Novel Insights into the Pathogenesis of Human Post-Primary Tuberculosis from Archival Material of the Pre-Antibiotic Era, 1931-1947.

OBJECTIVES: Primary and post-primary tuberculosis (TB) are distinct entities. The aim of this study was to study the histopathology of primary and post-primary TB by using the unique human autopsy material from the pre-antibiotic era, 1931-1947. MATERIAL AND METHODS: Autopsy data were collected from the autopsy journals, and the human tissue was collected from the pathology archives at the Department of Pathology, the Gades Institute. RESULTS: Histological presentations of TB lesions showed great diversity within a single lung. Post-primary TB starts as a pneumonia forming early lesions, characterized by the infiltration of foamy macrophages containing mycobacterial antigens within alveoli, and progressing to necrotic pneumonias with an increasing density of mycobacterial antigens in the lesions. These necrotic pneumonic lesions appeared to either resolve as fibrocaseous lesions or lead to cavitation. The typical granulomatous inflammation, the hallmark of TB lesions, appeared later in the post-primary TB and surrounded the pneumonic lesions. These post-primary granulomas contained lesser mycobacterial antigens as compared to necrotic pneumonia. CONCLUSIONS: Immunopathogenesis of post-primary TB is different from primary TB and starts as pneumonia. The early lesions of post-primary TB may progress or regress, holding the key to understanding how a host can develop the disease despite an effective TB immunity.

The value of histological examination in the diagnosis of tuberculous lymphadenitis in the era of rapid molecular diagnosis.

Extrapulmonary tuberculosis often poses a diagnostic challenge. This study aimed to assess the value of histological examination in diagnosing tuberculous lymphadenitis (LNTB) when performed simultaneously with rapid molecular assay (Xpert MTB/RIF) testing. People presumed to have LNTB were prospectively enrolled in a tertiary care hospital. Excision biopsy was performed and tested by histology, Xpert, and culture. Of 390 lymph nodes, 11 (2.8%) were positive by AFB microscopy, 124 (31.8%) by Xpert, 137 (35.1%) by culture, and histopathology was consistent with TB in 208 (53.3%). Altogether, LNTB was diagnosed in 228 and bacteriologically confirmed TB in 178 cases. Against culture, histopathology versus Xpert had higher sensitivity (93 vs. 62%) but lower specificity (68 vs. 83%). In patients with short clinical history, a significantly higher number of Xpert-positive specimens were culture-positive. Among patients with histology suggestive of TB, no difference was seen in response to treatment between bacteriology positive and negative, but a significant slow response was noted in bacteriology confirmed TB with nonspecific histology. In a country like Pakistan, with high TB and low HIV prevalence, diagnosis is possible for more than 95% of LNTB when Xpert and histopathology examination is used in combination, compared to less than 60% by Xpert alone.

Diagnosis of extrapulmonary tuberculosis using the MPT64 antigen detection test in a high-income low tuberculosis prevalence setting.

BACKGROUND: Extrapulmonary tuberculosis (EPTB) poses diagnostic challenges due to the paucibacillary nature of the disease. The immunochemistry-based MPT64 antigen detection test (MPT64 test) has shown promising results for diagnosing EPTB in previous studies performed in low-resource settings, with higher sensitivity than microscopy and culture. The aim of this study was to investigate the performance of the MPT64 test in a routine clinical setting in a high-income low TB prevalence country. METHODS: Extrapulmonary samples sent for TB diagnostics to microbiology and pathology laboratories at three regional tertiary care hospitals in Norway in a one-year period were included and subjected to the MPT64 test in parallel to the routine TB diagnostic tests. RESULTS: Samples from 288 patients were included and categorised as confirmed TB cases (n = 26), clinically diagnosed TB cases (n = 5), non-TB cases (n = 243) and uncategorised (n = 14), using a composite reference standard (CRS). In formalin-fixed biopsies, the sensitivity (95% CI) of the MPT64 test, microscopy, PCR-based tests pooled, and culture was 37% (16-62), 20% (4-48), 37% (16-62) and 50% (23-77), respectively, against the CRS. The MPT64 test showed a good positive predictive value (88%) and an excellent specificity (99, 95% CI 92-100) in formalin-fixed biopsies. In fine-needle aspirates, pus and fluid samples, the test performance was lower. CONCLUSIONS: The MPT64 test was implementable in pathology laboratories as part of routine diagnostics, and although the sensitivity of the MPT64 test was not better than culture in this setting, the test supplements other rapid diagnostic methods, including microscopy and PCR-based tests, and can contribute to strengthen the diagnosis of EPTB in formalin-fixed biopsies in the absence of culture confirmation.

