Pneumocystis jirovecii (previously Pneumocystis carinii) – Molecular diagnosis (PCR); Resistance mutations to first-line treatment (TMP-SMX) (PCR and sequencing).

Information 26-08-2018.

Pneumonia caused by the non-culturable opportunistic fungus Pneumocystis jirovecii (previously Pneumocystis carinii) (PcP, due to its acronym in English, Pneumocystis pneumonia) is an important cause of morbidity and mortality among patients infected by the human immunodeficiency virus (HIV) and other immunosuppressed patients. The prophylaxis and treatment of this infection is based mainly on the use of the combination trimethoprim-sulfamethoxazole (TMP-SMX) that inhibits two sequential enzymes responsible for the synthesis of folic acid. Treatment with TMP-SMX is widely available and has effectively reduced the incidence of pneumonia caused by this fungus. However, as a consequence of its use, the appearance of strains with mutations in the genes of the two target enzymes, linked with a greater degree of resistance to treatment has been described.

Pneumocystis jirovecii is an ubiquitous, unicellular, extracellular fungus that hardly develops in cell cultures and is not cultivable in synthetic media. Pneumocystis jirovecii infection causes severe pneumonia in immunocompromised individuals, such as HIV-infected patients, recipients of organ transplants, patients receiving chemotherapy or long-term glucocorticoid treatment. However, it is not uncommon to find immunocompetent patients but with some alteration, such as cystic fibrosis, colonized by this microorganism. Although the fungus is usually restricted to the lungs, its presence has been demonstrated in organs such as lymph nodes, spleen, liver, bone marrow and heart.

A structural feature of interest of Pneumocystis jirovecii and that differentiates it from the rest of the fungi is the presence of cholesterol in the cell membrane. The absence of ergosterol explains its natural resistance to amphotericin B and azoles. Pneumocystis jirovecii, like other fungi, cannot acquire folic acid from the host and uses the enzymes of the folate synthesis pathway for its synthesis. Therefore, the prophylaxis and treatment of this infection is mainly based on the use of the combination trimethoprim-sulfamethoxazole (TMP-SMX) to inhibit the synthesis of folate. This combination inhibits two enzymes in the folate metabolism present in two different stages: dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR). The sulfamethoxazole component inhibits the activity of the enzyme dihydropteroate synthase and the trimethoprim component selectively inhibits the activity of the enzyme dihydrofolate reductase.

The use of these drugs, sulfonamides and trimethoprim has been associated with mutations in the DHPS and DHFR enzyme genes, and as a consequence, resistance to these drugs. Long-term exposure to low levels of TMP-SMX has been identified as a risk factor for developing an infection with DHPS mutants or for selecting DHPS mutants, suggesting that the drug selection pressure is the mechanism by which an increase of DHPS mutants occurs in P. jirovecii. Hospitals or outpatient clinics are probably the most relevant environments for the selection of mutant Pneumocystis since patients with chronic sulphonamide therapy can accumulate mutant genotypes and be a reservoir for the transmission of resistant P. jirovecii strains.

A growing number of studies have identified mutations in the DHPS gene, mainly in codons 55 and 57. These mutations consist of point changes of nucleotides, which translate into amino acid substitutions in the enzyme: 55 Thr-Ala and 57 Pro-Ser. The presence of such mutations is associated with a decrease in the efficacy of sulfonamides in vitro, with the failure of anti-Pneumocystis prophylaxis, and with higher rates of treatment failure in patients with PCP treated with intravenous trimethoprim-sulfamethoxazole. It has been published that there is a three times higher risk of death from pneumonia in patients infected with strains of P. jirovecii carrying mutations in the DHPS gene. In addition, the presence of both mutations in a strain seems to increase its resistance to drugs. Other studies indicate that the use of high doses of TMP-SMX for the treatment of PcP may be able to overcome the degree of resistance conferred by these mutations; however, the emergence of new mutations could change this situation. Other point mutations have been detected in codons 23, 60, 111, 171, and 248. Therefore, surveillance of the DHPS genotype in patients with PcP seems to be justified.

The importance of mutations in the enzyme DHFR, target of trimethoprim, in P. jirovecii has been less studied and therefore is less clear. However, there is strong evidence of resistance to trimethoprim due to mutations in the DHFR gene in Plasmodium falciparum and other pathogenic bacteria. Numerous mutations have been described in the DHFR gene of P. jirovecci, some of which (those located in codons 26, 36, 37, 65, 144) are predicted to be located in the active site of the enzyme. These mutations, are conserved in different isolates of P. jirovecci, and some of them, coincide with the mutations found in other microorganisms, therefore, it is believed that they may have been selected by the pressure of a DHFR inhibitor and may confer some level of resistance. Also, there is good molecular evidence that DHFR mutations, which are frequently found in clinical isolates of P. jirovecii, severely affect the inhibitory effect of trimethoprim on the enzymatic activity of DHFR in vitro.

It has been described that there are geographical differences in the prevalence of these mutations in the DHPS and DHFR genes. Thus, for example, the prevalence of mutations of codons 55 or 57 of the DHPS gene is much higher in the United States than in European countries, ranging from 69% to 0-36%, respectively, according to the studies available.

The diagnosis of Pneumocystis jirovecii pneumonia (PcP) is based on clinical and radiological manifestations in combination with microbiological findings. The complexity of the diagnosis of PCP is due to the presence of nonspecific signs and symptoms and to the sensitivity limitations of the staining techniques. Polymerase chain reaction (PCR) tests have improved the accuracy of microbiological diagnosis and can be especially useful in cases of false negative microscopic examinations. Since P. jirovecii cannot be cultured in vitro, and therefore conventional methods cannot be used to analyze drug resistance. At the same time, that the molecular diagnosis is made, the detection of mutations in the genes coding for the DHPS and DHFR enzymes seems to be justified by the data indicating their association with the efficacy of the TMP-SMX first-line treatment.

Tests carried out in IVAMI:

  • Molecular diagnosis of Pneumocystis jirovecii infections by PCR.
  • Detection of mutations in the DHPS and DHFR genes of Pneumocystis jirovecii by PCR and sequencing.

Recommended sample:

  • Preferably samples of the lower respiratory tract (endotracheal aspirate, bronchoalveolar lavage or biopsy). Sputum samples can be accepted but have low sensitivity, so they are not recommended.

Conservation and shipment of the sample:

  • Refrigerated (preferred) for less than 2 days.
  • Frozen: more than 2 days.

Delivery of results:

  • Molecular diagnosis of Pneumocystis jirovecii infections by PCR: 24 to 48 hours.
  • Detection of mutations in the DHPS and DHFR genes of Pneumocystis jirovecii by PCR and sequencing: 3-5 days.

Cost of the test:

  • Detection of mutations in the DHPS and DHFR genes of Pneumocystis jirovecii by PCR and sequencing: consult