In a breakthrough, a blood test can diagnose TB disease

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This is an animation depicting an active TB infection targeting the lungs. — Jason Drees, Biodesign Institute at Arizona State University

In a marked departure, researchers have used a rapid blood test that relies on two Mycobacterium tuberculosis-specific peptide fragments for diagnosis of TB disease and monitoring treatment. Currently, sputum samples are used for diagnosing TB disease in the case of pulmonary TB and tissue samples in the case of extra-pulmonary TB. The blood test accurately detects minute levels of two biomarkers — CFP-10 and ESAT-6 — that are “actively secreted” by the bacteria when it causes TB disease. Currently, the assay costs less than $10.

In a pilot study, the new blood test was able to diagnose active TB cases with “high sensitivity and specificity”. It was able to diagnose active TB even in people who were coinfected with HIV. The results were published in a paper published today (March 28) in the Proceedings of the National Academy of Sciences (PNAS) by Chang Liu from Houston Methodist Research Institute. Dr. Liu is currently at Arizona State University.

The blood-based assay was able to provide quantitative results that will help in knowing the severity of active TB and in monitoring treatment outcomes. Unlike Xpert, it cannot detect rifampicin resistance.

The blood-based assay was able to diagnose both pulmonary and extra-pulmonary TB cases with high sensitivity — over 91% in the case of culture-positive pulmonary TB (PTB) and above 92% extra-pulmonary TB (EPTB), and 82% in culture-negative PTB and 75% in EPTB in HIV-positive patients. In the case of HIV coinfected cases, the sensitivity was 87.5% for PTB and 85.7% for EPTB cases. It also had high specificity (87-100%) in both healthy and high-risk groups.

“We want to detect only active TB, but not latent TB, so we selected CFP-10 and ESAT-6. However, we believe these two biomarkers are capable of detecting early activation of latent TB, but we are conducting more experiments to confirm that,” says Dr. Liu in response to a question on the choice of the two peptide fragments.

According to a 2014 WHO report, there is a need for a “rapid biomarker based non-sputum-based diagnostic test that uses an easily accessible sample and is able to accurately diagnose pulmonary TB (and ideally also extrapulmonary TB)”.

Obtaining sputum samples is not always easy and is particularly difficult in the case of little children and people who are HIV positive. About 15-25% of all TB cases are extrapulmonary and biopsy samples are needed in such cases. Even Gene Xpert, introduced a few years ago to improve sensitivity and specificity, relies on sputum samples, and as per a 2014 WHO update, Xpert has “very low quality evidence” for EPTB diagnosis. Also, cerebrospinal fluid or other samples are needed for diagnosing EPTB using Xpert.

The blood-based TB diagnostic assay will be go a long way in the war against TB, particularly in diagnosing TB in little children, people with HIV and extra-pulmonary TB cases.

The blood sample is first microwave irradiated for about 20 minutes, and the target peptides are enriched using a nanoparticle enrichment platform and a high-throughput mass spectroscopy for enhancing the detection of Mycobacterium-specific biomarkers. It take some about four hours to prepare a serum sample and 10 minutes to know if the two peptides are present in the blood.

“CFP-10 and ESAT-6 are also expressed by some other mycobacteria, they cause NTM infection, not TB. We discovered peptides in CFP-10 and ESAT-6 that are specific to TB. So we digest the two proteins [using microwave irradiation] before diagnosis,” Dr. Liu, who is the first author of the paper, says in an email. “NanoDisk is functionalized with antibody. Their capture and isolate the peptide targets from patient serum sample. In addition, due to their special material and nanostructure, they can enhance the signal of mass spectrometry during detection.”

“We are particularly excited about the ability of our high-throughput assay to provide rapid quantitative results that can be used to monitor treatment effects, which will give physicians the ability to better treat worldwide TB infections,” said Prof. Ye Hu from the Houston Methodist Research Institute and the Corresponding author of the paper said in a release. Prof. Hu  is currently at Arizona State University. “Furthermore, our technology can be used with standard clinical instruments found in hospitals worldwide.”

According to the authors, the NanoDisk-MS assay meets several of the WHO criteria for a noninvasive TB assay — “it uses a small, non-invasive specimen; does not require bacterial isolation; has high sensitivity and specificity for active TB cases in extrapulmonary, culture-negative, and HIV-infected TB patients; and directly quantifies Mtb antigens for rapid monitoring of anti-TB therapy effects”.

The assay was able to detect marked reduction in the peptides levels in both HIV-positive and HIV-negative TB cases that were started on TB treatment. But more studies need to be carried out to evaluate the performance of NanoDisk-MS assay in treatment monitoring as the pilot study was not designed to measure the rate of decline of TB bacteria with treatment.

