key: cord-0979277-95wkmwvp
authors: Oliveira, Manuela; Mason-Buck, Gabriella; Ballard, David; Branicki, Wojciech; Amorim, António
title: Biowarfare, Bioterrorism and Biocrime: a historical overview on microbial harmful applications
date: 2020-06-20
journal: Forensic Sci Int
DOI: 10.1016/j.forsciint.2020.110366
sha: e2c894b5e53df86fec564c6d2166e3d8ca143379
doc_id: 979277
cord_uid: 95wkmwvp

Microbial Forensics is a field that continues to grow in interest and application among the forensic community. This review, divided into two sections, covers several topics associated with this new field. The first section presents a historic overview concerning the use of microorganisms (or its product, i.e. toxins) as harmful biological agents in the context of biological warfare (biowarfare), bioterrorism, and biocrime. Each case is illustrated with the examination of case reports that span from prehistory to the present day. The second part of the manuscript is devoted to the role of MF and highlights the necessity to prepare for the pressing threat of the harmful use of biological agents as weapons. Preventative actions, developments within the field to ensure a timely and effective response and are discussed herein.

Microbial forensics (MF or forensic microbiology) is a recent and forthrightly expanding multidisciplinary branch of research that lays its foundations upon classical sciences such as forensic genetics, microbiology, epidemiology, medicine, molecular, and evolutionary biology [1] [2] [3] . Scientists within the field of MF endeavor to detect, identify, and trace the origin (i.e.: assigning to a source) of life-threatening pathogenic agents (bacteria, viruses, and toxins) 4 . This field has applications in a multitude of forensic casework scenarios, including bioterrorism 5; 6 , biocrime 7; 8 , fraud 9; 10 , outbreaks and transmission of pathogens 11; 12 , or accidental release of a biological agent, and/or a toxin 13; 14 . Traditionally, neither pathogens' outbreak monitoring nor toxicology is regarded as MF topics. Nonetheless, in our opinion, these two topics are fundamental to MF. The dispersion of pathogens can be either natural or incidental. If incidental, it can be either intentional or due to medical malpractice. Therefore, the application of reliable and robust surveillance protocols for pathogen monitoring would provide valuable information to distinguish between the spontaneous and harmful spread of microorganisms (i.e., related to biowarfare-BW, bioterrorism -BT, or biocrime -BC). Besides the differences between forensic investigations and epidemiological studies in terms of protocols and objectives, it is our opinion that the investigations of the sources of outbreaks fall in the domain of MF due to the shared aim of determining the source of the microorganisms involved (Figure 1) 2; 3; 12 .

As such, one of the priorities in MF is to distinguish between an accidental and an intentional release of a given pathogenic agent. Healthcare personnel should be constantly alert for unusual, unexpected, or unexplained illnesses. According to the United States Centers for Disease Control and Prevention (CDC) (http://emergency.cdc.gov/Documents/Planning/PlanningGuidance.PDF), several diagnostic indicators might point out an infectious disease outbreak associated with the intentional release of a biologic agent. Among them are: i) an unusual temporal or geographic clustering of illness, such as persons who attended the same public event or gathering or patients presenting with clinical symptoms suggesting an unexplained infectious disease outbreak (e.g., febrile illness, sepsis, pneumonia, respiratory failure, or rash);

ii) an unusual age distribution for common diseases (e.g., an increase of a children-characteristic illness among the adult population);

iii) a large number of cases of acute flaccid paralysis with prominent bulbar palsies, suggestive of a release of botulism toxin.

An intentional release of a given biological agent can constitute either a covert or an overt action. In the case of covert action, this release remains unannounced and concealed, going unnoticed for days or even weeks. The first sign of the spread is the occurrence of sick individuals, that may unknowingly be infecting others. An infected person may search for medical care, possibly at a distance from the release area. Contrarily, in the case of overt action, this release is immediately noticed and may even be announced. Contrarily, in overt actions, public health officials, and healthcare and communication systems are readily informed by perpetrators and overwhelmed by requests for information and treatment. Overt action aims to cause widespread panic.

The use of pathogenic agents as biological weapons (also known as bioweapons), present several advantages over other more conventional weapons (e.g., chemical weapons):

i) microorganisms are relatively inexpensive and can be easily mass-produced;

ii) large quantities of biological agents can be effortlessly concealed in small vials and easily transported;

iii) some agents can become water or airborne resulting in a widespread area of dissemination within a short time frame; iv) some of these pathogenic agents present person-to-person transmission.

Although plant materials can also be used for similar purposes (e.g., abrin and ricin), this work focuses on topics related to the use of microorganisms as bio-weapons, from exploring the historical perspectives of BW to the most recent applications in BT, and BC.

Biowarfare (BW) refers to the intentional use of biological agents (e.g., bacteria, viruses, fungi, and toxins) as weapons in war scenarios 15 . BW agents can be deadlier than other conventional weapon systems as even minute quantities can cause mass casualties and/or fatalities depending on the agent used 16; 17 .

The intentional use of microorganisms (or their toxins) as weapons is almost as old as humanity itself. Since pre-historic and ancient Greek and Roman times there have been reported examples such as the use of poisoned darts or contaminating water springs and wells with corpses or cadavers. From this early stage, BW has become more sophisticated, leaning towards the capability of being a weapon of mass destruction when associated with an appropriate delivery system, as specialized munitions on the battlefield and for covert use. Such developments are a direct result of advances in both the fields of microbiology and biotechnology 18; 19 .

As such, the historical evolution of BW can be divided into three distinct periods: i) from prehistory to 1900: except for a few well-documented cases, before to the foundation of Microbiology as a science -as a direct result of the studies of Louis Pasteur and Robert Koch culminating in the acceptance of the germ theory of disease -it is difficult to clarify if these BW attacks constituted real threats or were a part of political hoaxes; ii) from 1900 through 1945: this period is characterized by the emergence of small and unsophisticated national BW programs (e.g., Germany, Japan, the Soviet Union, and the United States; in detail below) and the use of biological weapons in both World War I and II; iii) after 1945: the broader access to biological agents and the progress made in the fields of biotechnology and biochemistry allowed the BW programs to become democratized and accessible even to small groups and individuals. In this period, the lethal potential of BW agents increased due to the developments in genetic engineering (Table 1) 20; 21 .

