Feasibility and acceptability of undertaking postmortem studies for tuberculosis medical research in a low income country.

Introduction: If we are to break new ground in difficult-to-treat or difficult-to-vaccinate diseases (such as HIV, malaria, or tuberculosis), we must have a better understanding of the immune system at the site of infection in humans. For tuberculosis (TB), the initial site of infection is the lungs, but obtaining lung tissues from subjects suffering from TB has been limited to bronchoalveolar lavage (BAL) or sputum sampling, or surgical resection of diseased lung tissue. Methods: We examined the feasibility of undertaking a postmortem study for human tuberculosis research at Mulago National Referral Hospital in Kampala, Uganda. Results: Postmortem studies give us an opportunity to compare TB-involved and -uninvolved sites, for both diseased and non-diseased individuals. We report good acceptability of the next-of-kin to consent for their relative's tissue to be used for medical research; that postmortem and tissue processing can be undertaken within 8 hours following death; and that immune cells remain viable and functional up to 14 hours after death. Discussion: Postmortem procedures remain a valuable and essential tool both to establish cause of death, and to advance our medical and scientific understanding of infectious diseases.

Microglia are not protective against cryptococcal meningitis.

Microglia provide protection against a range of brain infections including bacteria, viruses and parasites, but how these glial cells respond to fungal brain infections is poorly understood. We investigated the role of microglia in the context of cryptococcal meningitis, the most common cause of fungal meningitis in humans. Using a series of transgenic- and chemical-based microglia depletion methods we found that, contrary to their protective role during other infections, loss of microglia did not affect control of Cryptococcus neoformans brain infection which was replicated with several fungal strains. At early time points post-infection, we found that microglia depletion lowered fungal brain burdens, which was related to intracellular residence of C. neoformans within microglia. Further examination of extracellular and intracellular fungal populations revealed that C. neoformans residing in microglia were protected from copper starvation, whereas extracellular yeast upregulated copper transporter CTR4. However, the degree of copper starvation did not equate to fungal survival or abundance of metals within different intracellular niches. Taken together, these data show how tissue-resident myeloid cells may influence fungal phenotype in the brain but do not provide protection against this infection, and instead may act as an early infection reservoir.

Correlation of the ultrasound thyroid imaging reporting and data system with cytology findings among patients in Uganda.

BACKGROUND: Ultrasonography is a noninvasive modality for the initial assessment of thyroid nodules. The American College of Radiology Thyroid Imaging Reporting and Data System (ACR TI-RADS) has demonstrated good performance in differentiating malignant thyroid nodules. However, the combination of ACR TI-RADS categories and cytology has not been studied extensively, in Uganda. The study aims to correlate ACR TI-RADS with cytology among patients referred for US-guided fine-needle aspiration at Mulago National Referral Hospital. METHODS: This was a hospital-based cross-sectional study that recruited 132 patients with thyroid nodules. Spearman's correlation was used to establish a relationship between TI-RADS and cytology findings. The diagnostic accuracy of TI-RADS was assessed using sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios. RESULTS: Of 132 study participants, 90% (n = 117) were females, and the mean age was 41 ± 13 years. One hundred sixty-one thyroid nodules were analyzed. More than half of the thyroid nodules (54.7%, n = 87) were solid or almost solid, 96.9% (n = 154) were shaped wider than tall, 57.2% (n = 91) had smooth margins, 83.7% (n = 133) were hyperechoic or isoechoic, and 88.7% (n = 141) had no echogenic foci. TI-RADS 3 was the most common at 42.9% (n = 69). The proportions of malignancy for TI-RADS 4 and TI-RADS 5 were 73.3% and 85.7%, respectively. The correlation between ACR TI-RADS and the Bethesda system of thyroid classification scores was r = 0.577. The sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios of ACR TI-RADS were 90.9%, 98.5%, 90%, 99.3%, 62.3, and 0.1, respectively. CONCLUSION: We found that ACR TI-RADS classification is an appropriate and noninvasive method for assessing thyroid nodules in routine practice. It can safely reduce the number of unnecessary fine-needle aspiration in a significant proportion of benign thyroid lesions. Thyroid nodules classified as TI-RADS 3 should be followed routinely. ACR TI-RADS should be standardized as the screening tool in resource-limited areas.

