Tuesday, March 7, 2023

Can low carbohydrate diet make you slim?

Obesity research is basically an attempt to explain attitude of slim people towards obese. Some doctors don’t want to deal with obese patients because they think; they are dealing with someone non sensitive towards his/her own health. Obesity is beyond control of obese person. There could be genetic, hormonal or metabolic reasons behind altered physiology.

Intake of carbohydrates (sugar and starches) and fat generally blamed for getting obese or overweight. My great grandfather died at the age of 105 year and his diet was always rich in fat and protein with optimal amount of carbohydrates, but he remained slim and active throughout his life. The narrative that dietary fat causes heart attack is an absurd idea. I wonder to learn that Dr. Atkin’s New Diet Revolution also suggested high-fat diet to minimize the risk of heart disease. On the contrary low-fat and low-carbohydrate (low calorie) diet being recommended by the physicians could be more harmful for your heart. Diet rich in fat and low in carbohydrates improves the level of HDL and reduces the level of LDL in our blood, which is said to be healthy environment for our heart. Fat rich and low-carb diet also controls blood pressure and insulin level. The low-fat diet which people have been eating with hopes of protecting heart has been bad for heart ‘actually’ as it is rich in carbohydrates.

Our bodies burn carbohydrates along with fat to generate energy. Carbohydrates are burned first and fat later by our bodies. So, by drastically reducing carbohydrates and eating more fat and proteins, our bodies would ‘definitely’ reduce weight by burning stored body fat more efficiently for fuel and energy. When your body changes from carbohydrate burning engine to fat burning engine, our fat storing spots (belly, hips and thighs) become a source of energy; and we lose weight in spite of eating fat rich food with little or no carbohydrates. When this happens, we feel less hungry and tend to eat less.

Low carbohydrate diet would ‘definitely’ make you slim.

Friday, March 3, 2023

Migraine: An episodic headache

Migraine is an episodic headache disorder associated with autonomic symptoms with or without aura. Episodes of migraine could lead to loss of productivity in young professionals/individuals. Pathophysiological studies exhibit migraine to be a neurovascular headache. Migraine is caused by abnormal activation and modulation of trigeminocervical neurons. Impaired modulation of central nervous system aminergic pathways especially of the brainstem and activation of trigeminoparasympathetic system leads to vasodilation of meningeal arteries and dural venous sinuses.

 Migraine was considered to be vascular headache until 2010. The aura phase of migraine has been demonstrated to be a neuronal event, with electroencephalogram. A cortical wave showing cortical depression could be observed in electroencephalogram. So, physiologically migraine is an heterogenous entity.

Migraine treatment comprises two arms with an effort to address components of pathophysiology: (i) Strategy to terminate acute attacks and (ii) Strategy to prevent further attacks.

Ergotamine was the earliest analgesic known to be effective in acute migraine since 1920s. Now large number of ‘Nonsteroidal Anti-inflammatory Drugs’ (NSAID) and non-NSAID analgesics have been used as rescue medications. The targeted treatment was launched in 1990 with the advent of Triptans. Advances in preventive therapies have been very few. Beta-blockers and tricyclic antidepressants remain the main component of treatment of migraine,

Monday, June 29, 2020

Diagnostic dilemma in COVID-19: Identical twin of Pangolin-CoV


Outbreak of pneumonia in the third week of December-2019, in Wuhan City of Hubei Province of China led to the isolation of a new type of coronavirus in January 2020. The same was named as novel coronavirus-2019 (2019-nCoV) by Chinese Health Authorities. Pathogen free 'Human Airway Epithelial (HAE) cells' in tissue culture bottles were apically inoculated with bronchoalveolar lavage fluids and/or throat swabs from 9 patients. Apical supernatants from these cultures were the source of viral RNA. Genomic sequencing of 2019-nCoV was done by Chinese Scientists using ‘Sanger Sequencing’ facility and made available to GenBank and is registered under GenBank accession ID: MN908947. In 5 cases out of 9 Illumina and Oxford Nanopore Sequencing was also done. All the 9 patients whose samples were used to isolate virus for genomic sequencing had exposure to Huanan Seafood Market in Wuhan City. One of these patients stayed near Huanan Seafood Market.

Genomic implications:

There exists homology in genetic sequences of six strains on 2019-nCoV. The GenBank accession IDs of these strains are: EPI_ISL_402019, EPI_ISL_402020, EPI_ISL_402021, EPI_ISL_402022, EPI_ISL_402023 and EPI_ISL_402024. These epidemic strains of COVID-19 virus are just like human siblings.

