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The kits improve the sensitivity of smear microscopy and transport of sputum samples

Improved detection: The three kits (bottom) improve the sensitivity of smear microscopy by about 5% and make transporation of sputum samples easy as TB bacteria are killed.

To address the TB diagnostic challenges, a multi-institutional team has developed three cost-effective kits that improve the sensitivity of smear microscopy, enable transport of sputum samples at ambient temperature without using bio-safe containers, and extract DNA for diagnosing drug-resistant TB. The three kits are — TB Detect, TB Concentration & Transport, and TB DNA Extraction.

The TB Detect kit is for diagnosis using LED fluorescence microscopy, while the TB Concentration & Transport, and the TB DNA Extraction kits together are for detection of drug-resistance. The TB Detect kit helps increase the positivity of LED fluorescence microscopy by about 5%, while the TB DNA Extraction kit allows the detection of drug-resistant TB bacteria with a high level of sensitivity. The results of the study were published in PLOS ONE.

The TB Detect kit currently costs Rs.100 per sample, Rs.100 for the TB transport kit and Rs.85 for the DNA extraction kit.

In the case of the TB Detect kit, the team led by Prof. Jaya Sivaswami Tyagi from the Department of Biotechnology at AIIMS used a simple filter-based system to separate TB bacteria from the sputum to improve the sensitivity of LED fluorescence microscopy. In this, the sputum, which is viscous, is first liquefied using a proprietary reagent and pre-filtered. The bacteria in the liquefied sputum pass through a paper-like filter while some of the cell debris are retained on the filter.

The liquefied sputum containing the TB bacteria then comes in contact with a highly porous, plastic membrane filter underlain by multiple fibrous pads kept at the base of the filter device. While the sputum gets sucked by the fibrous pad due to capillary forces, the bacteria remain on the membrane.

“The membrane concentrates the bacteria in a small surface, which is about 1 cm in diameter. This leads to an increase in the detection limit,” says Mr. Nalini Kant Gupta from the Advanced Microdevices Pvt Ltd, Ambala, who developed the membrane filter, the filter device and the kits. He is a co-author of the paper.

The limit of detection was found to be higher when the bacteria were separated from sputum prior to examination under a microscope. “By separating the bacteria from sputum, it becomes possible to detect TB bacteria even when they are fewer in number. Even when only 1,000 bacteria are present per ml of sputum it is possible to detect them compared with 10,000 bacteria per 1 ml of sputum in the conventional system,” says Prof. Tyagi.

It also takes less time to examine a sample under a microscope — one minute vis-a-vis three-five minutes when the sputum is spread on a glass slide. This is because the sputum on a glass slide covers a large area requiring more number of fields to be viewed.

Diagnosis of drug-resistant TB is carried out only at central laboratories, and so samples have to be transported. The TB concentration & transport kit now makes it easy and simple to transport samples — transport it in sealed covers at ambient temperature making containment at low temperature redundant. This is because the bacteria are killed using a disinfectant.

“Since the bacteria are killed it is not possible to culture them. But for molecular testing to diagnose drug-resistant TB, only the DNA is needed and so it does not matter if the bacteria are dead or alive,” says Prof. Tyagi.

The third kit — TB DNA Extraction — allows DNA to be isolated from the bacteria present on the filter paper. “To extract the DNA, the filter paper containing the bacteria are heated at 90 degree C in a lysis solution. The cell membrane ruptures and the DNA gets released from the bacteria, which is purified for molecular testing,” says Dr. Sagarika Haldar from the Translational Health Science and Technology Institute (THSTI) and another corresponding author of the paper.

“Since the bacteria are dead it cannot be cultured for drug-susceptibility testing, so we used molecular testing. Here we looked for mutations in the resistance-conferring genes by amplifying and sequencing the DNA,” says Divya Anthwal from THSTI and first author of the paper.

Compared with culture, the sensitivity of the kit for drug-resistance was found to be high — 90% for rifampicin, 84% for isoniazid, and 83% for fluoroquinolones. Test specificity was 88-93%. “More importantly, 89-92% of samples tested positive for drug-resistance also tested positive when a standard method was used,” says Prof. Tyagi.

“The developed kits are to be evaluated for operational feasibility and performance in field settings under RNTCP. If found suitable, they can be used in the programme,” says Prof. Tyagi.

The kit for TB diagnosis was tested at the TB Hospital, Ambala and the National Institute of Tuberculosis and Respiratory Diseases (NITRD), Delhi using 1,190 samples. The kit for diagnosing drug-resistance was tested on 148 samples at NITRD and THSTI.

