Modulation of povidone-iodine (PVP-i) activity; antimicrobial efficacy and molecular diagnostic compatibility

Abstract

The present invention relates to kits, methods and systems for modulating free iodine in PVP-I, facilitating antimicrobial action while also ensuring compatibility with diagnostic assays.

Description

[0001] Modulation of Povidone-iodine (PVP-I) Activity; Antimicrobial Efficacy and Molecular Diagnostic Compatibility

[0002] Background of the Invention

[0003] Povidone-iodine (PVP-I) is a widely recognised antiseptic agent and has been extensively used for its broad-spectrum efficacy against bacteria, viruses, fungi, and protozoa. Traditionally, PVP-I has been applied topically for wound care and disinfection. Its lysing properties suggest its application could be extended to diagnostic and therapeutic devices, especially where rapid microbial lysis is required without interference with downstream molecular diagnostic techniques such as PCR (Polymerase Chain Reaction) or LAMP (Loop- mediated Isothermal Amplification). A key challenge in applying PVP-I in molecular diagnostic contexts is mitigating the interference of free iodine, the key antimicrobial active agent, with the amplification reaction. The present invention addresses this challenge by providing a system that modulates the iodine in PVP-I so that it has both antimicrobial action while also ensuring compatibility with diagnostic processes such as PCR and LAMP.

[0004] Summary of the Invention

[0005] The present invention provides a system and method for using PVP-I to achieve rapid microbial lysis in biological samples while also providing compatibility with diagnostic assays, particularly those that use fluorescence-based detection. The invention regulates the release of iodine species such as molecular iodine (l2), hypoiodous acid (HOI), and triiodide ion (l3“), ensuring broad-spectrum antimicrobial efficacy. The system can optionally employ a porous carrier-based sample collection device that is pre-treated with PVP-I and dried. A post-lysis buffer then neutralises iodine to ensure that downstream diagnostics are not affected for example through fluorescence attenuation due to residual molecular iodine.

[0006] In a first aspect of the invention, there is provided a sample collection device for biological sample processing and microbial inactivation comprising: a. A porous carrier pre-treated with a dried Povidone-iodine (PVP-I) formulation; b. A buffer system that rehydrates the PVP-I upon sample absorption, releasing antimicrobial iodine species for microbial lysis; c. A post-lysis buffer configured to neutralise free molecular iodine (l2), preventing interference with diagnostic assays. In a preferred embodiment, the PVP-I formulation is dried into the carrier material and rehydrated by the biological sample to release iodine species, including molecular iodine (l2) and hypoiodous acid (HOI), for broad-spectrum antimicrobial action.

[0007] In some embodiments, the buffer system maintains a pH range of 5.0 to 9.0, preferably, a pH in the range of 6.0 to 6.5 during the lysis phase, optimising the balance between elemental iodine (l2) and hypoiodous acid (HOI).

[0008] In an embodiment, the post-lysis buffer, also referred to herein as the neutralising buffer, comprises sodium thiosulfate to neutralise molecular iodine (l2) species, ensuring diagnostic compatibility by minimising fluorescence attenuation in PCR or LAMP assays or interreference of enzymatic processivity. Preferably, the neutralising buffer raises the pH of the sample, thereby shifting the iodine species equilibrium toward non-interfering forms such as iodide (I").

[0009] In another aspect of the invention, there is provided method for collecting and processing biological samples for diagnostic purposes, comprising: a. Collecting a sample with a porous carrier pre-treated with PVP-I; b. Rehydrating the PVP-I to release iodine species for microbial inactivation; c. Neutralising free molecular iodine (l2) with a buffer to prevent interference with diagnostic assays.

[0010] In another aspect of the invention there is provided a method for processing biological samples for microbial inactivation and diagnostic testing, comprising: a. Directly adding a Povidone-lodine (PVP-I) solution to the biological sample; b. Allowing the iodine species to lyse microorganisms within the sample; c. Adding a post-lysis neutralising buffer to convert residual molecular iodine (l2) into non-interfering species.

[0011] In a preferred embodiment, the PVP-I releases iodine species, including molecular iodine (l2), hypoiodous acid (HOI), and triiodide ion (l3“), upon addition to the sample, providing broad-spectrum antimicrobial action.

[0012] In an embodiment, the post-lysis buffer, or neutralising buffer, comprises sodium thiosulfate to neutralise molecular iodine (l2) species, ensuring diagnostic compatibility by minimising fluorescence attenuation in PCR or LAMP assays or interreference of enzymatic processivity. Preferably, the neutralising buffer raises the pH of the sample to above 8.0, thereby shifting the iodine species equilibrium toward non-interfering forms such as iodide (I’).

[0013] In another aspect of the invention, there is a provided a system for diagnostic processing of biological samples, comprising: a. A Povidone-lodine (PVP-I) solution is added to the sample, initiating microbial lysis through iodine activity; b. A post-lysis buffer configured to neutralise free iodine, preventing interference with diagnostic assays.

[0014] In a preferred embodiment, the antimicrobial efficacy of PVP-I is regulated by adjusting the pH of the buffer to ensure the sustained release of hypoiodous acid (HOI) and molecular iodine (l2), thereby maximising the inactivation of a broad spectrum of microbial species. In an embodiment the antimicrobial efficacy of PVP-I is alternatively or additionally regulated by inclusion of sodium thiosulphate into the neutralising buffer to remove molecular iodine (l2).

[0015] In another embodiment, the neutralising buffer reduces or eliminates the absorbance of molecular iodine (l2) in the 350-450 nm range, preventing inner filter effects that attenuate fluorescence signals in PCR and LAMP assays.

[0016] Brief Description of the Drawings

[0017] Figure 1- Free molecular iodine dependence on PVP-I solution concentration and pH (Zachar, 2021).

[0018] Figure 2- LIV-VIS spectra of povidone-iodine (PVP-I) standard solutions (from Kida et al, 2020).

[0019] Figure 3- LIV-VIS absorption spectra of PVP-I before and after oxidation of iodine by Na2S2O3 (0.1 M). Note x-axis scales are different for each chart (From Mannan, 2019).

[0020] Figure 4- Observed inner filter effect for LAMP amplification of genomic DNA from Mycobacterium tuberculosis, with 104copies per reaction diluted in (a) water and (b) saliva are shown and processed using a push buffer of (i) water, (ii) 20mM TRIS-CI pH 9 and iii) 20mM TRIS-CI pH 11. A control amplification using 104genomic copies without any treatment is also shown for comparison (shown in red).

