Bovine Lactoferrin and Current Antifungal Therapy Against Candida Albicans: A Systematic Review and Meta-Analysis

Abstract

Candida albicans is an important causative organism of opportunistic fungal infection, and it is a growing medical concern due to the increasing usage of broad-spectrum antibiotics, immunosuppressant agents, and other immunocompromising conditions. Currently, bLf and antifungal drugs have been known to have synergistic effects, increasing the drug’s efficacy. This study aims to investigate the efficacy of the synergistic effect of bLf and antifungal drugs. This review addressed the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. We conducted literature searches to assess the association of lactoferrin and current antifungal therapy against Candida albicans in ProQuest, PubMed, MEDLINE, EBSCOhost, SAGE, JSTOR, GARUDA, and Open Gray with no date restriction (until March 5^th, 2021). We used Jeffry’s Amazing Statistical Program (JASP) to measure the overall size effect of MIC (minimum inhibitory concentration) between studies. A total of 7 studies retained were experimental in vitro studies. Based on the available data, 4 out of 7 studies were included in the quantitative analysis. This systematic review showed that bovine lactoferrin could help inhibit the development of azole-susceptible and azole-resistant C. albicans. Furthermore, there was synergistic activity between lactoferrin and various antifungals. Our meta-analysis showed that lactoferrin could significantly inhibit the C. albicans growth than the control group. Bovine lactoferrin and its peptide derivatives isolated from bovine milk can significantly inhibit the growth of C. albicans, both susceptible to azoles and those with azole resistance.

Introduction

Candida albicans is an important causative organism of opportunistic fungal infection, and it is a growing medical concern due to the increasing usage of broad-spectrum antibiotics, immunosuppressant agents, and other immunocompromising conditions. A recent study estimated around 700,000 cases of invasive candidiasis worldwide annually.[1] Candidemia, the most common form of invasive candidiasis, is associated with high mortality rates ranging from 19.6% to 67% worldwide. Among the patients, they also experience prolonged hospital stays and increased healthcare costs.[2]

According to the World Health Organization, antimicrobial resistance has already become one of humanity’s top 10 global public health threats.[3] Even today, around 700,000 people die yearly because of antimicrobial resistance. This also poses significant problems for Candidiasis treatment as the frequency of azole-resistant Candida increases.[4] One of the modalities to tackle this antimicrobial resistance is an antimicrobial peptide (AMP). AMP’s advantages are that it is not prone to resistance development and has a broad spectrum.[5]

Lactoferrin (LF), a glycoprotein found in saliva, milk, vagina secretions, tears and other exocrine secretions, has been found to have antimicrobial activity and belongs to the AMP group.[6,7,8] Bovine milk is one of the natural sources of LF and has several advantages, such as inexpensive production and good tolerance.[6,8] Bovine lactoferrin (bLf) has been shown to have fungicidal activity against various Candida species, including C. albicans.[9] There is potential for oral bLf supplementation to affect oral candidiasis positively. The murine model revealed that C. albicans cell counts and tongue lesions significantly reduced in LF-treated mice.[10]

Currently, bLf and antifungal drugs have been known to have synergistic effects, increasing the drug’s efficacy. The success of developing new therapy against antifungal-resistant C. albicans will depend on our understanding of underlying resistance and how to exploit it.[11] Because of that, this study aims to investigate the efficacy of the synergistic effect of bLf and antifungal drugs, especially the mechanism to overcome the resistance and assess its potential to be used in a clinical setting.

Methods

This review addressed the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. Supplementary Table 1 shows the PRISMA 2020 checklist.

Supplementary Table 1.

