An assessment of the impact of environmental factors on the quality of post-mortem DNA profiling.

DNA profiling has ignited public interest and consequently their expectations for the capabilities of forensic criminal and science investigations. The prospect of characterising the genetic makeup of individuals or trace samples from a wide variety of depositional and post-mortem circumstances raises the question of how reliable the methods are given the potential for prolonged exposure to variation in environmental factors, i.e. temperature, pH, UV irradiation and humidity, that are known to induce damage to DNA. Thus, it is crucial to verify the validity of the DNA profiling for characterising the genetic makeup of post-mortem tissues. This project aimed to assess the reliability of sequence and microsatellite based genotyping of tissues (muscle, hair and bone) sampled from carcasses over a two year post-mortem period. This assessment investigated the impact of environment induced DNA degradation in the local geographic region that is typical of the circumstances that confront forensic practitioners in southern Australia and to utilise rigorous controls by studying animals whose time of death and burial was known and for which we had pre-decay tissue samples available. A ‘body farm’ with 12 pig carcasses on the northern Adelaide plains, ~60km north of Adelaide, which has a typical southern Australian Mediterranean climate, i.e. cold wet winters and hot dry summers. Pigs (Sus scrofa) were used as an experimental analogue for human subjects because of the logistical and ethical reasons. The pig carcasses were allocated among three treatments: four were left on the surface, four were buried at 1m depth, and four were buried at 2 m depth. These ‘burial’ conditions mimic a range of conditions encountered typically in forensic and archaeological studies. Cortical bone samples were taken from each pig carcass at one week, one month, three months, six months, one year and two years post-mortem and muscle and hair over the same sampling period for as long as those tissue types were present. A set of PCR primers to amplify two (short and a long) fragments from the hypervariable part of the mitochondrial control region (HVRI) that is used in forensic and evolutionary studies of humans and many other mammal species were developed. Also a panel of four pig microsatellite loci with fluorescent labels to facilitate automated multiplex genotyping. These loci matched as closely as possible the core motifs and allele lengths typical of the commercially available microsatellite marker kits used in Australian forensic science labs so that our experiments were as good a model as possible of the human forensic DNA technology. In this study it was possible to retrieve samples from muscle tissue up to 90 days, hair up to one year and bone at two years post-mortem. The analyses showed that the long and short HVRI region PCR fragments were only amplifiable up to 30 days from muscle tissue and that these fragments were amplifiable up to one year from hair. In contrast, in cortical bone both PCR fragments were amplifiable up to two years. The long fragment disappeared in muscle tissue completely after 30 days and in hair after six months. However, the long fragment was present in cortical bone even at two years. Overall, there was a general trend of loss of concentration of both the long and short fragments over time. Comparisons of the HVRI nucleotide sequences among tissues sampled from individual animals showed substitution changes in muscles as early as 30 days (3 out of 6 individuals) and hair at six months (1 out of 6 individuals). In contrast, in cortical bone substitutions first appeared at 365 days (1 out of 6 individuals). The most common substitution observed in all tissues types was the C-T transition, with A-G transversions observed in two episodes and C-A transversion observed in one episode. Analyses of microsatellite genotypes in muscle tissues showed high allele peaks on chromatograms up to day seven samples. However, by three months PCR was not successful from muscle tissue. While, bone tissue had lower allele peak heights compared to the muscle tissues, alleles were detectable up to six months. Allele drop out occurred for one animal (at 2 meters) in muscle tissue at the dinucleotide locus and for another animal (kept on surface) also in muscle tissue at a tetranucleotide locus. Stuttering was observed for a single animal at dinucleotide locus in muscle tissue (buried sample 2 meter depth). No stuttering or allele drop outs were seen in the bone tissue. Overall the four loci completely disappeared after 30 days in muscle tissue and after 180 days in bone tissue. In summary, analyses showed that post-mortem DNA degradation was present in all the three tissue types (muscle, hair and bone). The types of damage identified were DNA fragmentation, nucleotide substitutions and DNA loss, which resulted in a diminished frequency of successful PCR for mitochondrial and nuclear markers over time and stuttering and allele drop out in microsatellite genotyping. In addition, two nucleotide substitutions were concentrated in ‘hotspots’ that correlate with sites of elevated mutation rate in vivo. Also the frequency of successful PCR of longer nuclear and mitochondrial PCR products declined markedly more quickly than for shorter products. These changes were first observed at much shorter post mortem intervals in muscle and much longer post mortem intervals in hair and bone tissue. When considering the carcass deposition treatments, tissues that were retrieved from buried carcases showed higher levels of DNA degradation compared to tissues retrieved from carcases left on the surface. Overall, muscle tissue is a good source for DNA analysis in immediate post mortem samples, whereas hair and bone tissue are good source for DNA analysis from older samples. When comparing the microsatellite genotyping and mtDNA analyses, mtDNA is a reliable source for DNA analysis from tissue recovered from bodies that had decayed for longer post-mortem durations such as months to years, whereas microsatellite genotyping gives reliable results for tissue from shorter post mortem intervals (hours to few days). Therefore it is recommended that when analysing mtDNA sequences, cloning and sequencing PCR products can help to identify the base pair substitutions especially for tissue retrieved from longer post mortem intervals. In addition, increasing the template DNA concentrations and "neutralising" co-extracted DNA inhibitors should be considered when dealing with tissue from longer post mortem intervals. Finally, the more stringent protocols used in ancient DNA studies should be considered when dealing with tissue with much longer post mortem intervals in forensic settings.Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 200

