2020 College Annual Report and Year Book 2020-21

The 2020 College Annual Report and Year Book are available for download

Annual Report 2020

Year Book 2020/21

 

Diagnosis of COVID-19

Volume 16, Issue 1, January 2021  (download full article in pdf)

Editorial note

Coronavirus disease 2019 (COVID-19) is undoubtedly the most topical subject not only in the medical field, but also for humanity globally. In this issue of the Topical Update, Dr. Derek Hung and Prof. Kwok Yung Yuen present an overview on the diagnosis of COVID-19, which underpins effective disease control. We welcome any feedback or suggestion. Please direct them to Dr. Janice Lo (e-mail: ), Education Committee, The Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists.

Dr. Derek HUNG and Prof. Kwok Yung YUEN

Resident, Department of Microbiology, Queen Mary Hospital, Hospital Authority and

Professor, Department of Microbiology, Faculty of Medicine, The University of Hong Kong

Overview

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since December 2019 has infected 54 million population in all six major continents, resulting in over 1.3 million deaths by mid-November 2020. One of the most important aspects in curbing the spread of the virus is rapid yet accurate diagnosis of infection followed by timely isolation and contact tracing. Molecular testing is now the mainstay of diagnosis, supplemented by viral antigen testing. Antibody detection aids in assessment of immunity and disease prevalence in the population. As the disease progresses, there are worldwide efforts in developing a multitude of diagnostic platforms, both in-house and commercial. Studies also endeavour to assess optimal types and timing of specimen collection to enhance diagnostic yield. In this review, we would look at some of the knowledge and practices in making a diagnosis of COVID-19.

Specimen collection

Obtaining the best specimen optimizes the possibility of getting the correct diagnosis based on clinical suspicion. Being a predominantly respiratory pathogen, obtaining respiratory specimens for viral detection remains the primary modality for making a diagnosis of acute infection by SARS-CoV-2. The viral load is highest at or soon after symptom onset, with the viral load in the upper respiratory tract peaking earlier than the lower respiratory tract. The viral load decreases in the respiratory tract at a rate of 1 log10 per week. The World Health Organization (WHO) suggests that testing upper respiratory tract specimens is adequate for early stage infection, especially asymptomatic or mild cases. The Centers for Disease Control and Prevention (CDC) recognizes nasopharyngeal swab, nasopharyngeal wash, nasal wash obtained by health care professionals; nasal mid-turbinate swab, nasal swab obtained by either health care professionals or supervised self-collection on site; and posterior oropharyngeal saliva (POS) by supervised self-collection as valid specimens. Patients with lower respiratory tract symptoms such as productive cough, shortness of breath, or suspicious radiological findings should send sputum to enhance sensitivity. Induced sputum is not recommended due to increased risk of aerosol transmission,. Among different respiratory specimens, broncho-alveolar lavage (BAL) showed the highest positive rate.

For the upper respiratory tract specimen, comparing combined nasal swab/throat swab with nasopharyngeal swab, Vlek et al showed high concordance between these two methods (kappa coefficient 0.95) despite the cycle threshold value (Ct value) obtained from nasopharyngeal swab being lower. Another study suggested nasal swab alone also has good concordance with nasopharyngeal sampling. In contrast, oropharyngeal swab alone has inferior performance. Wang et al showed the sensitivity of oropharyngeal swab was 21.1% and meta-analysis by Bwire et al suggested the positive rate is as low as 7.6% in suspected cases, comparing with 69.6% and 71.3% for nasopharyngeal swab and lower respiratory tract specimen respectively. POS is increasingly studied as an alternative respiratory tract specimen for diagnosis. Theoretically well produced POS can concentrate secretions dripping down from nasopharynx and lower respiratory secretion moved up by ciliary activity of respiratory epithelium. It can be saved by patients themselves with instructions, thus reducing discomfort in specimen collection and minimizing aerosol exposure for health care professionals. The cost of collecting POS could be 2.59-fold lower than nasopharyngeal specimen, which could be significant in resource limited setting. The concordance between POS and nasopharyngeal swab is high, especially in the first 7 days of infection, up to 96.6% positive percent agreement. The sensitivity is comparable with nasopharyngeal swab in properly collected specimen. The sensitivity does not vary much between early morning and at least 2 hours after meal, which provides a convenient option for specimen collection. CDC and Hospital Authority of Hong Kong have adopted POS as an alternative option for upper respiratory specimen collection.

Viral shedding is also found in other specimens with stool being more studied. Meta-analysis showed viral shedding was found in faecal material in 40.5% of patients. The viral shedding in stool is more prevalent in those with gastrointestinal symptoms and may last longer than the shedding in respiratory tract. Viral RNA detected in blood and urine is relatively uncommon, respectively only 1% and 0% in one study with more than 200 patients10. Even without ocular symptoms, the conjunctival secretion may contain a small amount of SARS-CoV-2 RNA in around 8% of patients, warranting appropriate infection control measure in ophthalmological assessment.

Molecular testing

Detection of nucleic acid remains the backbone of diagnosing COVID-19 for treatment and public health purposes. Reverse-transcriptase polymerase chain reaction (RT-PCR) is the most widely used technique. After transcribing the viral RNA into complementary DNA (cDNA) with reverse transcriptase, the cDNA would be amplified and detected by real-time PCR. Potential molecular targets for SARS-CoV-2 include genes encoding structural proteins, e.g. spike (S), envelop (E), helicase (hel), nucleocapsid (N-N1 and N2), transmembrane (M); and non-structural regions, e.g. RNA-dependent RNA polymerase region (RdRp), haemagglutinin-esterase (HE), and open reading frame 1a (ORF1a) and ORF1b. Most scientific institutes and commercial platforms would design primers to target more than one gene, or to target multiple loci of the same gene to enhance diagnostic sensitivity and specificity. Though N gene RNA is shown by nanopore direct RNA sequencing study to be the most abundantly expressed transcript in SARS-CoV-2 infected cells, there is no consensus on which gene confers the best diagnostic performance. Presently, one conserved and one specific target region are recommended to mitigate effect of random mutation or genetic drift while maintaining specificity25. Various regimens for testing are proposed in the literature. Corman et al recommended the Charité protocol, which was to use E gene for screening and RdRp gene for confirmation. CDC used N1 and N2 genes as their diagnostic panel. Chu et al used N gene as screening test and ORF1b as confirmatory assay because the screening N gene assay is 10 times more sensitive than ORF1b. As an alternative confirmatory assay, Chan et al developed a real-time RT-PCR assay locally, targeting RdRp/Hel. This COVID-19-RdRp/Hel assay demonstrated significantly higher sensitivity and specificity for the detection of SARS-CoV-2 RNA than the RdRp-P2 assay in clinical evaluation.

Multiple commercial platforms were developed for molecular SARS-CoV-2 diagnosis for their high throughput, rapid turnaround time and ease of use with automation. Examples are Roche Cobas 6800/8800 system (targets ORF1a and E genes) and Abbott Alinity m SARS-CoV-2 assay (targets RdRp and N genes), where sample preparation, genetic material extraction, target amplification and result reporting are automated inside the system. Molecular point-of-care testing (POCT) refers to diagnostic platform that is portable (often desktop-size), requires minimal sample preparation steps and can provide reliable molecular results within 2 hours. POCT like Cepheid GeneXpert (Xpert Xpress SARS-CoV-2 assay, targets E and N2 genes) enables rapid testing near the site of collection in areas with little laboratory support. Fewer steps in manipulation reduce risk of cross contamination and laboratory error in processing. Many evaluation studies have been published to compare the performance of these commercial platforms against in-house diagnostic tests and for head-to-head comparison between platforms. For example, Cobas system is shown to have high diagnostic agreement with in-house molecular assays,, as well as with other commercial platforms such as Hologic Panther Fusion system and Cepheid GeneXpert. Cepheid GeneXpert reaches an agreement of 100 % compared to three in-house RT-PCRs in a multicentre evaluation in the Netherlands. Among commercial platforms there might be minor discordance between assays at very high Ct values and the viral load of clinical samples used in evaluative studies should be noted in particular.