PRISMA 2020 Checklist

Section and Topic	Item #	Checklist item	Reported on Page
TITLE
Title	1	Identify the report as a systematic review.	1
ABSTRACT
Abstract	2	See the PRISMA 2020 for Abstracts checklist.	N/A
INTRODUCTION
Rationale	3	Describe the rationale for the review in the context of existing knowledge.	1-2
Objectives	4	Provide an explicit statement of the objective (s) or question (s) the review addresses.	2
METHODS
Eligibility criteria	5	Specify the inclusion and exclusion criteria for the review and how studies were grouped for the syntheses.	2
Information sources	6	Specify all databases, registers, websites, organisations, reference lists and other sources searched or consulted to identify studies. Specify the date when each source was last searched or consulted.	2
Search strategy	7	Present the full search strategies for all databases, registers and websites, including any filters and limits used.	2
Selection process	8	Specify the methods used to decide whether a study met the inclusion criteria of the review, including how many reviewers screened each record and each report retrieved, whether they worked independently, and if applicable, details of automation tools used in the process.	2
Data collection process	9	Specify the methods used to collect data from reports, including how many reviewers collected data from each report, whether they worked independently, any processes for obtaining or confirming data from study investigators, and if applicable, details of automation tools used in the process.	2
Data items	10a	List and define all outcomes for which data were sought. Specify whether all results that were compatible with each outcome domain in each study were sought (e.g. for all measures, time points, analyses), and if not, the methods used to decide which results to collect.	2
	10b	List and define all other variables for which data were sought (e.g. participant and intervention characteristics, funding sources). Describe any assumptions made about any missing or unclear information.	2
Study risk of bias assessment	11	Specify the methods used to assess risk of bias in the included studies, including details of the tool (s) used, how many reviewers assessed each study and whether they worked independently, and if applicable, details of automation tools used in the process.	2
Effect measures	12	Specify for each outcome the effect measure (s) (e.g. risk ratio, mean difference) used in the synthesis or presentation of results.	2
Synthesis methods	13a	Describe the processes used to decide which studies were eligible for each synthesis (e.g. tabulating the study intervention characteristics and comparing against the planned groups for each synthesis (item #5)).	2
	13b	Describe any methods required to prepare the data for presentation or synthesis, such as handling of missing summary statistics, or data conversions.	2
	13c	Describe any methods used to tabulate or visually display results of individual studies and syntheses.	2
	13d	Describe any methods used to synthesize results and provide a rationale for the choice (s). If meta-analysis was performed, describe the model (s), method (s) to identify the presence and extent of statistical heterogeneity, and software package (s) used.	2
	13e	Describe any methods used to explore possible causes of heterogeneity among study results (e.g. subgroup analysis, meta-regression).	2
	13f	Describe any sensitivity analyses conducted to assess robustness of the synthesized results.	2
Reporting bias assessment	14	Describe any methods used to assess risk of bias due to missing results in a synthesis (arising from reporting biases).	2
Certainty assessment	15	Describe any methods used to assess certainty (or confidence) in the body of evidence for an outcome.	2
RESULTS
Study selection	16a	Describe the results of the search and selection process, from the number of records identified in the search to the number of studies included in the review, ideally using a flow diagram.	2-3
	16b	Cite studies that might appear to meet the inclusion criteria, but which were excluded, and explain why they were excluded.	N/A
Study characteristics	17	Cite each included study and present its characteristics.	2-3
Risk of bias in studies	18	Present assessments of risk of bias for each included study.	2;4
Results of individual studies	19	For all outcomes, present, for each study: (a) summary statistics for each group (where appropriate) and (b) an effect estimate and its precision (e.g. confidence/credible interval), ideally using structured tables or plots.	4-5
Results of syntheses	20a	For each synthesis, briefly summarise the characteristics and risk of bias among contributing studies.	5-6
	20b	Present results of all statistical syntheses conducted. If meta-analysis was done, present for each the summary estimate and its precision (e.g. confidence/credible interval) and measures of statistical heterogeneity. If comparing groups, describe the direction of the effect.	4-5;7
	20c	Present results of all investigations of possible causes of heterogeneity among study results.	4;7
	20d	Present results of all sensitivity analyses conducted to assess the robustness of the synthesized results.	5
Reporting biases	21	Present assessments of risk of bias due to missing results (arising from reporting biases) for each synthesis assessed.	2;4
Certainty of evidence	22	Present assessments of certainty (or confidence) in the body of evidence for each outcome assessed.	7
DISCUSSION
Discussion	23a	Provide a general interpretation of the results in the context of other evidence.	5-7
	23b	Discuss any limitations of the evidence included in the review.	8
	23c	Discuss any limitations of the review processes used.	8
	23d	Discuss implications of the results for practice, policy, and future research.	8
OTHER INFORMATION
Registration and protocol	24a	Provide registration information for the review, including register name and registration number, or state that the review was not registered.	Not registered
	24b	Indicate where the review protocol can be accessed, or state that a protocol was not prepared.	N/A
	24c	Describe and explain any amendments to information provided at registration or in the protocol.	N/A
Support	25	Describe sources of financial or non-financial support for the review, and the role of the funders or sponsors in the review.	N/A
Competing interests	26	Declare any competing interests of review authors.	N/A
Availability of data, code and other materials	27	Report which of the following are publicly available and where they can be found: template data collection forms; data extracted from included studies; data used for all analyses; analytic code; any other materials used in the review.	N/A

