A Case History in Cooperative Biological Research: Compendium of Studies and Program Analyses in Kazakhstan

Kazakhstan and the United States have partnered since 2003 to counter the proliferation of weapons of mass destruction. The US Department of Defense (US DoD) has funded threat reduction programs to eliminate biological weapons, secure material in repositories that could be targeted for theft, and enhance surveillance systems to monitor infectious disease outbreaks that would affect national security. The cooperative biological research (CBR) program of the US DoD’s Biological Threat Reduction Program has provided financing, mentorship, infrastructure, and biologic research support to Kazakhstani scientists and research institutes since 2005. The objective of this paper is to provide a historical perspective for the CBR involvement in Kazakhstan, including project chronology, successes and challenges to allow lessons learned to be applied to future CBR endeavors. A project compendium from open source data and interviews with partner country Kazakhstani participants, project collaborators, and stakeholders was developed utilizing studies from 2004 to the present. An earlier project map was used as a basis to determine project linkages and continuations during the evolution of the CBR program. It was determined that consistent and effective networking increases the chances to collaborate especially for competitive funding opportunities. Overall, the CBR program has increased scientific capabilities in Kazakhstan while reducing their risk of biological threats. However, there is still need for increased scientific transparency and an overall strategy to develop a capability-based model to better enhance and sustain future research. Finally, we offer a living perspective that can be applied to further link related studies especially those related to One Health and zoonoses and the assessment of similar capability-building programs.


Background and Introduction
After the Soviet Union breakup, the Nunn-Lugar Cooperative Threat Reduction program began in 1991 which funded work through the United States Department of Defense (US DoD) in several former Soviet Union (FSU) republics, including Kazakhstan, to dismantle, secure, and prevent proliferation of nuclear, chemical, and biological weapons of mass destruction (WMD). Since the early 2000s, the US DoD has funded and implemented a Biological Threat Reduction Program (BTRP) through the Defense Threat Reduction Agency (DTRA) in Kazakhstan.
After the BTRP addressed the biosecurity risks resulting from the breakup of the Soviet Union that included accounting for biological WMD and re-training scientists, the BTRP continued to fund the construction of a central reference laboratory (CRL), upgrades of other laboratories, and biological research. BTRP, which now runs programs in over 40 countries, has three lines of work: biosafety and biosecurity (BS&S); biosurveillance (BSV); and cooperative biological research (CBR). As discussed in our earlier manuscripts [1,2], Kazakhstan and the Central Asia region are interesting "hotspots" for infectious disease occurrence and surveillance due in part to its history, geography, and its diversity of vertebrate and invertebrate host species. Many of the BTRP supported research and epidemiologic studies involved One Health approaches, especially those studies related to zoonotic diseases. The objective of this project was to develop a historical perspective for the CBR program in Kazakhstan, including project chronology, successes and challenges, so that lessons learned might be applied to other CBR endeavors.

Historical Perspective
Kazakhstan has long maintained an infrastructure and tiered network for infectious disease surveillance that was a product of the Russian anti-plague (AP) system designed to combat seasonal epidemics through a multi-sectoral approach that recognized its zoonotic nature since the time of the Tsars [3,4]. The AP system consisted of a central administrative point which oversaw regional AP stations that, in turn, oversaw local field stations. The Russians recognized the need for "sentries" which served in the network to respond and mitigate outbreaks by deploying teams to the field by horse, covered wagon, and rail [4]. Dr. Aleksandr Gradzhanov, who was the director of the Uralsk Anti-Plague Station in western Kazakhstan, suggested these deployments were the first examples of mobile laboratories [5]. Later, some of the institutes from the AP system in Kazakhstan, which included the lead Almaty AP station, supported the Soviet bioweapons program [6]. Many of the foundational AP system organization, infrastructure, and resources still exist today at these institutes.

