Polymerase chain reaction (PCR)

PCR

Definition :

PCR is a laboratory method used to make many copies of a specific DNA sequence. This method serves many purposes including disease diagnosis, detection of difficult-to-isolate pathogens (any disease-producing microorganism) , mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships.

Polymerase chain reaction (PCR) is a molecular biology technique for enzymatically replicating DNA without using a living organism, such as E. coli or yeast. Like amplification using living organisms, the technique allows a small amount of DNA to be amplified exponentially. As PCR is an in vitro technique, it can be performed without restrictions on the form of DNA and it can be extensively modified to perform a wide array of genetic manipulations.ِ

Inventor :

PCR was invented by Kary Mullis. At the time he thought up PCR in 1983, Mullis was working in Emeryville, California for Cetus, one of the first biotechnology companies. There, he was charged with making short chains of DNA for other scientists. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway 1 one night in his car. He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region. Mullis has said that before his trip was over, he was already savoring the prospects of a Nobel Prize. He shared the Nobel Prize in Chemistry with Michael Smith in 1993.

Patent wars :

The PCR technique was patented by Cetus Corporation, where Mullis worked when he invented the technique in 1983. The Taq polymerase enzyme is also covered by patents. There have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit brought by DuPont. The pharmaceutical company Hoffmann-La Roche purchased the rights to the patents in 1992 and currently holds those that are still protected.

What does PCR need in work ?

PCR is used to amplify specific regions of a DNA strand. This can be a single gene, just a part of a gene, or non-coding sequence. PCR typically amplifies only short DNA fragments, usually up to 10 kilo base pairs (kb). Certain methods can copy fragments up to 47 kb in size, which is still much less than the chromosomal DNA of a eukaryotic cell - for example, a human cell contains about three billion base pairs.

PCR, as currently practiced, requires several basic components. These components are:

• DNA template, which contains the region of the DNA fragment to be amplified
• Two primers, which determine the beginning and end of the region to be amplified (see following section on primers)
• Taq polymerase (or another durable polymerase), a DNA polymerase, which copies the region to be amplified
• Deoxynucleotide triphosphates, (dNTPs) from which the DNA polymerase builds the new DNA
• Buffer, which provides a suitable chemical environment for the DNA Polymerase

The PCR process is carried out in a thermal cycler. This is a machine that heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction. To prevent evaporation of the reaction mixture (typically volumes between 15-100µl per tube), a heated lid is placed on top of the reaction tubes, or a layer of oil is put on the surface of the reaction mixture. These machines cost more than $2,500 USD, as of 2004.

What are primers ?

The DNA fragment to be amplified is determined by selecting primers. Primers are short, artificial DNA strands — often not more than 50 and usually only 18 to 25 base pairs long — that are complementary to the beginning or the end of the DNA fragment to be amplified. They anneal by adhering to the DNA template at these starting and ending points, where the DNA polymerase binds and begins the synthesis of the new DNA strand.

The choice of the length of the primers and their melting temperature (T_m) depends on a number of considerations. The melting temperature of a primer -- not to be confused with the melting temperature of the template DNA -- is defined as the temperature at which half of the primer binding sites are occupied. Primers that are too short would anneal at several positions on a long DNA template, which would result in non-specific copies. On the other hand, the length of a primer is limited by the maximum temperature allowed to be applied in order to melt it, as melting temperature increases with the length of the primer. Melting temperatures that are too high, i.e., above 80°C, can cause problems since the DNA polymerase is less active at such temperatures. The optimum length of a primer is generally from 15 to 40 nucleotides with a melting temperature between 55°C and 65°C.

Procedure :

The PCR process usually consists of a series of twenty to thirty-five cycles. Each cycle consists of three steps (Fig. 2).