From: Page MJ, McKenzie JE, Bossuyt PM, Boutron I, Hoffmann TC, Mulrow CD, et al. The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. BMJ 2021;372:n71. doi: 10.1136/bmj.n71. For more information, visit: http://www.prisma-statement.org/

Literature search

We conducted literature searches to assess the association of LF and current antifungal therapy against Candida albicans on ProQuest, PubMed, MEDLINE, EBSCOhost, SAGE, JSTOR, GARUDA and OpenGrey with no date restriction (until 5 March 2021). The search phrases applied on PubMed are as follows: (((“lactoferrin”) AND (“antifungal”)) AND (“synergis*”)) AND (“candida albicans”). The search limitation was experimental studies published in English. Furthermore, we also conducted a manual search to obtain the potential studies.

Study selection

The criteria of inclusion for the studies were: (1) reported as original article; (2) experimental in vitro studies; (3) the experimental group received bLF and antifungal combination therapy; (4) the control group accepted only nonfunctional solutions or no treatment; and (5) the primary outcome was the C. albicans growth inhibition. Criteria for exclusion were: (1) no group of control; (2) review, clinical trial and case report; (3) repeated publication; and (4) lack of available data.

Data extraction

Four authors retrieved detailed information independently from the included studies, and the fifth author resolved the disagreements between authors. These were the following data collected: (1) the first author and year of publication; (2) the type of LF; (3) the susceptibility test; (4) the antifungal combination; and (5) the outcome.

Quality assessment

Two independent reviewers evaluated the risk of bias. The quality assessment was adopted based on the adaptations applied in previous systematic reviews.[12] These were the parameters: (i) sample size; (ii) similar dimensions samples; (iii) control group; (iv) procedures standardiaation; (v) statistical analysis; and (vi) other risk of bias. Each parameter for the included studies was defined as ‘low,’ ‘high’ or ‘unclear.’ Disagreements between authors were resolved through discussion with the fifth author during the quality assessment.

Statistical analysis

We used Jeffry’s Amazing Statistical Program (JASP) to measure the overall size effect of minimum inhibitory concentration (MIC) between studies. JASP is an open-source and free statistical analysis program supported by the University of Amsterdam. We used a 95% confidence interval, which meant that MIC between studies would be statistically significant when the probability (p) is less than 0.05 (5%). The confidence interval did not pass the middle line (or 0 value). Furthermore, the research was practically significant if the overall correlation coefficient reached the researchers’ expectations. This expectation for the study was that the combined correlation was ≥0.5. Before the analysis model determination, we conducted the heterogeneity test. The heterogeneity test would indicate the I² and P value. When the I² value was considerable and P values less than 0.05, we could state that the data were heterogeneous. Thus, the random-effects model should be utilized as the analysis model. We also conducted trim and fill effects to identify and correct funnel plot asymmetry from publication bias.

Result

The primary search yielded 485 articles from across eight databases during the initial steps of the literature research. After checking for duplicates, 117 articles were eliminated. The other 368 articles then went through a screening process to check the title and abstract for their relevance to the topic and design study. The initial screening by title, keywords, and abstract resulted in 42 eligible articles. The 42 articles were read in total, and 33 articles were excluded. A total of seven studies retained were experimental in vitro studies. Based on the available data, four out of seven studies were included in the quantitative analysis. The PRISMA flow diagram 2020 for systematic reviews is presented in Figure 1.

Figure 1.

PRISMA flowchart

Risk of bias

Regarding evaluating the risk of bias, six out of seven studies only have one high risk of bias component. In contrast, a survey by Wakabayashi et al. (1996)[13] has two high risks of bias components. Sample size calculation is infrequently reported and done in an in vitro experimental study [Figure 2]. In addition, none of the studies reported any conflict of interest statement. Although some authors mentioned applying statistical analysis, only some adequately specified the methodology. It is worth noting that not all 10 items were discussed clearly in the studies we gathered. Several of them only mentioned partially the information we need to determine the degree of bias.

