In vitro production of infectious Plasmodium falciparum sporozoites.

An effective vaccine is needed for the prevention and elimination of malaria. The only immunogens that have been shown to have a protective efficacy of more than 90% against human malaria are Plasmodium falciparum (Pf) sporozoites (PfSPZ) manufactured in mosquitoes (mPfSPZ)1-7. The ability to produce PfSPZ in vitro (iPfSPZ) without mosquitoes would substantially enhance the production of PfSPZ vaccines and mosquito-stage malaria research, but this ability is lacking. Here we report the production of hundreds of millions of iPfSPZ. iPfSPZ invaded human hepatocytes in culture and developed to mature liver-stage schizonts expressing P. falciparum merozoite surface protein 1 (PfMSP1) in numbers comparable to mPfSPZ. When injected into FRGhuHep mice containing humanized livers, iPfSPZ invaded the human hepatocytes and developed to PfMSP1-expressing late liver stage parasites at 45% the quantity of cryopreserved mPfSPZ. Human blood from FRGhuHep mice infected with iPfSPZ produced asexual and sexual erythrocytic-stage parasites in culture, and gametocytes developed to PfSPZ when fed to mosquitoes, completing the P. falciparum life cycle from infectious gametocyte to infectious gametocyte without mosquitoes or primates.

Moringa oleifera Gum-Assisted Synthesis and Characterization of CoAgxFe2-xO4: Insight into Structural, Magnetic, Optical, and Biomedical Properties.

The sol-gel method was employed to prepare nano CoFe2O4 and silver-substituted CoFe2O4 nanohybrids (CoAgxFe2-xO4, x = 0, 0.1, 0.2, 0.3, 0.4) utilizing Moringa oleifera gum as biofuel. The morphology, size, shape, magnetic, optical, and functional groups of the crystallites were determined using various techniques such as UV-visible, Fourier transform infrared, X-ray diffraction, Rietveld, scanning electron microscopy, transmission electron microscopy, vibrating sample magnetometry, and photoluminescence. The produced nanoferrite has a spherical shape with cubic spinal structures. The optical properties were investigated in two different bands in the photoluminescence emission spectra at 469 and 493 nm. Saturation magnetization (Ms) and coercivity (Hc) decrease as the Ag content increases significantly. Furthermore, antibacterial (Gram-positive bacteria bacterial strains, Bacillus subtilis and Staphylococcus aureus, and Gram-negative bacterial strains, Pseudomonas aeruginosa, and Escherichia coli), antibiofilm activity (E. coli), and antioxidant (DPPH) activities were investigated. The substantial increase in the silver content offers a constructive impact on studied biomedical activities. These findings encourage additional research into the use of hybrid nanoparticles (an amalgamation of ferrite and a noble metal) in biomedical and pharmaceutical applications.

A replication competent Plasmodium falciparum parasite completely attenuated by dual gene deletion.

Vaccination with infectious Plasmodium falciparum (Pf) sporozoites (SPZ) administered with antimalarial drugs (PfSPZ-CVac), confers superior sterilizing protection against infection when compared to vaccination with replication-deficient, radiation-attenuated PfSPZ. However, the requirement for drug administration constitutes a major limitation for PfSPZ-CVac. To obviate this limitation, we generated late liver stage-arresting replication competent (LARC) parasites by deletion of the Mei2 and LINUP genes (mei2-/linup- or LARC2). We show that Plasmodium yoelii (Py) LARC2 sporozoites did not cause breakthrough blood stage infections and engendered durable sterilizing immunity against various infectious sporozoite challenges in diverse strains of mice. We next genetically engineered a PfLARC2 parasite strain that was devoid of extraneous DNA and produced cryopreserved PfSPZ-LARC2. PfSPZ-LARC2 liver stages replicated robustly in liver-humanized mice but displayed severe defects in late liver stage differentiation and did not form liver stage merozoites. This resulted in complete abrogation of parasite transition to viable blood stage infection. Therefore, PfSPZ-LARC2 is the next-generation vaccine strain expected to unite the safety profile of radiation-attenuated PfSPZ with the superior protective efficacy of PfSPZ-CVac.

Repeat controlled human Plasmodium falciparum infections delay bloodstream patency and reduce symptoms.

