First detection of bovine tuberculosis by Ziehl-Neelsen staining and polymerase chain reaction at dairy farms in the Lekok Sub-District, Pasuruan Regency, and Surabaya region, Indonesia.

Background and Aim: Bovine tuberculosis (TB) is a zoonotic disease of great public health importance, particularly in Indonesia, where control measures are limited or are not implemented. This study aimed to detect the presence of Mycobacterium pathogens in milk samples from dairy cattle in Pasuruan regency and Surabaya City, East Java, using Ziehl-Neelsen acid-fast staining and polymerase chain reaction (PCR). Materials and Methods: Milk samples were aseptically collected from 50 cattle in the Lekok Subdistrict, Pasuruan Regency, and 44 from dairy farms in the Lakarsantri Subdistrict, Wonocolo Subdistrict, Mulyorejo Subdistrict, and Kenjeran Subdistrict, Surabaya, East Java. To detect Mycobacteria at the species level, each sample was assessed by Ziehl-Neelsen staining and PCR using the RD1 and RD4 genes. Results: The results of PCR assay from 50 samples in Lekok Subdistrict, Pasuruan Regency showed that 30 samples (60%) were positive for Mycobacterium tuberculosis and two samples (4%) were positive for Mycobacterium bovis, although Ziehl-Neelsen staining did not show the presence of Mycobacterium spp. In the Surabaya region, 31 samples (70.45%) were positive for M. tuberculosis and three samples (6.8%) were positive for M. bovis. Six samples (13.63%) from all PCR-positive samples could be detected microscopically with Ziehl-Neelsen. Conclusion: The presence of bovine TB in this study supports the importance of using a molecular tool alongside routine surveillance for a better understanding of the epidemiology of bovine TB in East Java.

Whole-genome sequence and genesis of an avian influenza virus H5N1 isolated from a healthy chicken in a live bird market in Indonesia: accumulation of mammalian adaptation markers in avian hosts.

Background: Influenza A viruses are a major pathogen that causes significant clinical and economic harm to many animals. In Indonesia, the highly pathogenic avian influenza (HPAI) H5N1 virus has been endemic in poultry since 2003 and has caused sporadic deadly infections in humans. The genetic bases that determine host range have not yet been fully elucidated. We analyzed the whole-genome sequence of a recent H5 isolate to reveal the evolution toward its mammalian adaptation. Methods: We determined the whole-genome sequence of A/chicken/East Java/Av1955/2022 (hereafter, "Av1955") from a healthy chicken in April 2022 and conducted phylogenetic and mutational analysis. Results: Phylogenetic analysis revealed that Av1955 belonged to the H5N1 clade 2.3.2.1c (Eurasian lineage). The six gene segments (PB1, PB2, HA, NP, NA, and NS) out of the eight segments derived from viruses of H5N1 Eurasian lineage, one (PB2) from the H3N6 subtype and the remaining one (M) from the H5N1 clade 2.1.3.2b (Indonesian lineage). The donor of the PB2 segment was a reassortant among three viruses of H5N1 Eurasian and Indonesian lineages and the H3N6 subtype. The HA amino acid sequence contained multiple basic amino acids at the cleavage site. Mutation analysis revealed that Av1955 possessed the maximal number of mammalian adaptation marker mutations. Conclusions: Av1955 was a virus of H5N1 Eurasian lineage. The HA protein contains an HPAI H5N1-type cleavage site sequence, while the virus was isolated from a healthy chicken suggesting its low pathogenicity nature. The virus has increased mammalian adaptation markers by mutation and intra- and inter-subtype reassortment, gathering gene segments possessing the most abundant maker mutations among previously circulating viruses. The increasing mammalian adaptation mutation in avian hosts suggests that they might be adaptive to infection in mammalian and avian hosts. It highlights the importance of genomic surveillance and adequate control measures for H5N1 infection in live poultry markets.

Molecular identification of blaTEM gene of extended-spectrum beta-lactamase-producing Escherichia coli from healthy pigs in Malang district, East Java, Indonesia.

