Section 4 -

Role Of Cytopathology In Patient Care And Disease Surveillance Andrew Field

Medical infrastructure and skilled medical personnel are in short supply in developing countries, and this is particularly true in diagnostic pathology with shortages of microbiological laboratories, infectious disease services, laboratory scientists and pathologists. In many cases the shortage of treatment services limits the usefulness of diagnostic services, but the correct diagnosis is required for the efficient treatment of individual patients and the best use of limited treatment resources. In this setting, cytology has the potential to provide accurate and inexpensive diagnostic procedures, testing and results. The HIV epidemic has further stressed medical systems in developing countries and is associated with a resurgence of endemic infections and a wide range of opportunistic infections, some of which are AIDS defining illnesses. Again cytology offers a great potential for the diagnosis of these infectious diseases. There are well established protocols developed during the AIDS epidemic in developed countries, for instance, in the fine needle aspiration cytological diagnosis of infections and other lesions such as Kaposi's sarcoma (KS) and non-Hodgkin's lymphoma (NHL), which are increased in the HIV positive population [1]. Antiretroviral drug therapy has reduced the incidence of opportunistic infections and KS in the HIV positive patients in developed countries [2, 3, 4] but these drugs are still to become widely available in developing countries ravaged by AIDS. Cytology offers a range of non-invasive and minimally invasive diagnostic procedures, which include examination of sputum, urine and stools, as well as, fine needle aspiration biopsy (FNAB) cytology, cerebrospinal fluid cytology (CSF), skin and conjunctional scrapings, pleural, pericardial and ascitic fluids and cervical. Papanicolaou (PAP) stained smears. In each of these specimen types, routine cytology can exclude or diagnose malignancy, diagnose distinctive infection patterns, such as, granulomatous and suppurative lymphadenitis, diagnose specific viral infections by their cytotoxic effects and inclusions, and identify specific organisms under routine staining with Giemsa (G) and Papanicolaou (P) stains or specialised stains for particular infections, such as the Ziehl Neelsen (ZN) stain for mycobacteria and the methenamine silver (MS) stain for fungi. The cytological procedures also provide material for routine microbiological culture and drug sensitivity studies, if these are available. It is of benefit to know the immune status of the patient since the host response will vary with immune competency, for example, caseating granulomas associated with Mycobacterium tuberculosis may not occur in an HIV positive patient. Multiple infections can be recognised in the same material, the diagnosis often can be provided more rapidly than by traditional culture or tissue processing techniques, and the cytological specimen may allow visualisation of infectious agents that are not able to be grown in the microbiology laboratory, for example, Mycobacterium leprae, Pneumocystis carinii and Treponema pallidum. [5] In all cytological specimens, viral infections can be diagnosed by the characteristic inclusion bodies, which are aggregated viral synthesis products or particles in the cytoplasm or nucleus of the cell, or by multinucleated giant cells [5]. Simple skin or conjunctival scrapings wet fixed in alcohol and stained with the P stain or air dried and stained with G stain, show Herpes simplex or Varicella zoster infections in the Tzank smear, and, as well herpetic inclusions can be seen in the CSF in G and P stains. Cytomegalovirus has a characteristic "owl's eye" intranuclear inclusion and basophilic intracytoplasmic inclusions in G and P stained smears. The respiratory syncytial virus, particularly in immunocompromised patients, presents with multinucleated giant cells with eosinophilic cytoplasmic inclusions. Adenovirus has a characteristic intranuclear inclusion. The cervical PAP smear demonstrates a broad range of infectious agents including the koilocytes of human papilloma virus infection. In the developed world, the cytological characteristics of viral infections are supplemented by the use of (IF) staining with virus specific antibodies, and in infections where the cytological features are not diagnostic such as Influenza A and B and parainfluenza viruses, the immunofluorescent stain (IF) offers a rapid diagnosis and high specificity [5]. Bacterial, fungal and protozoan infections can be routinely diagnosed in cytological specimens, usually associated with a typical inflammatory pattern that alerts the cytopathologist, for example, plentiful neutrophils with macrophages in a proteinaceous background with debris, form the typical background for infections such as pneumococcus in a sputum or gonorrhoea in an urothelial discharge and the Gram stain will show the positive staining pneumococcus and negative intracellular diplococci of gonorrhoea. Actinomycetes or Nocardia Spp are seen in skin abscess smears