Another molecular technique is reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) test. Using multiple primers for the genetic target, RT-LAMP amplified nucleic acid by strand displacement in an isothermal condition of around 60- 65oC. It allows synthesis of large amount of genetic material up to 106 to 109 copies of target DNA within 30-60 minutes2. Without the need of thermal cycler as in RT-PCR, RT-LAMP facilitates development of rapid molecular POCT and has an expanding market in commercial diagnostic platform. On the down side, since multiple primers over a relatively small genetic region are needed for amplification, there are constraints in properly designing the primers. Abbott ID NOW is a commercial POCT platform using RT-LAMP, allowing real time detection of SARS-CoV-2 within 15 minutes targeting RdRp gene. Evaluation of ID NOW against other RT-PCR based platforms appears suboptimal in terms of diagnostic sensitivity. Compared to Cobas, ID NOW achieved only 73.9% positive agreement while GeneXpert achieved 98.9% positive agreement. In samples with Ct values greater than 30, positive agreement was 34.3% for ID Now and 97.1% for GeneXpert. A lower sensitivity of ID NOW over GeneXpert was also reported in another evaluation by Basu et al. In contrary, good diagnostic utility has been demonstrated in many other centres including Hong Kong that have designed their own RT-LAMP for COVID-19. Chow et al reported sensitivity of 95% at 60 minutes using RT-LAMP targeting a region across ORF3a/E gene as compared to RT-PCR. Lu et al achieved concordance rate of 93% against RT-PCR using in-house E gene RT-LAMP assay.

In order to improve the diagnostic sensitivity of molecular assays, clustered regularly interspaced short palindromic repeat (CRISPR)-based technology has been employed by coupling with Cas enzyme. The enzyme would be directed to the target DNA/RNA by a guide RNA complementary to the target sequence. Once bound, the collateral nuclease activity of the Cas enzyme would cleave surrounding reporter fluorophore and lead to signal amplification. DETECTR technology uses Cas12a enzyme to bind target DNA; while SHERLOCK technology uses Cas13a enzymes to bind target RNA. This technology can be incorporated in molecular techniques especially RT-LAMP to enhance the sensitivity and to lower the detection limit.

Next generation sequencing (NGS) enables sequencing of the entire genome in a relatively short period of time. Sharing of genetic data facilitates tracking of disease spread, understanding of disease transmission route, monitoring viral genome evolution and detecting emergence of mutation that may escape detection or enhance virulence. The cost and infrastructure required of NGS and the need of bioinformatics expertise limit its use to larger hospital and research centres.

Antigen detection

Like other respiratory viruses such as influenza and respiratory syncytial virus (RSV), direct antigen detection from respiratory specimen especially nasopharyngeal sample is another way of making a diagnosis of COVID-19. N protein was found previously to be the predominant structural protein released in large amount in nasopharyngeal aspirate during infection of SARS-CoV, and the same phenomenon is also shown in SARS-CoV-2 where the abundantly expressed N protein is widely used as an antigen detection target in COVID-19. Detection is achieved by capturing viral antigen in clinical specimens by monoclonal antibodies or monospecific polyclonal antibody fixed on a membrane, usually indicated by colour change of the strip in colorimetric lateral flow immunoassay. The assay can be delivered as POCT in an office setting since no complex laboratory support is required and the result can be available within a short period of time, usually <30 minutes. The major setback is the suboptimal sensitivity as compared to molecular diagnosis especially in samples with high Ct values. Evaluation by Lambert-Niclot et al using COVID-19 Ag Respi-Strip CORIS, a nitrocellulose membrane technology with colloidal gold nanoparticles sensitized with monoclonal antibodies directed against SARS-CoV-2 nucleoprotein (NP) antigens, showed sensitivity of only 50% when compared against multiple RT-PCR platforms. For samples with Ct value <25, the sensitivity is higher at 82.2%. In a local evaluation using Biocredit COVID-19 Ag test, the antigen test is 105 fold less sensitive than RT-PCR and it yielded a positive result in 45.7% RT-PCR positive combined nasopharyngeal swab/throat swab specimens only. There are attempts to improve sensitivity of rapid antigen assay. Porte et al evaluated an immune-chromatographic antigen assay using fluorescence signal showing sensitivity of 93% but the Ct value of the sample included in this study is relatively low with mean of 20. Other approaches by concentrating the antigen in specimens before testing with monoclonal antibodies targeting multiple different epitopes of the antigen were also reported. Based on a meta-analysis by Dinnes et al, the average sensitivity is around 56.2% for antigen assay with a high average specificity of 99.5%. Further refinement in antigen detection employs the detection of the change in bioelectric property by antigen binding to the antibody coated membrane. In Seo et al, anti-S antibody binds to SARS-CoV-2 particles to fabricate graphene-based field-effect-transistors (FET) biosensors and can respond down to 16 pfu/mL of virus. One challenge to this advance is the high background noise which can reduce sensitivity of detection. Overall, rapid antigen detection serves only an adjunctive role to molecular assay in making a diagnosis especially in outbreak situation where prevalence is high and molecular assay is not available. WHO has issued interim guidance of use of rapid antigen immunoassays.

Antibody detection

While antibody testing may not be useful in acute setting for COVID-19, it helps establish retrospective diagnosis, predict immunity and understand seroprevalence in a defined community. Commonly employed techniques are lateral flow immunoassay, chemiluminescent immunoassay, immunofluorescent assay, and enzyme-linked immunosorbent assay (ELISA). Median seroconversion times following symptom onset are 11 days for total antibodies, 12 days and 14 days for IgM and IgG respectively. Detection rate for IgM ranges from 11-71% in the first 7 days of infection, 36-87% between 8-14 days, and 56-97% after 14 days. For IgG, it ranges from 4-57% in first 7 days, 54-88% between 8-14 days, and 91-100% after 14 days. For SARS-CoV-2, there does not seem to have significant time difference between IgM and IgG response. IgM peaked at around 3 weeks after symptom onset and fell to baseline level at around day 36. The duration of IgG seropositivity remains unknown and longer longitudinal studies are required. Study from Iceland involving over 1200 confirmed patients showed no evidence of antiviral antibody decline by 4 months after diagnosis; and most other studies showed persistently detectable antibodies by 2-3 months after infection60. On the other hand, there are some evidences that the IgG level may decline faster in mild and asymptomatic61 COVID-19 cases.

S protein is an important antigen for neutralizing antibody production. The S1 domain is responsible for receptor binding while the S2 domain is responsible for fusion. The receptor binding domain (RBD) is located at S1. NP, which is a structural component of the helical nucleocapsid, also appears to be an important antigen for the development of serological assays to detect COVID-19. Earlier in the pandemic, using sera collected more than 14 days after symptom onset from 16 patients, To et al showed rates of seropositivity were 94% for anti-NP IgG, 88% for anti-NP IgM, 100% for anti-RBD IgG, and 94% for anti-RBD IgM. Another study compares sensitivity and specificity in testing anti-S and anti-NP IgG for evidence of immunity across multiple platforms, which shows they are comparable by day 37 after infection though seroconversion of anti-NP IgG may precede anti-S IgG by around 2 days (day 9-10 v day 11-12). Caruana et al observed that the decline of anti-NP antibody may be faster than anti-S and thus could be less sensitive longer after infection. Also titre of anti-S antibody may better reflect protection against reinfection67. Multiple commercial platforms were developed for high-throughput antibody testing in clinical laboratory. Automatic platforms such as Abbott SARS-CoV-2 IgG, which is a chemiluminescent micro-particle immunoassay, are also used in public hospital of Hong Kong for a shorter turnaround time.