For both microbiologists and historians it is challenging to distinguish between natural epidemics and deliberate biological attacks (e.g., plague outbreak during the siege of Caffa, malaria outbreak by the Napoleonic armies, yellow fever, and smallpox outbreaks during American Civil War; Table 1 ), mainly due to:

i) the lack of reliable scientific data regarding an alleged bioterrorism attack, especially before the advent of modern microbiology;

ii) the secretive nature and polemical conditions surrounding any putative biological attack, within which the available documents are susceptible to multiple political manipulations, and thus difficult to interpret objectively;

iii) the chronological distance from the ancient reports on these biological attacks, and the possible misunderstanding if they are read through a contemporary lens 21; 22 .

At the beginning of World War I (WWI), between late 1914 and early 1915, Germany implemented the first documented state BW program. The country was also a pioneer in the dissemination of biological weapons and for launching a true BW campaign, employing different biological pathogens against several neutral countries -including Argentina, Finland (by the time under Russian control), France, Norway, Romania, Russia, Spain, and the United States 20 . At the time, the main aim was to target livestock being shipped from neutral countries to the Allies in the anticipation of gaining a tactical advantage on the battlefield. This relied heavily on the collaboration of covert operatives (e. g., Anton and Carl Dilger, Frederick Hinsch, and Paul Hilken) in seaport cities to disseminate the pathogens 23 (Table 1 ). An example of such biological sabotage programs included the inoculation of allied horses and mules with glanders (Burkholderia mallei) and anthrax (Bacillus anthracis). However, such efforts did not achieve the expected military consequences.

After WWI, this program continued; it has been suggested that the bacterial agents causing typhoid (Salmonella enterica serovar Typhi), paratyphoid fever (S. enterica serovar Paratyphi), dysentery (Shigella spp.), cholera (Vibrio cholera), plague (Yersinia pestis), glanders (B. mallei), anthrax (B. anthracis), and wound infections (e.g., Staphylococcus aureus, Streptococcus spp., Bacteroides spp., Clostridium spp.)

were the most suitable for the German war efforts, mainly when these microorganisms were used for the production of aerosols or the contamination of water sources 24 .

During World War II (WWII), in 1941, despite Adolf Hitler's recurrent and stringent commands against the use of biological weapons, Heinrich Himmler founded an entomological institute, under the cover of the concentration camp at Dachau. Under the supervision of Eduard May, allegedly this institute was devoted to defensive research for Waffen-Schutzstaffel (commonly known as the SS) guards and staff protection against insect-borne diseases, such as malaria (Plasmodium spp.) and louse-vectored typhus (Rickettsia prowazekii). However, according to May's reports, it has been suggested that the true objectives of this institute were associated with the German BW program, including offensive purposes. Also, in 1943, Hermann Göring ordered the creation of a unit of "cancer research" at a new institute in Nesselstedt (near Poznan, Poland), under the supervision of Kurt Blome. It has been suggested that both Rudolf Mentzel and Erich Schumann were interested in the development of biological weapons for the Third Reich 25 .

Besides the Dachau facility, other institutions were involved in biological weapons development for defensive proposes; activities were restricted and few scientists were involved in these studies. Some of this workforce was devoted to the development and production of vaccines (e.g., for the foot and mouth disease virus, plague, and yellow fever) or sera (e.g., anthrax, botulism, and tularemia), while other activities focused on the dissemination of aerosolized pathogenic agents (e.g., aerosols dispersed using aircraft, dissemination using glass containers, and insect-borne dissemination). Field-tests were conducted by emulating non-pathogenic bacteria and aerial spraying of the foot and mouth disease virus 24 . On the 29 th of April 1945, American troops liberated Dachau and dissolved the entomological institute 25 .

The Japanese BW program began in 1932 under the command of Major General Shiro Ishii with the creation of the first biological weapons research facility at the Tokyo army's medical school, concealed as part of the Kwantung Army's Water Purification Department. Although considered as quite primitive in the level of technical sophistication, this program enrolled 3000 military personnel and hundreds of the foremost medical doctors and technicians and included more than 150 research centers, including the infamous Unit 731 (known as the Epidemic Prevention and Water Purification Department, this research center was located in Pingfang, Manchuria -responsible for human experimentation during the Second Sino-Japanese War), scattered throughout the Japanese empire, from Manchuria to Indonesia 26 .

Organisms and diseases of interest to the Japanese program were possibly tularemia (Francisella tularensis), glanders (B. mallei), meningitis and other forms of meningococcal disease (Neisseria meningitidis), brucellosis (Brucella spp.), typhoid fever (S. enterica serovar Typhi), dysentery (Shigella spp), cholera (V. cholerae), plague (Y. pestis, disseminated via the Uji bomb), anthrax (B. anthracis, disseminated via the Ha bomb), botulism (Clostridium botulinum; toxin) and smallpox (Variola major) ( Table 1) This program remains throughout History as one of the most atrocious by its dimension and the experiments performed on humans. It is estimated that at least 3,000 prisoners of war (Chinese, Koreans, Mongolians, Soviets, Americans, British, and Australians) were allegedly used as test subjects in Unit 731.

These tests included inoculation with several pathogens, vivisection, weapons tests, and germ warfare attacks 21 . No prisoner left the Japanese death factories alive 27 . As a consequence of the dissemination of pathogens during the Japanese BW program, it is supposed that thousands of civilians perished as a direct consequence of the attacks 27 . Moreover, due to the difficulty in controlling the dissemination, several Japanese soldiers were also victims of these attacks 21 .

Immediately after the end of WWII, the Soviets convicted Japanese scientists engaged in these activities for war crimes. However, the United States government covertly granted immunity to these scientists, possibly to gain exclusive access to the work developed and scientific breakthroughs from the BW program 28 .

The American BW program was secretly implemented, in 1942, under the auspices of President Franklin Roosevelt after the suspicion of the use of BW agents by Germany and Japan 29 . In 1947, President Harry Truman withdrew the US from the Geneva Protocol with Senate approval and conducted open-air field testing using non-pathogenic bacteria (Bacillus globigii and Serratia marcensces) on naval vessels near the Virginia Coast and San Francisco Bay, along with the dissemination of bacterial aerosols in bus stations and airports. At its peak, this program involved nearly 3,400 people and several research and production facilities and was responsible for investigations on more than 30 agents, including bacteria (e.g., B. anthracis, Brucella suis, Coxiella burnetti, Francisella tularensis), toxins (e.g., botulin and staphylococcal enterotoxin B), and viruses (e.g.,Venezuelan equine encephalitis and yellow fever virus) 30 .

The program was officially dismantled by President Richard Nixon, in 1969 and all facilities involved were converted for defense purposes only. In 1972, the Biological Weapon and Toxin Convention (BWTC) was signed 30 . However, before this decision, ten different BW agents have been weaponized, sophisticated delivery systems had been developed, and a huge research program was in place in the country 31 .

