Saturday, September 30, 2017

Which viruses maybe seen in human semen

One of the least understood questions in virology in a recent turn of outbreaks like Ebola and Zika are that there are reports of these viruses being transmitted sexually. Now that we know that semen by itself possibly harbours a microbiome (Link) there definitely is a good chance that many different microbes can harbour in reproductive tissues such as testis. But there is no comprehensive literature on all possible viruses infecting the male reproductive tract. 

A meta-analysis which searched for all the literature available on evidence of virus presence in semen turned up 3,818 PubMed search results. Screening all the literature, the authors concluded that 26 viruses can survive in human semen which can seed a viremia. A summary is shown in Table 1.
Table 1: Viruses that are capable of causing viremia and found in human semen. Source
It should be noted that not all viruses listed have a defined capability to be sexually transmitted. But their biological effects on variables such as sexual health is not known. It is also known what is the duration of residence and how far they can seed viremia, serve as latency and what is the likely concentration. As Alex Salam puts it, "Clinicians need to consider the possibility that traditionally non-sexually transmitted viruses can persist in semen, and this, therefore, raises the possibility of sexual transmission. Detection means that evidence of viral genetic material or viral protein was found in semen. It’s important to note that this does not mean that the virus is viable, i.e., capable of replicating. To prove this, the virus needs to be isolated and grown in cells or animals. For many of the viruses, this test has not been done, so we don’t know whether virus is viable or not."

This paper needs to be followed up with laboratory tests to establish the results. Further, this list is definitely not complete. But at least we have a more likely and possible catalogue of which viruses may be found in semen.

References:

Salam AP, Horby PW. The breadth of viruses in human semen. Emerg Infect Dis. 2017. https://doi.org/10.3201/eid2311.171049

Friday, September 29, 2017

Zika Virus- Update III

Photo 1: Digitally-colorized TEM of
Zika virus. Source
The last I wrote about Zika was almost more than an year ago (Link). Ever since, literature on Zika Virus (ZIKV) has grown tremendously. It is too much to ask for a summary of everything that is known about zika to date. For all my posts on Zika, refer this link. In this post I will give the most recent updates of interest.

Zika virus has roughly a global distribution and 79 countries are in the spotlight. The geographical regions affected are categorised into 4 categories (Category 1-4). Category 1 represent countries with new introduction of Zika virus since 2015 with ongoing transmission. Category 2 are areas with either the evidence of virus circulation before 2015 or with ongoing transmission of the virus that is no longer in the new or re-introduction phase, but where there is no evidence of interruption. Category 3 are regions with interrupted transmission and with potential for future transmission. Category 4, where the Aedes aegypti mosquito that spreads the disease exists, but there has been no reported transmission of the virus.
Fig 1: Global map of ZIKV infection categorised by country. Source
There are 2 hypothesis on why the ZIKV infection has emerged. The first is that the ZIKV infection was indeed causing infection earlier. But there was no targeted testing to detect them. The other is genetic hypothesis. As per this view, genetic change might have resulted in emergence of a virus strain with greater epidemic potential and virulence, causing epidemics with more severe disease. In a just published study a team of researchers have explored this hypothesis. The team strated with analysis of genetic differences between modern Zika strain and an ancestral strain isolated from a patient in Cambodia in 2010. Based on the initial analysis, a single serine to asparagine substitution (S139N) in the viral polyprotein was predicted as important. This was then proven with a mouse model which showed that virus with the S139N mutation caused the most damage to neuronal cells. As the author of the paper Chen-Feng Qin comments, "Besides host factors, such as low immunity to the virus in affected communities, there are definitely some other unknown viral proteins or amino acids that may contribute to the complex pathogenesis of microcephaly—independently or synergistically. Our study identified a unique genetic determinant that links to severe microcephaly".

The exact mechanism of pathogen invasion is not known. There is some indication that the virus targets neuronal progenitors in the developing brain. Research has suggested that AXL receptor tyrosine kinase is the cellular receptor for the virus. But most probably this is not the only receptor. Given that the ZIKV infection targets glial cells, researchers have tried to use Zika strain to attack glioblastoma in a laboratory model.

Photo 2: PET  brain images post-infection
with Zika virus. Source
In a recent research using PET scanning (using a probe [18F] DPA-714) scientists have imaged the brain inflammation following Zika virus infection in mice. They found that levels of Zika virus in the mouse brain increased from day 3 to day 10 post-infection. During the period, the mice showed a 2- to 6-fold increase in global brain neuroinflammation. The study highlited that despite Zika affecting local brain tissue parts, the inflammation extends to all parts of the brain.

The focus of research currently is the cross reactivity between Zika and Dengue. Zika and Dengue are closely related viruses. Both the viruses show antibody enhanced infection and hence there is the question as to how does dengue vaccine affect Zika infection. There is no clear answer to this question. There is some opinion that dengue infection enhances zika infection. More recently, it has been suggested that antibodies against DENV E-dimer epitope (EDE) not only neutralizes the dengue virus in mice, but also protects both adults and fetuses from Zika infection by neutralisation. The different findings in various papers is probably based on the various types of targetting antibody that is tested.

