth K N, Murthy S I, Pappuru R R, Mallick A, Sharma S. Ocular infections caused by Candida species: Type of species, in vitro susceptibility and treatment outcome. Indian J Med Microbiol [serial online] 2015 [cited 2019 Dec 15];33:538-46. Available from: http://www.ijmm.org/text.asp?2015/33/4/538/167331
| ~ Introduction|| |
Fungi are associated with opportunistic infections of the eye. The epidemiological pattern of fungal infections varies from country to country and even in different regions of the same country. Systemic and immune status of the individual may also determine predisposition towards fungal infection. Of all tissues of the eye, cornea is most susceptible to fungal infection. In India, prevalence of fungal keratitis varies from 7.3% to 44.8% in microbial keratitis reported from different parts of India. Fungal endophthalmitis is less common than fungal keratitis accounting for 8% to 16% of infective endophthalmitis in different reports and varies from place to place., In the Indian subcontinent, filamentous fungi such as Aspergillus species and Fusarium species are more commonly associated with keratitis and endophthalmitis compared to infection by yeast or yeast-like fungi.,, Amongst the yeasts, Candida remains the most common etiological agent of ocular infections world-wide. Risk factors for ocular Candida infections include excessive use of corticosteroids and antibiotics, contact lens wear, surgical procedures and diseases associated with immunodeficiency apart from trauma and co-existence of systemic or local underlying diseases. These infections are associated with significant morbidity and visual impairment and require early diagnosis and treatment with appropriate antifungal drugs.
Fungal infections continue to be challenging to ophthalmologists, both in terms of diagnosis and treatment. Therapeutic failures are high and poor outcome is attributed to factors such as delay in diagnosis, underlying host factors, poor drug penetration and resistance to antifungal drugs among some fungal species.,,
Candida infections are generally treated with amphotericin B, although flucytosine and fluconazole have also been recommended for the treatment of Candida infections of the eye.,, Although several drugs are used for treatment, very limited literature is available regarding antifungal susceptibility of Candida isolated from ocular infections., There is only one study that has reported type of Candida species prevalent in Indian patients with ocular infections and their antifungal susceptibility. The species identification was based on biochemical tests and the authors used microbroth dilution test to determine minimum inhibitory concentration (MIC). No attempt was made for clinical correlation. In order to formulate guidelines for the treatment of ocular candidiasis in Indian patients such a study would be useful. At our institute, all patients with ocular infection are investigated in the microbiology laboratory following a uniform guideline. This study was undertaken to test all isolates of Candida to determine the species and their susceptibility to various antifungal agents. We also analyzed the treatment that these patients received and their outcome.
| ~ Materials and Methods|| |
Forty-two clinical samples (corneal scrapings-29, vitreous fluid-12, pus-1) from patients with various eye infections, seen at L. V. Prasad Eye Institute between September 2010 and March 2014 were processed as per the protocol described previously. The samples were examined by direct microscopy as well as cultured in standard media. A culture isolate was considered significant if confluent colonies grew on solid medium, and/or if the isolate grew on two media and/or the organism was seen in direct microscopy and grew in even one medium. Significant isolates of Candida species were preserved on Sabouraud dextrose agar slopes at room temperature until tested further. All isolates were subjected to species identification by Vitek 2 compact system using YST strips (bioMérieux, France).
The isolates were tested for their susceptibility to various antifungal agents such as amphotericin B, itraconazole, voriconazole, fluconazole, caspofungin and natamycin, which are in use for treating eye infections. MIC of the drugs was determined by agar diffusion E test (ET) method on Muller Hinton agar containing 2% glucose with 0.005% methylene blue (Himedia, M1825, Mumbai, India) using E strips (bioMèrieux, France) of amphotericin B (0.002–32 μg/ml), itraconazole (0.002–32 μg/ml), voriconazole (0.002–32 μg/ml), fluconazole (0.016–256 μg/ml) and caspofungin (0.002–32 μg/ml) using manufacturer's instructions [Figure 1]. E test strips are not available for natamycin, therefore, 5% eye drop (Natacin, Entod Pharmaceutical Ltd., Mumbai, India) was used by microbroth dilution for testing the MIC of natamycin (final drug concentrations ranging from 128 to 0.25 μg/ml) by the method described earlier. ATCC strain of C. albicans (ATCC 90028) was used for standardization of the E test.
