Poliomielitis, una historia de inicio triste y final feliz / Poliomyelitis, a story with a sad beginning and a happy ending

Se desarrollan los principales elementos históricos en el estudio y la lucha contra la poliomielitis, su aislamiento por Karl Landsteiner en 1909, la primera vacuna con virus muerto (Jonas Salk, 1955), la segunda vacuna con virus vivo atenuado (Albert Sabin, 1961) y la reducción paulatina de la polio en todo el mundo, hasta llegar a menos de 200 casos al año (virus salvaje)(AU)

The main historical events in the study and fight against polio are shown, its isolation by Karl Landsteiner in 1909, the development of the first vaccine with dead virus (Jonas Salk, 1955), the second vaccine with live attenuated virus (Albert Sabin, 1961) and the gradual reduction of polio worldwide, reaching less than 200 cases a year (wild virus)(AU)

Narrativas vacunales y políticas de vacunación contra la poliomielitis en el contexto de la provincia de Alicante (1963-1978) / Vaccine narratives and polio vaccination policies in the context of the province of Alicante (1963-1978)

El análisis a pequeña escala es una vía de abordaje historiográfico que abrió, en su momento, nuevos caminos en la investigación. La proximidad del objeto de estudio, permite profundizar en los determinantes propios y específicos y en las realidades y prácticas, por ejemplo, de las campañas de inmunización masiva contra la poliomielitis diseñada a nivel nacional pero implementadas localmente, como en el estudio de caso que abordamos. Utilizando como modelo el ámbito sanitario provincial de Alicante, el trabajo añade datos para completar la información de proximidad sobre estas campañas y de ese modo, completar una visión de conjunto y un análisis comparado tanto en un ámbito externo como en un ámbito interno interterritorial, nacional y provincial, así como el seguimiento de las directrices dadas por los organismos nacionales e internacionales. En segundo término, dotar de significado al conjunto de actividades llevadas a cabo por diferentes actores, instituciones y medios de comunicación, implicados en el proceso de implantación, cobertura y seguimiento de las campañas de vacunación contra la polio en el ámbito provincial alicantino. El uso de narrativas de personas afectadas por la enfermedad permite añadir elementos relevantes sobre experiencias personales en el contexto de las campañas vacunales

Small-scale analysis is a path of historiographical approach that opened, at the time, new paths in research. The proximity of the study object allows us to delve into our own and specific determinants and the realities and practices of, for example, mass immunization campaigns against polio, designed at the national level but implemented locally, as in the case study that we discuss. Using as a model the provincial sanitary area of Alicante, the work adds data to complete the proximity information on these campaigns and thus, complete an overview and a comparative analysis both in an external and in an internal interterritorial, national scope and provincial level as well as the follow-up of the guidelines given by the national and international organisms. Secondly, to give meaning to the set of activities carried out by different actors, institutions and media, involved in the process of implementation, coverage and follow-up of polio vaccination campaigns in the province of Alicante. The use of narratives of people affected by the disease allows to add relevant elements about personal experiences in the context of the vaccine campaigns

Stopping live vaccines after disease eradication may increase mortality.

Several live vaccines may have beneficial non-specific effects (NSEs) reducing mortality more than can be explained by the prevention of the target infection, a phenomenon which has been linked to innate immune training. Most randomised controlled trials (RCTs) of oral polio vaccine (OPV) and measles vaccine (MV) have shown a large reduction in mortality that must have been at least partly nonspecific because it was much larger than the reduction explained by prevention of the target disease. Hence, stopping a live vaccine after disease-eradication could have negative health effects if the potential beneficial NSEs are not considered. We reviewed one eradicated disease, smallpox, and two infections likely to be eradicated in coming decades, polio and measles. No study was made of unintended effects of stopping smallpox vaccination when it happened in 1980. We have subsequently documented in both Guinea-Bissau and Denmark that smallpox-vaccinated individuals continued to have a survival advantage long after smallpox had been eradicated. The few studies which have examined the effect of OPV on survival all suggest strong beneficial NSEs; in RCTs, OPV compared with inactivated polio vaccine (IPV) has been associated with non-specific reductions in morbidity. RCTs, natural experiments and observational studies have found strong beneficial NSEs for MV. Hence, the imminent eradication of polio and the planned stop of OPV in 2024 and the subsequent eradication of measles infection and the possible stop to live MV could have negative effects for child survival. Before live vaccines are phased out, potential unintended effects of stopping these vaccines should be thoroughly studied.