[0021] Figure 5- Observed inner filter effect for PCR amplification using genomic DNA from Mycobacterium tuberculosis, with 104copies per reaction. The DNA template was processed using push buffers of (i) water, (ii) 20mM TRIS-CI pH 9 and iii) 20mM TRIS-CI pH 11. A control amplification using 104genomic copies without any treatment is also shown for comparison (shown in red).

[0022] Figure 6- PCR amplification plots for genomic DNA from Mycobacterium tuberculosis at 104copies per reaction, using the intercalating reporter dye Eva Green. The DNA template was processed using push buffers of (i) water, (ii) 20mM TRIS-CI pH 9 and iii) 20mM TRIS-CI pH 11. A control amplification using 104genomic copies without treatment was included for comparison. All amplifications gave the same result regardless of treatment.

[0023] Description of the Preferred Embodiments

[0024] Embodiment 1 : Carrier-Based Sample Collection Device

[0025] The device comprises of a porous carrier that has been pre-treated with a dried formulation of PVP-I. The porous carrier may be attached to a body (for example, an elongate pen-like body) to allow for ease of handling and manipulation. Upon absorbing a biological sample (e.g., saliva), the dried PVP-I is rehydrated. A lysis buffer including a non-ionic surfactant and a pH regulating buffer agent is then added. A preferred lysis buffer comprises 0.1% APG (alkyl polyglucoside) and 20mM Tris-CI, pH 6. The lysis buffer facilitates the release of active molecular iodine species that rapidly lyse microorganisms present in the sample. The sample is then treated with a post-lysis buffer or neutralising buffer (also referred to as a push or chase buffer) that comprises a pH regulating buffer agent and neutralises free iodine to prevent interference with fluorescence-based assays. Preferably, the post-lysis, or neutralising buffer comprises around 0.1 mM sodium thiosulfate, 20mM Tris-CI, pH 9, Other pH buffer regulating agents may be used; for example L-H istidine. The post-lysis buffer may optionally further comprise a chelating agent (for example, EDTA, and preferably 0.5 mM EDTA) In this embodiment, the porous carrier can comprise porous plastics, porous metals, porous ceramics, porous textiles and fibers or extruded plastic or metal with internal channels. In a specific embodiment, the porous plastic is a sintered porous plastic. The carrier can be made from a single material or multiple materials. In some preferred embodiments, the carrier is made of plastic. Said plastic carrier can be made from a single plastic, or multiple plastics. It is understood that plastic carriers may be made from a variety of plastics such as polyethylene. Polyethylenes which may be employed include but are not limited to high density polyethylene (HDPE), low density polyethylene (LDPE) and ultra-high molecular weight polyethylene (LIHMWPE). Carriers may also be made from polypropylene (PP), polyvinylidene fluoride (PVDF), polyamides, polyacrylates, polystyrene, polyacrylic nitrile (PAN), ethylene-vinyl acetate (EVA), polyesters, polycarbonates, or polytetrafluoroethylene (PTFE). Plastic carriers may be made from more than one of the aforementioned plastics. In one embodiment, a plastic carrier is made from about 30% PP and about 70% PE (wt:wt %). In other embodiments when PP and PE are combined, PP may be present in a range of from about 100% to about 0% and PE may be present in a range of from about 0 to about 100% (100% to 0%:0% to 100% wt:wt %). When PE is combined with other polymers, the PE is present in at least about 50% (wt %). In one embodiment the plastic is HDPE. In other embodiments the plastic is LIHMWPE, PP, polyamides, or polyacrylic nitrile.

[0026] In some embodiments, the carrier in the form of a sheathed fibre. A sheathed fibre comprises an outer sheath covering one or more inner fibres in the core. In some embodiments, the sheathed fibre may comprise PP and PE. Preferably, the outer sheath comprises PE and the core comprises PP.

[0027] The carrier can be manufactured in a range of densities to absorb and retain biological specimens of different viscosities. The precise detail of the carrier construction may vary depending on the specific application for use of the invention, and the skilled person will be able to select appropriate materials and densities. For example, where the sample to be collected is saliva, one such suitable material is a cylindrical PE / PP wick of -90% porosity. However, alternative carrier compositions may be employed.

[0028] Preferably, the porous form of the carrier provides capillary spaces for absorption of liquid biological sample. The porous structure can be designed to draw a known volume of sample fluid, e.g. saliva, into its capillary spaces, thereby regulating the amount of sample taken and making the test more consistent. Embodiment 2: Direct PVP-I Addition to Biological Samples

[0029] In this embodiment, PVP-I is directly added to the biological sample. The formulation rapidly releases iodine species to inactivate microorganisms. After lysis, a neutralizing buffer is introduced to convert the residual iodine species to inactive forms, such as iodide (I"), preventing interference with downstream diagnostic techniques. A similar or the same postlysis or neutralising buffer may be used as used for embodiment 1. Preferably, the neutralising buffer is comprised of around 0.1 mM sodium thiosulfate, 20mM Tris-CI, pH 9. This method offers flexibility by eliminating the need for a carrier, making it suitable for liquidbased handling systems.

[0030] In this embodiment, PVP-I can be added directly to a biological sample. For example, a similar lysis buffer as used in embodiment 1 may be used, which additionally comprises PVP-I. For example, a lysis buffer comprising 0.5% PVP-I, 0.1 % APG, and 20mM TRIS-CI pH 6 can be added directly to a biological sample. Alternatively, the lysis buffer may comprise 0.5% PVP-I, 0.1 % APG, and 20mM L-histidine. These values are for the final concentration in use; in some embodiments, the lysis buffer may be 10X concentrated, 5X concentrated or at an alternative concentration. The skilled person would be able to adjust the volume of lysis buffer added to the biological sample accordingly.

[0031] For example, a 10X lysis buffer at pH 6 may comprise 5% PVP-I, 1 % APG, and 200mM Tris- CI pH 6 and can be added directly to a biological sample. Alternatively, a 10X concentrated lysis buffer comprising 5% PVP-I, 1 % APG, and 200mM L-histidine can be added directly to a biological sample.

[0032] Alternatively, a 5X lysis buffer at pH 6 may comprise 2.5% PVP-I, 0.5% APG, and 100mM Tris-CI, and can be added directly to a biological sample. Alternatively, a 5X concentrated lysis buffer at pH 6 may comprise 2.5% PVP-I, 0.5% APG, and 100mM L-histidine can be added directly to a biological sample.