Evaluation of treatment response in extrapulmonary tuberculosis in a low-resource setting.

BACKGROUND: Diagnosing extrapulmonary tuberculosis (EPTB) is challenging and many patients are initiated on empirical anti-TB treatment without a laboratory confirmed diagnosis. Monitoring treatment response is thus important to ensure correct diagnosis and proper disease management. The definition of satisfactory response to treatment in EPTB remains unclear. The objectives of this study were to describe the clinical presentation of EPTB and the effect of treatment on clinical parameters. Further, to assess if simple clinical parameters, without laboratory data, could evaluate treatment response. METHODS: Prospective cohort study of presumptive EPTB patients at Mnazi Mmoja Hospital, Zanzibar. By using a composite reference standard, patients were categorized as TB or non-TB cases. The TB patients were followed during anti-TB treatment. RESULTS: There were 64 TB and 62 non-TB cases. The frequency of symptoms at baseline were comparable in TB and non-TB patients, with lymphadenitis and pleuritis as the most common manifestations. Among TB cases, there was a trend towards regression of lymphadenopathy after 2 months, and at treatment completion 24/28 (86%) cases showed full regression. Weight gain ≥5% was reported in 36/49 (73%) of the TB patients at 2 months and in 38/46 (83%) at treatment completion. After 2 months of treatment, a combination of clinical parameters; improvement of symptoms (50/50), ≥5% weight gain (36/49) and regression of physical signs (45/49) correlated with the treatment response. CONCLUSIONS: An algorithm including only simple clinical parameters could be used as an easy tool to assess treatment responses in low-resource settings. However, this needs to be tested on a larger sample size.

Epidemiological and histopathological profile of malignant melanoma in Malawi.

BACKGROUND: Studies on malignant melanoma have largely focused on Caucasian populations due to higher incidence in lighter-skinned individuals. While there is a well developed body of literature describing melanoma in African-Americans, much less is known about melanoma in black Africans. Prior reports have suggested that it is reportedly extremely rare in black Africans who are considered to mostly have the acral lentiginous subtype. However, an accurate understanding of melanoma in this part of the world is hindered by the very limited nature of prior publications. The aim of this study was to determine the epidemiological profile, anatomical distribution and histopathological features of melanoma presenting in Africans at a tertiary referral hospital in Malawi. METHODS: This is a retrospective study that characterized melanoma cases diagnosed from January 2012 to December 2017, at a cancer referral centre in Malawi. All confirmed, malignant melanoma cases during the study period were retrieved. Data abstracted included age, sex, anatomic site and whether it was a primary or metastatic site. Breslow thickness in millimetres, Clark level of invasion, presence of ulceration and melanoma subtype were also evaluated. RESULTS: One hundred thirty-two cases were included in the study, 81 (61%) were female and 26 (20%) were from a metastatic site. The mean age was 57 years (sd = 15) with the majority in the age group 60-69 years. Males presented at an older age than females. Ninety five percent of cutaneous melanomas were located on acral sites, most commonly the foot (87%) and the most common histopathological subtype was acral lentiginous. Eighty four percent presented with a Breslow thickness over 4 mm (median 9 mm). CONCLUSION: Our study shows that malignant melanoma occurs in black people in Malawi and may be an under-appreciated malignancy. While long term clinical follow-up was not available, most patients presented at late stages of the disease, supporting a poor prognosis. These results suggest that increased awareness of melanoma in black Africans and earlier intervention may have meaningful impacts on outcomes and survival.

