Role of Laboratory in Emerging Infectious Disease Control in Iran, Pasteur Institute of Iran, and national laboratory network.

Strengthening surveillance systems is a key aspect of outbreak response and was particularly important during the COVID-19 pandemic. Respiratory pathogens spread rapidly, and laboratory capacity is key to monitoring the spread. Prior to the pandemic, Iran had established a rapid response team and laboratory network to provide identification, monitoring, and detection of emerging infectious diseases, but did not have the laboratory capacity to respond to COVID-19. Following the announcement of the COVID-19 pandemic, the rapid response team diverted all attention to supporting COVID-19 surveillance. Iran built on the existing national laboratory infrastructure to incorporate SARS-CoV-2 surveillance into the response network. Based on existing international protocols, in-house molecular diagnosis capacity was operationalized, and commercial controls and assays were acquired and validated to national standards. The first COVID-19 laboratory was operational by January 25, less than 4 weeks before the initial detection of SARS-CoV-2 was announced. Assays and support were expanded and rolled out to form the COVID-19 National Laboratory Network, which consists of 560 multi-sectoral laboratories covering all provinces of Iran. The national laboratory network supports a wide range of operational capacities, including assay validation and protocol development, quality assurance, respiratory pathogen diagnosis and surveillance, and variant identification and assessment using multiple sequencing platforms. This network has supported the testing of over 55 million samples over the past 36 months using RT-qPCR and has sequenced approximately 2200 samples across the country, contributing the data to international databases, including GISAID.

Molecular detection of Crimean-Congo Haemorrhagic Fever (CCHF) virus in hard ticks from South Khorasan, east of Iran.

BACKGROUND & OBJECTIVES: Crimean-Congo Hemorrhagic Fever (CCHF) is a deadly viral infection reported from more than 30 countries. It is considered a zoonosis↱ and tick bites are the main route of transmission in nature. So far, the virus has been identified in 31 species of hard (Ixodidae) and soft (Argasidae) ticks. The aim of this study was to determine the rate of CCHF virus infection in hard ticks from South-Khorasan province, east of Iran. METHODS: In this study, 684 livestock including 302 sheep, 344 goats, 16 cows and 22 camels were sampled from Birjand, Qaen, Khusf, Darmian and Sarbisheh counties. Genus and species of the ticks were diagnosed under stereomicroscope according to valid morphological keys. Reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect the CCHF virus genome based on S segment in 100 ticks. RESULTS: RT-PCR detected CCHF virus genome in 7 out of 100 ticks. Positive ticks belonged to Hyalomma and Rhipicephalus genera. CCHF virus infected species were Rhipicephalus sanguineus, Hyalomma detritium and Hyalomma asiaticum. All the infected ticks were isolated from goat and sheep and were from Birjand county. INTERPRETATION & CONCLUSION: Our results suggest that Hyalomma and Rhipicephalus may be the main vectors of CCHF virus in the study area.

The ins and outs of SARS-CoV-2 variants of concern (VOCs).

SARS-CoV-2, a newly emerging coronavirus that caused the COVID-19 epidemic, has been spreading quickly throughout the world. Despite immunization and some fairly effective therapeutic regimens, SARS-CoV-2 has been ravaging patients, health workers, and the economy. SARS-CoV-2 mutates and evolves to adapt to its host as a result of extreme selection pressure. As a consequence, new SARS-CoV-2 variants have emerged, some of which are classified as variants of concern (VOC) because they exhibit greater transmissibility, cause more-severe disease, are better able to escape immunity, or cause higher mortality than the original Wuhan strain. Here, we introduce these VOCs and review their characteristics, such as transmissibility, immune escape, mortality risk, and diagnostics.

Serosurvey of Crimean-Congo hemorrhagic fever virus in livestock, Kohgiluyeh and Boyer-Ahmad, Iran, 2017.