Neutralization antibody test is important in assessing in vitro the functional capacity of the humoral response of COVID-19 patients to prevent reinfection by the virus. Traditional neutralization assay such as microneutralization and plaque reduction assay require manipulation of live virus and necessitate biosafety level 3 laboratories. As a result, pseudovirus neutralization assay has been developed. Vesicular stomatitis virus (VSV) expressing S protein of SARS-CoV-2, containing the RBD, is used so that the assay can be performed in biosafety level 2 facilities. SARS-CoV-2 neutralizing antibody starts to rise at around 7-10 days after symptom onset and the median peak time is 33 days after symptom onset. The neutralization titres then decline in 93% of the patients and by a median level of 35% over 3 months. Patients with more severe disease requiring ICU admission have accelerated and augmented neutralizing antibody response compared with non-ICU cases. In non-severe cases who have low peak neutralizing antibody titre, neutralizing antibody level might return to baseline within 2 months. Another clinical use of neutralization assay would be to confirm potentially false positive SARS-CoV-2 serology result. Three children with Kawasaki disease without symptoms or epidemiological linkage to COVID-19 were tested positive to anti-RBD and anti-NP antibodies by a microparticle-based immunoassay but were confirmed negative by microneutralization test.

Studies have shown there are serological cross-reactivity between SARS-CoV-2 and SARS-CoV. Testing sera taken from COVID-19 patients by ELISA, cross-reactivity is seen against S protein and RBD of SARS-CoV, though the intensity of cross-reaction against RBD is weaker than S protein. For the full length S protein, the amino acid sequence homology between SARS-CoV-2 and SARS-CoV is around 75%. The homology between them for RBD which is located in S1 domain is around 74%. For the receptor binding motif (RBM) of the RBD where the virus directly binds to angiotensin-converting enzyme 2 (ACE2), the homology is only 50%. The degree of amino acid homology explains the difference in the level of cross-reaction between them on ELISA. Chia et al showed even more significant cross-reactivity between SARS-CoV-2 and SARS-CoV antibody against NP by Luminex assay than antibody against S1 or RBD as the homology between the NP of these 2 viruses is around 90%. Despite some cross-reaction between antibodies against RBD on ELISA, there does not seem to have significant cross neutralization effect73. Only 1 out of 15 COVID-19 sera showed cross neutralization with SARS-CoV at very low titre. Overall the effect of cross-protection in vaccination and whether antibody-dependent enhancement effect would be seen between these 2 closely related viruses remains unknown.

Cross-reactivity against other human coronaviruses in SARS-CoV-2 infection has been investigated in a few trials. In a study by Wölfel et al, using immunofluorescence assay against recombinant S protein, cross-reactivity of SARS-CoV-2 sera is found against human coronaviruses OC43, NL63, HKU1 and 229E on comparing the titres between admission and convalescence samples, especially HKU1 and OC43 which are both betacoronavirus. In Shrock et al, deep serological profiling of sera from SARS-CoV-2 patients and pre-COVID sera are performed. Antibodies against S and NP are the most specific assay to differentiate SARS-CoV-2 and pre-COVID sera. Those with dramatic increase in anti-S antibody after COVID-19 infection also have increase in the intensity of cross-reactivity against other human coronaviruses, especially over more homologous regions of the S protein e.g. at residue 811-830 and 1144-1163. It could be novel antibodies of SARS-CoV-2 that cross-react or boost the anamnestic response against SARS-CoV-2 infection due to existing memory towards other human coronaviruses from past exposure. Moreover, pre-COVID sera also show some cross-reaction towards the homologous region of SARS-CoV-2 S protein and ORF1 in the same study.

Viral culture

Demonstration of live SARS-CoV-2 in cell culture requires biosafety level 3 facilities and are not routinely performed in most of the clinical laboratories. However, live virus isolation is still important for some diagnostic and research purposes so as to determine whether the amount of virus present is infectious to others, to evaluate therapeutic efficacy of potential antiviral compound, to develop viral neutralization assay for testing convalescent sera, to provide positive control for molecular assay development, and to develop vaccine strains. The host cell receptor for SARS-CoV-2 is ACE2. Non-human cell lines such as Vero E6 and Vero CCL-61 which have abundant ACE2 expression are commonly used for isolation. Cytopathic effect is seen by 3 days after inoculation. SARS-CoV-2 also grows in human continuous cell lines such as Calu3 (pulmonary cell line), Caco2 (intestinal cell line), Huh7 (hepatic cell line), and 293T (renal cell line). It grows modestly on U251 (neuronal cell line) which is not seen in SARS-CoV81. Confirmation of SARS-CoV-2 replication in the cell line can be done by molecular testing or immunostaining techniques. Cell lines can be engineered to express a transmembrane serine protease TMPRSS2 for priming of S protein and to facilitate the entry of SARS-CoV-2 into host cell. Organoid systems such as bat and human intestinal organoids are susceptible to SARS-CoV-2 and are developed to better study tissue tropism, the dynamics of infection and testing of therapeutic targets.

Radiological diagnosis and artificial intelligence

There are no pathognomonic radiological features on chest imaging for COVID-19 and the disease should not be ruled in or ruled out based on imaging alone. However, presence of suggestive imaging features can prompt further investigations in suspicious cases, such as lower respiratory tract viral testing for confirmation. Reports in literature have suggested that in some patients, radiological findings may precede the detection of SARS-CoV-2 in clinical specimen,. Chest X-ray (CXR) is a less sensitive modality than computed tomography of the thorax (CT thorax) with a reported CXR sensitivity of 69%85. As in other viral pneumonia, COVID-19 typically presents with multifocal air-space disease, especially with a bilateral lower lung distribution. More specific to COVID-19, it tends to have peripheral lung involvement, seen in 58% of CXR in one study. CT thorax has a higher sensitivity than CXR, quoted at around 60-98%. CT thorax often demonstrates the typical findings of peripheral bilateral ground glass opacities (GGO) with or without consolidation or ‘crazy-paving pattern’. Sometimes the GGO would arrange in a rounded pattern. Isolated lobar or segmental consolidation without GGO, centrilobular shadows, cavitory changes, lymphadenopathy and pleural effusions are rare86. As the disease advances, the opacities might coalesce, affecting central and bilateral upper lobes and may manifest as ‘white lung’ with diffuse infiltrate. The abnormalities usually peak by 2 weeks after symptom onset, replaced by scar tissue with recovery. In the COVID-19 pandemic, artificial intelligence (AI) programme is increasingly studied for screening abnormal radiological result which would be particularly useful for mass screening strategy in outbreak situation. The performance of AI is dependent on the radiological imaging algorithm being fed into the system for deep learning process. So far the result of this research has been promising with reported area under receiver operating characteristic curves greater than 0.9,. However, there are still lots of technical and ethical issue to resolve which include dataset bias, data privacy, and the distribution of ultimate accountability of result.

