A major challenge in the management of antiretroviral therapy (ART) is to improve the patient’s adherence, reducing the burden caused by the high number of drugs that compose the treatment regimens for human immunodeficiency virus positive (HIV+) patients. Selection of the most appropriate treatment regimen is responsible for therapeutic success and aims to reduce viremia, increase the immune system response capacity, and reduce the incidence rate and intensity of adverse reactions. In general, protease inhibitor (PI) is one of the pillars of regimens, and darunavir (DRV), in particular, is frequently recommended, along with low doses of enzyme inhibitors as cobicistat (COBI) or ritonavir (RTV), by the international guidelines. The potential of clinically significant drug interactions in patients taking COBI or RTV is high due to the potent inhibitory effect on cytochrome CYP 450, which attracts significant changes in the pharmacokinetics of PIs. Regardless of the patient or type of virus, the combined regimens of DRV/COBI or DRV/RTV are available to clinicians, proving their effectiveness, with a major impact on HIV mortality/morbidity. This study presents current information on the pharmacokinetics, pharmacology, drug interactions, and adverse reactions of DRV; it not only compares the bioavailability, pharmacokinetic parameters, immunological and virological responses, but also the efficacy, advantages, and therapeutic disadvantages of DRV/COBI or DRV/RTV combinations. The human immunodeficiency virus (HIV), responsible for the development of acquired human immunodeficiency syndrome (AIDS), is today one of the most serious public health challenges that humanity has to deal with . Nearly Another major benefit of ART is the possibility of preventing virus transmission, as this therapy manages to significantly reduce the number of maternal–fetal transmissions . Highly Patients with HIV infection may face increased risk of cardiovascular disease, neurocognitive disorders, liver disease, cancer, and bone demineralization. These comorbidities are not only attributed to the disease per se, but are considered, in some cases, side effects of ART. Comorbidities can also be caused by continued viral replication in so-called “sanctuaries,” or they can be consequences of chronic HIV inflammation. Nowadays, only a third of deaths among people infected with HIV are caused by the natural course of the disease. The rest of the deaths occur due to comorbidities, complications induced by drug interactions and toxicity of ART .ART can inhibit viral replication, controlling the infection and thus prolonging the patient’s life. Moreover, people infected with HIV can lead a normal life for decades. However, even patients who initially responded well to the treatment may experience moments or periods of therapeutic failure. This failure can be caused by many factors: low/lower patient adherence, too low potency of the drug, viral resistance, cellular characteristics, and pharmacokinetic considerations .There is an apparently large number of medicinal substances (30 molecules) available to HIV infected patients. Treatment regimens always include combinations of ARV drugs . The treIt is known that HIV has the great ability to make genetic mutations. There are significant zonal differences in the type of virus, which requires in-depth study related to its evolution in the body, depending on the geographical region and genetics of the infected population.This study aims to provide the main information on the topic related to ART usage in HIV-infected patients, especially regarding those in whose regimen a protease inhibitor (PI)—namely darunavir (DRV)—is introduced in combination with an enzyme inhibitor (a booster), which increases the ARV effect of PI. The pharmacokinetic of the PIs is significantly influenced when combined with ritonavir (RTV) or cobicistat (COBI) (both are boosters); therefore, a detailed analysis of changes in pharmacokinetic parameters is required in patients with this regimen. Management of ART involves choosing the best treatment regimen for the therapeutic success . The article reviews information from the literature relevant to the topic. At this time, there is not much comparative data between the advantages and disadvantages of using one inhibitor or another, or to strictly compare the values of boosted pharmacokinetic parameters.fixed dual combinations—emtricitabine/tenofovir, COBI/DRV, lamivudine/zidovudine, lopinavir/RTV, and abacavir/lamivudine;fixed triple combinations—abacavir/lamivudine/zidovudine and abacavir/lamivudine/dolutegravir;fixed quadruple combinations—elvitegravir/COBI/emtricitabine/tenofovir.The main classes of ARV drugs comprise 30 molecules active against HIV-1 as follows: nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), integrase inhibitors, PIs, and pharmacokinetic enhancers (boosters). In addition, there are new fixed dose combinations between various molecules, designed to improve administration and thus achieve adherence:In the life cycle of HIV, the protease is an essential element for viral maturation. HIV protease is a homo dimeric aspartyl protease, each monomer being composed of 99 amino acid residues with a catalytic aspartic acid (Asp) at position 25. HIV-1 protease cleaves the precursor of Gag (Gag) and Gag-Pol polyproteins encoded by the HIV genome-1 at nine processing sites to produce mature active proteins. Pol-polyproteins are first cleaved from Gag-Pol polyproteins and then further digested into protease, reverse transcriptase (p51), H ribonuclease (RNase H) (p15), and integrase .The active site is not fully exposed, being covered by two flaps, which must be opened to allow the substrates to access it. HIV-1 protease activity can be inhibited by blocking its active site. The indispensable role of HIV protease in viral maturation makes it a major target of ARV drugs. A large number of HIV protease protein structures have facilitated the formulation of new and improved inhibitors .The first PI approved by the United States was RTV (1996), shortly followed by indinavir (1996), nelfinavir (1997), saquinavir (1997), amprenavir (1999), lopinavir/RTV (2000), atazanavir (2003), fosamprenavir (2003), tipranavir (2005), and DRV (2006). The potency of these agents is similar, the choice of one of them in therapy is made taking into account the pharmacokinetics (regardless of how many times they are administered/day), tolerance, and the presence of antiviral resistance. Most HIV PIs are metabolized by the liver through enzymes that metabolize the drug cytochrome P450 (CYP450). Importantly, all approved HIV PIs have a high potential for drug–drug interactions due to their ability to inhibit drug metabolizing enzymes, most commonly cytochrome P450 3A4 (CYP 3A4) ,11.RTV is the strongest inhibitor of CYP 3A4 and, for this reason, is often combined in small doses (100 to 200 mg daily) with other PIs to produce a booster effect, increasing plasma levels and the half-life of the protease, without significantly increasing side effects ,13.COBI is a newly introduced pharmacological enhancer that has inhibitory activity against several drug metabolizing enzymes in addition to CYP 3A4 including CYP 2D6 and the P-glycoprotein (P-gp) transporter, making it an important agent. It is used in therapy when the increase in active substance levels of agents metabolized by the cytochrome P450 system is desired, being available as 150 mg tablets .Ten HIV PIs are currently available: saquinavir, indinavir, RTV, nelfinavir, amprenavir, fosamprenavir, lopinavir, atazanavir, tipranavir, and DRV. PIs represent the largest class of drugs used in the fight against HIV. These substances block the activity of HIV protease, an enzyme that the virus uses to break down high-mass polyproteins into smaller units needed to assemble new viral particles .PI acts in the late stage of the HIV replication process. When the virus infects a cell, its replication systems are forced to produce long viral precursor proteins. These precursors are grouped so that they are able to leave the cell, causing cell lysis and its death. Even before proteins leave the cell, they must be cleaved by an enzyme called “HIV protease” . The protease acts in the budding virion, causing the morphologically distinguishable immature virions to mature. At such a late stage in replication, the benefit of this action is the relatively small locus of activity. The requirement that all viral proteins be enzymatically cleaved at this location creates a blockage and involves a very specific and effective action of disrupting viral replication. However, there is a disadvantage of this mechanism, namely that these substances are not potentiated and acted upon at the initial time of infection. Thus, PIs have a virustatic rather than a virucidal effect .However, HIV can still replicate (even in the presence of PIs), but the resulting virions are immature and do not have the ability to infect new cells. All molecules currently known to act by inhibiting HIV protease are metabolized by CYP450, especially by the CYP3A4 isoenzyme group.0, then at 1, 2, 3, and 4 h) may be helpful to clinicians in understanding whether the maximum concentration and prolonged exposure exceed the therapeutic safety limits.If a CYP 3A4 inhibitor (RTV or COBI) is administered associated with both fluconazole  and atazanavir (PI), the rate of metabolization of all ARVs decreases, leading to their increased concentration in plasma. The action of other drugs also metabolized by CYP 3A4 isoenzymes  will be potentiated, increasing the risk of toxicity or, conversely, blocking the activity of other substances by hepatic metabolism. CYP3A4 inducers decrease the PI concentrations although this effect has been shown to counterbalance the effect of stimulators. When concentration problems occur, monitoring plasma concentrations  have some structural similarities and a similar binding pattern, which may be responsible for some of the common side effects of the regimens containing them.ART promotes the development of resistant viral variants extremely fast, within a few days or weeks from the moment of incomplete suppression of replication . The virHIV-1 protease is prone to resistance development largely due to the process in which it is synthesized in vivo. Reverse transcriptase creates viral deoxyribonucleic acid (DNA) from viral ribonucleic acid (RNA) that is embedded in the virus host cell during infection. This process, unlike eukaryotic DNA synthesis, is highly prone to errors due to the lack of correction mechanisms, causing HIV-1 to develop many polymorphic mutations ,31.The high error rate of this process increases the probability that genetic mutants will be introduced into the system being influenced by the evolutionary pressures of PIs. Consequently, the resistance development is exacerbated and persistent due to the limited ability of many PIs to maintain effective concentrations in the body and/or to reach the desired sites of action such as the central nervous system (CNS) . The CNSTo improve the effective penetration of PIs, strategies have been used to increase the ability to cross the blood–brain barrier (BBB) by increasing the lipophilic nature of inhibitors and introducing fluoride into the structure in different positions ,34.Resistance to PIs occurs due to mutations in the HIV protease. Currently, about 10–20 such mutations are known. Frequently, multiple mutations can occur/exist in the same patient, which leads to the appearance of totally different enzymatic structures . PublishUnique amino acid substitutions in the cavity of the active site of the enzyme, altering the size and shape of the side chains, which may alter the affinity of the drug and preserve the catalytic activity. The known mutations of this type are D30N, V32I, I47V, G48V, I50V, Val82, I84V, and G48T/L89M .Mutations that reduce the stability of the protease dimer, increasing dissociation between these portions and releasing limited inhibitor. The mutations associated with this mechanism are L24I, I50V, F53L, and Ile50 .Distal mutations, also related to PI resistance, may occur in different ways and may be difficult to detect, but