BA.5 sub-lineages associated with higher severity of COVID-19 infection: A cross-sectional study in Indonesia.

Objectives: We aimed to compare the clinical severity and outcome among laboratory-confirmed Omicron variant cases admitted between January and December 2022. Methods: This is a cross-sectional study conducted in Hasan Sadikin General Hospital between January and December 2022. We enrolled patients aged ≥18 years with laboratory-confirmed Omicron infection. Data were collected from clinical records and a whole genome sequencing database. We compared the risk of severe symptoms and mortality using a logistic regression analysis adjusted for sex, age, comorbidities, and vaccination status. Results: We enrolled 255 patients and the main sub-lineages were BA.1 (16.1%), BA.2 (11.4%), BA.5 (35.7%), XBB (22.7%), and BQ.1 (14.1%). Compared with BA.1/BA.2, BA.5 sub-lineages were associated with severe symptoms (adjusted odds ratio of 2.9, 95% confidence interval 1.1-8.2, P <0.05). The highest risk of severe symptoms and mortality was linked with a high number of comorbidities (adjusted odds ratio of 7.8, 95% confidence interval 1.7-22.4, P <0.05). Booster vaccination was protective of severity and mortality. Conclusions: Disease severity was associated with BA.5 sub-lineages and multiple comorbidities. Good management is particularly important for people with comorbidities. Furthermore, booster vaccination is also required to reduce severity and mortality.

Macrophage-2-Binding Protein Glycosylation Isomer (M2BPGi) and AGAP Score as Markers of Noninvasive Test for Liver Fibrosis versus FibroScan in Chronic Hepatitis B Patients: A Retrospective Observational Study.

Background: Liver biopsy as the gold standard for assessing the degree and diagnosis of fibrosis still has significant drawbacks, which make the emergence of a much less invasive diagnostic marker possible. M2BPGi levels and the AGAP score, the two newest serological markers, are known to have good sensitivity for detecting liver fibrosis. This study is aimed at determining the validity of examining M2BPGi levels and AGAP scores on the Fibroscan examination as markers of noninvasive test for liver fibrosis in chronic hepatitis B patients. Methods: This is an observational, descriptive study with a retrospective design. This study used secondary data taken from medical records and blood specimen research materials of outpatients at the Hepatology Gastroenterology Polyclinic at a tertiary general hospital in West Java, Indonesia, with a diagnosis of chronic hepatitis B. Results: There were 109 research subjects included. There were 73 (66.9%) subjects with no- or low-grade fibrosis and 36 (33.1%) with advanced fibrosis. The sensitivity and specificity of the M2BPGi were 88.9% and 61.6% (PPV 55.3%; NPV 91.8%; AUC 0.753), while the AGAP score was 47.2% and 100% (PPV 100%; NPV 79.3%; AUC 0.736). The combined M2BPGi level and the AGAP score showed a sensitivity of 80.9% and a specificity of 100% (PPV 100%; NPV 91.8%; AUC 0.905). Conclusion: The AGAP score and M2BPGi levels together are a better way to measure the degree of liver fibrosis in people with chronic hepatitis B than either M2BPGi or the AGAP score alone.

Expression of soluble moloney murine leukemia virus-reverse transcriptase in Escherichia coli BL21 star (DE3) using autoinduction system.

Autoinduction systems in Escherichia coli can control the production of proteins without the addition of a particular inducer. In the present study, we optimized the heterologous expression of Moloney Murine Leukemia Virus derived Reverse Transcriptase (MMLV-RT) in E. coli. Among 4 autoinduction media, media Imperial College resulted the highest MMLV-RT overexpression in E. coli BL21 Star (DE3) with incubation time 96 h. The enzyme was produced most optimum in soluble fraction of lysate cells. The MMLV-RT was then purified using the Immobilized Metal Affinity Chromatography method and had specific activity of 629.4 U/mg. The system resulted lower specific activity and longer incubation of the enzyme than a classical Isopropyl ß-D-1-thiogalactopyranoside (IPTG)-induction system. However, the autoinduction resulted higher yield of the enzyme than the conventional induction (27.8%). Techno Economic Analysis revealed that this method could produce MMLV-RT using autoinduction at half the cost of MMLV-RT production by IPTG-induction. Bioprocessing techniques are necessary to conduct to obtain higher quality of MMLV-RT under autoinduction system.

High-level expression of codon-optimized Taq DNA polymerase under the control of rhaBAD promoter.