1. The double-stranded DNA has to be heated to 94-96°C (or 98°C if extremely thermostable polymerases are used) in order to separate the strands. This step is called denaturing; it breaks apart the hydrogen bonds that connect the two DNA strands. Prior to the first cycle, the DNA is often denatured for an extended time to ensure that both the template DNA and the primers have completely separated and are now single-strand only. Time: usually 1-2 minutes, but up to 5 minutes. Also certain polymerases are activated at this step (see hot-start PCR).
2. After separating the DNA strands, the temperature is lowered so the primers can attach themselves to the single DNA strands. This step is called annealing. The temperature of this stage depends on the primers and is usually 5°C below their melting temperature (45-60°C). A wrong temperature during the annealing step can result in primers not binding to the template DNA at all, or binding at random. Time: 1-2 minutes.
3. Finally, the DNA polymerase has to copy the DNA strands. It starts at the annealed primer and works its way along the DNA strand. This step is called elongation. The elongation temperature depends on the DNA polymerase. Taq polymerase elongates optimally at a temperature of 72 Celsius. The time for this step depends both on the DNA polymerase itself and on the length of the DNA fragment to be amplified. As a rule-of-thumb, this step takes 1 minute per thousand base pairs. A final elongation step is frequently used after the last cycle to ensure that any remaining single stranded DNA is completely copied. This differs from all other elongation steps, only in that it is longer, typically 10-15 minutes. This last step is highly recommendable if the PCR product is to be ligated into a T vector using TA-cloning.

Figure 2: Schematic drawing of the PCR cycle. (1) Denaturing at 94-96°C. (2) Annealing at (eg) 68°C. (3) Elongation at 72°C (P=Polymerase). (4) The first cycle is complete. The two resulting DNA strands make up the template DNA for the next cycle, thus doubling the amount of DNA duplicated for each new cycle (a total of three cycles is shown above).

Steps of PCR process :

The PCR process consists of the following steps:

1. Initialization. The mixture is heated at 96°C for 5 minutes to ensure that the DNA strands as well as the primers have melted. The DNA Polymerase can be present at initialization, or it can be added after this step.
2. Melting, where it is heated at 96°C for 30 seconds. For each cycle, this is usually enough time for the DNA to denature.
3. Annealing by heating at 68°C for 30 seconds:The primers are jiggling around, caused by the Brownian motion. Short bondings are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the Tm of the double-stranded region between the template and the primer is greater than the annealing or extension temperature.
4. Elongation by heating 72°C for 45 seconds:This is the ideal working temperature for the polymerase. The primers, having been extended for a few bases, already have a stronger hydrogen bond to the template than the forces breaking these attractions. Primers that are on positions with no exact match, melt away from the template (because of the higher temperature) and are not extended.

The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template)

1. Steps 2-4 are repeated 25 times, but with good primers and fresh polymerase, 15 to 20 cycles is sufficient.
2. Mixture is held at 7°C. This is useful if one starts the PCR in the evening just before leaving the lab, so it can run overnight. The DNA will not be damaged at 7°C after just one night.

The PCR product can be identified by its size using agarose gel electrophoresis. Agarose gel electrophoresis is a procedure that consists of injecting DNA into agarose gel and then applying an electric current to the gel. As a result, the smaller DNA strands move faster than the larger strands through the gel toward the positive current. The size of the PCR product can be determined by comparing it with a DNA ladder, which contains DNA fragments of known size.

PCR optimization :

Since PCR is very sensitive, adequate measures to avoid contamination from other DNA present in the lab environment (bacteria, viruses, lab staff's skin etc.) should be taken. Thus DNA sample preparation, reaction mixture assemblage and the PCR process, in addition to the subsequent reaction product analysis, should be performed in separate areas. For the preparation of reaction mixture, a laminar flow cabinet with UV lamp is recommended. Fresh gloves should be used for each PCR step as well as displacement pipettes with aerosol filters. The reagents for PCR should be prepared separately and used solely for this purpose. Aliquots should be stored separately from other DNA samples. A control reaction (inner control), omitting template DNA, should always be performed, to confirm the absence of contamination or primer multimer formation .

Difficulties with polymerase chain reaction :

Polymerase chain reaction is not perfect, and errors and mistakes can occur. These are some common errors and problems that may occur.

1- Polymerase errors

Taq polymerase lacks a 3' to 5' exonuclease activity. This makes it impossible for it to check the base it has inserted and remove it if it is incorrect, a process common in higher organisms. This in turn results in a high error rate of approximately 1 in 10,000 bases, which, if an error occurs early, can alter large proportions of the final product.

2-Size limitations

PCR works readily with DNA of lengths two to three thousand base pairs, but above this length the polymerase tends to fall off, and the typical heating cycle does not leave enough time for polymerisation to complete. It is possible to amplify larger pieces of up to 50,000 base pairs with a slower heating cycle and special polymerases. These special polymerases are often polymerases fused to a DNA-binding protein, making them literally "stick" to the DNA longer.

3- Non specific priming

The non specific binding of primers is always a possibility due to sequence duplications, non-specific binding and partial primer binding, leaving the 5' end unattached. This is increased by the use of degenerate sequences or bases in the primer.

