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What is Hepatitis?

Hippocrates was the first to describe benign epidemic jaundice in book ‘De Morbis Internis’ which resembled hepatitis A. The definition is accurate even today. Hepatitis is commonly defined as the inflammation of the liver. It might be due to a wide variety of causes, such as heavy alcohol consumption, autoimmunity, drugs or toxins; however, viral infection is the most frequent cause. Virally mediated liver inflammation, called viral hepatitis, is a significant burden. On the basis of severity, the condition can range between mild and self-limiting to a severe illness that requires liver transplantation. Based on the duration of the inflammation to the liver, hepatitis is categorised as acute inflammation (lasting for <6 months) or chronic inflammation (lasting for >6 months). Acute hepatitis is usually self-resolving but can cause fulminant liver failure. Contrastingly, chronic hepatitis can induce liver damage, fibrosis, cirrhosis, hepatocellular carcinoma and characteristics of portal hypertension that lead to significant morbidity and mortality.²

Table 1: Viral Hepatitis At a Glance

Aetiology & Epidemiology of Viral Hepatitis

Viral hepatitis is considered a major public health issue. Chronic infection with HBV and HCV can cause liver damage.^2,3

• Hepatitis A Virus

HAV, a spherical 27-nanometer particle, was first discovered by Feinstone in 1973. It is a non-enveloped single-stranded RNA virus and the only species from the Hepatovirus genus that infects humans.⁴ The global infection rate of HAV is considerably high, but only 1.5 million cases are reported annually. Generally, people with access to safe drinking water and stronger socioeconomic regions have very low cases of HAV infection with <50% of the population being endemic, whereas people without access to safe drinking water and low-income regions have high levels of HAV infection with >90% of the population being endemic. In highly endemic countries, HAV infection at an early age with asymptomatic exposure acquires lifelong immunity in children.

• Hepatitis B Virus

HBV is a double-stranded DNA virus belonging to the Hepadnaviridae family and has ten genotype variants (A-J). The intact HBV virion is called the ‘Dane particle’. The viral core of HBV consists of nucleocapsid, hepatitis B core antigen that surrounds the viral DNA and DNA polymerase. The nucleocapsid is coated with hepatitis B surface antigen (HBsAg), a viral surface polypeptide. The gene coding for core antigen also codes for hepatitis B envelope antigen. Nearly one-third of the global population has had an HBV infection and around 5% of them remain carriers, whereas about 25% of these carriers develop chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Variations in the genotype distribution and risk factors rely on common factors in high-risk populations like vertical transmission which is associated with an increased risk of chronic disease and hepatocellular carcinoma. History of blood transfusion, intravenous drug or paraphernalia use, contaminated piercing instruments, sexual intercourse with an infected person and organ transplantation from HBV-positive donors are all risk factors for HBV transmission.

• Hepatitis C Virus

HCV, an RNA virus, was first discovered in 1989. It belongs to the Flaviviridae family with one serotype, minimum six major genotypes and 80+ subtypes. The extensive genetic variability poses a major challenge in developing a vaccine for the prevention of HCV infection. Globally, the most prevalent cause of parenteral hepatitis is HCV. High-risk groups for HCV infection include people who need frequent blood transfusions and people who receive organs from infected donors. With the development of safer screening and viral elimination techniques for blood transfusion, transfusion-associated HCV incidence is decreasing.

• Hepatitis D Virus

HDV, a single-stranded RNA virus, was first described in 1977 by Rizzetto et al in Italy by direct immunofluorescence. It contains the hepatitis D antigen and has HBsAg as its envelope protein. There are 8 different HDV genotypes; however, HDV-1 is responsible for most cases in North America, Europe and the Middle East. It is assumed as a defective virus as HDV requires the existence of HBsAg for full expression and replication. HDV infection occurs in HBsAg-positive patients as a coinfection with HBV or as a superinfection in chronic HBV carriers. About 10.58% of HBV carriers were coinfected with HDV, which is two-fold higher than the previously estimated. Western and middle Africa, the Amazon Basin, eastern and Mediterranean Europe, the Middle East and parts of Asia are the areas with the highest HDV infection carriage.

• Hepatitis E Virus

HEV, a small single-stranded RNA virus, is considered one of the most common yet underdiagnosed aetiologies. As per the World Health Organization, approximately 20 million HEV infections are detected annually, of which 3.3 million lead to symptomatic cases of acute hepatitis.² HEV has been associated with outbreaks of food and waterborne diseases and is common in developing countries with limited access to sanitation, clean water and hygiene.