Detection of host inflammatory reaction

In COVID-19, there are studies to diagnose and predict severe diseases by the host inflammatory response. Apart from direct viral damage, uncontrolled cytokine storm triggered by the virus leads to tissue damage and multiorgan failure. Mean interleukin-6 (IL-6) concentration in serum was found to be 2.9 fold higher in patients with complicated COVID-19 disease than non-complicated disease. It became one of the markers clinicians could use to predict progression into severe disease. Roche Elecsys IL-6 immunoassay received FDA Emergency Use Authorization to help identify patients at high risk of requiring intubation with mechanical ventilation. Molecules targeting IL-6 such as tocilizumab are also studied as therapeutic to prevent disease progress by blocking the inflammatory pathway. It does not show efficacy in preventing intubation or death in moderately ill hospitalized patients in the BACC Bay trial. Elevated CRP is associated with worse outcome, as well as elevated IL-10 which may be related to compensatory anti-inflammatory response and secondary infections. Haematologically, severe disease is associated with higher absolute neutrophil count, D-dimer and LDH but lower absolute lymphocyte101 and platelet count.

Conclusion

Global COVID-19 pandemic stimulates global effort in development of rapid yet accurate diagnostic techniques. Diagnosis is often limited by the low level of viral particles in the specimen and the subtle clinical features in early infection. Though traditional methods like RT-PCR are still the mainstay, we see expanding endeavours to strive for higher speed and lower limit of detection at an earlier time. Molecular techniques such as RT-LAMP, CRISPR/Cas, biosensor technology in antigen detection, AI operating system for image interpretation are pushing the diagnostic ability to the limit. Despite these scientific advances, there are still a lot of gaps to fill especially in understanding the nature and duration of humoral immunity response and its protection against re-infection. All these require continuous global cooperation and information exchange to make them possible.

Trainee Presentation Session 2020

Venue: Pao Yue Kong Auditorium, Hong Kong Academy of Medicine Jockey Club Building

Time: 28 Nov 2020 13:45 - 18:00

Detail schedule in pdf

Introduction

Dr. YAU Tsz Wai Derek

13:45 - 13:55

Platform Oral Presentation

13:55 - 15:55

Dr. FUNG Ka Kin Ben

Risk Factors and Antibiotic Susceptibility to Cephalosporin/Beta-Lactamse Inhibitor Combinations for Multidrug Resistant Pseudomonas aeruginosa

Dr. LEE Lok Hang Alfred

Diagnostic Stewardship Program for Urine Culture - the Impact on Antimicrobial Prescription in a Multi-Centre Cohort

Dr. LI Jing Xi Joshua

Cytomorphological Parameters in Correlation of Growth Pattern and Prediction of Grading of Ductal Carcinoma-in-situ of the Breast by Fine-Needle Aspiration

Dr. LIAO Jiawei Gary

"Pauci-Hemosiderotic" Fibrolipomatous Tumor: A Mimicker of Various Lipomatous Lesions

Dr. YUEN Ka Wan Karen

Thyroid Adenoma of Probable Ultimobranchial Body Origin: A Case Report

Dr.YEUNG Chun Fai Maximus

Importance of Alternative Promoter Usages in Cancers Revealed by Pan-Cancer Transcriptome Analysis

Dr. LING Tsz Ki Jacky

NT-proBNP for the Diagnosis and Monitoring in the First Local Case of Kawasaki-Like Multisystem Inflammatory Syndrome (MIS) Associated with COVID-19 Infection

Dr. HO Man Kit Mark

Collagenofibrotic Glomerulopathy: A Case Report

Poster Presentation

15:55 - 17:25

Dr. CHOW Kin Yi CHristina

Evaluation of BioFire FilmArray Respiratory Panel for Detection of Viruses

Download poster in pdf

Dr. NG King Man Kevin

Molecular Detection of Mycoplasma genitalium in Endocervical Swabs and Associated Macrolide and Fluoroquinolone Resistance in Hong Kong

Download poster in pdf

Dr. LI Xiu Ling Vivian

Data Review on a New Flow Cytometry Panel for the Diagnosis of Low Grade B-Cell Lymphoproliferative Disease

Download poster in pdf

break

Dr. KWOK Lok Ming Angie

Adenofibromatous Solitary Fibrous Tumor: a New Morphologic Variant Occurring in the Sinonasal Tract

Download poster in pdf

Dr. LUI Yin Wing

Dr. Cytology May be the First Clue to a Perforated Oesophagus: Stay Vigilant Even When it is 'Negative for Malignant Cells'

Download poster in pdf

Dr. HAU Man Nga

Oncocytic Variant of Secretory Carcinoma: Expanding the Morphological Spectrum of Secretory Carcinoma in Salivary Gland - a Case Report

Download poster in pdf

Dr. FONG Tsun Nestor

An Autopsy Case Report of Intravascular Large B-Cell Lymphoma with Initial Neurologic Presentation

Download poster in pdf

Dr. SO Yik Ka

Myxoid Spindle Cell Sarcoma with LMNA-NTRK Fusion: Expanding the Morphologic Spectrum of NTRK- Rearranged Tumors

Download poster in pdf

Dr. TSSNG Cheuk Ho Jason

Glomus Tumor of Sella Turcica with Synaptophysin Expression Mimicking Pituitary Adenoma

Download poster in pdf

Dr. NG Ka Man Joanna

Trichoblastic Carcinosarcoma Arising from the Vagina: A Case Report with Comprehensive Immunophenotypic Analysis

Download poster in pdf

Dr. CHAN Cheong Kin Ronald

Digitized Library of Pathology Specimens

Download poster in pdf

Dr. LAI Shun Wun Billy

The Clinical Significance of Neuroendocrine Features in Invasive Breast Carcinomas

Download poster in pdf

Dr. CHAN Angela Zaneta

Case Report: Malignant Sinonasal Solitary Fibrous Tumour with BCOR Immunoexpression

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Dr. WONG Wing Fung Wilson

A Rare Case of Constitutional Mismatch Repair Deficiency Syndrome in a 2-year-old Girl with Medulloblastoma and Signs of Neurofibromatosis Type 1

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Dr. CHANG Lik Chun John

A Sarcoma with MXD4-NUTM1 gene fusion - a Case Report

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Dr. CHEUNG Ka Chun Kevin

A Case Report on Monomorphic Epitheliotropic Intestinal T- Cell Lymphoma (MEITL)

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Judges Meeting and Words from Judges

17:25 - 17:40

Prize and Certificates Presentation

17:40 - 18:00

Results of College Exam 2020 (Council Meeting 16 October 2020)

 

Fellowship Assessment:

 

EXAM NO.	RESULT
E20221	FAIL

 

Membership Examination:

 

EXAM NO.	RESULT
E20117	PASS

 

 

 

Results of College Exam 2020 (Council Meeting 29 September 2020)

 

Fellowship Assessment:

 

EXAM NO.	RESULT
E20201	PASS
E20202	PASS
E20203	FAIL
E20204	PASS
E20205	PASS
E20206	FAIL
E20207	PASS
E20208	FAIL
E20209	PASS
E20210	PASS
E20211	PASS
E20212	PASS
E20213	PASS
E20214	PASS
E20215	PASS
E20216	FAIL
E20217	FAIL
E20218	PASS
E20219	FAIL
E20220	FAIL
E20222	PASS

 

Membership Examination:

 

EXAM NO.	RESULT
E20101	FAIL
E20102	PASS
E20103	PASS
E20104	PASS
E20105	PASS
E20106	PASS
E20107	PASS
E20108	PASS
E20109	PASS
E20110	PASS
E20111	FAIL
E20112	PASS
E20113	PASS
E20114	PASS
E20115	PASS
E20116	FAIL

 

 

 

Volume 29, Issue 1 (click here to download the full pdf version)

Message from the President

In this issue of the College Newsletter, I am saddened to bring the news to all Members and Fellows that one of our Honorary Fellows, Professor WU Bing Quan passed away peacefully on 21 June 2020. Professor WU was a pioneer in the development of Pathology in China. He was one of the first group of scientists to be sent to the United States for training where he met Professor Joseph CK LEE, Former Dean and current Honorary Clinical Professor of The Chinese University of Hong Kong. They became good friends during their time together in the United States and hence Professor WU became associated with our College. Professor LEE has kindly written an obituary for Professor WU and our College also sends its condolences to Professor WU’s family.