Presence of plantations, livestock significantly increases parasite diversity

Repercussions: When land use changes the internal niche is affecte too.

While we humans are known to destroy wildlife habitats and make life hard for animals, a new research has now pointed out that by changing the land use patterns and introducing livestock in their ecosystem we are indirectly increasing the parasite diversity in their gastrointestinal system. However, more studies are needed to understand if this might affect their population decline.

The team looked at over 4,000 mammalian faecal samples collected from 19 forest fragments at the Anamalai Hills of the Western Ghats. These samples belonged to 23 mammalian species including tigers, deer, porcupines, lion-tailed macaques, giant squirrels and otters. By analysing the faecal samples, they concluded that the presence of plantations and livestock significantly increased the parasite diversity. The paper published in Scientific Reports says that this could be due to spillover from the livestock and humans.

“By looking at the Natural History Museum parasite database, we noted that many of the parasites are known to infect humans. However, this doesn't necessarily mean that the people currently living in Anamalai Hills are currently infected,” explains Dr. Debapriyo Chakraborty, an independent disease ecologist and first author of the paper.

One Health approach

“We need to understand that these parasites can cause a broad range of infectious diseases and it is important to study the host-parasite interactions at a community level. Also, one host can have multiple parasites and vice versa,” says Dr. Chakraborty. “Before any new interventions like land use changes or habitat modifications are being carried out complex health consequences of these large-scale human activities should be kept in mind.”

He adds that the One Heath approach needs to be used as parasites, their animal hosts and humans are a complex system which needs to be studied together.

Human settlements

Also, some studies have shown that wildlife tends to congregate near human settlements. Herbivores such as deer may find the human settlements as a source of regular food and also as a safe zone where carnivores don't enter. This could also be a reason for pathogen spillover from livestock to wildlife. The group had shown previously that high parasite prevalence was one of the reasons for the decline of endangered lion-tailed macaque populations in small and degraded forest fragments in the Western Ghats.

“When the land use changes — coffee plantation to tea plantation or when degraded forests are converted to a coffee estate — the internal niche is affected. Many epidemic diseases including the Ebola virus started finding humans as a better new host when their natural habitats were disrupted. Similarly unknown diseases could occur as a result of this anthropogenic land use changes,” explains Dr. Govindhaswamy Umapathy at the Laboratory for the Conservation of Endangered Species, Centre for Cellular & Molecular Biology (CSIR-CCMB), Hyderabad. He is the corresponding author of the work.

“By changing the existing landscape we are not just affecting the wildlife but also taking a toll on the weather and rainfall patterns. The identified eco-sensitive zones must be conserved for the wellness of all,” adds Dr. Umapathy.

Bacteria form biofilms when exposed to low concentrations of antibiotic

Weathering stress: Bacteria sticking together to form pellicles.

It is well known that low concentrations of antibiotics can cause resistance to evolve among bacteria. Now, a group of researchers from IISER Pune has taken this further to explore how exactly this happens. They have studied how resistance to the antibiotic rifampicin evolves in E. coli under two conditions — when the antibiotic is present in low or high concentrations, and when there is steady or pulsed supply of antibiotics.

Bacteria develop drug resistance both when they are within the body and outside. The fact that antibiotics are unevenly distributed within the body or intake of drugs could be stopped midway can lead to evolution of drug resistance. Similarly, low doses of such drugs available intermittently in the environment can also cause drug resistance to evolve in the bacteria.

According to a study published in the journal Genetics, the process of evolution of drug resistance appears to be rapid. “We found that E. coli can evolve resistance to rifampicin within a few generations of drug exposure. That’s less than half a day,” says Nishad Matange from the Biology department of IISER, Pune, who studied emergence of resistance under laboratory conditions.

A characteristic of some drug-resistant strains of bacteria is that they do not live in isolation but get connected to each other, forming biofilms. Using genetics and biochemistry, the researchers found that when under exposure to low concentrations of rifampicin, the E. coli tend to form biofilms. This did not happen when they were exposed to high concentrations of antibiotic. “This is pretty dangerous since biofilms by themselves are a major challenge for hospitals and clinicians,” says Dr Matange.