Resistance to clinical malaria takes years to develop even in hyperendemic regions and sterilizing immunity has rarely been observed. To evaluate the maturation of the host response against controlled repeat exposures to P. falciparum (Pf) NF54 strain-infected mosquitoes, we systematically monitored malaria-naïve participants through an initial exposure to uninfected mosquitoes and 4 subsequent homologous exposures to Pf-infected mosquitoes over 21 months (n = 8 males) (ClinicalTrials.gov# NCT03014258). The primary outcome was to determine whether protective immunity against parasite infection develops following repeat CHMI and the secondary outcomes were to track the clinical signs and symptoms of malaria and anti-Pf antibody development following repeat CHMI. After two exposures, time to blood stage patency increases significantly and the number of reported symptoms decreases indicating the development of clinical tolerance. The time to patency correlates positively with both anti-Pf circumsporozoite protein (CSP) IgG and CD8 + CD69+ effector memory T cell levels consistent with partial pre-erythrocytic immunity. IFNγ levels decrease significantly during the participants' second exposure to high blood stage parasitemia and could contribute to the decrease in symptoms. In contrast, CD4-CD8 + T cells expressing CXCR5 and the inhibitory receptor, PD-1, increase significantly after subsequent Pf exposures, possibly dampening the memory response and interfering with the generation of robust sterilizing immunity.

TiO2-Doped Ni0.4Cu0.3Zn0.3Fe2O4 Nanoparticles for Enhanced Structural and Magnetic Properties.

TiO2 (0-10 wt %)-doped nanocrystalline Ni0.4Cu0.3Zn0.3Fe2O4 (Ni-Cu-Zn) ferrites were synthesized using the sol-gel route of synthesis. The cubic spinel structure of the ferrites having the Fd3m space group was revealed from the analysis of Rietveld refined X-ray diffraction (XRD) data. The secondary phase of TiO2 with a space group of I41/amd was observed within the ferrites with doping, x > 3 wt %. The values of lattice parameter were enhanced with the addition of TiO2 up to 5 wt % and reduced further for the highest experimental doping of 10 wt %. Field emission scanning electron microscopy (FESEM) images exhibit the spherical shape of the synthesized particles with some agglomeration, while the compositional purity of prepared ferrite samples was confirmed by energy-dispersive X-ray spectroscopy (EDX) and elemental mapping. The cubic spinel structure of the prepared ferrite sample was confirmed by the Raman and Fourier transform infrared (FTIR) spectra. UV-visible diffuse reflectance spectroscopy was utilized to study the optical properties of the ferrites. The value of band gap energy for the pristine sample was less than those of the doped samples, and there was a decrement in band gap energy values with an increase in TiO2 doping, which specifies the semiconducting nature of prepared ferrite samples. A magnetic study performed by means of a vibrating sample magnetometer (VSM) demonstrates that the values of saturation magnetization of the ferrites decrease with the addition of TiO2 content, and all investigated ferrites show the characteristics of soft magnetic materials at room temperature. The Mössbauer study confirms the decrease in the magnetic behavior of the doped ferrites due to the nonmagnetic secondary phase of TiO2.

Changing characteristics of Vibrio cholerae: emergence of multidrug resistance and non-O1, non-O139 serogroups.

The serogroups and antimicrobial susceptibility patterns of V. cholerae isolated in Hubli, India during the years 2000 to 2004 were monitored. A total of 256 V. cholerae isolates were obtained during the study period, of which 129 (50.4%) belonged to serogroup O1 while the O139 and non-O1, non-O139 serogroups constituted 61 (23.8%) and 66 (25.8%) isolates, respectively. V. cholerae O1 Ogawa was the predominant isolate during the first 2 years of the study. However, this was replaced by V. cholerae non-O1, non-O139 serogroups in the following years. The V. cholerae, which was susceptible to most enteric antimicrobials in 2000, was found to be multidrug resistant in subsequent years, with the development of fluroquinolone resistance since 2002. Surveillance of the epidemiological and microbiological characteristics of V. cholerae provides useful information for managing cholera cases. The V. cholerae non-O1, non-O139 serogroups coupled with multiple antimicrobial resistance may form a group of emerging diarrheal pathogens in the tropics.

Listeria monocytogenes meningitis: an uncommon opportunistic infection in HIV/AIDS.