may promote structural changes until the interaction with the inhibitor changes ,45. DistThe evaluation of the structural changes and the activity of the mutant proteins highlighted three categories of mutations, as follows:The highest resistance to PIs is correlated with primary and secondary mutations in the protease gene. Primary mutations are caused by changes in the active locus of the protease; secondary mutations help restore viral protease activity, increasing viral replication capacity. Typical examples are V82A/F/T mutations, which contribute to the resistance to many PIs including lopinavir/RTV, atazanavir, fosamprenavir, nelfinavir, and saquinavir. Cross-resistance to other ARVs has also been studied in people infected with HIV using more than 6000 clinical isolates. These studies indicated that 59–80% of isolates had reduced susceptibility to indinavir (IDV), RTV, nelfinavir (NFV), and saquinavir (SQV), among other PIs .The most common and general approved PIs have lower than optimal pharmacokinetic properties. This is due to the fact that PIs are only substrates for the CYP3A4 isoenzyme  .As a result, numerous approved PIs have low plasma levels and short half-life. Interestingly, some PIs act as both inhibitors and inducers of isoenzymes. As it turns out, RTV is a potent inhibitor of CYP3A4 and the administration of a low dose of RTV with PI significantly improves the pharmacokinetic parameters of PIs. PIs such as DRV, fosamprenavir, and atazanavir can be given as a once daily dose when given with a low dose of RTV .However, the use of RTV as a pharmacokinetic booster may raise some issues. When RTV is used as an enzyme inhibitor for other classes of antiviral compounds in HAART that does not contain PI, a subtherapeutic dose of RTV may accelerate the onset of PI-resistant HIV-1 variants . Multi-rUnlike RTV, COBI will not contribute to the development of drug-resistant HIV-1 variants. The formulation of a PI with COBI resulted in an all-in-one pill, convenient for patients, resulting in increased adherence by reducing the pill burden. While this regimen has improved the pharmacokinetic properties of currently approved PIs, a PI design that does not require a pharmacokinetic booster is the ideal option.Pharmacokinetics describes the quantitative aspects of the pathway of the drug substance in the body after administration . Figure Absorption may take some time; the interval elapsed from the time of administration until the occurrence of a certain level of concentration in the blood is called “lag time”—the period of delay. Absorption determines the level of plasma concentration . The staIf the distribution takes place faster than elimination, a biphasic decline will be observed on the curve. The first curve that will decrease, much faster, results from the distribution of the drug in the compartments of the body. The second curve occurs later, is slower, and represents a decrease in the plasma concentration of the substance after elimination . The time when/at which the plasma concentration of the drug falls below 50% is called the half-life (t1/2). Due to the different distribution and elimination, there are two half-lives, but in practice, no distinction is made between them ,55.At the end of the dosing interval, before the ingestion of a new dose, a residual concentration (Ctrough) is reached, which is most often equal to the minimum concentration (Cmin). The area under the curve (AUC) is a pharmacokinetic parameter that measures drug exposure over the entire dosing interval, without providing information if these pharmacokinetics have a dynamic profile  and the minimum concentration (Cmin)) .In the case of ARV drugs, this link between active substance concentrations, efficacy, and toxicity levels has been demonstrated, therefore Cmax, Cmin, and AUC are significant parameters. They indicate the degree of ARV exposure, either at a certain point in time (Cmax and Cmin) or at a certain time interval. The pharmacokinetics of any drug change with a single dose or with repeated doses. When the pharmacokinetic profile does not change after the next dose, an equilibrium level is reached, called the steady state. The time to reach this threshold is different and depends on a number of factors. Among ARV drugs, those that are capable of inducing CYP 450 enzyme systems need 14 days to reach steady state plasma levels .Another important factor in reaching the level of therapeutically effective plasma concentration is the binding to plasma proteins. The most important plasma proteins are albumin and alpha I-acid glycoprotein (AAG). AAG mainly binds HIV PIs and has a wide variety depending on race, body mass index (BMI) (stronger in those with high body mass index), and the stage of HIV infection (strong binding in the acute phase of the disease). Most ARV drugs are bound to plasma proteins, sometimes even completely. Only the free, unbound fraction is pharmacologically active, so the factors that prevent this binding influence the effectiveness of the therapeutic agent. The determination of the plasma drug concentration takes into account both the bound and the unbound fraction of plasma proteins .It is known that pharmacodynamics studies explain and describe the response/concentration relationship. When this correlation exists, plasma levels can be manipulated by adjusting the dose in order to obtain concentration values within a desired necessary therapeutic window . In practice, therapy monitoring can be used to correctly adjust the dose. If low doses are given for long periods of time and the level of plasma concentration falls below the optimum concentration, there is an increased risk of therapeutic failure due to the appearance of viral resistance and ART will be ineffective. On the other hand, too high doses of drug are associated with an increased risk of toxic effects, influencing the patient’s quality of life, with immediate consequences on their adherence to treatment .The pharmacokinetics of ARV substances are extremely different, depending on the concentration but also on the patient, with multiple side effects and high toxicity. Clinical studies have shown that there is a great variety in terms of therapeutic response, from one patient to another or for the same patient, depending on many variables/circumstances ,54,55,56Oral bioavailability of PIs is generally considered to be weak or variable . On average, the time to Cmax among agents is 3 h , with a median half-life of approximately 6 h. The elimination half-life varies between 3 and 15 h. Except for indinavir , all PI agents are extensively bound to proteins (>90%). Of all the pharmacokinetic parameters, the metabolic considerations on PIs may be the most worrying. This is especially true for patients receiving poly-medicine . PIs are inducers and substrates of multiple cytochrome P450 (CYP) isoenzymes including CYP 3A4, CYP 2D6, CYP 2C9, and β-glycoprotein.Compared to other classes of ARVs, PIs have unique characteristics that justify their prescription compared to other classes of drugs .A recent study pointed out that the intrinsic antiviral potency  may be higher for PIs than for other classes of ARV drugs. This observation is based on the fact that nucleoside analogues and integrase inhibitors target reactions in which a single enzyme complex and viral nucleic acid mediate a key step. Instead, PIs target the entire number of enzymes .Studies have found that PIs show residual ARV activity, even in individuals who have already developed resistance to other antiretrovirals. A significant proportion of newly developed viruses will remain partially sensitive to PIs for some time. This explains the need to continue a failed treatment regimen with PIs instead of eliminating them, in the case of patients who have no other treatment options. This is because the use of PIs is associated with low viral load and viremia below 10,000 copies/mL, which does not lead to a decrease in CD4, at least for 1–2 years ,59.Several studies comparing NNRTIs and PIs have shown that cluster gains of differentiation 4 (CD4) are generally higher using PIs than NNRTIs, even without dose adjustment in the case of patients undergoing complete viral suppression ,61,62.Reduced immune activation ;Reduction of CD4+ cell apoptosis ,65;Decreased fitness of PIs-resistant viral variants ;Affected replication of PIs-resistant viruses in T-cells (tymocytes) .From a clinical point of view, these characteristics make PIs a preferred class of ARVs, especially in patients with very low CD4 counts and whose priority is rapid immune restoration.One of the main limitations of most PIs is the low penetration into the central nervous system (CNS). This feature may not be relevant in the case of ARV regimens in triple combinations .The availability of PIs marked an important step in the treatment of HIV infection. However, there was a significant cost in terms of quality of life because first-generation PIs required complex dosing and had significant side effects. The latest PIs have overcome these limitations with a more convenient dosage  and a favorable safety profile and greater potency. The pharmacokinetic profile has improved considerably with low doses of RTV, and PIs continue to be key agents in the treatment of HIV . The maiTo minimize the metabolism of these enzymes, pharmacokinetic enhancers (PK enhancers) like RTV and COBI are used as ART due to their ability to inhibit CYP3A4, CYP3A5, and CYP2C9; this reduces the dose and frequency of PI administration ,72. MoniPeople living with HIV must follow strict treatment regimens to prolong their lives and reduce mortality . Lack ofThe most common side effects reported by patients treated with PIs are as follows: sexual dysfunction, gastrointestinal disorders , liver disorders, pancreatitis, cardiovascular disease, rash, and metabolic disorders. ARV intolerance and toxicity are the cause of frequent changes in treatment regimens. Unfortunately, this change also leads to the emergence of resistance ,87,88,89Progress in treatment and individualization of doses has led to fewer side effects compared to the initial regimens. Today, less than 10% of naive patients (those who are in the first treatment regimen) on ART still experience adverse events that could limit the treatment. Even so, long-term complications persist, especially the appearance of insulin resistance and disorders related to lipid metabolism. Therefore, it is necessary to continue monitoring the complete blood count and metabolic profile of these patients. Tests should be performed every three to six months, being also necessary an annual analysis of the urine (in order to verify proteinuria) and a lipid profile; if the treatment schemes are changed, the tests must be done again, respecting the periodicity ,89,90.Insulin resistance is a common subject in HIV patients, contributing to dyslipidemia and impaired glucose metabolism . Immune Metabolic disorders associated with PI therapy are the most problematic adverse events that may occur, their incidence varying from drug to drug. These disorders include hyperlipidemia, abnormal fat distribution (lipodystrophy), and impaired glucose tolerance, which are thought to be due to peripheral insulin resistance. Clinicians caring for patients treated with PIs should be alert to adverse events, especially those involving changes in lipid and glucose metabolism . Lipid dTwo studies published in 2007 on 25,000 patients showed that the use of PIs is directly correlated with myocardial infarction ,97. The Lipodystrophy is the redistribution of fat, with accumulation on the throat and abdomen, occasionally forming a “buffalo hump” , being usually associated with fat loss from the face, buttocks, and extremities. This disorder is resistant to treatment, often not reducing even with the discontinuation of the triggering agent. Some patients become unrecognizable after the treatment because of the body and facial feature alterations, many of them resorting to plastic surgery for some relief. Glucose intolerance, which sometimes leads to diabetes and genuine diabetic ketoacidosis, have been reported more and more frequently .Hyperlipidemia is closely associated