and sputa, in a similar suppurative background, as delicate beaded gram positive filaments one micron in diameter, sometimes in granules, and the two species can be distinguished by the Kinyoun procedure involving mild acid discolourisation in the Gram stain: Nocardia retain carbol fuchsin and appear red, Actinomyces do not [5]. Chlamydia trachomatis is diagnosed on stained conjunctival scrapings showing intracytoplasmic inclusions, supplemented, in developed countries, by direct IF staining of elementary bodies with monoclonal antibodies. Encephalitozoon sp of microsporidia in HIV patients can also be seen in conjunctival scrapings in stained smears, with a blue spore containing a faint red nuclear dot [6]. There has been a marked increase in the number of cases of tuberculosis in the developed world associated with the HIV epidemic, but the impact in the developing world has been greater due to the already high prevalence of tuberculosis. Mycobacteria can be detected in any cytological specimen using the ZN stain or its variants: the slightly curved bacilli are upto 10 microns in length and 0.2 to 0.6 microns in width and stain red in the ZN stain. Disseminated mycobacteria can be seen in HIV and other immunocompromised patients in huge numbers as negatively stained curved bacilli within the cytoplasm of macrophages and within the background serum in G stained smears [7], and by autofluorescence in P stained smears [8]. If a fluorescent microscope is available the auramine stain allows for the rapid and accurate diagnosis of mycobacterial infection. Fungal hyphae can be recognised in Papanicolaou and Giemsa stained smears in all cytological specimens, where the fungi are frequently seen as negative images in a granulomatous, or in the HIV patient, suppurative background. The yeasts of candida and other fungi stain positively in the Gram stain, except Cryptococcus neoformans whose yeasts are Gram negative. Cryptococcus can be seen in CSF using the India ink stain or mucicarmine stain, and also the G stain where the cell body stains weakly and the capsule is negative against the pale background [1]. P. carinii in a sputum has characteristic eosinophilic frothy casts of the alveolar spaces, containing cysts recognisable on G or P stained smears. Dot like tachyzoites in the cysts and free tachyzoites, with a central red nucleus and pale cytoplasm, can be seen in the G or P stains, although the cyst wall is negative and only stains in silver stains such as the MS stain [5]. Many parasitic infections can be seen in simple stool preparations. The modified ZN stain detects Cryptosporidium parvum, Cyclospora cayetanensis and Isospora belli. Microsporidia, Enterocytozoon bieneusi and Encephalitozoon intestinalis, stain in the Webber modified trichrome stain. In the HIV population the G stain is recommended for detection of many other parasites including blood pathogens, Leishmania donovani in bone marrow aspirates and Leishmania Spp in aspirates or imprints of cutaneous or mucocutaneous lesions [5]. FNAB in the developed world has a well-established role in the diagnostic workup for any palpable lesion in the breast, thyroid, skin, liver or lymph node as well as deep seated lesions [9]. Of all the cytological methods, FNAB has the greatest potential to be a powerful diagnostic tool in an infrastructure poor medical system, where the diagnosis of primary and secondary malignancy can be achieved along with the definitive diagnosis of endemic and emerging infections and opportunistic infections in the HIV positive population. The procedure needs the minimum of equipment, and is rapid, inexpensive, well tolerated by the patient and safe [1, 9]. The impact of a FNAB service is only limited by the need for sufficient medical infrastructure to allow treatment of the infections and other lesions diagnosed. The equipment required for a fine needle aspiration service is minimal and includes 23 and 24 gauge needles, syringes, a syringe holder for aspiration, Coplin jars and a stain such as G or P, or preferably both. This equipment is easily transportable, can be taken to various clinics, and requires only very basic laboratory space and equipment. But it does require adequately trained medical staff. Other specimens such as sputa, urines, CSF, bronchial washes, skin and conjunctival scrapings and cervical PAP smears primarily require strained technicians and cytologists, although cytopathologists ultimately are required for diagnostic work and teaching. Providing trained laboratory staff and cytopathologists is the major challenge in establishing a FNAB, general non-gynaecological and cervical PAP smear cytopathology service in a resource-limited medical setting. There are multiple reports in the literature of the role of FNAB in diagnosing a wide range of infections [1, 9, 10, 11], including in more recent publications, disseminated rhinosporidiosis [12], aspergillosis in various organs including lymph nodes and skin [13], tuberculosis in breast [14], lymph nodes [15, 16, 17] and epididymis and testis [18], actinomycotic abscesses in the liver [19, 20] and cervicofacial regions [21], cutaneous cryptococcosis [22] and cryptococcal lymphadenitis [23], and leprosy [24]. Lymph node aspirates can diagnose specific infections such as pyogenic bacteria, mycobacteria, fungi, cat scratch disease, toxoplasmosis and infectious mononucleosis in routine G and P stains, with the use where necessary of more specific stains for organisms, such as the ZN for mycobacteria [1, 9, 25]. In HIV positive patients, the general aim of the FNAB is to exclude an infectious agent (Mycobacterium avium-intracellular (MAI), M. tuberculosis, fungi and specific suppurative bacteria), KS and NHL, along with metastatic carcinomas [1]. If available, culture and drug sensitivity studies of FNA material are of great benefit, and in developed countries, this is mandatory in immune suppressed patients with their unusual infectious and deficient or atypical inflammatory reactions to common agents [1, 9]. Most lymphomas seen in HIV-positive patients are intermediate to high-grade B-cell neoplasms, often diagnosable on cytomorphology alone, and these may present as widely disseminated disease in soft tissues, the central nervous system and the gastrointestinal tract, as well as in lymph nodes and as primary effusion lymphomas. However, the definitive diagnosis in some cases, requires ancillary testing such as flow cytometry, molecular studies including in situ hybridisation, and cell block preparation for immunoperoxidase studies [1, 9], which usually are not available in the resource poor setting. The differential diagnosis of the target lesion should be considered before aspirating, and a standard aspiration technique used to minimize potential infection risks to the operator, with a protocol for extra slides for ZN, Gram and MS stains, as well as putting aside material for bacteriologic cultures or cell block. Immediate assessment in the patient clinic using the G stain establishes specimen adequacy, and provides a provisional diagnosis, for immediate action, and allows for extra passes for material for special stains and cultures. In FNAB material, suppurative lymphadenitis produces highly cellular smears of neutrophils showing varying degrees of degeneration, admixed with a variable number of lymphocytes and macrophages, in a necrotic background, commonly with visible bacteria, such as Streptococcal or Staphylococcal Sp, particularly on the G stain. The differential diagnosis includes cat scratch disease due to Bartonella henselae or Alipia felis, which can be seen in the Warthin-Starry stain (WS), commonly used for T. pallidum. In HIV-positive persons, transplant recipients and patients with a hematologic cancer, suppurative lymphadenitis should suggest either a bacterial infection (such as Staphylococcus, Streptococcus, Serratia, Pseudomanas or Klebsiella) or a fungal infection (disseminated Aspergillus, Cryptococcus, Candida, or other rare fungi) [1]. Granulomatous lymphadenitis generally produces moderately cellular smears although in some cases of mycobacterial infection cellularity may be low with caseous necrosis present. Epithelioid histiocytes with copious, pale eosinophilic cytoplasm, single elongate, "sandshoe" nucleus, bland chromatin and small nucleolus aggregate in characteristic granulomas, with a variable number of lymphocytes and neutrophils. The MS stain for fungi and ZN stain for mycobacteria are required. Culture is extremely useful, if a laboratory is available. In HIV-positive patients, mycobacterial infections may show large numbers of plump histiocytes with cross-hatched cytoplasm representing "negative image" bacilli, which are also found as curved, beaded bacilli in the background G stained serum [7]. MAI can easily be seen in macrophages and in the background on ZN. If neutrophils are prominent and the patient is not HIV-positive, cat-scratch fever (neck or axillary nodes) and Lymphogranuloma venereum (inguinal lymph nodes) should be considered. Toxoplasmosis produces a highly cellular, follicular hyperplasia pattern with a mixed lymphoid population in which small lymphocytes predominate along with tingible-body macrophages, occasional small histiocytic clusters and even occasional tachyzooites in the background or cysts [1]. BCG vaccination produces localised lymphadenopathy and cytomorphology resembling mycobacterial infections except that necrotic debris with neutrophils is more prominent in the BCG cases [26, 27]. KS is seen in the skin and lymph nodes in AIDS patients, in whom it is commonly associated with gastrointestinal, oral cavity or respiratory involvement. KS is derived from Human Herpes Virus-8 infected endothelial cells, is an AIDS defining illness and the commonest neoplasm seen in homosexual AIDS patients, although in the last decade, its incidence has decreased due to anti-retroviral treatment [2, 3]. In lymph nodes, the spindle cell proliferation involves the subcapsular and other sinuses before replacing the entire node, while partially involved lymph node shows the various stages of HIV infection, from follicular hyperplasia to involution. The FNA usually shows blood with scattered irregular tissue fragments consisting of