BACKGROUND & OBJECTIVES: Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral zoonosis with up to 50% mortality rate in humans. In addition to the role of animals in the transmission of the virus and spread of infected ticks through livestock trade, they can be employed as sentinel hosts for monitoring the infection. Iran is one of the endemic counties for CCHF and the disease has been documented in all provinces. However, in some provinces such as Kohgiluyeh and Boyer-Ahmad, the rate of the disease has been dramatically low. Therefore, this serosurvey was designed to investigate CCHF virus (CCHFV) seroprevalence among livestock in different parts of Kohgiluyeh and Boyer-Ahmad Province. METHODS: This study was conducted in 2017, in which, blood samples were collected from 501 sheep and goats from eight different cities of Kohgiluyeh and Boyer-Ahmad Province. CCHFV IgG antibodies were detected in livestock serum samples by specific ELISA assays for detection of ovine and caprine antibodies. RESULTS: In total, four sheep serum of out of 501 (0.8%) livestock samples (3 from Gachsaran and 1 from Dena) had CCHFV IgG antibodies. No significant association was found between CCHFV seropositivity and sex or age of animals (P>0.05). INTERPRETATION & CONCLUSION: Our findings suggest a minor CCHFV circulation in Kohgiluyeh and Boyer-Ahmad Province, Iran. However, due to the notion that outbreaks of tick-borne infections is hard to predict; steady and comprehensive monitoring programs especially in ticks and animals would be critical for understanding the circulation on the pathogen in a region.

Corrigendum to 'How Iran responded to expanding need for laboratory services for COVID-19?'

[This corrects the article DOI: 10.1016/j.hlpt.2021.100506.].

Evidence of Hantavirus circulation among municipal street sweepers, southwest of Iran.

Hantaviruses are rodent-borne zoonosis pathogens that cause hemorrhagic fever with renal syndrome (HFRS) and Hantavirus cardiopulmonary syndrome (HCPS) in humans. Rodents spread the virus via their excretions. The outbreak of Hantaviruses pose a significant public health problem. The epidemiology and history of Hantaviruses in Iran is not clear and regardless of the data from the few available studies, little is known about its epidemiology in this country. Herein, we discuss the prevalence of IgG antibody against Hantavirus serotypes in 385 street sweepers from southwest of Iran. Serum samples were investigated, using Hantavirus Pool 1 "Eurasia" IgG kit and Pool 2 "America" ELISA IgG kit (Euroimmun, Germany) to detect IgG antibodies against Old and New World Hantaviruses. The results showed a specific IgG antibody in two samples (0.5%). Both of seropositive cases had specific IgG antibody against Old World Hantaviruses. The data of the current study along with the previous data, indicate the circulation of Hantaviruses in Iran. Hence, the risk of Hantavirus infection in high-risk groups should be considered as a serious health issue.

How Iran responded to expanding need for laboratory services for COVID-19?

After the emergence of SARS-CoV-2 in early 2020 in Iran, the rapid response team of Pasteur Institute of Iran was the first lab starting detection and report of suspected human samples. This article is a short summery of all actions from the preparedness for detecting the first cases of COVID-19, expanding the nationwide laboratory service, choosing the suitable laboratory tests and other challenges in laboratory detection during SARS-CoV-2 pandemic in Iran.

Developing a Vaccine to Block West Nile Virus Transmission: In Silico Studies, Molecular Characterization, Expression, and Blocking Activity of Culex pipiens mosGCTL-1.

BACKGROUND: Mosquito galactose-specific C-type lectins (mosGCTLs), such as mosGCTL-1, act as ligands to facilitate the invasion of flaviviruses like West Nile virus (WNV). WNV interacts with the mosGCTL-1 of Aedes aegypti (Culicidae) and facilitates the invasion of this virus. Nevertheless, there is no data about the role of mosGCTL-1 as a transmission-blocking vaccine candidate in Culex pipiens, the most abundant Culicinae mosquito in temperate regions. METHODS: Adult female Cx. pipiens mosquitoes were experimentally infected with a WNV infectious blood meal, and the effect of rabbit anti-rmosGCTL-1 antibodies on virus replication was evaluated. Additionally, in silico studies such as the prediction of protein structure, homology modeling, and molecular interactions were carried out. RESULTS: We showed a 30% blocking activity of Cx. pipiens mosGCTL-1 polyclonal antibodies (compared to the 10% in the control group) with a decrease in infection rates in mosquitoes at day 5 post-infection, suggesting that there may be other proteins in the midgut of Cx. pipiens that could act as cooperative-receptors for WNV. In addition, docking results revealed that WNV binds with high affinity, to the Culex mosquito lectin receptors. CONCLUSIONS: Our results do not support the idea that mosGCTL-1 of Cx. pipiens primarily interacts with WNV to promote viral infection, suggesting that other mosGCTLs may act as primary infection factors in Cx. pipiens.