The Soviet BW program was initiated in the mid-1920s and was secretly continued until at least the 1990s, even after singing the BWTC, in 1972. This program was divided into two phases: i) from its start to 1972 (including the WWII, Russian Civil War, and the beginning of the Cold War): this period was characterized by the research and use of naturally occurring microorganisms;

ii) from 1972 to 1991: during this period, even after the termination of the American BW program, the Soviets increased the level of sophistication of their program, investing in biotechnological developments to create novel or modify existing bacterial and viral strains. It has been hypothesized that many of the BW scientists remain in Russia providing basic know-how to launch a third phase of the program with the support of the current President of the Russian Federation, Vladimir Putin 33 . At its peak, the Soviet BW program included nearly 60,000 scientists and technicians dispersed across more than 50 military and research centers 34 .

Around 50 biological agents were developed using either naturally occurring or genetically engineered strains of several bacteria (e.g., B. anthracis, Brucella melitensis, B. mallei, Clostridium perfringens, J o u r n a l P r e -p r o o f Clostridium tetani, F. tularensis, and Yersinia pestis), toxins (e.g., botulinum neurotoxin) and viruses (e.g., Marburg virus, smallpox virus, Variola major, and Venezuelan Equine Encephalitis Virus) 34 .

After the disintegration of the former Soviet Union, many scientists (e.g., Vladimir A. Pasechnik and Kanatjan Alibekov) deserted and provided international intelligence agencies with details about the ongoing works of Biopreparat (Biological Substance Preparation -the Soviet Union's major BW agency from the 1970s onwards). This information forced President Boris Yeltsin to admit not only that the country had covertly sustained its BW program (after signing the BWTC) but also that they were responsible for the anthrax outbreak (accidental aerosol release from a military facility) at Sverdlovsk, in 1979 34 .

The Iraqi BW program was launched, in the early 1980s, by Saddam Hussein. Throughout the Gulf War, the Iraqi Biological Research Centre for Military defense, located at Salman Pak, studied the use of several bacteria (e.g., B. anthracis, B. melitensis, C. botulinum, and C. perfringens), toxins (e.g., aflatoxin, trichothecene), and viruses (e.g., camelpox virus, influenza virus, rotavirus, and West Nile virus) as bioweapons. This research culminated in the mass production of bioweapons either loaded into munitions (200 bombs and 25 ballistic missiles) or stored in spray tanks for later dissemination as aerosols. As a result, both troops on the battlefield and civilian populations in the region of conflict were threatened by the possible use of these weapons 35; 36 .

The United Nations Special Commission (UNSCOM), mandated to eliminate and prevent the revitalization of the Iraqi BW program, was expelled from Iraq, after seven years of work. As a consequence, Operation Desert Fox began. During its duration, the United States and Great Britain bombarded and destroyed three locations associated with the BW program 35; 36 .

Contrary to BW, in a BT attack, biological agents are intentionally released against a civilian population 15 . This spread is motivated or justified by ideological objectives (either political or religious) intending to cause panic, mass casualties, or economic loss 37 . The biological agents can be used as they naturally occur or be genetically modified to improve mass dissemination (e.g., higher mortality or resistance to currently available medicines and vaccines) 38 . When facing the possibility of a BT attack is crucial to identify the agent involved, not only to prevent panic among the population but also to control the morbidity and mortality associated with the spread of the agent 39 In a BT attack using bacteria as weapons the most frequent diseases are anthrax (Bacillus cereus biovar anthracis -a variant of the B. cereus bacterium that has acquired plasmids similar to those of B. anthracis) 42 , glanders (B. mallei) 43 , melioidosis (Burkholderia pseudomallei) 43 , brucellosis (Brucella spp.) 44 , diphtheria (Corynebacterium diphtheria) 45 , Q fever (C. burnetii) 46 , hemolytic-uremic syndrome (Escherichia coli 0157:H7) 47 , tularemia (F. tularensis) 48 , salmonellosis (Salmonella spp.) 49 , typhus fever (R. prowazekii) 50 , Rocky Mountain spotted fever (Rickettsia rickettsii) 50 , cholera (V. cholera) 51 , plague (Y. pestis) 52 . In an attack using bacterial toxins the most frequent diseases are botulism (C. botulinum, neurotoxins) 53 , gangrene (C. perfringens, Epsilon toxin) 54 , dysentery (Shigella dysenteriae -Stx1 and Stx2) 55 , and food poisoning (Staphylococcus aureus, enterotoxins T-2 toxin and tetrodotoxin) 56 .

In a BT attack using viruses as weapons the most frequent diseases are encephalitis (Eastern Equine Encephalitis, Tick-borne encephalitis-complex viruses -Central European tick-borne encephalitis, Far Eastern tick-borne encephalitis, Russian spring and summer encephalitis viruses -, and Venezuelan equine encephalitis virus) 57-59 , hemorrhagic fever (Chapare, Crimean-Congo, Ebola, Guanarito, Kyasanur Forest disease, Lassa fever, Lujo, Machupo, Marburg, Omsk, Rift Valley fever, and Sabia virus) 60; 61 , respiratory syndromes isolated (highly pathogenic strains of 1918 Influenza and Avian Influenza H5N1, and SARS virus) [62] [63] [64] [65] or in combination with neurological symptoms (Hendra and Nipah virus) 66 , immunodeficiency syndromes (Human immunodeficiency virus -HIV) 67 and rashes (Foot-and-mouth disease 68 , Monkeypox 69 , Variola major -Smallpox -and Variola minor virus -Alastrim [70] [71] [72] . Depending on the effortlessness of transmission, the morbidity and mortality rates associated, and their capability to be mass-produced, these biological agents are classified into three categories (A, B, and C) by the CDC (https://emergency.cdc.gov/agent/agentlist-category.asp) 73; 74 (Table 2 ).

In 1972, a group designated R.I.S.E. (the acronym is not fully known but it is believed that R was for Reconstruction, the S for Society, and the E for Extermination; the I remains a mystery 75 ) attempted to attack water treatment systems, mainly targeting the Chicago area. This group intended to relaunch a new society more toned with ecological values by killing off the already existing world population. This attempt failed since some of the group recruits denounced the plans to the Chicago Police Department. The attempt failed because some of the new recruits of the groups denounced the plans to the Chicago Police Department. Subsequent analysis, performed by the CDC, concluded that the group possessed viable cultures of S. enterica serovar Typhi, Shigella sonnei, C. botulinum, Neisseria meningitidis, and C. diphtheria.