Pathophysiology

A virus enters the host system either through the enteric system or blood. Irrespective of the entry, the virus eventually travels to the liver where it infects hepatocytes, replicates either by direct translation of RNA or via reverse transcription of DNA and sheds the virions. HAV transmits via faecal-oral route. The virus disseminates into the liver via the portal vein, after it traverses the mucosa of the small intestinal wall. In HBV-infected cases, after the virus uncoats, its DNA integrates into the host nucleus as a covalently closed circular DNA (cccDNA) that can persist indefinitely in the hepatocytes, explaining the reactivation possibility in chronic inactive disease. Reverse transcriptase aids in the assembly of new viral molecules from cccDNA that are released by exocytosis. HCV is transmitted percutaneously (needlestick injuries containing contaminated blood) or non-percutaneously (perinatal transmission, sexual intercourse, blood transfusions, organ transplantation, religious scarification, body piercings or tattoos). The host-derived factors facilitating HCV entry are:

1. Scavenger receptor class B type I
2. Occludin
3. Claudin-I
4. CD81

Hepatocyte injury can either be self-limited and acute or insidious and chronic. The mechanism of hepatocyte injury is mediated by the host immune response against viral antigens which are expressed by infected hepatocytes and not as much by the cytopathic effects of the virus itself. The progression to chronic infection, as observed with HBV and HCV, is associated with the attenuation of T-cells that are virus-specific. Research shows that exhaustion of these virus-specific T-cells leads to an inability to clear the viruses, thereby allowing them to dwell chronically in host hepatocytes.^3,4

Evaluating & Monitoring Hepatitis

Baseline evaluation of patients suspected with viral hepatitis begins with a hepatic function panel. Severely affected patients might have elevated aminotransferases and bilirubin levels. Acute hepatitis patients typically show aminotransferase levels in thousands. Chronic hepatitis varies in presentation with aminotransferase levels. Often, the levels are elevated to 2-10 times of the upper normal. In most cases, alkaline phosphatase levels remain within the reference range; however, significantly elevated levels should be considered as biliary obstruction or liver abscess. The values of prothrombin time and international normalized ratio may appear prolonged in advanced liver disease. Leukopenia and thrombocytopenia might also be observed. Advanced liver disease patients suffering from easy bruising, variceal bleed or hemorrhoidal bleed may have anaemia with low haemoglobin and hematocrit levels. For patients suspected to have advanced liver disease, blood urea nitrogen and serum creatinine tests might be necessitated to check for renal impairment. For patients with altered mental status, serum ammonia levels must be checked as these are usually elevated in hepatic encephalopathy.²

Enzyme-linked immunosorbent assay and polymerase chain reaction are the technologies that aid in the diagnosis of acute and chronic viral hepatitis. Other specific tests for evaluating the viral hepatitis type include:

• Hepatitis A

Immunoglobulin M (IgM) antibody against HAV is the standard test for diagnosing acute HAV infection as it disappears after a few months. The presence of immunoglobulin G (IgG) antibodies against HAV signifies past infection and most patients with IgG antibodies have lifelong immunity against HAV.^2,3

• Hepatitis B

The first serum marker to appear in HBV-infected patients is HBsAg, whereas the first antibody is IgM antibodies against hepatitis B core antigen. However, the presence of HBsAg does not signify acute or chronic infection in the absence of symptoms. In symptomatic patients, the presence of HBsAg strongly suggests acute infection but does not rule out chronic infection with an acute superinfection by another hepatitis virus.^2,3

The presence of HBsAg, IgM core antibody, envelope antigen and viral load indicate acute HBV infection. A ‘window period’ is the duration during which HBsAg disappears before IgG antibodies appear against HBsAg. The presence of HBV DNA, HBsAg for >6 months, IgG core antibody and absence of surface antibody are indicators of chronic HBV infection.^2,3

• Hepatitis C

The presence of HCV RNA with/without IgM antibody signifies acute HCV infection, whereas chronic infection is suggested by the presence of HCV RNA and IgG antibodies. HCV RNA would become undetectable once the patient has cleared the infection.^2,3

