Objective To develop a reversed-phase high-performance liquid chromatography(RP-HPLC) method for the determination of residual phenylmethanesulfonyl fluoride(PMSF) content in recombinant virus-like particle(VLP) vaccine stock solution,and to verify the method for the determination of PMSF residues in recombinant VLP vaccine stock solution.Methods A RP-HPLC method for the determination of PMSF residues was developed by screening detection wavelength,flow rate,injection amount and chromatographic column.The specificity,linearity and range,limit of quantitation(LOQ),limit of detection(LOD),accuracy,precision and durability of the method were verified.The developed method was used to detect PMSF residues in three batches of recombinant hepatitis E vaccine stock solution.Results The RP-HPLC method was developed,with ChromCore 300 C18(4.6 mm × 250 mm,5 μm) as the chromatographic column at the column temperature of 25 ℃,the detection wavelength of 210 nm,the injection amount of 100 μL,0.1% trifluoroacetic acid(TFA)/water and0.1% TFA/acetonitrile as mobile phase at a flow rate of 1 mL/min,and the detection time of 18 min.The absorption peaks of PMSF control and vaccine stock solution containing PMSF appeared around 5.0 min,while the vaccine stock solution and stock solution buffer showed no absorption peak.There was a good linear relationship between the concentration of PMSF control and peak area in the range of 0-5.0 μg/mL,with R~2 of 0.999.The LOQ and LOD of this method was 0.250 μg/mL and 0.125 μg/mL,respectively.The recovery rates of PMSF control with high,medium and low concentrations were all between 90% and 110%.The relative standard deviations(RSDs) of retention time,peak height and peak area for the determination of instrument repeatability,sample repeatability and intermediate precision were all less than 5%.The RSDs of retention time,peak height and peak area were all less than 5% under the TFA concentration of 0.09%,0.1% and 0.11% at column temperature of 20,25 and 30 ℃.PMSF was not detected in the three batches of recombinant hepatitis E vaccine stock solution.Conclusion The developed RP-HPLC method has good specificity,linearity and durability,high accuracy and precision,and can be used to detect residual PMSF content in recombinant VLP vaccine stock solution.
Phenylmethanesulfonyl fluoride(PMSF) ; Reversed-phase high-performance liquid chromatography(RP-HPLC) ; Virus-like particle(VLP) vaccine

BACKGROUND

Cervical cancer remains to be the second leading cancer and cause of cancer-related deaths among Filipino women despite the use of the Papanicolaou screening. Latest research has shown that the human papillomavirus (HPV) is a necessary cause of cervical cancer. With major morbidity and high mortality rates associated with HPV infection and cervical cancer, several modes of primary and secondary forms of prevention have to be implemented. Among the primary modes of prevention is the administration of the preventive vaccine, which has consistently shown to decrease substantially HPV disease and cervical cancer rates in developed countries. In our country, before a successful vaccination, program is implemented, several sociocultural issues have to be addressed. Knowledge, attitude, and motivational factors are vital in determining acceptance of the vaccine. One relevant setting is exploring the willingness of mothers to get their female children vaccinated even before they become sexually active.

The aim of the study was to determine the association of the knowledge, attitude, and motivational factors of mothers on their willingness for their female children aged 9–13 years to undergo HPV vaccination at a tertiary government hospital.

This was a cross-sectional study that was carried out at a government institution.

The population consisted of 352 mothers with female children aged 9–13 years consulting the outpatient clinics at the department of obstetrics and gynecology at a tertiary government hospital.

A pretested and validated survey was given to 352 respondents. They were asked to answer a self-administered questionnaire that included sociodemographic, reproductive, sexual history variables, knowledge, and attitude, and motivational factors toward the disease and the associated vaccine.