“We have tested 21 patients (HIV- and HIV+) with multiple longitudinal samples, and were able to see biomarker decrease correlated to treatment in 19 of them. Larger clinical validation is underway [to know treatment resistance and therapeutic efficacy],” he says.

The researchers note that larger, randomised studies are needed to confirm the results of the pilot study.

“Any blood-based, rapid TB diagnostic assay is ideal and has huge potential as it does not depend on sputum samples [for pulmonary TB], and tissue samples in the case of extra-pulmonary TB. But many studies based on blood-based assays have not been successful earlier,” says Dr. Soumya Swaminathan, Director-General of ICMR, Delhi. “High specificity [correctly identify those with the disease] is very important and so large-scale tests have to be carried out in countries like India where a large population has latent TB infection.”

Published in The Hindu on March 28, 2017

Indian researchers find a new bacterial target for drug development

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(From left) The study by Anshika Singhal, Andaleeb Sajid and Richa Misra helped understand how bacteria form biofilm.

Indian researchers have found a new target that can potentially be used for developing new antibiotics that will be effective against many bacteria. The new target is made of two proteins — which form a complex that is responsible for the formation of biofilm — that perform very important functions and are critical for bacterial ability to successfully infect humans. The results were published in the journal Biofilms and Microbiomes.

Bacteria form biofilms, a kind of matrix, during infection in plants and animals. Biofilm shields the bacteria from antibiotics and help bacteria to survive harsh conditions such as extreme temperature or stress. Now a study by Indian researchers has found the molecular signaling events that play a crucial role in biofilm formation in Bacillus anthracis, the causative agent of anthrax.

Till now, all attention has been on developing antibiotics that target disease-causing bacteria and not the biofilm itself.
One of the basic questions that scientists have been trying to answer is how and when bacteria decide to form biofilm. “One possibility is that bacteria has sensors on the surface which senses some signal and helps in biofilm formation,” says Andaleeb Sajid from the Institute of Genomics and Integrative Biology (IGIB), Delhi and one of the authors of the paper.

“It was serendipity. Our lab was working on signaling in bacteria and we were studying PrkC and similar proteins. When PrkC protein is deleted, Bacillus bacteria are unable to form biofilm. So we started studying the mechanism by which PrkC protein controls biofilm formation,” she says.

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Gunjan Arora says the GroEL-PrkC complex could be a target for developing new drugs.

“Our hypothesis is that PrkC senses some signal and transmits it from outside to inside the cell. This signal goes to other proteins like GroEL. PrkC adds phosphate group (phosphorylate) to different proteins. The mystery to biofilm formation lies with one chaperone protein called GroEL. The addition of phosphate to this tiny machine initiates a course of events within bacterial cells leading to complex biofilm formation,” Dr. Sajid says.

The team found several proteins receive signals from PrkC protein. Using cutting edge genetics, molecular biology and proteomics techniques, they confirmed that GroEL was regulated by PrkC.

“From other unrelated bacteria we already had a clue that GroEL has a role in biofilm formation. We looked at the molecular level and found six amino acid residues where phosphate was getting added to the GroEL protein. Through a series of steps, we ascertained how important phosphorylation was for proper functioning of GroEL,” says Gunjan Arora from IGIB and the first author of the paper.

“We wanted to know if the bacteria has any other compensation mechanism to form biofilm in the absence of PrkC. So we made PrkC mutant bacteria to produce more of GroEL. The bacteria were able to form biofilm even in the absence of PrkC. This experiment helped us understand that PrkC is the influencer and GroEL is key to biofilm formation,” Dr. Arora says.

Both PrkC and GroEL perform very important functions and are critical for bacterial ability to successfully infect humans. “We think GroEL-PrkC complex could be a target for developing new antibiotic that will be effective against many bacterial pathogens such as the ones that cause MRSA, TB and pneumonia. One strategy to tackle drug resistant bacteria will be to develop multi-drug regimen that combines traditional antibiotics with candidate drugs that can block bacterial signaling and prevent biofilm formation,” Dr. Arora says.

Published in The Hindu on March 26, 2017

Plastic waste found in fish meant for human consumption

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As per a 2015 paper, plastic waste was found in fish from Indonesia and textile fibre in fish from California.

In a first, researchers have found plastic debris in fishes in Indonesia and California, U.S. meant for human consumption, raising a red flag for human health. According to a paper published in September 2015 in the journal Scientific Reports, man-made debris was found in 28% of individual fish and in 55% of all species samples from markets in Makassar, Indonesia. In the case of the U.S., man-made debris was found in 25% of individual fish and in 67% of all species. Anthropogenic debris was also found in 33% of individual shellfish sampled.