• Hepatitis D

The presence of antibodies signifies HDV infection, whereas viral load is used for detecting current infection.^2,3

• Hepatitis E

The presence of HEV antigen, RNA viral load and IgM antibodies indicate acute HEV infection. The patient’s response to antiviral therapy is evaluated using RNA viral load. IgG antibodies against HEV confirm vaccine efficacy or natural protection.^2,3

Figure 1: Action Plans for Controlling Viral Hepatitis

Join the Fight

The early detection of viral hepatitis depends on understanding the peculiar features of the five hepatitis viruses. Advances in the healthcare and diagnostics field have enabled timely and effective diagnosis as well as helped in establishing the multisystemic impact on the population. The advent of newer and improved therapies coupled with advanced diagnosis would promisingly aid in achieving control over this affliction a reality!

References

1. World Health Organization. (2022, June 10). World Hepatitis Summit 2022 statement.https://www.who.int/news/item/10-06-2022-world-hepatitis-summit-2022-statement
2. Mehta, P., & Reddivari, A. K. R. (2021). Hepatitis. In StatPearls [Internet]. StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK554549/
3. Castaneda, D., Gonzalez, A. J., Alomari, M., Tandon, K., & Zervos, X. B. (2021). From hepatitis A to E: A critical review of viral hepatitis. World journal of gastroenterology, 27(16), 1691. https://doi.org/10.3748/wjg.v27.i16.1691
4. Zarrin A, Akhondi H. (2022). Viral Hepatitis. In: StatPearls [Internet]. StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK556029

Writer & Reviewer: Kayshu Grover & Dr Prachi Sinkar

The post Hepatitis A to E: A Critical Review of Viral Hepatitis appeared first on HealthScreen Magazine.

On October 12, 1492, three ships docked on the lands of North America. Aboard the ship was Christopher Columbus and his crew. Upon arrival, he came across Native Americans. To show that his exploration didn’t mean any harm, he offered them gifts, clothes and food. In return, the tribesmen handed over dried up tobacco leaves. Confused Columbus soon realised that the leaves were a prized possession and loaded bagfuls of them overboard. Smoking was thereby introduced to Europeans, and before long, cigarette smoking became an entrenched habit worldwide.¹

While loading his ship, Columbus didn’t know that tobacco smoke contains over 7000 chemicals, of which 250 are harmful and over 69 carcinogenic. He also didn’t know that tobacco would one day be responsible for 8 millions deaths/year and smoking would be the number one risk factor of lung cancer.^2.3

In the 1900s, technological advancement allowed large-scale cigarette production. By the end of the 19th century, mass marketing glamorised smoking, leading to the global lung cancer epidemic in the 1940s and 1950s. Before the 1900s, lung cancer was a very rare disease. Today, it has become one of the leading causes of cancer death, and tobacco has become the main cause of 90% of male and 79% of female lung cancers.^1,3

What’s in a cigarette?

The devastating link between tobacco smoke and lung cancer is due to toxic chemicals in cigarettes. As a tobacco plant grows, it absorbs chemicals, like cadmium (used in battery acid) and lead (used in batteries and house paint), from soil, both toxic and carcinogenic. During the curing process, more harmful compounds form, e.g. tobacco-specific nitrosamines.² While manufacturing, other chemicals are added as follows:

1. Nicotine – used in insecticides; it increases the release of neurotransmitters in the brain and elevates mood
2. Ammonia – common household cleaner
3. Acetone – used in nail polish remover
4. Acetic acid – used in hair dyes
5. Arsenic – used in rat poison
6. Benzene – used in gasoline
7. Butane – used in lighter fluid
8. Carbon monoxide – released in car exhaust fumes
9. Naphthalene – used in mothballs
10. Methanol – used in rocket fuel
11. Tar – used for paving roads

Once a cigarette is lit, more harmful chemicals are produced in the burning process that were not present initially in the curing and manufacturing processes.²

Tobacco and Lung Cancer Pathogenesis

These carcinogenic compounds activate/inactivate several signalling pathways in malignant cells. They allow cancer cells to proliferate and multiply by inducing epigenetic alterations, which help the development and pathogenesis of lung cancer.⁴

1. Tobacco and genetic changes

TP53

TP53 is the most important tumour suppressor gene. In nearly 50% of lung cancers, DNA repairment and apoptosis induction are deregulated and mutated in p53-harbouring cells. Polycyclic aromatic hydrocarbons in cigarettes increase thymine and guanine replacement frequency in p53. Nicotine-derived nitrosamine ketone (NNK) in cigarette smoke increases substitution of guanine to adenine in exon 5 of p53. Tobacco smoke also downregulates p16 expression. It methylates the promoter region of p16. This allows cancer cells to proliferate and escape from apoptosis. Nicotine and NNK increase the expression of neurotransmitters adrenaline and noradrenaline and inhibit the expression of GABA in lung cancer cells.