Severe acute respiratory syndrome-coronavirus-2 spike (S) protein based vaccine candidates: State of the art and future prospects.

Coronavirus disease 2019 (Covid-19) is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) which is responsible for a global pandemic that started in late 2019 in Wuhan, China. To prevent the worldwide spread of this highly pathogenic virus, development of an effective and safe vaccine is urgently needed. The SARS-CoV-2 and SARS-CoV share a high degree of genetic and pathologic identity and share safety and immune-enhancement concerns regarding vaccine development. Prior animal studies with first generation (whole virus-based) preparations of SARS-CoV vaccines (inactivated and attenuated vaccine modalities) indicated the possibility of increased infectivity or eosinophilic infiltration by immunization. Therefore, development of second and third generation safer vaccines (by using modern vaccine platforms) is actively sought for this viral infection. The spike (S) protein of SARS-CoVs is the main determinant of cell entry and tropism and is responsible for facilitating zoonosis into humans and sustained person-to-person transmission. Furthermore, 'S' protein contains multiple neutralizing epitopes that play an essential role in the induction of neutralizing antibodies (nAbs) and protective immunity. Moreover, T-cell responses against the SARS-CoV-2 'S' protein have also been characterized that correlate to the IgG and IgA antibody titres in Covid-19 patients. Thus, S protein is an obvious candidate antigen for inclusion into vaccine platforms against SARS-CoV-2 viral infection. This manuscript reviews different characteristics of S protein, its potency and 'state of the art' of the vaccine development strategies and platforms using this antigen, for construction of a safe and effective SARS-CoV-2 vaccine.

Comparison five primer sets from different genome region of COVID-19 for detection of virus infection by conventional RT-PCR.

BACKGROUND AND OBJECTIVES: The new beta-coronavirus, which caused Severe Acute Respiratory Coronavirus-2 Syndrome (SARS-CoV-2), a major respiratory outbreak in Wuhan, China in December 2019, is now prevalent in many countries around the world. Identifying PCR-based viruses is a well-known and relatively stable protocol. Unfortunately, the high mutation rates may lead to widespread changes in viral nucleic acid sequences, and so using specific primers for PCR can be recommended. In this study, we evaluated the power of a conventional RT-PCR to detect SARS-CoV-2 RNA among the five set primer sets. MATERIALS AND METHODS: The five genomic regions of the Coronavirus SARS-2 virus including Nucleocapsids (N), Envelope (E), RNA depended RNA Polymerase (RdRp), ORF1ab and Spike (S) were selected for primer designing. A conventional RT-PCR was performed to compare sensitivity, specificity and other analytical characteristics of primers designed against two Real Time PCR commercial kits. RESULTS: The result of the comparative analysis showed that the ORF1ab, N and RdRp primers had a sensitivity, specificity and positive predictive value higher than other primers. A significant difference in the analytical sensitivity between the studied primer sets in RT-PCR kits was observed. CONCLUSION: In this study, the ORF1ab, Nucleocapsid and RdRp regions have the best primers for identifying the SARS-CoV-2 RNA between different genes that have been suggested.

Molecular Assay on Detection of Crimean Congo Hemorrhagic Fever (CCHF) Virus in Ixodid Ticks Collected from Livestock in Slaughterhouse from South of Iran.