Practical modifications to the PCR technique :

• Nested PCR - Nested PCR is intended to reduce the contaminations in products due to the amplification of unexpected primer binding sites.
• Intersequence specific (ISSR) PCR
• Ligation-mediated PCR

• Inverse PCR - Inverse PCR is a method used to allow PCR when only one internal sequence is known.

• RT-PCR - RT-PCR (Reverse Transcription PCR) is the method used to amplify, isolate or identify a known sequence from a cell or tissues RNA library.

• Assembly PCR - Assembly PCR is the completely artificial synthesis of long gene products by performing PCR on a pool of long oligonucleotides with short overlapping segments.

• Asymmetric PCR - Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other.

• Quantitative PCR - Q-PCR (Quantitative PCR) is used to rapidly measure the quantity of PCR product (preferably real-time), thus is an indirect method for quantitatively measuring starting amounts of DNA, cDNA or RNA.

• Quantitative real-time PCR is often confusingly known as RT-PCR (Real Time PCR) and RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions. This method uses fluorescent dyes and probes to measure the amount of amplified product in real time.

• Touchdown PCR - Touchdown PCR is a variant of PCR that reduces nonspecific primer annealing by more gradually lowering the annealing temperature between cycles.

• Hot-start PCR is a technique that reduces non-specific priming that occurs during the preparation of the reaction components.

• Colony PCR - Bacterial clones (E.coli) can be screened for the correct ligation products.

• RACE-PCR - Rapid amplification of cDNA ends.

• Multiplex-PCR - The use of multiple, unique primer sets within a single PCR reaction to produce amplicons of varying sizes specific to different DNA sequences.

• Methylation Specific PCR - Methylation Specific PCR (MSP) is used to detect methylation of CpG islands in genomic DNA.

Uses of PCR :

PCR can be used for a broad variety of experiments and analyses. Some examples are discussed below.

1- Genetic fingerprinting

Genetic fingerprinting is a forensic technique used to identify a person by comparing his or her DNA with a given sample.

2- Detection of hereditary diseases

The detection of hereditary diseases in a given genome is a long and difficult process, which can be shortened significantly by using PCR.

Viral diseases, too, can be detected using PCR through amplification of the viral DNA. This analysis is possible right after infection, which can be from several days to several months before actual symptoms occur. Such early diagnoses give physicians a significant lead in treatment.

3- Cloning genes

4- Mutagenesis

Mutagenesis is a way of making changes to the sequence of nucleotides in the DNA. There are situations in which one is interested in mutated (changed) copies of a given DNA strand, for example, when trying to assess the function of a gene or in in-vitro protein evolution (also known as Directed evolution).

5- Analysis of ancient DNA

Using PCR, it becomes possible to analyze DNA that is thousands of years old. PCR techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian Tsar.

6- Genotyping of specific mutations

Through the use of allele-specific PCR, one can easily determine which allele of a mutation or polymorphism an individual has.

7- Comparison of gene expression

Researchers have used traditional PCR as a way to estimate changes in the amount of a gene's expression. Ribonucleic acid (RNA) is the molecule into which DNA is transcribed prior to making a protein.

References :

1- www.pcrstation.com

2- www.pcrlinks.com

3- www. en.wikipedia.org

4- www.genome.gov

5- www.dnalc.org

6- www.pcrnewsletter.com

7- www.horizonpress.com

8- www.uq.edu.au

Fungal infections

Candidal vulvitis can be treated locally with cream but is almost invariably associated with vaginal infection which should also be treated. Vaginal candidiasis is treated primarily with antifungal pessaries or cream inserted high into the vagina (including during menstruation). Single-dose preparations offer an advantage when compliance is a problem. Local irritation may occur on application of vaginal antifungal products.

Imidazole drugs (clotrimazole, econazole, and miconazole) are effective against candida in short courses of 3 to 14 days according to the preparation used. Vaginal applications may be supplemented with antifungal cream for vulvitis and to treat other superficial sites of infection.

Nystatin cream is used in cases of vulvitis and infection of other superficial sites. Nystatin stains clothing yellow.

Oral treatment of vaginal infection with fluconazole or itraconazole is also effective; oral ketoconazole has been associated with fatal hepatotoxicity

Immunocompromised patients

Immunocompromised patients are at particular risk of fungal infections and may receive antifungal drugs prophylactically; oral imidazole or triazole antifungals are the drugs of choice for prophylaxis. Fluconazole is more reliably absorbed than itraconazole and ketoconazole and is considered less toxic than ketoconazole for long-term use.