Free radicals and reactive oxygen species

Every cigarette pack produces nearly 5 × 10⁴ free radicals that induce oxidative stress and DNA damage, causing replication/transcription errors and genomic instability. In healthy cells, chemical-induced damages to single-stranded DNA are repaired by nucleotide excision repair and base excision repair (BER) systems. Xrcc1 protein in the BER system detects single-stranded breaks in DNA and acts as a scaffold for binding other repair enzymes. In smokers, polymorphisms in XRCC1 gene reduce this protein activity and disturb the DNA repair system.

β-gelatinase

β-gelatinase enzyme decomposes collagen, elastin, fibronectin, and non-matrix molecules, including pro-TNF-α, IL-8 and TGF-β. Considering its carcinogenicity, β-gelatinase releases pro-angiogenic factors and enhances tumour cell angiogenesis. It inhibits T cell proliferation and hinders immune system response by incising alpha interleukin-2 receptor, activating TGF-β and separating ICAM-1.⁴

Figure 1: Lung cancer progression and staging

2. Tobacco and epigenetic changes

Epigenetics modulations, including DNA methylation, histone modifications, nucleosome changes and microRNAs (miR)-associated gene regulation, refer to heritable alterations in gene expression that are not due to changes in DNA sequence. These mechanisms regulate gene expression by altering the chromatin structure.

In healthy cells, DNA methylation occurs mainly in regions with a high frequency of CG sites and at 5′ end of the genes. Being repressive to transcription, the modification is catalysed by DNA methyltransferases. Different methylation patterns are reported in infants whose mothers smoked during pregnancy. Nicotine changes the expression of DNA methyltransferases, causing demethylation of synuclein-gamma oncogene. Smoking can also cause the formation of methylated tumour suppressor gene p16.

The human genome contains nearly 1600 miR—non-coding small RNAs regulating expression of several genes. Chemicals in cigarette smoke alter the miR expression pattern. MiRs regulate the expression of oncogenes and tumour suppressor genes and act as oncogenes themselves. Cigarette smoke enhances miR-504 expression, an important miR for dopamine receptor gene DRD1 expression, ultimately inducing tobacco addiction.⁴

Figure 2: Healthy Lungs vs Smokers Lungs

3. Tobacco and Growth Signalling pathways

Nicotine binds to nicotinic acetylcholine receptors (nAchR), epidermal growth factor receptor (EGFR) and β-adrenergic receptor (AR-β). Owing to its ability to pass through the cell membrane, in non-neuronal settings, nAchR regulates growth, differentiation and migration of cancer cells. Several signal transduction pathways, such as MAPK, AKT and PKC, become activated, which further inhibit apoptosis, stimulate tumour cell proliferation and induce angiogenesis. However, the activation and secretion of neurotransmitters through nAchR is mediated by smoking. Nicotine binds to EGFR and AR-β and acts as a growth factor. EGFR is a tyrosine kinase receptor and crucial in cell growth, development and tumorigenesis. NNK contributes to cancer cell proliferation by synthesising thromboxane A2 (TXA2) and activating the TXA2 receptor. TXA2 activates the transcription factor cAMP response element-binding protein, enhances Bcl-2 expression and increases cell proliferation.⁴

4. Impact of Tobacco on Cell Survival, Growth and Apoptosis

Nicotine acts as a mitogen through cyclin D1 overexpression and cell cycle transition from G1 to S phase. It elevates the proliferative potential of cells by activating PI3K/AKT signalling pathway as a fundamental axis in tumorigenesis, tumour growth and drug resistance. Nicotine and NNK activate ERK and STAT pathways and disrupt anti-growth signals to increase cell growth. Cigarette chemicals suppress NK cell activity and proliferation. Defects in the apoptosis lead to cell survival and unlimited tumour cell growth. NNK and its metabolites inhibit apoptosis through upregulating hemeoxygenase (HO-1) and activating NF-κB and ERK pathway in lung tissue.⁴

5. Impact of Tobacco on Angiogenesis and Cancer Metastasis

Angiogenesis or blood vessel formation from endothelial cells is important for nutrition and oxygen delivery to tumour cells. In one mechanism, nicotine increases the expression and secretion of nitric oxide, a vasoconstrictor and angiogenesis mediator, as well as of endothelial growth factors.

