CHAPTER 16 Immunosuppression
Chapter contents
Introduction
Immunosuppression is seen in a diverse group of patients as a result of a number of unrelated aetiologies. The underlying causes, shown in Box 16.1, include:
In recent years, the clinical course of HIV infection in Europe, North America and Australasia has changed. Several factors are responsible for this, including an improved understanding of disease pathogenesis, the increasing use of prophylaxis against opportunistic infections and combination antiretroviral therapy (cART). Among populations with access to prophylaxis and cART there has been a significant reduction in AIDS-defining events due to opportunistic infections, hospital admissions, and all-cause HIV-associated mortality. Patients without access to these therapeutic interventions continue to present with advanced immunosuppression and with a variety of opportunistic infections and malignancies. This scenario continues to apply to many sub-Saharan and Asian populations among whom, for political and economic reasons, the HIV pandemic continues unabated. Following the introduction of cART in 1996, patients who are able to tolerate it live longer, have a dramatically lower incidence of opportunistic infections and Kaposi’s sarcoma but remain at high risk of malignancy. This is reflected in a relative increase in AIDS-related malignancy, such as non-Hodgkin lymphoma.1 Additionally, non-AIDS defining malignancies, including Hodgkin lymphoma and lung cancer, are observed with greater frequency than in the general population.2
Technical aspects
The nature of the specimen relates to the clinical problem to be investigated.
Bronchoalveolar lavage (BAL) and induced sputum (IS)
Respiratory problems are likely to be investigated by means of bronchoalveolar lavage, bronchial washings or induced sputum (see Ch. 2). These liquid samples are typically processed as cytospins, or possibly as ThinPrep (Hologic®) preparations depending on local practice. Rigorous attention should be paid to obtaining induced sputum samples. The patient should starve for several hours before the procedure, as it may provoke nausea and or vomiting. The patient should clean their mouth using a toothbrush and sterile water, then gargle using sterile water. This is to remove oral debris that may contaminate the sample. Expectoration is induced by 15–20 min inhalation of a mist of 3% saline from an ultrasonic nebuliser. Specimens are collected in sterile plastic containers for both cytology and microbiology. For diagnosis of Pneumocystis pneumonia (PCP), the highest yield is from specimens that are clear, and which resemble saliva; purulent specimens suggest PCP and/or a bacterial infection.
For BAL, a large volume (80–240 mL) of sterile saline is instilled in aliquots of 20–80 mL; aliquots are sequentially instilled and aspirated. Most clinicians collect the pooled ‘return’ and divide it up, with samples being sent for cytological, microbiological (including mycobacterial) and virological evaluation. Processing in the cytology laboratory is by standard techniques3 but strict attention to safety precautions is mandatory. Because of the wide range of possible diagnoses and since more than one disease process may coexist, material should be stained by the Papanicolaou (PAP), Ziehl Neelsen (ZN) and Grocott methods and additional slides prepared and retained for May-Grünwald Giemsa (MGG), Gram, Perl’s, immunocytochemistry or other special techniques.
Lymph nodes
Enlarged lymph nodes or other solid lesions can be examined by means of fine needle aspiration (FNA) (see Ch. 13). If the lesion is small or deep seated the aspiration may be performed under ultrasound guidance. The sample may consist of direct spreads and liquid preparations taken from needle rinse samples according to local practice. Liquid samples are particularly important as they can be used for the preparation of further cytospins and/or cell block preparations for additional investigations such as flow cytometry, immunocytochemistry and in situ hybridisation. The FNA technique is straightforward and has been described elsewhere.4 The capillary technique (without suction), using a 23G needle is recommended, A 20 mL syringe is usually attached in case of fluid samples to prevent spillage.
Cerebrospinal fluid (CSF)
CSF is usually obtained following lumbar puncture for investigation of neurological disease. The volumes vary from 1 to 10 mL. The sample is typically prepared as a cytospin preparation and stained according to the methods of PAP and MGG. Additional special stains for fungi and other infective agents should be performed and ancillary studies including flow cytometry may be helpful. For further details please refer to Chapter 34.
Health and safety issues
Health and safety precautions should be strictly observed with the use of protective gloves, masks, aprons and protective eye wear. Care should be taken when removing the needle; it is not recommended to re-sheath the needle before disposing of it. Instead it should be immediately placed together with the syringe in a suitable sharps container. It is important to follow local guidelines for decontamination and disposal of contaminated material with careful hand washing after the procedure. When taking FNA samples, a needle stick injury is a rare but significant hazard associated with this procedure. The risk of acquiring HIV infection following a percutaneous (needlestick) exposure to HIV from a patient with known HIV infection in a healthcare setting is approximately 3/1000 injuries. Post-exposure prophylaxis (PEP) is recommended by both the Chief Medical Officers’ Expert Advisory Group on AIDS in the UK and by the United States Public Health Service in the USA, if a healthcare worker has had a significant occupational exposure (from blood/other body fluid from a patient known to be HIV infected, or determined to be at high risk of HIV infection).1,2 PEP should be started as soon as possible after the exposure (ideally within 1 hour) and continued for 28 days: PEP is not recommended beyond 72 hours post-exposure. The currently recommended regimen of PEP in the UK is Truvada (245 mg tenofovir and 200 mg emtricitabine [FTC], one tablet) OD and Kaletra [film coated tablets] (200 mg lopinavir and 50 mg ritonavir, two tablets) BD. In the UK, these recommendations are consistent with guidelines for PEP following non-occupational exposure to HIV.