Allen A. Schwandne (the group leader) and Steve Pera (a biology expert working in collaboration with the group) were arrested and released on bail. Both activists retreated to Cuba; Mr. Schwandner was arrested for counterrevolutionary activities against the Cuban regime, sentenced to six years, and died during imprisonment. In 1974, Mr. Pera voluntarily returned to the United States, negotiated a plea agreement, and sentenced to five years imprisonment 76 .

In 1981, an activist group auto-denominated "Dark Harvest Commandos" dropped a package at the London-Exeter railway line, near the Porton Down campus, where the Chemical defense Establishment was located. The package contained soil collected from Gruinard Island, a testing site for anthrax spore bombs used by the British government during WWII (1941) . With this attack, the group claimed to be returning the "seeds of death" to their source. The analysis performed revealed low concentrations of B. anthracis spores (approx. 10 organisms/gram of soil), in a form that was highly unlikely to cause any harm. As such, following the test, in 1941, the levels of contamination had been decaying by natural means. This group sent a second package addressed to the Conservative Party Conference. This group sent a second package addressed to the Conservative Party Conference. However, in this second incident, no spores were identified in the soil analyzed. In 1986, after the initiative to decontaminate the soil, the activities of this group ceased 77; 78 .

In 1984, before a forthcoming election in Wasco County, the Rajneeshees -a religious cult -intended to sicken voters so they would be incapable of voting, hence enabling the cult to gain political control of the county seat and government. Salmonella enterica serovar Typhimurium bacteria (strain ATCC 14028) was isolated, cultured, and mass-produced from Bactrol discs -legitimately acquired from VWR Scientific (Seattle, Washington) to be used in the Rajneesh Medical Corporation (RMC) licensed laboratory -and used against inhabitants of The Dalles 76; 79 .

This remains the largest act of BT in the United States and included three separate incidents. The first incident consisted of contaminating the hand of several commune members. These members then greeted people with a handshake and contaminated doorknobs and urinals at the Wasco County courthouse. However, no casualties were registered as a result of this attack. The second incident, in August, was perpetrated against three county commissioners unfavorable to the Rajneeshees. While visiting the commune, the County commissioners were offered drinking water contaminated with the selected bacteria. Among these representatives, two became sick and one was hospitalized. In the third incident in September of the same year, members of the cult contaminated salad bars, salad dressing, and coffee creamers in local restaurants with the same bacteria. As a consequence of this attack, more than 750 people became sick and 45 were hospitalized 80 .

The cult was also studying the use of several other agents (S. enterica serovar Typhi, Enterobacter cloacae, Neisseria gonorrhoeae, S. dysenteriae, hepatitis, and AIDS) and the possibility of contaminating The Dalles' water supply system using dead rodents (beavers, rats, and mice) 79 .

The Aum Shinrikyo is a religious cult that became widely known for the attack perpetrated in the Tokyo subway system, on the 20 th March 1995, using Sarin gas (a deadly nerve agent) poisoning nearly 6,000 people. The cult claimed to have between 20,000 to 40,000 worldwide followers and a net worth of $1.5 billion dollars 81 .

Besides this chemical agent, the cult was also involved in BT activities using toxins and microorganisms, such as botulinum toxin, B. anthracis, V. cholera, and C. burnetii while attempted to obtain Ebolavirus 82;

J o u r n a l P r e -p r o o f 83 . To maintain its sufficiency, instead of buying cultures from external suppliers, the cult isolated their own cultures. This fact may explain the several unsuccessful BT attempts executed by the cult. Clostridium botulinum was harvested in the soil from Ishikarigawa Basin during the spring of 1990 81 . In April 1990, the cult attempted to release botulin toxin, using a vehicle driven around the Japanese Parliament and other government buildings in Tokyo. Three years later, by the occasion of the Crown Prince wedding, the cult sprayed this toxin from a truck circulating the Imperial Palace and other main government buildings in Tokyo. Another two years later, right before the Sarin gas attack in the subway, an attack with botulin toxin was prevented in the subway at Kasumagaseki Station 81 . Also, an anthrax strain presenting high homology to the Sterne 34F2 strain, the most commonly used for cattle and livestock vaccination, was sprayed on the roof of the Aum building in East Tokyo 84 .

On the 18 th September 2001, a week after the attacks on the Twin Towers of the World Trade Center in New York, letters containing spores of B. anthracis were mailed to media companies (ABC News, CBS News, NBC News, the New York Post, and the National Enquirer). These letters held the postmark from Trenton, New Jersey. Only two of these letters were recovered (one addressed to the New York Post and other to Tom Brokaw at NBC).

The second wave of letters began three weeks later, on the 9 th October. Two more letters with the same postmark were sent to the Senate offices of the Democratic U.S. Senators Tom Daschle (South Dakota) and Patrick Leahy (Vermont).

As a consequence of these attacks, between the 4 th October and 20 th November 2001, a total of 22 anthrax cases (11 by inhalation and 11 by cutaneous contact) were reported, five being fatal 85 . More than 30 people tested positive for B. anthracis spores' exposure and nearly 32,000 individuals underwent antibiotic prophylaxis 86 . On the 19 th February 2010, more than eight years after the anthrax letters, the "Amerithrax Investigative Summary" was published and officially closed the FBI's investigation. Bruce Ivins, a microbiologist and biodefense researcher from the US Army Medical Research Institute of Infectious Diseases (USAMRIID), in Fort Detrick, working on anthrax vaccines and treatment was incriminated as the sole perpetrator of these BT acts. However, on the 29 th July 2008, Dr. Ivins committed suicide through an overdose of Tylenol with codeine. Therefore, it has impossible to obtain a legal resolution concerning his guilt or innocence 87 . The government's case was considered circumstantial, based on the genetic similarity between the genetic features of the anthrax spores found in the letters and the cultures grown at Dr. Ivin's laboratory (strain RMR-1029) 88 . Curiously, during the early stages of the investigation, the FBI counted on the collaboration with Dr. Ivins 89 

Biocrime can be defined as the use of a disease-causing agent or toxin to kill, debilitate or cause panic for a specific individual or a limited group of individuals, motivated by personal reasons such as revenge, jealousy, or the desire for monetary gain by extortion 37; 39 . Therefore, the main differences between BC and BT are the number of people affected and the motivation behind the attack. Usually, the perpetrators of these crimes have both the required scientific knowledge and ready access to the biological agent to be used. The use of MF in BC investigations can be illustrated by the paradigmatic cases of the intended dissemination of S. dysenteriae Type 2 90 , HIV [91] [92] [93] , and hepatitis B (HBV) and C (HCV) viruses [94] [95] [96] . In such cases, it is of primordial importance to establish the source of the bioagent through the comparison of the isolate found in/on the victim and that associated with the perpetrator 39 .