Casein Kinase 2 Mediates HIV- and Opioid-Induced Pathologic Phosphorylation of TAR DNA Binding Protein 43 in the Basal Ganglia.

SUMMARY STATEMENT: HIV/HIV-1 Tat and morphine independently increase pathologic phosphorylation of TAR DNA binding protein 43 in the striatum. HIV- and opioid-induced pathologic phosphorylation of TAR DNA binding protein 43 may involve enhanced CK2 activity and protein levels.

LAMP-enabled diagnosis of Kaposi's sarcoma for sub-Saharan Africa.

Kaposi's sarcoma (KS) is an endothelial cancer caused by the Kaposi's sarcoma-associated herpesvirus (KSHV) and is one of the most common cancers in sub-Saharan Africa. In limited-resource settings, traditional pathology infrastructure is often insufficient for timely diagnosis, leading to frequent diagnoses at advanced-stage disease where survival is poor. In this study, we investigate molecular diagnosis of KS performed in a point-of-care device to circumvent the limited infrastructure for traditional diagnosis. Using 506 mucocutaneous biopsies collected from patients at three HIV clinics in Uganda, we achieved 97% sensitivity, 92% specificity, and 96% accuracy compared to gold standard U.S.-based pathology. The results presented in this manuscript show that LAMP-based quantification of KSHV DNA extracted from KS-suspected biopsies has the potential to serve as a successful diagnostic for the disease and that diagnosis may be accurately achieved using a point-of-care device, reducing the barriers to obtaining KS diagnosis while increasing diagnostic accuracy.

A post-mortem analysis of tenofovir, lamivudine, efavirenz and fluconazole penetration in female genital tissues.

BACKGROUND: Optimal penetration of anti-infectives in the female genital tract (FGT) is paramount in the treatment and prevention of infectious diseases. While exposure of anti-infectives in lower FGT tissues (e.g. cervix, vagina) has been described, little data exist on upper genital tissues (e.g. ovary, uterus). METHODS: Autopsies were performed and post-mortem tissues were collected within 24 h of death for female participants with advanced HIV in Uganda (n = 27). Tenofovir, lamivudine, efavirenz and fluconazole concentrations were measured using LC-MS/MS in plasma, ovarian, uterine, cervical and vaginal tissues. Tissue penetration was calculated as tissue-to-plasma concentration ratios (TPRs). RESULTS: TPRs of tenofovir, lamivudine and fluconazole were highest in vaginal tissue (medians 1.86, 1.83 and 0.94, respectively), while the TPR of efavirenz was highest in ovarian tissue (median 0.65). With cervix as a reference compartment, vaginal TPRs were significantly higher than cervical for all four drugs; TPRs of efavirenz in uterine and ovarian compartments were also significantly higher than cervical. Most of the post-mortem FGT samples had a TPR of greater than 1 for tenofovir and lamivudine, while less than 50% had a TPR of greater than 1 for both efavirenz and fluconazole. CONCLUSIONS: Penetration of anti-infectives was not homogeneous among the FGT compartments. Approximately 70% of FGT tissues had a TPR of greater than 1 for tenofovir and lamivudine, favouring the prevention of local HIV replication and transmission in the FGT.

Pathology for Thoracic Conditions in Low- and Middle-Income Countries.

Pulmonary disease in low- and middle-income countries is highly diverse and dependent on the population, background epidemiology, environmental exposures, and smoking status. Credible evaluation of lung diseases requires skilled clinicians, imaging infrastructure, microbiology, and pathologic diagnostics, including imaging-guided cytology and biopsy. When these tools are available, improvement in patient outcomes is feasible. Pathologic diagnostics of lung lesions, including histology, immunohistochemistry, and molecular testing, are critical to properly stratify patient risk and determine exact therapies for each patient. A critical focus on research and directed interventions in lung cancer treatment specifically is needed to downstage this disease and improve patient outcome.

High-resolution disease maps for cancer control in low-resource settings: A spatial analysis of cervical cancer incidence in Kampala, Uganda.