Opportunistic infection
Bacterial infection
Mycobacterial infection
Mycobacterial infection is strongly associated with immunosuppression. Worldwide HIV infection is the greatest risk factor for tuberculosis. Additionally immune suppressed patients are more likely to present with clinically significant infection with ‘atypical’ mycobacteria, which are rarely encountered as clinically important pathogens in the immune competent. This is exemplified by Mycobacterium avium-intracellulare (MAI), also known as M. avium complex (MAC), which in the context of profound HIV-associated immune suppression (CD4 count <100), may result in a disseminated infection. MAI may be identified by cytological examination of sputum, bronchoalveolar lavage fluid and lymph node FNA, by histological examination of liver, bone marrow and by culture of the above and of blood (Figs 16.1, 16.2).
Cytological findings: MAI infection
Patients who have recently commenced cART and who have an atypical mycobacterial infection, the so-called cART induced immune reconstitution inflammatory syndrome (IRIS) may confound the cytopathologist, as inflammatory responses may be modified, such that granulomatous response may be observed in response to M. xenopi, M. kansasii and MAC.5 The organisms can also be visualised with MGG, Grocott and periodic acid-Schiff (PAS) stains. Culture is necessary to distinguish MAI from mycobacterium tuberculosis (MTB). More recently, the use of so-called Papanicolaou induced fluorescence (PIF) has been described to detect acid fast bacilli in Papanicolaou-stained preparations.6
Mycobacterium tuberculosis (MTB) occurs in persons other than immune suppressed, lung and lymph nodes being the commonest sites, although other organs are also affected. An account of the FNA diagnosis of 574 non-immunosuppressed cases is given by Das et al.7 Evidence of epithelioid granulomata without necrosis was seen in 31.5% of FNAs and granulomata with necrosis in 31.9%. In the remaining cases (36.6%), the material consisted of acellular caseous debris. The sensitivity of FNA for diagnosing TB in the lymph nodes in other studies has been 62%, 38% of cases being false negative. Acid fast bacilli are found in less than half of the cases with classical morphology. In addition, morphology of mycobacterial infection in HIV-positive patients, particularly those receiving antiretroviral treatment, may be altered and may not show typical granulomata and necrosis. Instead, an appearance of abscess, with prominent acute inflammation, may be present. In all cases of suspected mycobacterial infection and negative ZN stain, microbiological confirmation is required before treatment. Moreover, acid fast staining has been described in Legionella pneumophila.8
Respiratory disease remains the commonest clinical presentation in the imm|unosuppressed and the clinical distinction of Pneumocystis jiroveci, the causative agent for Pneumocystis pneumonia (PCP) from other fungal or bacterial infection is sometimes difficult. Extrapulmonary disease and disseminated MTB are not uncommon, and are frequently associated with lymphadenopathy, when FNA may be diagnostic.9
Other bacterial infections
Staphylococcus aureus, Haemophilus pneumoniae, Streptococcus pneumoniae, Pseudomonas aeruginosa and Legionella pneumophila are all causes of pulmonary infection in immunosuppressed patients. In lung-allografted patients, such bacterial infections are a particular problem as those transplanted for cystic fibrosis may be colonized with multiple resistant Pseudomonas aeruginosa and Burkholderia cepacia.10 None is amenable to cytological identification but must be considered in the differential diagnosis of pneumonia especially in cases where BAL is cytologically non-diagnostic. Nocardia can be morphologically recognised in BAL fluid,11 but is uncommon (see Ch. 2).
Viral infection
Cytomegalovirus (Fig. 16.3)
Cytomegalovirus (CMV) is a DNA virus of the herpes virus group. It is a widely distributed opportunistic infectious agent in all classes of immunocompromised persons, particularly those with AIDS and organ transplant recipients, and usually presents with visceral manifestations, especially of the lung, brain, eye and gastrointestinal tract. CMV interstitial pneumonitis seen in bone marrow transplant patients is particularly aggressive compared with its counterpart in HIV infection12 and is associated with a high mortality rate.13 It is the most common cause of opportunistic infection in lung allograft recipients in the 4–8 week postoperative period.14 Indeed, in those who survive for two weeks or more, the prevalence of CMV infection may exceed 75%.15 However, it is not always associated with a pneumonitis. PCR on BAL fluid and peripheral blood is a sensitive method of detecting active infection, but together with cytological investigations cannot distinguish between simple and pathological infection.
