Characterization of outer membrane vesicles released by clinical isolates of Neisseria gonorrhoeae.

The sexually transmitted pathogen Neisseria gonorrhoeae releases membrane vesicles including outer membrane vesicles (OMVs) during infections. OMVs traffic outer membrane molecules, such as the porin PorB and lipo-oligosaccharide (LOS), into host innate immune cells, eliciting programmed cell death pathways, and inflammation. Little is known, however, about the proteome and LOS content of OMVs released by clinical strains isolated from different infection sites, and whether these vesicles similarly activate immune responses. Here, we characterized OMVs from four N. gonorrhoeae isolates and determined their size, abundance, proteome, LOS content, and activation of inflammatory responses in macrophages. The overall proteome of the OMVs was conserved between the four different isolates, which included major outer membrane and periplasm proteins. Despite this, we observed differences in the rate of OMV biogenesis and the relative abundance of membrane proteins and LOS. Consequently, OMVs from clinical isolates induced varying rates of macrophage cell death and the secretion of interleukin-1 family members, such as IL-1α and IL-1ß. Overall, these findings demonstrate that clinical isolates of N. gonorrhoeae utilize membrane vesicles to release proteins and lipids, which affects innate immune responses.

Neisseria gonorrhoeae-derived outer membrane vesicles package ß-lactamases to promote antibiotic resistance.

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea. The treatment of gonorrhoea is becoming increasingly challenging, as N. gonorrhoeae has developed resistance to antimicrobial agents routinely used in the clinic. Resistance to penicillin is wide-spread partly due to the acquisition of ß-lactamase genes. How N. gonorrhoeae survives an initial exposure to ß-lactams before acquiring resistance genes remains to be understood. Here, using a panel of clinical isolates of N. gonorrhoeae we show that the ß-lactamase enzyme is packaged into outer membrane vesicles (OMVs) by strains expressing blaTEM-1B or blaTEM-106, which protects otherwise susceptible clinical isolates from the ß-lactam drug amoxycillin. We characterized the phenotypes of these clinical isolates of N. gonorrhoeae and the time courses over which the cross-protection of the strains is effective. Imaging and biochemical assays suggest that OMVs promote the transfer of proteins and lipids between bacteria. Thus, N. gonorrhoeae strains secret antibiotic degrading enzymes via OMVs enabling survival of otherwise susceptible bacteria.

Bacterial outer membrane vesicles and host cell death signaling.

The programmed cell death pathways of pyroptosis and apoptosis protect mammals from infections. The activation of host cell death signaling depends on cell surface and cytosolic receptors that bind bacterial molecules or sense their activity. The formation of cytosolic protein complexes, such as the inflammasome and apoptosome, activates caspases, pore-forming proteins, and inflammatory cytokines. These pathways respond to bacteria and their released membrane vesicles. Outer membrane vesicles (OMVs) that emerge from the outer membrane of Gram-negative bacteria deliver a range of bacterial molecules, including lipids, proteins, polysaccharides and nucleic acids to host cells. Recent findings describe how OMV-associated molecules activate pyroptosis, apoptosis, and other inflammatory pathways. We discuss here how OMV-associated molecules are sensed by the immune system and how this contributes to infections and inflammatory diseases.

Mitochondrial dysfunction caused by outer membrane vesicles from Gram-negative bacteria activates intrinsic apoptosis and inflammation.

Sensing of microbes activates the innate immune system, depending on functional mitochondria. However, pathogenic bacteria inhibit mitochondrial activity by delivering toxins via outer membrane vesicles (OMVs). How macrophages respond to pathogenic microbes that target mitochondria remains unclear. Here, we show that macrophages exposed to OMVs from Neisseria gonorrhoeae, uropathogenic Escherichia coli and Pseudomonas aeruginosa induce mitochondrial apoptosis and NLRP3 inflammasome activation. OMVs and toxins that cause mitochondrial dysfunction trigger inhibition of host protein synthesis, which depletes the unstable BCL-2 family member MCL-1 and induces BAK-dependent mitochondrial apoptosis. In parallel with caspase-11-mediated pyroptosis, mitochondrial apoptosis and potassium ion efflux activate the NLRP3 inflammasome after OMV exposure in vitro. Importantly, in the in vivo setting, the activation and release of interleukin-1ß in response to N. gonorrhoeae OMVs is regulated by mitochondrial apoptosis. Our data highlight how innate immune cells sense infections by monitoring mitochondrial health.