Objective: The increase and prevalence of multidrug-resistant bacteria in livestock animals are serious public health concerns. This study aimed to identify the presence of the blaTEM gene in extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolated from rectal swabs of apparently healthy pigs in Malang District, East Java, Indonesia. Materials and Methods: A total of 120 rectal swab samples were collected from the pigs. The rectal swabs were screened for the presence of E. coli using standard microbiological identification procedures. The Kirby-Bauer disk diffusion method identified multidrug-resistant E. coli. Five different classes of antibiotics were used to identify multidrug-resistant isolates, including Ciprofloxacin, Trimethoprim, Tetracycline, Streptomycin, and Aztreonam. Multidrug-resistant E. coli isolates were characterized for the presence of ESBL using double-disk synergy test methods. The presence of blaTEM genes was determined using polymerase chain reaction methods. Results: The results of this study indicated that 107 (89.2%) out of 120 samples analyzed were positive for E. coli isolates. A total of 32 (29.9%) E. coli isolates were identified to be multidrug-resistant and further subjected to molecular testing. The molecular analysis revealed (5; 15.6%) E. coli isolates to harbor the blaTEM gene. Conclusion: The results of this study revealed that pigs and products of pork origin must be considered a source of transmission of ESBL-producing E. coli to public health important under the food chain.

Prevalence and antibiotic resistance of Staphylococcus aureus and Escherichia coli isolated from raw milk in East Java, Indonesia.

Background and Aim: Raw milk can be a source of food-borne disease transmission and a medium for spreading antibiotic-resistant bacteria. Staphylococcus aureus and Escherichia coli are bacteria that have the pathogenic ability to attack host cells and are capable of harboring antibiotic-resistant genes. This study estimated the prevalence and antibiotic resistance of S. aureus and E. coli isolated from raw milk in East Java, Indonesia. Materials and Methods: Two hundred and fifty raw milk samples were collected from five dairy farms in East Java. S. aureus and E. coli were isolated using their respective selective media, whereas antibiotic susceptibility testing was performed using the Kirby-Bauer disk diffusion method. The methicillin-resistant S. aureus (MRSA) was confirmed using the oxacillin resistance screen agar test, and extended-spectrum beta-lactamase (ESBL)-producing E. coli was determined using the double-disk synergy test. The presence of mecA and blaTEM genes were screened by the polymerase chain reaction method. Results: Results indicated that the prevalence of S. aureus was 138 (55.2%) and that E. coli was 176 (70.4%). Of the 138 S. aureus isolated, 27 (19.6%) were MRSA, and among the 176 E. coli isolates identified, 3 (1.7%) were ESBL producers. The mecA gene was observed in 2 (7.4%) MRSA and all 3 (100%) ESBL-producing E. coli isolated harbored blaTEM genes. Conclusion: The presence of MRSA and ESBL-producing E. coli in raw milk is a serious public health threat, and public awareness should be raised about the dangers posed by these pathogenic organisms.

Emerging threats and vaccination strategies of H9N2 viruses in poultry in Indonesia: A review.

Avian influenza virus subtype H9N2 was first documented in Indonesia in 2017. It has become prevalent in chickens in many provinces of Indonesia as a result of reassortment in live bird markets. Low pathogenic avian influenza subtype H9N2 virus-infected poultry provides a new direction for influenza virus. According to the latest research, the Indonesian H9N2 viruses may have developed through antigenic drift into new genotype, posing a significant hazard to poultry and public health. The latest proof of interspecies transmission proposes that, the next human pandemic variant will be avian influenza virus subtype H9N2. Manipulation and elimination of H9N2 viruses in Indonesia, constant surveillance of viral mutation, and vaccines updates are required to achieve effectiveness. The current review examines should be investigates/assesses/report on the development and evolution of newly identified H9N2 viruses in Indonesia and their vaccination strategy.

Detection of invA virulence gene of multidrug-resistant Salmonella species isolated from the cloacal swab of broiler chickens in Blitar district, East Java, Indonesia.