Figure 2.

Risk of bias for laboratory studies

Characteristic of eligible studies

All studies used in this review were published in English between 1996 and 2020. LF was used as the intervention in all studies, although the type of peptides differed between each study: LF (n = 7), lactoferricin (n = 2), apo-LF (n = 1), holo-LF (n = 1), LF 21 kDa (n = 1), LF 38 kDa (n = 1) and LF 45 kDa (n = 1).[11,13,14,15,16,17,18] Three out of seven studies performed LF characterization before being used with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).[14,16,17] There are four methods of susceptibility test that were used: microdilution (n = 5), macrodilution (n = 1), disk diffusion (n = 1) and growth inhibition assay (n = 1), which resulted in MIC, except disk diffusion that resulted in inhibition zone.[13,14,15,17,18]

Drug interaction tests [Table 1] were conducted with checkerboard assay (n = 4) and microdilution (n = 2).[11,13,14,15,17,18] Various current antifungals were used in these studies: amphotericin B (AMB) (n = 1), fluconazole (FLZ) (n = 3), itraconazole (ITZ) (n = 3), nystatin (NYS) (n = 1), 5-fluorocytosine (5FC) (n = 1), voriconazole (VRZ) (n = 1), clotrimazole (CTZ) (n = 1), lactoperoxidase (LPO) (n = 1) and milk RNase of 15 kDa (MR15) (n = 1).[11,13,14,15,16,17,18]

Table 1.

Characteristic of the Included Studies

Author & Year	Lactoferrin Type	Characterization	Study Design	Methods		Antifungal Combination	Candida Species	Parameter
				Susceptibility Test	Drug Interaction Test
Fernandes et al. (2020)15	Lactoferrin (Sigma): Control (LF) Saturated (holo-LF) Dialyzed (apo-LF) Lactoferrin dairy 1 (LFD1): Control (LFD1) Saturated (holo-LF-D1) Dialyzed (apo-LF-D1) Lactoferrin dairy 2 (LFD2) Control (LFD2) Saturated (holo-LF-D2) Dialyzed (apo-LF-D2)	SDS-PAGE Mass- Spectrometry	In vitro	Microdilution	Checkerboard assay	Amphotericin B, nystatin, fluconazole, itraconazole, voriconazole, and 5-fluorocytosine	C. albicans SC5314	MIC (IC80)* FIC
Nakano et al. (2019)16	Lactoferrin (Morinaga, Tokyo, Japan)	N/A	In vitro	Microdilution	Checkerboard assay (crystal violet staining assay)	Lactoperoxidase	C. albicans TIMM 1768	MIC (IC50)** FIC
Rastogi, N. et al. (2014)17	Lactoferrin (Morinaga, Tokyo, Japan) LF21 LF38 LF45	SDS-PAGE PPSQ 21-A protein sequencer (Shimadzu, Japan)	In vitro	Macrodilution Disk diffusion	N/A	N/A	C. albicans ATCC 90028	MIC (IC90)*** Inhibition zone
Murata, et al. (2013)18	Lactoferrin Lactoferricin (Lfcin)	SDS-PAGE	In vitro	Microdilution	Microdilution	Milk RNase of 15 kDa (32 µg/mL)	C. albicans TIMM 1768	MIC (IC50)**
Kobayashi et al. (2011)19	Lactoferrin (Wako)	N/A	In vitro	Growth inhibition assay	Checkerboard assay	Fluconazole and itraconzole	C. albicans SC5314 C. albicans CAE3DU3 C. albicans Darlington C. albicans C26 C. albicans C40	MIC (IC50)** FIC
Wakabayashi et al. (1998)21	Lactoferrin LFcinB	N/A	In vitro	Crystal violet (CV) staining assay (microdilution)	Microdilution	Fluconazole, and itraconazole	C. albicans azole susceptible: ATCC 90028 TIMM 1768 C. albicans azole resistant: TIMM 3164 TIMM 3315 TIMM 3317	MIC (IC80)* FIC
Wakabayashi et al. (1996)14	Lactoferrin (Mo rinaga, Tokyo, Japan) LFcin B (LF-B)	N/A	In vitro	Microdilution	Checkerboard assay	Clotrimazole	C. albicans TIMM 1768	MIC (IC80)* FIC

