Assisting cytopathology training in medically under-resourced countries: defining the problems and establishing solutions.

Cytology is able to deliver rapid accurate diagnoses with minimal equipment and laboratory infrastructure at minimal cost, and this is especially so for fine needle biopsy (FNB), which is a powerful diagnostic tool in medically resource-poor environments, where histopathology laboratories are small in number and poorly supported financially. The crucial element in the development of cytology services is to train a sufficient number of well trained cytopathologists and cytotechnologists to create a 'critical mass' of personnel who not only provide routine diagnostic services, but also can train an ever expanding number of pathologists, cytotechnologists, and health workers. A review of practical programs to train cytopathologists and cytotechnologists in their own countries will be presented, including a recent series of FNB and cytology tutorials run in sub Saharan Africa. The need for local cytopathology programs and the potential for both local and visiting cytopathologists to provide a faculty will be discussed, as well as a range of possible programs which can bring African pathologists and trainee pathologists to Western institutions for periods of their training. Ideally, the regional Societies of Cytology, including the recently formed West African Society of Cytology, will establish their own diagnostic protocols, training programs, syllabuses, examinations and accreditation and career pathways for both cytopathologists and cytotechnologists, and organize tutorials where they will invite overseas faculty to contribute. Crucially, these new societies will empower cytopathologists and cytotechnologists to approach health services and governments to state the need for cytology services as a cost-effective accurate diagnostic service that enhances patient care.

University partnership to address the shortage of healthcare professionals in Africa.

The shortage of qualified health professionals is a major obstacle to achieving better health outcomes in many parts of the world, particularly in Africa. The role of health science universities in addressing this shortage is to provide quality education and continuing professional development opportunities for the healthcare workforce. Academic institutions in Africa, however, are also short of faculty and especially under-resourced. We describe the initial phase of an institutional partnership between the Muhimbili University of Health and Allied Sciences (MUHAS) and the University of California San Francisco (UCSF) centred on promoting medical education at MUHAS. The challenges facing the development of the partnership include the need: (1) for new funding mechanisms to provide long-term support for institutional partnerships, and (2) for institutional change at UCSF and MUHAS to recognize and support faculty activities that are important to the partnership. The growing interest in global health worldwide offers opportunities to explore new academic partnerships. It is important that their development and implementation be documented and evaluated as well as for lessons to be shared.

Kaposi's sarcoma before and during a human immunodeficiency virus epidemic in Tanzanian children.

BACKGROUND: With the onset of AIDS increased frequency of Kaposi's sarcoma (KS) has been reported. However, there is no case-based comparison of childhood (<14 years) KS before and during the HIV pandemic in sub-Saharan Africa. Here we report on the Tanzanian cancer registry data of pediatric KS in Tanzania and implications with regard to pathogenic factors. METHODS: One hundred fifty histologically confirmed pediatric KS (PKS) cases registered during 1968 through 1995 (28 years) were analyzed with regard to demographic and clinical characteristics before and during the AIDS epidemic. Statistical analysis was done with the Epi-Info program and chi square test. RESULTS: Of children with PKS 126 (84%) were male and 24 (16%) were female. The gender ratio was 5.1:1 and 5.4:1 during the endemic and epidemic periods, respectively. The highest occurrence of PKS was observed in the 0- to 5-years age group. Overall 73 (4.9/year) of these cases were registered during the pre and 77 (5.9/year) during the AIDS period. Over time a significant increase in anatomically disseminated KS cases was evident during the AIDS epidemic (P = 0.003). CONCLUSIONS: These observations indicate that children younger than 5 years are at high risk for developing KS, possibly reflecting low resistance to human herpesvirus (HHV) 8 infection. It is also likely that an increased susceptibility to HHV8 infection and morbidity is related to progressive immunodeficiency. The increase in AIDS PKS incidence appears to reflect a direct or indirect promoting effect of HIV on the development of KS lesions. Recognition of the high KS risk in small children warrants considerations of possible prevention measures including HIV/HHV8 vaccination and therapeutic options.