|Figure 1: E test result of Candida albicans isolated from keratitis (Serial no. 1) on Mueller Hinton agar with glucose and methylene blue showing minimum inhibitory concentration (MIC) of amphotericin B - 0.094 µg/ml, itraconazole - 1.0 µg/ml, caspofungin - 0.064 µg/ml, voriconazole - 0.064 µg/ml and fluconazole - > 256 µg/ml|
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The isolates were labeled as susceptible (S), susceptible dose-dependent (SDD) or resistant (R) based on the CLSI M27-S4 document interpretative guidelines for MIC breakpoints: Breakpoints for itraconazole were noted and interpreted in accordance with published literature, as CLSI has not described the interpretative criteria for this antifungal. Based on earlier reports, an isolate was considered susceptible to natamycin if the MIC value for the isolate was ≤16 μg/ml.,,
| ~ Results|| |
During the study period (September 2010 and March 2014), 3,006 patients with culture-positive fungal keratitis and 80 patients with culture-positive fungal endophthalmitis were diagnosed. Majority of the culture isolates were filamentous fungus with yeast-like fungi involved in 29 (0.96%) keratitis and 12 (15%) endophthalmitis patients. With an additional patient with fungal orbital cellulitis, a total of 42 isolates of Candida were included in the study [Table 1]. The most common species isolated was Candida albicans (12-keratitis, 4-endophthalmitis, 1-orbital cellulitis). The other species included C. parapsilosis (10-keratitis, 1-endophthalmitis), C. guilliermondii (3-keratitis), C. ciferrii (2-keratitis), C. glabrata and C. famata (1-keratitis, 1-endophthalmitis) and one isolate each of C. tropicalis, C. pelliculosa and C. utilis. Two isolates from endophthalmitis cases could not be identified to the species level and were labelled as Candida species [Table 1]. The direct microscopy procedures used in the study were able to detect yeast-like budding cells suggestive of Candida species in 18 out of 38 (47.36%) of the samples.
|Table 1: Distribution of various species of Candida isolated from different ocular infections|
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The mean MIC of amphotericin B for the isolates from 29 patients with keratitis was 0.2 µg/ml (range 0.032–0.75 µg/ml). MICs of all drugs tested against the 29 Candida isolates from patients with keratitis are shown in [Table 2]. Taking CLSI break point of ≤1 µg/ml to determine susceptibility, 29 (100%) isolates were susceptible to amphotericin B. MIC of caspofungin was in the susceptible range (≤1 µg/ml) in 28 (96.5%) isolates while one isolate (C. albicans) showed high MIC value (0.38 µg/ml) indicating susceptible dose-dependant.
|Table 2: Minimum inhibitory concentration (MIC) of various antifungal drugs against Candida species isolated from patients with keratitis (n = 29)|
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According to CLSI guidelines, 12 out of 29 (41.3%) corneal isolates were sensitive to fluconazole, 2 were susceptible dose-dependent and 15 isolates were resistant. Resistance to voriconazole was seen in four isolates. On testing with itraconazole two of the isolates were susceptible, remaining being either resistant or susceptible dose-dependent. At break point value of 16 µg/ml, all isolates from patients with keratitis were susceptible to natamycin.
[Table 3] shows the MICs of the drugs against Candida species isolated from patients with endophthalmitis and orbital cellulitis. Twelve out of 13 isolates were susceptible to amphotericin B while all were susceptible to natamycin. They were either resistant or susceptible dose-dependent to itraconazole. Resistance to voriconazole, caspofungin and fluconazole was seen in 2 (15.3%), 3 (23%) and 5 (38.4%) isolates, respectively. The MIC50 and MIC90 of all antifungals against C. albicans and C. parapsilosis, that were the most common isolates, are shown in [Table 4].