*IC80 is described as the concentration of protein/protein fragment (mg/ml) resulting in 80% reduction in viability of the microorganisms determined in vitro. **IC50 is described as the concentration of protein/protein fragment (mg/ml) resulting in 50% reduction in viability of the microorganisms determined in vitro. ***IC90 is described as the concentration of protein/protein fragment (mg/ml) resulting in 90% reduction in viability of the microorganisms determined in vitro

The following C. albicans strains were tested: TIMM 1768, ATCC 90028 and SC5314 as azole-susceptible strains; TIMM 3164, TIMM 3315 and TIMM 3317 as azole-resistant strains; CAE3DU3, the disrupted mutant of erg3; Darlington strain, a clinical strain bringing mutations of erg3 and erg11; C26, a CDR1 overexpressing mutant; and C40, a CaMDR overexpressing mutant [Table 1].[11,13,14,15,16,17,18] There were three media used in these studies: RPMI 1640 (n = 4), sabouraud dextrose (n = 2), and yeast extract peptone dextrose medium (n = 1) [Table 2].[11,14,15,16,17,18] The result was assessed after 24–72 hours on 37°C.[11,13,14,15,16,17,18]

Table 2.

Summary of the results

Author & Year	Media	MIC	FIC	Outcome
Fernandes et al. (2020)15	RPMI 1640 (Sigma-Aldrich) supplemented with 0.165 M MOPS (morpholine propanesulfonic acid) and 2% D-glucose)	>256 µg/mL (LF) >256 µg/mL (holo-LF) 256 µg/mL (apo-LF) 16 µg/mL (LFD1) >256 µg/mL (holo-LF-D1) 32 µg/mL (apo-LF-D1) 16 µg/mL (LFD2) >256 µg/mL (holo-LF-D2) 32 µg/mL (apo-LF-D2)	0.375 (LFD1; AMB) 1.5 (LFD1; NYS) 1.5 (LFD1; FLZ) 1.0 (LFD1; ITZ) 1.5 (LFD1; VRZ) 2.0 (LFD1; 5FC) LF with AMB: 0.5 (LF-S) 0.5 (holo-LF-S) 0.5 (apo-LF-S) 0.375 (LFD1) 0.375 (holo-LF-D1) 0.375 (apo-LF-D1) 0.375 (LFD2) 0.375 (holo-LF-D2) 0.375 (apo-LF-D2)	LF was synergistic with amphotericin B.
Nakano et al. (2019)16	Fetal calf serum-RPMI 1640 (FCS-RPMI) medium	>2000 µg/mL	0.134 (7.8 µg/mL LF; 50 µg/mL LPO)	LF enhanced the growth-inhibitory activity of FLZ and increased the activity of ITZ against all the strains tested in the study.
Rastogi et al. (2014)17	Yeast extract peptone dextrose medium	80.85 µg/mL (LF) 25.12 µg/mL (LF21) 40.32 µg/mL (LF38) 47.52 µg/mL (LF45)	N/A	All the three fragments were found to exhibit more antifungal activities than the native molecule. LF21 had maximum activity against C. albicans, followed by LF38 and LF45. The same value for the native protein was much higher. In disk diffusion, FLZ, LF21, LF38, LF45 and LF respectively with corresponding diameters as 25 mm, 20 mm 17 mm, 13 mm and 7 mm. These data indicate that the antifungal activities of all the three fragments are substantially more than that of native LF. In this case, even LF38 showed a higher potency than the native molecule.
Murata et al. (2013)18	Sabouraud dextrose broth	1000 µg/mL (LF) >32 µg/mL (Lfcin)	125 µg/mL (LF MIC with 32 µg/mL MR15) 32 µg/mL (Lfcin MIC with 32 µg/mL MR15)	The growth was not inhibited by the addition of MR15 (32 µg/mL). With the addition of MR15, MIC of LF were reduced 2-fold to 16-fold. In the same manner as LF, the MIC of Lfcin B were also reduced 2-fold to 4-fold when used together with MR15 (32 µg/mL).