Using the survey proportion estimation methods, the prevalence of women who were willing to enroll their daughters for HPV vaccination was 97.18% (n = 42, 95% confidence interval [CI]: 94.91 to 98.46%). It can be noted that only a third of the sample had high knowledge on the vaccine and its use 34.93% (n = 124, 95% CI: 30.25 to 39.92%). More women who reached college level (χ2: 5.67) and also those whose youngest child was between 11 and 13 years old (χ2: 8.82)-had higher knowledge scores than otherwise. Those who have an annual income of greater than or equal to P 60,000 (χ2: 16.55) and are non-Catholic (χ2: 18.77) – also appeared to have higher knowledge ratings on the questionnaire. Women who never to a few times a year attend church-related activities had higher knowledge scores compared to women who were more frequent goers (χ2: 16.33). For the attitude toward the vaccine, more mothers believed that getting the vaccine would not have an effect on a girl’s sexual activity and most agreed that they would not be viewed as bad parents. Most women also did not believe that religion would affect their willingness to vaccinate their children. There was an association in the degree of agreement between negative and positive attitudes from the Chi-square test performed (χ2: 7.44, P: 0.01). There were more agreeing responses from factors determining positive attitude and more disagreeing responses in the factors determining negative attitude. With regard to motivational factors, more women agreed that the cost was prohibitive and that they were more willing if only two doses would be required for their daughters. They were also not concerned about what other parents may think about getting the vaccine. Most answered that they were willing to follow their doctors’ recommendations and they have trust in vaccine manufacturers. Most women were also concerned that their daughters may get cervical cancer in the future. There was no difference in the proportion of agreeing responses between positive and negative motivating factors among the study participants (Z: 0.30, P: 0.79). This suggested that these factors could be important predictors of willingness to use vaccination on their children. Based on the crude odds ratios from the logistic regression, the likelihood of being willing to administer HPV vaccine to their children was almost twice as the knowledge score and scores on the positive attitude items increased, and was found to be statistically significant. At the same time, the odds of willingness increased by more than twice as the score on the negative attitude items decreased, and was also significant. There was no noted association for the other predictors of the association.

The role of knowledge and attitudes on the negative perceptions on the vaccine were important predictors of the willingness of mothers to have their female children vaccinated against HPV infection.

Cough is a very common symptom causing medical consult. Several remedies are readily available in the market however these are currently not recommended among the pediatric population due to a few reasons which include the benign nature of acute cough, limited effectivity and lack of support from the United States Food and Drug Administration (USFDA) due to abuse potential.

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We report a case of a 2-year-old male, no known co-morbidities with a 2 week history of upper respiratory tract infection. Initial assessment showed viral infection hence patient was given medications for symptomatic treatment. However, l week after, patient still presented with symptomatic persistent coughing that disrupted his activities of daily living, hence antitussive medication was already prescribed. After another 7 days, there was still persistence of symptoms, hence patient was given a trial medication of Pregabalin 0.7 milligram/kg/dose which noted instant cough relief one hour after the initial intake. Patient also reported to be more playful, improved sleep at night and improved appetite. Patient received total of 2 doses of Pregabalin in the span of 48 hours. On the third day, patient was still coughing but reported to be significantly less frequent and more productive, hence medication was then put on hold. Patient continuously improved after 5 more days and was eventually cough free.

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This case report demonstrates the adequacy of Pregabalin as a supportive antitussive medication in a patient with an acute cough secondary to a viral infection.

Objective To prepare mouse monoclonal antibodies against the ectodomain of E2 (E2ecto) glycoprotein of Western equine encephalitis virus (WEEV). Methods A prokaryotic expression plasmid pET-28a-WEEV E2ecto was constructed and transformed into BL21 (DE3) competent cells. E2ecto protein was expressed by IPTG induction and presented mainly as inclusion bodies. Then the purified E2ecto protein was prepared by denaturation, renaturation and ultrafiltration. BALB/c mice were immunized with the formulated E2ecto protein using QuickAntibody-Mouse5W as an adjuvant via intramuscular route, boosted once at an interval of 21 days. At 35 days post-immunization, mice with antibody titer above 1×10⁴ were inoculated with E2ecto intraperitoneally, and spleen cells were fused with SP2/0 cells three days later. Hybridoma cells secreting specific monoclonal antibodies were screened by the limited dilution method, and ascites were prepared after intraperitoneal inoculation of hybridoma cells. The subtypes and titers of the antibodies in ascites were assayed by ELISA. The biological activity of the mAb was identified by immunofluorescence assay(IFA) on BHK-21 cells which were transfected with eukaryotic expression plasmid pCAGGS-WEEV-CE3E2E1. The specificity of the antibodies were evaluated with E2ecto proteins from EEEV and VEEV. Results Purified WEEV E2ecto protein was successfully expressed and obtained. Four monoclonal antibodies, 3G6G10, 3D7G2, 3B9E8 and 3D5B7, were prepared, and their subtypes were IgG2c(κ), IgM(κ), IgM(κ) and IgG1(κ), respectively. The titers of ascites antibodies 3G6G10, 3B9E8 and 3D7G2 were 10⁵, and 3D5B7 reached 10⁷. None of the four antibody strains cross-reacted with other encephalitis alphavirus such as VEEV and EEEV. Conclusion Four strains of mouse mAb specifically binding WEEV E2ecto are successfully prepared.
Horses ; Animals ; Mice ; Encephalitis Virus, Western Equine ; Ascites ; Immunosuppressive Agents ; Antibodies, Monoclonal ; Immunoglobulin M