BACKGROUND: Ticks are vectors of a wide variety of pathogens that can be transmitted to humans, and tick-borne diseases are a significant public health issue worldwide. The present study was carried out on the hard tick infestation of livestock transported to Rafsanjan slaughter house in the southeast of Iran. METHODS: A cross-sectional survey was carried out biweekly from April to September 2016 to determine tick infestation of the meat-producing animals. All the livestock included in our study were thoroughly inspected for the presence of hard ticks on different parts of their bodies. RESULTS: A total of 258 hard ticks were collected from the body of livestock hosts. The ticks that were sampled were classified into two genera and five species: Hyalomma marginatum, Hy. anatolicum, Hy. asiaticum, Hy. dromedarii, and Rhipicephalus sanguineus. Hyalomma dromedarii was the most abundant species in the study area. More than 50 percent of the sampled ticks were collected from the body of camels brought to the slaughter house however molecular analysis showed no Crimean Congo Hemorrhagic Fever (CCHF) virus infection in tick specimens. The Sex ratio of the sampled hard ticks shows that female tick infestation was more common among the study livestock. CONCLUSION: Due to the crucial role of hard ticks in the transmission of different pathogens to humans, additional investigations are necessary to determine the risk of consumption of infested meat-producing animals in the study area.

Imported cases of Chikungunya virus in Iran.

BACKGROUND: Chikungunya virus (CHIKV) is a widespread mosquito-borne virus representing a serious challenge to public health. The largest outbreak in the Middle-East was recorded in 2016-2017 in Pakistan. Sistan and Baluchistan Province of Iran shares a wide border with Pakistan; accordingly, introduction of CHIKV from Pakistan to Iran seems to be probable. The current study is aimed at investigating CHIKV infection in Sistan and Baluchistan Province. METHODS: Between April 2017 and June 2018, a total of 159 serum samples of CHIK suspected cases from 10 cities of Sistan and Baluchistan Province were tested by molecular and serological assays. Samples obtained up to 4 days after onset of illness were tested by real time PCR (n = 8). Samples collected 5-10 days after disease onset were subjected to ELISA, as well as real time PCR tests (n = 72). Samples obtained after the 10th day of disease onset were tested by only ELISA (n = 79). Phylogenetic analysis of real time PCR positive samples was carried out by sequencing of a 1014-bp region of Envelope 1 gene (E1 gene). Chi-square and independent t tests were used to evaluate the association between variables and CHIKV infection. RESULTS: In total, 40 (25.1%) out of 159 samples tested positive either by real time PCR or ELISA tests.Out of 151 samples serologically analyzed, 19 (12.6%) and 28 (18.6%) cases were positive for anti-CHIKV IgM and anti-CHIKV IgG antibodies, respectively. Of 80 samples tested by real time PCR, CHIKV RNA was detected in 11 (13.7%) sera, all of them had recent travel history to Pakistan. Additionally, phylogenetic analysis of 5 samples indicated their similarity with recent isolates of Pakistan outbreak 2016-2017 belonging to Indian Ocean sub-lineage of ECSA genotype. A significant correlation between abroad travel history and CHIKV infection was observed (P < 0.001). The most common clinical symptoms included fever, arthralgia/arthritis, myalgia, headache, and chill. CONCLUSIONS: These results present substantial evidence of CHIKV introduction to Iran from Pakistan and emphasize the need for the enhancement of surveillance system and preventive measures.

Hantavirus infection in Iranian patients suspected to viral hemorrhagic fever.

BACKGROUND: Hantaviruses are a group of emerging pathogens causing hemorrhagic fever with renal syndrome and Hantavirus cardiopulmonary syndrome in human. This study was conducted to investigate Hantavirus infection among Iranian viral hemorrhagic fever suspected patients. METHODS: From April 2014 to June 2016, 113 cases from 25 different provinces of Iran were analyzed for Hantavirus infection by IgM/IgG ELISA and pan-Hantavirus RT-PCR tests. RESULTS: Although, viral genome was detected in none of the subjects, IgM and IgG antibodies were detected in 19 and 4 cases, respectively. Differentiation of the anti-Hantavirus antibodies according to virus species by EUROLINE Anti-Hantavirus Profile Kit revealed three Puumala virus IgM positive, one Hantaan virus IgM positive, one Hantaan virus IgM borderline, and two Puumala virus IgG borderline cases. CONCLUSIONS: This study demonstrates the circulation of Hantaviruses in Iran and calls for further investigations of these life-threatening viruses in the country.