[0033] In this example, the biological sample should be left to incubate in the lysis buffer for a few minutes before the neutralising buffer is added. The biological sample may incubate in the lysis buffer for 5 seconds - 15 minutes, 5 seconds -10 minutes, 5 seconds - 5 minutes, 5 seconds - 4 minutes, 5 seconds - 3 minutes, 5 seconds - 2 minutes, or 5 seconds - 1 minute, 30 seconds - 1 minute, 30 seconds - 2 minutes, 30 seconds - 3 minutes, 30 seconds - 4 minutes, 30 seconds - 5 minutes, 30 seconds - 10 minutes or 30 seconds - 15 minutes . Preferably the biological sample is left to incubate in the lysis buffer for less than 5 minutes, more preferably for 30 seconds - 1 minute before the neutralising buffer is added.

[0034] The skilled person would be able to alter the incubation time depending on the concentration of lysis buffer and sample volume.

[0035] The neutralising buffer is preferably at pH 9 and comprises 0.1 mM sodium thiosulfate and 20mM Tris-CI. Alternatively, the neutralising buffer may comprise 0.1 mM Sodium thiosulfate, 20mM L-Histidine at pH 9 ,and optionally 0.05 mM EDTA. In some embodiments, the neutralising buffer may be 10X concentrated or 5X concentrated, or at an alternative concentration, and the volume of neutralising buffer to added to the mixture of biological sample in lysis buffer should be adjusted accordingly.

[0036] For example, a 10X neutralising buffer at pH 9 comprising 1 mM sodium thiosulfate and 200mM Tris-CI can be added directly to the mixture of biological sample and lysis buffer. Or an alternative neutralising buffer comprising 1 mM sodium thiosulfate, 200mM L-histidine and 0.5 mM EDTA can be added directly to the mixture of biological sample and lysis buffer.

[0037] Alternatively, a 5X neutralising buffer at pH 9 may comprise 0.5 mM sodium thiosulfate, and 100mM Tris-CI and can be added directly to the mixture of biological sample in lysis buffer. Or an alternative neutralising buffer comprising 0.5mM sodium thiosulfate, 100mM L- histidine and 0.25 mM EDTA can be added directly to the mixture of biological sample and lysis buffer.

[0038] In some embodiments the neutralising buffer does not comprise sodium thiosulfate.

[0039] The skilled person would be able to adjust the amount of neutralising buffer to be added to a biological sample based on the concentration of said neutralising buffer. After addition of the neutralising buffer, the sample can be used in diagnostic assays such as PCR and LAMP.

[0040] Buffer System for Iodine Neutralisation

[0041] Both embodiments use a specifically designed neutralising buffer to neutralise free iodine post-lysis. Said neutralising buffer preferably comprises around 0.1 mM sodium thiosulfate, 20mM Tris-CI and has a pH of approximately 9. The neutralising buffer shifts the equilibrium of iodine species to a non-reactive state, by raising the pH to alkaline levels, the pH may be raised above 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12. 5, 13.0, 13.5, or 14.0. Preferably, the pH is raised above 8.0. The pH may be in the range of 7.5-14.0, 8.0- 14.0, 8.5-14.0, 9.0-14.0, 9.5-14.0, 10.0-14.0, 7.5-10.0 or 8.0-10.0. Alternatively, or additionally, the neutralising buffer can optionally include a reducing agent such as sodium thiosulfate, preferably at up to 0.1% to ensure the complete removal of residual molecular iodine (l2) and triiodide ions (l3“), allowing fluorescence detection in diagnostic tests such as PCR or LAMP.

[0042] Detailed Description of the Invention

[0043] This invention describes interacting chemistries which require modulation for specific activities and function.

[0044] The activity of PVP-I is regulated by modifying the pH of the buffer keeping PVP-I in a state such that it acts as a reservoir for iodine in several states, each having a specific activity toward different microbe groups. A neutralisation buffer is used to dilute out inhibitory substances, and to convert remaining molecular iodine (l2) into I’ ions since molecular form interferes with fluorescence measurements used in downstream detection methods such as PCR and LAMP.

[0045] The system therefore must consider several complex interchanges between stabilisation of active groups of the PVP-I complex, and compatibility with downstream diagnostic processes by modifying these groups after they have completed their lysis step.

[0046] PVP-I

[0047] PVP-I is a widely used antiseptic with a long history of effectiveness against a broad spectrum of pathogens, including bacteria, viruses, fungi, and protozoa. Its rapid action and broad applicability make it a staple in medical settings, particularly for skin disinfection before surgery, wound cleaning, and general antisepsis. It was introduced as an antiseptic agent to the pharmaceutical market in the 1950s and is as effective as iodine in combating a wide range of disease-causing microorganisms (WHO, 2018; Shelanski & Shelanski, 1956; Siggia, 1957).

[0048] Rapid Microbial Lysis

[0049] The process of microbial lysis by PVP-I occurs within seconds. The high reactivity of iodine ensures that it quickly binds to and oxidises cellular components. The rapid release of iodine from the PVP-I complex ensures that microorganisms are exposed to a high concentration of iodine almost immediately.

[0050] This quick action is particularly important in clinical settings where preventing the spread of infection is critical. For instance, during surgical preparation, the immediate lysis of bacteria on the skin reduces the risk of infection at the incision site. In wound care, the rapid action of PVP-I helps to quickly reduce the microbial load, allowing for a better healing environment.

[0051] Effectiveness Across Different Microbial Species and Clinical Uses

[0052] PVP-l’s effectiveness is not limited to any single class of pathogens. It has been shown to be effective against both Gram-positive and Gram-negative bacteria, including antibioticresistant strains such as MRSA (Methicillin-resistant Staphylococcus aureus). It also effectively inactivates viruses, including enveloped viruses like SARS-CoV-2, and fungi. The broad-spectrum nature of PVP-I, combined with its rapid action, makes it an indispensable tool in the prevention and management of infections.

[0053] The rapid lysis of microbes by PVP-I translates into significant clinical benefits. In surgical settings, where time is of the essence, the ability to quickly and effectively disinfect the skin reduces the window of opportunity for pathogens to enter the body. In emergency care, where wounds may be contaminated with a variety of microorganisms, the immediate application of PVP-I can prevent infections from taking hold, leading to better patient outcomes.

[0054] Moreover, in everyday healthcare, the use of PVP-I in antiseptic washes or scrubs provides a quick and effective means of reducing microbial contamination, helping to prevent the spread of infections in healthcare settings.

[0055] Mode of Action

[0056] The active component of PVP-I is iodine, which is released slowly from the povidone-iodine complex. This release mechanism allows for a sustained antimicrobial effect, but it is the initial rapid release of iodine that is primarily responsible for the immediate, potent antimicrobial activity.