While all anthropogenic debris found in the digestive tracts of fish and whole shellfish sampled in Indonesia contained plastic, it was fibre from textiles in the case of fish from California (there was only 20% plastic waste). The difference in the type of man-made found in fish in the two countries reflects the waste-management practices in the two countries.

In Indonesia, of the 76 fish from 11 different species collected from a market, 21 (28%) had anthropogenic debris in the digestive tract. Of the 11 fish species collected, plastic waste was found six species. At 56%, the Indian mackerel had the most amount of debris, followed by herring at 29%.  Totally 105 plastic pieces were removed from the fish. The average size was 3.5 mm in length and up to 4.5 mm in width.

In the case of fish from 64 fish from 12 different species taken from California, 16 (25%) fish and four of 12 shellfish had man-made fibre from textiles waste inside the digestive tract; six fish had plastic waste. Totally, 30 individual fibre pieces were removed from the fish. The number of anthropogenic particles in individual fish was up to 10 pieces. The researchers were unable to know if the textile fibre found inside fish was synthetic or natural. The average length of fibre 5.5 mm and width was up to 0.05 mm.

“As anthropogenic debris is associated with a cocktail of priority pollutants, some of which can transfer to animals upon ingestion, this work supports concern that chemicals from anthropogenic debris may be transferring to humans via diets containing fish and shellfish, raising important questions regarding the bioaccumulation and biomagnification of chemicals and consequences for human health,” notes the paper.

Small-sized man-made debris has been shown to cause “physical damage leading to cellular necrosis, inflammation and lacerations of tissues in the gastrointestinal (GI) tract. As such, anthropogenic marine debris may cause physical harm to humans when debris is ingested via seafood,” the paper says.

Plastic debris is widely present in oceans. According to a February 2015 study published in Science, eight million tonnes of plastic entered the ocean from around the world in 2010. China was the worst offender contributing 8.82 million tonnes of plastic per year; India was ranked 12th with 0.60 million tonnes.

It was always known that of the huge plastic waste that enters the world oceans, certain percentage of degraded plastic in the form of tiny particles would be consumed by fishes and other marine animals and ultimately enter the food chain.

TB diagnosis, treatment sub-optimal in Indian prisons

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Screening, diagnosis and treatment of people with tuberculosis is “sub-optimal” in Indian prisons, says a study published recently. Only 79 prisons (50%) screened new inmates at the time of entry, and 92 prisons (59%) carried out periodic or regular screening. As a result, researchers from the Delhi-based International Union Against Tuberculosis and Lung Disease (The Union) were able to diagnose 80 new TB cases by screening nearly 5,100 prisoners. These people “could have been missed in the existing [TB testing] system” in Indian prisons.

157 prisons studied

The study was conducted in 157 prisons — central, district and sub-district — that housed 0.2 million inmates. There were 342 inmates with TB in 92 prisons when the study was carried out in 2013. The results were published in the International Journal of Infectious Diseases.

The study found an association between periodic screening and TB patients but no such association between the entry-level screening and TB patients.

“Entry-level screening helps in identifying TB patients among those prisoners/inmates who are new in the prison. Regular screening identifies TB patients among those who have been in the prison for certain duration and are at higher risk [owing to prison conditions]. Our study indicates that entry-level screening alone is not sufficient to diagnose all TB patients in prisons and needs to be supplemented with regular screening,” Banuru Muralidhara Prasad from The Union and the first author of the study says in an email.

The WHO and The Union advocate regular screening. “In this study, regular screening was limited to a few central and district prisons,” the paper notes.

Diagnostic facility

Entry-level screening is more in prisons which had a doctor and was the least in sub-district prisons. Though doctors are available in 129 (89%) prisons, only 65% were trained under the national tuberculosis programme.

Though the availability of diagnostic facility in prisons ensures early diagnosis of TB, the study found the availability of diagnostic and treatment services had “no significant” relation to TB diagnosis. Central prisons, where inmates serve more than two years of imprisonment, had better facilities — doctors trained in TB programme (90%), periodic screening (73%) and availability of TB services (65%) — compared with district and sub-district prisons.

India has 0.37 million inmates housed in 1,400 prisons across the country. Overcrowding, malnutrition, lack of infection control, to name a few makes prisons a high-risk environment for spread of TB. “Prisoners very often originate from the most vulnerable sectors of society. They already have an increased risk of diseases such as TB. In prison, these problems are amplified by poor living conditions and overcrowding, poorly ventilated spaces,” a 2001 WHO document says.