It has been documented that Genomic sequences of 2019-nCoV obtained from nine patients of severe pneumonia at Wuhan City of Hubei Province of China had 88% identity with two bat-derived 'Severe Acute Respiratory Syndrome’ (SARS) like coronaviruses with genomic IDs: bat-SL-CoVZC45 and bat-SL-CoVZXC21. Genetic sequence of 2019-nCoV has been found very distinct from SARS-CoV (2002-03). Findings were published by Chinese group of scientists in the Lancet, volume 395 in the month of February 2020.


A group of veterinary scientists working under the guidance of Yongyi Shen of the College of Veterinary Medicine, South China Agricultural University, Guangzhou-510642, China, have explored through extensive study on 25 Malayan pangolins (Manis javanica) and 4 Chinese pangolins (Manis pantadactyla) that 2019-nCoV has very close relationship with coronavirus isolated from 17 out of 25 Malayan pangolins. However, Chinese pangolins were found negative for 2019-nCoV infection by RT-PCR. Coronavirus isolated from Malayan pangolins showed 100%, 98.2%, 96.7% and 90.4% amino acid identity with 2019-nCoV in E, M, N and S genes, respectively. The study reflects that Malayan pangolins are the natural host of this zoonotic coronavirus. Yongyi Shen and his team has proved through this marvelous study that 2019-nCoV is an identical twin of Pangolin-CoV.

Analysis of plasma samples from 8 of the Malayan pangolins showed high positivity for antibodies against 2019-nCoV by double antigen sandwich ELISA. Plasma sample of one of the Malayan pangolins showed positivity for antibodies against 2019-nCoV even at 1:80 dilution of plasma.

Diagnostic dilemma:

The Real Time-Reverse Transcription-Polymerase Chain Reaction (rRT-PCR) was developed for early detection of 2019-nCoV infection using nasopharyngeal and/or oropharyngeal swabs from patients having clinical symptoms of viral infection. RT-PCR has limitations that it could not be used for detecting past infections.

Diagnostic dilemma is that rRT-PCR covers all the six strains of 2019-nCoV. There could be more unknown strains of 2019-nCoV having identical genomic expression. Molecular diagnostic technology is very new and sensitive but has its limitations. It provides indirect evidence of a causative organism. Real Time RT-PCR for 2019-nCoV does not distinguish between active infection or positive results due to unknown SARS Related Coronaviruses (SARSr-CoV).  

The only validated method for diagnosis of 2019-nCoV by World Health Organization (WHO) is rRT-PCR and nobody could question its accuracy. Collection of sample, extraction and purification of viral RNA, storage of reagents of RT-PCR Kits, setting up of rRT-PCR Assay, operation of PCR-Machine and interpretation of rRT-PCR results need extensive training and experience.

Information Brochures/Manuals of all the Real Time RT-PCR Kits used for detecting 2019-nCoV immaculately exhibit that the kits are meant for Research use only (RUO) . Information is available online regarding recommended use of kits. I would like to share an adapted image from the Brochure of RealStar SARS CoV-2 RT-PCR Kit 1.0 of Altona Diagnostics GmbH, Hamburg (Figure-1) and another image from the Manual of Liferiver Novel Coronavirus (2019-nCoV) Real Time RT-PCR Kit of Shanghai ZJ Bio-Tech Co. Ltd., China (Figure-2), for attesting my point that diagnostic use of RT-PCR kits for 2019-nCoV (SARS CoV-2) is not valid or ethical.



Figure-1 For Research use only (RUO)
Figure-2: For Research use only (RUO)
What happens to your sample?

Extraction of RNA is the start point of all the kits for RT-PCR analysis of nasopharyngeal or oropharyngeal swabs as well as bronchoalveolar lavage samples. The purity of RNA extracted could only ensure the accuracy of results. It is highly important to use the RNA Extraction Kit compatible with the kit being used for rRT-PCR. Some most extensively used RNA Extraction Kits and Systems are:


§  AltoStar Automation System AM 16 (Altona Diagnostics)
§  QIAamp Viral RNA Mini kit (QIAGEN)
§  QIAsymphony (QIAGEN)
§  MagNA Pure 96 System (Roche)
§  Maxwell 16 IVD Instrument (Promega)
§  VERSANT kPRC Molecular System SP (Siemens Healthcare)

If you use spin column based, RNA extraction procedure, extra precaution should be taken to get rid of any traces of ethanol in the RNA purified; since ethanol is an inhibitor of rRT-PCR.