The first documented case of the use of toxins with criminal intentions is associated with the murder of Captain Vassilli Buturlin, in 1910 (St. Petersburg, Russia). Patrick O'Brien de Lacy (his brother-in-law), with the assistance of Vladimir Pantchenko (a medical doctor), injected Captain Buturlin with diphtheria toxin as a consequence of an inheritance dispute. The initial plan was to inject the victim with cholera (at that time, endemic in the city) but instead, they administered the diphtheria toxin 97 .

During nine years (1909 -1918) the French insurance broker Henri Girard convinced his victims to buy life insurance policies and to make him their primary beneficiary. To obtain the death benefits, Mr. Girard and his collaborators spiked the victims' food with S. enterica serotype Typhi and natural toxins from the mushrooms belonging to the Amanita genus. Girard was studying bacteriology at the time of the first murder. Mr. Girard was responsible for the murder of Louis Pernotte and Madame Monin and for infecting or poisoning six others. Arrested in August 1918, Mr. Girard committed suicide while awaiting trial by ingesting one of his microorganism cultures (most likely typhoid) secretly hidden among his personal belongings. Before his death, Mr. Girard confessed to these crimes and implicated four accomplices that were then convicted and given sentences ranging from two years imprisonment to life imprisonment with ward work 20 .

From 1935 to 1936, the Japanese physician Dr. Tei-Sabro Takahashi was involved in five incidents of infecting several competing colleagues, their families, and his wife using pastries spiked with S. enterica serovar Typhi. As a result of these incidents, 17 colleagues were infected and three subsequently died. Dr. Tei-Sabro Takahashi was arrested by the Saitama Prefecture Police Department in 1937 and a year later he was convicted and sentenced to the death penalty 20; 98 .

Three years later, in 1939, the Japanese physician Dr. Kikuko Hirose, offered Ritsuo Kato (her former husband) pastries spiked with S. enterica serovar Typhi and S. enterica serovar Paratyphi. The pastries were then shared with his family and colleagues from Kawaike Elementary School, resulting in twelve individuals becoming infected and one death. Dr. Hirose was found guilty by the Supreme Court and sentenced to eight years imprisonment 20; 51 .

In the ten years comprehended between 1902 and 1912, the German variety artist Karl Hopf (also known as Athos) attempted to poison his father, mother, two children, and three former wives. Mr. Hopf studied chemistry and worked as a pharmacist in London, having experience in drugs' handling. When his third wife, Wally Siewec, became severely sick due to gastrointestinal illness, suspicions were raised against Mr. Hopf. A police search in his dwellings discovered morphine, opium, cyanide, digitalis, arsenic, and live cultures of typhus, cholera, and other pathogenic agents. Mr. Hopf was detained in April 1913 and judged in January 1914. He was found guilty of poisoning his two children and his first wife, and of attempting to poison his second and third wives. Mr. Hopf was sentenced to death and guillotined in March 1914 at the Royal Prison Preungesheim in Frankfurt 20 .

In 1916, the New Yorker Magazine asserted that Arthur Warren Waite, a dentist who later become known as "The Playboy Poisoner", made several attempts to acquire pathogens, such as those causing pneumonia, tuberculosis, influenza, and diphtheria. Trying to obtain a financial advantage, Dr. Waite first killed Mrs. Hannah Peck (his mother-in-law) by spiking her food with a mixture of diphtheria and influenza. The same procedure was unsuccessfully adopted in the attempt to kill John Peck (his father-in-law), using a nasal spray spiked with tuberculosis and then with influenza and typhoid. When John Peck survived, Dr. Waite used chlorine gas and arsenic. Dr. Waite was found guilty and was sentenced to death in the electric chair at Sing Sing Correctional Facility, in New York, on 24 May 1917 99 .

In 1933, in Calcutta (India), Dr. Taranath Bhatacharyna (a physician trained in bacteriology) assisted by Benoyendra Chandra Pandey injected the arm of Amarendra Pandey (Benoyendra´s half-brother) with a lethal dose of Y. pestis. The dispute was initiated by the division of their father's estate and because of Amarendra´s flamboyant lifestyle. Three years after, both perpetrators were convicted and sentenced to death 20 .

In 1964, Mitsuru Suzuki, a Japanese medical doctor with training in bacteriology, was detained for infecting four of his colleagues using dysentery-contaminated sponge cake. Subsequently, Dr. Suzuki was associated with a series of dysentery and typhoid fever outbreaks involving nearly 200 patients and four deaths. The team of prosecutors argued that Dr. Suzuki's dissertation included studies of S. enterica serovar Typhi recovered from several patients and that a culture of these bacteria was reported as stolen from Japan's National Institutes of Health 102; 103 .

In 1996, an outbreak of S. dysenteriae type 2 took place at a large medical center in Texas. Twelve members of the laboratory staff became indisposed after eating baked goods (muffins and doughnuts) anonymously left in the lunchroom, between the night and morning shift. Identification, serotyping, and pulsed-field gel electrophoresis revealed that the stool isolates from nine of the victims were identical to the S. dysenteriae strain retrieved from an uneaten muffin in the lunchroom and to a laboratory's stock culture of the same pathogen that was partially-missing from the laboratory stockpile 90 . On 28 August 1997, Diane Thompson (a member of the lab staff) was accused of infecting 12 of her colleagues and was later sentenced to 20 years imprisonment 104 .

In 1990, Graham Farlow, an asymptomatic HIV-positive inmate from a New South Wales (Australia) prison, stabbed officer Geoffrey Pearce with a needle containing HIV-positive blood. A few months later, the officer tested HIV-positive and died seven years after the attack 20 .

In 1992, Jennifer Jackson informed Brian Stewart (her former husband) that their 11-month old son Brryan Jackson was hospitalized after an asthma attack. After several arguments concerning child support and paternity between the couple, Mr. Stewart injected Brryan with HIV-positive blood. Mr. Stewart, a phlebotomist working at a laboratory in Barnes Hospital (St. Louis, Missouri), has drawn extra tubes of blood from patients and took home all the equipment necessary to inject the child. Four years after, Brryan was diagnosed with acquired immunodeficiency syndrome (AIDS) and three opportunistic infections. Although Mr. Stewart pleaded innocent, in 1998, he was convicted and sentenced to life imprisonment for attempted murder 100 .