Proliferation and apoptosis in the evolution of endemic and acquired immunodeficiency syndrome-related Kaposi's sarcoma.

Kaposi's sarcoma (KS) is a multifocal lesion that occurs predominantly in the skin, most frequently in people infected with HIV-1, and that evolves through early stages (patch and plaque) to a tumor-like late stage (nodular). Both, endemic African (EKS) and AIDS-associated (AKS) KS expressed human herpesvirus 8 (HHV-8) as shown by PCR. By immunohistochemistry the expression of cellular Bcl-2 and c-myc was confined in early stages of both EKS and AKS to relatively few endothelial cells (EC) whereas in nodular KS most of spindle cells (SC) strongly expressed both genes. CD40 was usually strongly expressed in SC at all KS stages as well as in EC of non-involved tissue whereas CD40L (CD154) was not demonstrable. Fas (CD95) was moderately to weakly expressed by SC whereas p53 and Waf-1 were found in less than 5% of the SC. In both AKS and EKS at nodular stage almost no apoptotic SC were detected. In most AKS and EKS low levels of cell proliferation were seen but AKS showed consistently higher values compared to EKS. All clinical types and stages of KS showed a diploid cellular DNA content by flow cytometric analysis of microselected lesions. Thus, we conclude that KS during evolution represents diploid, probably reactive, cell proliferation, which progressively increases the expression of strong cellular and also viral (HHV-8) antiapoptotic factors.

Breast cancer before and during the AIDS epidemic in women and men: a study of Tanzanian Cancer Registry Data 1968 to 1996.

Kaposi's sarcoma and malignant lymphoma are two cancers that are most often associated with human immunodeficiency virus (HIV) infection. Recently, other cancers, including cervical cancer, have been associated with AIDS. The role of HIV in the pathogenesis of these malignancies is not well understood, and few studies have been done to determine any general increase in cancers after the onset of the HIV epidemic. This study compared breast cancer before and during the AIDS period by studying the total Tanzanian Cancer Registry data (1968 to 1996). The mean age among males increased from 50.88 to 52.63 years (p = 0.45) and among females decreased from 44.79 to 43.23 years (p = 0.005) before and during the AIDS epidemic, respectively. A statistically significant decrease in the incidence of breast cancer was observed during the AIDS epidemic period in both males (p = 0.001) and females (p = 0.021). The male-to-female ratio widened significantly from 0.09:1 to 0.03:1 (p = 0.0001). Further studies are needed to determine the incidence and observed changes of different cancers, including breast among patients with HIV/AIDS.

Immunological profile of endemic and epidemic Kaposi's sarcoma patients in Dar-es-Salaam, Tanzania.

Kaposi's sarcoma (KS) presents in four clinicopathological types namely classical/sporadic (CKS), endemic African (EKS), iatrogenic (IKS) and that associated with AIDS (AKS). Recently a putative herpes virus (HHV-8) was described and shown to be present in all four types of KS. The immunological status of patients with EKS has been conflicting. In this study total leucocyte counts, total lymphocyte counts and lymphocyte subsets of patients with EKS and AKS were determined by flow cytometry and compared to those of healthy HIV-1 seronegative controls. Results show that 50% of EKS lesions were of nodular type. Patients with EKS had significantly lower levels of CD4+ T- lymphocytes and CD4:CD8 ratio but significantly higher CD8+ T-lymphocytes compared to controls. Patients with AKS had significantly lower levels of CD4+ T-lymphocytes and also CD4:CD8 ratios but significantly higher percentage of CD8+ T-lymphocytes when compared with EKS patients. These findings indicate that in both forms of KS there is a certain degree of immunological disturbance which is more conspicuous in AKS because of HIV infection and suggests that HIV-1 acts synergistically with the aetiological agent (HHV-8) to cause a more aggressive type of KS.

Paediatric malignancies in Tanzania.