Asymptomatic thyroiditis presenting as pyrexia of unknown origin: a case report

Abstract Background Pyrexia of unknown origin is a difficult and challenging problem for the physician. Endocrine disorders, such as subacute thyroiditis, rarely present with pyrexia of unknown origin. Subacute thyroiditis can have a broad spectrum of clinical presentations including fever and biochemical thyrotoxicosis without overt signs or symptoms. Case presentation A previously healthy 42-year-old Sri Lankan Sinhalese man was extensively investigated for a prolonged fever of 3 weeks with high inflammatory markers. He had mild tenderness over his neck with cervical lymphadenopathy with no thyrotoxic symptoms or signs. An ultrasound scan revealed an enlarged thyroid with increased vascularity and he had suppressed thyroid-stimulating hormone with elevated free thyroxine and free triiodothyronine hormone levels. Fine-needle aspiration cytology confirmed thyroiditis. He responded well to low-dose steroids. Conclusion Subacute thyroiditis should be considered in the diagnostic workup of pyrexia of unknown origin even in the absence of overt toxic symptoms of thyroid hormone excess

Fever with lymphadenopathy – Kikuchi Fujimoto disease, a great masquerader: a case report

Abstract Background Kikuchi Fujimoto disease is an uncommon benign condition of necrotizing histiocytic lymphadenitis commonly seen in East Asian and Japanese populations. It commonly presents with fever, cervical lymphadenopathy, and elevated inflammatory markers. Diagnosis of Kikuchi Fujimoto disease is based on histopathological studies of the involved lymph nodes. The presentation of Kikuchi Fujimoto disease can mimic many sinister conditions including lymphoma. Treatment is mainly supportive provided that accurate diagnosis is made and sinister conditions like lymphoma ruled out. Case presentation We report the case of an 18-year-old Sri Lankan Moor woman who presented with fever and cervical lymphadenopathy for 1 month. She had elevated inflammatory markers with high lactate dehydrogenase and ferritin levels. She had an extensive work-up including an excision biopsy of an involved lymph node and bone marrow biopsy. Finally, a diagnosis of Kikuchi Fujimoto disease was based on histopathology of the lymph node and negative bone marrow biopsy. Conclusions Although Kikuchi Fujimoto disease is a self-limiting condition, it is a great masquerader which mimics the clinical features of many sinister conditions including tuberculosis, lymphoma, and adult-onset Still’s disease. Early recognition of the disease is of crucial importance in minimizing potentially harmful and unnecessary evaluations and treatments

