Remo Lobetti BVSc (Hons) MMedVet (Med) PhD Dipl ECVIM
Bryanston Veterinary Hospital, South Africa.
Presentation ACVIM 2008
Pneumocystis carinii is a saprophyte of low virulence that primarily occurs in the mammalian lung. The taxonomy of P. carinii is uncertain: it has been classified as a unicellular protozoan as it is sensitive to drugs used to treat protozoan infections; its reproductive behavior is similar to that of yeast cells; and phylogenetic classification based on 16S‑like rRNA sequences indicates that P. carinii is most closely related to a fungus. The morphology of the organism and the histopathology of the lesions produced by both human and animal isolates throughout the world are similar. Only a single species name has been assigned to the genus Pneumocystis, but antigenic differences suggest that several strains may exist. Although currently controversial, 4 species have been described: 2 species that infect dogs and rats, P. carinii and P. wakefieldiae; 1 species that infects mice P. murina; and P. jirvecii, which infects humans. Biologic differences between isolates from different hosts are suggested by the relative difficulty of experimental interspecies transmission.
Pneumocystis appears to be maintained in nature by transmission from infected to susceptible animals within a species with the primary mode of spread being air‑borne droplet transmission between hosts. The entire life cycle of P. carinii is completed within the alveolar spaces, where organisms adhere in clusters to the pneumocytes. Two main forms, the trophozoite and cyst, are found. Although Pneumocystis infections are usually limited to the lung, in humans and in a single dog various organisms have been reported in extra-pulmonary sites. Severe immunodeficiency states in people, such as AIDS, can be associated with lymphatic or hematogenous dissemination of the organisms from the lungs to other tissues.
Pneumocystis can be inhaled from the environment and colonize the lower respiratory tract of clinically healthy mammals, however, organisms rarely multiply to large numbers in the lungs of clinically healthy hosts. In conditions where there is impaired host resistance or pre‑existing pulmonary disease proliferation of organisms can occur. Alveolo-capillary blockage and decreased gaseous exchange occurs secondary to the overgrowth and clustering of P. carinii within the alveolar spaces. Intra‑alveolar organisms are often accompanied by thickening of alveolar septa, but they seldom invade the pulmonary parenchyma and are rarely found in alveolar macrophages. The inflammatory response that the organism provokes contributes to the pulmonary alveolar damage.
Most canine cases have been in miniature dachshunds younger than 1 year, although cases of pneumocystosis have been reported in Cavalier King Charles spaniels, a Shetland sheepdog, and a Yorkshire terrier. The typical clinical history in dogs is that of gradual weight loss and variable polypnea progressing over a period of time. The weight loss, which occurs in spite of a good appetite in most dogs, may be associated with diarrhea and occasional vomiting. Coughing is not always present, but exercise intolerance is consistent. Infected animals may show some response to antibiotic or cortisone therapy. Affected dogs generally remain relatively alert and afebrile. Abnormalities on clinical examination include polypnea, tachycardia, and pulmonary crackles on thoracic auscultation. Animals are cachectic and often show dermatological changes such as superficial bacterial pyoderma and demodecosis. Although the mucous membranes are generally of normal color, in severely affected animals they may be cyanotic.
Hematological abnormalities are usually nonspecific, with left shift neutrophilic leukocytosis seen most consistently and less frequently, eosinophilia and monocytosis. The white cell response often appears inadequate in light of the pulmonary changes. Polycythemia may occur secondarily to arterial hypoxemia from impaired gaseous exchange. Thrombocytosis is often also present. Serum proteins are usually normal with a low-to-low normal globulin level, which correlates with low gamma globulin levels on serum protein electrophoresis. Decreased lymphocyte function and low levels of serum IgA, IgG, and IgM have been reported. Arterial hypoxemia, hypocapnia, and alkalemia indicate an uncompensated respiratory alkalosis. The PO2 is often lower than would be expected from the clinical signs and thoracic radiographs.
Findings on survey thoracic radiography include diffuse, bilaterally symmetric, and alveolar to interstitial lung disease. Solitary lesion, unilateral involvement, cavitary lesion, spontaneous pneumothorax, or lobar infiltrate may occasionally be present. Cor pulmonale may also be evident.
A specific diagnosis requires direct demonstration of P. carinii in either lung biopsy specimens or respiratory fluids. Transtracheal aspirates and broncho-alveolar lavage are very effective in identifying organisms in dogs. Samples for cytology may also be obtained by endobronchial brushing, and fine needle lung aspirates. None of the cytological techniques are as reliable or as definitive as lung biopsy for documenting active pneumocystosis. Unfortunately lung biopsy has the greatest risk of complications such as hemorrhage, pneumothorax, and death from anesthesia. Antimicrobial therapy can begin 24-48 hours before specimen collection without masking the presence of organisms in the sample.