with both hyperglycemia and redistribution of fat and may put HIV patients receiving HAART at increased risk for cardiovascular disease . PIs supMany patients experience gastrointestinal symptoms at the beginning of treatment with PIs or when switching from one regimen to another. These symptoms frequently lead to discontinuation of therapy and can be a major obstacle to efficient treatment . The stuSexual dysfunctions occur more frequently in patients treated with PIs than in patients treated with other substances . There aMild to moderate increases in serum aminotransferase and alkaline phosphatase levels that occur in a large proportion of patients taking ARV regimens containing PI. Moderate to severe increases in serum aminotransferase levels  are found in 2% to 18% of patients, depending on the agent, the frequency of monitoring, and most importantly, the presence of virus coinfection with hepatitis B (HBV) or C (HCV). In HIV patients, without HCV or HBV infection (“mono infection”), increases in alanyl aminotransferase (ALT) and aspartate aminotransferase (AST) are uncommon; levels of more than five times the upper normal limit were reported in only 1–4% of patients. These increases are usually asymptomatic and self-limiting and can be resolved even with continued medication. Therefore, this should not be a reason to discontinue the medication.Hyperbilirubinemia, without other manifestations of liver damage, is caused by the inhibition of glucuronyl transferase UDP, the liver enzyme responsible for conjugating bilirubin that is deficient in Gilbert’s syndrome. Hyperbilirubinemia is usually mild, averaging 0.9–1.5 mg/dL, but may be more severe in patients with Gilbert’s syndrome with increases of 2.5 mg/dL or more and clinical jaundice. Jaundice, however, does not indicate liver damage. However, jaundice caused by PIs can be painful for the patient and is a reason to discontinue treatment.Acute, idiosyncratic liver damage has been reported in most agents not very frequently. The few reported cases usually occurred within one to 12 weeks after starting therapy, and the pattern of serum enzyme growth ranged from hepatocellular to mixed and cholestatic. Signs of allergy or hypersensitivity  may also occur as well as the appearance of autoantibodies. Acute liver damage from PIs is usually self-limiting but severe and isolated cases of acute liver failure have been reported, especially in patients with pre-existing underlying liver disease.Exacerbation of chronic underlying hepatitis B or C in co-infected individuals. Hepatitis usually occurs in 2–12 months after starting therapy, being associated with a hepatocellular pattern of serum enzyme growth, and also by increases, followed by decreases in the serum levels of HBV DNA or HCV RNA. These fluctuations can be severe, and even fatal cases have been reported .All drugs in the class of PIs have been associated with liver damage. About a third of patients treated with PIs have liver problems associated with high levels of enzymes. Most of the time, these reactions are mild and transient . PIs areDrug interactions are the cause of many of HAART complications. Among them, an important role is played by the inhibition, respectively the enzymatic induction produced by PIs as well as the interactions of the P-glycoprotein.Enzymatic inhibition occurs when a substance blocks or reduces the activity of metabolic enzymes. The net effect is to increase plasma levels and the effects of the parent compound, while preventing the formation of metabolites and reducing elimination. All PIs are inhibitors of cytochrome 450, especially in the CYP 3A4 isomorph. From this point of view, PIs behave similarly to ciprofloxacin, erythromycin, clarithromycin, diltiazem, itraconazole, or ketoconazole. They all inhibit CYP 3A4 and affect the pharmacokinetics of low therapeutic index substances. For example, in patients treated with carbamazepine, vomiting and dizziness, increased liver enzyme levels, and increased serum anticonvulsant levels were observed 12 h after a dose of RTV . Even otThe metabolism of the PIs is, in turn, affected by other potent 3A4 inhibitors, because all PIs are metabolized by the enzyme 3A4. Any substance with a stronger inhibitory effect on 3A4 can slow down the metabolism of PIs. The other potent inhibitors that influence the rest of the PIs include ciprofloxacin, clarithromycin, diltiazem, erythromycin, itraconazole, nefazodone, and grapefruit juice. Inhibition of PI metabolism by these agents may increase their efficacy or reduce the dose required (as seen when RTV is used to potentiate other PI). However, there is also an adverse reaction such as worsening of side effects and toxicity, which leads to an increased risk of events such as headaches, nausea, diarrhea, hepatitis, and pancreatitis. All lead, in the end, to non-adherence. It should be mentioned that the interruption of the potent inhibitor, other than PIs, entails a return to the uninhibited state, but also a rapid reduction in circulating levels of PI, which puts the patient in danger of developing viral resistance to the entire class of PIs.This represents the phenomenon of increase or acceleration of metabolic enzyme activity, under the action of a substance or a compound. The net effect is to decrease the levels of the parent compound and its effects, while increasing the production of metabolites and the amount available for elimination. Rifampicin, carbamazepine, phenytoin, ethanol, and barbiturates are potent inducers of many enzymes belonging to the CYP 450 family . InductiThese glycoproteins are responsible for the outflow or “pumping” of substances outside the cells and are the “guardians” that influence the bioavailability and pharmacokinetics of drugs in the intestine and liver and prevent the penetration of the drug into the brain, testicles, and placenta. PIs are all substrates of P-glycoproteins, which may explain why it is difficult for ARVs to cross the blood–brain barrier or other sanctuaries. When P-glycoproteins are inhibited, the concentration of the drug increases, especially if it is a substrate for the P-glycoprotein. RTV initially inhibits the P-glycoprotein, which may allow other substrates to reach higher serum levels, either in the intestinal wall or in the blood–brain barrier. This inhibition of P-glycoprotein, together with 3A4 inhibition, would have an additive inhibitory effect and would increase the level of substrate drugs for P-glycoprotein and CYP 3A4. In vitro, long-term RTV exposure induces P-glycoprotein expression, reducing the drug ability to ‘cross the barrier’, and effectively reducing the bioavailability of P-glycoprotein as a substrate ,111.ART combined with two or more PIs has changed the standard of care in the treatment of HIV infection. Two PIs regimens such as DRV/RTV are commonly used as initial therapy with PIs. As viral resistance increases and the development of new PIs decreases, clinicians resort to dual RTV-boosted PIs to treat patients. The potency of these combination therapies is increased, while the burden of pills, dietary restrictions, and often side effects are reduced. These clinical advantages lie in the improvement of the pharmacological properties of the substances, in particular, through changes in pharmacokinetics (absorption and metabolism). Changes are occurring in the absorption and metabolism of PIs when co-administered with a cytochrome P450 enzyme inhibitor (CYP) . The ability of RTV to increase concomitant plasma concentrations (Cmin) of co-administered PI is probably the greatest clinical benefit of dual therapy, as inadequate ARV concentrations may support long-term ARV resistance.The fact that there is a close link between the therapeutic response and the plasma concentration of the drug substance is certain. Studies show that inadequate levels of circulating substance play an important role in therapeutic failure and the emergence of viral resistance . Low adhRTV also has tolerability problems in a large number of patients. Other disadvantages of RTV are that it inhibits/induces other enzymes that metabolize drugs, resulting in a significant number of drug-drug interactions . Based oCOBI’s CYP 3A4 inhibitory potency is similar to that of RTV . A totalCOBI is marketed as a single agent or in fixed combinations, in association with atazanavir, DRV, or elvitegarvir. The two boosters, COBI 150 mg/day and RTV 100 mg/day, may be interchangeable with similar therapeutic results. However, clinicians prefer the administration of 150 mg COBI as a boosting agent for CYP 3A4 metabolites rather than RTV due to its higher specificity and the fact that it has no enzyme induction effect. Hence, the profile of interaction with other drugs is different.RTV, marketed as Norvir; it is included in Kaletra—lopinavir/RTV;COBI, commercially called Ybost; it comes in many combinations: atazanavir/COBI (Evotaz), elvitegravir/COBI/emtricitabine/tenofovir (Genvoya), DRV/COBI (Resolve), elvitegravir/covetousness/emtricitabine/tenofovir disoproxil (Stribild), DRV/COBI/emtricitabine (Symtuza).Although both COBI and RTV have similar boosting profiles for CYP3A4 and can be used with the same therapeutic efficacy, COBI is preferred due to its greater specificity and the absence of inducing properties. In addition, the two substances interact differently with other biochemical/medicinal substances metabolized by the following enzymes: CYP1A2, CYP2B6, CYP2C8, and CYP2C9. In this context, one of the biggest challenges for clinicians is to adjust doses in patients with multi-drug therapy:37H48N6O5S2, is an ARV drug in the class of systemic PIs used in combination with other drugs for the treatment of HIV/AIDS. It was approved for commercialization on 1 March 1996, becoming the seventh ARV and the second PI entering the U.S. market. This class of ARVs has played an extremely important role in reducing the number of deaths caused by HIV/AIDS. The introduction of HAART at that time in the United States led to a decrease in the annual death rate from 50,000 to 18,000 . T. T127]. From the analysis of the 2D diagram, it is revealed that RTV binds to the following amino acids of the wild type HIV-1 protease: GLY27, ALA28, VAL32, ASP29, ALA82, ALA28, PRO81, ARG8, and ILE47. ASP25 forms hydrogen (H) bonds to the oxygen in the HIV protein side chain, ALA28 and PRO81 form π–alkyl bonds via chains A and B. The amino acids ALA28 and ILE50, in the HIV-1 chain A and B establish π–alkyl and alkyl bonds with RTV. ILE47 and ALA28 form a π–alkyl bond with the 1,3-thiazole group of the ligand, ASP30 through C–H, and ALA82 through both the C–H and the π–alkyl bonds. ARG8 bonds via π–cationic with the protein.In RTV docking with the B-chain of HIV-1, the main link is π–alkyl. PRO81 is formed by the pyrolidine protein bond through the benzene ring. The amino acid ASN25 in the A chain as well as in the B chain  bonds and interacts at the same oxygen atom in the ligand structure.ILE84 forms a π–alkyl bond with benzene and C-alkyl in the ligand side chain. VAL32 binds by alkyl, and GLY27 and ASP25 by the H bond. ASP29 and GLY49 to O, in the side chain, form the C–H bond, and ASP29 also forms the H bond. Absorption. According to the summary of product characteristics (SPC), a study of HIV-infected volunteers evaluated the pharmacokinetics of RTV multiple-dose regimens during fasting. RTV accumulation was observed to be slightly lower than that calculated on a single dose basis due to an increase in apparent dose-dependent clearance. Over time, the plasma concentration of RTV before the next dose decreases, possibly due to enzyme induction, but appears to stabilize after two weeks of treatment. The time to peak plasma concentration (Tmax) remained constant—approximately 4 h as the dose increased. Renal clearance was on average less than 0.1 L/h and relatively constant over the entire dosing range. It has been observed that foods slightly decrease the bioavailability of RTV film-coated tablets, especially foods with moderate and high lipid content .Distribution. The apparent volume of distribution (VB/F) of RTV is approximately 20–40 L after a single 600 mg dose. In humans, RTV has been shown to be 98–99% bound