Establishment of reference range of CD4 T-lymphocyte in healthy Nepalese adults.

OBJECTIVE: CD4 T lymphocytes are the most widely used cellular markers to assess the course of HIV infection, clinical staging and, monitoring the effect of antiretroviral therapy. The regional reference range for Eastern, Central and Western development region of Nepal had already been established whereas the same was still lacking in Mid-western and Far-western development region. The objective of this study was to establish reference range of CD4 T lymphocyte in the remaining two development regions and finally the national reference range using data from previous study. RESULTS: The average values (mean ± SD) of CD4 and CD3 T cell in present study was (819 ± 294) cells/µl and (1546 ± 532) cells/µl, respectively. The absolute CD4 T cell (914 ± 303) and CD3 T cell (1671 ± 560) count in female were significantly higher than those from male, CD4 (757 ± 270) and CD3 (1465 ± 499) (p value-0.000). National reference value of CD4 was determined to be (798 ± 335) cells/µl for healthy Nepalese adults.

Evaluation of Five International HBV Treatment Guidelines: Recommendation for Resource-Limited Developing Countries Based on the National Study in Nepal.

Hepatitis B virus (HBV) infects the liver, causing cirrhosis and cancer. In developed countries, five international guidelines have been used to make a decision for the management of patients with chronic HBV infection. In this review, since the guidelines were established by clinical and epidemiological data of developed countries, we aimed to evaluate whether (1) HBV patient profiles of developing countries are similar to developed countries, and (2) which guideline can be applicable to resource-limited developing countries. First, as an example of the most recent data of HBV infections among developing countries, we evaluated the national HBV viral load study in Nepal, which were compared with the data from other developing countries. In Nepal, the highest number of patients had viral loads of 20-2000 IU/mL (36.7%) and belonged to the age group of 21-30 years; HBV epidemiology in Nepal, based on the viral loads, gender, and age groups was similar to those of not only other developing countries but also developed countries. Next, we reviewed five international HBV treatment guidelines of the World Health Organization (WHO), American Association for the Study of Liver Diseases (AASLD), National Institute for Health and Care Excellence (NICE), European Association for the Study of the Liver (EASL), and Asian Pacific Association for the Study of the Liver (APASL). All guidelines require the viral load and alanine aminotransferase (ALT) levels for decision making. Although four guidelines recommend elastography to assess liver cirrhosis, the WHO guideline alternatively recommends using the aspartate aminotransferase (AST)-to-platelet ratio index (APRI), which is inexpensive and conducted routinely in most hospitals. Therefore, in resource-limited developing countries like Nepal, we recommend the WHO guideline for HBV treatment based on the viral load, ALT, and APRI information.

Efficacy and Safety of Injectable and Oral Antibiotics in Treating Gonorrhea: A Systematic Review and Network Meta-Analysis.

Gonorrhea is the second most frequently reported sexually transmitted infectious disease of bacterial origin in the world. Current empiric therapies rely on broad-spectrum antibiotics. However, treatment options are becoming limited due to the rise of drug-resistant gonorrhea. To control the rise of drug-resistant gonorrhea and to identify alternative treatment options, clinicians will have to increasingly rely on experimental evidence for the treatment of gonorrhea patients. Thus, we performed a systematic review and network meta-analysis of all randomized clinical trials about the efficacy and safety of various antibiotic regimens in adults with gonorrhea. We searched all references in Embase and PubMed from the date of their inception to January 2019, and then an updated search was performed in March 2019. Of the 28,843 identified references, 44 fulfilled our selection criteria. We used a network meta-analysis based on a frequentist approach to evaluate the efficacy and safety of 12 injectable and 11 oral antibiotics. The efficacy of treatments was ranked by p score and inconsistency was assessed by a back-calculation method. Certainty of evidence was evaluated by the GRADE system. For injectable drugs, there was no difference in efficacy between a reference antibiotic and other drugs. However, ceftriaxone had significantly better efficacy than cefuroxime (OR, 12.03; 95% CI 3.73-38.79), cephaloridine (OR, 42.41; 95% CI 8.77-205.07), kanamycin (OR, 5.45; 95% CI 1.25-23.70), penicillin (OR, 13.11; 95% CI 4.48-38.37), and spectinomycin (OR, 4.70; 95% CI 1.62-13.62). Thus, ceftriaxone was the most effective injectable drug (p score of 0.924). As for oral drugs, azithromycin was the most effective compound (p score of 0.8633). There were no significant differences in safety between injectable and oral treatments. In our systematic review of randomized controlled trials, we found azithromycin and ceftriaxone to be the most effective antibiotics for the treatment of gonorrhea. This is in line with current guidelines which recommend a combination therapy of azithromycin and ceftriaxone for the treatment of gonorrhea due to increased antimicrobial resistance. Our analysis identified gentamicin and ofloxacin as alternative therapeutics to treat drug-resistant gonorrhea.