Background: The global burden of cervical cancer is concentrated in low-and middle-income countries (LMICs), with the greatest burden in Africa. Targeting limited resources to populations with the greatest need to maximize impact is essential. The objectives of this study were to geocode cervical cancer data from a population-based cancer registry in Kampala, Uganda, to create high-resolution disease maps for cervical cancer prevention and control planning, and to share lessons learned to optimize efforts in other low-resource settings. Methods: Kampala Cancer Registry records for cervical cancer diagnoses between 2008 and 2015 were updated to include geographies of residence at diagnosis. Population data by age and sex for 2014 was obtained from the Uganda Bureau of Statistics. Indirectly age-standardized incidence ratios were calculated for sub-counties and estimated continuously across the study area using parish level data. Results: Overall, among 1873 records, 89.6% included a valid sub-county and 89.2% included a valid parish name. Maps revealed specific areas of high cervical cancer incidence in the region, with significant variation within sub-counties, highlighting the importance of high-resolution spatial detail. Conclusions: Population-based cancer registry data and geospatial mapping can be used in low-resource settings to support cancer prevention and control efforts, and to create the potential for research examining geographic factors that influence cancer outcomes. It is essential to support LMIC cancer registries to maximize the benefits from the use of limited cancer control resources.

Neuropathological Changes in Nakalanga Syndrome-A Case Report.

Nakalanga syndrome is a clinical manifestation of onchocerciasis-associated epilepsy characterized by stunting, delayed or absent secondary sexual development and skeletal deformities, and is often accompanied by epileptic seizures. The pathophysiology of Nakalanga syndrome is unknown. Here, we describe the post-mortem findings of a 17-year-old female who died with Nakalanga syndrome in northern Uganda. Macroscopic and histopathological examination of all major organs (liver, lungs, kidney and heart), including the brain and the pituitary gland, was performed. The suspected cause of death was malaria, and all major organs and pituitary gland appeared normal, except the lungs, which were edematous consistent with the malaria. Neuropathological changes include signs of neuro-inflammation (gliosis and activated microglia), which co-localized with tau-reactive neurofibrillary tangles and threads. The pathology was most abundant in the frontal cortex, thalamic and hypothalamic regions, and mesencephalon. The choroid plexus showed psammoma bodies. These findings indicate accelerated aging, probably due to repeated seizures. The neuropathological findings were similar to other persons who died with onchocerciasis-associated epilepsy. Examination of the pituitary gland did not reveal new information concerning the underlying pathophysiological mechanism of Nakalanga syndrome. Therefore, more post-mortem studies should be performed.

Rates of refusal of clinical autopsies among HIV-positive decedents and an overview of autopsies in Uganda.

Background: Human immunodeficiency virus (HIV)-related mortality remains high in sub-Saharan Africa. Clinical autopsies can provide invaluable information to help ascertain the cause of death. We aimed to determine the rate and reasons for autopsy refusal amongst families of HIV-positive decedents in Uganda. Methods: We consented the next-of-kin for post-mortem examinations among Ugandan decedents with HIV from 2017-2020 at Kiruddu National Referral Hospital. For those who refused autopsies, reasons were recorded. Results: In this analysis, 165 decedents with HIV were included from three selected wards at Kiruddu National Referral Hospital.  Autopsy was not performed in 45% of the deceased patients; the rate of autopsy refusal was 36%. The most common reasons for autopsy refusal were time constraints (30%), family satisfaction with clinical diagnosis (15%), fear of disfigurement of the remains (15%), and lack of perceived benefit (15%). By seeking consent from multiple family members and clearly explaining to them the purpose of performing the autopsy, we found a reduction in the rate of autopsy refusal among relatives of the deceased patients at this hospital compared to previous studies at the same site (36% vs. 60%). Conclusions: We found lower rates of autopsy refusal compared to previous studies at the same site. This underscores the importance of clearly explaining the purpose of autopsies as they increase active sensitization about their relevance and dispel myths related to autopsies among the general population. Good, culturally sensitive, and timely explanations to the family of the benefits of autopsy increase the rate of obtaining permission. Building capacity for performing autopsies by training more pathologists and increasing laboratory resources to decrease the turn-around-time for autopsy reports and extending these services to peripheral health facilities could improve autopsy acceptance rates.