“Prisoners disproportionately come from disadvantaged backgrounds. They can, therefore, be at a higher risk of acquiring TB infection even before they arrive in prison, as well as of suffering from comorbidities, such as HIV infection, hepatitis and diabetes. Thus, prisoners are a key population to be covered by the End TB Strategy,” says the WHO’s  Ethics guidance for the implementation of the End TB strategy report.

Published in The Hindu on March 23, 2017

Indian researcher develops smartphone-based device for male infertility screening

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The device has 98% accuracy in identifying sperm concentration and motility. – Photo: Dr. Deepak Modi

An Indian and a team of researchers from Harvard Medical School, Boston have developed a smartphone-based semen analyser that will go a long way in identifying the cause of infertility in men — low sperm concentration and motility —with nearly 98% accuracy. The portable, easy to use, automated device can be used by untrained individuals, is highly inexpensive, and provides results in about five seconds.

The device measures sperm concentration and motility based on unwashed, unprocessed semen samples. The accuracy of the device was similar to computer-assisted, lab-based analysis even when performed by untrained users. Male infertility affects over 30 million (12%) of men in the world. The stigma against male infertility prevents many men from getting tested; the new device may help many men to undertake semen analysis at home and in low-resource settings. The results were published on March 22 in the journal Science Translational Medicine.

The smartphone-based platform made by Manoj Kumar Kanakasabapathy, the first author of the paper, has an optical attachment for image magnification and a disposable microfluidic device for loading the semen sample. A disposable microchip with a capillary tip and a rubber bulb is used for simple, power-free semen sample handling. The software has a user-friendly interface that guides the user through each step of testing. The results can be stored on the phone for monitoring over time.

Fifty-six cryopreserved semen samples (with semen count of about 100,000 sperm per ml) were first used to evaluate the device. Though the difference between manual and smartphone-based semen analysis increased as sperm concentration increased, the accuracy was over 98%. The device was evaluated for sensitivity, specificity and accuracy by testing 164 semen samples collected from patients. The device was unable to measure samples that had over 100 million sperm per ml, but was able to accurately detect abnormal semen samples — sperm count less than the WHO threshold of 15 million sperm per ml.

The sensitivity, specificity and accuracy of the device to detect abnormal samples based on semen count alone were 91%, 97% and 96% respectively. In the case of motility, the sensitivity, specificity and accuracy of the device were 99%, 87% and 98% respectively. The sensitivity, specificity and accuracy for both sperm concentration and motility were 99%, 89% and 98% respectively.

The “current version” uses the smartphone camera for sample imaging and different smartphones can be used with minor modifications to the optical arrangement. Three different smartphones (Moto X, Moto G4, and LG G4) were used by the researchers and got the same results. The team also evaluated how well untrained users performed the test using the device.

The device has certain limitations: accuracy suffers when samples contain higher-than-average number of nonsperm cells such as white cells. The algorithm counts sperm based on sperm-head measurement and so cannot differentiate between sperm and other larger cells. It is possible to address this by using more image-intensive image analysis but that would increase the processing time. The device also cannot evaluate sperm morphology.

Besides being used for infertility testing, the device can be used by men for home-based monitoring after undergoing vasectomy. As per guidelines, sperm count should be less than 100,000 sperm per ml between eight and 16 weeks after the vasectomy procedure. Earlier studies have shown that compliance for postvasectomy follow-up semen analysis is “extremely poor”. The device finds application in animal breeding as well.

Published in The Hindu on March 23, 2017

Overweight, obese men at higher risk of severe liver disease, liver cancer

Men with more than normal body weight from an early age (17-19 years) are at higher risk of developing severe liver disease and even liver cancer in later life. The risk is increased when such men develop diabetes.

The association between body weight and higher risk of severe liver disease has become clear in a study based on data of more than 1.2 million Swedish men enlisted for military conscription between 1969 and 1996. Compared with men who have normal weight, those who are overweight (body mass index more than 25 kg/m²) have about 50% greater risk and obese men (BMI greater than 30 kg/m²) are more than twice likely to develop liver disease and even liver cancer in later life.

An earlier study involving nearly 50,000 men showed an association between high BMI in early age was associated with increased risk of end-stage liver disease. The study was based on data collected between 1969-70, when overweight and obesity were relatively uncommon — only 0.8% were overweight. So the study was not statistically powered to confirm an association between high BMI and increased risk of end-stage liver disease.