Using the Tanzania Cancer Registry, the pattern of childhood malignancies up to the age of 15 years over a period of 22 years (1973-1995) was analysed. Lymphomas were most frequent (38.9%) followed by soft tissue sarcomas (13.1%), retinoblastomas (11.1%) and squamous cell carcinoma (6.7%). The frequency of soft tissue sarcomas is relatively high but appears not to be related to human immunodeficiency virus (HIV) infection pandemic. The high frequency of squamous cell carcinoma also needs further investigation.

Expression of HHV-8 latency-associated T0.7 RNA in spindle cells and endothelial cells of AIDS-associated, classical and African Kaposi's sarcoma.

Analysis by polymerase chain reaction (PCR) and serological studies have demonstrated a close association between the novel human herpes virus, Kaposi's sarcoma-associated herpes virus (KSHV) or human herpes virus-8 (HHV-8) and the development of Kaposi's sarcoma (KS). To clarify the role of HHV-8 in KS pathogenesis, we investigated at the cellular level by in situ hybridization the expression of a recently described 0.7-kb HHV-8-encoded mRNA (T0.7 mRNA) in KS tissues of different epidemiological origin (AIDS-KS, African endemic KS and classical KS). The T0.7 mRNA likely encodes a small membrane protein, supposedly expressed in latently HHV-8-infected cells. Indeed, we detected T0.7 mRNA in virtually all cells of the cell line BCBL-1 established from a body cavity-based lymphoma (BCBL) and latently infected with HHV-8. In all KS biopsies examined, independent of their epidemiological type, the late-stage (nodular) KS tissues showed a high level of T0.7 mRNA expression in typical KS spindle cells but also in endothelial cells lining blood vessels, indicating latent HHV-8 infection of these cells. The presence of T0.7-expressing cells was restricted to KS tumor tissue and therefore appears to indicate an important role of latent HHV-8 infection in KS pathogenesis.

Kaposi's sarcoma before and during the HIV epidemic in Tanzania.

The Tanzania cancer registry recorded 39,920 cancer cases from 1968 to 1995. Kaposi's sarcoma (KS) constituted 4% of the cases of which 79.9% were males. During the AIDS period changes in the demographics of KS were noted showing an increase in the diagnosis of KS (p=0.0001). There was an overall decrease in the mean age from 41.7 years before to 37.3 years during the AIDS epidemic (p=0.002). In males this decreased from 42.7 to 38.8 years (p=0.01) but was not statistically significant in females (p=0.06). In both periods the cases were observed in the sexually active age groups. The narrowing of the male/female ratio during the AIDS period (p=0.0004), and an increase in extra-limb lesions from 19.7% before to 48.7% during the AIDS epidemic indicates the emergence of an aggressive form of KS in Tanzania, suggesting a co-factor role of HIV in KS pathogenesis.

Expression of adhesion molecules in endemic and epidemic Kaposi's sarcoma.

Spindle cells and vascular endothelium in nodular lesions of AIDS associated (epidemic) and endemic Kaposi's sarcoma showed similar immunohistochemical patterns of expression for cell adhesion molecules and extracellular matrix proteins. Spindle cells as well as endothelium also expressed both alpha 5 and alpha V integrin subunits and ICAM-1 suggesting a possible role for inflammatory cytokines in spindle cell formation. The spindle cell compartment was rich in collagen, laminin, fibronectin and tenascin suggesting an important reactive component in the evolution of Kaposi's sarcoma. The lack of thrombospondin expression in the spindle cells favours the contention that they could be transitional, proliferating cells of endothelial origin. Specific expression of tat protein was not seen suggesting minimal if any HIV replication in these lesions. Our findings suggest similar histopathogenetic mechanisms for endemic and epidemic Kaposi's sarcoma. The clinically more malignant features of most AIDS related cases may reflect an important effect of systemic and focal cytokines in HIV patients and possibly other cofactor(s), i.e. tat protein in the induction and growth of the lesions.

Oncogenesis in HIV-infection.