Real Time RT-PCR:

The rRT-PCR is a complex assay for detection of target genes: Target-E Gene (for B βCoV specific RNA), Target-S Gene (for SARS CoV-2 specific RNA) and Target-IC (for Internal Control). Different companies have different reporters (fluorescent dye labels) with or without quencher. The RealStar SARS CoV-2 RT-PCR Kit has used FAMTM, Cy5 and JOETM without any quencher respectively for above mentioned target genes.

The RT-PCR comprises three steps, which are completed in Thermal Cycler at different temperatures ranging from 55oC to 95oC. The dye acquisition takes place during amplification cycle. In brief:

Step-I is Reverse Transcription; it is done at 55oC at hold for single cycle of 20 minutes.

Step-II is Denaturation; it is done at 95oC at hold for single cycle of 2 minutes.

Step-III is Amplification of DNA; it is a multicycle process (at least 30-45 cycles). One cycle of Amplification is completed in holds at three different temperatures: 95oC for 15 seconds, 55oC for 45 seconds and 72OC for 15 seconds. So, one amplification cycle is completed in 1 minute and 15 seconds. 45 cycles for amplification will be completed in 56 minutes and 25 seconds (75x45=3375 seconds). So, the total turnaround time for rRT-PCR is 3 hours including sample preparation. However, it would take longer as the kit is meant for 14 samples and 6 controls. Preparation of multiple specimens and setting up machines needs extra time and precautions to avoid contaminations.

Some most commonly used RT-PCR machines are:
  • ABI Prism 7500 SDS (Applied Biosystems)
  • CFX96TM Deep Well Real-Time PCR Detection System (Bio-Rad)
  • CFX96TM Deep Well Dx System (Bio-Rad)
  • CFX96TM Real-Time PCR Detection System (Bio-Rad)
  • CFX96TM Dx System (Bio-Rad)
  • LightCycler 480 Instrument II (Roche)
  • Rotor-Gene 6000 (Corbett Research)
  • Rotor-Gene Q5/6 Plex Platform (QIAGEN)
  • Mx 3005TM QPCR System (Stratagene)
  • VERSANT kPCR Molecular System AD (Siemems Healthcare)


The data analysis on Real Time-PCR machines needs expertise as per instrument manufacturer’s instructions. Qualitative Analysis of fluorescence signals captured for genes under detection is done to conclude the outcome of rRT-PCR assay.


Each amplification cycle doubles the copies of DNA molecule. If we start with single copy of cDNA after transcription from viral RNA; after completion of 31 amplification cycles; there will be more than one billion (1x109) copies of DNA. After completion of 41 cycles of amplification; more than one trillion (1X1012) copies of DNA will be available for detection and analysis.

Past infection with 2019-nCoV could be detected trough tests for detecting IgM and IgG type antibodies against the virus. Double antigen sandwich ELISA could be very sensitive and specific assay for detecting both the IgM and IgG type antibodies.

Saturday, June 20, 2020

COVID-19 casualties: Embalming of dead bodies



Massive publicity of Coronavirus Disease (COVID-19) has inflamed human psyche and emotions. Our existence on this planet is ever since our conception though we celebrate our physical birth days. Human form of life is said to be the best of 2.1 million (2.1x106) living species born through placenta. We have been better than other living creatures due to possession of superior mind, memory, intellect and emotions. We have social bonding with our family and friends. Coronavirus Disease (COVID-19) has taken the shape of a pandemic due to human to human spread of its causative agent. The Chinese authorities isolated this new type of coronavirus from patients of outbreak of pneumonia in December-2019, in Wuhan City of Hubei Province of China during laboratory investigations on 7th January 2020 and named it 2019-nCoV.

Symptoms of COVID-19 are like influenza and include fever, headache, shivering (rigors), myalgia, malaise, cough and sometimes diarrhea also. In elderly and immunocompromised patients, it is causing severe respiratory distress due to pneumonia and causing death due to comorbidities in-spite of best treatment. Lack of immuno-prophylaxis is the cause of casualties as vaccines for this disease are not available. However, precautionary measures are in force to avoid catching nCoV infection. Personal hygiene, washing of hands frequently with soap and water, wearing of face mask and maintaining physical distance (social distance) from other individuals are the ways to save ourselves from COVID-19. Human to human spread of the disease is through micro-droplets of nasal secretions of infected person.

Need of the hour is to take necessary precautions to avoid catching nCoV infection. COVID-19 could be fatal in 2-3% patients only. Just being positive for 2019-nCoV by RT-PCR does not warrant any panic. Viral infections are self-curing in 95-97 cases with symptomatic treatment. Only 3 to 5% cases would need hospitalization. Imposition of stricter control measures by administration to avoid community transmission of COVID-19 has spread great scare of death in the minds of entire population. People have been avoiding receiving the bodies of their near and dear ones, who unfortunately died of COVID-19 complications.