A DNA polymerase from Thermus aquaticus remains the most popular among DNA polymerases. It was widely applied in various fields involving the application of polymerase chain reaction (PCR), implying the high commercial value of this enzyme. For this reason, an attempt to obtain a high yield of Taq DNA polymerase is continuously conducted. In this study, the l-rhamnose-inducible promoter rhaBAD was utilized due to its ability to produce recombinant protein under tight control in E. coli expression system. Instead of full-length Taq polymerase, an N-terminal deletion of Taq polymerase was selected. To obtain a high-level expression, we attempted to optimize the codon by reducing the rare codon and GC content, and in a second attempt, we optimized the culture conditions for protein expression. The production of Taq polymerase using the optimum culture condition improved the level of expression by up to 3-fold. This approach further proved that a high level of recombinant protein expression could be achieved by yielding a purified Taq polymerase of about 8.5 mg/L of culture. This is the first research publication on the production of Taq polymerase with N-terminal deletion in E. coli with the control of the rhaBAD promoter system.

Immunogenicity, Safety, and Efficacy of Influenza Vaccine in T2DM and T2DM with Chronic Kidney Disease.

Patients with Type 2 diabetes mellitus (T2DM) and Chronic Kidney Disease (CKD) face an increased risk of morbidity and mortality after influenza infection. Several studies have shown that the influenza vaccine effectively prevents morbidity and mortality in T2DM patients. However, there has been limited research aimed at assessing the effectiveness of the trivalent influenza vaccine in T2DM-CKD patients. This study aimed to identify Geometric Mean Titers (GMTs), seroprotection, seroconversion, safety, and efficacy. This open-label clinical trial was conducted at AMC Hospital in Bandung, West Java, Indonesia between June 2021 and July 2022. The study subjects consisted of 41 T2DM and 26 T2DM-CKD patients who were administered the trivalent influenza vaccine. There was a significant difference in the average age, with the T2DM-CKD patients being older. Median titers post-vaccination for the B/Washington virus were higher in the T2DM patients compared to the T2DM-CKD patients, and this difference was statistically significant. A majority, comprising 75.6% of the T2DM and 80.8% of the T2DM-CKD patients monitored post-influenza-vaccination, did not experience any adverse reactions. The most common reaction was the sensation of fever, with incidence rates of 12.2% in the T2DM patients and 15.4% in the T2DM-CKD patients. Furthermore, we observed that the incidence of Influenza-like Illness was highest at 7.3% in the T2DM patients and 7.7% in the T2DM-CKD patients. The trivalent influenza vaccine demonstrated equivalent safety and effectiveness in both groups.

Codon optimization of a gene encoding DNA polymerase from Pyrococcus furiosus and its expression in Escherichia coli.

BACKGROUND: DNA polymerase is an essential component in PCR assay for DNA synthesis. Improving DNA polymerase with characteristics indispensable for a powerful assay is crucial because it can be used in wide-range applications. Derived from Pyrococcus furiosus, Pfu DNA polymerase (Pfu pol) is one of the excellent polymerases due to its high fidelity. Therefore, we aimed to develop Pfu pol from a synthetic gene with codon optimization to increase its protein yield in Escherichia coli. RESULTS: Recombinant Pfu pol was successfully expressed and purified with a two-step purification process using nickel affinity chromatography, followed by anion exchange chromatography. Subsequently, the purified Pfu pol was confirmed by Western blot analysis, resulting in a molecular weight of approximately 90 kDa. In the final purification process, we successfully obtained a large amount of purified enzyme (26.8 mg/L). Furthermore, the purified Pfu pol showed its functionality and efficiency when tested for DNA amplification using the standard PCR. CONCLUSIONS: Overall, a high-level expression of recombinant Pfu pol was achieved by employing our approach in the present study. In the future, our findings will be useful for studies on synthesizing recombinant DNA polymerase in E. coli expression system.

High Positivity Rate of Urinary Mycobacterium tuberculosis Antigens Cocktail (ESAT-6, CFP-10, and MPT-64) in Active Tuberculosis Patients With and Without Human Immunodeficiency Virus Infection: A Cross-Sectional Study.