Epidemiology of West Nile Virus in the Eastern Mediterranean region: A systematic review.

BACKGROUND: West Nile Virus (WNV), a member of the genus Flavivirus, is one of the most widely distributed arboviruses in the world. Despite some evidence for circulation of WNV in countries summarized by the World Health Organization as the Eastern Mediterrian Regional Office (EMRO), comprehensive knowledge about its epidemiology remains largely unknown. This study aims to provide a concise review of the published literature on WNV infections in the Eastern Mediterranean Regional Office of WHO (EMRO). METHODOLOGY/PRINCIPAL FINDINGS: A systematic review of WNV prevalence studies on humans, animals and vectors in the EMRO region was performed by searching: Web of Science, Science Direct, Scopus, PubMed, Embase and Google Scholar. Finally, 77 citations were included, comprising 35 seroprevalence studies on general population (24460 individuals), 15 prevalence studies among patients (3439 individuals), 22 seroprevalence studies among animals (10309 animals), and 9 studies on vectors (184242 vector species). Of the 22 countries in this region, five had no data on WNV infection among different populations. These countries include Kuwait, Bahrain, Oman, Syria and Somalia. On the other hand, among countries with available data, WNV-specific antibodies were detected in the general population of all investigated countries including Djibouti (0.3-60%), Egypt (1-61%), Iran (0-30%), Iraq (11.6-15.1%), Jordan (8%), Lebanon (0.5-1%), Libya (2.3%), Morocco (0-18.8%), Pakistan (0.6-65.0%), Sudan (2.2-47%), and Tunisia (4.3-31.1%). WNV RNA were also detected in patient populations of Iran (1.2%), Pakistan (33.3%), and Tunisia (5.3% -15.9%). WNV-specific antibodies were also detected in a wide range of animal species. The highest seropositivity rate was observed among equids (100% in Morocco) and dogs (96% in Morocco). The highest seroprevalence among birds was seen in Tunisia (23%). In addition, WNV infection was detected in mosquitoes (Culex, and Aedes) and ticks (Argas reflexus hermanni). The primary vector of WNV (Culex pipiens s.l.) was detected in Djibouti, Egypt, Iran and Tunisia, and in mosquitoes of all these countries, WNV was demonstrated. CONCLUSIONS: This first systematic regional assessment of WNV prevalence provides evidence to support the circulation of WNV in the EMRO region as nearly all studies showed evidence of WNV infection in human as well as animal/vector populations. These findings highlight the need for continued prevention and control strategies and the collection of epidemiologic data for WNV epidemic status, especially in countries that lack reliable surveillance systems.

Seroprevalence of West Nile virus in Khuzestan province, southwestern Iran, 2016-2017.

BACKGROUND & OBJECTIVES: West Nile virus (WNV) is a neurotropic Flavivirus transmitted to humans through mosquito bites. As there is no specific antiviral treatment or approved vaccine against WNV, control and prevention of the infection is the best strategy to reduce the burden of WNV-related diseases. The circulation of WNV has been indicated in several regions of Iran including the Khuzestan province. Considering the complex ecology of WNV, the latest data are necessary for the implementation of preventive measures. Therefore, the present study was designed to provide updated information on the seroepidemiology of WNV in Khuzestan province. METHODS: A total of 408 sera were taken from volunteers living in Khuzestan. The presence of specific immunoglobulin G (IgG) antibody against WNV was tested by the enzyme-linked immunosorbent assay (ELISA) method. All the data and participants' demographic information were analyzed by SPSS and Esri's ArcMap GIS software programs. RESULTS: Anti-WNV IgG antibody was detected in 97 (23.8%) out of the 408 tested sera. The highest seropositivity rate was observed in cases aged between 20-29 yr and the lowest seropositivity rate was seen in those <19 yr of age (p = 0.001). There was no statistically significant association between WNV infection and gender, occupation, and educational level. The majority of positive cases were from the city of Ahvaz (47 cases, 48.4%) and Andimeshk (32 cases, 33%). INTERPRETATION & CONCLUSION: This study supports the earlier findings suggesting the circulation of WNV in Khuzestan province. Therefore, the implementation of an integrated surveillance system and training of health care workers and general population regarding the infection would be valuable to reduce the burden of possible outbreaks.