Kobayashi et al. (2011)19	RPMI 1640 (with l-glutamine, without NaHCO3, and supplemented with 2% glucose; pH 7.0 from Gibco BRL, Paisley, Scotland)	C. albicans SC5314 ⟶ >6400 µg/mL C. albicans CAE3DU3 ⟶ >6400 µg/mL C. albicans Darlington ⟶ >6400 µg/mL C. albicans C26 ⟶ >6400 µg/mL C. albicans C40 ⟶ >6400 µg/mL	C. albicans SC5314 ⟶ 2 (FLZ); 0.28 (ITZ) C. albicans CAE3DU3 ⟶ <0.016 (FLZ); <0.035 (ITZ) C. albicans Darlington ⟶ <0.25 (FLZ); <0.13 (ITZ) C. albicans C26 ⟶ 2 (FLZ); <0.03 (ITZ) C. albicans C40 ⟶ 2 (FLZ); <0.03 (ITZ)	LF significantly enhanced the growth inhibitory effects of FLZ only against CAE3DU3 and Darlington strain while LF significantly enhanced the growth inhibitory activity of ITZ against all strains tested.
Wakabayashi et al. (1998)21	RPMI 1640 medium supplemented with 2.5% heat-inactivated fetal calf serum, 20 mM HEPES, 2 mM L-glutamine, and 16 mM sodium hydrogen carbonate (pH 7.0)	LF ATCC 90028 ⟶ >6400 µg/mL TIMM 1768 ⟶ 6400 µg/mL TIMM 3164 ⟶ 1600 µg/mL TIMM 3315 ⟶ 200 µg/mL TIMM 3317 ⟶ 1600 µg/mL LfcinB ATCC 90028 ⟶ 400 µg/mL TIMM 1768 ⟶ 400 µg/mL TIMM 3164 ⟶ 400 µg/mL TIMM 3315 ⟶ 50 µg/mL TIMM 3317 ⟶ 200 µg/mL	LF ATCC 90028 ⟶ 2.06 (FLZ); 1.06 (ITZ) TIMM 1768 ⟶ 1.13 (FLZ); 1.13 (ITZ) TIMM 3164 ⟶ 1.13 (FLZ); 0.63 (ITZ) TIMM 3315 ⟶ 0.13 (FLZ); 0.13 (ITZ) TIMM 3317 ⟶ 0.25 (FLZ); 0.25 (ITZ) LfcinB ATCC 90028 ⟶ 1.06 (FLZ); 0.56 (ITZ) TIMM 1768 ⟶ 1.06 (FLZ); 1.06 (ITZ) TIMM 3164 ⟶ 2.06 (FLZ); 0.56 (ITZ) TIMM 3315 ⟶ 0.13 (FLZ); 0.13 (ITZ) TIMM 3317 ⟶ 0.13 (FLZ); 0.13 (ITZ)	From examination of the FIC indices for both strains, it was interpreted that the combination of FLZ and LF, as well as the combination of FLZ and LfcinB, was synergistic [Table 2]. With strain TIMM3164 growing in the yeast form, a reduction of the MIC of FLZ was not evident after the addition of LF or LFcin B. In tests with ITZ, a decrease in the MIC and a synergistic effect were observed when ITZ was combined with LF or LfcinB in the case of strains TIMM3164, TIMM3315, and TIMM3317.
Wakabayashi et al. (1996)14	Sabouraud dextrose broth	200 µg/mL (LF) 12.5 µg/mL (LfcinB)	0.187 (LF; CTZ) 0.19 (LF-B; CTZ)	LF and LfcinB with CTZ exhibited synergistic effect.

MIC: minimum inhibitory concentration; FIC: fractional inhibitory concentration; LF: lactoferrin; LFD1: lactoferrin dairy 1; LFD2: lactoferrin dairy 2; holo-LF: iron saturated lactoferrin; apo-LF: dialyzed lactoferrin; holo-LF-D1: iron saturated lactoferrin dairy 1; apo-LF-D1: dialyzed lactoferrin dairy 1; holo-LF-D2: iron saturated lactoferrin dairy 2; apo-LF-D2: dialyzed lactoferrin dairy 2; AMB: amphotericin B; NYS: nystatin; FLZ: fluconazole; ITZ: itraconazole; VRZ: voriconazole; 5FC: 5-fluorocytosine; CTZ: clotrimazole; LPO: lactoperoxidase; LF21: lactoferrin 21 kDa; LF38: lactoferrin 38kDa; LF45: lactoferrin 45 kDa; Lfcin: lactoferricin; LfcinB: lactoferricin B; MR15: milk RNase 15 kDa; BLF: bovine lactoferrin; LGG: Lactobacillus rhamnosus GG