Prevalence and Mother-to-newborn Transmission of Hepatitis B Virus in Tertiary Care Hospital in Nepal.

BACKGROUND: The prevalence of hepatitis B infection is heterogeneous and ubiquitous. This study aimed to identify the prevalence of the infection among the pregnant women who attended in a tertiary care hospital, transmission of the infection in their newborn and associated risk factors. METHODS: A one year prospective study was conducted. Mother's venous blood was collected for the hepatitis B serological test during the antenatal care or before delivery; the newborn's cord blood was also collected for the HBsAg. RESULTS: A total 16400 pregnant women were tested; of them 53 were HBsAg positive. The total prevalence of the infection was 0.32% among the pregnant: 0.5% among the indigenous and 0.2% in other than indigenous. The infection was significantly high in the indigenous group compare to other than indigenous [2.596 (1.475-4.569), p=.001]. Thirty-two out of 53 hepatitis B positive pregnant were delivered in the hospital, of them 75% (n=24) were indigenous and 25% (n=8) were other than indigenous. Eight out of 32 were highly infectious (HBeAg+), of them majority (75%) were indigenous ethnicities. Twenty-one out of 32 were anti-HBe reactive, among which majority were indigenous ethnicities (76.2%). Six out of eight babies, born with HBeAg reactive mothers, were infected (75%), of which majority were indigenous ethnicities (67%). In total, one-fifth of the newborn delivered were HBsAg positive (18.8%). CONCLUSIONS: The prevalence of hepatitis B infection among the total pregnant was low. The proportion of the infection in the indigenous ethnicity was significantly high compared to other ethnic group, which shows that the infection was clustered among the indigenous people.

Sustained Viremic Control in HIV-Infected Patient: Case Report from Nepal.

A case of human immunodeficiency virus (HIV) infection is described from Nepal with constant maintenance of CD4 count and HIV-RNA level below the lower detection threshold for more than ten years. The case was diagnosed of HIV positive in the year 2008. He had his viral load estimation performed every year since then which was always below lower detection limit and remained healthy without treatment. The patient also had not any kinds of opportunistic infection till date. He is married now and has not transmitted the disease to his wife.

Antimicrobial susceptibility pattern of Shigella spp. isolated from children under 5 years of age attending tertiary care hospitals, Nepal along with first finding of ESBL-production.

BACKGROUND: Shigella is an important cause of bacterial gastroenteritis in resource-poor countries. The treatment of shigellosis mostly requires antibiotics. However, the increase of multidrug resistance along with emergence of extended-spectrum ß-lactamase and ciprofloxacin resistance among Shigella spp. has challenged the situation. This study was conducted to determine the distribution of species and antibiotic susceptibility pattern of Shigella species isolated from stool specimen among children less than 5 years of age in Nepal. RESULTS: Out of total 717 stool samples collected, 15 cases of Shigella spp. was isolated which includes 12 S. flexneri and 3 S. sonnei. Multidrug resistance was found among 13(86%) of the isolates. One of the isolates of S. flexneri was found to be ESBL-producer with MIC >256 mg/L for cefixime. CONCLUSION: The high occurrence of multidrug resistance among Shigella spp. along with a case of ESBL-production for the first time in Nepal alarms the concerns about dissemination of the resistant isolates. So, systemic monitoring of the antimicrobial susceptibility pattern of Shigella spp. is becoming crucial to guide therapy.

Isolation, speciation and antifungal susceptibility testing of Candida isolates from various clinical specimens at a tertiary care hospital, Nepal.