Lessons learnt from managing a case of dengue hemorrhagic fever complicated with acute liver failure and acute kidney injury: a case report

Abstract Background Dengue is a common arboviral infection with a diverse spectrum of clinical manifestations. Dengue hemorrhagic fever is a more severe form of infection characterized by plasma leak and hemoconcentration. Although hepatic dysfunction is common in dengue illness, massive liver necrosis is rarely reported. Lactic acidosis is a poor prognostic marker in liver failure related to dengue. Management of acute renal injury in dengue hemorrhagic fever due to prolonged shock is challenging as the fluid reabsorption during the recovery phase expands the intravascular volume and precipitates heart failure and pulmonary edema. Case presentation We report the case of a 43-year-old Sri Lankan Sinhalese woman with serologically confirmed dengue fever presenting with evidence of plasma leakage developing acute liver failure evidenced by deranged liver functions, coagulopathy, and altered sensorium and acute kidney injury with anuria. She had elevated serum lactate levels. In addition to the “standard care,” she was managed with intravenously administered N-acetyl cysteine and blood transfusions, even in the absence of bleeding or dropping packed cell volume, targeting a higher packed cell volume anticipating a better oxygenation at tissue level. Continuous veno-venous hemodialysis was employed and continued for 138 hours removing the fluids reabsorbed during the recovery phase to prevent her from developing heart failure and pulmonary edema. She made full recovery with no sequelae. Conclusions N-acetyl cysteine and packed cell transfusion aiming at a higher packed cell volume to maintain adequate tissue perfusion during shock may be beneficial in acute liver failure due to dengue virus. The use of a continuous form of renal replacement such as continuous veno-venous hemodialysis is of paramount importance in managing fluid states in the recovery phase of dengue hemorrhagic fever in those with renal impairment. Interesting observations made in the fluid dynamics during the reabsorption phase need further studies preferably with an animal model

Fever with pancytopenia: unusual presentation of extrapulmonary tuberculosis: a case report

Abstract Background Tuberculosis is a major health problem in the developing world. Diagnosis of extrapulmonary tuberculosis is delayed because the presentation is nonspecific. Extrapulmonary tuberculosis can present with various hematological manifestations, including pancytopenia. Pancytopenia could be due to hypersplenism, maturation arrest, hemophagocytic lymphohistiocytosis, or infiltration of the bone marrow by caseating or noncaseating granulomas causing reversible or irreversible fibrosis. Case presentation We report a case of a 56-year-old Sri Lankan Sinhalese man who presented with pyrexia of known origin with significant loss of weight and loss of appetite. He had mild pallor with mild hepatosplenomegaly. He had high inflammatory markers with pancytopenia in a peripheral blood smear. His chest radiograph was unremarkable, and he had a negative Mantoux test result. A diagnosis of disseminated tuberculosis was made on the basis of caseating tuberculous granulomas in the bone marrow. Conclusions Disseminated tuberculosis remains a diagnostic challenge because the presentation is vague and nonspecific. In case of pyrexia of unknown origin with peripheral cytopenia, the possibility of disseminated tuberculosis should be considered, particularly in endemic areas. Simultaneous culture and histopathological examination of the bone marrow is important in such instances, because results of common tests such as chest radiography or Mantoux tests can be negative

Dengue hemorrhagic fever complicated with transient diabetic ketoacidosis: a case report

Abstract Background The increasing global prevalence of both dengue and diabetes may warrant closer observation for glycemic control and adapted fluid management to diminish the risk for a severe clinical presentation of dengue. Dengue illness is rarely known to precipitate diabetic ketoacidosis among diabetic patients. Both type 1 and type 2 diabetes increase the release of pro-inflammatory cytokines by various mechanisms and increase the risk of plasma leak in dengue fever. Acute pancreatitis is an atypical and rare presentation of dengue fever. We report a case of transient diabetic ketoacidosis in a previously well man which was challenging for the treating physician. Case presentation A 26-year-old previously healthy Sri Lankan Sinhalese man presented to hospital with dengue hemorrhagic fever in compensated shock. He was found to have diabetic ketoacidosis and was managed with hydration and insulin infusion. Following recovery from dengue shock, his sugars normalized and ketogenesis stopped without exogenous insulin. Conclusions Transient hyperglycemia with ketoacidosis, such as in our patient, has not been reported in the literature. Dengue virus inducing a transient pancreatitis during the viremic phase, however, is a possibility