Amphotericin by intravenous infusion is used for the empirical treatment of serious fungal infections. Fluconazole is used for treating Candida albicans infection. Caspofungin is licensed for the empirical treatment of systemic fungal infections (such as those involving Candida spp. or Aspergillus spp.) in patients with neutropenia.

Recurrent vulvovaginal candidiasis

Recurrence of vulvovaginal candidiasis is particularly likely if there are predisposing factors such as antibacterial therapy, pregnancy, diabetes mellitus and possibly oral contraceptive use. Reservoirs of infection may also lead to recontamination and should be treated; these include other skin sites such as the digits, nail beds, and umbilicus as well as the gastro-intestinal tract and the bladder. The partner may also be the source of re-infection and, if symptomatic, should be treated with cream at the same time.

Treatment against candida may need to be extended for 6 months in recurrent vulvovaginal candidiasis. Some recommended regimens [all unlicensed] include:

· fluconazole by mouth 100 mg (as a single dose) every week for 6 months

· clotrimazole vaginally 500-mg pessary (as a single dose) every week for 6 months

· itraconazole by mouth 400 mg (as 2 divided doses on one day) every month for 6 months.

Imidazole antifungals

The imidazole antifungals include clotrimazole, econazole, ketoconazole, sulconazole, and tioconazole. They are used for the local treatment of vaginal candidiasis and for dermatophyte infections.

KETOCONAZOLE

Indications

skin, hair, and mucosal mycoses that cannot be treated with other antifungals (including dermatophytoses, pityrosporum folliculitis, cutaneous candidiasis, chronic mucocutaneous candidiasis, oropharyngeal and oesophageal candidiasis, chronic recurrent vaginal candidiasis); systemic mycoses that cannot be treated with other antifungals (including histoplasmosis, blastomycosis, coccidioidomycosis, paracoccidiodomycosis)

Cautions

the CSM has advised that prescribers should weigh the potential benefits of ketoconazole treatment against the risk of liver damage and should carefully monitor patients both clinically and biochemically. It should not be used by mouth for superficial fungal infections.

predisposition to adrenocortical insufficiency; avoid in porphyria ; pregnancy .

Hepatotoxicity

Potentially life-threatening hepatotoxicity reported very rarely; risk of hepatotoxicity greater if given for longer than 14 days. Monitor liver function before treatment, then on weeks 2 and 4 of treatment, then every month. Avoid or use with caution if abnormal liver function tests (avoid in active liver disease) or if history of hepatotoxicity with other drugs.

Counselling

Patients should be told how to recognise signs of liver disorder and advised to seek prompt medical attention if symptoms such as anorexia, nausea, vomiting, fatigue, abdominal pain, jaundice, or dark urine develop

Contra-indications

hepatic impairment; breast-feeding

Side-effects

nausea, vomiting, abdominal pain; pruritus; less commonly diarrhea, headache, dizziness, drowsiness, and rash; very rarely fatal liver damage, dyspepsia, raised intracranial pressure, adrenocortical insufficiency, erectile dysfunction, menstrual disorders, azoospermia (with high doses), gynaecomastia, thrombocytopenia, photophobia, and alopecia

Dose

200 mg once daily, increased if response inadequate to 400 mg once daily; continued until symptoms have cleared and cultures negative (usually for 4 weeks in dermatophytoses, 2–3 weeks for oral and cutaneous candidiasis, 1–2 months for hair infections); child body-weight 15–30 kg, 100 mg once daily; body-weight over 30 kg, adult dose

Chronic, recurrent vaginal candidiasis, 400 mg once daily for 5 days

ITRACONAZOLE

Cautions

absorption reduced in AIDS and neutropenia (monitor plasma-itraconazole concentration and increase dose if necessary); susceptibility to congestive heart failure (see also CSM advice, below); renal impairment; pregnancy and breast-feeding; interactions: (antifungals, triazole)

Hepatotoxicity

Potentially life-threatening hepatotoxicity reported very rarely. Monitor liver function—discontinue if signs of hepatitis develop; avoid or use with caution if history of hepatotoxicity with other drugs or in active liver disease; use with caution in patients receiving other hepatotoxic drugs

Counselling

Patients should be told how to recognise signs of liver disorder and advised to seek prompt medical attention if symptoms such as anorexia, nausea, vomiting, fatigue, abdominal pain or dark urine develop

CSM advice (heart failure)

Following rare reports of heart failure, the CSM has advised caution when prescribing itraconazole to patients at high risk of heart failure. Those at risk include:

· patients receiving high doses and longer treatment courses;

· older patients and those with cardiac disease;

· patients receiving treatment with negative inotropic drugs, e.g. calcium channel blockers.