OBJECTIVE: To report an interesting case of meningitis caused by Listeria monocytogenes meningitis in an HIV seropositive individual. MATERIALS & METHODS: A previously healthy 45 years old HIV seropositive man, presented with atypical clinical features of meningitis. Blood and Cerebrospinal fluid (CSF) were obtained for biochemical and microbiological investigations. RESULTS: CSF analysis showed pleocytosis with lymphocytic predominance. Gram stain of CSF was negative; however culture yielded growth of gram positive bacilli with tumbling motility. Based on relevant biochemical tests the isolate was identified as Listeria monocytogenes. Patient was treated with i.v. ampicillin and recovered completely. CONCLUSION: Listeriosis is relatively rare in HIV/AIDS among the immunodeficient populations. Atypical clinical and laboratory findings make the diagnosis difficult and these infections may go undiagnosed. Since it is easily treated with readily available antibiotics, it is important to diagnose them at the earliest and thereby prevent treatment failure.

Fluoroquinolone-resistant Vibrio cholerae isolated during a cholera outbreak in India.

During the cholera epidemic of 2002 in and around Hubli, south India, Vibrio cholerae strains resistant to fluoroquinolones were isolated. Among the isolates of V. cholerae non-O1, non-O139 serogroups, 55.9% and 47.1% were resistant to norfloxacin and ciprofloxacin, respectively. However, only 12.5% of the O1 serogroup strains were resistant to both norfloxacin and ciprofloxacin. Though the O139 serogroup strains were susceptible to these antibiotics, they exhibited multidrug resistance. Emergence of fluoroquinolone-resistant V. cholerae that also exhibited multidrug resistance is of great significance in the epidemiology and control of cholera.

Neonatal sepsis caused by Salmonella enterica serovar Weltevreden.

Salmonella enterica serovar Weltevreden is an uncommon cause of gastroenteritis occurring worldwide. For the first time, we report 2 cases of neonatal sepsis caused by S. Weltevreden from Hubli, India. In the first case, the neonate had features of septicemia and S. Weltevreden was isolated from a blood culture. The other neonate had omplalitis and clinical features of septicemia. S. enterica serovar Weltevreden was isolated from the umbilical swab culture of this neonate. Even though extensive investigations were conducted, the source of infection could not be identified. Both neonates recovered completely after appropriate antibiotic and supportive therapy.

Community-acquired methicillin-resistant Staphylococcus aureus infections in a south Indian city.

There are increasing numbers of reports of community-acquired Staphylococcus aureus being resistant to methicillin. The present study was undertaken as no such reports are available for the developing nations. In a prospective study, between June to December 2001, at the Karnataka Institute of Medical Sciences, Hubli, Karnataka, India, methicillin-resistant S. aureus (MRSA) isolates were tested for clindamycin-susceptibility, a surrogate marker for community-acquired strains. Patients with clindamycin-susceptible isolates were interviewed to determine if they had acquired them in the community and also to identify any risk factors. Of the 116 patients with S. aureus infection, 18.1% had infection with methicillin-resistant strains. Clindamycin-susceptible MRSA accounted for 61.9% of cases. Among these, 46.1% patients were confirmed to have acquired the MRSA from the community, based on inclusion criteria. The community-acquired MRSA were susceptible to multiple antibiotics, as compared to nosocomial isolates. Except for one patient with diabetes mellitus, no other patient had any known risk factor for acquiring MRSA. As significant numbers of MRSA infections are being acquired from the community, treatment options for S. aureus infections may need to be reviewed. Effective infection control programs for the community should be considered to prevent the spread of these infections.

Incidence of Vibrio cholerae serogroup O139 infection with low virulence in Hubli, Karnataka (India).

384 stool samples from patients with acute gastroenteritis were processed by standard culture techniques and antibiogram of V. cholerae was performed. Stool samples from 93 (24.22%) patients yielded V. cholerae, 58 (62.37%) of which were V. cholerae, El Tor O1 Ogawa, 31 (33.33%) V. cholerae O139 and 4 (4.30%) V. cholerae non O1 non O139. Of the culture proven cholera cases watery diarrhoea was observed in 79 (84.95%), vomiting in 57(61.29%), muscle cramps in 21 (22.58%) and sweating in 18 (19.35%). Majority of these patients presented with moderate dehydration 57 (61.29%). Mild dehydration was found in 19 (20.43%) and severe dehydration in 17 (18.28%). While majority of patients with O139 infection had mild to moderate dehydration 25 (80.65%), severe dehydration was more common with O1 infection 11 (64.71%). This study reflects the importance of monitoring the V. cholerae by serogrouping, antibiogram typing, which keep on varying constantly.