Disease burden of varicella versus other vaccine-preventable diseases before introduction of vaccination into the national immunisation programme in the Netherlands.

IntroductionEstimating burden of disease (BoD) is an essential first step in the decision-making process on introducing new vaccines into national immunisation programmes (NIPs). For varicella, a common vaccine-preventable disease, BoD in the Netherlands was unknown.AimTo assess national varicella BoD and compare it to BoD of other vaccine-preventable diseases before their introduction in the NIP.MethodsIn this health estimates reporting study, BoD was expressed in disability-adjusted life years (DALYs) using methodology from the Burden of Communicable Diseases in Europe (BCoDE)-project. As no parameters/disease model for varicella (including herpes zoster) were available in the BCoDE toolkit, incidence, disease progression model and parameters were derived from seroprevalence, healthcare registries and published data. For most other diseases, BoD was estimated with existing BCoDE-parameters, adapted to the Netherlands if needed.ResultsIn 2017, the estimated BoD of varicella in the Netherlands was 1,800 (95% uncertainty interval (UI): 1,800-1,900) DALYs. Herpes zoster mainly contributed to this BoD (1,600 DALYs; 91%), which was generally lower than the BoD of most current NIP diseases in the year before their introduction into the NIP. However, BoD for varicella was higher than for rotavirus gastroenteritis (1,100; 95%UI: 440-2,200 DALYs) and meningococcal B disease (620; 95%UI: 490-770 DALYs), two other potential NIP candidates.ConclusionsWhen considering the introduction of a new vaccine in the NIP, BoD is usually estimated in isolation. The current approach assesses BoD in relation to other vaccine-preventable diseases' BoD, which may help national advisory committees on immunisation and policymakers to set vaccination priorities.

Childhood health shocks, comparative advantage, and long-term outcomes: Evidence from the last Danish polio epidemic.

This paper examines the long-term effects of childhood disability on individuals' educational and occupational choices, late-career labor market participation, and mortality. We merge medical records on children hospitalized with poliomyelitis during the 1952 Danish epidemic to census and administrative data, and exploit quasi-random variation in paralysis incidence in this population. While childhood disability increases the likelihood of early retirement and disability pension receipt at age 50, paralytic polio survivors are more likely to obtain a university degree and to go on to work in white-collar and computer-demanding jobs than their non-paralytic counterparts. Our results are consistent with individuals making educational and occupational choices that reflect a shift in the comparative advantage of cognitive versus physical skills. We also find that paralytic polio patients from low socioeconomic status backgrounds are more likely to die prematurely than their non-paralytic counterparts, whereas there is no effect on mortality among polio survivors from more advantaged backgrounds.

Quality of life assessment scales in polio survivors: a scoping review.

BACKGROUND: Quality of life evaluation is essential to explore the effect of paralytic polio on the daily life experience of the polio survivor. Researchers have employed a range of assessment instruments to evaluate quality of life among polio survivors. Hence, to select the appropriate scale, it is crucial to compare the contents and psychometric properties of these instruments. PURPOSE: This scoping review explores quality of life instruments that are used in polio literature and analyzes their contents and psychometric properties using the Consensus-based Standards for the Selection of Health Measurement Instruments (COSMIN) criteria. METHOD: Using the Arksey and O'Malley framework, we conducted a literature search in the following electronic databases Medline, CINAHL, Web of Science, Embase, and Google Scholar to identify relevant studies that focused on quality of life of polio survivors. Of the 88 articles that qualify for full-text screening, 34 studies met our inclusion criteria. Two independent reviewers extracted data from the selected studies via Covidence, a reference manager that allows for blinding of reviews. RESULTS: Most of the instruments included in this review are generic, self-reported, and multidimensional. Despite having mostly adequate psychometric properties, these properties were not evaluated in polio survivors. CONCLUSION: The information provided in this review could be used to guide instrument selection and identify the need to develop a new tool or to adapt a pre-existing scale for measuring quality of life among polio survivors.

Comparison of Common Monogenic Defects in a Large Predominantly Antibody Deficiency Cohort.