Zika has gained strong interntational attention and there is a great focus on studying the Zika transmission, pathogenesis, treatment and vaccine.

References:

Yuan etal. A single mutation in the prM protein of Zika virus contributes to fetal microcephaly. Science (2017). DOI: 10.1126/science.aam7120

David Baud, Duane J Gubler, Bruno Schaub, Marion C Lanteri, Didier Musso. An update on Zika virus infection. Lancet (2017). http://dx.doi.org/10.1016/S0140-6736(17)31450-2

Estefania Fernandez, Wanwisa Dejnirattisai, Bin Cao, Suzanne M Scheaffer, Piyada Supasa, Wiyada Wongwiwat, Prabagaran Esakky, Andrea Drury, Juthathip Mongkolsapaya, Kelle H Moley, Indira U Mysorekar, Gavin R Screaton, Michael S Diamond. Human antibodies to the dengue virus E-dimer epitope have therapeutic activity against Zika virus infection. Nature Immunology, 2017; DOI: 10.1038/ni.3849.

Kyle Kuszpit, Bradley S. Hollidge, Xiankun Zeng, Robert G. Stafford, Sharon Daye, Xiang Zhang, Falguni Basuli, Joseph W. Golden, Rolf E. Swenson, Darci R. Smith, Thomas M. Bocan. [18F]DPA-714 PET Imaging Reveals Global Neuroinflammation in Zika Virus-Infected Mice. Molecular Imaging and Biology, 2017; DOI: 10.1007/s11307-017-1118-2

Sunday, September 17, 2017

Compound isolated from C difficile act against C difficile: Avidocin-CDs


Clostridium difficile represents a unique problem in health care system. C difficile is not affected by a wide range of antibiotics and they start showing up when gut microbiome is depleted due to antibiotics. C difficile is often treated with antibiotics such as vancomycin, metronidazole and fidaxomicin which cause further disruption of the resident microbiota leading thus increasing chances of relapse. A faecal microbiome transplant (FTM) is an option but it comes with some challenges. For example, It is nearly impossible to rule out every possible infection from a healthy donor and we are technologically incompetent yet to find the right best combination of microbiome for the gut. The best answer would be an antibiotic that kills only and only C diff. That means we have to be extremely specific.

Fig 1: Electron micrograph of negatively stained
purified diffocin particles isolated from CD4 strain.
C difficile is a highly competitive bacteria. C difficile strains compete with each other and they produce an R-type bacteriocin (High-molecular-weight or phage tail-like) which is called as diffocins. In a generalised sense, R-type bacteriocins attack target cells by specific receptor-binding on the surface. This is followed by sheath contraction and insertion of the core through the envelope of the target bacterium. The diffocins are ultra specific in their activity and their receptor binding activity can be modified to attack C difficile in general by modifying its binding to specific receptors. 

Table 1: Sensitivity of various bacteria to modified diffocins.
Source
Table 1 shows a summary of the sensitivity of various bacterial strains to heterologously expressed, recombinant diffocins. A company that is currently working on using these diffocins and their modified version- Avidbiotics Corp (California based Biotech company) have named them as Avidocin-CDs. One of such constructs Av-CD291.2 had been found to have wide spectrum activity against a range of hypervirulent C difficile strains tested, without affecting the microbiome.

There is sufficient literature evidence to indicate that avidocins are highly specific for C difficile and able to survive transit through mouse GI tract.

In the latest paper, the story becomes better now. The team of scientists from the University of Sheffield, AvidBiotics Corp, and the University of Glasgow have published a study on Avidocin-CD291.2 on how it could be extended for clinical use. Analysis of rare Av-CD291.2–resistant mutants enabled identification of S-layer protein A (SlpA) as the target. The paper showed that Av-CD291.2–resistant mutants lack an S-layer. The lack of S-layer also introduces a high sensitivity to innate immune molecules combined with sporulation defects. These S-layer mutant strains survived poorly in the standard charcoal medium which is used to transport C difficile strains. Interestingly, acquisition of Avidocin-CD resistance results in loss of toxin production and complete loss of virulence.

As Dr Robert P. Fagan, senior corresponding author comments, "We discovered that the weapons naturally produced by C difficile and those engineered by our colleagues at AvidBiotics were using certain proteins in the S-layer to identify which strains to target. The C difficile S-layer is unique to these bacteria, which explains why Avidocin-CD killing is so specific. Scientists at AvidBiotics Corp were then able to engineer different versions of Avidocin-CD to target 12 of the 14 known types of S-layer."