Introduction: Human immunodeficiency virus (HIV) infection is a risk factor for the occurrence of a large of Mycobacterium tuberculosis (Mtb) antigen load in the body. The antigens cocktail namely early secretory antigenic target protein 6-kDa (ESAT-6), Culture filtrate protein 10 kDa (CFP-10), and Mycobacterium tuberculosis protein 64 (MPT-64) are secreted by Mtb during replication, hence, their concentration increase in patients with active Tuberculosis (TB). This increased levels facilitates their entry into the systemic circulation, followed by secretion by the glomerulus into the urine. The aim of this study was to determine the positivity rate of the urinary Mtb antigens cocktail between TB patients with and without HIV infection. Methods: This is an observational descriptive comparative study conducted with a cross-sectional design. Random urine samples were collected from patients diagnosed with active TB in Dr. Hasan Sadikin Bandung Hospital in 2021. The subjects were divided into 2 groups, TB-HIV group and TB without HIV group. The samples were tested using the quantitative immunochromatography method. Result: Sixty active TB patients consisting of TB patients with HIV infection (n = 30) and TB patients without HIV infection (n = 30). The positivity in the urinary Mtb antigens cocktail was 93.3% for TB-HIV group and 100% for TB without HIV group (P = .492). The median concentration of urinary Mtb antigens cocktail in TB patients without HIV infection was higher than that of TB patients with HIV infection (137.73 ng/mL vs 96.69 ng/mL, respectively; P = .001). Conclusion: There was no significant difference in the positivity rate, meanwhile, there was a significant difference in concentration of the urinary Mtb antigens cocktail between active TB patients with and without HIV infection. Interestingly, this urinary Mtb antigens cocktail can be found in both groups without being affected by the patient's immune condition, thus becoming a test to assist diagnose active TB.

Production of codon-optimized Human papillomavirus type 52 L1 virus-like particles in Pichia pastoris BG10 expression system.

Cervical cancer caused by Human papillomavirus (HPV) is one of the most common causes of cancer death in women worldwide. Even though the disease can be avoided by immunization, the expensive price of HPV vaccines makes it hard to be accessed by women in middle-low-income countries. Thus, the development of generic HPV vaccines is needed to address inequalities in life-saving access. This study aimed to develop the HPV52 L1 VLP-based recombinant vaccine using Pichia pastoris expression system. The l1 gene was codon-optimized based on P. pastoris codon usage resulting CAI value of 0.804. The gene was inserted into the pD902 plasmid under the regulation of the AOX1 promoter. The linear plasmid was transformed into P. pastoris BG10 genome and screened in YPD medium containing zeocin antibiotic. Colony of transformant that grown on highest zeocin concentration was characterized by genomic PCR and sequencing. The positive clone was selected and expressed using BMGY/BMMY medium induced with various methanol concentrations. The SDS-PAGE and Western blot analyses showed that 55 kDa L1 protein was successfully expressed using an optimum concentration of 1% methanol. The self-assembly of HPV52 L1 protein was also proven using TEM analysis. Moreover, we also analyzed the B-cell epitope of HPV52 L1 protein based on several criteria, including antigenicity, surface accessibility, flexibility, and hydrophilicity. We assumed that epitope 476GLQARPKLKRPASSAPRTSTKKKKV500 could be developed as an epitope-based vaccine with a neutralizing antibody response toward HPV52 infection. Finally, our study provided the alternative for developing low-cost HPV vaccines, either VLP or epitope-based.

Optimized expression of large fragment DNA polymerase I from Geobacillus stearothermophilus in Escherichia coli expression system.

Bst DNA polymerase is a DNA polymerase derived from Geobacillus stearothermophilus, has a strand-displacement activity, and is used in loop-mediated isothermal amplification (LAMP) for rapid detection of COVID-19. Despite its potential to be employed in the detection of COVID-19, using commercially available enzymes is not economically feasible. The use of noncommercial enzyme for routine use is desirable. However, research on Bst DNA polymerase is still limited in Indonesia. For those reasons, a preliminary study of scale-up production of recombinant Bst polymerase was conducted. Therefore, the optimization of expression conditions was performed. The optimum conditions for Bst polymerase expression were as follows: 1 mM of IPTG, post-induction incubation time of 6 h, and induction at OD600 1.1. Employing optimum conditions could result in 2.8 times increase in protein yield compared to the initial conditions. Subsequently, an operation in 1 L working volume by a lab-scale bioreactor had been performed, followed by purification and dialysis. The optimum result for a 1 L lab-scale bioreactor was achieved by applying 100 rpm and 3 vvm, giving 11.7 mg/L of protein yield. Bst polymerase was successfully purified showing 813.56 U/mg of polymerase activity.