to plasma proteins and is consistent at concentrations between 1.0–100 µg/mL. RTV binds with comparable affinities to both alpha-1 acid glycoprotein (GAA) and human plasma albumin (APU). Studies in rats have shown that the highest concentrations of RTV are achieved in the liver, adrenal glands, pancreas, kidneys, and thyroid. The tissue/plasma ratio was approximately 1 in the lymph nodes in rats, indicating that RTV is distributed in the lymph tissue. It slightly penetrates into the brain .Drug metabolism. RTV is extensively metabolized by hepatic cytochrome P450, mainly by CYP 3A4 isoenzymes and, to a lesser extent, by CYP 2D6 isoforms. Animal and in vitro studies with human liver microsomes have shown that RTV is oxidatively metabolized. In humans, four metabolites of RTV have been identified. The major metabolite is isopropyl-thiazole (M-2), with antiviral properties similar to the parent compound. However, the AUC of metabolite M-2 was approximately 3% of that of RTV .Clearance. RTV is eliminated primarily by the hepatobiliary route. Approximately 86% of the radiolabeled molecules were found in feces, some of which is thought to be unabsorbed RTV. In studies, the renal route has not been shown to be a major route of elimination of ritonavir .The RTV target organs are the liver, retina, thyroid gland, and kidneys. Hepatic changes involved hepatocellular, biliary, and phagocytic structures and were accompanied by increases in liver enzymes. Human clinical trials did not reveal RTV-induced ocular changes. All thyroid changes were reversible upon discontinuation of RTV. Clinical investigations did not show significant impairment of the thyroid function tests or significant renal impairment in humans. There were observed various side effects: common (at low doses): diarrhea and increased lipid levels; and rare: changes in heart rate, severe allergies (hypersensitivity reactions) and eczema (Steven-Johnson syndrome) ,72,129.40H53N7O5S2) is a structural analogue of RTV, in which the valine fragment was replaced with a 2-morpholinethyl group and the hydroxyl group was removed. These changes cancel out the anti-HIV activity per se of COBI, retaining only the highly inhibitory and selective effects on the CYP 3A protein isoenzyme family [Cobicistat -2- methyl) (methyl) carbamoyl- amino)-4-(morpholin-4-yl) butanoic acid with the amino group of 1,3-thiazol-5-ylmethyl -5-amino-1,6-diphenylhexane-2-yl) carbamate, COBI has a molecular weight of 776 (g/mole), good solubility, and terminal half-life of 3–4 h. Plasma protein binding is 97–98% and CSF to plasma ratio is 1.1% .It acts as a pharmacokinetic enhancer in the treatment of HIV-1 by inhibiting the CYP P450 enzymes responsible for the metabolism of other drugs. It works as a CYP450 inhibitor. It belongs to the class of 1,3-thiazoles, but also to the class of morpholines, urea, is a carbamate ester and an amide of monocarboxylic acid. The chemical structure of the substance is shown in Selective inhibitor of cytochrome P450 (subfamily CYP3A enzymes) with RTV-like potency, and COBI inhibits the mechanism of action of these enzymes. Inhibition of CYP3A-mediated metabolism enhances systemic exposure to drugs that are substrates for CYP3A (atazanavir or DRV), which have limited bioavailability after oral administration and a reduced half-life due to CYP3A-mediated metabolism. The inhibitory effect of COBI is also manifested on other enzymes: CYP 3A4, CYP2D6, P-gp, OATP1, MATE1, and BCRP . COBI doProvided in the form of a film-coated tablet, COBI assures 150 mg active substance concentration. It is indicated as a pharmacokinetic enhancer for atazanavir 300 mg once daily, or DRV 800 mg once daily, as one of the components of ART combination in HIV-1 infected adults. It is recommended to be co-administered orally, both food and the drug. The tablet should not be chewed or crushed. COBI is formulated in fixed doses with atazanavir, DRV, and tenofovir/emtricitabine/elvitegravir .n = 68) were 1.2 ± 0.3 µg/mL, 10.9 ± 0.8 µg h/mL, and 0.07 ± 0.07 µg/mL, respectively [Absorption. After oral administration of COBI with food to HIV-1 infected subjects, peak, plasma concentrations were reached 4 h post-dose. Mean steady-state Cmax, AUC, and Cper-dose values  after administration of multiple doses of COBI to HIV-1 infected subjects  by CYP3A and (minor pathway) by CYP2D6 and is not subject to glucuronidation. After oral administration, 99% of the circulating radioactivity detected in plasma was due to unmodified COBI. Concentrates of metabolites are low in urine and feces and do not contribute to the CYP3A inhibitory activity of COBI .Clearance. After oral administration, 86% and 8.2% of the dose were found in feces and urine, respectively. The median elimination half-life of COBI after administration of Tybost (trade name) is approximately 3–4 h ,134.Pharmacotoxicology. Conventional studies on repeated dose toxicity, genotoxicity, and toxicity to reproduction and development have shown no risk to humans.Side effects. As common side effects can be highlighted the following: high blood sugar, increased appetite, insomnia, drowsiness, nightmares, headache, dizziness, altered taste, nausea, diarrhea, abdominal pain, bloating, flatulence, dry mouth, jaundice, eczema and itch; rare side effects include kidney stones ,136.DRV is the latest formulated PI  providinThe compound was licensed for marketing in the United States of America in 2006 and in 2007 in Europe. Since 2016, DRV is a treatment option for children over three years of age, adolescents, and naive adults, infected with HIV-1, but also for critical patients . Since iLow doses of RTV or COBI are used to increase DRV plasma level. It can also be used in prophylaxis. The administration is done once or twice a day, only according to a schedule established by the specialist. This regimen may include 800 mg of DRV and 150 mg of COBI, 800 mg of DRV and 100 mg of RTV (once daily). When given twice a day, 600 mg of DRV in combination with 150 mg COBI or 100 mg RTV should be taken .DRV is marketed as a 100 mg/mL oral suspension or as 75, 150, 300, or 600 mg film-coated tablets. In fixed doses, it is formulated with COBI (800 mg DRV/150 mg COBI). As with other ARVs, DRV does not cure HIV/AIDS .DRV is an inhibitor of dimerization and catalytic activity of HIV-1 protease, thus inhibiting proteolytic activity and subsequent replication of HIV-1. It also selectively inhibits the cleavage of Gag-Pol-encoded HIV polyproteins in virus-infected cells, preventing the formation of infectious mature viral particles .have not been previously treated with ART; and6/L. In deciding to start DRV treatment in these patients previously treated with ART, the administration should be guided by genotypic testing [have been previously treated with ART, have no DRV resistance mutations (DRV-RAMs) and have HIV-1 plasma RNA <100,000 copies/mL and CD4 + cell count ≥100 cells × 10 testing .In therapy, a low dose of RTV or COBI, in combination or not with other ARV medicines, should be used to treat patients infected with HIV-1, adult, adolescent, and pediatric patients over three years old and with body weight ≥10–15 kg ,149, whoAfter initiating treatment with DRV, patients are advised not to change the dose, dosage form, or discontinue treatment without talking to their healthcare professionals. The drug interaction profile of DRV depends on the use of RTV or COBI as a pharmacokinetic enhancer. Therefore, DRV may have different contraindications and recommendations for concomitant use depending on the use of RTV or COBI for potentiation .The efficacy and safety of DRV alone or in combination with potentiating drugs have been demonstrated in numerous studies ,155,156.If the affinity for wild-type strains is increased, species that have developed resistance to PIs are less sensitive to DRV, and ARV activity is compromised .The major complication of ART is the evolution of drug-resistant viral strain variants. This resistance occurs due to rapid viral replication. Most PIs select viral species with one or more specific mutations in the coding region that confer resistance. DRV appears to have a high genetic barrier against developing viral resistance in the case of wild-type virus as well as a large group of clinical isolates resistant to other PIs . FollowiAlthough structurally, DRV is similar to amprenavir, DRV has two intertwined tetrahydrofuran rings. This difference is important in the antiviral activity of DRV, because the bistetrafuran fragment reverses stereochemistry at the point that binds it to the rest of the molecule and allows additional interactions of DRV with ASP 29, a key amino acid of the HIV protease .Pharmacokinetic studies in which DRV was administered alone showed a bioavailability of 37%. In combination with RTV, a potent CYP3A inhibitor, the plasma concentration increased, the bioavailability reaching 82%. This demonstrates that DRV is sensitive to a presystemic metabolism or to an efflux effect or even to both. Potentiation drugs reduce these effects. According to CPR, DRV is primarily metabolized by CYP3A. RTV and COBI inhibit CYP3A, thereby significantly increasing plasma DRV concentrations ,169,170.DRV is rapidly absorbed after oral administration. The maximum plasma concentration in the presence of a low dose of RTV is generally reached within 2.5–4 h ,149. TheP-glycoprotein from intestinal epithelial cells may decrease oral absorption of PIs by mediating drug efflux. Low levels of intestinal absorption, together with CYP 450 activity, are major factors in the low bioavailability of DRV. The combination of a boosting drug inhibits both the action of CYP 450 and the efflux transport systems in the intestinal mucosa, thus improving the bioavailability of DRV ,149.DRV is strongly bound to plasma proteins in a proportion of approximately 95%, binding in particular to plasma α1-acid glycoprotein ,167. It In vitro experiments with human liver microsomes (HLM) indicated that DRV mainly undergoes oxidative metabolism. DRV is extensively metabolized by the hepatic system CYP 450 and almost exclusively by the CYP3A4 isoenzyme. In humans, at least three oxidative metabolites of DRV were identified, with 10-fold lower activity on the wild-type HIV than the parent substance ,161.A study conducted in 2009  demonstrAfter a dose of 400/100 mg, DRV with RTV, approximately 79.5 and 13.9% of the administered dose is found in the feces and urine, respectively. Unchanged DRV was approximately 41.2 and 7.7% of the administered dose in feces and urine, respectively. The terminal clearance half-life of DRV was approximately 15 h when combined with RTV. The intravenous clearance of DRV alone (150 mg) and in the presence of a low dose of RTV was 32.8 and 5.9 L/h, respectively ,149.DRV is generally well tolerated in humans. The most common side effect is rash 7% of cases. This is followed by diarrhea (2.3%), headache (3.8%), abdominal pain (2.3%), constipation (2.3%), and vomiting (1.5%). DRV can also cause allergic reactions, and people allergic to RTV can also have a reaction to DRV ,176. HigAs common side effects of DRV are as follows: diarrhea, vomiting, nausea, indigestion, eczema, itching, abdominal pain, bloating, flatulence, headache, fever, peripheral neuropathy, lipodystrophy, insomnia, weakness, fatigue, diabetes, increased creatinine levels, cholesterol, and liver enzymes. Rare side effects  may also occur. It must not be used in patients with severe hepatic impairment and should be used with caution in cases of moderate hepatic impairment. Drug–drug interactions are a permanent concern for clinicians treating HIV-infected patients. In addition to HAART, which requires combinations of multiple ARV drugs, patients should frequently take other medications to prevent or treat opportunistic infections, associated diseases, and to manage ARV side effects. Many of these drug interactions can lead to dose adjustments or even contraindications to their use . HoweverThe emergence of PIs was an important step in the treatment of HIV-infected patients. Their use, together with two nucleoside analogues, has radically changed, in a positive way, the life prognosis of patients. If the initially