MPT64 antigen detection test improves routine diagnosis of extrapulmonary tuberculosis in a low-resource setting: A study from the tertiary care hospital in Zanzibar.

BACKGROUND: Extrapulmonary tuberculosis (EPTB) is a diagnostic challenge. An immunochemistry-based MPT64 antigen detection test (MPT64 test) has reported higher sensitivity in the diagnosis of EPTB compared with conventional methods. The objective of this study was to implement and evaluate the MPT64 test in routine diagnostics in a low-resource setting. METHODS: Patients with presumptive EPTB were prospectively enrolled at Mnazi Mmoja Hospital, Zanzibar, and followed to the end of treatment. Specimens collected were subjected to routine diagnostics, GeneXpert® MTB/RIF assay and the MPT64 test. The performance of the MPT64 test was assessed using a composite reference standard, defining the patients as tuberculosis (TB) cases or non-TB cases. RESULTS: Patients (n = 132) were classified as confirmed TB (n = 12), probable TB (n = 34), possible TB (n = 18), non-TB (n = 62) and uncategorized (n = 6) cases. Overall, in comparison to the composite reference standard for diagnosis, the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of the MPT64 test was 69%, 95%, 94%, 75% and 82%, respectively. The MPT64 test performance was best in TB lymphadenitis cases (n = 67, sensitivity 79%, specificity 97%) and in paediatric TB (n = 41, sensitivity 100%, specificity 96%). CONCLUSIONS: We show that the MPT64 test can be implemented in routine diagnostics in a low-resource setting and improves the diagnosis of EPTB, especially in TB lymphadenitis and in children.

Rapid and Specific Diagnosis of Extrapulmonary Tuberculosis by Immunostaining of Tissues and Aspirates With Anti-MPT64.

BACKGROUND: Extrapulmonary tuberculosis (EPTB) constitutes about 15% to 20% of all cases of tuberculosis (TB). The confirmation of EPTB has always been a challenge to laboratory personnel. We aim to evaluate the diagnostic potential of immunostaining with anti-MPT64 in various EPTB specimens. MATERIALS AND METHODS: We studied a total of 51 TB cases and 38 non-TB control specimens comprising of fine-needle aspirates and formalin-fixed biopsies. These were investigated using a combination of the Ziehl-Neelsen method, the Lowenstein-Jensen culture, immunostaining with anti-MPT64 and anti-BCG, and nested-polymerase chain reaction (PCR) for IS6110. Results of all the tests were compared using nested-PCR as the gold standard. RESULTS: Diagnostic validation of immunostaining for anti-MPT64 was performed using nested-PCR as the gold standard. The overall sensitivity, specificity, and positive and negative predictive values for immunostaining with anti-MPT64 were 100%, 97%, 97%, and 100%, respectively. CONCLUSIONS: Immunostaining using anti-MPT64 is a rapid and sensitive method for establishing an early and specific diagnosis of Mycobacterium tuberculosis infection. The technique is simple to be incorporated into routine pathology laboratories.

Differential in vivo expression of mycobacterial antigens in Mycobacterium tuberculosis infected lungs and lymph node tissues.