Disseminated Coccidioidomycosis in Africa.

BACKGROUND: Coccidioidomycosis is an endemic disease in the Americas. No cases have been reported in Africa. PATIENT: A 23-year-old HIV seronegative Ugandan man was referred to Mulago National Referral Hospital in Kampala, Uganda with a 10-month history of haemoptysis and difficulty breathing, and a 6-month history of localized swellings on the extremities. He had associated weight loss and drenching sweats, but no fevers. He had taken anti-tuberculosis medicine for 2 months with no improvement. He had never travelled out of Uganda. On physical examination, he had cystic swellings and ulcerated lesions on the extremities. He had tachypnoea, crackles in the chest and mild hepatomegaly. Bronchoscopic examination showed two masses occluding the right main bronchus. Bronchoscopic biopsy showed findings consistent with coccidioidomycosis. The patient improved with antifungal treatment and was discharged. CONCLUSION: We report the first case of disseminated coccidioidomycosis with pulmonary and cutaneous manifestations in Africa. LEARNING POINTS: Coccidioidomycosis is an endemic disease in the Americas and may now be present in Africa.The patient had taken anti-tuberculosis medicine for 2 months with no improvement.Coccidioidomycosis should be considered in the differential diagnosis of tuberculosis.

Ovarian cancer: diagnostic accuracy and tumor types distribution in East Africa compared to North America.

BACKGROUND: Ovarian cancer is a spectrum of several histologically distinct tumor types that differ in etiology, response to therapy, and prognosis. In resource-limited settings, the diagnosis of ovarian cancer can be challenging. This study describes the distribution of ovarian cancer tumor types in East Africa as well as assessing the diagnostic accuracy by using contemporary methods. METHODS: Data from 210 women identified from the records with a diagnosis of ovarian cancer in a period of 15 years were included. Two tissue microarrays were constructed and stained with 20 antibodies relevant to ovarian cancer subtyping. An integrated diagnosis was reached by the review of full Haematoxylin and Eosin stained sections, with consideration of immunohistochemical results. The integrated diagnoses were compared with the original diagnoses, and the degree of agreement was evaluated by percentage and Kappa statistics. RESULTS: Though limited by selection bias, the results suggest lower rates of ovarian cancer in East Africa compared to a North American population from Alberta, Canada. There was a higher proportion of sex cord stromal tumors and germ cell tumors in the East African population. Diagnostic accuracy for main ovarian tumor type categories was substantial (Kappa 0.70), but only fair for specific ovarian carcinoma histotypes (Kappa 0.34). Poor Haematoxylin and Eosin stain was the main factor hindering the correct diagnosis, which was not related to tissue processing. CONCLUSIONS: In a resource-limited setting, where immunohistochemistry is not routinely carried out, diagnostic accuracy for the main categories of ovarian carcinoma is substantial and could be further improved by standardization of the basic Haematoxylin and Eosin stain.

Establishing the College of Pathologists of East, Central and Southern Africa - The Regional East Central and Southern Africa College of Pathology.

ISSUES: The scarcity of pathologists in sub-Saharan Africa is a well established fact that is attributable to few training programmes in the region; this is further compounded by the lack of harmonised curricula, training and exams within and without member countries. DESCRIPTION OF THE INTERVENTION: Through the Association of Pathologists of East, Central and Southern Africa, the College of Pathologists of East, Central and Southern Africa (COPECSA) was formed with the clear-cut goal of establishing a regional and internationally recognised college to support and inform good quality medical and laboratory practice by promoting leadership, mentorship and excellence in the safe practice of pathology through training, exams, accreditation, advocacy and professional development for health. LESSONS LEARNT: Since its inception in 2010, COPECSA has conferred fellowships to 120 practising pathologists in the East, Central and Southern Africa in partnership with international organisations; the college has been awarded five competitive grants and conducted several quality improvement workshops. RECOMMENDATIONS: This paper describes the journey that COPECSA has made towards standardising the practice and training of pathology in the East Central and Southern Africa region.

Mycetoma in Uganda: A neglected tropical disease.