|Table 3: Minimum inhibitory concentration (MIC) of various antifungal drugs against Candida species isolated from patients with endophthalmitis and orbital cellulitis|
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|Table 4: Minimum inhibitory concentration (MIC50 and MIC90) of various antifungal drugs against Candida albicans and Candida parapsilosis|
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[Table 5] outlines the visual acuity at presentation, treatment given and outcome in the patients with Candida keratitis. In 17 out of 29 patients, 5% topical natamycin was started based on either clinical suspicion (9 patients) or observation of filamentous fungal elements (8 patients) in the corneal scrapings. Smear examination was not done for four patients, two of whom were also started on natamycin. Of the remaining 7 patients, three received topical 0.15% amphotericin B (Onco division, Wintac Ltd., Bangalore, India), two patients received 1% topical voriconazole (Vozole, Aurolab, Madurai, India) and one patient received fluconazole (0.3%, Zocon, FDC, Proxima, India) eye drops. One patient each received a combination of amphotericin B with either fluconazole or natamycin. Presence of budding yeast-like cells in the direct microscopy [Case 22, [Table 5] accounted for initial treatment with antifungals other than natamycin. Four patients [Table 5], patient no. 6, 16, 21, 29] did not receive antifungal treatment. Seventeen patients were given oral antifungal in the form of either ketoconazole (200 mg twice a day) or fluconazole (200 mg once a day) or itraconazole (100 mg twice a day). Visual acuity at the time of presentation was better than or equal to 20/60 in 3 out of 29 (10.3%) patients. Surgical intervention in 11 patients (11/29, 37.9%) included penetrating keratoplasty (PK) in 7/29 (24.1%) and evisceration in 4 (13.7%) patients. On medical therapy, with or without tissue adhesive application, outcome of healed ulcer was achieved in 12 out of 18 (66.6%) patients with visual acuity better than or equal to 20/60 in 7 out of 18 (38.8%) patients.
|Table 5: Corneal scraping microscopy results, treatment given and outcome in patients with Candida keratitis (n = 26)|
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Eleven patients with clinical endophthalmitis underwent parsplana vitrectomy with intraocular antibiotics (vancomycin and ceftazidime) while one patient was suspected with panophthalmitis and required evisceration [Table 6]. None of the patients were clinically suspected to be fungal. Direct microscopy of vitreous from 4 out of 12 (33.3%) patients showed presence of budding yeast-like cells with rest of them being negative on microscopy. Based on culture report, 10 out of 11 patients were given intravitreal antifungal injection [amphotericin B (5 µg/0.1 ml)-7, voriconazole (50 µg/0.1 ml)-2, both-1]. The lone patient with orbital cellulitis improved with incision and drainage with betadine packing/washing and antibacterial antibiotics because of mixed infection (C. albicans, S. aureus, Klebsiellapneumoniae) but was left with no vision because of long-term orbital infection. Visual acuity at presentation and final visual acuity are shown in [Table 6].
|Table 6: Microscopy results of vitreous/exudate, treatment given and outcome in patients with Candida endophthalmitis (n = 9) and Candida orbital cellulitis (n = 1)|
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| ~ Discussion|| |
The Candida species tested in this study are derived from a large series of patients with ocular infections. The eye infections that may be caused by Candida species range from extraocular (keratitis, orbital cellulitis) to intraocular (endophthalmitis, panophthalmitis). Failure to treat them with specific drugs may lead to irreversible consequences in the form of vision loss. An increased awareness of possible infection with yeast-like fungi is important to initiate appropriate investigation and treatment. This study shows that Candida albicans was the predominant species associated with ocular infection and accounts for 40.4% of all the Candida species isolated from ocular specimens, followed by C. parapsilosis (26.1%) and C. guilliermondii (7.1%). A study from Brazil reports 53.3% of yeast isolates from corneal infections to be C. albicans, followed by C. parapsilosis (20%) and C. tropicalis (20%). On the contrary, a study from South Florida states C. parapsilosis to be the most common species associated with Candida keratitis accounting for 68.7% of Candida keratitis, followed by C. albicans (25%) and C. tropicalis (6.3%).