BACKGROUND: The clinical course of tuberculosis (TB) infection, bacterial load and the morphology of lesions vary between pulmonary and extrapulmonary TB. Antigens expressed in abundance during infection could represent relevant antigens in the development of diagnostic tools, but little is known about the in vivo expression of various M. tuberculosis antigens in different clinical manifestations. The aim of this study was to study the differences in the presence of major secreted as well as somatic mycobacterial antigens in host tissues during advanced rapidly progressing and fatal pulmonary disease with mainly pneumonic infiltrates and high bacterial load, and to compare this to the presence of the same antigens in TB lymphadenitis cases, which is mainly chronic and self-limiting disease with organised granulomas and lower bacterial load. METHODS: Human pulmonary (n = 3) and lymph node (n = 17) TB biopsies, and non-TB controls (n = 12) were studied. Ziehl-Neelsen stain, nested PCR 1S6110 and immunohistochemistry were performed. Major secreted (MPT32, MPT44, MPT46, MPT51, MPT53, MPT59, MPT63, and MPT64) and somatic mycobacterial antigens (Mce1A, Hsp65, and MPT57) were detected by using rabbit polyclonal antibodies. RESULTS: Plenty of bacilli were detectable with Ziehl-Neelsen stain in the lung biopsies while no bacilli were detected in the lymph node biopsies. All the cases were shown to be positive by PCR. Both secretory and somatic antigens were expressed in abundance in pulmonary infiltrates, while primarily somatic antigens were detected in the lymphadenitis cases. Of the secreted antigens, only MPT64 was consistently detected in both cases, indicating a preferential accumulation of this antigen within the inflammatory cells, even if the cells of the granuloma can efficiently restrict bacterial growth and clear away the secreted antigens. CONCLUSIONS: This study shows that major secreted mycobacterial antigens were found in high amounts in advanced pulmonary lesions without proper granuloma formation, while their level of staining was very low, or absent, in the lymph node TB lesions with organised granulomas and very low bacillary load, with one exception of MPT64, suggesting its role in the persistence of chronic infection. These findings have implication for development of new diagnostic tools.

CD11c(+) dendritic cells rather than Langerhans cells are reduced in normal skin of immunosuppressed renal transplant recipients.

The increased risk of squamous cell carcinomas (SCC) in renal transplant recipients (RTR) is related to impaired immunosurveillance as a consequence of immunosuppressive therapy. Since dendritic cells (DC) play an important role in immunosurveillance, we investigated the quantity of DC subsets and macrophages in normal skin of RTR and immunocompetent controls by immunohistochemistry. In this comparative study Langerhans' cells (LC) were present in similar numbers in RTR and controls. The number of CD11c+ DC was significantly reduced in RTR, particularly in patients on triple treatment therapy, compared with controls. Macrophages were significantly increased. Plasmacytoid DC were not detected in normal skin. The reduced quantity of CD11c+ DC and increased number of macrophages in normal skin of immunosuppressed RTR may contribute to the increased incidence of SCC in RTR. This finding underlines the role of DC subsets in immunosurveillance, and may have implications for our understanding of the effect of immunosuppression on DC subsets.

Rapid diagnosis of tuberculosis in aspirate, effusions, and cerebrospinal fluid by immunocytochemical detection of Mycobacterium tuberculosis complex specific antigen MPT64.

The aim of the study was to evaluate the diagnostic potential of immunocytochemical staining for the detection of Mycobacterium tuberculosis complex-specific antigen MPT64, in tuberculous lymph node aspirates, cerebrospinal fluid, and effusions from pleura and abdomen. One hundred ninety patients with a diagnosis of tuberculosis (cases) and 80 patients with nontuberculous lesions (controls) were enrolled and differentiated on the basis of clinical features, histology, cytology, clinical biochemistry, Ziehl-Neelsen staining, Lowenstein-Jensen culture, and response to antituberculous therapy. Cervical lymph nodes fine-needle aspirate (n = 150), cerebrospinal fluid (n = 27), pleural fluid (n = 41), and peritoneal fluid (n = 52) were collected and stained with anti-MPT64 and anti-BCG antibodies using immunocytochemistry. Nested-PCR for IS6110 was done for comparison and to calculate the diagnostic indices of the ICC staining. ICC staining with anti-MPT64 was positive in 128/190 (67.4%) tuberculous smears and in 4/80 (5%) control smears thus giving sensitivity of 67.4% and the specificity of 95%, while anti-BCG was positive in 112 (58.9%) tuberculous smears and in 16/80 (20%) control smears with sensitivity of 58.9% and specificity of 80%. When diagnostic validation of ICC was done using PCR as the gold standard, the overall sensitivity, specificity, positive- and negative-predictive values for ICC staining in smears with anti-MPT64 was 96, 96, 95, and 97%, respectively, while the corresponding values for anti-BCG were 87, 88, 86, and 88%. ICC staining using anti-MPT64 represents a robust and simple method for establishing an early etiological diagnosis of M. tuberculosis complex infection in extrapulmonary tuberculosis.