Minimum inhibitory concentration

Microdilution, macrodilution and growth inhibition assay resulted in inhibitory concentration (IC), while disk diffusion resulted in inhibition zone diameter [Table 1]. Different antifungal susceptibility testing methods may not be comparable across studies, with various measurements, such as complete inhibition, substantial inhibition, 90% growth inhibition (IC90) (n = 1), 80% growth inhibition (IC80) (n = 3), 50% growth inhibition (IC50) (n = 3) or CFU quantification, being used.[11,13,14,15,16,17,18] The lowest MIC for LF was 16 μg/mL, and the highest >2000 μg/mL for azole-susceptible C. albicans. For azole-resistant C. albicans, the lowest MIC was ranging from 200 μg/mL to >6400 μg/mL. For lactoferricin B, the MIC was ranging from 12.5 μg/mL to 400 μg/mL for azole-susceptible C. albicans and 50 μg/mL to 400 μg/mL for azole-resistant C. albicans.[11,13,14,15,16,17,18]

Fractional inhibitory concentration

The fractional inhibitory concentration (FIC) indexes were calculated as a summation of the IC for drug A in the combination/IC for drug A alone and the IC for drug B in the combination/IC for drug B alone. When the IC was greater than the highest concentrations tested, the highest concentrations were substituted for the IC. The effects of the drugs were interpreted to be indicative of synergy, indifference or antagonism when the FIC indexes were <1, 1–4 or >4. The FIC calculation requires at least a fourfold reduction in the MIC of both agents to produce an FIC of <0.5. However, in the case of antifungals and LF, reducing the MIC of the drug is more critical than reducing the MIC of LF.[11,13,14,15,16,17,18]

Most of the experiments reported synergistic results between LF (native LF, apo-LF, holo-LF) with AMB (n = 10), LPO (n = 1), ITZ (n = 1) and CTZ (n = 1) against azole-susceptible C. albicans; with ITZ (n = 7) and FLZ (n = 4) against azole-resistant C. albicans.[11,13,14,15,18] While Lfcin also exhibited synergistic activity with ITZ (n = 1) and CTZ (n = 1) against azole-suspceptible C. albicans, with ITZ (n = 3) and FLZ (n = 2) against azole-resistant C. albicans.[13,18]

Statistical analysis

There were four studies by Fernandes et al.,[14] Rastogi et al.,[16] Wakabayashi et al.[13] and Wakabayashi et al.[18] that included quantitative analysis. The articles reported MIC between the studies. The residual heterogeneity estimates test between studies is 29.554%, meaning that these articles have the same results. The P value of this study is 0.023, meaning this study was significant [Table 3].

Table 3.

Residual Heterogeneity Estimates

	Estimate
τ²	70.919
τ	8.421
I² (%)	29.554
H²	1.420
Coefficients
	Estimate	Standard Error	z	P
Intercept	18.035	7.937	2.272	0.023
Note. Wald test

We analysed these four studies using JASP. The combined correlation was 18.4 with CI (95%) = 2.48–33.59. CI of this study showed that the data were statistically significant. The classical meta-analysis revealed that P value <0.005 indicated there was a correlation MIC between the studies [Figure 3].

Figure 3.

Forest plot

The funnel plot showed a possible publication bias. Four articles have a low risk of bias. The articles are asymmetry (to the right). The trim and fill analysis suggested one article (on the right of the funnel plot). We concluded that the sensitivity analysis was good enough, and the publication bias of this study is low [Figure 4].

Figure 4.

Funnel plot

Discussion

We have assessed the bLF antifungal and synergistic activity on C. albicans. A total of seven in vitro experimental research of the systematic review, along with four out of seven studies by Fernandes et al.,[14] Rastogi et al.,[16] Wakabayashi et al.[13] and Wakabayashi et al.[18] have been analysed in this meta-analysis. This systematic review showed that bLF could help inhibit the development of azole-susceptible and azole-resistant C. albicans.[11,13,14,15,16,17,18] Furthermore, there was synergistic activity between LF and various antifungals. Our meta-analysis showed that LF could significantly inhibit the C. albicans growth more than the control group.[11,13,14,15,16,17,18]