BACKGROUND AND AIM: The increasing number of multidrug-resistant (MDR) Salmonella species on poultry farms in Indonesia has caused concern regarding human health. This study was conducted to determine the presence of the virulence gene invA in MDR Salmonella species isolated from the cloacal swab of broiler chickens in Blitar district, East Java Province, Indonesia. MATERIALS AND METHODS: Cloacal swab samples were collected by purposive sampling from 15 farms in four districts. Isolation and identification of bacteria were performed using standard microbiological techniques. Confirmation of MDR isolates was done using five different classes of antibiotics, including the beta-lactam, aminoglycoside, fluoroquinolone, phenicol, and monobactam groups. An antibiotic susceptibility test was conducted using the Kirby-Bauer disk diffusion method, and a polymerase chain reaction method was used to screen for the presence of invA. RESULTS: It was observed that 32.26% (50/155) of the samples were positive for Salmonella species. Of these 50 Salmonella isolates, 7 (14%) were identified as MDR strains. An important finding was the detection of invA in all the seven MDR Salmonella strains (100%) isolated from the cloacal swab of broiler chickens in Blitar district, East Java Province. CONCLUSION: Veterinarians have an extremely important role in monitoring the use of antibiotics in farm animals to mitigate the rapid spread of MDR organisms in our environment, which can otherwise cause serious economic losses and also public health issues.

The Dominance of CRF01_AE and the Emergence of Drug Resistance Mutations Among Antiretroviral Therapy-Experienced, HIV-1-infected Individuals in Medan, Indonesia.

BACKGROUND: human immunodeficiency virus type 1 (HIV-1) infection is a serious public health threat worldwide. Medan is one example of big cities in Indonesia with a high prevalence of HIV-1 infection; however, quite a limited study had conducted for detecting the circulation of HIV-1 subtypes in Medan. In addition, a serious factor that can implicate the treatment of HIV-1-infected individuals is the emergence of drug resistance mutations. Thus, the information on HIV-1 infection is important to improve the treatment for infected individuals. METHODS: sixty-seven antiretroviral therapy-experienced, HIV-1-infected individuals were recruited for this study. HIV-1 pol genes encoding protease (PR genes) and reverse transcriptase (RT gene), as well as env and gag genes, were amplified from DNA derived from peripheral blood samples. HIV-1 subtyping was conducted to study the dominant HIV-1 subtype circulating in the region. In addition, the emergence of drug resistance mutations was analyzed based on the guidelines published by the International Antiviral Society-United States of America (IAS-USA). RESULTS: the dominant HIV-1 subtype found in Medan was CRF01_AE (77.6%). In addition, another subtype and recombinant viruses such as recombinants between CRF01_AE and subtype B (12.2%), subtype B (4.1%), and CRF02_AG (4.1%) were also found. Drug resistance-associated major mutations were found in 21.6% (8/37) of RT genes and 3.1% (1/32) of PR genes studied. CONCLUSION: our study showed that the dominant subtype found in ART-experienced, HIV-1-infected individuals residing in Medan was CRF01_AE. The emergence of drug resistance mutations in RT and PR genes indicated the importance to monitor the prevalence of drug resistance mutations among HIV-1-infected individuals in Medan.

Detection of human immunodeficiency virus type 1 transmitted drug resistance among treatment-naive individuals residing in Jakarta, Indonesia.

The presence of transmitted drug resistance (TDR) in human immunodeficiency virus type 1 (HIV-1) infected individuals naive to antiretroviral therapy, may affect the effectiveness of treatment. Jakarta, the capital city of Indonesia, recorded the highest number of cumulative HIV infection cases in the country. This study aimed to identify on the appearance of TDR, as well as to identify HIV-1 subtypes circulating among treatment-naive individuals in Jakarta. Whole blood samples collected from 43 HIV-1 infected, treatment-naive individuals. Viral subtyping and drug resistance testing were performed on HIV-1 pol genes amplified using nested polymerase chain reaction. CRF01_AE was detected most frequently in Jakarta (73.08%). Drug resistance-related major mutation was not detected in protease fragments of pol gene, but two major mutations, K103N (6.67%) and Y181C (6.67%), were detected in reverse transcriptase fragments of pol gene. Our results suggest that TDR was emerged in Jakarta at a certain extent, thus further surveillance study to monitor the TDR prevalence and circulating HIV-1 subtypes in this region is considered to be necessary.