Humans ; Herpesvirus 4, Human ; Epstein-Barr Virus Infections ; Carcinoma, Small Cell ; Nasopharynx/pathology* ; Carcinoma, Neuroendocrine/pathology* ; Nasopharyngeal Neoplasms/pathology*

Background and Objectives: Municipal solid waste workers (MSWWs) are important in the city’s waste management. With these vital contributions, they face unique occupational hazards and health risks. This study aims to determine the prevalence of occupational infections, such as soil-transmitted helminth infections (STHI) and hepatitis A virus (HAV), as well as the occurrence of Helicobacter pylori infection among the MSWWs of Baguio City. Methods: This cross-sectional analytic study collected data from volunteer MSWWs using a questionnaire to gather information on age, duration of employment, use of gloves in the workplace, and hand hygiene practices. Stool samples were obtained from participants and were analyzed for STHI using the Formalin Ether Concentration Technique (FECT). H. pylori infection was detected using the SD Bioline rapid antigen test kit on stool samples while blood samples were collected and tested for HAV antibodies using the Aria IgG/IgM rapid test kit. Results: Of the 44 volunteer MSWWs tested, 25 were infected with hazardous pathogens. Specifically, six workers (13.6%) were infected with STHI, four (9.1%) were infected with HAV and 15 (34.1%) were infected with H. pylori. Among those infected with STHI, Ascaris lumbricoides and Endolimax nana were the predominant species, each with a prevalence rate of 33.3%. In contrast, Blastocystis hominis and hookworm infections each had a prevalence rate of 16.7%. A significant association was found between STHI prevalence and the preference for alcohol hand rubs over hand washing, with a p-value of 0.008. Conclusion The analysis revealed a significant associat ion between the prevalence of STHI and the preference for alcohol hand rubs over hand washing, suggesting that MSWWs may have a false sense of security regarding their hygiene practices. The findings revealed the critical importance of proper hand washing in preventing STHI. Future research should expand data collection to encompass a broader range of socio-demographic, environmental, and lifestyle factors that may influence infection rates. Additionally, including a control group of individuals not exposed to waste management could help differentiate between factors specific to waste handling and those related to other occupations. This study emphasizes the need for collaborative efforts among researchers, public health authorities, and waste management agencies to enhance the health and safety of MSWWs while addressing broader public health concerns related to waste management practices.
Human ; Hepatitis A virus (HAV) ; Helicobacter pylori

People living with human immunodeficiency virus (PLHIV) are 100 times more at risk for cutaneous adverse drug reactions (ADRs). Acute generalized exanthematous pustulosis (AGEP) is a rare and severe cutaneous ADR associated with systemic involvement in 20% of cases.

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This is a case of a 30-year-old male living with HIV admitted for acute gastroenteritis. Eight hours after initiation of intravenous ciprofloxacin and metronidazole, the onset of generalized monomorphic asymptomatic pustules was observed with associated weakness, fever, thrombocytosis, and neutrophilia. Ciprofloxacin was shifted to piperacillin-tazobactam. The patient was managed with intravenous hydrocortisone and oral cetirizine. Thereafter, the lesions remained stable in size and no new lesions occurred. The patient was referred to the dermatology service for further evaluation and management. A diagnostic workup was done which revealed subcorneal pustular dermatitis on histopathology, no fungal elements on periodic acid-Schiff stain, negative Gram stain, and no growth on culture. This case was diagnosed as AGEP secondary to ciprofloxacin. Dermatologic management consisted of oral antihistamines and topical steroids. The patient experienced generalized desquamation and gradual resolution of pustules over a two-week period with the eventual appearance of normal skin.