Rare presentation of rickettsial infection as purpura fulminans: a case report

Abstract Background Purpura fulminans is an acute life-threatening disorder characterized by intravascular thrombosis and hemorrhagic infarction of the skin complicated with disseminated intravascular coagulation. It is commonly seen in acute infections following meningococcal and streptococcal infections. Few cases of purpura fulminans following rickettsial infections have been described in the literature. Case presentation We report a case of a 55-year-old Sri Lankan woman who presented to Teaching Hospital Peradeniya with a febrile illness, headache, and myalgia that progressed to an erythematous rash starting over the bilateral lover limbs and hands and that became black and necrotic with a few hemorrhagic blebs. She had normocytic anemia, platelet clumps, and monocytosis as well as a deranged clotting profile. The result of immunofluorescence antibody testing for rickettsial immunoglobulin G was strongly positive for Rickettsia conorii with a rise in titer convalescent sera, and a diagnosis of purpura fulminans following rickettsial infection was made. The patient made an excellent recovery with chloramphenicol treatment. Conclusions The treating physician should consider the rare but very treatable condition of rickettsial infection as a differential diagnosis in the etiological diagnostic workup of patients presenting with severe purpuric and hemorrhagic rash with fever

Rare Presentation of Self-Limiting Kikuchi–Fujimoto Disease in Relapsing Nature

Background. Kikuchi–Fujimoto disease (KFD) is a rare, benign, and self-limiting disease that is commonly associated with cervical lymphadenopathy and fever. The disease has a wide spectrum of clinical manifestations, and definitive diagnosis is based on the histological appearance in the excision biopsy of the lymph nodes. Recurrence of KFD is reported rarely. Case Presentation. A 56-year-old Iranian woman with a background history of thrombocytopenia presented with fever, malaise, loss of appetite, and weight loss with cervical lymphadenopathy. The excision biopsy of the cervical lymph nodes confirmed the diagnosis of KFD, and she made a full recovery with improvement of symptoms, regression of cervical lymph nodes, and normalization inflammatory markers. One year after remission, she presented with similar clinico-biochemical profile, and repeat biopsy confirmed KFD. Conclusion. Although the rate of recurrence of the disease is very low, the treating physician should consider the possibility and confirm it histologically

Confirmed Ceylon krait (Bungarus ceylonicus) envenoming in Sri Lanka resulting in neuromuscular paralysis: a case report

Abstract Background Ceylon krait (Bungarus ceylonicus) is a venomous elapid snake endemic to Sri Lanka. It inhabits shaded home gardens and forests in the wet zone of Sri Lanka and might creep into houses in the night. Despite frequent encounters with humans, reports of envenoming are very rare. Case presentation We report a case of a 26-year-old Sri Lankan Sinhalese man with confirmed Ceylon krait envenoming presenting with bilateral partial ptosis, ophthalmoplegia, facial muscle weakness, and dysphagia. Single fiber electromyography and repetitive nerve stimulation confirmed neuromuscular paralysis. He was administered polyvalent anti-venom serum immediately following admission without a prompt clinical response. Complete recovery was observed 3 days following the bite. Conclusions Because of the rarity of envenoming, precise and detailed information on the clinical manifestations following envenoming is lacking. However, Ceylon krait bite can be potentially fatal; so, treating physicians should be aware of species identification, habitat, and biting habits and clinical presentation of envenoming of Ceylon krait. This case report adds knowledge to the existing limited literature available on Ceylon krait envenoming; a rare but potentially fatal clinical entity