Arthrostoma supriatnai sp. nov. (Nematoda: Ancylostomatidae) parasitic in Mydaus javanensis (Carnivora: Mephitidae) from Mount Ciremai, Java, Indonesia.

Arthrostoma supriatnai sp. nov. was described from Mydaus javanensis obtained from Mount Ciremai, Java, Indonesia. It is characterized by having a buccal capsule with ten articulated plates including a pair of additional lateral plates. To date, the genus Arthrostoma consists of eleven species that are native to Asia. Of them, only A. miyazankiense and A. tunkanati have ten articulated plates including the lateral plates. However, the present species has a much stouter body, being readily distinguishable from these two species. Moreover, its male has much shorter spicules than A. miyazakiense, and an arrow-shaped gubernaculum, differing from A. tunkanati, in which gubernaculum is distally bifid. In the female, the present species is readily distinguished from these two species in the shape and number of vulval swellings.

Delivery structure of nationalism message on Twitter in the context of Indonesian netizens.

Nationalism has emerged to be discussed in this modern era due to the emergence of a globalized society. Its delivery structure of nationalism message is significant to be investigated due to its impact on the self-determination of a nation so that nationalism spreads among civil society. In the Indonesian context, discussions of Indonesia's Independence Day arise as a response to this self-determination. Its discussion came up in a face-to-face conversation, as well as on Twitter through the #HUTRI76 network. As a microblogging platform, Twitter was able to facilitate discussion of Indonesian in articulating their Independence Day. The concept of nationalism was utilized to sharpen the analysis. Hence, the social network analysis method has been applied to explore various metrics in the #HUTRI76 Twitter network. The results showed some influencers who varied in their profession, emerged and played a role in sparking discourse on Indonesia's Independence Day. The discussion of Indonesian Independence Day on Twitter was a reproduction message of the government. Moreover, users engaged the varied media to cross-posting their messages as regards Indonesia's Independence Day, which means Twitter acts as a hub through hyperlinks embedded in tweets.

The helminth endoparasite type specimens of the Museum Zoologicum Bogoriense, Indonesia.

A list of the type specimens of helminth endoparasites in the Museum Zoologicum Bogoriense (MZB), Indonesia, is presented. There are types of 93 helminth endoparasite species consisting of 63 species of nematodes, 22 species of cestodes, and 8 species of trematodes that have been deposited in MZB. Altogether, there are currently 211 deposited numbers of type-specimens at MZB, which contain 817 specimens wet preserved and 85 specimens mounted. The list has now been added to the scientific publication record and museum catalog.

Expression of Codon-Optimized Gene Encoding Murine Moloney Leukemia Virus Reverse Transcriptase in Escherichia coli.

Moloney murine leukemia virus reverse transcriptase (MMLV-RT) is the most frequently used enzyme in molecular biology for cDNA synthesis. To date, reverse transcription coupled with Polymerase Chain Reaction, known as RT-PCR, has been popular as an excellent approach for the detection of SARS-CoV-2 during the COVID-19 pandemic. In this study, we aimed to improve the enzymatic production and performance of MMLV-RT by optimizing both codon and culture conditions in E. coli expression system. By applying the optimized codon and culture conditions, the enzyme was successfully overexpressed and increased at high level based on the result of SDS-PAGE and Western blotting. The total amount of MMLV-RT has improved 85-fold from 0.002 g L-1 to 0.175 g L-1 of culture. One-step purification by nickel affinity chromatography has been performed to generate the purified enzyme for further analysis of qualitative and quantitative RT activity. Overall, our investigation provides useful strategies to enhance the recombinant enzyme of MMLV-RT in both production and performance. More importantly, the enzyme has shown promising activity to be used for RT-PCR assay.

Impact of social activity restriction and routine patient screening as a preventive measurement for tertiary referral hospital staff in a country with high COVID-19 incidence.