Going by the published literature, the recommended treatment for Candida keratitis is amphotericin B or voriconazole. Therese et al., tested 50 Candida species in vitro and showed susceptibility to amphotericin B with MICs of <2 µg/ml. This study found low MIC50 and MIC90 of amphotericin B, voriconazole and caspofungin compared to other antifungals for the common Candida isolates from ocular infections. There are no reports of use of natamycin in the treatment of Candida keratitis although natamycin is the most widely available topical antifungal formulation in the world. Our study shows that not only were all Candida species sensitive to natamycin in vitro but also a large number of patients responded to treatment with natamycin. Of the 12 patients who responded to medical therapy, eight were treated with natamycin alone. Based on our observation we believe that natamycin, considered the drug of choice for fungal keratitis caused by filamentous fungi, may be administered for keratitis caused by Candida species and further alteration in drug may be warranted only in case of no response or worsening after 1 week of therapy. This has the added advantage of not having to use amphotericin B that needs to be formulated as eye drop from parenteral drug and is more expensive (natamycin-Rs. 118/vial, amphotericin B-Rs. 330/vial). With current availability of voriconazole eye drop (Aurolab, Madurai, India), however, the alternative drug could be voriconazole (Rs. 265/vial) rather than amphotericin B. With low MICs, both voriconazole and amphotericin B remain promising anti-Candida drugs. Both of these drugs are amenable to intraocular use as well with their margin of safety for retina and are preferred drugs for the treatment of Candida endophthalmitis. Caspofungin emerges as an alternative drug for the treatment of Candida keratitis with 28 out of 29 (96.5%) isolates being sensitive to it. With its limited availability, however, it is out of reach for ophthalmologists at the moment.
This study suffers with many drawbacks inherent in a retrospective study. The results of in vitro antifungal susceptibility were not available at the time of treatment of the patient. It is also obvious that in some cases probably the microbiology report did not reach the physician on time. This delay may account for change of topical treatment in keratitis patients to amphotericin B in some but not all patients with positive microscopy of the corneal scraping for yeast-like cells. It is also to be noted that Candida species may appear as filamentous fungi in corneal scrapings, thereby leading to institution of natamycin as the first line of treatment. A delay of reporting is inherent in the culture method involved but all efforts should be made by the laboratory to convey the result of microscopic examination to the treating physician as early as possible. In contrast to patients with keratitis, patients with endophthalmitis received intraocular antifungal injection, in majority of the cases; however, useful vision was achieved only in 4 out of 12. Poor visual outcome despite treatment has been reported in Candida endophthalmitis which is attributed to immunocompromised state, delayed diagnosis and virulence factors of the micro-organism.
Although laborious and time-consuming, the gold standard for fungal susceptibility tests continues to be broth microdilution method. To overcome the difficulties, several easy-to-use commercial methods such as Fungi test, disk diffusion test and E test have come in to the market. E test results may be affected by the type of media used and the inoculum size and duration of incubation; nevertheless, with proper standardization and quality control, these factors can be controlled. In the current study, E test was used for its simplicity, accuracy and reliability in testing antifungal drugs against yeast.
With due diligence on the part of the microbiology laboratory, it may be possible to obtain an early diagnosis of Candida infection of the eye. Candida species are highly virulent with morphological transition between yeast and hyphal forms (phenotypic switching), expession of adhesins and invasins on the cell surface, formation of biofilms and secretion of hydrolytic enzymes. An early intervention is essential to achieve a favourable outcome. None of the species have developed resistance to the available antifungal drugs except for fluconazole and itraconazole; therefore, the need of the hour is to diagnose and treat early.
Financial support and sponsorship
Hyderabad Eye Research Foundation, Hyderabad.
Conflicts of interest
| ~ References|| |
Rautaraya B, Sharma S, Kar S, Das S, Sahu SK. Diagnosis and treatment outcome of mycotic keratitis at a tertiary eye care center in eastern India. BMC Ophthalmol 2011;11:39.
Kunimoto DY, Das TP, Sharma S, Jalali S, Majji AB, Gopinathan U, et al
. Microbiologic spectrum and susceptibility of isolates. Part I. Postop endophthalmitis. Am J Ophthalmol 1999;128:240-2.
Kunimoto DY, Das TP, Sharma S, Jalali S, Majji AB, Gopinathan U, et al
. Microbiologic spectrum and susceptibility of isolates. Part II. Post traumatic endophthalmitis. Endophthalmitis Research Group. Am J Ophthalmol 1999;128:242-4.
Malathi J, Parameswaran SK, Lysa S, Selvaraj M, Madhavan HN. A study on the incidence, microbiological analysis and investigation on the source of infection of postoperative infectious endophthalmitis in a tertiary care ophthalmic hospital: An 8-year study. Indian J Ophthalmol 2010;58:297-302.
Gupta A, Srinivasan R, Kaliaperumal S, Saha I. Post-traumatic fungal endophthalmitis-a prospective study. Eye (Lond) 2008;22:13-7.
Safneck JR. Endophthalmitis: A review of recent trends. Saudi J Ophthalmol 2012;26:181-9.