Tumour metastasis, the major cause of lung cancer mortality, is the spread of tumour cells from one organ to others. The progression and metastasis of cancer in smokers is faster than non-smokers. Prolonged nicotine use decreases the expression of adhesion molecules, such as E-cadherin and β-catenin, in lung cancer cells. Additionally, breaking the extracellular matrix using matrix metalloprotease enzymes is important for tumour cell metastasis and invasion. Also, cyclooxygenase-2 (COX-2) elevates both angiogenesis and aggressive potential of tumour cells by increasing the production of prostaglandins and converting the pre-cancerous agents to carcinogens. Nicotine enhances the metastasis of esophageal carcinoma in that it upregulates and increases the activity of MMP-2 and COX-2.⁴

Beyond lung cancer…

Smoking is associated with over 15 types of cancer, including breast, bladder, colorectal, larynx, liver, pancreas, kidney, ovary, mouth, pharynx, and nose and sinuses cancer. It induces cardiac and lung diseases, stroke, diabetes, and chronic obstructive pulmonary disease, including emphysema and chronic bronchitis. Smoking increases the risk of tuberculosis, eye ailments, and immune system-related illnesses, e.g., rheumatoid arthritis.

Lipid Profile Modifications

Smokers demonstrate high serum cholesterol, triglyceride, and low-density lipoprotein levels, but low high-density lipoprotein. Nicotine increases blood sugar levels, thus increasing the risk of diabetes. Diabetic smokers require high insulin doses to maintain blood sugar levels.⁵

Blood Cell Parameter Changes

Carbon monoxide in cigarette smoke leads to high Hb levels, increased capillary permeability, increased erythrocytes and hematocrit values and decreased plasma volume. The irritating effect of the tobacco smoke on the respiratory tree as well as nicotine-induced release of catecholamine and steroid hormones increase the leukocyte count. Smoking-induced oxidative stress causes increased platelet activation and aggregation, atherothrombotic cardiovascular events and endothelium injury caused by nicotine.

Figure 3: Smoking, lung cancer and associated comorbidities

Is there a safe level of smoking?

‘One cigarette a day is not harmful’.

‘Passive smoking won’t kill you’.

‘Cutting back is just as good’.

‘Mild cigarettes are less risky’.

‘I only smoke occasionally’.

No matter the excuses, there is no denying that ‘smoking is injurious to health’. There is no safe level of smoking. Smoking 1-5 cigarettes daily is as harmful as smoking 20+ cigarettes daily. For daily heavy smokers, the risk of lung cancer mortality is 23 times in men and 13 times in women than non-smokers.

Ways to Quit:

Tobacco urges and cigarette cravings can be strong, but if you are determined, you can stand up against these.

• Cold turkey and gradual withdrawal
• Nicotine-replacement therapy
• Avoid triggers like alcohol
• Eat fruits and vegetables
• Exercise, meditate and relax
• Delay ‘the next cigarette’
• Lean on your loved ones

Quit for Good

Cigarettes come at a great cost: your life. Irrespective of the amount and times you smoke, it’s never too late to quit.⁵ As soon as you stop smoking, your body starts healing itself:

• In 20 minutes, heart rate and blood pressure drop.
• In 12 hours, carbon monoxide in blood drops to normal.
• In 1-3 months, blood circulation and lung function improve.
• In 1 year, the risk of heart disease is half that of smokers.
• In 5-10 years, the risk of mouth, throat and larynx cancer is halved.
• In 10 years, the risk of lung cancer is nearly half that of smokers.

Don’t wait, quit today!