BACKGROUND: Predominantly antibody deficiencies (PADs) are the most common primary immunodeficiencies, characterized by hypogammaglobulinemia and inability to generate effective antibody responses. OBJECTIVE: We intended to report most common monogenic PADs and to investigate how patients with PAD who were primarily diagnosed as suffering from agammaglobulinemia, hyper-IgM (HIgM) syndrome, and common variable immunodeficiency (CVID) have different clinical and immunological findings. METHODS: Stepwise next-generation sequencing and Sanger sequencing were performed for confirmation of the mutations in the patients clinically diagnosed as suffering from agammaglobulinemia, HIgM syndrome, and CVID. RESULTS: Among 550 registered patients, the predominant genetic defects associated with agammaglobulinemia (48 Bruton's tyrosine kinase [BTK] and 6 µ heavy chain deficiencies), HIgM syndrome (21 CD40 ligand and 7 activation-induced cytidine deaminase deficiencies), and CVID (17 lipopolysaccharides-responsive beige-like anchor deficiency and 12 atypical Immunodeficiency, Centromeric instability, and Facial dysmorphism syndromes) were identified. Clinical disease severity was significantly higher in patients with µ heavy chain and CD40 ligand mutations compared with patients with BTK (P = .003) and activation-induced cytidine deaminase (P = .009) mutations. Paralysis following live polio vaccination was considerably higher in patients with µ heavy chain deficiency compared with BTK deficiency (P < .001). We found a genotype-phenotype correlation among patients with BTK mutations regarding clinical manifestation of meningitis and chronic diarrhea. Surprisingly, we noticed that first presentations in most patients with Immunodeficiency, Centromeric instability, and Facial dysmorphism were respiratory complications (P = .008), whereas first presentations in patients with lipopolysaccharides-responsive beige-like anchor deficiency were nonrespiratory complications (P = .008). CONCLUSIONS: This study highlights similarities and differences in the clinical and genetic spectrum of the most common PAD-associated gene defects. This comprehensive comparison will facilitate clinical decision making, and improve prognosis and targeted treatment.

[Child mortality for children under five years of age declined seven fold in Guinea-Bissau over the latest 40 years].

Observations on child mortality in Guinea-Bissau developed the idea of vaccines having non-specific effects (NSEs) not being explained by the prevention of the targeted infections. The live vaccines against measles, tuberculosis, polio and smallpox have beneficial NSEs, but the inactivated vaccines are associated with negative effects for female survival. The repeated campaigns with polio and measles vaccines have been particularly effective in reducing child mortality. Hence, stopping live vaccines after eradication could have negative consequences for child survival.

Campaigns with oral polio vaccine may lower mortality and create unexpected results.

Three studies from Guinea-Bissau found conflicting effects of OPV-at-birth (OPV0) on child survival. One study from 2004 suggested excess male mortality among children receiving OPV0 compared with children receiving NoOPV0 during a period of shortage of OPV. However, two subsequent studies showed beneficial effects of OPV0. In 2004, two national OPV-campaigns had been conducted in Guinea-Bissau. In a reanalysis of the 2004-study, in a survival analysis the age-adjusted mortality rate of study participants was 67% (95% CI=42-81%) lower after the OPV-campaigns than before the campaigns. In the OPV0 group only 22% (655/3031 person-years (pyrs)) of follow-up time was "after" the OPV-campaigns whereas 55% (473/859 pyrs) of the time in the NoOPV0 group was post-campaign (p<0.0001, Chi2). Censoring for OPV-campaigns in the original study removed excess male mortality and made the three studies more homogeneous. Overall, there is now considerable evidence that OPV, like other live vaccines, has important beneficial non-specific effects.

Morbidity and mortality following poliomyelitis - a lifelong follow-up.

BACKGROUND AND PURPOSE: In the world today 10-20 million people are still living with late effects of poliomyelitis (PM), but the long-term consequences of the disease are not well known. The aim of this study was to describe lifelong morbidity and mortality among Danes who survived PM. METHODS: Data from official registers for a cohort of 3606 Danes hospitalized for PM in the period 1940-1954 were compared with 13 762 age- and gender-matched controls. RESULTS: Compared with controls, mortality was moderately increased for both paralytic as well as non-paralytic PM cases; Hazard Ratio, 1.31 (95% confidence interval, 1.18-1.44) and 1.09 (95% confidence interval, 1.00-1.19), respectively. Hospitalization rates were approximately 1.5 times higher among both paralytic and non-paralytic PM cases as compared with controls. Discharge diagnoses showed a broad spectrum of diseases. There were no major differences in morbidities between paralytic and non-paralytic PM cases. CONCLUSIONS: Poliomyelitis has significant long-term consequences on morbidity and mortality of both paralytic and non-paralytic cases.