haphazardly arranged spindle cells with enlarged, mildly irregular oval hyperchromatic nuclei, resembling granulomas [1, 28]. The cytoplasm of the cells is often poorly seen, but metachromatic stroma often surrounds cells delineating the pale blue cytoplasm in the G stain. The nuclei lack the "sand shoe" indentations of epithelioid histiocytes. Occasionally, KS produces highly cellular smears with large tissue fragments in which slit-like spaces and hemosiderin can be seen [1]. Single spindle cells with elongate cytoplasm occur in the background. If neutrophils are admixed with spindle cells and histiocytes, then bacillary angiomatosis should be considered and a WS used to stain the cat scratch organisms Bartonella henselae andBartonella quintana. Cytology can provide specimens for diagnosis and screening with repeat specimens over a period of time. This is a powerful tool for epidemiological and disease prevalence studies comparing different countries, different socioeconomic groups and distinct populations. This so-called "geographic cytopathology" allows for the analysis of disease distributions and variations in specific tumours or infections, providing information that may help our understanding of the pathogenesis of different diseases, including infections, in different socio-economic and ethnic groups [29]. The cervical Pap smear is the classic example of a successful cancer prevention program based on exfoliative cytology, and of a cytological diagnostic method that can determine the prevalence of an infectious agent [30, 31, 32, 33, 34, 35, 36]. Pilot studies are being used to assess the potential impact of cervical screening in reducing the incidence of cervical carcinoma in different areas of the world in which cervical cancer varies greatly in prevalence [37, 38, 39]. Cervical screening programs have proved in developed countries to reduce greatly the instance of cervical carcinoma. However cervical screening programs based on the cervical smear can only impact in the resource limited medical settings, if there are treatment facilities to follow up and treat the detected lesion, and this currently masks assessment of the impact of cervical screening on the incidence of cervical cancer in developing countries [39]. But the feasibility of cervical screening with the manually read cervical Pap smear has been established in numerous low resource settings throughout Asia, Africa and America [40], and this appears the most sustainable, cost effective cervical cancer prevention service available. An example of this, is the program run by the Vietnamese in South Vietnam where there is an established high risk of cervical carcinoma, far greater than in North Vietnam [39]. Public health resources need to target individuals in geographic areas with a high rate of cervical carcinoma, with cytology based screening programs. Cervical PAP smear programs have reduced cervical cancer in developed countries, and reproducing this result in the developing world will rely on developing and maintaining high quality cytological reading of cervical smears, and establishing the infrastructure for follow-up and treatment [39, 40]. It appears far less appropriate to wait for an HPV vaccine, or to experiment in the developing world with radical new and poorly established "screen and treat" protocols using inspection of the cervix after application of acetic acid or Lugol's Iodine, followed by immediate ablative treatment of high grade lesions and cancer without a pathological diagnosis [39, 41]. In conclusion, FNAB, general nongynaecological cytology and the cervical PAP smear are less expensive and require less medical infrastructure than surgical pathology and microbiological studies, and cytology specimens provide material for a wide range of ancillary studies where these are available, including culture, IF, PCR, viral typing and drug sensitivities. Cytopathology and surgical pathology are complementary, but in a resource poor setting, cytology offers distinct advantages in cheaper costs, portability and less need for laboratory space and equipment. This can extend to postmortems on AIDS patients, where FNAB and core biopsy samples backed up by microbiological studies can deliver diagnoses [42]. The challenge is to train sufficient laboratory staff and cytopathologists and equip them with adequate laboratories to provide this service. This is where medical practitioners in the developed world can best support the establishment of a cytology service in developing countries: visits by cytopathologists to centres providing tutorials and teaching; funding medical practitioners and laboratory scientists from developing countries to train and attend study programs and tutorials in the developed world; establishing educational courses in developing countries and assisting with staffing; and providing regional and nationwide quality assurance programs and continuing education. This crucial interaction can be on a doctor to doctor, or hospital to hospital or national body to national body level, but ideally should be coordinated. 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