[0057] Iodine penetrates microbial cell walls and reacts with key cellular components, including proteins, nucleotides, and fatty acids. This reaction leads to the disruption of vital cellular structures. One of the most critical effects is the oxidation of thiol groups (-SH) in proteins, leading to their denaturation. As proteins become denatured, the structural integrity of the microbial cell is compromised, leading to cell lysis.

[0058] Iodophors are complexes formed by iodine and a solubilising agent or carrier, which serves as a reservoir for the active "free" iodine (Gottardi, 1991). "Free iodine" refers to the amount of iodine present in the solution, while "available iodine" denotes the portion not bound in the iodine reservoir. The concentration of free iodine is the most critical factor for both the chemical and microbiological activity of iodophors. PVP-lodine is a stable chemical complex of polyvinylpyrrolidone (PVP), which lacks microbicidal properties on its own (Ripa & Reder, 2002), and elemental iodine (Schenck et al., 1979; Eel & Sebille, 1961).

[0059] The iodine in PVP-lodine (PVP-I) exists primarily in three forms and each contribute differentially to its antimicrobial activity and to the UV-vis absorption spectra characteristics which impacts fluorescence /

[0060] • Molecular Iodine (l2)

[0061] Molecular iodine is the diatomic form of iodine. It is a neutral, non-charged molecule and is considered the most active form in terms of antimicrobial properties. I2can penetrate microbial cell walls, leading to disruption of cellular functions and ultimately cell death.

[0062] • Hypoiodous Acid (HOI)

[0063] Hypoiodous acid is formed when molecular iodine reacts with water. HOI is particularly effective as a virucide, and its potency is significantly higher than that of l2against viruses. It is more prevalent at slightly acidic to neutral pH levels.

[0064] • Triiodide Ion (l3“)

[0065] The triiodide ion is formed when molecular iodine reacts with iodide ions (I") present in the solution. The triiodide ion acts as a reservoir for molecular iodine. It is less active directly as an antimicrobial agent but can dissociate back into l2and I", providing a sustained release of molecular iodine over time.

[0066] These three forms of iodine co-exist in equilibrium within the PVP-I solution, contributing to its broad-spectrum antimicrobial properties. The balance among these forms is influenced by factors such as pH, concentration, and the presence of other ions or compounds in the solution. The PVP portion itself has no biocidal effect, but owing to its affinity for cell membranes can deliver the iodine-containing preparation to the target.

[0067] Once elemental iodine (I2) is added to water, it hydrolyses in a pH-dependent manner to form hypoiodous acid (HIO) and iodide (I-). The overall stoichiometry of iodine hydrolysis between pH 2 and 7 is given below; for a more detailed description of iodine hydrolysis, the reader is referred to Lengyel et al. (1993):

[0068] (PVP)n • l2(PVP)n + l2... Equation 1 l2+ H2O ^ HIO + |- + H+... Equation 2

[0069] HIO ^ H++ Or ... Equation 3

[0070] 3 • HIO ^ 3 • H++ IO3 + 2 • I’ ... Equation 4

[0071] I2 + I" «-> I3' ... Equation 5 pH and Buffering Capacity

[0072] Both the hydrolysis and the subsequent equilibrium between elemental iodine and hypoiodous acid are pH dependent. Table 1 shows the proportions of elemental iodine, hypoiodous acid and hypoiodite for a pH range between pH 5 and 9.

[0073] A higher pH leads to a gradual reduction in elemental iodine, causing a shift in the equilibrium toward hypoiodous acid. Since the primary active disinfectants are elemental iodine and hypoiodous acid, the optimal pH for using iodine as a disinfectant is close to neutral or mildly alkaline (pH 7-7.5). This pH range ensures sufficient levels of both elemental iodine and hypoiodous acid (Table 1).

[0074] When the pH is > 8.0, hypoiodous acid becomes unstable and gradually breaks down into iodate and iodide (Ellis & van Vree, 1989). However, without a stronger oxidant like chlorine, iodate formation is unlikely (Black et al., 1970). For more detailed information on speciation and its pH dependence, refer to Gottardi (1999). Table 1: Effect of pH on the speciation of iodine

[0075] There is a seemingly misguided common wisdom that the target active ingredient is just the elemental I2 content and that therefore, low pH (pH<5) useful for high elemental I2 content is the current commercial market products standard. In contrast, a neutral pH~7, is preferred for an antiviral medication formulation for respiratory infections to increase antiviral potency with higher content of the HIO (Table 2).

[0076] Since the primary active disinfectants are both elemental iodine and hypoiodous acid, the optimal pH for using iodine as a disinfectant should be close to neutral or mildly alkaline (pH 7-7.5). Note, at pH 8 and above the activity of PVP-I might be slightly or completely reduced due to the decreased concentration of free iodine and the instability of HOI at this pH.

[0077] It is crucial to consider the chemistry of iodine and the conditions that favour the stability of elemental iodine and hypoiodous acid (HOI), which are the main antimicrobial agents. The goal is to dry down PVP-I (Povidone-lodine) in formulations that maintain both the antibacterial and antiviral properties by maintaining access to both elemental iodine and hypoiodous acid. This is achieved at neutral pH 7.0.

[0078] Table 2: Disinfection power of different iodine species when applied to different microbial groups and pH range where the most effective disinfectant prevails (Adapted from Taylor and Butler, 1982) pH 7.0 / s still effective e.g. influenza A virus, poliovirus type 1 and adenovirus type 3 (Wada et al., 2016)

[0079] The different chemical species of iodine vary in their disinfection power. The active disinfectants are elemental iodine and hypoiodous acid (Backer & Hollowell, 2000). Other species including iodide, iodate (IOT) and hypoiodite have mild or little antimicrobial activity (Chang, 1958). Comparing the two disinfection-active chemical species, the oxidizing power of hypoiodous acid is nearly twice that of elemental iodine (West, 1984).

[0080] • Hypoiodous Acid (HIO); Highest

[0081] HIO is the most effective due to its ability to penetrate cells and oxidise critical components.

[0082] • Molecular Iodine (l2); High l2is highly effective due to its strong oxidising properties and ability to disrupt cell and viral structures.

[0083] • 3. Triiodide (l3_); Moderate to High l3“ is an effective antimicrobial agent because it exists in equilibrium with l2and I", contributing to the antimicrobial activity of l2. It is particularly useful in solutions where it maintains a reserve of active iodine such as PVP-I.

[0084] • Hypoiodite (Ol_); Effectiveness: Moderate

[0085] Ol" is an effective oxidising agent but less potent than HIO and l2.