Mycetoma is considered a neglected tropical disease globally. However, data on its burden and the associated complications in Uganda are limited. Hence we aimed to estimate its burden in Uganda. Firstly, a systematic PubMed search for all studies of any design on mycetoma in Uganda without restriction to the year of publication was conducted. A retrospective review of all the biopsy reports at the Pathology Reference Laboratory, Department of Pathology, Makerere University, Kampala, Uganda from January 1950 to September 2019 was conducted to identify any reports on mycetoma histological diagnosis. During the 70-years study period, 30 cases were identified by the literature review, with 249 additional cases identified by review of biopsy reports (total of 279 cases). The average incidence was estimated at 0.32/100,000 persons and prevalence of 8.32/100,000 persons per decade. However, there was a general decline in the number of cases detected recently. Males and the age group of 21-30 years were the most affected by mycetoma in Uganda, and only 7% of the cases were children. The highest number of cases was recorded from Kampala (n = 30) and Jinja (n = 19) districts. The majority of the cases (68%) were referred from surgical units. The foot was the most affected part of the body (72%). Ten per cent of the cases had bone involvement of which 58% required amputation. Fungi were the most common causative agents (89%) followed by Nocardia species (5%) and Actinomycetes (4%). The index of clinical suspicion of mycetoma was low (45%) with a very large differential diagnosis. Mycetoma is a relatively rare disease in Uganda, mostly caused by fungi, and there is a big gap in data and epidemiological studies. More systematic studies are warranted to define the true burden of mycetoma in Uganda.

Deep fungal infections diagnosed by histology in Uganda: a 70-year retrospective study.

Fungal infections cause substantial morbidity and mortality. However, the burden of deep fungal infections is not well described in Uganda. We aimed to estimate the burden and etiology of histologically diagnosed deep fungal infections in Uganda. We retrospectively reviewed histology reports at the Pathology Reference Laboratory, Department of Pathology, Makerere University, Kampala, Uganda from January 1950 to September 2019 to identify any reports that had a fungal infection as the diagnosis. Over the study period, 697 cases of deep fungal infections were identified with an average incidence of 0.73/100,000 persons per decade. There was a general decline in the number of cases detected. Median age of the cases was 28 years (IQR: 11-40) and majority (59%) were male. The age group of 0-10 years were the most affected. The foot was the most affected part of the body (26%). Deep mycoses identified include eumycetoma (32%), subcutaneous phycomycosis (26%), histoplasmosis (9.2%), chromoblastomycosis (4.6%), aspergillosis (3.3%), cryptococcosis (3.3%), blastomycosis (1.6%), subcutaneous mycosis (1.4%), dermatomycosis (1.3%), coccidioidomycosis (0.6%), mucormycosis (0.6%), and sporotrichosis (0.1%). Histoplasma was the commonest causative agent (9.2%) followed by Aspergillus (3.4%) and Cryptococcus (3.3%), while 81% of the fungal pathogens were not identified to genus/species level. Only 31% of the cases were diagnosed clinically as deep fungal infections. There is a substantial burden of deep fungal infections caused by multiple fungal pathogens in Uganda. There is need to build local capacity for mycology so as to improve on the index of clinical suspicion and diagnostic capabilities.

Neuroinflammation and Not Tauopathy Is a Predominant Pathological Signature of Nodding Syndrome.

Nodding syndrome (NS) is an epileptic disorder occurring in children in African onchocerciasis endemic regions. Here, we describe the pathological changes in 9 individuals from northern Uganda who died with NS (n = 5) or other forms of onchocerciasis-associated epilepsy (OAE) (n = 4). Postmortem examinations were performed and clinical information was obtained. Formalin-fixed brain samples were stained by hematoxylin and eosin and immunohistochemistry was used to stain astrocytes (GFAP), macrophages (CD68), ubiquitin, α-synuclein, p62, TDP-43, amyloid ß, and tau (AT8). The cerebellum showed atrophy and loss of Purkinje cells with hyperplasia of the Bergmann glia. Gliosis and features of past ventriculitis and/or meningitis were observed in all but 1 participant. CD68-positive macrophage clusters were observed in all cases in various degrees. Immunohistochemistry for amyloid ß, α-synuclein, or TDP-43 was negative. Mild to sparse AT8-positive neurofibrillary tangle-like structures and threads were observed in 4/5 NS and 2/4 OAE cases, preferentially in the frontal and parietal cortex, thalamic- and hypothalamic regions, mesencephalon and corpus callosum. Persons who died with NS and other forms of OAE presented similar pathological changes but no generalized tauopathy, suggesting that NS and other forms of OAE are different clinical presentations of a same disease with a common etiology.