Economic Perspective
The infrastructure from the AP system also shaped the research culture among Kazakhstan and its neighbors in Central Asia which presented limited work often only in Russian language journals which explains why they are underrepresented in current scientific literature [7]. After the dissolution of the Soviet Union, Kazakhstan continued the AP system, but lost much of its funding and mission, resulting in an institutional "brain drain," decay of infrastructure, and the biosecurity risk of unsecure pathogen material. Since Kazakhstan has become a republic, it has built a robust economy with its wealth of substantial natural resources: natural gas, minerals, and oil. In that regard, the International Monetary Fund, World Bank, and United Nations rank Kazakhstan in the middle fifty countries according to gross national product.
Research outputs are often measured in bibliometrics such as published and cited peer-reviewed articles. Kazakhstan maintains a high literacy rate among the general population, which reflects their historic scientific record of publications. The 2018 Nature Index ranks the top 50 countries for life sciences according to their research outputs and Kazakhstan is not ranked in the Nature Index, but it is ranked in the second tier of 50 countries at #76 according to the Scimago Institution under agricultural and biological sciences [8,9]. National public funding amounts and the number of publications are easily tracked and the public funding also influences the policies that shape the scope and definition of research [10]. Though the number of publications did increase in Kazakhstan during the period from 1996-2014, it can be argued that Kazakhstan's current national policies for funding scientific research has not caught up with the current gross national product [7]. Thus, challenges remain among research programs in Kazakhstan including limited funding, improvement of infrastructure, need for development of programs that focus on merit-based advancement, fostering peer-reviewed quality research, and enhancing scientific transparency.