The 15th Trainee Presentation Session, the 28th Annual General Meeting and Conferment Ceremony, the 28th T.B. TEOH Foundation Lecture and Dinner were successfully held on 23rd November 2019 when the social unrest was quietening down. All attending Members, Fellows and their families enjoyed a wonderful evening. Professor KWONG Yok Lam, our 28th T.B. TEOH Foundation Lecturer, delivered a lecture entitled “An amazing journey from micro to macro and back” with a human touch of his interest in reading science fiction. The winner of the 15th Trainee Presentation Session was Dr. CHENG Hua Tse Timothy who presented his work on ‘Comprehensive characterization and resolution of eltrombopag interference on bilirubin measurement’.

The Academies of Medicine for Singapore and Malaysia organised the 53rd Singapore Malaysia Congress of Medicine in Singapore in January 2020. I attended the Congress as well as the Joint Academy Council Meeting on behalf of the College. In the Congress Symposium, a fascinating talk on how big data and social media affects the hierarchical relationship and massive manipulation was presented by Mr. George YEO, the Former Cabinet Minister of Singapore.

The Topical Update in this issue was on “Liver Injury associated with Immune Checkpoint Inhibitors – An Update on Clinicopathological Features“, by Dr. LO Cheuk Lam Regina of The University of Hong Kong. In response to the recent COVID-19 pandemic, Professor LAI Koon Chi Christopher of The Chinese Unive rsity of Hong Kong, and Professor Siddharth SRIDHAR of The University of Hong Kong, jointly published an article on “Coronavirus Diversity and Infection through Host Receptor” in Ming Pao on 7th April 2020. On 29th April 2020, our College also released a press statement to the general public on the “Use of Over-the-Counter COVID-19 Test Kits”, explaining the risks associated with false positives and false negatives of the point-of-care test kits based on IgG and IgM antibodies. Apart from numerous reports in various newspapers, our College also noted about 40,000 ‘shares’ on social media.

Different parts of College Examinations have been or will be conducted from July to September 2020. It is challenging to conduct examinations in a pandemic situation, but with the approval from the Academy Council, Colleges are allowed to conduct examinations using telecommunication technology where appropriate and possible. I would like to thank our External Examiners, Chief Examiners, Deputy Chief Examiner and Local Examiners in advance for their tremendous effort in making it possible for the College Examinations to be conducted as scheduled.

Finally, allow me to wish you all ‘good health’ going forward !

Dr. CHAN Ho Ming
President

July 2020

Drowning: A Rational Approach to its Diagnosis

Volume 15, Issue 2, July 2020  (download full article in pdf)

Editorial note:

Drowning often presents in various scenarios depending on the circumstances. This Topical Update provides a proper approach to the diagnosis. We welcome any feedback or suggestions. Please direct them to Dr. FOO Ka-chung of Education Committee, the Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists.

Dr. FOO Ka-chung

Specialty Coordinator (Forensic Pathology), Education Committee

The Hong Kong College of Pathologists

Introduction

Drowning is referred as “death occurring within 24 hours of a submersion incident”. Definition by World Health Organization is "the process of experiencing respiratory impairment from submersion or immersion in liquid ". [1,2] It is a form of asphyxia with a distinct pathophysiology and mechanism of death. It is also a diagnosis by exclusion, and therefore every piece of information should be regarded as crucial. Pathologists are obliged to work under Coroner’s jurisdiction in interviewing the next-of-kin (if available), reviewing antemortem medical records and preliminary findings provided by investigating officer, performing an autopsy as directed and compiling reports capable of addressing anticipated issues.

As Forensic Pathologists mostly deal with sudden and unexpected deaths, cases of drowning with unsalvageable outcome are often encountered. Hospital Pathologists, on the other hand, are dealing with patients presenting a clinical picture in which death eventually occurred after vigorous cardiopulmonary resuscitation followed by development of various systemic complications, e.g. pneumonia, acute respiratory distress syndrome, multi-organ failure, disseminated intravascular coagulopathy, hypoxic-ischaemic encephalopathy etc.

Manner of Death

Information derived from the Coroner’s Report [3] and the Centre for Health Protection [4] suggested that majority of cases were accidental or suicidal in nature. Only a few were homicides. However, it should be remembered that a body found immersed in water does not necessarily imply a diagnosis of drowning. Nor its manner be automatically presumed basing on the prevalent trend. The deceased can die of natural conditions preceding or during submersion as well as unnatural elements that contributed to the drowning process, explaining the failure of extrication from water versus genuine lethal trauma before or while in water. [5,6]

Pathophysiology

The mechanism of death is complex involving changes to viscera, biochemical alterations and also at a cellular level. The culprit is the medium imposing hydrostatic and osmotic effect to the lungs. [7] The acute change in intravascular volume with electrolyte imbalance is the consequence. Several stages of drowning present in response to the rising levels of carbon dioxide and decreasing oxygen tension in blood. Voluntary breath holding for about 1 to 2 minutes is followed by a stage of involuntary urge to breath with aspiration of fluid for about 1 to 3 minutes. Tonic-clonic seizures, together with some degree of respiratory activity, will occur in the next 1.5 minutes with eventual involuntary breath holding and terminal gasping before cessation of cardiac activity. [1]

It has even been mentioned that only a few inches of water is sufficient to drown a person, as in the case of sudden incapacitation by onset of acute illness while standing close to a washbasin or bucket. [5,6] It was reported that about 1 mL/kg to 11 mL/kg of water aspirated can result in drowning. [7]

A rare entity underdiagnosed in daily practice, or seldom made by pathologists, is referred as "dry drowning" or "immersion syndrome", with negative autopsy findings of typically drowned lungs due to severe laryngeal spasm, therefore preventing further intake while stimulating the sensitive receptors and subsequently triggering cardio-inhibitory reflexes (Ebbecke reflex, Aschner reflex, Hering reflex). [1,5,6,7,8]

Diagnosis of Drowning

The possibility of drowning should always be considered when a deceased was recovered from a body of fluid or the head was found submerged inside a medium of fluid. The deceased could be found near a body of fluid where it could be washed onto the rocky shore, beach, or riverbank. Domestic environments such as bathtub also house this potential danger affecting all walks of life, especially for those who have chronic illness with sudden unexpected precipitation or the young. While the diagnosis of drowning could be straightforward one, such as a witnessed fall into water with subsequent submersion, it can be extremely difficult when critical information derived from the case is absent or inconclusive. Challenging scenarios can appear with unclear circumstances preventing proper formulation of the manner of death. Moreover, while findings derived from postmortem and ancillary investigations may collaborate with the diagnosis, it can be equally confusing when concomitant conditions are unveiled.

Presence of a natural condition which may contribute to death

Let’s consider the following case:

A 51 year-old female was found collapsed underwater in a public swimming pool of about 1.4 meters deep and was certified dead despite intensive resuscitation. There was no eye witness leading to her collapse. Autopsy revealed severe ischaemic heart disease with no evidence of acute infarction. Both lungs were congested and oedematous but frothy fluid was absent probably due to suction during resuscitation. Cause of death is labelled as drowning as the overall features were compatible with drowning.