Kinetics of lymphocyte reconstitution after allogeneic bone marrow transplantation: markers of graft-versus-host disease.

GVHD causes extensive morbidity and mortality in patients who receive alloHCT. Predictive and reliable markers for GVHD are currently lacking but required to improve the safety and accessibility of alloHCT. We present an experimental rat model of myeloablative total body irradiation and fully mismatched major and minor histoincompatible, T cell-depleted BMT, followed by delayed infusion of donor lymphocytes. This treatment, in contrast to marrow transplantation alone, resulted in severe aGVHD and 100% lethality within 2-6 weeks. We investigated the reconstitution kinetics and phenotypes of donor leukocyte subpopulations as well as the histopathology of selected organs that may correlate with GVHD, with the goal to find potential disease-related markers. We observed histological changes mainly confined to the skin, with degenerative changes in the basal layer. LNs and spleen showed deranged architecture with markedly increased accumulation of lymphocytes, whereas the gut, liver, and lungs appeared normal. Of the lymphocyte markers tested, donor-derived CD62L(+) T cells were markedly decreased in animals suffering from GVHD. Furthermore, we observed peripheral depletion of CD4(+)CD25(hi)FoxP3(+) T(reg), which was in contrast to controls. The relative frequency of these lymphocyte subpopulations in blood may therefore serve as accessible cellular markers of aGVHD. We propose that the animal model presented is instructive for the identification of clinically relevant markers of GVHD, which could improve disease diagnosis and management in alloHCT.

Expression profiling of major histocompatibility and natural killer complex genes reveals candidates for controlling risk of graft versus host disease.

BACKGROUND: The major histocompatibility complex (MHC) is the most important genomic region that contributes to the risk of graft versus host disease (GVHD) after haematopoietic stem cell transplantation. Matching of MHC class I and II genes is essential for the success of transplantation. However, the MHC contains additional genes that also contribute to the risk of developing acute GVHD. It is difficult to identify these genes by genetic association studies alone due to linkage disequilibrium in this region. Therefore, we aimed to identify MHC genes and other genes involved in the pathophysiology of GVHD by mRNA expression profiling. METHODOLOGY/PRINCIPAL FINDINGS: To reduce the complexity of the task, we used genetically well-defined rat inbred strains and a rat skin explant assay, an in-vitro-model of the graft versus host reaction (GVHR), to analyze the expression of MHC, natural killer complex (NKC), and other genes in cutaneous GVHR. We observed a statistically significant and strong up or down regulation of 11 MHC, 6 NKC, and 168 genes encoded in other genomic regions, i.e. 4.9%, 14.0%, and 2.6% of the tested genes respectively. The regulation of 7 selected MHC and 3 NKC genes was confirmed by quantitative real-time PCR and in independent skin explant assays. In addition, similar regulations of most of the selected genes were observed in GVHD-affected skin lesions of transplanted rats and in human skin explant assays. CONCLUSIONS/SIGNIFICANCE: We identified rat and human MHC and NKC genes that are regulated during GVHR in skin explant assays and could therefore serve as biomarkers for GVHD. Several of the respective human genes, including HLA-DMB, C2, AIF1, SPR1, UBD, and OLR1, are polymorphic. These candidates may therefore contribute to the genetic risk of GVHD in patients.

Serodiagnosis of tuberculous lymphadenitis using a combination of antigens.