Overview of the CBR Program in Kazakhstan
Since 2005, DTRA BTRP has funded over $20 M in CBR alone in Kazakhstan mainly through single, large integrating contracts where contractors have implemented over 25 studies through their science partners (Figure 1). These studies were initially framed by the DTRA science leads, ministerial heads of Kazakhstan and subsequently the contractors developed a country science plan reflective of these leaders' needs, which detailed the projects and studies, participating institutes, research collaborators, and stakeholders. Often the country plan described what studies would be implemented rather than describing an overall research strategy. Through the implementation of the CBR projects and studies, project outputs included developing abstracts and presentation of results, documenting progress in routine reports, and authoring peer-reviewed publications. In addition, outputs related to optimizing scientific methods were used to inform and revise standard operating procedures for infectious disease surveillance.  While the primary BTRP objective is related to US national security by reducing global health security threats by working with partner countries, CBR enables those partner countries to focus on broader capacity-building for biosurveillance and enhancing BS&S [11]. These capabilities include priorities for biosurveillance and pathogen research based on the US Department of Health and Human Services (DHHS) and the US Department of Agriculture (USDA) Select Agents list plus those pathogens of pandemic concern that include emerging infectious diseases; and promote hypothesis-based research projects. Earlier BTRP work in Kazakhstan and other FSU republics was implemented in a top-down fashion, through ministries and related state institutes such as public and veterinary health, which have disease surveillance responsibilities similar to the AP system. The list of participating institutes in Kazakhstan has grown along with the number of projects and studies (Table 1) since the BTRP program started, which is a positive aspect of the program. The objectives for the three lines of BTRP's work BS&S, BSV, and CBR are often achieved in consensus for capacity-building which is traditionally associated with international development, but are inherently difficult to measure. A major accomplishment of DTRA BTRP funding has been building research networks to better implement the CBR program in Kazakhstan, which can be measured in simple metrics such as conference presentations and peer-reviewed publications, both in the English language, and outcomes such as results from testing suspect samples and operational enhancements. The cooperation between Kazakhstan and the US continues to show intent for reducing biological threats and the CBR program has also increased cooperation among institutes in Kazakhstan that often did not previously communicate their respective infectious disease surveillance results. The overall capacity-building and capability-maturing has been harder to assess and quantify.
DTRA BTRP funded several CBR projects and smaller studies which contractors implemented during 2005-2007, 2009. In 2005-2007, the initial CBR projects centered on studies involving select agents including zoonoses: anthrax, plague, tularemia, highly pathogenic avian influenza, and brucellosis. The typical CBR projects funded researchers in Kazakhstan and project collaborators in the US and UK who mentored and guided those researchers to develop and test their hypotheses. From 2009-2014, five CBR projects were funded with up to $ 1M each, which covered labor hours, materials, travel, and infrastructure overhead. In parallel, several smaller 12-month studies were funded with approximately $100 k called Threat Agent Detection and Response (TADR) Activity Projects (TAP) and were implemented in parallel to the CBR projects. The TAP studies were intended to support TADR which was the earlier name for the biosurveillance line of work and only funded material and did not fund labor hours. From 2015-2018, the trend for smaller studies continued as TAP studies evolved to Central Reference Laboratory (CRL) Activity Projects (CAP), which were designed and implemented to support studies for the new CRL that DTRA funded and constructed in Almaty, Kazakhstan.
CBR has served as a foundation for incorporating standard operating procedures into working practice under multiple research studies, especially related to BS&S to ensure that the work is performed in a safe and secure environment while supporting tasks to enhance infectious disease surveillance for human and animal health. More importantly, growing a quality research program especially in terms of capability maturity, that is the state "as is" and "to be", requires a strategic vision. The evolution and progression of the Kazakhstan CBR program are linked and illustrated in an iterative fashion (Figure 1).
The projects were largely assigned by the need and individual discussions among project participants, collaborators, and implementers, as this program was not developed through a traditional, competitive grant-based system. In 2014, from the original funding for 12 CBR projects and studies, the work completed lead to over six follow-up studies (Figure 1). From 2015-2018, later CBR projects were also developed and funded without earlier direct project links. There was a common focus to address research projects and studies that addressed zoonotic diseases and employed a One Health approach. All of these institutions provided various skill sets and techniques from viral and rickettsial disease research fields, as well as data analysis and mapping. The project was successful, in obtaining many tick and human samples made available by the Kazakhstani scientists. Moreover, during the study a number of Kazakhstani institute staff was trained in modern diagnostic and data management techniques. Infectious disease agents were detected in multiple different areas of the country, several for the first time at specific locations, and the results were written up for presentations at a number of international meetings [12][13][14][15]. The downside for the project was a lack of sequencing capability on site and collaborators were unable to transfer any genomic material (including PCR products) out of the country, where sequence data could be generated. This lack of sequence data meant that otherwise important and novel data on new and suspected infectious agents could not be determined and therefore the epidemiology of the rodent-and vector-borne infectious diseases caused by these agents could not be properly defined. There were also issues of access to laboratories and difficulty in sharing data, while minor issues have potential to delay project schedule. Lastly, the incomplete investigation of the infectious agents made it difficult to obtain meaningful data and publish the results in peer-reviewed international journals. Nevertheless, the project revealed many potential infectious disease issues in Kazakhstan that will provide a starting point for future, in-depth investigations [2,16]. One of the solutions to these problems will be introduction on a national scale of identification workflows centered around rapid state-of-the-art genomic sequencing capability.
Additionally, some of the smaller TAP and CRL (CAP projects) studies demonstrated higher impact and success than the larger CBR projects because the project activity could be performed with a smaller number of researchers and required less coordination that would be necessary with researchers from various institutes in Kazakhstan. KZ-29 and TAP-2 were performed concurrently and leveraged the same project resources to fulfill both studies. The TAP-2 tick identification study which was designed to apply molecular methods, including using quantitative real-time polymerase chain reaction (qPCR) assays, to confirm the identification of ticks to the level of genus and species when the conventional visual identification was questionable due to inconclusive morphology and life stage presentation. Questing adult ticks were collected by tick drag during spring and summer from 2012-2014 in seven administrative rayons (county level) of West Kazakhstan oblast (province level). The total number of ticks collected (n = 2232) were identified according to visual morphological methods which resulted in four genera: Dermacentor (n = 2097), Hyalomma (n = 4), Rhipicephalus (n = 126), and Ixodes (n = 5). After DNA extraction, Dermacentor and Ixodes tick pools were tested using tick genus-specific assays that produced results in concordance with conventional visual identification [2]. Additional tests were run to exclude cross-reaction and ensure fidelity of the Dermacentor and Ixodes assays. A partner country scientist from UAPS presented this data in a poster at the 2013 Conference of Research Workers in Animal Diseases (CRWAD) and demonstrated the utility of a molecular PCR-based method to complement and enhance tick identification [12]. The KZ-29 and TAP-2 examples demonstrated synergy through linking the studies in a practical manner.
In terms of simple metrics, from 2009-2014, the CBR, TAP, and CAP work resulted in at least 12 peer-reviewed publications, 60 conference presentations, and 30 research collaborator visits to Kazakhstan. Overall, the Kazakhstan CBR projects have resulted in a few hundred conference presentations and several peer-reviewed publications. The benefit of conference presentations is the practice of developing and presenting results while meeting and networking with peers in the scientific community. While the number of peer-reviewed publications is often used to judge scientific merit, publications are often developed well after a project has ended. In addition, the number of citations from these publications also grew over time beyond the time of the initial publications (Tables 2 and 3).   More recent CBR work has engaged applied science and academic institutes in Kazakhstan to take advantage of their capabilities and resources for next-generation workforce training and tapping into their basic research programs. These resources also include individuals who have been educated through the current system that includes more exposure to US and Western science and technology and better knowledge of the English language associated with major publications. This approach also reinforces similar examples of public-private partnerships where a "triad" is formed when academic and university sectors are engaged [17]. In Kazakhstan, these capabilities and resources complemented the CBR program while their peer institutes, already engaged in this program, have the required infrastructure to perform research activities on Select Agents which require higher biological containment measures. As the CBR program has evolved, the next-generation workforce has also developed through experience and incorporating various participants such as academics. It has become apparent to the US participants that levels of English language proficiency among Kazakhstani collaborators has markedly increased. This is an obvious factor in improving better communications and has a larger role in project planning and implementation. In the long term, this will have been one of the most useful by-products from BTRP, enabling Kazakhstani personnel access to international literature, scientific meetings, and personal relationships with international scientists and physicians. This ability and practice to consistently and effectively network increases the chances to collaborate especially for competitive funding opportunities.