The presence of human Kaposi's sarcoma associated herpesvirus-like sequences (KSHV) was examined in different epidemiological variants of Kaposi's sarcoma (KS) and in KS-derived cell cultures by polymerase chain reaction (PCR). KSHV DNA was present in all tumor biopsies of AIDS-associated KS (59 biopsies), endemic KS (26 biopsies; 21 African endemic KS, 5 Greek endemic KS), sporadic/classical KS (28 biopsies) and post-transplant/iatrogenic KS (6 of 7 biopsies). On the contrary, these sequences were only detected rarely in non-involved skin of KS patients (3 positive specimens of 12), in the peripheral blood mononuclear cells of HIV-infected patients (3 positive specimens of 54) and in lymphoma-biopsies (3 positive specimens of 47). Cell cultures derived from KS skin lesions were positive for KSHV DNA only in the first two passages. However, two longer-term positive cultures from a biopsy of a patient affected with sporadic KS and a biopsy of a patient affected with epidemic KS was identified. A strong association of KSHV with KS tissue was observed in all the different epidemiological variants of KS. Long-term positive KS-derived cell cultures will be an important tool to study the herpesvirus-like agent and to investigate its functional role in the initiation and progression of KS.

Heterogeneity of spindle cells in Kaposi's sarcoma: comparison of cells in lesions and in culture.

The immunophenotype of spindle cells in epidemic, endemic, and classic (sporadic) Kaposi's sarcoma (KS) lesions was defined by the demonstration of various cell markers and compared with that of KS-derived cell lines. No significant histological or immunophenotypic differences were observed between the three clinical types of KS at comparable stages. The spindle-cell compartment of the different KS types was composed predominantly of a mixture of proliferating CD45+/CD68+ bone-marrow-derived monocytes and TE7+/collagen+ fibroblastic cells with varying expression of EN4/PAL-E/CD31/CD34/CD36 endothelial-associated antigens and/or smooth-muscle-specific alpha-actin (alpha-actin). The latter cells appeared to represent transitional forms of fibroendothelial and fibromyocytic cells. The in vitro cultured KS-derived cell lines (KS-3, KS-6, and KS-8) expressed the fibroblastic antigen TE7 and smooth-muscle-specific alpha-actin but not leukocytic or endothelial-associated antigens consistent with the phenotype of fibromyoid spindle cells of primary lesions. Neither HIV antigen nor provirus DNA was demonstrable in the epidemic KS lesions. The observed heterogeneity of the spindle-cell compartment further substantiates the view that Kaposi's sarcoma, irrespective of clinical setting, expresses salient features more compatible with reactive, tumor-like lesion than clonal sarcoma.

A histological and immunohistological study of malarial placentas.

Sections of 18 malaria-infected placentas were stained with haematoxylin and eosin, periodic acid and methenamine silver, and immunohistochemically with monoclonal antibodies against human common leukocyte antigen, CLA (CD 45), B cells (CD 20, L 26), T cells (CD 45RO, UCHL-1) and collagen IV. Parasitized erythrocytes accumulated in the maternal villous spaces, with none in the foetal circulation. These were found in association with inflammatory leukocytes and pigments. Fibrinoid necrosis was more prevalent in the heavily infected placentas. Thickening and reduplication of foetal capillary basement membranes, and a decrease in leukocytes, including B and T cells, were seen in the heavily infected placentas. These findings are in keeping with previously reported depression of cellular and humoral immunity in patients with heavy parasitaemia.

A role for a new herpes virus (KSHV) in different forms of Kaposi's sarcoma.

Kaposi's sarcoma (KS) is a previously rare, tumour-like lesion of controversial biological nature. KS has since the early 1980s become frequent in patients with AIDS, particularly in homosexuals. KS is also endemic in Central Africa predominantly in otherwise healthy men but also in women and children. Recently, evidence for the presence of novel, herpes virus DNA sequences in more than 90% of AIDS Kaposi lesions (AKS) was presented. This DNA was identified using representational difference analysis (RDA) generating short, unique sequences with variable homology to several herpes virus, but no intact virus was recovered. If these DNA-sequences are also present in other, non-HIV-associated forms of Kaposi's sarcoma this would strongly suggest a specific, aetiopathological involvement of this putative new herpes virus in the pathogenesis of Kaposi's sarcoma, rather than a contamination of yet another opportunistic virus in immunosuppressed AIDS patients.