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Ciprofloxacin is commonly used to treat opportunistic infections in the setting of HIV but it has never been documented to cause AGEP in such settings. Decreased CD4+ T-cell count (460 cells/µL) are factors associated with drug eruptions. Despite its toxic presentation, AGEP has a good prognosis with prompt withdrawal of the offending drug and supportive management.

The outbreak of monkeypox has become a global matter of concern since last year. Monkeypox is a zoonotic disease caused by monkeypox virus（MPXV）infection，and in addition to the typical symptom of rash，MPXV infection can cause a series of neurological manifestations，with the potential mechanisms of immune-mediated neurological damage after infection and direct invasion of the virus into the nervous system. This article reviews the neurological manifestations of MPXV infection，so as to facilitate the early identification and diagnosis of the neurological complications of MPXV infection and adopt appropriate prevention and treatment measures in a timely manner.
Monkeypox virus

Cytomegalovirus（CMV）ventriculoencephalitis is a type of severe encephalitis caused by CMV infection of the ependyma and brain parenchyma，with the main clinical manifestations of fever，headache，disturbance of consciousness，and convulsions. The opportunistic infection of CMV often occurs in immunocompromised people，mainly in patients with acquired immune deficiency syndrome（AIDS），while nervous system infection is extremely rare in people with competent immune function. This article reports a case of CMV ventriculoencephalitis in a non-AIDS young male patient，with pyrexia and disturbance of consciousness as the initial presentation. Contrast-enhanced magnetic resonance imaging showed a typical enhanced signal shadow of the ventriculomeninges，and a confirmed diagnosis was made based on cerebrospinal fluid CMV examination. We hope that this case of non-AIDS-related CMV ventriculoencephalitis will provide help for clinicians to improve their understanding of the clinical and imaging manifestations of this disease.
Cytomegalovirus

Objective To perform quality control in live attenuated yellow fever vaccine(chicken embryo cell)virus seed bank at the genomic level using the new generation Illumina/Solexa sequencing platform.Methods The live attenuated yellow fever vaccine strain YF17D-204 was inoculated into primary chicken embryo cells,and the chicken embryo cell adapted strains of live attenuated yellow fever vaccine were screened to establish YFV17D-CEC tertiary virus seed bank. The genome RNA of virus seeds was extracted,and the RNA library was prepared. The new generation Illumina/Solexa sequencing platform was used for high-throughput RNA sequencing. The whole genome nucleic acid sequence of yellow fever virus was systematically analyzed by using biological softwares such as FastQC,Trimmomatic,SPAdes,GapFiller,PrInSeS-G,Prokka,RepeatMasker,CRT,NCBI Blast~+,KAAS,HMMER3,TMHMM,SignalP,LipoP,ProtCamp and MegAlign.Results The whole genome of YFV17D-CEC tertiary virus seed bank contained 10 862 nucleotides,including an open reading frame(ORF)from 119 to 10 354(10 236 bp),encoding 3 412 amino acids. Sequence alignment analysis showed that the sequence of YF17D-CEC tertiary virus seed bank was 100% identical with YFV17D RKI(JN628279.1),YF/Vaccine/USA/Sanofi-Pasteur-17D-204/UF795AA/YFVax(JX503529.1)and YFV17D-204(KF769015.1),and no mutation occurred in the whole genome of the tertiary virus seed bank. Comparison of the sequences of different live attenuated yellow fever vaccine strains showed that yellow fever virus had multiple polymorphic sites.Conclusion YFV17DCEC has good genetic stability in primary chicken embryo cells. High-throughput RNA sequencing technology can quickly detect the whole genome information of YF17D-CEC virus seed bank,and the sequence analysis data can be used in the gene level quality control of yellow fever vaccine virus seed banks.
High-throughput RNA sequencing ; Live attenuated yellow fever vaccine ; Gene expression ; Virus seed bank ; Quality control