Transmission dynamics of HIV-1 subtype B strains in Indonesia.

Human immunodeficiency virus type 1 (HIV-1) and acquired immunodeficiency syndrome (AIDS) represent a major public health concern in Indonesia. Although circulating recombinant form (CRF) 01_AE is a predominant subtype in Indonesia, HIV-1 subtype B (HIV-1B) is also widely prevalent. However, the viral genetic evolution, spatial origins, and patterns of transmission of HIV-1B in Indonesia remain unclear. In the present study, we described the evolutionary characteristics and spatial-temporal transmission networks of HIV-1B in Indonesia. To elucidate the epidemiological link between HIV-1B epidemics in Indonesia and those in the remainder of the world, we conducted phylogenetic analyses of HIV-1B strains in Indonesia. Based on the results obtained, at least three epidemic clades [the Indonesia, United States (US), and China clades] of HIV-1B were found to be prevalent in Indonesia. In order to identify the potential source and transmission route of Indonesian HIV-1B strains, we performed Bayesian analyses and constructed Maximum clade credibility trees of each clade. Although some HIV-1B strains in Indonesia were introduced from Thailand, the prevalent HIV-1B strains appeared to have been directly introduced from Europe or America. Indonesian HIV-1B may have spread via the main dispersal of pandemic HIV-1B strains via the US from the Caribbean region rather than being directly introduced from Africa.

Genetic Diversity and Drug Resistance of HIV-1 Circulating in North Sulawesi, Indonesia.

Manado, the capital city of North Sulawesi, is a unique region in Indonesia because of its religion. We collected peripheral blood samples from 63 individuals on antiretroviral therapy. The amplification of viral genomic fragments, viral subtyping, detection of HIV drug resistance-associated mutations (DRAMs), and phylogenetic analyses were performed. Viral subtyping revealed that the most prevalent HIV type 1 (HIV-1) subtype/circulating recombinant form (CRF) was CRF01_AE (84.1%), followed by subtype B (6.8%) and recombinants between CRF01_AE and CRF02_AG (4.5%). Although no major DRAMs were present in protease genes, they were detected in reverse transcriptase (RT) genes. Nine of 38 samples (23.7%) had major DRAMs against nucleoside RT inhibitors (NRTIs) and/or non-NRTIs. The results of phylogenetic analyses indicated that CRF01_AE in North Sulawesi is related to that in Bali. Therefore, Bali may play an important role in circulating CRF01_AE in North Sulawesi.

Appearance of Drug Resistance Mutations Among the Dominant HIV-1 Subtype, CRF01_AE in Maumere, Indonesia.

BACKGROUND AND OBJECTIVES: Human Immunodeficiency Virus (HIV) is still a major health issue in Indonesia. In recent years, the appearance of drug resistance-associated mutations has reduced the effectiveness of Antiretroviral Therapy (ART). We conducted genotypic studies, including the detection of drug resistance-associated mutations (from first-line regimen drugs), on HIV-1 genes derived from infected individuals in Maumere, West Nusa Tenggara. Maumere, a transit city in West Nusa Tenggara, which has a high HIV-1 transmission rate. METHOD: We collected 60 peripheral blood samples from 53 ART-experienced and 7 ART-naive individuals at TC Hillers Hospital, Maumere between 2014 and 2015. The amplification and a sequencing analysis of pol genes encoding protease (the PR gene) and reverse transcriptase (the RT gene) as well as the viral env and gag genes were performed. HIV-1 subtyping and the detection of drug resistance-associated mutations were then conducted. RESULTS: Among 60 samples, 46 PR, 31 RT, 30 env, and 20 gag genes were successfully sequenced. The dominant HIV-1 subtype circulating in Maumere was CRF01_AE. Subtype B and recombinant viruses containing gene fragments of CRF01_AE, subtypes A, B, C, and/or G were also identified as minor populations. The major drug resistance-associated mutations, M184V, K103N, Y188L, and M230I, were found in the RT genes. However, no major drug resistance-associated mutations were detected in the PR genes. CONCLUSION: CRF01_AE was the major HIV-1 subtype prevalent in Maumere. The appearance of drug resistance-associated mutations found in the present study supports the necessity of monitoring the effectiveness of ART in Maumere.