The presence of a co-existing medical condition, be it undiagnosed or known to the deceased, has to be evaluated carefully to attribute its extent of contribution to death. A sudden precipitation into cardiac arrhythmia explained the reason why a habitual swimmer is incapacitated and eventually succumbed in the water. From the investigator’s point of view, possible legal issues regarding adequate supervision of the swimming environment may be raised which could lead to possible lawsuit and inquest. As such, the pathologist should be ready to address the extent of contribution of medical condition to the tragic outcome.

Medical background of the deceased has to be thoroughly reviewed including conditions such as asthma, epilepsy, cardiovascular conditions (e.g. Long QT syndrome type 1). Psychiatric history including substance abuse should also be elicited.

Presence of trauma which may be related to death

Another case is presented here:

An 87 year-old female was found floating off shore from a pier. She was known to be a habitual swimmer and there was no known chronic illness. There were multiple lacerations on chest and right upper limb. The thoracic cavity was breached and right lung had collapsed. Tinge of frothy fluid was noticed briefly by paramedics before transportation to mortuary. Autopsy revealed severe coronary stenosis and the left lung was mildly hyperinflated.

The presence of trauma may or may not be related to death as injuries inflicted can be produced ante-mortem or post-mortem. Assessment for vital reactions at the wound margin may be helpful to determine its nature.

All forms of injuries must be explained correlating inanimate objects in the environment. Sliding abrasions may be inflicted upon skidding down a slope while blunt force injuries may be a genuine assault. Self-inflicted injuries may occur in suicide as a back-up technique, for example, a stab to the chest or incised wound on the neck, yet it might at times mimic a homicide.

Dragging effects as a result of contact with river bed or ocean floor propelled by sea waves or tidal current is not uncommon and should be interpreted in light of such movement in water. Abrasions or lacerations may be found on forehead, dorsum, knees and toes. In addition, aquatic animal activity, such as crustaceans, will produce bites and nipping around orifices. The body, on the other hand, may be struck by watercraft or its parts including the propeller, predominantly located below the waist and over the extremities while the subject is maintaining a vertical position. It should be located posteriorly upon floating postmortem. [5,6] At times injuries could be severe enough to hinder the diagnosis by producing serious disruption of the viscera. The presence of postmortem mutilation further complicate the diagnosis, let alone in jeopardizing the facial features and hindering identity as often encountered in mass fatalities.

Healthy adults who can swim rarely drown unless there is an intervening reason such as superimposed injury, fatigue or dangerous environment. The level of fitness, history of risk-taking behaviour, pre-swim activities, swimming ability and experience should be explored.

In the present case, the cause of death is labelled as drowning and suggested an accidental manner with sudden precipitation of undiagnosed cardiac condition, complicated by postmortem propeller injuries by marine traffic, evidenced by lack of blood infiltration at the site of traumatic amputation.

Let’s consider another case:

An 80 year old male, who was an inmate of old aged home with multiple comorbidities confined to a wheelchair, was found submerged underneath river. He was last seen swaying around a footbridge about 3 meters above the river several hours earlier. Probable suicidal intention was identified. Autopsy revealed extensive comminuted fractures of the vault, subarachnoid haemorrhages and cortical contusions. Both lungs did not appear to be waterlogged.

Injuries may also be produced before or upon entering water and their extent have to be assessed. This could be related to subsequent question of survivability. In this case, considering the severity of the head injuries, it would appear that the deceased was unable to survive in water (or at most only a transient period) and succumbed rapidly. The cause of death is therefore attributed to head injuries upon falling with his top of head bumping the river bed.

Another case to ponder:

A 33 year-old female was found submerged about 20 meters off shore. Linear reddish bruising was found on the anterior neck. The face and eyes were congested with petechiae. Small amount of frothy fluid was present. Both lungs were congested and oedematous. Dissection also revealed deep bruising of strap muscles suggestive of pressure applied to neck. Subsequent investigation revealed spouse’s involvement with manual strangulation during a quarrel.

Suspicious injuries should be noticed which may be an act of homicidal drowning. In the present case, the cause of death is a combination of drowning and pressure on neck, with latter being a significant event rendering the deceased unconscious when pressure was applied and succumbed to the effects of immersion.

Presence of drugs which may be related to death

Let’s consider the following case:

A 29 year-old male was found floating in the river reported by local residents. No personal property could be found. No suicide note was present. He was last seen alive by wife 3 days ago and was believed to have quarreled with a female acquaintance, exhibiting violent behavior and soon disappeared afterwards. Wife reported missing to Police the next day and his personal belongings were discovered in a shopping mall. Autopsy revealed features of drowning. Postmortem toxicology analysis showed presence of cocaine and its metabolite benzoylecgonine in blood. It was not known to the family whether he had a history of drug abuse.

Toxicology samples are crucial to exclude conditions that may mimic autopsy features of drowning, such as pulmonary oedema. It may help to exclude an accident, explain for failure to extricate or survival in water, as well as inferring an intention to end one’s life or a deliberate intoxication. In the present case, analysis of hair samples was performed to address the issue whether he was exposed to illicit drug on a chronic basis and therefore exhibiting tolerance.

Presence of decomposition features may obscure the effects of drowning

Let’s consider the following case:

A 32 year-old male was found in the reservoir exhibiting moderate decomposition changes. Suicide note was found in personal property placed neatly on the shore. Autopsy did not reveal any significant trauma or lethal disease conditions. Both lungs were not hyperinflated but huge amount of serosanguinous effusion was present in chest cavities. Police investigation also revealed a strong suicidal intention and third party was not involved.

Typical findings of drowning are often masked by decomposition changes. In addition, the time of death has to be determined during investigation. For fresh bodies examined at scene, corrective factors should be applied while measuring the core temperature against ambient temperature as the rate of cooling in flowing and still water are different. Casper's dictum refers to the rate of putrefaction after 1 week in air being equivalent to 2 weeks in water and 8 weeks burial in soil. The varying features of decomposition hint to the postmortem interval and is generally slower in cold water than a body discovered on land, but may be accelerated in bacterial laden stagnant water. As micro-organisms continue to disseminate and distribute throughout various body compartments, decomposition will be accelerated upon retrieval.

While the cause of death can remain unascertainable due to decomposition, the pathologist could nonetheless leave a remark stating the overall findings was not inconsistent with that of drowning. This is dependent on the degree of diagnostic certainty dictated by the available circumstances and likelihood of other intervening events, such as injuries (which could also be obscured by decomposition).

Let’s consider another case:

A 69 year-old female with a history of psychotic illness was found floating in the sea, three days after her husband had reported missing to Police. No suicide note was found. Body exhibited early decomposition changes. Postmortem toxicology analysis revealed a toxic level of amisulpride in the blood samples. As there was no concrete evidence about the suicidal intention or actual clinical progress on the psychiatric condition, it remained unclear whether the deceased fell into the water out of her intention.

Destruction of micro-architecture by decomposition permit considerable degree of postmortem redistribution of drugs which possibly account for the elevated levels in the specimen. The cause of death and manner can remain inconclusive.

Mysterious circumstances

Let’s consider a case with apparently suspicious circumstances:

A 32 year-old male with a known history of mood disorder was found floating near a port. His leg was tied to a dumbbell. Suicide note was found at home. He was last seen alive two days ago and reported missing by family another two days later. The body exhibited early decomposition changes but the lungs appeared hyperinflated. Further Police investigation tracked the last whereabouts of the deceased including the use of surveillance camera in the vicinity and revealed no evidence of third party involvement. The shopkeeper selling the dumbbell clearly recalled visit by the deceased on the day of death.