The Estimated Health and Economic Benefits of Three Decades of Polio Elimination Efforts in India.

OBJECTIVE: In March 2014, India, the country with historically the highest burden of polio, was declared polio free, with no reported cases since January 2011. We estimate the health and economic benefits of polio elimination in India with the oral polio vaccine (OPV) during 1982-2012. METHODS: Based on a pre-vaccine incidence rate, we estimate the counterfactual burden of polio in the hypothetical absence of the national polio elimination program in India. We attribute differences in outcomes between the actual (adjusted for under-reporting) and hypothetical counterfactual scenarios in our model to the national polio program. We measure health benefits as averted polio incidence, deaths, and disability adjusted life years (DALYs). We consider two methods to measure economic benefits: the value of statistical life approach, and equating one DALY to the Gross National Income (GNI) per capita. RESULTS: We estimate that the National Program against Polio averted 3.94 million (95% confidence interval [CI]: 3.89-3.99 million) paralytic polio cases, 393,918 polio deaths (95% CI: 388,897- 398,939), and 1.48 billion DALYs (95% CI: 1.46-1.50 billion). We also estimate that the program contributed to a $1.71 trillion (INR 76.91 trillion) gain (95% CI: $1.69-$1.73 trillion [INR 75.93-77.89 trillion]) in economic productivity between 1982 and 2012 in our base case analysis. Using the GNI and DALY method, the economic gain from the program is estimated to be $1.11 trillion (INR 50.13 trillion) (95% CI: $1.10-$1.13 trillion [INR 49.50-50.76 trillion]) over the same period. CONCLUSION: India accrued large health and economic benefits from investing in polio elimination efforts. Other programs to control/eliminate more vaccine-preventable diseases are likely to contribute to large health and economic benefits in India.

Vaccine-Derived Polioviruses and Children with Primary Immunodeficiency, Iran, 1995-2014.

Widespread use of oral poliovirus vaccine has led to an ≈99.9% decrease in global incidence of poliomyelitis (from ≈350,000 cases in 1988 to 74 cases in 2015) and eradication of wild-type poliovirus serotypes 2 and 3. However, patients with primary immunodeficiency might shed vaccine-derived polioviruses (VDPVs) for an extended period, which could pose a major threat to polio eradication programs. Since 1995, sixteen VDPV populations have been isolated from 14 patients with immunodeficiency in Iran. For these patients, vaccine-associated paralysis, mostly in >1 extremity, was the first manifestation of primary immunodeficiency. Seven patients with humoral immunodeficiency cleared VDPV infection more frequently than did 6 patients with combined immunodeficiencies. Our results raise questions about manifestations of VDPVs in immunodeficient patients and the role of cellular immunity against enterovirus infections. On the basis of an association between VDPVs and immunodeficiency, we advocate screening of patients with primary immunodeficiency for shedding of polioviruses.

Anesthesia and Poliomyelitis: A Matched Cohort Study.