Genotypic Characterization of Human Immunodeficiency Virus Type 1 Prevalent in Kepulauan Riau, Indonesia.

Kepulauan Riau is a famous tourist destination in Indonesia. The epidemic of human immunodeficiency virus type 1 (HIV-1) infection is gradually increasing in this region. We collected peripheral blood samples from 62 antiretroviral therapy-experienced individuals. The amplification of viral genomic fragments, HIV-1 subtyping, and the detection of HIV drug resistance (HIVDR) were performed. Viral subtyping revealed that the most prevalent HIV-1 subtype/circulating recombinant form (CRF) was CRF01_AE (55.6%), followed by recombinants between CRF01_AE and subtype B (17.8%) and then subtype B (15.6%). Recombinants containing CRF02_AG gene fragments were also detected (11.1%). Regarding HIVDR, no drug resistance-associated major mutations were found in pol genes encoding protease, although minor mutations were frequently detected. Furthermore, major mutations, including M184V (2.2%) and Y188L (2.2%), were identified in the viral pol gene encoding reverse transcriptase derived from a study participant, suggesting that the prevalence of HIVDR is low in the region.

Sero- and Molecular Epidemiology of HIV-1 in Papua Province, Indonesia.

BACKGROUND: human immunodeficiency virus (HIV) infection and acquired immune deficiency syndrome (AIDS) cause serious health problems and affect the Indonesian economy. Papua province has the highest prevalence of HIV infection in the country; however, epidemiological data are limited. Therefore, in order to reveal the current situation of HIV/AIDS in Papua province, sero- and molecular epidemiological studies of HIV were conducted. METHODS: serological tests were conducted on 157 healthy individuals from the general population residing in Paniai, Papua. In addition, a molecular epidemiological study was then conducted on HIV type 1 (HIV-1) genes derived from infected individuals. Peripheral blood samples from HIV-1-positive individuals and 15 additionally enrolled, previously confirmed HIV-1-positive individuals were subjected to a genotypic analysis. RESULTS: serological tests revealed that 2 out of 157 (1.27%) healthy individuals were HIV-positive. In addition, HIV-1 subtyping revealed that subtype B and CRF01_AE were the major subtype and circulating recombinant form (CRF) of HIV-1 prevalent in the region, while subtype A1 and a recombinant form including viral gene fragments of CRF01_AE and subtype B was also detected. In addition, HIV drug resistance-associated major mutations were detected in the reverse transcriptase gene derived from infected individual on antiretroviral therapy. CONCLUSION: these results provide important information for clearer understanding on the current situation of HIV/AIDS in Papua province in Indonesia.

Genotypic Characterization of Human Immunodeficiency Virus Type 1 Derived from Antiretroviral Therapy-Naive Individuals Residing in Sorong, West Papua.

Papua and West Papua provinces have the highest prevalence rate of human immunodeficiency virus type 1 (HIV-1) infection in Indonesia; however, data on the molecular epidemiology of HIV-1 are limited. We conducted a genotypic study on HIV-1 genes derived from antiretroviral therapy-naive individuals residing in Sorong, West Papua. HIV-1 genomic fragments were amplified from 43 peripheral blood samples, and sequencing analysis of the genes was carried out. Of the 43 samples, 41 protease (PR), 31 reverse transcriptase (RT), 26 gag, and 25 env genes were sequenced. HIV-1 subtyping revealed that CRF01_AE (48.8%, 21/43) and subtype B (41.9%, 18/43) were the major subtypes prevalent in the region, whereas other recombinant forms were also detected. Major drug resistance-associated mutations for PR inhibitors were not detected; however, mutations for the RT inhibitors, A62V and E138A, appeared in a few samples, indicating the possible emergence of transmitted HIV-1 drug resistance in Sorong, West Papua.