[0086] Iodate (IO3_); Effectiveness: Low IO3has very limited direct antimicrobial properties. It is much less reactive and does not have the same oxidative capacity as HIO or l2.

[0087] • Iodide (I-); Lowest

[0088] 1“ is the least effective antimicrobial agent on its own but can be converted into more active species.

[0089] Antimicrobial Effects

[0090] • Bacteria o Gram-Positive Bacteria

[0091] PVP-I is generally effective against a broad range of Gram-positive bacteria, such as Staphylococcus aureus and Streptococcus pyogenes. o Gram-Negative Bacteria

[0092] Similarly, PVP-I is effective against Gram-negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa.

[0093] • Sporicidal Activity o PVP-I has sporicidal properties, effective against endospore-forming bacteria like Clostridium difficile.

[0094] • Viruses

[0095] Hypoiodous acid (HOI) is an effective virucide, with studies suggesting it is up to 40 times more effective against viruses than molecular iodine (l2). o Enveloped Viruses

[0096] Enveloped viruses (e.g., coronaviruses, influenza viruses) are highly susceptible to PVP-I. o Non-Enveloped Viruses

[0097] Although non-enveloped viruses are generally less susceptible to antiseptics like iodine compared to enveloped viruses, PVP-I has been shown to be effective toward HPV and BPV (Capriotti, 2015).

[0098] • Fungi and Protozoa

[0099] PVP-I is effective against fungi and protozoa, such as Candida albicans and Trichomonas vaginalis. Tris(hydroxymethyl)aminomethane (TRIS) Buffer

[0100] TRIS is commonly used as a buffering agent to maintain a stable pH. However, TRIS is a weak reducing agent, which could pose a problem in maintaining the desired levels of HOI. Since iodine is a mild oxidizing agent, the reducing nature of TRIS could potentially reduce iodine species, including HOI, to iodide (I"), thereby diminishing the antimicrobial efficacy. To mitigate this risk TRIS is used in minimal concentrations, preferably of no more than 50mM, 40mM, 30 mM or 20 mM. Preferably TRIS can be used at concentrations in the range of 5- 50mM, 10-50 mM, 20-50 mM, 5-40 mM, 10-40 mM, 20-40 mM, 5-30 mM, or preferably 10- 30mM. More preferably around 20mM of TRIS is used to maintain the desired pH without significantly reducing iodine species. Alternative non-reducing buffers could also be considered as a means to maintaining the integrity of the iodine species. For example, instead of TRIS, L-Histidine can be used at a concentration of 10-70 mM, 10-60 mM, 15-60 mM, 45-55 mM, 15-25mM, preferably around 50 mM, more preferably around 20 mM.

[0101] In an embodiment, the lysis buffer comprises, 0.5% PVP-I, 0.1% APG, 20mM TRIS-CI, in sterile water and is adjusted to a pH of around 6 with sodium hydroxide and / or hydrochloric acid. The pH of the lysis buffer can be adjusted, for example with sodium hydroxide and / or hydrochloric acid until the required pH is reached.

[0102] In an alternative embodiment, the lysis buffer comprises 0.5% PVP-I, 0.1% APG, 20mM L- histidine, in sterile water and is adjusted to a pH of around 6 with sodium hydroxide and / or hydrochloric acid. L-Histidine is a zwitterionic amino acid with a buffering range centred around pH 5.5-7.5, ideal for maintaining the balance of l2and HOI species in a PVP-I system.

[0103] Note that where the lysis buffer is used in conjunction with a porous carrier which included dried down PVP-I, the lysis buffer need not contain additional PVP-I (eg, it may comprise 0.1% APG, 20 mM Tris-CI pH 6, or 0.1% APG, 20mM L-histidine pH 6).

[0104] Sublimation Considerations During Drying

[0105] Sublimation refers to the transition of a substance from the solid phase directly to the gas phase without passing through the liquid phase. For iodine to sublimate from PVP-I the PVP- I complex needs to be heated or subjected to a reduced pressure environment. Under these circumstances the iodine molecules within the complex can gain enough energy to break free from the PVP matrix and enter the gaseous phase as iodine vapor.

[0106] During the drying process, it is essential to ensure that the PVP-I complex remains intact to provide a continuous release of iodine. The PVP-I complex acts as a reservoir for iodine, releasing it slowly over time. Therefore, the drying process should not disrupt the PVP-I complex or cause excessive loss of iodine through sublimation.

[0107] Iodine can sublimate under certain conditions, especially during drying processes that involve heat or reduced pressure. Sublimation of iodine could lead to a loss of active iodine from the formulation, reducing the overall effectiveness of the dried PVP-I product. To prevent this, drying should be performed at lower temperatures and pressures that minimise iodine sublimation.

[0108] In an embodiment, drying is carried out at temperatures below 75°C 70°C, 65°C, 60°C, 55°C, 50°C, 45°C, 40°C, 35°C, 30°C or 25°C. Preferably, drying is carried out below 65°C, more preferably below 40°C. In a preferred embodiment, drying is carried out at room temperature (that is, typically temperatures in the range of 18-22°C).

[0109] The characteristic brown colour of the PVP-I complex is due to iodine. If sublimation of iodine occurs, the iodine transitions from the solid phase (or complexed phase) directly into the gas phase, leaving the PVP matrix behind. When iodine sublimates, the brown colour associated with the iodine diminishes or disappears as the iodine leaves the complex. If the complex still retains its colour this suggests that the iodine is still present in the complex. If the process is incomplete or the conditions (temperature, pressure) were not sufficient to cause sublimation, the iodine remains in the complex, and thus, the colour remains. However, it’s important to note that even after sublimation, some colour might persist if only a portion of the iodine has sublimated or if there are other coloured components in the mixture. Incomplete sublimation or the presence of residual iodine bound more tightly in the matrix provides a persistent colour.

[0110] Avoiding Reducing Agents

[0111] Avoid the use of reducing agents in the formulation, as they can diminish the antimicrobial activity by reducing iodine species.

[0112] Controlling Drying Conditions Use gentle drying techniques to prevent the loss of iodine through sublimation and ensure the stability of HOI.

[0113] Post-Drying Stability

[0114] After drying, store the product in a cool, dry environment to minimize the risk of iodine sublimation and to maintain its antimicrobial properties over time.

[0115] Concentration Effects

[0116] One of the distinctive characteristics of PVP-I solutions is their "concentration anomaly." Within a specific range of PVP-I concentrations, the concentration of free iodine paradoxically increases as the solution becomes more diluted. This effect follows a "bell curve" pattern (Figure 1): at concentrations below 0.05%, PVP-I loses its reservoir complex properties and behaves like aqueous iodine. As the concentration increases, the free iodine level rises, peaking at 0.1% PVP-I, and then decreases with further increases in PVP-I concentration. Correspondingly, in vitro studies have shown that the bactericidal effectiveness of povidone-iodine improves at more dilute concentrations, with the fastest killing rates observed between 0.1% and 1%.