BACKGROUND: Poliomyelitis is a viral infectious disease caused by 1 of the 3 strains of poliovirus. The World Health Organization launched an eradication campaign in 1988. Although the number of cases of poliomyelitis has drastically declined, eradication has not yet been achieved, and there are a substantial number of survivors of the disease. Survivors of poliomyelitis present a unique set of challenges to the anesthesiologist. The scientific literature regarding the anesthetic management of survivors of poliomyelitis, however, is limited and primarily experiential in nature. Using a retrospective, matched cohort study, we sought to more precisely characterize the anesthetic implications of poliomyelitis and to determine what risks, if any, may be present for patients with a history of the disease. METHODS: Using the Mayo Clinic Life Sciences System Data Discovery and Query Builder, study subjects were identified as those with a history of paralytic poliomyelitis who had undergone major surgery at Mayo Clinic Rochester between 2005 and 2009. For each case, 2 sex- and age-matched controls that underwent the same surgical procedure during the study period were randomly selected from a pool of possible controls. Medical records were manually interrogated with respect to demographic variables, comorbid conditions, operative and anesthetic course, and postoperative course. RESULTS: We analyzed 100 cases with 2:1 matched controls and found that the peri- and postoperative courses were very similar for both groups of patients. Pain scores, postanesthesia care unit admission, length of postanesthesia care unit stay, intensive care unit admission, length of intensive care unit stay, and initial extubation location were not significantly different between the 2 groups. Looking at pulmonary complications in our primary outcome, there was no significant difference between the 2 groups (17% vs 14% for polio versus control, respectively; conditional logistic regression odds ratio = 1.5; 95% confidence interval, 0.7-3.3; P = 0.33). In addition, no difference was noted in those requiring a code or rapid response team intervention (4% vs 3% for polio versus control; P = 0.46) and the 30-day mortality rate was also not significantly different, with 2% of polio patients dying compared with 3% of controls (P = 0.79). The analysis of the primary outcome was repeated for the subset of patients with a history of poliomyelitis who had persistent neurologic deficits preoperatively (n = 36) and their matched controls (n = 72). In this subset analysis, there were 4 (11%) polio patients and 8 (11%) control patients who experienced pulmonary complications (conditional logistic regression odds ratio = 1.00; 95% confidence interval, 0.27-3.72; P = 1.00). The percentage of patients experiencing specific pulmonary complications of interest was similar between groups (postoperative mechanical ventilation: 6% vs 8% for polio and control patients, respectively; prolonged mechanical ventilation: 0% vs 1%; reintubation: 8% vs 4%; pulmonary infection: 6% vs 6%; and aspiration: 0% vs 1%). CONCLUSIONS: This study suggests that patients with a history of poliomyelitis do not seem to have an increased risk of pulmonary complications in the perioperative period. However, an odds ratio as great as 3.3-fold may be present.

Robustness against serum neutralization of a poliovirus type 1 from a lethal epidemic of poliomyelitis in the Republic of Congo in 2010.

In 2010, a large outbreak of poliomyelitis with unusual 47% lethality occurred in Pointe Noire, Republic of Congo. Vaccine-mediated immunity against the outbreak virus was never investigated. A wild poliovirus 1 (WPV1) isolated from a fatal case (termed PV1-RC2010) showed a previously unknown combination of amino acid exchanges in critical antigenic site 2 (AgS2, VP1 capsid protein positions 221SAAL → 221PADL). These exchanges were also detected in an additional 11 WPV1 strains from fatal cases. PV1-RC2010 escaped neutralization by three different mAbs relevant for AgS2. Virus neutralization was tested in sera from fatal cases, who died before supplementary immunization (n = 24), Gabonese recipients of recent oral polio vaccination (n = 12), routinely vaccinated German medical students (n = 34), and German outpatients tested for antipoliovirus immunity (n = 17) on Vero, human rhabdomyosarcoma, and human epidermoid carcinoma 2 cells. Fatal poliomyelitis cases gave laboratory evidence of previous trivalent vaccination. Neutralizing antibody titers against PV1-RC2010 were significantly lower than those against the vaccine strain Sabin-1, two genetically distinct WPV1s isolated in 1965 and 2010 and two genetically distinct vaccine-derived PV strains. Of German vaccinees tested according to World Health Organization protocols, 15-29% were unprotected according to their neutralization titers (<1:8 serum dilution), even though all were protected against Sabin-1. Phylogenetic analysis of the WPV1 outbreak strains suggested a recent introduction of virus progenitors from Asia with formation of separate Angolan and Congolese lineages. Only the latter carried both critical AgS2 mutations. Antigenetically variant PVs may become relevant during the final phase of poliomyelitis eradication in populations with predominantly vaccine-derived immunity. Sustained vaccination coverage and clinical and environmental surveillance will be necessary.

Bacterial lipopolysaccharide binding enhances virion stability and promotes environmental fitness of an enteric virus.