developed PIs had many side effects and thus imposed limitations in administration, recent generations are well tolerated, significantly improving the quality of life of the patients. Today, PIs have a convenient dosage, are administered in a single dose, daily, with high potency and a favorable safety profile. Co-administration of a pharmacokinetic enhancer has greatly contributed to this effective and safe therapeutic profile of PIs. To this, it is added the increased barrier to the occurrence of viral resistance of PIs and the specific immune benefit of this class. All these features make PIs key agents in the fight against HIV. Dual therapeutic regimens were initially designed to be administered to patients in whom monotherapy had failed. However, the practice has proved unquestionable qualities; in addition to the much higher potency, there has been better patient compliance due to the decrease in the pill burden and the reduction in the number and intensity of side effects.DRV is part of the new generation of PIs, bringing a great advantage in the treatment of HIV/AIDS because it has superior efficacy on wild-type HIV but also on already resistant strains. DRV co-administered with a booster—RTV or COBI—has a higher pharmacokinetic profile due to booster inhibition of metabolic pathways. Boosted DRV is well tolerated by patients, with fewer side effects than other regimens. Therefore, DRV co-administered with RTV or COBI is a viable treatment option for both naive and experienced patients.COBI and RTV are considered equivalent pharmacokinetic stimulants with similar inhibitory effects on CYP3A4, CYP2D6, P-gp, and other transporters. Similar exposures to elvitegravir, atazanavir, and DRV are made when combined with either substance. However, in the presence of inducers, etravirine, rifamycin, or anticonvulsants, COBI may not be as effective as RTV in inhibiting CYP3A4. However, COBI is a more specific inhibitor of CYP3A than RTV and has no inducing properties. Thus, it is anticipated that drugs metabolized primarily by CYP1A2, CYP2B6, CYP2C8, CYP2C9, and CYP2C19 or subject to glucuronidation reactions will be affected differently by RTV and COBI. Additionally, data on drug–drug pharmacokinetic interactions for COBI are often extrapolated from RTV. However, separate studies are needed in the case of COBI. Although they have similar profiles, problems can also occur when changing a treatment regimen that contains RTV with another that includes COBI. Clinicians need to know the differences in the pharmacokinetic profile of the two boosters, especially when evaluating and managing potential drug interactions, with most HIV+ patients having multiple comorbidities and co-medications. In these situations, monitoring the therapy, by various means, is absolutely necessary in order to be able to act promptly by adjusting the doses or by modifying the therapeutic regimens.


BackgroundAround 80-85% of coronavirus disease 2019 (COVID-19) cases were reported to have mild disease and home treatment of such patients was proved to be effective without significant morbidity or mortality. Therefore, the aim of this study was to assess the outcome of home management of non-severe COVID-19 infection in healthcare providers in the developing world.MethodsThis observational cohort study was conducted at the National Institute of Cardiovascular Diseases from June 2020 till January 2021. It included health care workers who tested positive for COVID-19 with non-severe infection and received home treatment. The COVID-19 management team monitored their symptoms and oxygen saturation over the phone. Need-based lab tests, X-rays, home proning, steroids, and oxygen were administered along with the standard intuitional management strategies. Study outcomes included duration of recovery, need for hospitalization, and expiry.ResultsA total of 128 patients were included, out of which 98 (76.6%) were male, and the mean age was 32.9 ± 5.9 years. Fever was the most common symptom, seen in 89.8% of patients. Most of the patients (85.9%) had no pre-existing comorbidities. Five patients received home oxygen therapy, seven received steroid therapy, and one received home pruning. The average time of recovery was 13.8 ± 8.1 days with no mortality; however, 14 (10.9%) patients were hospitalized due to worsening of symptoms.ConclusionHome treatment for COVID-19 patients with mild to moderate disease after appropriate risk assessment can be a safe and effective option to preserve hospital capacities for more needy and severely ill patients. Coronavirus disease 2019 (COVID-19) became a global pandemic with its overwhelmingly high transmission rates after its outbreak in Wuhan in late 2019 [Since the start of the disease from 2019 till June 2021, more than 175,676,457 people were infected with COVID-19, and out of them, more than 3,790,320 people lost their lives [The good thing in this scenario was that around 80% of the patients were presenting with non-severe disease ,8 but onThis was an observational cohort study conducted at the COVID-19 care unit of the National Institute of Cardiovascular Diseases (NICVD), Karachi, Pakistan. It was conducted from June 2020 till January 2021. It was started after approval from the ethical review committee of the NICVD . All those doctors and paramedics who gave consent and had symptoms of fever, cough, or shortness of breath and were diagnosed as COVID by a polymerase chain reaction (PCR) test were included in the study. Those patients who were asymptomatic and tested positive because of exposure were also included. All the patients who were included belong to the non-severe COVID group. All those patients were excluded who have symptoms but PCR was negative, or have a severe or critical disease at presentation, or were not health care providers.Patients were classified, managed, and selected for study by the COVID-19 management team of the hospital, which included selected doctors from the departments of infectious diseases, critical care, and cardiology. This team was created to deal with patients with suspected and confirmed COVID. On the basis of World Health Organization (WHO) criteria, COVID patients were classified into non-severe, severe, and critical groups by this team. In the non-severe COVID category, those patients were included who were either asymptomatic or have symptoms but maintained oxygen saturation above 94% at room air and had clear lung fields on X-ray chest. The diagnosis of COVID-19 was made on the basis of a positive COVID PCR test via a nasal swab. After initial evaluation by the above team, patients were discharged and were followed at home via phone calls. On phone calls, the COVID management team assessed symptoms, temperature spikes, and oxygen saturation by thermometers and oxygen saturation probes, respectively, for each patient. Until they maintained an oxygen saturation of more than 94%, they were kept on symptomatic therapy, and X-rays and labs were only done if the patients' saturation dropped to less than 94%.Data collection was done on a structured questionnaire, which included demographic data (gender and age), reported symptoms , and gastrointestinal (GI) symptoms), and preexisting conditions  of all the enrolled patients (see Appendices). Laboratory and imaging assessments, such as chest X-ray (CXR), C-reactive protein (CRP), complete blood count (CBC), absolute lymphocyte count, ferritin, and lactate dehydrogenase (LDH) levels, were obtained for patients where ever needed based on clinical assessment and dropping of oxygen saturation. Study outcomes included the duration of recovery, need for hospitalization, and expiry.Data were collected on a structured proforma and analysis was performed using Statistical Package for the Social Sciences (SPSS) version 19 . Categorical data were summarized as frequency and percentages and mean ± standard deviations were computed for quantitative data.A total of 128 doctors and paramedical staff of the institution who tested positive for COVID-19 and received home management during the first and second waves of the COVID-19 outbreak were included. Out of 128, 98 (76.6%) patients were male, the age range was from 21 to 58 years, with a mean age of 32.9 ± 5.9 years. The mean duration of symptoms was 10.0 ± 6.7 days. The most commonly reported symptom was fever (89.8%), as shown in Table Oxygen saturation monitoring was the main tool of home management. It remained >94% in 108 (80.5%) of patients. No further hematological testing was done in these patients who were managed symptomatically. In the rest of the cases, lab investigations were conducted; the results are shown in Table Among those patients who dropped their oxygen saturation, five patients (3.9%) received home oxygen therapy, seven patients (5.4%) received steroid therapy, and one patient received home proning. The average time of recovery was 13.8 ± 8.1 days; however, 14 patients (10.9%) were hospitalized due to worsening of symptoms. Of these re-hospitalized patients, chest X-ray was normal in nine patients, bilateral infiltrates were observed in two patients, and mild bilateral perihilar infiltrates, bilateral haziness, and mild right-sided pleural effusion were reported for one patient each, both of whom were managed at home or moved to hospital with no mortality in either case.The COVID-19 outbreak proved to be the ultimate test of healthcare systems around the world, especially in resource-limited healthcare systems of low- and middle-income countries like Pakistan with weak governance, insufficient health policies, improper basic health facilities, and poor response of the general public towards protective measures and SOPs . Under tAppropriate risk assessment of the patients is crucial. Patients with severe characteristics such as room air oxygen saturation of <90% and hypotension requiring inotropic support should be considered for intensive care admission. Patients with moderate characteristics, such as a room air oxygen saturation of <95%, heart rate of >110 bpm, respiratory rate of <24/min, fatigue, and systolic blood pressure >90 mmHg should be considered for in-hospital management, and patients with mild symptoms can be considered for home treatment for symptoms, such as fever, sore throat, or cough while maintaining adequate nutrition and hydration -15.Patients included in this study were all healthcare professionals, presumably with a better understanding of disease progression, however, to adopt such a strategy for the general population, more comprehensive telemedicine infrastructure would be needed. The effectiveness of such initiatives during the current pandemic has been witnessed in various parts of the world. Zhai Y et al. reported substantial benefits of the Telemedicine Consultation System (ETCS) introduced by the National Telemedicine Center of China (NTCC) in remote patient monitoring, consultations, multidisciplinary care, prevention, spreading awareness education, and training . Crane SThe limitation of this study was the small sample size along with the inclusion of only health care workers of a single center. But this gave confidence that this system can be implemented on a larger scale in the society to offload non-severe COVID patients’ burden on the health care system of developing countries. The cost of a pulse oximeter is far less than the cost of hospitalization, so it is the most effective tool in the home management of these patients. This also highlights the need for an integrated futuristic telemedicine infrastructure.The home treatment option of COVID-19 patients with a mild disease by implementing proper monitoring is safe and effective. Our experience of the home management of patients with close monitoring showed promising results with no mortality reports and a low hospitalization rate. Monitoring oxygen saturation via a pulse oximeter is the best tool in the home monitoring model to detect a deterioration in the condition before it worsens. While in a healthcare system with limited resources, a home treatment strategy after appropriate risk assessment can help make better use of available resources and preserve hospital capacity for needier and severely ill patients.