Background: Measuring COVID-19 incidence among hospital staff and the influencing factors and preventative measures affecting outcomes is important given their high risk of exposure and potential impacts on health service provision. Method: Study participants included all hospital staff with COVID-19 confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) from March 2020 to July 2021. Data were collected on age, gender, occupation, working area, symptoms and vaccination status. We also collected data on pediatric oncology patients and their caregivers to review the hospital screening policy. Results: Approximately 59% of positive cases among hospital staff occurred in the green zone; 75% were fully vaccinated. Whole-genome sequencing indicated that staff infections in June 2021 were Delta variant. A decrease in cases coincided with government implementation of social activity restriction. When RT-PCR was performed in suspected cases, 3 of 36 pediatric oncology patients and 10 staff tested positive. After routine screening, 8 of 121 patients, 3 patient caregivers, and 5 staff tested positive, all were asymptomatic, and all were infected in the community. Conclusions: Routine testing for staff, patients and caregivers, vaccination booster programs, continuing education of health care workers, and government policy, such as social activity restriction, are needed to protect frontline workers.

Titer disparity of anti-Spike receptor binding domain SARS-CoV-2 antibody between vaccinated and naturally infected individuals.

In conjunction with other health promotion strategies, vaccination of coronavirus disease 2019 (COVID-19) is a strategy to alleviate the burden of infection. The aim of this study was to determine the differences in antibody response strength between individuals who received COVID-19 vaccination and those who had a natural infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A cross-sectional study was conducted among post-natural confirmed COVID-19 infection and immunized people in Bali, Indonesia. The vaccination was using Sinovac-CoronaVac with two-weeks interval between the two vaccine doses. To measure the level of anti-Spike receptor binding domain (SRBD) of SARS-CoV-2 antibody, we used Roche electro- chemiluminescence immunoassay (ECLIA) platform. Blood samples were obtained before and 28 days after first immunization in the vaccinated group, as well as two weeks after hospital discharge in the confirmed COVID-19 patients based on real-time reverse transcription polymerase chain reaction (RT-PCR). A total of 58 confirmed COVID-19 patients and 60 vaccinated individuals were included. On the 28th day after the initial vaccination, the seroconversion rate among vaccinated individuals was 91.67%. The mean titer of anti-SRBD SARS-CoV-2 antibody among vaccinated participants was 63.62±82.57 IU/mL (ranged between 0 IU/mL and 250 IU/mL). The mean titer among naturally infected group was 188.47±94.57 IU/mL (ranged between 4.25 IU/mL to 250 IU/mL) regardless the severity of COVID-19. Our data suggested that the titer of anti- SRBD SARS-CoV-2 antibody was significantly higher in naturally infected individuals compared to those who received COVID-19 vaccination (p<0.001). These data suggest that not all individuals vaccinated with Sinovac COVID-19 had protective level of anti- SRBD SARS-CoV-2 antibody and booster dose of heterologous vaccine maybe required.

A new species of Spinicauda (Nematoda: Heterakidae) from Gonocephalus kuhlii (Reptilia: Agamidae) of Ciremai Mountain, Java-Indonesia.

Spinicauda ciremaiensis sp. nov. (Nematoda: Heterakidae) from the large intestine of Gonocephalus kuhlii (Reptilia: Agamidae) collected in Gunung Ciremai National Park, West Java, Indonesia is described and illustrated. Of the five described species of Spinicauda from the Oriental region, Spinicauda ciremaiensis sp. nov. comes closest to S. sumatrana in having 14 pairs of caudal papillae and the presence of a tail filament. However, the new species differs from S. sumatrana in the longer spicula, smaller eggs, and weak sclerotization of the gubernaculum. S. ciremaiensis sp. nov. is also the only species belonging to the genus Spinicauda which has a sclerotized accessories piece of gubernaculum. A key to the species of Spinicauda is also provided. [http://zoobank.org/urn:lsid:zoobank.org:act:785F2D63-E5EB-4E4C-A7E1-7F7E7AD7C392].

Improving the Expression of Human Granulocyte Colony Stimulating Factor in Escherichia coli by Reducing the GC-content and Increasing mRNA Folding Free Energy at 5'-Terminal End.