Other diagnostic tests that have been utilized in the detection of organisms in broncho-alveolar lavage specimens from people and lung tissue from dogs include direct or indirect FA test; immunoperoxidase staining, and PCR. In humans serologic tests have been developed, however, their diagnostic value is uncertain as antigenemia is found in up to 15% of clinically normal humans.
Specific therapy is most beneficial in cases in which the disease has been diagnosed during the early stages. Although a number of drugs have been used in the treatment of P. carinii, the two drugs that have successfully been used to treat pneumocystosis are pentamidine isethionate and the combination of trimethoprim and sulfamethoxazole, the latter being the most effective in the dog.
Pentamidine isethionate is an aromatic diamidine used in humans. Its major side effects include impaired renal function, hepatic dysfunction, hypoglycemia, hypotension, hypocalcemia, urticaria, and hematological disorders. Intra-muscular administration of this drug has been successful in treating a dog with pneumocystosis with the only side effect being localized pain at the injection site. Pentamidine has also been used at a reduced dosage, in combination with sulfonamides to lower its toxic side effects.
The combination of trimethoprim and sulfamethoxazole has been found more effective and less toxic than pentamidine in treating and preventing Pneumocystis pneumonia in immunosuppressed humans. A dose of 15 mg/kg tid or 30 mg/kg bid for 3 weeks has been successfully used in miniature dachshunds with pneumocystosis. Folic acid supplementation should be given if side effects such as leukopenia and anemia are observed or if long‑term therapy is required. Although atovaquone is licensed for the treatment of people with pneumocystosis it is not as effective as pentamidine or trimethoprim‑sulfamethoxazole but has a lower toxicity. Bioavailability is increased when the drug is given with food with a high fat content.
Combination therapy using clindamycin and primaquine has been effective both in vivo and in vitro, but neither drug is effective alone. Aromatic diamidines, such as diminazene, imidocarb, and amicarbalide, have been more effective than pentamidine in treating experimental P. carinii pneumonia. Dapsone and trimethoprim or pyrimethamines, in combination, have been effective in experimental animals and clinical trials in immunosuppressed people with pneumocystosis. Trimetrexate, a lipid‑soluble antifolate, has been given concomitantly with leucovorin in people with Pneumocystis pneumonia and AIDS. As with most of the other drugs, neutropenia with or without thrombocytopenia has been the main side effect. In experimentally infected animals, P. carinii is resistant to imidazole antifungal drugs, but the anthelmintics benzimidazole and albendazole have been shown to have some effect.
Supportive care is essential for any patient with pneumocystosis. Oxygen therapy administered by cage, mask, or intubation is needed, and ventilatory assistance may also be required. Bronchodilators may help reduce airway resistance. If a patient is receiving immunosuppressive agents, they should be temporarily discontinued; however, anti‑inflammatory drugs may be indicated. In humans the successful treatment of pneumocystosis results in a decline in arterial oxygen related to the inflammatory reaction to dying organisms and the administration of anti‑inflammatory doses of cortisone has been shown to improve pulmonary function and survival. Nonspecific immuno-stimulants such as cimetidine and levamisole have been given adjunctively to treat affected miniature dachshunds but in all probability have limited effect.
It is very important that once a diagnosis of pneumocystosis is made that a predisposing immune deficiency state be investigated.
IMMUNOLOGICAL STUDIES IN THE DACHSHUND
Total leukocyte count is usually elevated, as a result of a mature neutrophilia and monocytosis. Lymphocyte numbers can be elevated, normal, or depressed. Lymphocyte blast transformation assay using phytohemagglutinin and pokeweed mitogen shows severe immunosuppression, especially when compared to controls, even though a normal lymphocyte count can be present. Immunoglobulin fraction quantification is consistent with deficiencies of IgA, IgM, and IgG. In dogs with P. carinii, the absence of immunoglobulins in the presence of the pathological changes is a significant finding as cases are presented with chronic ongoing infections, which should result in an immunoglobulin response. Immunoglobulin fraction quantification repeated once the P. carinii and the skin infections are resolved still show deficiencies. From the lymphocyte transformation studies and the immunoglobulin fraction quantification it would appear as though there is both a T- and B-cell abnormality in affected dogs. Lung tissue stained with anti-canine IgG immunoperoxidase staining shows positive staining of organisms within the air spaces stain, whereas those within the cytoplasm of alveolar macrophages stain poorly, implying that the amount of available IgG is insufficient. Various mechanisms appear to be involved in P. carinii adherence to alveolar macrophages and lung epithelial cells. Mannose receptors play a role in this adherence because cells expressing a cloned macrophage-mannose receptor are able to bind and internalize P. carinii organisms through this receptor. However, the act of binding P. carinii to alveolar macrophages does not result in either phagocytosis or macrophage activation. Complement is not essential for this activity but does enhance antibody mediated phagocytosis. Serum complement activity measured in affected miniature dachshunds has been normal. CD3 and CD79a lymphocyte staining of lymph nodes and spleen shows marked absence of B-cells with the presence of T-cells. T-cells stained positively with the CD3 marker and negative with CD79a marker; whereas B-cells stained positive to CD79a and negative with CD3.