Cerebrospinal Fluid and Brain Tissue Penetration of Tenofovir, Lamivudine, and Efavirenz in Postmortem Tissues with Cryptococcal Meningitis.

The central nervous system (CNS) is a known HIV reservoir, yet little is known about drug exposure in the brain. Our primary objective was to quantify exposure of three common antiretrovirals in brain tissue and compare exposures to plasma and cerebrospinal fluid (CSF). We also sought to identify pockets of brain most vulnerable to inadequate drug exposures and examine the role of meningitis in drug penetration into the CNS. Tenofovir, lamivudine, and efavirenz concentrations were measured using liquid chromatography and tandem mass spectrometry in plasma and CSF from 14 individuals with HIV, 7 with cryptococcal meningitis. In four individuals (three with meningitis) drug concentrations were also measured in 13 distinct brain tissue regions. In subjects with meningitis, geometric mean ratio (95% confidence interval) of tenofovir CSF to plasma was 66% (7-598%) and 14% (6-31%) in subjects without meningitis. Lamivudine CSF penetration was 100% (25-409%) in subjects with meningitis and 30% (24-37%) in subjects without meningitis. Tenofovir brain tissue concentrations were 36% (14-124%) of plasma and 49% (1-572%) of CSF. Lamivudine brain concentrations were 37% (23-64%) of plasma and 27% (1-104%) of CSF. Efavirenz brain tissue concentrations were 128% (108-179%) of plasma. Tissues collected postmortem provide a unique opportunity to assess drug distribution in tissues difficult to sample in living subjects. CSF is a poor surrogate for drug exposure throughout the CNS. Antiretrovirals differentially penetrate into the CNS and penetration may be enhanced by meningitis.

Breast diseases histologically diagnosed at a tertiary facility in Uganda (2005-2014).

BACKGROUND: The prevalence and distribution of histologically diagnosed breast disease are not well documented in low income countries, Uganda inclusive. Although the greater majority of breast lesions globally are benign, breast cancer is the most frequently diagnosed cancer all over the world. We aimed at documenting the prevalence of different breast diseases histologically diagnosed at the histopathology laboratory of the Department of Pathology of the Makerere University College of Health Sciences (MakCHS Lab) over a decade (2005-2014). We also describe the demographic characteristics of the patients in Uganda diagnosed with breast disease at the MakCHS Lab during the same period. METHODS: This was a 10 year retrospective study of histologically diagnosed breast disease between 2005 and 2014 inclusive at the MakCHS Lab. We extracted information from hard copies of all 2510 histopathology reports retrieved from archives of the Department of Pathology at the MakCHS Lab. 640 records that were either damaged beyond recognition of key details, were duplicated, were implausible or had no conclusive diagnosis made were excluded. Information to be analyzed was then entered into Epidata (version 3.1) on a password protected laptop. Data analysis was done using SPSS software (v16 for Windows × 64). RESULTS: From the 1870 patients' records eventually analyzed, breast disease was most diagnosed in female patients (97.1%). The overall mean age for breast disease diagnosis was 33 years (S.D ± 16.46) and median age 26 years (IQR: 20-43). Fibroadenoma (40.1%) was the most diagnosed breast disease overall. We noticed steadily increasing frequency of diagnosis of cancerous breast diseases over the last half of the study period. Invasive ductal carcinoma was the most diagnosed breast cancer (326 cases, 55.6%). A high female to male breast cancer ratio of 48:1 was observed. The highest regional breast cancer proportion was from the Western region of the Country. CONCLUSIONS: There is need for more research into the picture of breast disease in the country, covering various demographic characteristics of the country's population for all regions and informing about its incidence rates and prevalence and also the breast cancer risk estimate for benign breast disease.