Evaluation of first rapid diagnostic kit for Anti-Crimean-Congo Hemorrhagic Fever virus IgM antibody using clinical samples from Iran.

Crimean-Congo Hemorrhagic Fever (CCHF) is a potentially fatal tick-borne viral disease, which is in the WHO list for emerging infections likely to cause major epidemics in the near future. Early diagnosis of CCHF is very important for both patient treatment and infection control. An efficient CCHF rapid test therefore is of great significance. This study was conducted to evaluate the sensitivity and specificity of the first CCHF rapid diagnostic kit (CCHF Sero K-SeT, CORIS BioConcept, Belgium) for detection of IgM specific antibody in patients' sera or plasma, using 87 clinical serum samples from Iranian patients. Although the assay showed an acceptable specificity of 92.9% (13/14), a low sensitivity rate of 39.7% (29/73) was observed. There was no association between the results of CCHF rapid diagnostic kit and the genotype of CCHF virus. This evaluation revealed that the CCHF Sero K-SeT is not suitable for screening of CCHF suspected cases due to its poor sensitivity.

Current status of Crimean-Congo haemorrhagic fever in the World Health Organization Eastern Mediterranean Region: issues, challenges, and future directions.

Crimean-Congo haemorrhagic fever (CCHF) is the most widespread, tick-borne viral disease affecting humans. The disease is endemic in many regions, such as Africa, Asia, Eastern and Southern Europe, and Central Asia. Recently, the incidence of CCHF has increased rapidly in the countries of the World Health Organization Eastern Mediterranean Region (WHO EMR), with sporadic human cases and outbreaks of CCHF being reported from a number of countries in the region. Despite the rapidly growing incidence of the disease, there are currently no accurate data on the burden of the disease in the region due to the different surveillance systems used for CCHF in these countries. In an effort to increase our understanding of the epidemiology and risk factors for the transmission of the CCHF virus (CCHFV; a Nairovirus of the family Bunyaviridae) in the WHO EMR, and to identify the current knowledge gaps that are hindering effective control interventions, a sub-regional meeting was organized in Muscat, Oman, from December 7 to 9, 2015. This article summarizes the current knowledge of the disease in the region, identifies the knowledge gaps that present challenges for the prevention and control of CCHFV, and details a strategic framework for research and development activities that would be necessary to curb the ongoing and new threats posed by CCHFV.

Crimean-Congo hemorrhagic fever among children in Iran.

Crimean-Congo hemorrhagic fever (CCHF) is a viral zoonotic disease which is endemic in Iran. The etiological agent of CCHF is an RNA virus belonging to the genus Nairovirus of the family Bunyaviridae. CCHF virus (CCHFV) can be transmitted to humans through bites from infected ticks and direct contact with infected blood or tissues. Although the disease has been observed in different age groups, the rate of disease is lower in children and elderly. This study was designed to characterize CCHFV-infected children in Iran. Between 2000 and 2016, a total of 908 CCHF suspected cases (in children less than 19 years old) were evaluated for CCHFV infection by CCHF IgM ELISA and RT-PCR. CCHFV infection was observed in 161 (17.73%) of subjects. Most CCHF positive children were male (70.8%) and >15 years of age (65.8%). Contact with livestock was the main risk factor (35.4%). Sistan and Baluchestan provinces had the highest frequency within the infected cohort (68.3%). The overall mortality rate was 11.8%. This study also revealed a significant reduction in CCHF-fatality rates in Iranian children when compared to earlier studies in Iran. Having contact with livestock was the major risk factor and CCHF was more common in male children of an older age.