These immunological abnormalities are similar to what has been described in people affected with common variable immunodeficiency syndrome (CVID), also known as acquired or adult onset hypogammaglobulinemia. It is a primary immunodeficiency disease characterized by little or no antibody production by the B-lymphocytes, normal or decreased numbers of B-lymphocytes, and abnormal T-lymphocyte function.
COMMON VARIABLE IMMUNE-DEFICIENCY
In humans CVID is a common primary immunodeficiency with symptoms occurring at any time, but with two major peaks of onset at 5-10 and 20-30 years. The disease was first described in 1953 in a 39-year-old woman with hypogammaglobulinemia. CVID is a heterogeneous group of diseases characterized by hypogammaglobulinemia and repeated, predominantly, bacterial infections. Most of the patients have the normal number of peripheral T and B lymphocytes. B lymphocytes, however, show defects of differentiation and proliferation after antigen stimulation. The number of plasma and memory cells is reduced. As a result of this, the values of serum IgG and IgA are reduced and also irregularly the values of IgM. The antibody response to protein and polysaccharide antigens is insufficient or completely missing in some cases. The attempts to classify CVID based on in vitro immunoglobulin production and B lymphocyte subsets have confirmed marked heterogeneity of this antibody defects. Differentiation of precursor cells into mature B lymphocytes and then into plasma cells is a process regulated by products of a number of genes. Mutation of genes for inducible costimulator (ICOS) has been described in patients with CVID. Subsequently, the mutations of genes encoding transmembrane activator and calcium-modulator and cytophilin ligand interactor (TACI) and CD19 have been found. Although gene mutations have been described, these defects are found in less than 15% of the patients and for this reason the etiology of most of the patients with CVID remains unknown. The described mutations and findings of other expected gene defects will probably cause disintegration of this heterogeneous antibody deficiency syndrome.
The homozygous deletion of ICOS causes an antibody deficiency syndrome in affected individuals. The clinical phenotype includes recurrent and serious bacterial infections, autoimmune phenomena, splenomegaly, lymphadenopathy and malignancy. Patients with CVID, based on ICOS defect, have mild B lymphopenia and significantly reduced number of memory CD19 and CD27 B lymphocytes. This laboratory finding is not, however, characteristic for ICOS mutations because up to 75% of patients with CVID have a reduced number of memory isotype-switched CD19/CD27/IgM B lymphocytes. ICOS deficiency, based on one point mutation, was the first example of an autosomal recessive disorder that occurs only in approximately 2% of patients with CVID.
TACI deficiency may manifest in autosomal dominant as well as in recessive traits in familial and in sporadic CVID. In the case of heterozygous mutations, the clear evidence that they are the cause of humoral deficiencies has not been shown. It is not possible to exclude that heterogeneous mutations may be the only modifying factor that leads to the manifestation of other defects. The TACI mutations show a range of clinical symptoms from no infection to very severe infections, suggesting that other genetic and environmental factors contribute to the variable disease spectrum. In addition, TACI deficiency may also represent a common genetic defect for CVID and selective IgA deficiency (sIgAD), which has been long proposed to be based on the clinical observations in CVID/sIgAD families.
Homozygous mutation in the CD19 gene has been reported in four members of a family with antibody deficiency that complies with classification criteria for CVID. The defect is autosomal recessive. Its real frequency is unknown in patients with CVID because the findings discovered up to now are rare. The numbers of peripheral B lymphocytes are normal or only slightly reduced in affected individuals. CD19 expression on B lymphocytes is missing and as a result of this, the expression of CD21 surface molecules is reduced. Calcium influx into cells is disturbed.
Keywords: pneumonia, Miniature dachshunds, immune-deficiency, decreased B-lymphocytes, abnormal T-lymphocyte function.
Beard CB, Carter JL, Keely SP, et al. Genetic variation in Pneumocystis carinii isolates from different geographic regions: Implications for transmission. Emerg Infect Dis 6: 265, 2000. Cunningham-Rundles C, Knight AK. Common variable immune deficiency: reviews, continued puzzles, and a new registry. Immunol Res 38:78-86, 2007. Kirberger RM, Lobetti RG. Radiographic aspects of Pneumocystis carinii pneumonia in the miniature dachshund. Vet Radiol Ultrasound 39:313, 1998. Kovacs JA, Gill VJ, Meshnick S, et al. New insights into transmission, diagnosis, and drug treatment of Pneumocystis carinii pneumonia. J Am Med Assoc 286: 2450, 2001. Lobetti R. Common variable immunodeficiency in miniature dachshunds affected with Pneumocystis carinii pneumonia. J Vet Diagn Invest 12:39, 2000.. Greene CE, Chandler F, Lobetti RG.Pneumocystosis In: Greene CE (Ed). Infectious diseases of the Dog and Cat. 3rd edition, Saunders, Elsevier, St Louis, 2005, pp 651.