In 1993, Iwan E. visited Gina O. (both perpetrator and victim were not identified in court using their surnames), his former girlfriend, to pay her some money she had lent him. In the middle of an argument in the apartment kitchen, E. was allegedly threatened with a knife and responded by injecting blood tainted with HIV (previously collected from E's friend) in the arm of O. Three months after this episode, O. tested HIV-positive. Although self-defense was claimed, the incriminating testimony of E.´s friend supported the conviction of attempted murder, and E was sentenced to 12 years imprisonment 101 .

In 1994, with the pretext of giving a vitamin B-12 injection, Richard J. Schmidt, a gastroenterologist, injected Janice Trahan with a mixture of blood or blood-products infected with HIV-1 and HCV, obtained from two patients under his care. Before the crime, Trahan tried to finish the 10 year-long extramarital affair with Dr. Schmidt. In 1995, Trahan was confirmed as HIV-positive and accused Dr. Schmidt of infecting her. During the following investigation, all men previously involved with Trahan were tested and found to be HIV-negative. The donor of the infected blood was identified, and the comparative phylogenetic analyses performed (env and reverse transcriptase genes) revealed this patient as the source for the virus. The case of The State of Louisiana vs. Richard J. Schmidt remains as the first example were phylogenetic analyses of HIV-1 sequences were admitted and used as evidence in a criminal proceeding in the United States 92 .

Microbial forensics is a relatively new scientific discipline, established in 2003 as a direct consequence of the necessity to identify the strain used in the US. MF relies on knowledge obtained from other basic and applied sciences along with non-traditional forensic science disciplines; microbiology, microbial ecology, public health epidemiology, microbial genomics, and toxicology, along with bioinformatic analysis and process engineering 81 .

In 2007, the National Research Council Committee on Metagenomics (US) highlighted metagenomics' effectiveness in the fields of bio-defense and MF 'to precisely identify and characterize microbes that have played a role in war, terrorism, and crime events, thus contributing to discovering the source of the microbes and the party responsible for their use' 105 (please see below for further information on metagenomics).

Therefore, MF efforts focus on the elucidation of the following questions:

 What was the pathogenic microbial agent used?  Where did it came from?  Where are the natural reservoirs located?  What are the possible transmission routes involved?  Are there any vectors involved?  What probable targets are affected?  How did the microorganism evolve?  Are there toxins involved?  To which antibiotics are the microorganisms sensitive or resistant to?  Was the microorganism genetically modified or chemically treated to enhance its features such as virulence or dispersion capacity?

These questions can be divided into two main domains: scientific and legal (or criminal) attribution. Scientific attribution aims to determine the origin of a given pathogenic agent to a known origin to the highest degree of scientific confidence (and exclude other possible source origins) 84; 106 . In the case of a BW, BT, and BC is crucial to detect the pathogen as soon as possible to minimize the health hazards associated with its dispersion. This can be achieved with the creation of biosecurity programs via international collaboration, with the collective aim to periodically monitor public places not only to define the baseline concentration of microorganisms, to include variations such as seasonal fluctuation but also to detect the sudden increase of non-expected microorganism 82; 83 . Furthermore, the information gathered can be applied to develop a better preparedness response plan and to discourage BT attacks. Legal attribution aims to determine who was responsible for the acts of interest (BW, BT, or BC) or who was otherwise implicated, according to the principles recognized and applied within a legal system 84; 106 .

Legal attribution intends to determine with respect who was responsible for the acts of interest (biocrime, bioterrorism or biowarfare) or who was otherwise implicated, according to the principles recognized and applied within a legal system 84; 106 .

Another fundamental factor to consider is sample collection and preservation as the entire process is reliant on appropriate and efficient sampling procedures; to maximize recovery of microorganisms and to maintain the integrity of the sample. The validation of the results is also an important factor to enable the J o u r n a l P r e -p r o o f use of such evidence to clarify the facts to determine, possibly in a court of law, whether a crime is likely to have been committed or not.

In most cases, MF is applied to entirely new situations (sampling area, location, type of agent). Hence, instead of applying the stringent standard operating protocols (SOP) to process the crime scene, it is desirable to adapt the existing rules to a combination of previous knowledge from specialists, their research expertise, and common sense 3, 4 .

Samples collection and preservation is a fundamental step of the entire process. It limits the validation of the results and may prevent samples from constituting the evidence necessary for the clarification of a given crime.

The value of the evidence, even when carefully collected and preserved, can be lost if the chain of custody is not properly constituted. The chain of custody is frequently recognized as the weakest link in a criminal investigation. It refers to the procedure of careful and chronological documentation of evidence, establishing its link to a criminal offense. From the beginning to the end of a judicial process, it is essential to demonstrate and document each step, ensuring evidence "tracking" and "integrity" from the crime scene to the courtroom. The collection of samples must be carefully performed by specialized technicians, using the appropriate equipment (e.g., suits, gloves, masks) to minimize contamination of the sample and to avoid the risk of infection. Samples must be accompanied by a record documenting; who collected it, under what conditions and the methods used for its collection, where and how the sample was preserved (e.g., temperature, relative humidity), and who had access and conducted any scientific work on the sample 3; 4 .

The first techniques for microorganism identification and detection were limited to phenotypic methods, associated with antigenic and/or antimicrobial resistance profiles. These methods only allow resolution at the genus and/or species level 3 . Then, in the late 1980s, these methods were complemented with nucleic acid-based methods (e.g., DNA fingerprinting, whole-genome sequencing, and microarray analysis). Such methods improved taxonomic resolution to the isolate/strain level, they are independent of the changes observed in the phenotypic characteristics due to the influence of environmental factors. Moreover, nucleic acid-based methods also decreased the turnaround time between sample collection and result availability 2, 12 .

In the early 2000s, high-throughput sequencing (HTS) [also referred to as massive parallel sequencing (MPS) technology or next-generation sequencing (NGS)] was introduced. Due to its increased multiplexing capacity, this method complies with different approaches, either allowing for whole-genome sequencing (WGS) of a single microorganism (e.g., viruses, bacteria, or fungi) or sequencing all the microbial species present in complex samples or matrices collected from a given environment or individual (metagenomics) 2 . In 2014, HTS approaches were introduced in routine diagnostics, for the investigation of outbreaks and transmissions, and used for genotyping of highly-resistant microorganisms. Therefore, clinical microbiologists or infectious disease specialists frequently resort to HTS, in collaboration with molecular microbiologists and infection control professionals. HTS has frequently been proven as suitable for studies of forensic epidemiology, concerning source identification, outbreak detection, transmission routes, pathogen evolution, and to identify the dynamics of multi-drug resistant pathogens, as reviewed in Oliveira et al, 2018 12 .