References:

1. Tobacco-Free Life. History of Tobacco. https://tobaccofreelife.org/tobacco/tobacco-history/. 2016.
2. American Lung Association. What’s in a cigarette? https://www.lung.org/quit-smoking/smoking-facts/whats-in-a-cigarette. July 13, 2020.
3. World Health Organization. Tobacco. https://www.who.int/news-room/fact-sheets/detail/tobacco. 24 May 2022.
4. Nooshinfar, E., Bashash, D., Abbasalizadeh, M., Safaroghli-Azar, A., Sadreazami, P., & Akbari, M. E. (2017). The molecular mechanisms of tobacco in cancer pathogenesis.Int J Cancer Manag. 2017;10(1):e7902. https://brieflands.com/articles/ijcm-7902.html. doi: 10.17795/ijcp-7902.
5. Centers for Disease Control and Prevention.  Smoking and Diabetes. https://www.cdc.gov/diabetes/library/features/smoking-and-diabetes.html. August 10, 2021.

Writer & Reviewer: Sharvari Indulkar & Dr Prachi Sinkar

The post Smoking-induced Lung Cancer appeared first on HealthScreen Magazine.

Making up more than two-thirds of the human body weight!

As vital as this fluid is for sustenance of life,

It can become a life-threatening noxious liquid!

Water is an abundant resource, but access to safe drinking water is still a luxury not available to one and all! People depend on water bodies, such as rivers, ponds, and wells, for abstraction of water, but these water bodies are often contaminated with noxious chemicals and/or harmful disease-causing pathogens. As per the World Health Organisation, at least 2 billion people around the world use faecal-contaminated water source for drinking purposes. Diseases like diarrhoea, cholera, dysentery, typhoid and poliomyelitis caused by a number of bacterial, viral, and parasitic organisms can be transmitted by consumption of microbiologically contaminated water and leads to approximately 4,85,000 diarrhoeal deaths every year.¹

Water-borne diseases are a global burden and claim millions of lives every year. In developing countries, these diseases are associated with physical scarcity of water, i.e., lack of available usable resources as well as economic scarcity of water, that is, lack of investment in water infrastructure. Water-borne infections are caused due to ingestion, airborne exposure or contact with water contaminated by a variety of agents like bacteria, viruses, protozoa and helminths. Based on the mode of transmission, water-borne diseases have been classified into four categories, explained in Figure 1.²

Caption– Figure- 1: Categories of Water-borne Diseases

There has been a sharp decline in water-borne outbreaks since the last century, but infectious water-borne diseases are still a cause for global concern. Protozoans belonging to genera, such as Cryptosporidium, Naegleria and Giardia, and bacterial genera, such as Salmonella, Vibrio, Legionella, Escherichia, Campylobacter, Shigella and Pseudomonas, are the major causative agents associated with outbreaks and infections. Various factors such as infrastructure, chemical coats on pipes, architecture of systems, treatment deficiencies as well as weather and temperature contribute to such outbreaks.^1,2 Starting with water-borne pathogens and diseases, let’s understand novel approaches in the detection of water-borne diseases.

Emerging Water-borne Pathogens

There are more than 1000 species of pathogens including bacteria, viruses, protozoa as well as fungi. The development of a disease depends on various factors such as minimal infectious dose, pathogenicity, host susceptibility and environmental characteristics. Enteric bacteria have minimal infectious dose value in the range of 10⁷-10⁸ cells, while certain species have it as low as 10¹ to 10^3. The species belonging to genera like Shigella, Campylobacter, Escherichia and Vibrio are known to have lower values. In case of protozoans, a few oocysts are sufficient for producing the disease, and a small number of viruses are enough for developing the disease. Viruses such as caliciviruses, enteroviruses, coxsackieviruses, polyomaviruses and cytomegalovirus are classified as water-transmitted viral pathogens. Influenza virus and coronaviruses have been suggested as organisms that can be transmitted via drinking water; however, the evidence is inconclusive.^2,3

Water-borne pathogens appear repeatedly due to the following reasons:²

• Contaminated water
• Increased population sensitive to the pathogens
• Altered drinking water treatment technology
• Globalization of commerce and travel
• Development of molecular methods

Certain water-borne pathogens such as protozoa Microsporidia, bacteria (like Mycobacterium avium, Helicobacter pylori, Tsukamurella and Cystoisospora belli) and viruses (like adenoviruses, parvoviruses, coronaviruses, and polyomavirus) are examples of emerging potential in water-borne pathogens. Majority of these organisms have certain resistance against common disinfectants like chlorine, ultraviolet light and heat used for treating water sources. This resistance presents a challenge in removal of these pathogens.³ Figure 2 depicts some major pathogens that are detected in drinking water systems.^1,2