Prader–Willi syndrome (PWS) is a complex genetic syndrome combining hypotonia, hyperphagia, a PWS-specific neurocognitive phenotype, and pituitary hormone deficiencies, including hypothyroidism. The low muscle mass associated with PWS causes a low energy expenditure due to a low basal metabolic rate. Combined with increased energy intake due to hyperphagia, this results in a high risk of obesity and associated cardiovascular disease. To reduce the high mortality in PWS (3% yearly), exercise is extremely important. As hypothyroidism can impair exercise tolerance, early detection is crucial. We performed a literature search for articles on hypothyroidism in PWS, measured thyroid hormone (TH) levels in 122 adults with PWS, and performed a medical file search for medication use. Hypothyroidism (low free thyroxin) was present in 17%, and often central in origin (80%). Triiodothyronine levels were lower in patients who used psychotropic drugs, while other TH levels were similar. One in six patients in our cohort of adults with PWS had hypothyroidism, which is more than in non-PWS adults (3%). We recommend yearly screening of free thyroxin and thyroid-stimulating hormone levels to avoid the negative effects of untreated hypothyroidism on basal metabolic rate, body mass index, and cardiovascular risk. Additionally, we recommend measuring TH concentrations 3–4 months after the start of growth hormone treatment. Prader–Willi syndrome (PWS) is a rare, complex, multisystem condition with an estimated prevalence of 1/10,000 to 1/30,000 ,3. FeatuDue to hypotonia and the low muscle mass associated with the syndrome, adults with PWS have a low basal metabolic rate (BMR) which, combined with hyperphagia, increases the risk of developing obesity. To increase energy expenditure and compensate for this low BMR, exercise is extremely important. However, if left untreated, hypothyroidism can cause fatigue and exercise intolerance ,7,8,9. TApart from affecting BMR, hypothyroidism can also have more direct cardiovascular effects. Low thyroid hormone (TH) levels have been associated with diastolic hypertension, increased systemic vascular resistance leading to decreased cardiac output, myocardial stiffness, left ventricular diastolic dysfunction, accelerated atherosclerosis, and coronary artery disease . Even suOur clinical experience is that hypothyroidism is frequently missed in adults with PWS. The PWS-specific behavioral phenotype, physicians’ unawareness of the PWS-specific diagnostic pitfalls, and the lack of medical guidelines for adults with PWS can cause both patients’ and doctors’ delay. The intellectual disability present in most PWS adults makes it difficult for patients to express symptoms such as fatigue and constipation. In addition, physicians often falsely assume that fatigue or excessive daytime sleepiness are inherent to the syndrome, and do not perform further investigations. Moreover, reduced appetite and weight gain—other important symptoms of hypothyroidism—are hard to recognize, as adults with PWS have hyperphagia and are often on a diet. Therefore, hypothyroidism can easily be missed if not actively screened for.In 122 adults with PWS, we performed TH measurements and reviewed medical files for clinical data, including use of medication. As few large studies have investigated TH measurements in adults with PWS, we provide a thorough exploratory analysis of the patient characteristics possibly associated with TH concentrations  treatment, and use of psychotropic drugs). Additionally, we searched the literature for the prevalence and mechanisms of hypothyroidism  in adults with PWS. Based on our findings, we present recommendations for the screening and management of hypothyroidism in adults with PWS.Ethical review and approval were waived for this study by the Medical Ethics Committee of the Erasmus University Medical Center, Rotterdam, the Netherlands. In this retrospective study, we reviewed the medical files of adults that visited the multidisciplinary outpatient clinic of our PWS reference center in the Erasmus University Medical Center, between January 2015 and December 2020, and underwent our routine systematic health screening. This systematic screening consists of a structured interview, a complete physical examination, a medical questionnaire, a review of the medical file including medication use, biochemical measurements and, if indicated and feasible, additional tests, as described previously (see ).® assay, Vitros ECI Immunodiagnostic System; Ortho-Clinical Diagnostics, Rochester, MI, USA) before 12 April 2019, and 13.5–24.3 pmol/L after that date (Fuijrebio Lumipulse® assay). Reference values in our center for T3 were 1.4–2.5 nmol/L before 12 April 2019, and 0.7–2.0 nmol/L after that date. TSH, fT4 and T3 measurements changed methods during the study, but they were calibrated similarly, as checked by external quality assessment schemes.During the visit, blood samples were taken for general medical screening, including evaluation of thyroid function , TSH). The reference values in our center for TSH were 0.4–4.3 mU/L before 1 February 2019, and 0.56–4.27 mU/L after that date. The reference values for fT4 were 11–25 pmol/L  or TH measurements , fT4, T3, free T3 (fT3), reverse T3 (rT3), and TSH) in methylation-positive individuals with PWS. Exclusion criteria were: meeting reports, workshop summaries, conference abstracts, guidelines, articles that included 10 or fewer subjects with PWS, articles that were not available online, and articles that were not available in English. When the same population was described in multiple articles, the population with the most laboratory values or the largest population was included. When an article described thyroid function before and after the start of GH treatment, only data at baseline were included in the table. Authors were contacted to clarify data when needed.n (%). We used a chi-squared test to compare the prevalence of hypothyroidism between males and females, between paternal deletion and mUPD, between patients who did and did not use GH treatment, and between patients who did and did not use psychotropic drugs. To investigate the relationship between hypothyroidism, and BMI and age, we used the Wilcoxon rank sum test. We also used the Wilcoxon rank sum test to investigate the relationships between gender, genotype, and use of GH treatment and psychotropic drugs on the one hand, and laboratory measurements  on the other hand. If there were ties, an exact calculation method was used. The Kendall rank correlation test was used to assess correlations between age and BMI on the one hand, and laboratory measurements  on the other hand. As this was an exploratory analysis, no correction for multiple testing was performed.Statistical analysis was performed using R version 3.6.3. Descriptive statistics for continuous variables are reported as median (interquartile range (IQR)). For dichotomous variables we display the number of patients and the percentage of the total number of patients, 2 (IQR 26–36). We included 58 males and 64 females. Paternal deletion was the most common genotype , followed by mUPD . A total of 63 patients (52%) had never received GH treatment, while 43 patients (35%) received GH treatment at the time of the study. Medication use before our systematic screening included use of hydrocortisone , estrogen replacement therapy , testosterone replacement therapy , and thyroid hormone replacement therapy . A total of 67 (55%) patients lived in a non-specialized facility, 24 (20%) in a specialized PWS group home, and 31 (25%) with family. Most patients had received special education .Baseline characteristics of the 122 adults with PWS participating in the study are shown in Hypothyroidism was present in 21 patients (17%). A total of 12 patients had central hypothyroidism, 3 patients had primary hypothyroidism, and in 6 patients it was unknown whether the hypothyroidism was central or primary. In 17 patients, the diagnosis of hypothyroidism was based on referral letters. TH concentrations were provided in the referral letter in five cases. Additionally, two patients were diagnosed during childhood by the pediatric endocrinology department at our reference center, and two patients were diagnosed during our systematic health screening. The median age at diagnosis of hypothyroidism was 18 years (IQR 13–27) (age at diagnosis was unknown in two patients). The median dose of levothyroxine in patients with hypothyroidism was 68.8 μg (IQR 50.0–100.0) daily. Additionally, one patient with very mild hypothyroidism did not receive any treatment.p = 0.051). There was no relationship with genotype, age, BMI, or GH treatment (Three patients (2%) had a normal fT4 concentration, with a TSH concentration above the reference range , while one patient was diagnosed with hyperthyroidism, treated with thiamazole. Although not statistically significant, hypothyroidism seemed to be more prevalent in females (23%) than in males  hypo- or hyperthyroidism, TH concentrations and the associations between TH concentrations and patient characteristics  are shown in p = 0.02). The mean age of patients using psychotropic drugs was 36 years, and the mean age of patients not using psychotropic drugs was 29 years. No associations for specific types of psychotropic drugs were found  or in mixed populations containing children and adults (n = 9). Paternal deletion was the most common genotype in all studies. The prevalence of hypothyroidism differed between 0% and 33% in most studies, with one study reporting a prevalence of 72%. However, this study only included 18 children with PWS who were only up to 2 years old and, in this study, thyroid axis dysfunction was defined as serum total T4 and/or serum fT4 levels below the 2.5th percentile of a reference population. The prevalence of hypothyroidism in studies that only included adults ranged between 5% and 13%. For mixed populations of both children and adults, this prevalence was between 0% and 26%. Only two studies that included adults reported whether the hypothyroidism was central or primary in origin [The results of our literature review are summarized in n origin ,32. AlthBased on the results of our cohort, the literature review, and our clinical expertise, we formulated practical clinical recommendations for the screening and treatment of hypothyroidism in adults with PWS .The prevalence of hypothyroidism detected in our cohort of 122 adults with PWS was 17%, compared to only 3% in non-PWS adults . The risOur prevalence of hypothyroidism was higher than that of most previous studies on hypothyroidism in adults and mixed cohorts of adults and children . HoweverCompared to the general population, there are several aspects of PWS that increase the complexity of the diagnosis and treatment of hypothyroidism in these patients. An increased vulnerability to the effects of untreated hypothyroidism of the patients, diagnostic challenges, and altered TH metabolism make hypothyroidism a complex issue in adults with PWS.The vulnerability of the patients makes the treatment of hypothyroidism an important topic. The common effects of hypothyroidism on the muscles and the brain can be especially harmful to adults with PWS, as they already have impaired exercise tolerance and brain function.Patients with PWS have a high risk of developing obesity due to hyperphagia and a low BMR inherent to the syndrome. Hypothyroidism can cause arthralgia, lethargy, exertion fatigue, shortness of breath, and muscle problems ,57; thisThyroid function is responsible for a variety of physiological processes