Conclusions
Reducing biological threats through CBR programs is still important even in the absence of biological weapons programs. BTRP has specific objectives for reducing biological threats especially WMD and emerging infectious diseases as it relates to US national security and the research funded is a hybrid of pathogen research and disease surveillance [11]. The cooperation and investments in CBR have led to improved infrastructure, resources, and operations for enhancing active surveillance for infectious disease outbreaks, whether natural, intentional, or accidental. In our analysis, findings related to the CBR program in Kazakhstan include successes, limitations, and continuing challenges that are summarized from recent discussions and comparative literature reviews. The CBR program, which so far has demonstrated data-sharing in Kazakhstan can be an effective model for enhancing cooperation and capability-building which in turn reduces the risk for potential future biological threats.
Scientific accomplishments and capability-building were also demonstrated in Kazakhstan through the CBR program despite challenges and limitations. In addition to simple metrics, the nature of the respective collaborations is important to consider as it often influences future outputs such as when publications are made and how further work is proposed and implemented. In Kazakhstan, we also observed that when work is published the impact on citations is not known for some time and often the publication was not the primary reason for collaboration [18]. Limited feedback from Kazakhstani CBR participants also stated the opportunity to receive external funding for research and publishing this work were likely the primary reason for engaging in the CBR program.
At a 2018 National Academies of Sciences, Engineering, and Medicine workshop called Enhancing Global Health Security through International Biosecurity and Health Engagement Programs, a committee was formed to assist BTRP and provide future guidance. One element related to CBR and capability-building in general is the need to exact expectations, which is important under a highly specific program that may have different views across the funder, implementers, and partner country participants [19]. Exacting expectations across multiple stakeholders is especially difficult when the program requirements and policy do not result in a one-size-fits-all approach. An agile approach that reinforces existing resources and uses a multi-disciplinary and multi-sectoral approach is effective for capability and development.
Continuing challenges for increasing scientific transparency and incorporating a capability maturity model in order to advance respective research programs were also noted. In addition to the KZ-29 and TAP-2 examples described, the importance and difficulty for increasing scientific transparency via sample material exchange was noted. While the exchange of genomic sequences has been demonstrated, working towards sample material exchange would further increase scientific transparency [19]. BTRP and other programs have funded work related to genomic sequencing and infectious disease modeling [20,21]. These programs funded by the US and other countries worldwide further encourage cooperation and may present opportunities to exchange sample material.
For long-term capability-building, the sustainability of CBR program in Kazakhstan needs to reach a maturity level where it can compete and acquire external research funding apart from BTRP; this threshold remains a challenge. When funding a scientific research program, it is difficult to balance immediate needs and manage risk with raising quality and innovation. Similarly, standardized evaluation techniques such as the capability maturity model (CMM) which are often used in business and technology sectors are also good for assessment at a scientific research program level. At the BTRP level, outputs for cost-effectiveness, which could be incorporated into their program lifecycle analysis, and measuring benefits to foreign relations would augment program metrics. A unified top-down and bottom-up approach that is focused on capability maturity also needs to be employed to ensure that funder and recipients agree on expectations and science objectives.
The CBR program continues to align outputs for long-term sustainability such as encouraging partner countries to establish their own external funding. From our literature review, sustainability and setting policy are two common challenges similar programs also face [22]. For BTRP, designing impactful projects, which would reinforce their targeted scientific engagement approach, is even more challenging given specific program objectives and short project durations. It is suggested that the overall impact is limited when a linear process starting with the science integrity and ending with applying those findings and outcomes to a real world [22]. It is well known that multi-sectoral collaborations are effective and that motivations among collaborators affect the research outputs [18]. Within specific program objectives, certain requirements for research proposals should be viewed more as opportunities rather than limitations [19]. Going forward, the ability for Kazakhstani partner participants to further develop research proposals for funding, leverage existing resources and networks will continue to enhance the CBR program.