Caption– Figure- 2: Pathogens in Drinking Water Systems (Elaborated as follows:)

Bacteria

• Campylobacter
• Yersinia enterocolitica
• Escherichia coli
• Burkholderia pseudomallei
• Legionella pneumophilaand related bacteria
• Nontuberculous mycobacteria
• Pseudomonas aeruginosa
• Salmonella
• Shigella
• Vibrio cholerae
• Helicobacter pylori

Virus

• Adenovirus
• Enteroviruses
• Hepatitis A and E viruses
• Rotavirus
• Sapoviruses
• Astroviruses
• Norovirus

Protozoa

• Acanthamoeba
• Cryptosporidium
• Entamoeba histolytica
• Giardia intestinalis
• Naegleria fowleri
• Toxoplasma gondii

Helminths

• Dracunculus medinensis
• Schistosoma

Diagnosing Water-borne Pathogens

Water-borne pathogens include bacteria, viruses as well as protozoans. Reliable analysis of these requires a method that has attributes such as automation, high specificity and sensitivity, result reproducibility, as well as affordability and the time required for analysis. Staring with molecular techniques, let’s acquaint ourselves with the cutting-edge methodologies used for detection of water-borne pathogens.²

• Molecular-based Methods

These methods can be species-specific and provide phylogenetic information. Alternative indicators can also be incorporated permitting distinction between human and animal pathogens as well as tracking the source of pollution.²

• Polymerase Chain Reaction

Polymerase chain reaction (PCR) is one of the most commonly used techniques that operates by amplifying a specific target DNA sequence in a cyclic three-step process for exponential amplification of the target sequence. Quick analysis is one of the major advantages of molecular techniques. However, they require accurate primers and optimal reaction mixture for avoiding false results and cannot differentiate viable and non-viable cells. Multiplex PCR, a type of PCR allows simultaneous detection of several target organisms of diverse pathogens in a single reaction. Quantitative real-time PCR is another version of PCR that provides high sensitivity and specificity, faster rate of detection as well as minimizes the risk of cross-contamination and does not require post-PCR analysis. Quantitative reverse-transcriptase PCR provides an estimation of the concentration of RNA viruses. It can detect viable cells as it detects messenger RNA which is present only in viable organisms but might be unable to detect damaged genomes.^1,2

• DNA Microarrays

Microarrays are glass slides or chips coated with specific chemically synthesized oligonucleotide probes that allow rapid detection of multiple genes of multiple organisms simultaneously owing to their capacity for screening large numbers of sequences. They have numerous advantages including high throughput capacity, automation, ability to detect antimicrobial resistance and host origin of contaminants. However, it comes with drawbacks, such as relatively high cost, low specificity and sensitivity due to non-specific hybridization and difficulty in distinguishing between viable and non-viable cells.

Oligonucleotide microarray, a powerful genomic technology, is widely used to monitor gene expression under different cell growth conditions, detect specific mutations in DNA sequences as well as characterise micro-organisms in environmental samples. DNA microarrays contain high-density immobilized nucleic acids in a two-dimensional ordered matrix that enables simultaneous detection of genes in a single assay via nucleic acid hybridization.^1,2

• Next-generation Sequencing

Next-generation Sequencing (NGS) is a DNA-sequencing technology that allows high-throughput parallel analysis of DNA sequences from PCR amplicons and determines specific microbial consortia in a water sample.¹

• Pyrosequencing

Pyrosequencing is a DNA sequencing technology that uses enzyme-coupled reaction and bioluminescence (polymerase, Adenosine-5 -triphosphate sulfurylase, luciferase and apyrase) to monitor pyrophosphate release in real time. This technology provides a large number of sequences read in a single run, but is limited due to cost, amount of DNA in the samples, complexity of analysis, availability of computing power and the efficiency of data generation.^1,2

• Fluorescence in Situ Hybridization

Fluorescence in situ hybridization (FISH) is a technique that involves the hybridization of sample with fluorescent-labelled rRNA oligonucleotide probes which allow enumeration of particular microbial cells using confocal microscopy, fluorescence microscopy or flow cytometry. It aids in the detection and identification of different micro-organisms in mixed populations like biofilms. Major drawbacks of this technique are low sensitivity and necessity of pre-enrichment and concentration steps that might increase inhibitor concentrations leading to false-negative results.^1,2