The main advantages of HTS over classical Sanger sequencing are:

(i) high-throughput capacity, hundreds of millions of sequencing reactions can be accomplished in parallel, enabling full sequencing of an entire bacterial genome in just one or two instrument runs;

(ii) a single protocol can be applied for all microorganisms for identification and genotyping;

(iii) DNA cloning is eliminated, exclusively depending on libraries preparation, in a cell-free system;

(iv) no prior knowledge about the sequence of a particular gene/genome is needed since HTS can read the DNA templates randomly distributed throughout the entire genome, and then de novo genome assembly can be applied;

(v) no need for isolation and culture of the microorganism of interest, a particularly critical aspect as many strains are unable to grow in culture media, allowing the identification of microorganisms present at trace levels and also those previously undetected by conventional methods;

(vi) cost (usually less than US$1000 (US) per genome, depending on the genome length) and the reduction in turnaround time (only a few hours) 2 .

The main disadvantage of HTS is associated with data storage and bioinformatics analysis. Depending on the platform used, in a single run, 30,000-500,000 sequence reads of 50-700 nucleotides can be generated. Due to the extremely sizeable quantity of data collected, the process deeply depends on storage ability and bioinformatics capacity to produce valuable data and to evaluate the quality of the HTS platform 2 .

During the last two decades, the advances in HTS technology, coupled with developments in machine learning, resulted in the production of extremely large amounts of high-quality sequence data that can be used for the prompt analysis of microbial communities from environmental samples 107; 108 . Metagenomics bypasses two of the major limitations associated with the "classical" studies of microbial communities: it is culture-independent, provides information on the true diversity of a given ecosystem (99% of microorganisms in nature that have not yet been cultivated), and offers insights into the complex metabolic pathways associated with these microorganisms (e.g., antimicrobial resistance, or virulence factors) [109] [110] [111] .

However, certain challenges must be addressed before the widespread application of metagenomics in the MF context. On one hand, the whole process -sample collection, maker selection, computational and bioinformatics, and metagenome analysis benchmarking and standards -presents several experimental biases [112] [113] [114] , limiting its application to MF. The development of validated experimental protocols and SOPs would ensure the required sensitivity, specificity, precision, accuracy, reproducibility, repeatability, the limit of detection, reportable range, false positive and negative ranges, and robustness of this method 107; 115 . Although there has been an increased interest in HTS [MiSeq™ (Illumina, CA) or the Ion S5™ (ThermoFisher Scientific, UK)] by some members of the forensic community, the vast majority of forensic laboratories are more familiar with and still using Sanger sequencing technologies 116 .

For the widespread application of MF, the forensic community needs to be further educated. Experts must address all the requirements of transparency (e.g., application of legal standards for genetic privacy and reliable standards) and accuracy of the data produced (e.g., correct use of databases and informatics J o u r n a l P r e -p r o o f tools). End-users (e.g., crime scene investigators, lawyers, judges, and juries) must be responsive to these new developments, recognizing both its possibilities and constraints.

Stable isotope [i.e., hydrogen ( 2 H/ 1 H), carbon ( 13 C/ 12 C), nitrogen ( 15 N/ 14 N), and oxygen ( 18 O/ 16 O)] ratios, can render unparalleled information in forensic investigations concerning the assignment of a source to a given microorganism. Microorganisms use these elements as water and nutrients supplies incorporating the isotopic variations in its nucleic acids. It has been suggested these variations can be used to trace specific batches of media or with specific sources of water, allowing to determine from which laboratory the microorganisms were produced. However, the high replication rate of microorganisms results in a rapid turnover of these elements 117; 118 .

Criminals and terrorists often consider biological agents (i.e., virus, bacteria, or bacterial toxin) as an attractive alternative to conventional weapons. Bioweapons production is associated with relatively low cost, microorganisms are relatively accessible, they can be simple to produce and to deliver while avoiding detection and even the threat of their use can induce fear among individuals and potential widespread social disruption.

The release of a biological weapon is intended to induce diseases or even death. Usually, these are naturally occurring microorganisms but, sometimes, they can be engineered to become harmful by increasing their ability to cause or spread disease or to resist known therapeutic approaches.

Throughout this review, it has been demonstrated that despite its form (either BW, BT, or BC) bioterror is a historical fact, almost present since the dawn of times. Therefore, facing this reality, experts must be aware that the correct question is not if we will have another attack using biological agents as weapons but rather when will be the next attack using biological agents as weapons.

With this in mind, it is essential to plan for a timely and effective response to the release and dissemination of a biological agent, including the ability to obtain a reliable and informative classification of the agent(s) being used. Controlled access to data held about global collections of representative strains, along with research to characterize biological agents that are less well studied and more difficult to culture, would aid in this effort. Preventative and early detection measures such as comprehensive environmental monitoring should also be implemented.

It is also essential to manage the expectations of law enforcement agencies, the general public, policymakers, and the scientific community along with highlighting the range of capabilities of microbial forensics.

Drafting and critical revision of the manuscript: MO, GMB, DB, WB, AA. Tables   Table 1. Examples of the use of microorganisms in biowarfare during the past millennia with landmarks of major advances changes in microbiology and legislation (shaded rows).

Pre-historic times Melanesian tribesman (actual Vanuatu) used arrowheads contaminated with tetanus 20 14th century BC The Hittite army send rams infected with tularemia to their enemies 120 6th century BC (Trojan War)

Scythian archers infected their arrows by dipping them into decomposing cadavers and human blood containing C. perfringens and C. tetani 121

Introduction to the Special Issue on Forensic Genetics

Microbial forensics: new breakthroughs and future prospects

Genética forense no humana

Forensic genetics and genomics: Much more than just a human affair

Building microbial forensics as a response to bioterrorism

Microbial forensics: building a national capacity to investigate bioterrorism

Duties and difficulties of investigating and prosecuting biocrimes

Biocrimes, microbial forensics, and the physician

Genomics of foodborne pathogens for microbial food safety

Applications of DNA-based methods in food forensics

Molecular epidemiology and evolution concepts in microbial forensics

New Trends in Microbial Epidemiology: Can An Old Dog Learn New Tricks?