Many genetic changes in the E.coli descendents were observed when the bacteria were exposed to low concentration of the antibiotic rifampicin. In order to understand the relevant genetic mutations that helped the bacteria form biofilms, the researchers engineered the individual mutations into the parent bacteria and studied which particular mutation was responsible for resistance against rifampicin. They found that biolfilm formation was mediated by the activation of particular gene called the fim operon promoter. Activation of the gene allowed the expression of a type of fimbriae — thread-like structures that help a bacterium attach itself to another bacterium. These are important in the formation of biofilms.

Though the researchers have specifically studied how E. coli evolve resistance against rifampicin, the results may be extrapolated to Gram-negative bacteria in general. This will be useful in studying drug resistance in other Gram-negative bacteria such as Klebsiella and Salmonella which cause hazardous infections.

However, the team is planning to extend this study to a host of other antibiotics. “Eventually, we aim to generate a multi-antibiotic low and high drug concentration genetic map that will point out significant genes and cellular pathways that are responsible for the evolution of resistance at low and high drug respectively,” says Dr Matange.

The bacteria have become resistant to all commonly used antibiotics. The highest resistance was against sulfamethaxozole

Grave situation: Vibrio cholerae pathogen isolated from patients is resistant to multiple drugs

A study of 443 clinical isolates of cholera-causing bacterium (Vibrio cholerae) isolated from the faecal sample of diarrohea patients from two sites — Kolkata and Delhi — reveals how extensively the bacteria have developed resistance against most routinely used antibiotics.

Using 22 antibiotics belonging to nine different classes, a team led by Bhabatosh Das from the Centre for Human Microbial Ecology at the Translational Health Science and Technology Institute (THSTI) found that 99% (438) of the isolates have developed resistance to more than two antibiotics, over 17% (76) to more than 10 antibiotics and 7.5% (33) to more than 14 antibiotics. The results were published in the journal Proceedings of the National Academy of Sciences (PNAS).

The highest resistance (99.8%) was seen against the antibiotic sulfamethaxozole, whereas resistance to neomycin was the least, with only 4% showing resistance.

Isolates collected from Kolkata showed relatively less resistance than isolates collected from Delhi. This might probably because the Kolkata isolates were collected between 2008 and 2013, while the Delhi samples were collected during 2014-2015.

The team sequenced the whole genome of the bacteria isolated during 1980, 2000s, 2014 and 2015. “In 1980, fewer antibiotics were used and resistance was minimal. With increasing usage of different antibiotics, the resistance against them has also increased. By 2014-2015 the bacteria have become extensively drug resistant (XDR) to all commonly used antibiotics,” says Dr. Das.

The team then studied if the resistant genes are still functional and found that all antibiotic genes that have been identified so far are indeed functional.

To check if the genes are functional, the team tested the susceptibility of E. coli to different antibiotics and then transferred the resistant gene to the E. coli. “We found that E. coli which was earlier susceptible to a particular antibiotic became resistant after the gene was transferred,” says Jyoti Verma from THSTI and the first author of the paper.

Secreation of proteins

Next the researchers studied whether the proteins responsible for antibiotic resistance get synthesised in the presence and absence of antibiotics. The scientists found that even in the absence of the antibiotics in the culture environment, the proteins get expressed.

“That was really surprising,” says Dr. Das. “The expression of the antibiotic resistance proteins even in the absence of the antibiotic suggests that it may contribute to other cellular metabolic processes.”

“We found that a single isolate has multiple resistant genes from the same class that can neutralise the effect of different antibiotics. So even if we introduce a new generation of antibiotic, the bacteria will most probably be able to neutralise it,” he says.

The resistant genes are genetically linked to different mobile genetic elements, which mean that resistance can spread very easily and quickly to other bacterial species through horizontal gene transfer.

Among more than 206 serogroups of V. cholerae, only two serogroups are pathogenic cholera-causing bacteria. They are generally not found in the environment but only in animal reservoirs such as water birds and crustaceans.

The mechanism

So how do the bacteria develop resistance to antibiotics if not naturally present in the environment at all times? “The toxigenic V. cholerae are detected in the gut of even healthy individuals. But they do not manifest the disease cholera as other bacteria present in the gut repress the expression of the toxigenic genes,” Dr. Das says.

Dr. G. Balakrish Nair from the Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram adds: “The V. cholerae are naturally competent to uptake DNA from the environment. So in the gut when the bacteria that have a resistant gene die the DNA gets released and are taken up by the V. cholerae.”

In a study published this year, a team led by Dr. Das demonstrated that when DNA is added in the growth medium, the V. cholerae bacteria tend to take up the resistant gene and become resistant in turn.

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