PIP: Samples were examined by polymerase chain reaction (PCR) for the presence of the putative Kaposi's sarcoma herpes virus (KSHV). KS DNA from HIV-negative, African, endemic (EKS) samples, and epidemic HIV-positive KS (AKS), and sporadic KS (SKS) samples were tested from Tanzania and Sweden. All of the HIV KS (18 African EKS and 4 Swedish SKS) as well as the HIV-positive AIDS-related KS (16 African and 7 Swedish AKS) biopsies were shown to contain the previously described DNA sequences. KS lesions from children, females, and males in various tissues were analyzed including skin, lymph nodes, gut and oral mucosa. All forms of KS showed a single PCR product of the expected size (233 base pairs). To exclude amplification of other types of herpes virus, virus preparations of Epstein-Barr virus (EBV), herpes simplex virus, cytomegalovirus, vesicular stomatitis, and human herpes virus type 6 (HHV6) were assayed, again by PCR, using the KSHV primers. No PCR products were obtained with any of these virus strains. However, most HIV-positive and HIV-negative KS DNA samples also contained either EBV and/or HHV6 sequences. All biopsies from non-KS tissues (cells) of HIV-positive and HIV-negative individuals were consistently negative for KSHV by PCR. The observation that the same herpes virus-like DNA sequence is present in endemic and sporadic, as well as AIDS-related, Kaposi's sarcoma cases suggests a possible pathogenic association between this putative novel, herpes-like virus and KS. The herpes virus-like DNA sequences described by Y. Chang in 1994 may indeed represent a novel herpes (KSHV), etiopathologically associated with various clinical forms of Kaposi's sarcoma. Its pathogenic importance is indicated by its presence in different KS tissues with various clinical types of KS and its absence from non-KS-involved tissues. Furthermore, the presence of KSHV in KS of children suggests a nonsexual mode of transmission.

Thymic immunopathology and progression of SIVsm infection in cynomolgus monkeys.

Thymuses from 22 cynomolgus monkeys infected with simian immunodeficiency virus (SIVsm) developed characteristic cortical and medullary changes including formation of B-cell follicles (8/21) and accumulation of virus immune complexes. Advanced thymic histopathology was correlated with more pronounced immunodeficiency. SIVsm provirus was detected by polymerase chain reaction (PCR) in most (16/18) thymuses and spliced viral env mRNA in 3 (3/7) thymuses with advanced histopathologic changes indicative of thymic SIVsm replication. By combined in situ hybridization (ISH) and immunohistochemistry, viral RNA was localized mainly to the follicular dendritic network, macrophages, multinucleated giant cells, and lymphocytes of the medullary regions. Latent infection by an Epstein-Barr-related herpesvirus (HVMF1) was also found by PCR and by ISH in medullary regions of three (3 of 8) thymuses with B-cell follicles, suggestive of an inductive role for B-cell proliferation in these thymuses. In a control group of HIV-2-infected nonimmunosuppressed monkeys, no comparable thymic changes were observed. Our results indicate that SIV, and probably by analogy HIV, can have direct and diverse pathogenic effects on the thymus that are important in the development of simian (human) AIDS.

B-cell lymphomagenesis in SIV-immunosuppressed cynomolgus monkeys.