[0117] Maintaining h and HOI During Drying

[0118] I2 and HOI are preserved during the drying process through careful control pH, avoiding conditions that could lead to the reduction of iodine species or unwanted side reactions. This involves maintaining the PVP-I complex so that it acts as a reservoir to supply these agents. pH Recommendation

[0119] The most effective pH is mildly acidic to neutral, typically around pH 6.0, 6.1, 6.2, 6.3, 6.4,

[0120] 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1 , 8.2, 8.3, 8.4, 8.5,

[0121] 8.6, 8.7, 8.8, 8.9, 9.0. Preferably, the pH is between 6.5 to 8.0, more preferably the pH is between 6.0 to 7.5, 6.0 to 6.5 or 7.0 to 7.5, most preferably 6.0 to 6.5. This pH range helps maintain the balance between l2and HOI, ensuring that both remain in a significant concentration to provide antimicrobial efficacy.

[0122] Spectral Overlap and Its Effects on FAM Fluorescence

[0123] The molecular iodine (l2) with absorbance in the 350-360nm range is the key peak causing the reduced fluorescence output. PVP-I has a characteristic absorbance spectrum and absorbs in the UV-VIS wavelengths (Figure 2); Kireev & Shnyrev (2015) give a good synopsis. Three absorptions maxima are observed at wavelength of 240, 290, and 360-390 nm. The peak at 240 nm is the indication of povidone while, wavelengths of 290 and 360- 390 nm are due to iodate ion and iodine, respectively.

[0124] These absorbencies have been shown to impact the intensity of fluorescence obtained in both PCR and LAMP for FAM based fluorophores. Blue LEDs excite in the range 350nm- 450nm with a peak excitation centred around 400nm. This matches the PVP-I absorbance which lowers the fluorescence signal.

[0125] In the UV-Vis spectrum of Povidone-lodine (PVP-I), the absorbance peaks typically correspond to the different iodine species present in the solution. These peaks are significant because they reflect the complex equilibrium between the different iodine species in PVP-I solutions, and their relative intensities can give insights into the concentration and stability of these species in the formulation.

[0126] • 190-200 nm, associated with PVP

[0127] PVP absorbs in the far UV region, typically around 190-200 nm. This peak is due to the n^TT* transitions of the carbonyl (C=O) groups in the pyrrolidone ring of PVP. While this peak is characteristic of the polymer, it is generally outside the range of interest when focusing on iodine-related absorbance in PVP-I formulations, which occurs at longer wavelengths.

[0128] • 220-230 nm, associated with Iodide ion (I )

[0129] This peak is due to the electronic transitions in the iodide ion. Iodide is formed as part of the equilibrium between molecular iodine (l2) and iodide (I") in the PVP-I solution.

[0130] • 290-300 nm, associated with: Triiodide ion (l3)

[0131] The triiodide ion (l3“) absorbs strongly in this region. Triiodide is formed when molecular iodine (l2) reacts with iodide (I") in the solution. This peak is indicative of the presence of triiodide in the PVP-I complex.

[0132] • 350-360 nm, associated with: Molecular iodine (l2)

[0133] The peak in this region corresponds to the absorption by molecular iodine. This peak is generally broad and indicates the presence of free iodine in the solution, which is responsible for the antiseptic properties of PVP-I. Fluorescein amidite (FAM) is a commonly used fluorescent dye that emits green fluorescence when excited by blue light. It has a strong emission peak around 520 nm, which makes it sensitive to spectral overlap with other substances that absorb light within this range.

[0134] • Reabsorption and Inner Filter Effect: If a substance in the solution has an absorption spectrum that overlaps with the emission spectrum of FAM, it can absorb the emitted fluorescence, leading to a decrease in the observed fluorescence intensity. For FAM, any substance that absorbs strongly around 520 nm could contribute to this effect.

[0135] • Inner Filter Effect Due to Excitation Light Absorption

[0136] When the excitation light is absorbed by iodine species before it can excite FAM, this phenomenon is part of what’s known as the inner filter effect. It leads to a decrease in the effective excitation light intensity that reaches the FAM molecules, resulting in reduced fluorescence emission. The inner filter effect is particularly significant in solutions with high concentrations of absorbing species like l2or l3“.

[0137] • FRET (Forster Resonance Energy Transfer): If another molecule or fluorophore is present that has an absorption spectrum overlapping with the emission spectrum of FAM and is in proximity, energy transfer could occur from FAM to this other molecule, reducing the fluorescence intensity of FAM.

[0138] Among the iodine species (molecular iodine, iodate ions, iodide ions, and triiodide ions), triiodide ions (l3“) and molecular iodine (l2) are likely to have the most pronounced effect on FAM fluorescence due to their absorption characteristics:

[0139] • Triiodide Ion (l3_)

[0140] The triiodide ion also absorbs light in the visible spectrum, though its absorption peaks are primarily at 288 nm and 352 nm. However, it has a tail extending into the visible range, and at higher concentrations, this could overlap slightly with the excitation wavelength of FAM, causing some degree of attenuation of the excitation light. This species has absorption peaks in the visible range, particularly around 288 nm and 352 nm, but it also has some absorption extending into the near-UV and visible regions. The presence of l3“ could lead to spectral overlap with FAM’s emission, potentially diminishing fluorescence through reabsorption or inner filter effects.

[0141] • Molecular Iodine (l2) l2has a broad absorption spectrum that extends into the visible region, including wavelengths around 461 nm, which is close to the excitation wavelength of FAM (488 nm). This means that l2can absorb some of the excitation light intended for FAM, thereby reducing the amount of light that reaches the FAM molecules. This reduction in excitation light results in lower fluorescence intensity.

[0142] I2has absorption bands in the UV and visible regions, including a broad absorption band cantered around 461 nm. While this is somewhat lower than FAM’s emission peak, any overlap in the absorption spectrum could still result in decreased fluorescence intensity due to reabsorption

[0143] Others in the literature have shown that it is possible to reverse this absorbance effect by removing the l2from solution using Na2S20s (Figure 3), or by shifting the pH significantly >pH 8.0 (Table 1).