BACKGROUND: Candida species are responsible for various clinical infections ranging from mucocutaneous infection to life threatening invasive diseases along with increased resistance to antifungal drugs has made a serious concern. Resistance to antifungal agents has increased during the last decade. Thus, identification of Candida up to species level and its antifungal susceptibility testing has a paramount significance in the management of Candidal infections. The aim of the study was to speciate Candida species and to determine antifungal susceptibility pattern of Candida species to antifungal agents. METHODS: A total of 100 consecutive Candida species were isolated from 1248 clinical specimens over 7 months period. Growths on Sabouraud dextrose agar were evaluated for colony appearance, macroscopic examination, Gram staining, germ tube test and urea hydrolysis test. Further, they were processed for Candida speciation on CHROMagar. Antifungal susceptibility testing was performed as recommended by Clinical and Laboratory Standards Institute (CLSI) M44-A document. RESULTS: Out of 100 Candida isolates, Candida albicans (56%) was the most common species. Among the non-albicans Candida species, Candida tropicalis (20%) was the predominant isolate followed by Candida glabrata (14%). Regarding antifungal susceptibility pattern, Candida species were more susceptible to clotrimazole (82%) followed by fluconazole (64%) and miconazole (44%). CONCLUSIONS: Candida albicans was the predominant species responsible for various Candidal infections. Among commonly used antifungal drugs clotrimazole, miconazole and fluconazole were most effective.

Molecular epidemiology of Rotavirus causing diarrhea among children less than five years of age visiting national level children hospitals, Nepal.

BACKGROUND: Rotaviruses are the major cause of diarrhea among the infants and young children all over the world causing over 500,000 deaths and 2.4 million hospitalizations each year. In Nepal Rotavirus infection positivity rates ranges from 17.0 to 39.0% among children less than 5 years. However, little is known about the molecular genotypes of Rotavirus prevailing. The objective of this study was to estimate the burden of Rotavirus gastroenteritis and determine the genotypes of Rotavirus among children less than 5 years. METHODS: The cross sectional study was conducted from January to November 2014 among children less than 5 years old visiting Kanti Children's Hospital and Tribhuvan University Teaching Hospital. Rotavirus antigen detection was performed by Enzyme Linked Immunosorbent Assay (ELISA) using ProSpecT Rotavirus Microplate Assay. Among the Rotavirus antigen positive samples, 59 samples were used for Rotavirus RNA extraction. Multiplex PCR was performed to identify G type comprising G1-G4, G8-G10 and G12 and P type comprising P[4], P[6], P[8], P[9], P[10], and P[11]. RESULTS: A total of 717 diarrheal stool samples were collected from patients ranging from 10 days to 59 months of age. Rotavirus antigen positive was found among (N = 164)22.9% of patients. The highest number of the diarrhea was seen in January. Molecular analysis of Rotavirus genotypes revealed that the predominant G-Type was G12 (36%) followed by G9 (31%), G1 (21%), G2 (8.6%). The predominant P- type was P6 (32.8%) followed by P8 (31%), P10 (14.8%), P4 (14.8%). A total of seven G/P type combinations were identified the most common being G12P [6] (35.8%), G1P [8] (15.1%), G9P [8] (15.1%). CONCLUSION: Rotavirus diarrhea is, mostly affecting children from 7 to 24 months in Nepal, mostly occurring in winter. The circulating genotypes in the country are found to be primarily unusual genotypes and predominance of G12P[6]. It is recommended to conduct genotyping of Rotavirus on large samples before starting vaccination in the country.

Clinicomycological Characterization of Superficial Mycoses from a Tertiary Care Hospital in Nepal.

Background. Superficial mycosis is a common fungal infection worldwide, mainly caused by dermatophytes. However, the prevalence of species varies geographically. In addition, fungal treatment is best guided according to species isolated. This study was carried out to determine the clinical as well as mycological profile of superficial mycoses in a tertiary care hospital, Nepal. Methods. This was a prospective case-control laboratory based study conducted over a period of six months from January to June 2014 at Tribhuvan University Teaching Hospital, Nepal. A total of 200 specimens were collected from the patients suspected of superficial mycoses. The specimens were macroscopically as well as microscopically examined. The growth was observed up to 4 weeks. Results. Out of total 200 specimens from the patients suspected of superficial mycoses, tinea corporis 50 (25%) was most common clinical types. KOH mount was positive in 89 (44.5%) and culture was positive in 111 (55.5%). Trichophyton mentagrophytes 44 (39.6%) was the most common isolate. Conclusions. The diagnostic yields of KOH mount and culture were found to be complementary to each other. Thus both the methods added with clinical findings are equally important to establish superficial mycosis.