Diagnosing AIDS and Emerging Infections in Resource-Limited Settings:
The Role of the Pathologist in Patient Care and Disease Surveillance
Section 4 -
Role Of Cytopathology In Patient Care And Disease Surveillance
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 . Antiretroviral drug therapy has reduced the incidence of opportunistic
infections and KS in the HIV positive patients in developed countries
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. 
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 . 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 .
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 .
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 .
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 , and by autofluorescence in P stained
smears . 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 . 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 .
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 .
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 . 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
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
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
including in more recent publications, disseminated rhinosporidiosis ,
aspergillosis in various organs including lymph nodes and skin ,
tuberculosis in breast , lymph
and epididymis and testis ,
actinomycotic abscesses in the liver
cervicofacial regions ,
cutaneous cryptococcosis 
and cryptococcal lymphadenitis , and
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
positive patients, the general aim of the FNAB is to exclude an infectious agent (Mycobacterium avium-intracellular (MAI), M. tuberculosis, fungi and specific
KS and NHL, along with metastatic carcinomas . 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
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
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)
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 . 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 .
BCG vaccination produces localised lymphadenopathy and cytomorphology resembling mycobacterial
infections except that necrotic debris with neutrophils is more prominent in the BCG cases
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
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
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 . 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 .
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
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
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 .
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 , 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 .
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
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
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
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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