Forensic Pathologists do not interpret a case relying solely on the autopsy findings. Circumstantial information can play a role to hint the pathologist appropriate features that should be looked for during scene and body examination. In the present case, there could be an underlying psychiatric vulnerability suggestive of a suicidal intent. A body with weight affixed to limbs can of course represent an unlawful disposal, but may as well indicate a determination to kill oneself. Examination of the knot tying at the involved body part is crucial.

For suspicious case a detailed investigation into the events before death is expected. The salient areas of such are briefly mentioned here.

Witness account

This is valuable and gives considerable weight to the case. For example, witnessed jumping into the water, signs of mental impairment, activities prior to submersion, the duration of immersion, bystander resuscitation with possibility of repositioning of body, accounts provided by lifeguard and nearby video surveillance, are all hints to the state of mind prior to drowning. [5,6] Homicidal drowning is rare unless one is being incapacitated by alcohol, drugs or physical weakness, or taken by an element of surprise such as being pushed unexpectedly into water. [9]

Scene and environment

Water temperature, current, terrain, water depth, underwater condition, floating objects, marine animal activities or plants, presence of safety and rescue measures are important to consider. A seemingly innocent river with slow volume of flow may harbor strong underwater currents creating significant eddies and vortex sucking the swimmer rapidly, coupled by additional injuries inflicted by submerged rocks and waterfalls, or falling log from trees nearby. [10]

Body floats owing to formation of putrefactive gas producing buoyancy and is affected by lung volume. It could even overcome weights added to the body in concealed homicide. The body will continue to sink as hydrostatic force exert pressure to the chest and abdominal compartments creating negative buoyancy. In extremely cold water with minimal bacterial activity, the body will never resurface and decompose through formation of adipocere. [1,5,6,7,8] Coupled together with witness account about the last seen at the point of immersion, an estimation of current speed and body drop rate (about 1.5 and 2 feet in salt and fresh water respectively) can allow back-calculation of the site of drowning in moving water, i.e. the distance from shore, which is useful for rescue and case reconstruction. [5,6]

For indoor environment a bathroom may present with wet, floor, wet towels and soap scum level in the tub (if water has been drained already). The presence of bucket and mop, and other cleansing material maybe an attempt to disturb the scene. [5,6] A discovery of electrical appliances would call for a proper investigation to the possibility of electrocution.

Location of body

The place where body is discovered does not necessarily indicate the site of drowning. A body can be brought by a receding tide to the shore and there is always a possibility of drowning in another place, such as an indoor environment. [5,6,7] The body maybe disposed into the sea as an act of mimicking suicide. Differentiation between genuine drowning versus other causes; as well as fresh versus salt water immersion would be helpful. The appropriateness of the subject to the location is important. A restricted access may suggest unauthorized entry to the premises and should be investigated.

State of body

The condition of body regarding to its state of dryness or wetness, any attachment by aquatic debris and clothing identified are important. [5,6,7] Minute pieces of evidence pertaining to the identity, drug habit, personal property, weapon and suicide note should not be overlooked. Clothing and status of equipment, especially in diving related fatality should be examined. A naked body may be a deliberate act of hindering proper identification, or could be linked to a sexually motivated homicide. The body composition, water temperature, current action, type of clothing, method of water entry may all affect the presence or absence of clothing on body and should be interpreted with care. [6]

The presence of sand, seaweed or other vegetation should be documented and described, with the possibility of sampling for trace evidence and hinting the location of drowning in doubtful situations. A pair of shriveled and pale hands or feet can be found regardless of whether the individual was alive or not. Commonly referred as "washerwoman's skin”, there is wrinkling and grayish white discoloration of skin at sites devoid of sebaceous glands. Histological features of swelling of epidermis keratinizing squamous epithelium, detachment of horny layer, fraying of keratin lamellae and vacuolation in the basal layer are observed. There are reports in older literature with reference to such histological changes in an attempt to determine the postmortem interval, though subjecting to environmental factors of water type, temperature, movement, pollution and dermal characteristics of the subject. [11]

Hospital Pathologists are familiar with the appearance of hypostasis but such phenomenon would be present on face, upper chest and distal end of extremities. This is explained by dangling position adopted by the body with head and limbs pointing downwards owing its specific gravity while the posterior trunk is floating backup. [1,5,6,7] On the other hand, hypostasis can be minimal when exposed to fast flowing water. [5,6] For bodies lying in bath tub there may be a line of demarcation corresponding to the water level. [7] The importance of visiting a scene cannot be emphasized more.

Clear or blood tinged oedema is usually described as a plume of froth around the nose and mouth. It is non-specific in nature and consists of bronchial mucus, oedematous fluid, air and the drowning medium. The redness is accounted by the ruptured capillaries exuding into the respiratory tract. [7,8,] And most importantly it is transient in nature. In addition, slit, mud, sand, vegetation, algae and shell fragments may be present in bronchi and bronchioles visible both grossly and microscopically. [5,6]

Autopsy Findings

The role of an autopsy is to retrieve relevant findings that support the diagnosis. Not all the features will be present, depending on the nature of drowning process. Interpretation is only meaningful when combined with sufficient circumstantial information.

Emphysema aquosum

A pair of waterlogged lungs is a result of over-distension due to strenuous effort in an attempt to overcome oxygen depletion upon water influx. It is more prominent in the periphery and a combination of both lungs with effusion weighing more than 1000 g is usual. [7,8] There is also overlapping of medial edges in the anterior mediastinum with indentation or imprints by the corresponding ribs. It is distinguished from chronic emphysema by protrusion of sectioned bronchial and vessels at the cut surface for the latter. Histology shows flattened inter-alveolar septa, dilated pulmonary alveoli and compression of septal capillaries. [11] Alveolar macrophages stained CD 68+ (smoker cells) may be washed from the alveoli to heart allowing its detection, as well as stimulation of certain subsets of myelomonocytes in lung tissues [8], though the validity of such remains low from a practical point of view. In addition, aspirated particles such as plant material in the distal bronchioles may be suggestive of ante-mortem aspiration.

Paltauf's spots

These are subpleural haemorrhages located in middle lobe fissure of about the size of a fingernail due to rupture of capillaries by overdistension and haemolysis by fresh water drowning.

Haemorrhage in neck muscles

The strap muscles and posterior occipital muscles may show tiny haemorrhages and altered histological appearance of the myofibrils with fiber degeneration, abnormal clumps of red material and ragged red fibers, owing to anoxic and ischaemic insult secondary to violent convulsive movements. At an ultra-structural level there is myofibrillar disruption and abnormal mitochondria. [12] A prudent approach is to exclude a mechanical cause before ascribing such to the effects of drowning.

Spleen

A contracted and anaemic spleen due to hypoperfusion and sympathetic stimulation with vasoconstriction is often nonspecific. [8]

Mastoid ear haemorrhages

Haemorrhage into ear compartment occurs as a result of pressure difference subsequent to blockage of Eustachian tube by water. [8,9]

Aspiration of fluid in the sphenoidal sinus:

“Svechnikov's sign” refers to presence of fluid (about 9 ml) in sphenoid and maxillary sinus by water penetration, which could also occur during postmortem. [7,8] It has been studied in literature with recent attempt to quantify and be detected by postmortem CT scan. [13]

Gastric dilatation

“Wydler's sign” refers to swallowing of water with resultant layer of sediment separating into three layers. This is also reported in recent postmortem imaging modalities with a certain degree of diagnostic confidence. [14] Oesophageal mucosal tears can be found occasionally due to distension by water. The presence of superficial radial ruptures of gastric mucosa is referred as "Sehrt's sign".