Glucose 6 phosphate dehydrogenase deficiency unmasked by diabetic ketoacidosis: an underrated phenomenon.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked disease responsible for moderate to severe hemolytic anaemia. Despite being the most common erythrocyte enzyme disorder, it is often overlooked in the regular diagnostic parlance. A 40-year-old male patient admitted to the casualty with an acutely exacerbated diabetic ketoacidosis, showed features of hemolytic anaemia on peripheral smear examination. Crucially, the spherocytes and bite cells suggested a possibility of G6PD deficiency. This was substantiated by an increased reticulocyte count (6.8%) and a reduced quantitative G6PD enzyme assay (7.2%). There was no significant family or prior medical/ drug history. Interestingly, the hemolytic features were evidenced when blood glucose levels were returning to normal values. The insulin mediated NADPH loss may have resulted in an increased erythrocyte oxidant sensitivity and a loss of sulfhydryl group availability; causing hemolysis to manifest. G6PD deficiency is conventionally affiliated with drug induced oxidative stress. But an association with a diabetes mellitus is seldom reported. This case is being presented as it highlights the lesser known complication of diabetic crisis such as hemolysis secondary to a G6PD deficiency.

Diagnostic value of latex agglutination test in diagnosis of acute bacterial meningitis.

OBJECTIVES: To know the incidence of bacterial meningitis in children below five years of age. To compare conventional culture and antigen detection methods (Latex agglutination test). MATERIALS AND METHODS: 100 CSF samples of clinically suspected meningitis cases in children below 5 years of age were included. The samples were subjected to cell count, Gram stain, culture and LAT. The organisms isolated in the study were characterized according to standard procedures. RESULTS: Of the 100 cases studied, 31 cases were diagnosed as ABM by Gram stain, culture and latex agglutination test as per WHO criteria. The hospital frequency of ABM was 1.7%. 15 (48.38) cases were culture positive. Gram stain was positive in 22(70.96) cases and LAT in 17(54.83) cases. Haemophilus influenzae was the most common causative agent of acute bacterial meningitis followed by S.pneumoniae. Case fatality rate was 45.16%. The sensitivity and specificity of LAT was 66.66% and 87.91% respectively. CONCLUSION: Bacterial meningitis is a medical emergency and early diagnosis and treatment is life saving and reduces chronic morbidity. LAT was more sensitive compared to conventional Gram stain and Culture technique in identifying the fastidious organisms like H.influenzae, S.pneumoniae and Group B Streptococcus. However, the combination of Gram stain, Culture and LAT proved to be more productive than any of the single tests alone.

Morganella morganii, subspecies morganii, biogroup A: An unusual causative pathogen of brain abscess.

Morganella morganii is a gram negative aerobe , found often as intestinal commensal. It is commonly implicated in Urinary tract infections and pyogenic infections, but rarely causes CNS infections especially brain abscess. There are very few published reports of Morganella morganii as a causative pathogen in brain abscess. High index of suspicion of this pathogen is important in cases of brain abscess secondary to otogenic infections. This paper reports an unusual case of Morganella morganii, subspecies morganii, biogroup A Brain abscess. The paper also reviews other infections caused by Morganell morganii.

Increasing burden of hospital acquired infections: resistance to cephalosporin antibiotics among klebsiella and Escherichia coli.