BACKGROUND: The diagnosis of extra-pulmonary tuberculosis (EPTB) by conventional methods such as culture and microscopy has low sensitivity and requires an invasive procedure. A simple rapid serological test would be of great value. METHODS: Six antigens (ESAT-6, Ag85A, TB10.4, Rv3881c, lipoarabinomannan (LAM) and Ara6-BSA) were tested in an ELISA to detect antigen specific IgG and IgM antibodies in sera from 54 culture and histology-confirmed tuberculous lymphadenitis (TBLN) patients, among whom four were HIV seropositive, sera from 25 smear positive pulmonary tuberculosis (PTB) patients, 15 culture and histology-negative lymphadenitis (non-TBLN) patients (n=15) and 22 healthy controls (HCs). RESULTS: The sensitivities of the antigens for the detection of IgG in sera of TBLN patients ranged from 4 to 30 %. Specificities ranged from 91 to 100 % with sera from HCs. Sensitivities of the antigens for detection of IgM ranged from 0 to 15 % and specificities ranged from 91 to 100 %. LAM was the most potent antigen followed by ESAT-6 and Rv3881c for detection of IgG. However, the sensitivity for antigen specific IgG antibody detection was improved when LAM was combined with ESAT-6 and Rv3881c.The sensitivity was 54 % and the specificity 91 %. CONCLUSIONS: The study suggests that the combined use of LAM, ESAT-6 and Rv3881c for the detection of IgG in sera of TBLN patients could be a supplement to microscopy of fine- needle aspirate (FNA) to diagnose EPTB.

[Hidradenitis suppurativa]. / Hidradenitis suppurativa.

BACKGROUND: Hidradenitis suppurativa is a chronic inflammatory skin disease characterized by recurrent tender nodules and boils, usually in the armpits and groins. Draining fistulas and hypertrophic scarring are hallmarks of more severe disease. The objective of this article is to review the clinical presentation, diagnostic considerations and treatment of the disease. MATERIAL AND METHODS: The article is based on a non-systematic literature search in PubMed, review of dermatology textbooks and the author's personal clinical experience. RESULTS: Hidradenitis suppurativa, also known as acne inversa, is a follicular occlusion disease that can severely reduce quality of life. Staphylococci and other pathogenic bacteria frequently colonize the lesions, but the disease is not primarily a bacterial infection. Smoking and obesity can worsen disease activity. Moderate and severe disease is usually treated with excisional surgery. Antibiotics, often tetracyclines, are indicated for mild disease and as an adjunct to surgery in more severe disease. Antibiotics, however, are not curative. New treatment options, such as TNF-alpha inhibitors and zinc gluconate should still be considered experimental. INTERPRETATION: Hidradenitis suppurativa is probably underdiagnosed. The disease is often recalcitrant to treatment. The effect of medical treatment is not supported by high quality evidence.

Gender differences in the clinical diagnosis of tuberculous lymphadenitis--a hospital-based study from Central India.

OBJECTIVE: Tuberculous lymphadenitis can be difficult to diagnose clinically, and as it is thought to be more common in females, we describe here the clinical characteristics of cervical tuberculous lymphadenitis in men and women and compare this with cytology to assess their diagnostic value. METHODS: Two hundred and nineteen patients with tuberculous lymphadenitis, aged 14 years or more, who presented with a neck mass to the Department of Pathology, Ujjain Hospital, Ujjain, India were included in the study. The presenting clinical symptoms and signs were compared between men and women and with the cytology of fine needle aspirates from the lymph nodes. RESULTS: Seventy-five percent of the patients were aged between 14 and 35 years, with a male to female ratio of 1:2.1. One or more constitutional symptoms were present in 56.6% of patients on presentation. There were more men with clinical symptoms than women. Fever was the most common manifestation in both gender groups. Fever for more than 30 days, cough, weight loss, and night sweats were significantly more common in men. On cytology, necrotic granulomas were found to be associated with constitutional symptoms. CONCLUSIONS: Constitutional symptoms were more frequently reported by men than by women and showed a correlation with necrotic granulomas on cytology.

Rapid and specific diagnosis of tuberculous pleuritis with immunohistochemistry by detecting Mycobacterium tuberculosis complex specific antigen MPT64 in patients from a HIV endemic area.