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Publicly posted funding calls through major national government funders such as BTRP broad area announcements are prime examples of external funding that Kazakhstan can apply for and pursue. While BTRP's fundamental research scope is on US Biological Select Agents, pathogens of pandemic potential, emerging, and re-emerging infectious diseases, partner country scientists and research collaborators have linked non-Select Agents to research topics through developing hypotheses for co-infection and differential diagnosis.

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Leveraging existing resources including knowledge of in-country organizational structure, workflow, related networks, and current studies are important to establish buy-in from a top administrative level to the working research level which further reinforces multi-sectoral approach and encourages communication.

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The degree of collaboration in Kazakhstan is emphasized especially in teamwork to develop strategies and cooperate effectively. Leveraging existing resources while reinforcing networks and maintaining relationships should be frequent and encouraged. The ability to link consecutive projects through effective collaboration and funding pursuits builds greater capability and avoids one-off studies.
Our experience suggests incorporating multi-sectoral partners that better leverage existing resources may offer higher impact especially among similar research studies of small scale and magnitude [19]. For example, initial studies such as those involving multi-institute participants in Kazakhstan were challenged with distributing project tasks and budgets equally. Often these initial studies were designed to demonstrate new cooperation among institutes in Kazakhstan, however they may have been performing similar tasks under their respective missions. Alternatively, a multi-sectoral approach such as those providing mutual benefits across different scientific, public health communities, and disadvantaged populations have benefits beyond generating research data [23]. An important effect of employing these types of studies is engaging the public and educating them on the purpose and objective of this work as related to larger programs which demonstrates transparency and counters any misperceptions and stigmas. Further incorporating approaches such as One Health that included joint animal and human health studies in Kazakhstan should also be encouraged to increase impact and reinforce cooperation in future CBR studies.
Our compendium provides a perspective that can help draw a roadmap for a scientific research program when the project outputs and metrics are incorporated. In addition, aligning program objectives and exacting expectations across participants and stakeholders contribute to the roadmap that ultimately builds that partner country's capability and capacity necessary for sustainability. Tracking the evolution of program projects and studies, especially collaborations and networks, is emphasized to capture important activities that are often omitted when large programs evolve and institutional memory is lost through turnover of human resources. To advance science through increased transparency and mature capabilities, a multi-sectoral approach, commitment, and vision is required to advocate a strategy and can be implemented through strong leadership.