Among HIV-infected patients with Pneumocystis jiroveci pneumonia, CMV may be detected in bronchoalveolar lavage fluid or induced sputum. however, unless there is cytopathological evidence of active infection (intra-cytoplasmic and into nuclear inclusions) – this is often not clinically significant. By contrast, identification of CMV in these samples from a patient immunosuppressed following an organ transplant frequently has clinical significance. Culture of CMV for diagnostic purposes is slow and expensive and estimation of serological titres is not particularly helpful and has been superseded by PCR on blood, fluid and sputum (see Ch. 34). Early diagnosis and aggressive treatment improve survival.16
Identification of the characteristic viral cytopathic effects by light microscopy of histological or cytological material from the respiratory or gastrointestinal tract still provides the most rapid and certain means of diagnosis.17–19
Cytological findings: CMV infection
Similar cells indicative of CMV oesophagitis have been identified in oesophageal brushings20 and in FNA from the salivary glands.21 Sensitivity of detection of CMV is enhanced by special techniques such as monoclonal antibody studies,22 in situ hybridisation23 and PCR, which is now a routine investigation in clinical practice.24 In situ hybridisation has been successfully carried out on decolourised Papanicolaou-stained slides.25
Herpes simplex and Herpes zoster
Recurrent Herpes simplex virus (HSV) types I and II infections are very troublesome to the immune suppressed, but are seldom life threatening. Mucocutaneous oral and facial lesions, oesophageal and cervical infection in women and anorectal involvement in homosexual men present similar clinical features to H. simplex infection in non-immunocompromised persons. In the earlier years of heart lung transplantation, 10% of patients were found to develop HSV pneumonia and ulcerative tracheobronchitis, but this has been minimised more recently by the introduction of prophylactic antiviral therapy with Acyclovir.26,27 In the context of HIV infection, HSV is a very rare cause of pneumonitis (see Ch. 2).
Cytological findings: Herpes simplex
The classical multiple moulded nuclei are particularly a feature of infections of squamous epithelium. Diagnosis by morphological appearance alone is therefore difficult on routine staining of BAL fluid. Additional investigation such as in situ hybridisation is helpful in suspected cases.28
Other viral infections
Both Epstein-Barr virus (EBV) and human papillomavirus (HPV) are associated with the condition of hairy leukoplakia, which produces a pearly-white raised lesion on the underside and lateral surface of the tongue.29 Oesophageal lesions are also possible. Koilocytosis and herpetic-type nuclear inclusions are reported to occur. EBV is also associated with post-transplant lymphoproliferative disorders and possibly other malignant tumours, such as gastric carcinoma. The cytomorphological features are no different to those described in non-immunosuppressed individuals.
It is well established that genital HPV infection is linked with cervical pre-neoplasia and cervical cancer in women and anorectal and penile carcinoma in immunosuppressed men. Women who are immunosuppressed due to infection by HIV have a higher incidence and grade of cervical pre-cancer.30 Pre-cancer in immunosuppressed men also follows a more aggressive course.31 In a prospective cohort study Ellerbrock et al. reported that 20% of HIV-positive participants developed squamous intraepithelial lesion (SIL) compared with 5% of HIV-negative matched controls over a 30 month follow-up (incidence of 8.3 and 1.8 cases per 100 person-years, respectively).32 It is interesting that enrolment in a well-managed cervical screening programme, such as the UK-NHSCSP, with contemporaneous access to cART, is associated with an incidence of invasive carcinoma no different from a non-HIV-positive cohort (see Chs 23, 25).33
Intranuclear inclusions due to Polyoma virus may occasionally be identified in exfoliated cells in urine from immunosuppressed patients, particularly transplant recipients. These are sometimes referred to as ‘decoy’ cells and can be mistaken for carcinoma-in-situ change (Fig. 16.4). JC virus (JCV) carries little clinical significance but BK viral infection is more likely to cause a viral nephropathy which can compromise graft survival.34 In some units, urine cytology is employed as a means to detect recurrence of polyoma BK virus (BKV) infection with consequent modulation of the degree of immunosuppression.35 Nested PCR is the most reliable means of diagnosis of polyoma virus in the urine and molecular biology remains the only means of distinguishing BK virus and JCV.36
Hepatitis B and C recurrent viral infections are common, especially in liver transplant patients allografted for hepatitis B (HBV) and hepatitis C (HCV) disease. Currently, antiviral therapy including lamivudine and its analogues may be effective in recurrent HBV disease, but therapy is not available for recurrent HCV.