Both LF and lactoferricin B showed better inhibitory activity in azole-susceptible C. albicans compared to azole-resistant C. albicans, which had azole resistance. This is thought to be related to the fungal cell wall’s surface structure and LF’s antifungal mechanism, which targets the cell wall proteins.[15,19,20] A study by Lee and Lee[21] showed that this cation peptide attaches to the charged plasma membrane negatively, which causes the transmembrane pore-like structure formation that resembles an ion channel. In this case, cell membrane permeabilisation leads to intracellular cell organelles leakage, ending in cell apoptosis.[21] Candida spp. with different resistance characteristics have various compensatory activities of the components that make up the structure of the cell wall when the cell is under stress conditions. In azole-sensitive C. albicans, there was a significant increase in mannan. But, in Candida spp. resistant to fluconazole, there was a very high increase in chitin. However, further research is still needed to determine the changes in cell walls that occur when LF is given to both C. albicans susceptible and resistant to azoles.[22]

Most of the studies showed synergistic activity between bovine LF (native LF, apo LF and holo LF) with amphotericin B, lactoperoxidase, itraconazole, and ketoconazole against azole-susceptible C. albicans. Meanwhile, fluconazole and itraconazole showed synergistic results with LF against azole-resistant C. albicans. Lactoferricin also showed synergistic behavior towards azole-susceptible C. albicans (with itraconazole and ketoconazole) and C. albicans of azole-resistant (with fluconazole and itraconazole).[11,13,14,15,16,17,18] Until now, the precise mechanism of how LF relates to the C. albicans cell wall is unclear. Thus, it had no opportunities to describe the sensitivity of C. albicans higher had isolated to LF pre-exposed to drugs through antifungal agents.[19,23]

LF showed better synergistic activity with azoles (fluconazole, itraconazole and clotrimazole) and polylines (amphotericin B and nystatin) than other antifungals.[11,13,14,18] This may be because both groups act at the exact location on the fungal cell membrane. For example, azoles affect by distorting the fungal cell membranes, blocking the step of 14α-demethylation inside the production of ergosterol. The disrupted ergosterol production and 14α-methyl sterols accumulation will alter fungal membrane-associated functions. Polylines can bind sterol components inside the Candida spp. Cell wall to help it become permeable enough. It also has been suggested that the polyenes may repress their precursor production of sterols. Hence, a compromised cell membrane will increase permeability and facilitate drug movement across the cell membrane to its targets. The increased susceptibility to LF led to exposure to azoles may also be related to these drug mechanisms on the Candida spp. Cell wall.[19,23]

In this study, the difference in test results is influenced by several technical matters. Microdilution, macrodilution and growth inhibition assays give results in the form of MICs. The antifungal susceptibility of different methods may not become comparable enough of the studies, using various parameters, such as 90% growth inhibition, 80% growth inhibition, 50% inhibition of growth, or CFU quantification.[14] Different test methods and media can give different results even though using the same substance. Inhibition testing of substances that have potential as antifungals is influenced by the assay type, the assay media’s composition and the tested isolates’ resistance profile. The substance with a high diffusion coefficient or solubility can penetrate the medium better so that although the importance has low antifungal activity, in small amounts, it can give the appearance of an inhibition effect similar to the substance with high antifungal activity and low penetration ability.[24] This may also shed light on why some potential antifungals may yield different results from inside the studies of in vitro in both in vivo and clinical trials. Isolates to be tested for synergistic activity should also be retested with confirmatory tests such as standardised macrodilution, microdilution or E-test. The microdilution method is more sensitive for susceptibility testing because it can determine the MIC, which becomes the lowest concentration required to help inhibit the growth of fungi, which is crucial for researchers in developing antifungal drugs.[25,26,27]

More clinical studies should be done to identify and maximise the antifungal effects of available drugs. These combination therapies aim to lower the antifungal agent dosage and decrease the development of drug resistance.

Conclusion

This systematic review and meta-analysis give a comprehensive overview and assessment of the available methodologies and LF antifungal activity against C. albicans. bLF and its peptide derivatives isolated from bovine milk can significantly inhibit the growth of C. albicans, both susceptible to azoles and those with azole resistance.

In addition, this glycoprotein also shows synergistic activity with various antifungals. The best antifungal activity and synergism were shown by bLF against azole-susceptible C. albicans. Further researche are needed to explore the mechanism of synergistic activity between various types of LF and currently existing antifungals against various Candida species so that they can be used as adjuvant therapy or main therapy in treating candidiasis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.