Side-effects

very rarely nausea, vomiting, dyspepsia, abdominal pain, diarrhoea, constipation, jaundice, hepatitis (see also Hepatotoxicity above), heart failure (see CSM advice above), pulmonary oedema, headache, dizziness, peripheral neuropathy (discontinue treatment), menstrual disorder, hypokalaemia, rash, pruritus, Stevens-Johnson syndrome, and alopecia; with intravenous injection, very rarely hypertension and hyperglycaemia

Indications & Dose

By mouth, oropharyngeal candidiasis, 100 mg daily (200 mg daily in AIDS or neutropenia) for 15 days; see also under Sporanox® oral liquid below

Vulvovaginal candidiasis, 200 mg twice daily for 1 day

Pityriasis versicolor, 200 mg daily for 7 days

Tinea corporis and tinea cruris, either 100 mg daily for 15 days or 200 mg daily for 7 days

Tinea pedis and tinea manuum, either 100 mg daily for 30 days or 200 mg twice daily for 7 days

Onychomycosis, either 200 mg daily for 3 months or course (‘pulse') of 200 mg twice daily for 7 days, subsequent courses repeated after 21-day interval; fingernails 2 courses, toenails 3 courses

Histoplasmosis, 200 mg 1–2 times daily

Systemic aspergillosis, candidiasis and cryptococcosis including cryptococcal meningitis where other antifungal drugs inappropriate or ineffective, 200 mg once daily (candidiasis 100–200 mg once daily) increased in invasive or disseminated disease and in cryptococcal meningitis to 200 mg twice daily

Maintenance in AIDS patients to prevent relapse of underlying fungal infection and prophylaxis in neutropenia when standard therapy inappropriate, 200 mg once daily, increased to 200 mg twice daily if low plasma-itraconazole concentration (see Cautions)

Prophylaxis in patients with haematological malignancy or undergoing bone-marrow transplant, see under Sporanox® oral liquid below

By intravenous infusion, systemic aspergillosis, candidiasis and cryptococcosis including cryptococcal meningitis where other antifungal drugs inappropriate or ineffective, histoplasmosis, 200 mg every 12 hours for 2 days, then 200 mg once daily for max. 12 days

child and elderly safety and efficacy not established

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martindale

Genital candidiasis responds well to topical antifungal treatment with creams or pessaries. Uncomplicated vaginitis responds to short-course topical therapy with azoles such as butoconazole, clotrimazole, miconazole, terconazole, or tioconazole, to topical nystatin, or to oral therapy with fluconazole, itraconazole, or ketoconazole. Complicated disease, which occurs in up to 10% of cases, requires at least 7 days of treatment with topical antifungals or an oral dose of fluconazole repeated 72 hours later.Vulvovaginal candidiasis with non-albicans species may not respond to topical azoles, in which case topical boric acid or topical flucytosine are often effective. In pregnancy, topical azoles are recommended and appear to be more effective than nystatin; treatment is given for 7 days. Recurrent vaginal candidiasis should be treated for at least 6 months with daily oral ketoconazole, daily or monthly oral itraconazole, weekly oral fluconazole, or weekly topical clotrimazole. Yogurt preparations containing Lactobacillus spp. have been used in the treatment and prevention of vaginal candidiasis in an attempt to restore the natural vaginal flora but evidence to support their use is limited.

Candidiasis

Candida spp. are commensal fungi commonly found in the gastrointestinal tract, mouth, and vagina; they may become pathogenic when natural defence mechanisms are impaired. C. albicans is the species most commonly associated with infection although infections with other Candida spp., notably C. glabrata (Torulopsis glabrata), C. krusei, C. lusitaniae, C. parapsilosis, and C. tropicalis, also occur and together are commonly referred to as non-albicans species. Predisposing factors for candidiasis include antibacterial therapy, skin or mucosal breach, abdominal surgery, debility, diabetes mellitus, pregnancy, and neutropenia and T-cell immunodeficiency; candidiasis often occurs in patients with HIV infection.