The drug susceptibility pattern of klebsiella species and Escherichia coli to cephalosporins was examined in the year 2004 and 2005. A total of 180 klebsiella and 220 E coli in 2004 and 95 klebsiella and 210. E coli in the year 2005, isolated from various clinical specimens were studied. Resistance rates among the klebsiella species, ranged from 50.00%-86.36% and 42.10%-84.21% in 2004 and 2005 respectively. Resistance rates among E coli ranged from 46.66%-90.8% and 50.0%-86.66% in 2004 and 2005 respectively. In the year 2005 resistance to cefixime and cefpodoxime significantly increased. Resistance marginally increased to cephalexin, cefuroxime and ceftazidime. Resistance to cefotaxime was considerably low (42.10%) in 2005 compared to 62.96% in 2004. The study highlights the importance of exercising caution in the choice of empirical therapy. It also points towards the need for regular monitoring of resistance pattern in the hospital and temporary withholding of the antibiotic with high resistance so as to prevent the spread of multidrug resistant strains in the hospital and the community.

Comparison of disc diffusion methods for the detection of extended-spectrum Beta lactamase-producing enterobacteriaceae.

BACKGROUND: Resistance to broad-spectrum ß lactams, mediated by extended-spectrum ß lactamases (ESBLs), is an increasing problem world wide. This resistance poses problems for in vitro testing and reporting. Increased prevalence of ESBLs among Enterobacteriaceae creates a great need for laboratory testing methods that will accurately identify their presence. MATERIALS AND METHODS: During the study, the Enterobacteriaceae isolated were tested for the presence of ESBL by the National Committee for Clinical Laboratory Standards (NCCLS) screening test, Jarlier double disc synergy (approximation) test (DDST) and NCCLS phenotypic confirmatory test (PCT), and compared their efficiency in detection. RESULTS: A total of 313 Enterobacteriaceae were isolated and tested for the presence of ESBL. NCCLS PCT identified 200 (63.89%) as ESBL producers and DDST identified 176 (56.23%), with a P-value of <0.001. Among the screening agents, ceftazidime had a better sensitivity (89.49%) and specificity (95.74%). CONCLUSIONS: Close monitoring of the susceptibility pattern of isolates and careful spacing with specific discs can identify many ESBL producers. Ceftazidime has a better sensitivity and specificity as a screening agent. A combination of different tests can be useful for accurate identification.

Extended spectrum beta lactamase producing Klebsiella pneumoniae in neonatal intensive care unit.

OBJECTIVE: As infections due to Extended Spectrun beta Lactamase (ESbetaL) producing Klebsiella pneumoniae were increasing in the NICU at Karnataka Institute of Medical Sciences Hospital, Hubli, India, the present study was carried out to identify any environmental sources and the mode of transmission. METHODS: Environmental samples from various sites were collected monthly for a period of six months. RESULTS: ESbetaL producing K. pneumoniae were isolated from all the sites except room air at least on one occasion. ESbetaL producing K. pneumoniae was always isolated from one of the incubators, medicine trolley and sink; while at least one of the health care workers carried it in the hands four out of six times tested. ESbeta L producing K. pneumoniae with similar antibiogram were also isolated from the clinical samples obtained from the neonates. CONCLUSION: Widespread use of third generation cephalosporins as a pre-emptive antibiotic for suspected cases of septicaemia may have contributed to emergence of ESbetaL producing K. pneumoniae in addition to other risk factors. ESbetaL producing K. pneumoniae have extensively colonised the environment of the NICU. Transmission of these pathogens to the neonates has probably occurred through the healthcare workers. Efforts to improve hand hygiene among the healthcare workers and mothers are urgently needed.

Alpha interferon inhibits translation mediated by the internal ribosome entry site of six different hepatitis C virus genotypes.

Certain genotypes of hepatitis C virus (HCV) respond less often than others to treatment with interferon (IFN). The mechanisms for this differential response are not known. In this report antiviral effects of IFN-alpha2b on translation were examined in a hepatic cell line using chimeric clones of internal ribosome entry site (IRES) sequences from six different HCV genotypes and the green fluorescence protein (GFP) gene. As a control, IFN action at the level of the IRES was examined in the presence of different cytokines. It was determined that IFN-alpha2b specifically inhibited the translation of GFP mediated by IRES sequences from six major HCV genotypes in a concentration-dependent manner. Other cytokines including tumour necrosis factor alpha, transforming growth factor beta 1, interleukin 1 and interleukin 6 have no inhibitory effect. The inhibition of translation in these experiments was not due to extensive intracellular degradation of IRES-GFP mRNA. These results suggest that the antiviral action of IFN-alpha2b blocks IRES-mediated translation and this effect is the same among HCVs of other genotypes.