O'Day DM, Ray WA, Robinson RD, Head WS, William TE. Differences in response in vivo
to amphotericin B among Candida albicans
strains. Invest Ophthalmol Vis Sci 1991;32:1569-72.
O'Day DM. Selection of appropriate antifungal therapy. Cornea 1987;6:238-45.
Sengupta J, Khetan A, Saha S, Banerjee D, Gangopadhyay N, Pal D. Candida
keratitis Emerging problem in India. Cornea 2012;31:371-5.
Hamada Y, Okuma R, Katori Y, Takahashi S, Hirayama T, Ichibe Y, et al
. Bibliographical Investigation (domestic and overseas) on the treatment of endogenous Candida
endophthalmitis over an 11-year period. Med Mycol J 2013;54:53-67.
Panda A, Sharma N, Angra SK. Topical fluconazole therapy of Candida
keratitis. Cornea 1996;15:373-5.
Mascaro VL, Hofling-Lima AL, Gompertz OF, Zorat Yu MC, Archimedes da Matta D, Colombo AL. Antifungal susceptibility testing of yeast isolated from corneal infections. Arq Bras Oftalmol 2003;66:647-52.
Therese KL, Bagyalakshmi R, Madhavan HN, Deepa P. In-vitro
susceptibility testing by agar dilution method to determine the minimum inhibitory concentrations of amphotericin B, fluconazole and ketoconazole against ocular fungal isolates. Indian J Med Microbiol 2006;24:273-9.
Sharma S. Ocular Microbiology. In: Choudhury Z, Vanathi M, editors. Ch. 5.3. Vol. 1. ed.1 Postgraduate Ophthalmology. New Delhi: Jaypee Highlights Medical Publishers, Inc; 2011. p. 218-40.
Sims CR, Paetznick VL, Rodriguez JR, Chen E, Ostrosky-Zeichner L. Correlation between microdilution, E-test and disk diffusion methods for antifungal susceptibility testing of posaconazole against Candida
spp. J Clin Microbiol 2006;44:2105-8.
Lalitha P, Vijaykumar R, Prajna NV, Fothergill AW.In vitro
natamycin susceptibility of ocular isolates of Fusarium
species: Comparison of commercially formulated natamycin eye drops to pharmaceutical-grade powder. J Clin Microbiol 2008;46:3477-8.
Clinical and Laboratory Standards Institute (CLSI). Reference method for broth dilution antifungal susceptibility testing of Yeasts; approved standard CLSI document M27-A3, 2014, Clinical and Laboratory Standards Institute, Wayne, Pennsylvania, USA.
Arthington-Skaggs BA, Lee-Yang W, Ciblak MA, Frade JP, Brandt ME, Hajjeh RA, et al
. Comparison of visual and spectrophotometric methods of brothmicrodilution MIC end point determination and evaluation of a sterol quantitation method for in vitro
susceptibility testing of fluconazole and itraconazole against trailing and nontrailing candida
isolates. Antimicrob Agents Chemother 2002;46:2477-81.
Pradhan L, Sharma S, Nalamada S, Sahu SK, Das S, Garg P. Natamycin in the treatment of keratomycosis: Correlation of treatment outcome and in vitro
susceptibility of fungal isolates. Indian J Ophthalmol 2011;59:512-4.
Rosa RH Jr, Miller D, Alfonso EC. The changing spectrum of fungal keratitis in South Florida. Ophthalmology 1994;101:1005-13.
Shah CP, McKey J, Spirn MJ, Maguire J. Ocular candidiasis: A review. Br J Ophthalmol 2008;92:466-8.
Guarner J, Brandt ME. Histopathologic diagnosis of fungal infections in the 21st
Century. Clin Microbiol Rev 2011;24:247-80.
Essman TF, Flynn HW Jr, Smiddy WE, Brod RD, Murray TG, Davis JL, et al
. Treatment outcomes in a 10-year study of endogenous fungal endophthalmitis. Ophthalmic Surg Lasers 1997;28:185-94.
Ranque S, Lachaud L, Gari-Toussaint M, Michel-Nguyen A, Malle M, Gaudart J, et al
. Interlaboratory reproducibility of Etest amphotericin B and caspofungin yeast susceptibility testing and comparison with CLSI method. J Clin Microbiol 2012;50:2305-9.
Mayer FL, Wilson D, Hube B. Candida albicans
pathogenicity mechanisms. Virulence 2013;4:119-28.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]