in the adult brain . Adult-oThe increased vulnerability of the patients, combined with diagnostic challenges and altered thyroid hormone metabolism, make the diagnosis and treatment of hypothyroidism an important issue in adults with PWS.Diagnostic challenges include patients’ delay, doctors’ delay, and unreliability of TSH.Due to the intellectual disability that is often present in PWS, patients are often unable to express their complaints. Especially when the symptoms associated with hypothyroidism are subtle , they will not be reported by the patients.In the general population, TH concentrations are usually measured when there is a clinical suspicion of hypothyroidism. Unexplained weight gain, reduced appetite, fatigue, and constipation are well-known clinical signs of hypothyroidism that will alert most physicians to measure TH concentrations . Howevern = 12) and primary hypothyroidism (n = 3) were present. Serum TSH concentrations in patients with central hypothyroidism are often normal [Apart from patients’ and doctors’ delay, there is another diagnostic challenge. Hypothyroidism can be both primary and central in PWS . In our n normal ,71,72. Fn normal ,20,21,22As symptoms of hypothyroidism are unreliable in patients with PWS, and hypothyroidism can be both primary and central, we recommend to screen for hypothyroidism by measuring serum TSH and fT4 concentrations every year.Prescription of endocrine and non-endocrine medication may disturb TH metabolism. Likewise, altered levels of “hunger hormones” may affect TH concentrations. Examples of these TH metabolism-altering factors in adults with PWS include use of psychotropic drugs, growth hormone treatment, and disturbed leptin and ghrelin levels.Psychotropic drugs can cause a disturbed synthesis and metabolism of TH ,74. CompGH treatment is often prescribed to children and young adults with PWS. In our population, one-third of the patients were treated with GH. Patients who used GH treatment had generally been receiving GH treatment for several years before visiting our outpatient clinic. It has been suggested that GH treatment enhances peripheral conversion of T4 to T3, resulting in decreased fT4 and increased T3 levels ,81,82. SHigh leptin levels caused by obesity in patients with PWS can lead to an increased conversion of T4 to T3 . This meAs with any study, our study has several strengths and limitations. One strength of our study is the population size, considering the rareness of the disease. Furthermore, we are the first to not only describe the prevalence of hypothyroidism and TH concentrations, but also the relationship with medication use. One of our limitations is that this was a retrospective study and, therefore, we had many missing values for T3, as this was not routinely measured in all patients. Another limitation is that thyroid peroxidase (TPO) antibodies were not measured; therefore, we were not able to distinguish autoimmune thyroid diseases. Furthermore, we did not measure thyroxine-binding globulin (TGB) and, therefore, we do not know whether free T3 levels were disturbed in our cohort. The last limitation is that laboratory measurements before the start of levothyroxine treatment were not available in 12 patients with hypothyroidism. In these cases we had to rely on referral letters that mentioned whether the patient had central or primary hypothyroidism.In conclusion, one in six patients in our cohort of 122 adults with PWS had hypothyroidism, which is more frequent than in non-PWS adults. Hypothyroidism is often central in origin, and it is therefore important to measure not only TSH, but also fT4. We recommend yearly screening of fT4 and TSH to prevent the negative effects of untreated hypothyroidism on BMR, BMI, cardiovascular risk, and brain function. Additionally, we recommend measuring thyroid hormone concentrations 3–4 months after the start of GH treatment.


Molybdenum (Mo) microbial bioreduction is a phenomenon that is beginning to be recognized globally as a tool for the remediation of molybdenum toxicity. Molybdenum toxicity continues to be demonstrated in many animal models of spermatogenesis and oogenesis, particularly those of ruminants. The phenomenon has been reported for more than 100 years without a clear understanding of the reduction mechanism, indicating a clear gap in the scientific knowledge. This knowledge is not just fundamentally important—it is specifically important in applications for bioremediation measures and the sustainable recovery of metal from industrial or mine effluent. To date, about 52 molybdenum-reducing bacteria have been isolated globally. An increasing number of reports have also been published regarding the assimilation of other xenobiotics. This phenomenon is likely to be observed in current and future events in which the remediation of xenobiotics requires microorganisms capable of degrading or transforming multi-xenobiotics. This review aimed to comprehensively catalogue all of the characterizations of molybdenum-reducing microorganisms to date and identify future opportunities and improvements. Human activities are endangering the environment. Due to the growth of the population, intensive industrialization, in addition to urbanization and agriculture, has caused significant damage to the environment. The overexploitation of natural resources and ignorance of the laws of nature have exacerbated these problems. Pollution caused by hydrocarbons and metal ions has increased in recent years ,2, repreToxicity cases ranging from acute to chronic, within occupational and environmental high-exposure settings, have been identified to be caused by toxic agents from metals and their compounds. Typically, heavy metals naturally exist in the environment. Since the pre-industrial period, the level of heavy metals has significantly increased due to anthropogenic activities ,4,5. A cMolybdenum (Mo) is an important trace element that acts as a micronutrient that is necessary as a co-factor for more than 50 enzymes ,19. WithFor over a century, the microbial molybdate reduction to Mo-blue has not been understood. This phenomenon has been proven to be enzymatic rather than abiotic . Previou2), wulfenite (lead molybdate), or powellite . The direct mining of molybdenite, which is commonly undertaken, enables the metal to be obtained. Furthermore, the metal can also be recovered as a byproduct of copper mining [Molybdenum is located in group VI, period V, of the transition series as a metallic element. The atomic number for this element is 42, and it has a relative atomic mass of 95.94 g/mol, with melting and boiling points of 2623 and 4639 °C, respectively. Because molybdenum metal does not freely exist in nature, it typically presents either as molybdenite  in nature, it can be combined to form an assortment of polymolybdate compounds [2MoO4), ammonium molybdate ((NH4)2MoO4), and ammonium paramolybdate ((NH4)6Mo7O24.4H2O). By comparison, molybdenum trioxide (MoO3) is sparingly soluble, and other compounds such as molybdenite (MoS2), calcium molybdate (CaMoO4), molybdenum chloride (MoCl5), and metallic molybdenum (Mo) are completely insoluble in an aqueous medium [Pure molybdenum naturally exists as a silvery-white metal with variable oxidation states between 2 and 6, the most stable of which are Moand Mo6+ . Becauseompounds ,29,30. Es medium ,28. Molys medium ,19; thes6+), which is readily absorbed following oral administration, can be seen in non-ruminant animals compared to ruminant animals. In contrast, tetravalent molybdenum (Mo4+) has been found to not be readily absorbed [Molybdenum and molybdenum compounds may enter the body by either the oral intake of food and drinking water or inhalation. Though absorption by the gastrointestinal tract is rapid and nearly complete, the efficiency and rate of absorption via inhalation are still unknown . The solabsorbed . Within absorbed . This ababsorbed ,36. Regaabsorbed ,37. Molyabsorbed ,34,37, wAnimals suffering from copper deficiency have been indicated to be more susceptible to molybdenum toxicity, and the complex interaction between molybdenum with copper and sulphate has yet to be discovered. Non-ruminants can be given dietary sulfate to minimize the symptoms of molybdenum poisoning, whereas dietary sulfate administered to copper-deficient animals will exacerbate the problem ,37.Acute or chronic exposure can be used to evaluate the effects caused by molybdenum compounds. Data obtained on human toxicity as a result of chronic exposure are currently limited, and no relevant data are currently available on the acute toxicity of molybdenum compounds. Thus, the toxicity of these compounds needs to be assessed for different species of organisms because most of the chemicals are specified to their target and the effects may differ. The existence of numerous chemical forms of the molybdenum compounds should also be considered. The toxicity of molybdenum compounds in different animal models has been reported in several studies. Hence, it is important to identify the toxicity to spermatogenesis and oogenesis, particularly those of ruminants.In one study, the effects of dietary molybdenum on pup growth during lactation and the reproductive ability of Long–Evans rats fed for 13 weeks with diets containing a graded dose of molybdenum  and 5–20 mg/kg copper showed noticeable infertility among males due to the degeneration of the seminiferous tubules. Furthermore, in the groups that received the two highest doses, more pronounced effects were seen. Lactating mothers exposed to the two highest doses and their pups experienced lower weight-gain during lactation compared with those in the lower-dose groups . The oraMolybdenum endorses cellular function by catalyzing a variety of hydroxylation and redox transfer reactions ,19 that One of the most important biological interrelationships between animal nutrition and medicine can be seen in the complex interaction between molybdenum, copper, and sulfate metabolisms regarding the kinetics, bioavailability, and utilization of copper. This interrelationship, however, is not yet fully understood. Among ruminants, different molybdenum compounds react with sulfides to form thiomolybdate compounds that then further react with copper to form an insoluble complex that is poorly absorbed. A decreased copper uptake impairs its utilization and results in the synthesis of several copper-dependent proteins ,33,37,43A primary manifestation of molybdenum toxicity can be presented in impaired copper utilization and metabolism resulting from a secondary copper deficiency (molybdenosis) ,45,46. TIn recent years, the bacterial-based remediation of environmental pollution has drawn significant interest as a promising innovative technology ,49,50. IThe diversity of molybdenum, which is used in superalloys, nickel-base alloys, lubricants, additives, glassworks, paint, pigments, electronics, and many other uses, has been proven to be valuable. Molybdenum can be present in the discharged effluent associated with these products . Due to In addition to industrial pollution, mining sites are a major cause of molybdenum pollution. Molybdenum is the main dissolved metal in the aqueous effluent from many uranium flotation mills, probably due to the high solubility of the molybdenum metal in the form of molybdate ions at the high pH values that are used, whereas most other metals are precipitated as hydroxides. For instance, in New Mexico, molybdenum and other toxic metals released from the Molycorp molybdenum mine resulted in levels of these metals exceeding the New Mexico environmental standard in the