Reference:

Joseph A. Kirk, Dana Gebhart, Anthony M. Buckley, Stephen Lok, Dean Scholl, Gillian R. Douce, Gregory R. Govoni, Robert P. Fagan. New class of precision antimicrobials redefines role of Clostridium difficile S-layer in virulence and viability. Science Translational Medicine, 2017; 9 (406): eaah6813 DOI: 10.1126/scitranslmed.aah6813

Tuesday, September 05, 2017

Hypervirulent Klebsiella pneumoniae

One of the most common ideas associated with clinical antibiotic resistance is the fitness factor. I have in multiple posts talking about this idea in my previous posts. The idea is that when a bacteria acquired genes for antibiotic resistance there is a fitness cost associated with it. Until and unless there is this constant antibiotic threat to the bacteria, it is not so useful for the bacteria to keep resistance genes, since there is a cost associated with maintaining that gene. There are several experiments that showed at least for some drugs that if you mix a sensitive and resistant phenotype of a given organism and allow it grow together on an antibiotic free condition, the sensitive strain tends to dominate easily.

The idea is all fine and good in laboratory conditions. In a series of 5 hospital acquired pneumoniae infection (All fatal) following surgery in a Chinese hospital, a Klebsiella pneuomoniae ST11 strain has been isolated which is not only an MDR (Multi drug resistant) but also a hypervirulent strain.

The study, in summary, basically went something like this. There were 5 back to back pneumoniae cases following surgery for traumatic cases who developed pneumoniae and died of it. The first one in the series was identified as the most probable patient zero (Index case). The other four patients were located in different wards with overlapping stays. In all the cases multiple samples were taken and all the Klebsiella pneumoniae strains isolated were fully characterised using phenotypic and genetic approaches. In total 21 non-repeated carbapenem-resistant K pneumoniae strains were recovered from various clinical specimens of the five patients

What was really interesting to me was that they did a string test, human neutrophil assay and a wax moth virulence test to establish its hypervirulence status. These tests are really valuable models and so I think I should explain these in a little bit of detail.

Photo 1: Positive “String test” on a hypervirulent
strain of K. pneumoniae. Source
String test (Don't confuse this with the string test done to identify Vibrio cholerae), is a kind of qualitative marker used to test the hypermucoviscous phenotype seen in Klebsiella pneumoniae which are an indicator or hypervirulence. Basically, you grow the organism on a 5% sheep blood agar at 37°C overnight. The string test is positive when a bacteriology inoculation loop or needle is able to generate a viscous string > 5 mm in length by stretching bacterial colonies on an agar plate. See Photo 1. In this paper, authors reported that the string test was positive for all five strains, each producing strings longer than 20 mm.

Fig 2: Neutrophil Survival of K pneumoniae strains.
Source
Neutrophil assay is a test to see how good the bacteria resist killing by the neutrophil in laboratory conditions. In principle,  neutrophils obtained from healthy volunteers. A well containing 10⁶ neutrophils and 10⁶ CFU of opsonised K pneumoniae in RPMI/H medium is prepared at 37°C. A small sample is taken at intervals and bacteria is plated on Luria broth agar. Survival is calculated as a percentage of CFUs with reference to controls. For the study, K pneumoniae 4 and 5 (representative strains), two classic ST11 strains FJ8 and FJ9 (known to be not hypervirulent strains) and two known K1 hypervirulent K pneumoniae strains 1088 and 91 along with PC K pneumoniae which was removed for its virulence plasmid were studied. Fig 1 shows the results. You can clearly see that the strains 4 and 5 were really evading the neutrophil killing.

Fig 2: Virulence potential of isolated strains.
Source
Another test for virulence was done using Galleria mellonella or honeycomb moth. Basically, the bacteria are incubated with the larvae and you look for the ability of the larva to survive. For this study, cultures of K pneumoniae strains were washed with PBS and larva was infected with the bacteria and survival rate of the larvae was studied. See Fig 2. 

The study does provide a compelling evidence that the strains they isolated were hypervirulent. Yes, they were also MDR strains but most of the K pneumoniae strains isolated globally are MDRs. But mind you they are not super bugs. I wasn't sure if these strains were resistant to drugs like Colistin and from the data they appear tigecycline sensitive.

I take this study as a proof that ST11 type which is a common circulating type in Asia is capable of hypervirulence. They have a 170 kbp plasmid (pVir- CR-HvKP4) which makes it hypervirulent, multidrug resistant, and transmissible. Considering so many people are dying from Klebsiella pneumoniae infections it may be worthwhile to test how many percentages of it is actually hypervirulent.

Reference

Danxia Gu, Ning Dong, Zhiwei Zheng, Di Lin, Man Huang, Lihua Wang, Edward Wai-Chi Chan, Lingbin Shu, Jiang Yu, Rong Zhang, Sheng Chen. A fatal outbreak of ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae in a Chinese hospital: a molecular epidemiological study. The Lancet Infectious Diseases, 2017. https://doi.org/10.1016/S1473-3099(17)30489-9