Ancillary Investigations

These tools can diagnose drowning with a higher degree of confidence, yet their limitations should be observed at the same time.

Histology

A differential staining of the intimal of aortic and pulmonary trunk is reported in the literature between saltwater and freshwater drowning. [15]

Immunohistochemical staining

Intrarenal aquaporin-2 (AQP2), intracerebral expression of aquaporin-4, aquaporin-5, HSP70, fibronectin are studied and reported with variable results. Surfactant protein A (SP-A) is produced by type II alveolar cells and showed increased expression with granular pattern in drowning case, despite that these stains could not readily differentiate between fresh and salt water drowning. AQP2, a channel protein for controlling flow of water molecules in the cellular interface, has shown apical expression in the apical membrane of the collecting in salt water drowning. [16] Arginine-vasopressin (AVP) was similarly expressed in the cytoplasm of renal tubules. Both have potentially served as markers to distinguish between salt and fresh water drowning, accounted by the increased binding and expression in a hyperosmolar environment. [17] While differentiation is necessary to exclude unlawful disposal of body, this can occur "naturally" when the body was dragged by sea currents from river in some regions.

Biochemistry

There are literatures studying derangement of electrolytes including sodium, chloride, and magnesium between left and right ventricles basing on the effect of hypertonic and hypotonic action of the aspirated water in drowning, referred as the “Getter’s test”. Results were not promising and appeared to be controversial and not adopted for routine use. Strontium was also studied to a certain extent as an indicator of drowning. It has been reported that a difference of 75 µg/L between cardiac chambers could be an indicator of drowning. This test also falls short if the drowning medium has relatively low strontium concentration. [7,8]

Diatom test

This test has often been quoted as a gold standard for some to prove that drowning has occurred. Diatoms are microscopic unicellular algae coated with silica that exist in soil, water and atmosphere. If an individual is drowned in fluid which contains diatoms, they may be identified in the lungs and other organs if circulation is maintained at the time of aspiration. The diatoms can reach various organs such as brain, kidney, liver and bone marrow (femur being the most protected bodily compartment therefore its detection is generally regarding as true positive). The technique in collection of proper bodily samples should be strictly free from environmental contamination. Aided by the oxidizing property of strong acids, detergent or enzyme, the rest of the diatom tissue is consumed leaving a pellet to be centrifuged and then examined microscopically. [1,7,8,11,18] A sample of water must be taken from the suspected site of drowning for comparison. One should notice that a negative result does not rule out drowning as the cause of death.

Its application in cases with advanced decomposition explained why it is often regarded as a gold standard. [19] The confounding factor is often the presence and concentration of diatoms in the environment plus the amount being aspirated. Unfortunately there is scanty environmental data about the species and frequency of their occurrence in local waters. Much data is needed for quantification for the profile of these algae in the environment, before designing an appropriate cut off value and proper positive species identification to achieve a reasonable sensitivity and specificity. Comparison may not be possible when the original site of drowning is unknown.

Postmortem imaging

Postmortem CT scan may show accumulation of aspirated fluid in the maxillary and sphenoidal sinuses (Svechnikov's sign), apart from detection of fluid in trachea and patchy ground glass opacities in the lung parenchyma. In another study, the presence of three layers consisting frothy material, fluid materials and dense component, visualized via different image contrasting features [13,14]. Care should be exercised during transportation as movement of body may result in reshuffling of content.

Conclusion

Despite ever expanding literature on the research about the pathophysiology and findings, as well as validity of ancillary investigations, pathologists are still facing challenges with vague circumstantial information, presence of ante/post-mortem trauma, decomposition changes, as well as non-specific autopsy findings. Nevertheless, as part of the indispensable team in death investigation, pathologists are obliged to take a proactive role in analyzing all available findings which might eventually shed light on any interpretable direction despite circumstantial evidence might still remain unclear. An inquest may be held after careful consideration by the Coroner and this has been the practice adopted to rebut unfounded allegations and refute rumors, when submitted evidence would be intensely examined. It is hoped that evidence presented and testimony of witnesses can address the appropriate issues and allow the next-of-kin to understand the circumstances before the final moment.

Reference

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3. Hong Kong Judiciary. Coroners’ Report 2018. Hong Kong; 2019. 62-73 p. Department of Health, HKSAR. Hong Kong Drowning Report. Hong Kong; 2019. 8-12 p.
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5. Armstrong EJ, Erskine KL. Water-Related Death Investigation Practical Methods and Forensic Applications. 1st edition. CRC Press; 2013. 27-149 p.
6. Shkrum MJ, Ramsay DA. Forensic Pathology of Trauma: Common Problems for the Pathologist. 1st ed. Humana Press; 2007. 243-293 p.
7. Lunetta P, Modell JH. Macroscopical, Microscopical and Laboratory Findings in Drowning Victims: A Comprehensive Review. Forensic Pathology Review Volume 3. 1st ed. Humana Press; 2005. 3-77 p.
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9. Byard RW. Drowning deaths in rivers. Forensic Sci Med Pathol. 2017 Sep;13(3):388-389. doi: 10.1007/s12024-017-9857-6. Epub 2017 Mar 11.
10. Dettmeyer RB. Forensic Histopathology: Fundamentals and Perspectives. 2nd ed. Springer; 2018. 60-65 p.
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12. Lo Re G, Vernuccio F, Galfano MC, Picone D, Milone L, La Tona G, Argo A, Zerbo S, Salerno S, Procaccianti P, Midiri M, Lagalla R. Role of virtopsy in the post-mortem diagnosis of drownig. Radiol Med. 2015 Mar;120(3):304-8. doi: 10.1007/s11547-014-0438-4. Epub 2014 Jul 11.
13. Gotsmy W, Lombardo P, Jackowski C, Brencicova E, Zech WD. Layering of stomach contents in drowning cases in post-mortem computed tomography compared to forensic autopsy. Int J Legal Med. 2019 Jan;133(1):181-188. doi: 10.1007/s00414-018-1850-4. Epub 2018 Apr 24.
14. Byard RW. Aortic intimal staining in drowning. Forensic Sci Med Pathol. 2015 Sep; 11(3):442-4. doi: 10.1007/s12024-014-9563-6. Epub 2014 Apr 22.
15. Barranco R, Castiglioni C, Ventura F, Fracasso T. Immunohistochemical expression of P-selectin, SP-A, HSP70, aquaporin 5, and fibronectin in saltwater drowning and freshwater drowning. Int J Legal Med. 2019 Sep;133(5):1461-1467. doi: 10.1007/s00414-019-02105-1. Epub 2019 Jun 20.
16. Barranco R, Ventura F, Fracasso T. Immunohistochemical renal expression of aquaporin 2, arginine-vasopressin, vasopressin receptor 2, and renin in saltwater drowning and freshwater drowning. Int J Legal Med. 2020 Apr 2. doi: 10.1007/s00414-020-02274-4. [Epub ahead of print]
17. Hürlimann J, Feer P, Elber F, Niederberger K, Dirnhofer R, Wyler D. Diatom detection in the diagnosis of death by drowning. Int J Legal Med. 2000;114(1-2):6-14.
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Results of College Written Exam in Chemical Pathology & Clinical Microbiology and Infection 2020
 

EXAM NO.	RESULT
E20114	PASS
E20115	PASS
E20116	FAIL
E20217	PASS
E20218	PASS
E20219	FAIL

Candidates who failed in written exam cannot proceed further in Membership or Fellowship Assessment in 2020.
Passed candidates can proceed further in Membership or Fellowship Assessment in 2020.