But a paper published today (March 21) in the journal Gut found a statistically significant association between BMI and severe liver disease. Men who have higher BMI even at an early age are at higher risk of developing severe liver disease and liver cancer as the “duration of being exposed to a high BMI” increases the risk.

Compared with men with BMI 18.5-22.5 kg/m², men with higher BMI had higher risk of severe liver disease, with the greatest risk in those with BMI greater than 30 kg/m². The non-alcoholic fatty liver disease is likely to be the “main driver” of severe liver disease in adolescent men, the study says.

Consumption of alcohol and smoking were taken into account and those who had developed alcoholic liver disease were excluded from the final analysis, but this did not change the overall finding. But the risk of severe liver disease was “highly affected” by development of type 2 diabetes during follow-up across all BMI categories.

It is well known that people with high BMI are at increased risk of developing diabetes, which in turn is associated with heightened risk of developing severe liver disease. Yet, increased risk of severe liver disease was seen even in overweight men who did not have diabetes. As the data was restricted to men, the researchers could not study the association between BMI and severe liver diseases in women. But they say that earlier studies of middle-aged women in the UK have shown found an association; but the risk was “uncertain” for younger women.

Therefore, even if the risk is increased in overweight and obese men who develop diabetes, the association of high BMI at an early age and severe liver disease at a later date “cannot solely be explained” by type 2 diabetes, the study says.

There should be targeted intervention to prevent diabetes and more than normal BMI at an early age.

Published in The Hindu on March 21, 2017

CCMB: Can alpha chain of clusterin protein control weight, fat gain in animals?

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Dr. Ramakrishnan (left), Dr. Mohan Rao (centre) and Suvarsha Matukumalli studied rats injected with beta chain and found fat accumulation from day two onwards.

The two chains of clusterin protein, which are normally expressed in several tissues and can be found in several body fluids, when present together tend to lower lipid levels but administration of one of its chains — alpha or beta — results in completely different outcomes. Cells treated with a recombinant beta chain tend to accumulate fat while cells treated with an alpha chain showed no increase in lipid accumulation. Rabbits administered with a recombinant beta chain showed nearly 40% increase in weight while animals given an alpha chain showed no such increase. The results were published in the journal Scientific Reports.

“Two chains of clusterin when present together tend to decrease body weight but one of the two chains (beta clusterin) increases body weight. This is quite unusual,” says Dr. Ch. Mohan Rao from the Centre for Cellular and Molecular Biology (CCMB), Hyderabad, and the corresponding author of the paper. “So the alpha chain should ideally be compensating for increase in body weight. But the alpha chain does not do that.”

Rats gained lean mass not fat

“While excess energy gets accumulated in the form of fat when beta chain was injected into rats, we did not see this in the case of alpha chain. One possibility is that the alpha chain helps in the metabolism of food in such a way that fat does not accumulate,” he says. “Dissected rats that were given alpha chain showed increased levels of lean mass.”

Apparently, there was no difference in the food intake between animals treated with alpha or beta chain. “It means that weight increase can happen even when there is no increase in food intake. It is the energy management by the body that is important. And alpha chain seems to modulate metabolism in such a way to promote energy expenditure and thus prevent fat accumulation,” he says.

The effect of alpha and beta chains were tested on myoblast cells, fibroblast and cancer cells. The individual chains were injected into rabbits as well. “In my lab we study the effect of small heat shock protein on health and disease. To raise antibody for clusterin we injected the chains separately into rabbits. One set of rabbits was gaining weight while the other did not. That’s when we investigated the reasons. The animal-house in-charge noticed the change in the animals,” recalls Dr. Rao.

Rats too gained weight

Though the effects of the two chains were seen in rabbits, the researchers turned to rats as more animals were required for investigating the effect of individual chains on animals.

“We could see fat accumulation in cells from day two onwards. We observed for 10 days and fat accumulation continued for all the 10 days; we could study cells continuously only for 10 days,” says Suvarsha Rao Matukumalli from CCMB and the first author of the paper. “In the case of animals injected with beta chain, fat accumulation continued for four-five months. The controls and animals given alpha chain did not show weight or fat gain.”

When cells were administered both the chains simultaneously, the cells did not accumulate fat for two-three days but started thereafter. “Fat accumulation was not as much as when only the beta chain was given but fat accumulation nevertheless continued,” says Ms. Matukumalli. But the effect of both the chains in animals was quite different. “When we introduced both alpha and beta chains together in animals we did not see any weight gain. The animals were very much like the controls,” she says. “Only large-scale, in-depth studies can reveal if alpha chain prevents weight gain.”

Published in The Hindu on March 19, 2017