Comparison of different concentration methods for the detection of hepatitis A virus and calicivirus from bottled natural mineral waters

Accidental and deliberate microbiological contamination in the feed and food chains-how biotraceability may improve the response to bioterrorism

Overview of biological terrorism: potential agents and preparedness

Combating the threat of biowarfare and bioterrorism: defending against biological weapons is critical to global security

Use of biological weapons

History of chemical and biological warfare agents

Biological warfare: a historical perspective

A Short History of Biological Warfare: From from Pre-History to the 21st Century

History of biological warfare and bioterrorism

Biological warfare and bioterrorism

Biological weapons, war crimes, and WWI

The role of scientific discovery in the establishment of the first biological weapons programmes

The Entomological Institute of the Waffen-SS: evidence for offensive biological warfare research in the third Reich

The price of alliance: Anglo-American intelligence cooperation and Imperial Japan's criminal biological warfare programme

Historical overview of biological warfare. Medical aspects of chemical and biological warfare

The history of biological warfare

The US biological warfare and biological defense programs

Biological warfare in a historical perspective

Overview: cause and prevention in biowarfare and bioterrorism

Biological weapons in the former Soviet Union: an interview with Dr

The Soviet Biological Weapons Program and Its Legacy in Today's Russia

Nuclear blindness: An overview of the biological weapons programs of the former Soviet Union and Iraq

Iraq's biological weapons: the past as future

After Saddam: regional insecurity, weapons of mass destruction, and proliferation pressures in postwar Iraq

Biological warfare, bioterrorism, and biocrime

Epidemiology of bioterrorism

Forensic microbiology

Bacillus anthracis comparative genome analysis in support of the Amerithrax investigation

The Microbial Rosetta Stone Database: a compilation of global and emerging infectious microorganisms and bioterrorist threat agents

Possession, Use, and Transfer of Select Agents and Toxins--Addition of Bacillus Cereus Biovar Anthracis to the HHS List of Select Agents and Toxins. Interim final rule and request for comments

Burkholderia mallei and Burkholderia pseudomallei as bloterrorism agents: national aspects of emergency preparedness

Laboratory exposures to brucellae and implications for bioterrorism. Emerging Infectious Diseases 11

Diphtheria, other corynebacterial infections, and anthrax

Coxiella burnetii genotyping. Emerging Infectious Diseases 11

E Coli 0157: h7. Clinical Guide to Bioweapons and Chemical Agents

Tularaemia: bioterrorism defense renews interest in Francisella tularensis

Review of Salmonella detection and identification methods: aspects of rapid emergency response and food safety

Pathogenic rickettsiae as bioterrorism agents

Non-O1 Vibrio cholerae septicemia: case report, discussion of literature, and relevance to bioterrorism

Plague: from natural disease to bioterrorism

Botulism: the many faces of botulinum toxin and its potential for bioterrorism

Toxins of Clostridium perfringens as a natural and bioterroristic threats

Bacteria as potential tools in bioterrorism, with an emphasis on bacterial toxins

Trends in detection of warfare agents: detection methods for ricin, staphylococcal enterotoxin B and T-2 toxin

Viral agents as biological weapons and agents of bioterrorism

Category C potential bioterrorism agents and emerging pathogens

Viruses of the Bunya-and Togaviridae families: potential as bioterrorism agents and means of control

Rift Valley fever virus: a real bioterror threat

Hemorrhagic fever viruses

The potential use of influenza virus as an agent for bioterrorism

Influenza as a bioweapon

Unconventional biological threats and the molecular biological response to biological threats

Bioterrorism and emerging infectious disease-antimicrobials, therapeutics and immune-modulators

Nipah virus-a potential agent of bioterrorism?

A pandemic of terror and terror of a pandemic: American cultural responses to HIV/AIDS and bioterrorism

The foot-and-mouth disease epidemic of 2001 in the UK: implications for the USA and the "war on terror

Poxvirus dilemmas-monkeypox, smallpox, and biologic terrorism

Smallpox and bioterrorism

Smallpox: a potential agent of bioterrorism

Smallpox: historical review of a potential bioterrorist tool

Threats in bioterrorism I: CDC category A agents

Threats in bioterrorism. II: CDC category B and C agents

RISE: A Case Study

RISE, the Rajneeshees, Aum Shinrikyo and Bruce Ivins. Biological Threats In The 21st Century: The Politics, People, Science And Historical Roots

Dark Harvest

From Biological Warfare to Healthcare: Porton Down

US Biodefense & Homeland Security: Toward Detection & Attribution. In. (NAVAL POSTGRADUATE

The Science of Forensic Epidemiology in Detecting a Biological Attack (Bioterrorism)

Science needs for microbial forensics: developing initial international research priorities

A long-term survey unveils strong seasonal patterns in the airborne microbiome coupled to general and regional atmospheric circulations

Bioattribution needs a coherent international approach to improve global biosecurity

Designing an effective microbial forensics program for law enforcement and national security purposes

Microbial forensic investigation of the anthrax-letter attacks

Review of the scientific approaches used during the FBI's investigation of the 2001 anthrax letters

The anthrax attacks 10 years later

The Anthrax Letters: Challenges and Lessons. Medical Response to Terror Threats 65

The mirage man: Bruce Ivins, the anthrax attacks, and America's rush to war

An outbreak of Shigella dysenteriae type 2 among laboratory workers due to intentional food contamination

Source identification in two criminal cases using phylogenetic analysis of HIV-1 DNA sequences

Molecular evidence of HIV-1 transmission in a criminal case

Molecular testing of multiple HIV-1 transmissions in a criminal case

Former dentist faces another lawsuit

Molecular evolution in court: analysis of a large hepatitis C virus outbreak from an evolving source

Viral phylogeny in court: the unusual case of the Valencian anesthetist

Toxin: the cunning of bacterial poisons

Microbial forensics: the next forensic challenge

Poisoning the Pecks of Grand Rapids: The Scandalous 1916 Murder Plot

Son, 24, forgives dad who injected him with HIV: 'That's all I can do

The history of bioterrorism, biowarfare, and biocrimes

Historical perspectives of microbial bioterrorism

Diane Thompson: A Case Study

The new science of metagenomics: revealing the secrets of our microbial planet

Forensic public health: epidemiological and microbiological investigations for biosecurity

Validation of high throughput sequencing and microbial forensics applications

Forensic identification with environmental samples

Metagenomics: the next culture-independent game changer

Tax4Fun: predicting functional profiles from metagenomic 16S rRNA data

Metagenomic Approaches for Understanding New Concepts in Microbial Science

Forensic human identification with targeted microbiome markers using nearest neighbor classification

Influence of swabbing solution and swab type on DNA recovery from rigid environmental surfaces

Consistent and correctable bias in metagenomic sequencing experiments

Expansion of microbial forensics

DNA Based Methods in Intelligence-Moving Towards Metagenomics. Preprints 2020

Microbe forensics: oxygen and hydrogen stable isotope ratios in Bacillus subtilis cells and spores

Stable isotope ratios and forensic analysis of microorganisms

This work was financed by FEDER -Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 -Operational Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT-Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the project "Institute for Research and Innovation in Health Sciences" (POCI-01-0145-FEDER-007274).