Red River and caused the extinction of many of the river’s floras and fauna . A complIn Armenia, nearly 300 square kilometers of land are polluted with heavy metals such as lead, copper, and molybdenum from the Alaverdi copper–molybdenum mine in the area. Land adjacent to the mining activities suffers from the worst pollution, with levels of these heavy metals at 20–40 times the limit . The con6+) reduction was first reported in Escherichia coli by Capaldi and Proskauer [E. coli K12 [Thiobacillus ferrooxidans (now Acidithiobacillus ferrooxidans) was then continued on Enterobacter cloacae strain 48 or EC 48 [Desulfovibrio desulphuricans and Desulfovibrio vulgaris, where molybdenum is reduced to MoS2 (Mo4+). Molybdenum disulfide is insoluble and precipitates out of solutions, which is another avenue for molybdenum bioremediation [Microorganisms utilize the mechanisms of bioreduction, bioprecipitation, bioaccumulation or sequestration, efflux pumping, and biosorption to detoxify and immobilize metal ions of molybdenum, chromium, copper, and mercury ,66,67,68roskauer . Howevercoli K12 . The worcoli K12  on Thiobor EC 48  and otheediation .The earliest documented report on molybdenum bioremediation was in Tyrol, Austria. A plant-microbial consortium was used to treat large pasture areas that had been contaminated with molybdenum from industrial discharge. Soluble toxic molybdenum was detoxified using microbes from sewage, which transformed soluble molybdenum into insoluble compounds . The useE. cloacae strain 48 [6+ to M5+) before phosphate joined to form Mo-blue. Similar Mo-blue spectra were produced in both the bacterial molybdate reduction and ascorbate-reduce phosphate determination methods, indicating the possible formation of the phosphomolybdate intermediate [The mechanism of microbial molybdate reduction to Mo-blue was first proposed for train 48 ,83. Enzyrmediate . At thisrmediate ,86.24+). Molybdate ions, however, form a unique complex structure called polymolybdate anions in an acidic solution. This is a hallmark of molybdenum chemistry. The polymolybdate ions then further combine with other anions, such as sulfate, phosphate, or silicate, to form a mega-complex (12O3+: 12-molybdophosphate), hence converting it to molybdenum blue. Both of these donated electrons are spread across the entire polymetallate sphere by a thermally-activated hopping process, as shown in electron spin resonance (ESR). The two reduced electrons are in constant motion, as shown by nuclear magnetic spectroscopy, and therefore account for the valency of all of the twelve molybdenum atoms. The movement of electrons across the Keggin complex causes a mixed valency of molybdenum oxidation states, is between 6+ and 5+ [In an aqueous medium (pH 7), molybdenum exists as molybdate ions  isolated from different soils and polluted waters around the world. From the isolated molybdenum-reducing bacteria (52 bacteria), 45% are from Malaysia (23 bacteria), followed by Nigeria (eight bacteria), Egypt (seven bacteria), and Indonesia (five bacteria). The remainder are from Sudan (two bacteria) and Pakistan (two bacteria), as well as Iraq, China, USA, and Antarctica, at one bacterium each (E. coli K12 was the only standard ATCC-type strain (strain ATCC 10798). A molybdenum-tolerant bacterium (Bacillus subtilis LM 4-2) from China was isolated [Bacillus spp. and Staphylococcus spp. are gram-positive rod and cocci, respectively. The remaining genera are gram-negative rods, with the majority belonging to the Enterobacteriaceae family , and Bacillus tequilensis strain Pharon2 (MK078034) [Statistical-based optimization, such as the response surface method (RSM), is a composition of statistical and mathematical techniques for empirically building a model. It is a well-known and current approach for constructing approximation models based on experimental observations ,141,142.ain MIE2 , Serratiain HMY1 , BacilluK078034) , and it maxμ) in a certain period, thus producing a lag time (λ). Within the growth curves, a final phase is involved in which the rate diminishes and ultimately reaches zero. Therefore, an asymptote (A) is obtained. Changes in the growth rate frequently result in a sigmoidal curve with the characteristic lag phase just after t = 0. An exponential phase then follows, and it is succeeded by a stationary phase and, finally, the death phase [Bacterial growth-linked processes often demonstrate a unique phase. The specific growth rate commences at a value of zero, after which it accelerates to a maximal value . This is commonly used in the development of secondary models, such as of the effects of substrate, product, pH, and temperature on the growth rate of the organism [mμ, can be demonstrated by the slope of the line when the activity or organisms grow or expand exponentially [Bacillus sp. strain A.rzi [maxμ, λ and A from the model itself [In addition to the asymptotic value and the lag period, another valuable parameter of a growth curve is the maximum specific growth rate , bias factor (BF), accuracy factor (AF), and corrected Akaike information criterion (AICc) [Once the maximum specific reduction rate values are obtained from the primary modelling exercise, the values can be used to model the effect of the substrate (molybdate) on the rate of reduction. Several important parameters, such as the specific reduction rate, theoretical maximum reduction, and determination of the reduction at high molybdenum concentrations , can be shown by the mathematical modeling of the reduction process . In addin (AICc) . It appeE. cloacae strain 48 [+6) reduction was shown to increase with the concentration and halted as the concentration reached 100 mM. The mK (apparent) was 16.5 mM, and maxV was 0.33 mU mg−1. A second attempt at purifying the Mo-reducing enzyme [E. cloacae strain 48 used phosphomolybdate (PM) as an electron acceptor using ammonium sulfate (40%–50%) fractionation, and ion exchange on DEAE-cellulose and gel filtration on Sephacryl S-200. An excellent ammonium sulfate fraction was then obtained with 6.5-fold enzyme purification and about 97% recovery of the initial value. A single peak was produced from ion exchange chromatography on DEAE-cellulose. Though the level of purification was outstanding, a significant decrease in the activity was recorded in both the gel filtration and ion exchange steps. About 1.6% of the final yield was found in the initial crude, indicating that a large degree of enzyme denaturation or loss occurred, and the final specific activity was 240 mU mg−1 protein. A 40-fold purification was achieved after the ion exchange and gel filtration chromatographic steps. From the SDS-PAGE of the concentrated gel filtration step, three protein bands with estimated molecular weights of 95, 100, and 105 kDa were obtained, indicating that the purification of the enzyme was not successful [The first attempt to purify the Mo-reducing enzyme was first carried out in train 48 . The purg enzyme  from E. ccessful .Serratia sp. Dr.Y5 [The first purification of the molybdenum-reducing enzyme was reported from the bacterium p. Dr.Y5 . The enzp. Dr.Y5 . maxV for NADH of 12 mU mg−1 protein and an apparent mK of 0.79 mM, whereas at 5 mM NADH, the apparent maxV and apparent mK values for 12-MoP (phosphomolybdate) were 12.05 mU mg−1 protein and 3.87 mM, respectively. A higher maxV for NADH was reported in strain Dr.Y5 than EC 48 at 6.28 mU mg−1 protein [mK for NADH was lower than that of EC 48 at 1.65 mM, thus indicating a better affinity to a Mo-reducing enzyme in strain Dr.Y5. Both strains produced similar apparent maxV values for 12-MoP. A higher apparent mK for 12-MoP in Dr.Y5, relative to 0.32 mM for EC 48, was obtained. which might suggest that the enzyme from EC 48 has a higher affinity for 12-MoP than strain Dr.Y5. In addition, the catalytic efficiency (cat/Kmk) of the Mo-reducing enzyme from Dr.Y5 was found to be 5.47 M−1s−1, which is about 2 × 105 less efficient than chromate reductase from Thermus scotoductus, a related metal reductase [maxV and comparatively very low mK for NADH and phosphomolybdate [Characterizing the purified fraction from strain Dr.Y5 showed that the optimum pH was 6 and that temperature stability occurred between 25 and 35 °C. At 15 mM 12-phosphomolybdate, a plot of initial rates against concentrations of substrate showed an apparent  protein . Howevereductase . To dateolybdate .Serratia sp. strain MIE [mK and maxV for NADH and phosphomolybdate were 0.859 mM and 16.11 mU mg−1 and 6.02 mM and 6.89 mU mg−1, respectively. The catalytic efficiency measured as the ratio of cat/KmK was 7.89 M−1s−1. The most recent purification of the Mo-reducing enzyme came from a cyanide-degrading bacterium Serratia sp. strain UPM-FR1 [mK and maxV for NADH and phosphomolybdate were 1.81 mM and 21.2 mU mg−1 and 4.53 mM and 21.66 mU mg−1, respectively, and the catalytic efficiency (cat/KmK) was 5.35 M−1s−1  number. The only established toxic metal reductases are mercury reductase [The molybdenum-reducing enzyme was also successfully purified from rain MIE , with st UPM-FR1 . The pur5 M−1s−1 . The woreductase  and seleeductase . Hence, E. cloacae 48 indicates that the location occurred downstream from cytochrome [E. cloacae strain 48 was revisited by adjusting the reaction mixture after addition of cyanide to a neutral pH [The electron transport chain (ETC) is a series of electron transfer reactions taking place within the inner mitochondrial membrane. Several heme-proteins, soluble enzymes, coenzymes, Fe–S clusters, and a host of several metal cofactors are involved in the electron transfer process and subsequently form the components of the system. The components of the electron transport chain are then organized in a unique manner to form complexes , with ATPase serving as the fifth complex (complex V) . Exampletochrome . Howevertochrome ,102,129.utral pH . TherefoThe optimization of Mo-blue reduction for the purpose of bioremediation offers a method for the removal of molybdenum pollutants in addition to physical and chemical methods. Presently, molybdenum pollutants are chemically treated as a remediation technique . MicrobiThis review aimed to provide an update on the current knowledge regarding the molybdenum-reducing bacteria that have been isolated to date. This review also provided an update on the ability of some molybdenum-reducers to degrade other xenobiotics, which is a feature that needs to be studied and optimized in the future. A number of these xenobiotics, such as amides, can be used as electron donors for molybdenum reduction, and the ability of other xenobiotics to act as electron donors should be studied in the future. The inhibitory effect on the molybdenum reduction of cationic heavy metals, such as mercury, copper, and silver, is also seen in other anionic reductions, such as chromate and arsenate microbial reductions. Thus, means to combat this issue need to be studied for real remediation applications in the future. The true identity of the molybdenum-reducing enzyme is currently unknown, and the future sequencing of the purified enzyme could reveal the underlying mechanism behind the reduction process. Inhibition kinetics studies have shown that molybdate is toxic at high concentrations. Thus, the limits of the capability of bacteria to remediate sites highly contaminated by molybdenum, in addition to molybdenum-rich effluent from mine tailings, should be studied. The activation energy for molybdenum reduction also needs to be explored in future studies to distinguish molybdenum from other anionic metal reductions and other xenobiotic degradation processes.