Purpose: Strategy for improving the production of biopharmaceutical protein continues to develop due to increasing market demand. Human granulocyte colony stimulating factor (hG-CSF) is one of biopharmaceutical proteins that has many applications, and easily produced in Escherichia coli expression system. Previous studies reported that codon usage, rare codon, mRNA folding and GC-content at 5'-terminal end were crucial for protein production in E. coli. In the present study, the effect of reducing the GC-content and increasing the mRNA folding free energy at the 5'-terminal end on the expression level of hG-CSF proteins was investigated. Methods: Synonymous codon substitutions were performed to generate mutant variants of open reading frame (ORF) with lower GC-content at 5'-terminal ends. Oligoanalyzer tool was used to calculate the GC content of eight codons sequence after ATG. Whereas, mRNA folding free energy was predicted using KineFold and RNAfold tools. The template DNA was amplified using three variant forward primers and one same reverse primer. Those DNA fragments were individually cloned into pJexpress414 expression vector and were confirmed using restriction and DNA sequencing analyses. The confirmed constructs were transformed into E. coli NiCo21(DE3) host cells and the recombinant protein was expressed using IPTG-induction. Total protein obtained were characterized using SDS-PAGE, Western blot and ImageJ software analyses. Results: The result showed that the mutant variant with lower GC-content and higher mRNA folding free energy near the translation initiation region (TIR) could produce a higher amount of hG-CSF proteins compared to the original gene sequence. Conclusion: This study emphasized the important role of the nucleotide composition immediately downstream the start codon to achieve high-yield protein product on heterologous expression in E. coli.

Three new species of Cloacininae (Nematoda: Strongyloidea) parasitic in Dorcopsis muelleri (Schlegel, 1866) from Papua and Salawati island, Indonesia.

Three new species of Cloacininae are described from Dorcopsis muelleri obtained from Salawati Island and Kaimana, Papua. Cloacina woworae sp. nov. differs from all congeners by having postero-lateral and medio-lateral rays not fused and the esophagus wider anterior to the nerve ring. Compared to its congeners, C. beveridgei sp. nov. resembles C. caballeroi Mawson, 1977, C. cretheis Beveridge, 2002, C. erigone Beveridge, 2002, and C. syphax Beveridge Speare, 1999 in the morphology of the dorsal ray.  However, C. beveridgei has distal segments of sub-median papillae longer than the proximal segments. This character differentiates it from C. caballeroi, C. cretheis, and C. erigone, which have the distal segment of the sub-median papilla shorter than the proximal. Furthermore, this new species can be distinguished with C. syphax in having a shorter vagina vera and in the shape of the anterior region of the vagina. Cervonemella kaimanaensis sp. nov. differs from Ce. reardoni, the sole species in the genus Cervonemella, in the shape and longer vagina, shorter and slender tail, shorter position of vulva from posterior end and longer spicules.

Description of New Species of Coronostrongylus and Dorcopsistrongylus (Strongylida: Chabertiidae) from Dorcopsis muelleri (Macropodidae) from Kumawa Mountains, West Papua, Indonesia.

New species of Coronostrongylus and Dorcopsistrongylus (Strongyloidea: Chabertiidae) are described from Dorcopsis muelleri (Macropodidae) from Kumawa Mountains, West Papua, Indonesia. Coronostrongylus hasegawai n.sp .is most similar to C. spearei, the only other species described from New Guinea, in having 24 longitudinal pleats in the buccal cavity and spicules less than 1,400 long. Coronostrongylus hasegawai differs from C. spearei, in a suite of characters including the shape of the cephalic collar, the proportions of the buccal capsule, the disposition of the bursal rays, the length of the spicules and the proportions of the ovejector. Dorcopsistrongylus supriyatnai n.sp differs from all congeners in lacking large anteriorly directed intestinal diverticula. The genus Coronostrongylus is found in Australia and New Guinea while the genus Dorcopsistrongylus appears to be endemic to New Guinea.

Sinistral, Dextral, and Mixed Coiling Patterns Observed in Heligmonellid Nematodes (Trichostrongyloidea).

Coiling patterns of heligmonellid nematodes were examined for 520, 208, and 33 individuals of Nippostrongylus brasiliensis, Orientostrongylus tenorai, and Sabanema sp., respectively, collected from murine rodents of Indonesia. Besides typical sinistral coiling, complete dextral coiling was found in 3.3% of N. brasiliensis and 12.1% of Sabanema sp. Mixed coiling with partial sinistral and dextral patterns was also observed in 38.8% of N. brasiliensis, 60.7% of Sabanema sp., and 3.4% of O. tenorai. In dextral coils, the left ventral area with large ridges was located inside as in sinistral coils, keeping the ability to cling to intestinal villi. The cuticular dilatation at left to left dorsal area was located caudally in sinistral coils but rostrally in dextral coils. Presence of mixed coiling indicates that the coiling patterns can change. As the transition of coiling pattern accompanies a change in direction of coil axis, it is surmised that the dextral coiling may be chosen when a worm leaves a villus to move to another villus.