• Immunology-based Methods

Immunological detection with antibodies methods are based on antibody-antigen interaction that have been employed for the detection of multiple pathogens. Serum neutralization tests, enzyme-linked immunosorbent assays, immunofluorescence and immunomagnetic separation are some examples of immunology-based methods. The limitations associated with these methods are low sensitivity, false-negative results, cross-reactivity and need for pre-enrichment.^1,2

• Biosensors

Biosensors are made of a bioreceptor (recognising target) and a transducer (converting biological interactions into measurable electrical signal). They have numerous advantages like automation, short analysis time, portability, real-time measurements, miniaturisation of biological analytical techniques and no need for pre-enrichment. The drawbacks include great sensitivity to pH, mass and temperature. Biosensors are of various types, namely:^1,2

• Optical biosensors
• Electrochemical biosensors
• Mass-sensitive biosensors

Caption– Figure- 3: Molecular Methods and Detected Micro-organisms

Hurdles in Diagnosing Water-borne Pathogens

The major challenge for the detection of water-borne pathogens is the absence or lack of a unified method encompassing water analysis for every pathogenic micro-organism of interest, such as bacterium, virus or protozoan. Some of the other major challenges in detection of water-borne pathogens include:²

• Physical differences between the major pathogen groups
• Low pathogen concentration necessitating enrichment and concentration
• Presence of inhibitors
• Absence of general protocols for sample collection
• Absence of culture-independent detection methods
• Detection of the host origin of pathogens

Quantitative Microbial Risk Assessment

Quantitative microbial risk assessment (QMRA) is a systematic quantitative assessment process that combines information about the dose response of an infectious agent with the distribution of exposure routes. QMRA aids in estimating the health risks or rates of illness due to exposure to particular pathogens. It aids in predicting the burden of water-borne diseases, setting tolerable limits, identifying means of reducing the risk and determining measures to protect water safety. There are 5 steps in risk assessments, namely:

1. Hazard identification
2. Exposure assessment
3. Dose-response assessment
4. Risk characterization
5. Risk management and communication

Major drawbacks of QMRA approach are its dependency on the method used for monitoring pathogens in water and the lack of available data (from cultivation methods) for completing the assessment. QMRAspot, a computational tool based on QMRA, was developed for rapid and automatic quantitative microbial risk assessment of drinking water.²

Expanded Threat!

Water is a basic necessity, but a large population lacks access to safe clean and contamination-free water. Water-borne pathogens present a critical global health concern, especially in developing countries, making it imperative to develop different approaches for detection of pathogens. Various methods, such as cultural, molecular as well as biosensor-based, have been implemented, but they come with certain drawbacks. Culture-dependent methods are extensively used for the detection of pathogens but are time-consuming and have low sensitivity. Molecular or biosensor-based methods are dependent on lab methods but might require pre-treatments and are expensive.²

Water-borne diseases are directly related to environmental deterioration and pollution. Despite continued efforts for maintaining the safety of water, outbreaks related to water-borne pathogens are still being reported globally. As per the World Health Organisation, more than 2 billion people reside in water-stressed areas, which are expected to aggravate in the view of aggravating climate change and increasing population worldwide. With the arrival of the monsoon season, water-borne pathogens and resulting infections would be on the rise. The world is already employing precautions against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but prevention of water-borne as well as air-borne pathogens is also of the essence!

References

1. Srivastava, K. R., Awasthi, S., Mishra, P. K., & Srivastava, P. K. (2020). Biosensors/molecular tools for detection of waterborne pathogens.Waterborne Pathogens, 237-277. https://doi.org/10.1016/B978-0-12-818783-8.00013-X
2. Ramírez-Castillo, F. Y., Loera-Muro, A., Jacques, M., Garneau, P., Avelar-González, F. J., Harel, J., & Guerrero-Barrera, A. L. (2015). Waterborne pathogens: detection methods and challenges.Pathogens, 4(2), 307-334. https://doi.org/3390/pathogens4020307
3. Gall, A. M., Mariñas, B. J., Lu, Y., & Shisler, J. L. (2015). Waterborne viruses: a barrier to safe drinking water.PLoS pathogens, 11(6), e1004867. https://doi.org/1371/journal.ppat.1004867

Writer & Reviewer: Kayshu Grover & Dr Vaishali Khudkhudia

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