Let me instill confidence in you that the dead would never spread any infection if bodies are embalmed. We need to maintain social distancing from the living persons and not from the dead. Dead are not exhaling any infectious droplets. Dead need our love and care for proper send off from this world. We could take necessary precautions to sanitize and temporarily preserve the bodies of our near and dear ones who unfortunately bid goodbye to us due to COVID-19 complications. Medical colleges and Medical Research Institutes have been embalming and preserving dead bodies for very long periods for dissection and demonstration to medical students. The history of embalming dates-back to 4000 BC. Egyptians were the first to master the methods of embalming. Why can’t we do embalming of dead bodies before handing over to the relatives?

Provincial governments all over the world should issue advisories to all the hospitals to essentially do hypodermic and surface embalming of dead bodies before handing over to relatives and friends of the diseased. Embalming would ensure safe transportation of the dead body for cremation without any risk of infection. Pathologists and Anatomists should be assigned the task of onsite embalming even if the death occurs at home. Dead bodied of army warriors are always preserved by Arterial embalming along with coloring and masking agents before transportation to native places of martyrs.
 
We have experienced that face mask and alcoholic sanitizers have potential to keep away any infection. Sodium hypochlorite at a concentration of 0.04% in tap water can kill bacteria, fungi and all the viruses including coronavirus. The bleach we use for fabric bleaching at home, contains 4% Sodium hypochlorite (see Figure-1). Sodium hypochlorite at a concentration of 0.04% in tap water is sprayed to sanitize roads and buildings in Containment Zones. One cap full (10 ml) of fabric bleach in 1000 ml (1 L) of tap water gives 0.04% Sodium hypochlorite solution. This solution could be used to sanitize dead body before short distance transportation. Dead body could be wrapped in a bed sheet soaked in 0.04% solution of Sodium hypochlorite. Body could be given bath of this solution.

Figure-1



We trust that eye/ear drops containing just 0.01% (w/v) Benzalkonium Chloride are free of any infectious agents and could be instilled in eyes or ears (see Figure-2).  People can use solution of Benzalkonium Chloride at the universally accepted concentration (0.01% w/v) to surface sanitize dead bodies of their dear ones without any risk of infection. We mop our floors with germicide Lyzol which contains very high percentage of Benzalkonium Chloride (2.4580% w/w: see Figure-3). Just one cap full (15 ml) of Lyzol (500 ml Pack) in 4000 ml (4L) of tap water would give 0.01% solution of Benzalkonium Chloride which could be used for surface sanitization of the dead body. Dead body could be wrapped in a bed sheet soaked in the solution of Lyzol at optimum concentration as cited above.

Figure-2























Figure-3
All relatives and friends of a diseased person should be provided counselling for handling the dead body. People should be educated and encouraged to accept the dead bodies of their loved ones without any fear of infection. Any disrespect to the corpse (dead body) invites applicability of section 297 IPC in India and similar laws must be available in other countries too. Section 297 IPC or similar sections should be invoked both for hospitals and people to show respect to the dead. I would like to appeal to World Health Organization (WHO) for issuing necessary advisory to all the countries to take necessary steps for embalming of the dead bodies during the pandemic for safe transportation for funeral.

Thursday, June 18, 2020

Evolution of a pandemic: Coronaviruses to COVID-19


Coronavirus is not an exclusive finding of 2020 though it has been projected as “Novel Coronavirus” (nCoV). The so called nCoV has been designated as etiological agent of “Coronavirus Disease-2019” (COVID-19) that took shape of pandemic with its epicenter at Wuhan City of Hubei Province of China. Bovine and avian Coronaviruses are known since 1976 and a lot of research has already been done on their structure and pathogenicity.

The well characterized members of coronavirus family are positive-stranded RNA viruses. Coronaviruses possess a single-stranded, non-segmented, polyadenylated infectious RNA genome. The size of viral particles (virions) vary from 80-120 nm in diameter. Nanometer (nm) is ultra-small unit of measurement of length. There are one billion (1000,000,000) nanometers in a meter. These viruses could only be revealed through Transmission Electron Microscope (TEM) or Scanning Electron Microscope (SEM). Coronaviruses have pleomorphic look due to 10-20 nm long surface peplomers. Coronaviruses can cause diarrhea, hepatitis and bronchitis in lower mammals and birds. Three species of coronavirus are known to cause hepatitis in mice and seven species of coronavirus are known to cause bronchitis in birds.