[0144] Modifications with Push Buffer to Counter the Inner Filter Effect

[0145] One of the key benefits of the system is the ability to modify the push buffer (post-lysis buffer; neutralisation buffer) that is applied to remove the collected sample from the collection wick. This introduces the possibility for having two independently buffered reactions; one for collection and one for delivery. The push buffer is used to neutralise the absorbance effects of iodine as well as diluting out any inhibitory substances from the collected sample, this is achieved by modulating the pH. The molecular iodine in the wick will approach 0% when the pH is >8.0 (Table 1) and its absorbance in the blue wavelengths will be reduced. This is shown in figure 4, where the effect of push buffer pH on fluorescence is shown.

[0146] At pH 9 and above, in a PVP-I solution Iodine (l2) is partially hydrolysed into hypoiodous acid (HIO) and iodide ions (I"), Hypoiodous acid (HIO) can deprotonate into hypoiodite ions (IO-), which can then disproportionate into iodate (IO3“) and iodide ions (I"). The overall environment becomes increasingly favourable for the formation of iodate (IO3“) and iodide ions (I-), reducing the availability of free iodine (l2) in the solution. The conversion of l2and HIO into iodate (IO3“) and iodide (I") at pH 9 and above is effectively irreversible, meaning that once these reactions occur, the iodine species are not easily returned to their original forms.

[0147] Where the sample prepared in water and has a low pH <7.0, the signal to noise (height of the signal growth above the background) is significantly reduced compared to the unprocessed control. As the pH of the sample is increased by increasing the pH of the pushbuffer the inner filter effect is reduced and the signal to noise increases to the same level as the PVP-I minus control, Figure 4 (a).

[0148] The effect of increasing the pH of the push buffer on probe fluorescence output was similar for PCR (Figure 5). As the pH of the sample is increased by increasing the pH of the pushbuffer the inner filter effect is reduced resulting in an increase in the signal to noise ratio which can be restored to the same level as the PVP-I minus control.

[0149] The inner filter effects were not observed with Eva green, where there was no impact on overall fluorescence between treatments compared with the PVP-I minus control (Figure 6).

[0150] The same restoration of fluorescence was obtained by using sodium thiosulphate. When sodium thiosulfate is introduced, preferably at around 0.1 mM, to a solution of PVP-I it rapidly neutralises the free iodine.

[0151] Common Uses:

[0152] • Neutralisation: Sodium thiosulfate can be used to neutralize PVP-I after its application, such as in certain medical or chemical processes where you want to stop the action of iodine.

[0153] • Staining and Skin Reactions: It’s sometimes used in cases where PVP-I staining (from iodine) needs to be removed or if there’s a need to mitigate a reaction to iodine.

[0154] Here we add it to the chase buffer (also referred to as the post-lysis or neutralising buffer) after the iodine has lysed the bacteria and viruses, the make the lysed reaction compatible with PCR or isothermal assays. In an alternative embodiment, the chase buffer comprises 0.1 mM sodium thiosulfate, 20mM TRIS-CI, in sterile water and is adjusted to pH 9 with sodium hydroxide and / or hydrochloric acid.

[0155] In a preferred embodiment, the chase buffer comprises 0.1 mM sodium thiosulfate, 20 mM L-H istidine, 0.05 mM EDTA, in sterile water and is adjusted to approximately pH 9 with sodium hydroxide and / or hydrochloric acid. One advantage of the inclusion of EDTA is that this can prevent oxidation by chelating metal ions that catalyse thiosulfate oxidation.

[0156] In some embodiments, thiocyanate, found in saliva can be used for neuralisation.

[0157] Saliva and Fluorescence Attenuation

[0158] Where template is prepared in saliva Figure 4 (b), the signal to noise is maintained at the PVP-I minus control levels indicating that saliva is modifying the levels of molecular iodine. Saliva can remove iodine from a solution primarily through reduction to iodide ions, breakdown of starch-iodine complexes by amylase, or by binding to iodine, all of which reduce the detectable concentration of iodine in the solution. This reaction reduces the amount of molecular iodine in the solution, effectively also irreversibly “removing” its visible form. The effect of this would be the removing the brownish yellow colour which is observed when saliva is taken up into the wicks instantly removing the colour from the wick.

[0159] Among the possible reducing agents present in saliva thiocyanate is the most likely to remove the colour from a 0.2% PVP-I (povidone-iodine) solution instantly as observed empirically. Saliva contains reducing agents, like thiocyanate (SCN“), that can reduce iodine (l2) to iodide ions (I"). Hypoiodous acid (HIO) is a less reactive species compared to iodine (l2) and does not readily react with thiocyanate ion (SCN“).

[0160] Natural levels of thiocyanate in saliva have been established by others (Tenovuo, 1989; Pruitt and Tenovuo, 1985; Rai et al., 2007; Madiyal et al., 2018); The levels of thiocyanate naturally present in saliva would be sufficient to convert all the iodine present in 0.2% PVP-I, since the thiocyanate concentration exceeds the iodine concentration that can be derived from the PVP-I even when thiocyanate is present at modest levels.

[0161] • Presence of SCN" in Saliva SCN is naturally present in saliva at significant levels, especially in individuals exposed to certain dietary sources and is readily available to react with iodine o Non-smokers: 500 to 3,000 pM. o Smokers: 3,000 to 6,000 pM (or even higher in heavy smokers).

[0162] • Reaction Speed

[0163] Thiocyanate reacts very rapidly with iodine, reducing it to iodide ions, which are colourless. This reduction would lead to the immediate loss of the brownish colour associated with povidone-iodine.

[0164] • Concentration and Effectiveness

[0165] The concentration of thiocyanate in saliva, even at lower levels, is sufficient to react with iodine in a dilute PVP-I solution like 0.2%. This makes it the most plausible agent for causing an immediate colour change in such a solution.

[0166] Comparison with Other Reducing Agents and enzymatic mechanisms, the removal of iodine results is an instantaneous colour change which is more likely due to the reaction with thiocyanate rather than the breakdown through other reducing agents or of the starch-iodine complex:

[0167] • Ascorbic Acid: While a strong reducing agent, ascorbic acid is typically not present in significant amounts in saliva to cause a rapid colour change unless introduced externally.

[0168] • Hydrogen Sulphide (H2S) and Cysteine: These could reduce iodine, but their concentration in saliva is generally low, making them less likely to cause a rapid colour change.

[0169] • Glutathione and Uric Acid: Present in saliva but in lower concentrations, and their reaction with iodine would be slower compared to thiocyanate.

[0170] • Starch-iodine Complex: The starch-iodine reaction involves the enzymatic breakdown of starch, which can be a bit slower because it depends on enzyme activity, substrate concentration, and environmental conditions. The breakdown of the starch-iodine complex by amylase in saliva, while relatively quick, usually takes a few seconds to several minutes depending on the concentration of starch and amylase. It is not as instantaneous as the reduction reaction involving thiocyanate.