Enteric viruses, including poliovirus and reovirus, encounter a vast microbial community in the mammalian gastrointestinal tract, which has been shown to promote virus replication and pathogenesis. Investigating the underlying mechanisms, we find that poliovirus binds bacterial surface polysaccharides, which enhances virion stability and cell attachment by increasing binding to the viral receptor. Additionally, we identified a poliovirus mutant, VP1-T99K, with reduced lipopolysaccharide (LPS) binding. Although T99K and WT poliovirus cell attachment, replication, and pathogenesis in mice are equivalent, VP1-T99K poliovirus was unstable in feces following peroral inoculation of mice. Consequently, the ratio of mutant virus in feces is reduced following additional cycles of infection in mice. Thus, the mutant virus incurs a fitness cost when environmental stability is a factor. These data suggest that poliovirus binds bacterial surface polysaccharides, enhancing cell attachment and environmental stability, potentially promoting transmission to a new host.

Poliomyelitis and its elimination in Cuba: an historical overview.

INTRODUCTION: Polio was first detected in Cuba in the late 19th century among residents of the US community on the Isla de Pinos (Isle of Pines, now Isle of Youth), apparently introduced through migration from the USA. The first outbreak was reported in 1906 on the Isle, with the first epidemic reported in the former province of Las Villas in 1909. The epidemics subsequently intensified, by 1934 becoming periodic every four to five years, and accompanied by high morbidity, mortality and crippling sequelae, primarily among children. OBJECTIVE: To review and analyze the history of polio and its control in Cuba, from the disease's first appearance in 1898 until WHO/PAHO certification of elimination in 1994. METHODS: The historiological method was used; archival documents, medical records, and available polio morbidity and mortality statistics from the Ministry of Public Health's National Statistics Division before 1959 and from 1959 through 2000 were reviewed. Crude morbidity and mortality rates were calculated using population estimates at mid-period. Reports and scientific publications describing polio vaccination campaigns and their results were also reviewed, and key informants were interviewed. RESULTS: After initial introduction of polio in Cuba, five major epidemics occurred between 1932 and 1958: in 1934 (434 cases, 82 deaths); 1942 (494 cases, 58 deaths); 1946 (239 cases, 33 deaths), 1952 (492 cases, 15 deaths) and 1955 (267 cases, 8 deaths). Between 1957 and 1961 the disease's endemicity reached epidemic levels, with the last outbreak occurring in 1961, with 342 cases, 30% of them in children aged >4 years. In 1962, Cuba launched a nationwide polio vaccination campaign, the first of annual campaigns thereafter carried out in the framework of a coherent national program aimed at polio elimination. Using the Sabin oral vaccine and targeting the entire pediatric population in a single time period, five million doses were administered in the first campaign, reaching 87.5% of the target population aged 1 month through 14 years, constituting 109.4% of planned coverage. Since that year, no deaths from polio have been recorded (there were ten cases between 1963 and 1989) and WHO/PAHO certified polio elimination in Cuba in 1994. CONCLUSIONS: Cuba controlled polio with effective vaccination strategies and appropriate epidemiological measures, in the context of social, financial and political support. KEYWORDS History, poliomyelitis, epidemiology, disease control, vaccination, Sabin vaccine, Cuba.

An outbreak of wild poliovirus in the Republic of Congo, 2010-2011.

BACKGROUND: The Republic of Congo has had no cases of wild poliovirus type 1 (WPV1) since 2000. In October 2010, a neurologist noted an abnormal number of cases of acute flaccid paralysis (AFP) among adults, which were later confirmed to be caused by WPV1. METHODS: Those presenting with AFP underwent clinical history, physical examination, and clinical specimen collection to determine if they had polio. AFP cases were classified as laboratory-confirmed, clinical, or nonpolio AFP. Epidemiologic features of the outbreak were analyzed. RESULTS: From 19 September 2010 to 22 January 2011, 445 cases of WPV1 were reported in the Republic of Congo; 390 cases were from Pointe Noire. Overall, 331 cases were among adults; 378 cases were clinically confirmed, and 64 cases were laboratory confirmed. The case-fatality ratio (CFR) was 43%. Epidemiologic characteristics differed among polio cases reported in Pointe Noire and cases reported in the rest of the Republic of Congo, including age distribution and CFR. The outbreak stopped after multiple vaccination rounds with oral poliovirus vaccine, which targeted the entire population. CONCLUSIONS: This outbreak underscores the need to maintain high vaccination coverage to prevent outbreaks, the need to maintain timely high-quality surveillance to rapidly identify and respond to any potential cases before an outbreak escalates, and the need to perform ongoing risk assessments of immunity gaps in polio-free countries.