AIM: The aim of the study was to evaluate the diagnostic potential of immunohistochemistry using an antibody to the secreted mycobacterial antigen MPT64, specific for Mycobacterium tuberculosis complex organisms, on formalin-fixed biopsies from patients with pleural tuberculosis (TB) from a high TB and HIV endemic area. METHODS AND RESULTS: Pleural biopsies from 25 TB cases and 11 non-TB cases were studied. Ziehl-Neelsen staining for acid-fast bacilli and immunohistochemistry with anti-MPT64 and anti-Bacille Calmette-Guérin (BCG) antibodies was performed. Nested polymerase chain reaction (N-PCR) for IS6110 was performed for comparison. Acid-fast bacilli were detected in only 2 cases and 3 biopsies showed granulomas with caseous necrosis. Immunostaining with anti-MPT64, anti-BCG, and N-PCR were positive in 20 (80%), 12 (48%), and 16 (64%) of the cases, and 0, 3 (27%), and 2 (18%) of the non-TB controls, respectively. The diagnostic validity of immunohistochemistry was calculated by comparison with N-PCR-positive TB cases and N-PCR-negative non-TB controls. The sensitivity of immunohistochemistry with anti-MPT64 and anti-BCG were 81% and 56% respectively, and the corresponding specificities were 100% and 78%. CONCLUSIONS: Detection of the MPT64 antigen by immunohistochemistry improves the diagnosis of TB pleuritis caused by M. tuberculosis complex organisms in patients living in HIV-endemic areas with atypical histology and negative staining for acid-fast bacilli.

Correlation of Hsp70-1 and Hsp70-2 gene expression with the degree of graft-versus-host reaction in a rat skin explant model.

BACKGROUND: Graft-versus-host disease (GVHD) is the most serious complication after allogeneic hematopoietic stem-cell transplantation. A human skin explant assay has been used to predict the risk of GVHD in patients by histological grading of graft-versus-host reactions (GVHR). New molecular markers of GVHR might help to further increase the predictive value of the assay. METHODS: A rat skin explant assay has been developed to further aid in identifying potential novel molecular markers. RESULTS: In inbred rat strains GVHR were observed in skin explants co-cultured with allogeneic lymphocytes stimulated against minor or major histocompatibility antigens. The histological signs of GVHR were similar to those observed in human skin explant assays and acute GVHD lesions occurring in rats after experimental bone marrow transplantation. Heat shock protein (HSP) 70 has been shown to be expressed during GVHR. We therefore investigated the expression of the three major histocompatibility complex (MHC)-linked HSP70 genes in rat skin explants. The two major stress-inducible genes Hsp70-1 and Hsp70-2 were found to be upregulated in the allogeneic rat skin explant assays. The increase in mRNA correlated with the GVHR grade (I-IV). Interestingly, the expression of the third MHC-linked Hsp70 gene Hsp70-3 was not found to be augmented during GVHR. CONCLUSION: The observed induction of the MHC-encoded Hsp70-1 and Hsp70-2 genes might serve as new markers of GVHR and as potentially novel diagnostic tools for GVHD.

Detection of Mycobacterium tuberculosis by polymerase chain reaction with DNA eluted from aspirate smears of tuberculous lymphadenitis.

We have developed and evaluated a polymerase chain reaction (PCR) assay suitable for the detection of Mycobacterium tuberculosis DNA from fine needle aspirate smears of patients with tuberculous lymphadenitis. Air-dried fine needle aspirates of cervical lymph nodes from 98 patients with tuberculous lymphadenitis were studied for cytomorphology, detection of acid fast bacilli by Ziehl-Neelsen staining, culture and nested PCR with IS6110 for mycobacteria on DNA eluted from the dried unstained cytology smear. Twenty aspirate smears with diseases other than tuberculosis were similarly tested as controls. Mycobacterial-DNA was amplified by PCR in 84 (85%) cases and in 1 (5%) control. The mycobacteria could be detected by Ziehl-Neelsen stain and culture in 15 (15.3%) and 24 (24.4%) cases, respectively, whereas both tests were negative in controls. When results were compared with nested PCR on DNA from biopsies from the same case as the gold standard, the sensitivity, specificity, positive and negative predictive values of smear PCR were 85%, 95%, 96%, and 59%, respectively. In conclusion, PCR using dried cytology smear material is feasible and is a simple and sensitive technique for an early and specific diagnosis of M. tuberculosis complex. This is particularly useful when cytology is equivocal and can obviate the use of more invasive procedures.