Species of coronaviruses are characterized on the bases of molecular weight of surface polypeptides (glycoproteins). The molecular weight of surface glycoproteins of coronaviruses could be ranging from 15,000 to 200,000. They possess a large genome of 5x106 to 8x106 molecular weight. Massive research work has been done on coronaviruses in various virology laboratories all over the world to ascertain their genomic structure.

In the year 2002 “Severe Acute Respiratory Syndrome (SARS) was caused in humans by avian coronavirus (SARS-CoV). The animal reservoir of this coronavirus was perhaps bats. The avian coronavirus spread to other animals like cats and first infected humans in the Guangdong Province of southern China in 2002. An epidemic of SARS-CoV affected 26 countries in 2002-2003 and >8000 cases were reported as per World Health Organization (WHO). The transmission of SARS-CoV is primarily from human to human in the second week of disease due to excretion of viruses in respiratory secretions. So, infection spreads trough droplets of nasal secretion.

The current pandemic with Coronavirus has deliberately been given new nomenclature due to its genomic distinction from SARS-CoV known earlier. This new type of coronavirus causing SARS is called 2019-nCoV.

Symptoms of disease:

Symptoms of COVID-19 are like influenza and include fever, headache, shivering (rigors), myalgia, malaise, cough and sometimes diarrhea also. Fever is most frequently reported symptom, but it may be absent in initial stage especially in elderly and immunocompromised patients.

Cough may be dry initially with shortness of breath. Fever and diarrhea may progress in second week of disease. Elderly and people with associated illnesses like diabetes, hypertension, heart disease or organ transplant, may need special care to protect themselves from COVID-19.

Personal protection measures:

There is no immuno-prophylaxis for nCoV as vaccines are still under development. Personal hygiene, exercise and protein rich diet and vitamin-C are key to avoid coronavirus infection. Fresh fruits or juices rich in vitamin-C, high protein diet, coffee drinks, frequent drinking of warm water could be of great help to keep away COVID-19.

Frequent washing of hands with soap and water. Hand sanitization, wearing of face mask and maintaining physical distance (social distance) of approximately 6 feet would be of great help to avoid 2019-nCoV.

The need of the hour is not to panic but to follow the instructions and guidelines issued by local and national health authorities.

Diagnostic criterion:

Real Time - Reverse Transcription - Polymerase Chain Reaction (Real Time RT-PCR) is the only specific investigation to ascertain 2019-nCoV infection. The Chinese authorities isolated a new type of coronavirus from patients of outbreak of pneumonia in Wuhan City during laboratory investigations on 7th January 2020 and named it 2019-nCoV.  Nasopharyngeal or oropharyngeal swabs are taken from suspected persons and transported to Laboratories in specially constituted transportation medium.

The principle of RT-PCR detection method is based on ‘Fluorogenic 5’ Nucleage Assay’. During the PCR reaction, the DNA polymerase cleaves the probe at 5’ end and separates the reporter dye from quencher dye only when the probe hybridizes to the target DNA. This cleavage results in the fluorescent signal generated by the cleaved reporter dye, which is monitored almost Real-Time by PCR detection system.

Warnings:

1.      This assay needs to be carried out by skilled personnel only.

2.      Clinical samples should be treated as highly infectious and samples should be prepared in biosafe laminar flow hoods.

3.      Do not use the kit after its expiry date.

4.      Carry out the test as per global standard procedure only.

5.      Avoid repeated freezing and thawing of reagents.

Care and treatment of infected persons:

Infected patients should be managed as per conditions. Patients should be home quarantined or admitted to COVID-19 Hospitals, as per clinical assessment. Symptomatic treatment should be started immediately.  Every patient needs to be instructed to wear face mask and wash/sanitize his/her hands frequently and maintain physical distance from other family members and friends. Proper counselling needs to be provided to patients with adequate supply of diet recommended by nutritionist. Health professionals need to wear ‘personal protection equipment’ (PPE) with adequate sanitization to avoid any cross infection.

Enforcement measures by administration:

Lockdown of public transport, schools, colleges, universities, offices and places of worships may be needed to restrict community transmission and spread of COVID-19. Medical infrastructure needs to be upgraded efficiently for optimum treatment of COVID-19 patients. Thermal screening at Airports, Seaports, Railway Stations and Bus Terminals is the first and foremost step to identify persons carrying virus. Proper arrangements for compulsory quarantine for 14 days of suspects with infection.

 

Investigations should be done as per WHO guidelines for pandemic surveillance. Containment Zones with large number of COVID-19 patients should be put under strict surveillance to avoid community spread of the disease. Strict enforcement of health & hygiene-related discipline could save the citizens and the nation.