[0171] References

[0172] Backer H, Hollowell H. (2000). Use of iodine for water disinfection: Iodine toxicity and maximum recommended dose. Environmental Health Perspectives, 108, 679-684.

[0173] Black AP, Kinman RN, Keirn MA, et al. (1970). The disinfection of swimming pool waters. Part I: Comparison of iodine and chlorine as swimming pool disinfectants. American Journal of Public Health and the Nation's Health, 60(3), 535-545.

[0174] Capriotti K, Stewart KP, Pelletier JS, Capriotti J. (2015). A novel topical 2% povidone-iodine solution for the treatment of common warts: A randomized, double-blind, vehicle-controlled trial. Dermatology and Therapy (Heidelb), 5(4), 247-252.

[0175] Chang SL. (1958). The use of active iodine as a water disinfectant. Journal of the American Pharmaceutical Association, 47, 417-423.

[0176] Eel J, Sebille E. (1961). Spectrophotometric study of polyvinylpyrrolidone-iodine complex. Comptes Rendus, 252, 405.

[0177] Ellis KV, van Vree HBRJ. (1989). Iodine used as a water disinfectant in turbid waters. Water Research, 23(6), 671-676.

[0178] Gottardi W. (1991). Iodine and iodine compounds. In: Block S, ed. Disinfection, Sterilization and Preservation. 4th ed. Philadelphia (PA): Lea & Febiger; pp. 152-166.

[0179] Kida D, Gladysz O, Szulc M, Zborowski J, Junka A, Janeczek M, Lipihska A, Skalec A, Karolewicz B. (2020). Development and evaluation of a polyvinyl alcohol-cellulose derivative-based film with povidone-iodine predicted for wound treatment. Polymers, 12(6), 1271. Kireev, S. V., & Shnyrev, S. L. (2015). Study of molecular iodine, iodate ions, iodide ions, and triiodide ions solutions absorption in the UV and visible light spectral bands. Laser Physics, 25(7), 075602.

[0180] Lengyel I, Epstein IR, Kustin K. (1993). Kinetics of iodine hydrolysis. Inorganic Chemistry, 32(25), 5880-5882.

[0181] Madiyal A, Ajila V, Babu SG, Hegde S, Kumari S, Madi M, Achalli S, Alva P, Ullal H. (2018). Status of thiocyanate levels in the serum and saliva of non-smokers, ex-smokers, and smokers. African Health Sciences, 18(3), 727-736.

[0182] Mannan A, Khan AJ, Khan M, Abbas G, Roohullah S, Hussain M. (2019).

[0183] Spectrophotometric estimation of polyvinylpyrrolidone, iodate and iodine simultaneously in povidone-iodine complex in pure and pharmaceutical preparations.

[0184] Pruitt KM, Tenovuo J. (1985). The Lactoperoxidase System: Chemical and Biological Significance. New York (NY), USA: Dekker.

[0185] Rai B, Kharb S, Anand SC. (2007). Salivary enzymes and thiocyanate: Salivary markers of periodontitis among smokers and non-smokers; a pilot study. Advances in Medical and Dental Sciences, 1 , 1-4.

[0186] Ripa S, Bruno R, Reder R. (2002). Clinical applications of povidone-iodine as a topical antimicrobial. In: Handbook of Topical Antimicrobials: Industrial Applications in Consumer Products and Pharmaceuticals. Boca Raton (FL): CRC Press.

[0187] Schenck HV, Simak P, Haedicke E. (1979). Journal of Pharmaceutical Sciences, 68(12), 1505.

[0188] Shelanski HA, Shelanski MV. (1956). PVP-lodine: History, toxicity, and therapeutic uses. Journal of the International College of Surgeons, 25(6), 727.

[0189] Siggia S. (1957). The chemistry of polyvinylpyrrolidone-iodine. Journal of the American Pharmaceutical Association, Scientific Edition, 46(3), 201. Taylor GR, Butler MA. (1982). A comparison of the virucidal properties of chlorine, chlorine dioxide, bromine chloride, and iodine. Journal of Hygiene (Cambridge), 89, 321-328.

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Claims

CLAIMS:

1. A kit for biological sample processing and microbial inactivation said kit comprising: a. A sample collection device comprising a porous carrier pre-treated with a dried Povidone-lodine (PVP-I) formulation; b. A lysis buffer that rehydrates the PVP-I upon sample absorption, releasing antimicrobial iodine species for microbial lysis; c. A neutralising buffer configured to neutralise free molecular iodine (l2).

2. The kit of claim 1 , wherein the PVP-I formulation is dried into the carrier material.

3. The kit of any preceding claim, wherein the lysis buffer maintains a pH range of 5.0 to 9.0 during the lysis phase.

4. The kit of claim 3, wherein the lysis buffer maintains a pH range of 6.0 to 6.5 during the lysis phase.

5. The kit of any preceding claim, wherein the neutralising buffer comprises sodium thiosulfate.

6. The kit of any preceding claim, wherein the neutralising buffer raises the pH of the sample to above 8.

7. A method for collecting and processing biological samples for diagnostic purposes, comprising: a. Collecting a sample with a porous carrier pre-treated with PVP-I; b. Rehydrating the PVP-I with a lysis buffer to release iodine species for microbial inactivation; c. Neutralising free molecular iodine (l2) with a neutralising buffer.

8. A method for processing biological samples for microbial inactivation and diagnostic testing, comprising: a. adding a Povidone-lodine (PVP-I) lysis buffer solution to the biological sample which releases iodine species; b. Allowing the iodine species to lyse microorganisms within the sample; c. Adding a post-lysis neutralising buffer.

9. The method of claim 7 or 8, wherein the iodine species, includes molecular iodine (l2), hypoiodous acid (HOI), and triiodide ion (l3“).

10. The method of claim 7 or 8, wherein the neutralising buffer comprises sodium thiosulfate.

11. The method of claim 7, 8 or 10, wherein the neutralising buffer raises the pH of the sample to above 8.0.

12. A system for diagnostic processing of biological samples, comprising: a. A Povidone-lodine (PVP-I) lysis buffer solution which is added to the sample, initiating microbial lysis through iodine activity; b. A neutralising buffer configured to neutralise free iodine.

13. The system of claim 12, wherein the pH of the lysis buffer is in the range of 6.0-6.5.

14. The system of claim 12, wherein the neutralising buffer comprises sodium thiosulphate.

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