B-cell lymphomas developed frequently (approx. 40%) in SIVsm (SMM3) immunosuppressed monkeys and were mostly extranodal, aggressive and all associated with an EBV-related simian herpes virus operationally designated herpes virus Macaca fascicularis (HVMF-I). Lymphoma tissues from 21 monkeys were studied by PCR and DNA PAGE for mono/oligoclonality of the VDJ-rearranged IgH genes. Most lymphomas (n = 15) showed a monoclonal and approximately 1/3 (n = 6) an oligoclonal VDJ rearrangement pattern. The time after infection to tumor presentation was significantly shorter for oligoclonal than for monoclonal lymphomas, suggesting that oligoclonal selection frequently precedes the outgrowth of a single malignant clone. Comparison of the VDJ rearrangements in an established lymphoma cell line and the original, oligoclonal lymphoma tissue indicated in vitro selection of one HVMF-infected clone. Longitudinal studies of sequential lymph-node biopsies showed that the malignant lymphoma clone in 3 out of 8 lymphomas could be identified as a predominant clone in lymph nodes 2-12 months after SIV infection and 6-10 months before clinical presentation of the lymphomas. VDJ-rearranged DNA corresponding to that of the lymphomas was also detected in most sera at the time of lymphoma manifestation but not in corresponding PBL preparations. Clearly, the SIVsm AIDS model in cynomolgus monkeys represents a powerful tool for biological and clinical studies of herpes-virus-associated lymphomagenesis in immunosuppressed states.

AIDS-associated kaposis-sarcoma in tanzanians of Indian origin - report of cases and review of literature.

Kaposi's sarcoma (KS) was up to the 1980's seen as a rare indolent sporadic disease in Southern Europe and as an endemic disease in East and Central Africa. With the onset of the HIV/AIDS epidemic a more aggressive, disseminated type of KS was recognized in HIV infected people with AIDS. Interestingly, KS has not been reported in Indians living in Africa for several generations. Recently KS was however, diagnosed in two Tanzanian Indians, both infected with HIV. Clinical and pathological studies of these two cases showed the characteristic hallmarks of KS in both HIV infected and uninfected people. From the literature and cancer registry data it appears that KS has been even more rare in India and other Far Eastern countries, compared to Europe and the Americas, with only a few cases reported with and without HIV association. The present data and that reported earlier in the literature support the notion of an infectious agent transmitted sexually in the pathogenesis of KS. Ethnic/genetic factors could also be of importance.

Expression of Epstein-Barr-virus-related nuclear antigens and B-cell markers in lymphomas of SIV-immunosuppressed monkeys.

Simian-immunodeficiency-virus(SIV)-infected cynomolgus monkeys develop B-cell lymphomas in approximately one third of the cases. We have now studied the expression of cynomolgus-Epstein-Barr-virus(cyno-EBV) nuclear antigens in 13 cyno-EBV-carrying SIVsm-associated monkey lymphomas and established cell lines from 3 of these tumors. Immunoblots of cell lysates were probed with polyspecific and monospecific reagents directed against human EB-virus EBNAI-6, and against the membrane protein LMPI. An EBNA2-cross-reacting protein was demonstrated in 8 lymphoma tissues (8/13) and in the 3 cell lines derived from the tumors. All tumors expressed a polypeptide with 50 to 55 kDa molecular weight, which cross-reacted with some antibodies to EBNAI. Absorption experiments with normal monkey tissue showed that this polypeptide was specific for the cyno-EBV-carrying lymphoma cells. Equivalents of EBNA3-6 and LMPI could not be detected. Immunophenotypical characterization showed that the monkey lymphomas were similar to human HIV-associated B-cell lymphomas. Malignant B-cell lymphomas in experimentally SIVsm-infected cynomolgus monkeys can be a model for EBV-associated lymphomagenesis in immunodeficiency states.

Immunohistochemical distribution of leucocyte antigens in lymphoid tissues of cynomolgus monkeys (Macaca fascicularis).

Crossreactivity of antibodies to human leucocyte antigens with lymphoid tissues of cynomolgus monkeys was studied by immunohistochemistry and immunoblotting. Of a total of 54 clusters of differentiation (CD) antigens, 39 were expressed essentially with the same immunostaining patterns in the monkey as in human lymphoid tissues. By immunoblotting L26 (CD20) detected a 35 Kd molecule in the monkey lymph node. Our observations indicated that most of the CD antigens are expressed and can be studied in lymphoid tissues of cynomolgus monkeys.