Differential expression of mycobacterial antigen MPT64, apoptosis and inflammatory markers in multinucleated giant cells and epithelioid cells in granulomas caused by Mycobacterium tuberculosis.

The development of granulomas is a major histopathological feature of tuberculosis. Very little information is available concerning the physiology and functions of different cell types in the tuberculous granulomas. The aim of this study was to compare the epithelioid cells (ECs) and multinucleated giant cells (MGCs) in the granulomas caused by Mycobacterium tuberculosis complex organisms. Lymph node biopsies from 30 cases of lymphadenitis were studied for expression of the secreted mycobacterial protein MPT64, caspase 3 as a marker of apoptosis, apoptosis-related proteins (Fas Ligand, Fas and Bax) and inflammatory cytokines (interleukin-10, transforming growth factor-beta (TGF-beta), tumour necrosis factor-alpha and interferon-gamma) by immunohistochemistry. MGCs more often contained M. tuberculosis secretory antigen MPT64 (p < 0.001) and expressed more TGF-beta (p = 0.004) than ECs. The total number of apoptotic MGCs was higher than the number of apoptotic ECs (p = 0.04). Interestingly, there was a significant negative correlation between apoptosis and MPT64 expression in MGCs (r = -0.569, p = 0.003), but not in ECs, implying that the heavy antigen load would lead to inhibition of apoptosis in these cells. When compared with ECs, higher percentage of MGCs expressed Fas Ligand and Fas (p < 0.004). The role of MGCs may thus be different from surrounding ECs and these cells by virtue of higher mycobacterial antigen load, more TGF-beta and reduced apoptosis may contribute towards persistence of infection.

Immunohistochemistry using a Mycobacterium tuberculosis complex specific antibody for improved diagnosis of tuberculous lymphadenitis.

The clinical and histological criteria used to diagnose lymphadenitis caused by Mycobacterium tuberculosis complex organisms have poor specificity. Acid-fast staining and culture has low sensitivity and specificity. We report a novel method for diagnosis of tuberculosis that uses immunohistochemistry to detect the secreted mycobacterial antigen MPT64 on formalin-fixed tissue biopsies. This antigen has not been detected in non-tuberculous mycobacteria. Polymerase chain reaction (PCR) for amplification of IS6110 from DNA obtained from the biopsies was used as a gold standard. Fifty-five cases of granulomatous lymphadenitis with histologically suspected tuberculosis obtained from Norway and Tanzania were evaluated. Four known tuberculosis cases were used as positive controls, and 16 biopsies (12 foreign body granulomas and four other non-granulomatous cases) as negative controls. With immunohistochemistry, 64% (35/55) and with PCR, 60% (33/55) of granulomatous lymphadenitis cases were positive. Using PCR as the gold standard, the classical tuberculosis histology had sensitivity, specificity, positive and negative predictive values of 92, 37, 60, and 81%, respectively, and immunohistochemistry had sensitivity, specificity, positive and negative predictive values of 90, 83, 86, and 88%, respectively. The observed agreement between PCR and immunohistochemistry was 87% (kappa = 0.73). Immunohistochemistry with anti-MPT64 antiserum is a rapid, sensitive, and specific method for establishing an etiological diagnosis of tuberculosis in histologic specimens. Immunohistochemistry has the advantages over PCR of being robust and cheap, and it can easily be used in a routine laboratory.