HIV-1 transmitted drug resistance mutations among antiretroviral therapy-Naïve individuals in Surabaya, Indonesia.

BACKGROUND: The emergence of transmitted drug resistance (TDR) compromises the effect of antiretroviral therapy (ART), resulting in treatment failure of human immunodeficiency virus (HIV) disease. Although more than a decade has passed since ART was introduced into Indonesia, information on TDR is limited. Here, a genotypic study of TDR among ART-naïve individuals was conducted in Surabaya, Indonesia. METHOD: HIV-1 seropositive participants were recruited from the communities of commercial sex workers and intravenous drug users as well as from the university teaching hospital in Surabaya. Protease (PR) and reverse transcriptase (RT) genes were sequenced in order to conduct HIV-1 subtyping and phylogenetic analysis and to detect TDR. TDR was defined as the presence of at least one surveillance drug resistance mutation on the WHO list or major drug resistance mutations in the International AIDS Society-USA panel. RESULT: Fifty two and 47 of the PR and RT genes, respectively, were successfully sequenced in the 58 samples. HIV-1 subtyping revealed that 86.3% (50/58) of the sequenced samples were classified as CRF01_AE, 8.6% as subtype B, 3.4% as B/CRF01_AE, and 1.7% as A/G/CRF01_AE. TDR of PR inhibitors was not detected in this study. In contrast, TDR of RT inhibitors was detected in 4.3% (2/47) of samples. In addition, minor drug resistance mutations were detected in 98.1% (51/52) and 12.8% (6/47) of PR and RT genes, respectively. CONCLUSION: This study clarified the predominance of the CRF01_AE strain in Surabaya, Indonesia. The prevalence of TDR was below 5%, indicating that the currently available first-line regimen is still effective in Surabaya. However, the prevalence might be underestimated since we detected only major population of HIV-1 in individuals. Therefore, continuous surveillance is required in order to detect the emergence of TDR in the early phase.

Appearance of drug resistance-associated mutations in human immunodeficiency virus type 1 protease and reverse transcriptase derived from drug-treated Indonesian patients.

Although HIV-1 drug resistance is a major obstacle in Indonesia, information on drug resistance is limited. In this study, the viral subtype and appearance of drug resistance mutations in the HIV-1 protease (PR) and reverse transcriptase (RT) genes were determined among drug-treated, HIV-1-infected patients in Surabaya. HIV-1 patients who received antiretroviral therapy (ART) more than 2 years were randomly recruited regardless of the viral load or ART failure. Fifty-eight HIV-1 PR genes and 53 RT genes were sequenced. CRF01_AE viruses were identified as the predominant strain. Major drug resistance mutations were not detected in the PR genes. In contrast, 37.7% (20/53) of the participants had one or more major drug resistance mutations in the RT genes, predominantly M184V (28.3%), K103N (11.3%), and thymidine analogue mutations (TAMs) (20.8%). The high prevalence of drug resistance mutations in RT genes indicated the necessity of monitoring the effectiveness of ART in Indonesia.

Detection of drug resistance-associated mutations in human immunodeficiency virus type 1 integrase derived from drug-naive individuals in Surabaya, Indonesia.

Although human immunodeficiency virus type 1 (HIV-1) infection causes serious health problems in Indonesia, information in regard to drug resistance is limited. We performed a genotypic study on HIV-1 integrase derived from drug-naive individuals in Surabaya, Indonesia. Sequencing analysis revealed that no primary mutations associated with drug resistance to integrase inhibitors were detected; however, secondary mutations, V72I, L74I/M, V165I, V201I, I203M, and S230N, were detected in more than 5% of samples. In addition, V201I was conserved among all samples. Most integrase genes were classified into CRF01_AE genes. Interestingly, 40% of the CRF01_AE genes had an unusual insertion in the C-terminus of integrase. These mutations and insertions were considered natural polymorphisms since these mutations coincided with previous reports, and integrase inhibitors have not been used in Indonesia. Our results indicated that further studies may be required to assess the impact of these mutations on integrase inhibitors prior to their introduction into Indonesia.