Candidiasis (or candidosis) may be classified as mucocutaneous or superficial, invasive (deep-organ), or disseminated. Mucocutaneous candidiasis includes infections of the oropharynx, genital organs, and skin. Oropharyngeal and vulvovaginal candidiasis are commonly termed thrush. Most superficial infections may be effectively treated with topical antifungals, although the rare chronic mucocutaneous candidiasis syndrome usually requires systemic treatment. The choice of antifungal should be guided by the availability of an appropriate formulation for the site of infection, toxicity, and the duration of treatment.

_________________________________________________________

FLUCONAZOLE

Cautions

renal impairment; pregnancy and breast-feeding; concomitant use with hepatotoxic drugs, monitor liver function with high doses or extended courses—discontinue if signs or symptoms of hepatic disease (risk of hepatic necrosis); susceptibility to QT interval prolongation; interactions: Appendix 1 (antifungals, triazole)

Side-effects

nausea, abdominal discomfort, diarrhoea, flatulence, headache, rash (discontinue treatment or monitor closely if infection invasive or systemic); less frequently dyspepsia, vomiting, taste disturbance, hepatic disorders, hypersensitivity reactions, anaphylaxis, dizziness, seizures, alopecia, pruritus, toxic epidermal necrolysis, Stevens-Johnson syndrome (severe cutaneous reactions more likely in AIDS patients), hyperlipidaemia, leucopenia, thrombocytopenia, and hypokalaemia reported

Dose

vaginal candidiasis (see also Recurrent Vulvovaginal Candidiasis) and candidal balanitis, by mouth, a single dose of 150 mg

Mucosal candidiasis (except genital), by mouth, 50 mg daily (100 mg daily in unusually difficult infections) given for 7–14 days in oropharyngeal candidiasis (max. 14 days except in severely immunocompromised patients); for 14 days in atrophic oral candidiasis associated with dentures; for 14–30 days in other mucosal infections (e.g. oesophagitis, candiduria, non-invasive bronchopulmonary infections); child by mouth or by intravenous infusion, 3–6 mg/kg on first day then 3 mg/kg daily (every 72 hours in neonate up to 2 weeks old, every 48 hours in neonate 2–4 weeks old)

Tinea pedis, corporis, cruris, pityriasis versicolor, and dermal candidiasis, by mouth, 50 mg daily for 2–4 weeks (for up to 6 weeks in tinea pedis); max. duration of treatment 6 weeks

Invasive candidal infections (including candidaemia and disseminated candidiasis) and cryptococcal infections (including meningitis), by mouth or intravenous infusion, 400 mg on first day then 200–400 mg daily; max. 800 mg daily in severe infections [unlicensed dose]; treatment continued according to response (at least 8 weeks for cryptococcal meningitis); child 6–12 mg/kg daily (every 72 hours in neonate up to 2 weeks old, every 48 hours in neonate 2–4 weeks old); max. 400 mg daily

Prevention of relapse of cryptococcal meningitis in AIDS patients after completion of primary therapy, by mouth, 200 mg daily or by intravenous infusion, 100–200 mg daily

Prevention of fungal infections in immunocompromised patients, by mouth or by intravenous infusion, 50–400 mg daily adjusted according to risk; 400 mg daily if high risk of systemic infections e.g. following bone-marrow transplantation; commence treatment before anticipated onset of neutropenia and continue for 7 days after neutrophil count in desirable range; child according to extent and duration of neutropenia, 3–12 mg/kg daily (every 72 hours in neonate up to 2 weeks old, every 48 hours in neonate 2–4 weeks old); max. 400 mg daily

Resistance

The emergence of strains of Candida spp. resistant to fluconazole has become increasingly important, particularly in immunocompromised patients receiving long-term prophylaxis with fluconazole. In addition to resistance in C. albicans, infections with C. dubliniensis, C. glabrata, and C. krusei, all of which may be less sensitive to fluconazole than C. albicans, have been noted in these patients, and secondary resistance of C. glabrata has been reported during fluconazole therapy. Resistance to fluconazole has been reported to occur more frequently than resistance to either ketoconazole or itraconazole and may be related to the widespread use of this drug. Cross-resistance with other azoles and with amphotericin B has been reported.

Fluconazole resistance has also been reported in Cryptococcus neoformans and Histoplasma capsulatum. Histoplasmosis developed during treatment with fluconazole in a patient with HIV infection. Fluconazole-resistant C. neoformans has been isolated from an immunocompetent patient who had not been exposed to azole antifungals previously.