Comparison of real-time PCR and hemagglutination assay for quantitation of human polyomavirus JC

Human polyomavirus JC (JCV), the etiological agent of the disease progressive multifocal leukoencephalopathy (PML) affects immunocompromised patients particularly patients with AIDS. In vitro studies of JCV infection are hampered by the lack of sensitive JCV quantitation tests. Although the hemagglutination (HA) assay has been routinely employed for in vitro quantitation of JCV, its sensitivity is severely limited. We have employed a real-time PCR assay which compares favorably with the HA assay for the in vitro quantitation of JCV. JCV(Mad1), propagated in primary human fetal glial (PHFG) cells in two independent laboratories, was purified and quantitated by the HA assay. Both batches of purified JCV(Mad1) were then serially diluted in Dulbecco's Modified Eagle's Medium to obtain HA titers ranging from 64 to 0.001 HA units (HAU) per 100 μL of virus suspension. DNA was extracted from 100 μL of virus suspension and eluted in 50 μL of buffer, and DNA amplification and quantitation were performed in the Bio-Rad iCycler iQ Multicolor Real-Time PCR Detection System using T-antigen as the target gene. Real-time PCR for quantitation of JCV was sensitive and consistently detected 1.8 × 10(1 )copies of JCV DNA, and as low as 0.001 HAU equivalent of JCV. Moreover, there was a strong linear correlation between the HA assay and the DNA copy number of JCV(Mad1). The intra-run and inter-run coefficients of variation for the JCV standard curve were 0.06% to 4.8% and 2.6% to 5.2%, respectively. Based on these data, real-time PCR can replace the less-sensitive HA assay for the reliable detection, quantitation and monitoring of in vitro JCV replication

Neonatal Septicemia in Nepal: Early-Onset versus Late-Onset

Introduction. Neonatal septicemia is defined as infection in the first 28 days of life. Early-onset neonatal septicemia and late-onset neonatal septicemia are defined as illnesses appearing from birth to three days and from four to twenty-eight days postnatally, respectively. Methods. In this cross-sectional study, blood samples from the suspected infants were collected and processed in the bacteriology laboratory. The growth was identified by standard microbiological protocol and the antibiotic sensitivity testing was carried out by modified Kirby-Bauer disk diffusion method. Results. Among total suspected cases, the septicemia was confirmed in 116 (12.6%) neonates. Early-onset septicemia (EOS) was observed in 82 infants and late-onset septicemia (LOS) in 34 infants. Coagulase-negative staphylococcus (CoNS) (46.6%) was the predominant Gram-positive organism isolated from EOS as well as from LOS cases followed by Staphylococcus aureus (14.6%). Acinetobacter species (9.5%) was the predominant Gram-negative organism followed by Klebsiella pneumoniae (7.7%). Conclusions. The result of our study reveals that the CoNS, Staphylococcus aureus, Acinetobacter spp., and Klebsiella pneumoniae are the most common etiological agents of neonatal septicemia. In particular, since rate of CoNS causing sepsis is alarming, prompting concern to curb the excess burden of CoNS infection is necessary

Ceftazidime-avibactam has potent sterilizing activity against highly drug-resistant tuberculosis.

There are currently many patients with multidrug-resistant and extensively drug-resistant tuberculosis. Ongoing transmission of the highly drug-resistant strains and high mortality despite treatment remain problematic. The current strategy of drug discovery and development takes up to a decade to bring a new drug to clinical use. We embarked on a strategy to screen all antibiotics in current use and examined them for use in tuberculosis. We found that ceftazidime-avibactam, which is already used in the clinic for multidrug-resistant Gram-negative bacillary infections, markedly killed rapidly growing, intracellular, and semidormant Mycobacterium tuberculosis in the hollow fiber system model. Moreover, multidrug-resistant and extensively drug-resistant clinical isolates demonstrated good ceftazidime-avibactam susceptibility profiles and were inhibited by clinically achievable concentrations. Resistance arose because of mutations in the transpeptidase domain of the penicillin-binding protein PonA1, suggesting that the drug kills M. tuberculosis bacilli via interference with cell wall remodeling. We identified concentrations (exposure targets) for optimal effect in tuberculosis, which we used with susceptibility results in computer-aided clinical trial simulations to identify doses for immediate clinical use as salvage therapy for adults and young children. Moreover, this work provides a roadmap for efficient and timely evaluation of antibiotics and optimization of clinically relevant dosing regimens

Childhood Septicemia in Nepal: Documenting the Bacterial Etiology and Its Susceptibility to Antibiotics

Introduction. Children are among the most vulnerable population groups to contract illnesses. The varying microbiological pattern of septicemia warrants the need for an ongoing review of the causative organisms and their antimicrobial susceptibility pattern. Therefore, the objective of this study was to document the bacterial etiology of childhood septicemia and its antibiotic susceptibility profile. Methods. Cross-sectional type of study in 1630 suspected patients was conducted at CMCTH from January 2012 to December 2013. Blood samples were collected aseptically for culture. The organisms grown were identified by standard microbiological methods recommended by American Society for Microbiology (ASM) and subjected to antibiotic susceptibility testing by modified Kirby-Bauer disk diffusion method. Methicillin resistance was confirmed using cefoxitin and oxacillin disks methods. Results. Septicemia was detected in 172 (10.6%) cases. Among Gram-positive organisms, coagulase negative staphylococci (CoNS) were leading pathogen and Acinetobacter spp. were leading pathogen among Gram-negative isolates. Vancomycin, teicoplanin, and clindamycin were the most effective antibiotics against Gram-positive isolates while amikacin was effective against Gram-positive as well as Gram-negative isolates. Methicillin resistance was detected in 44.4% of Staphylococcus aureus. Conclusions. This study has highlighted the burden of bacterial etiology for septicemia among children in a tertiary care center of central Nepal

Role Of Nested Polymerase Chain Reaction (PCR) In The Clinico-Epidemiological Diagnosis Of Malaria In Nepal

Malaria poses a diagnostic challenge to laboratories of both developed and developing countries. Microscopic examination of Giemsa stained blood smears is the most commonly used method of malaria diagnosis in Nepal with recent introduction of Quantitative Buffy Coat (QBC) at B.P. Koirala Institute of Health Sciences (BPKIHS). Eighty-five randomly selected samples among 3182 patients referred to Clinical Laboratory Services (CLS) at BPKIHS for QBC malaria testing from June 18, 2002 to July 17, 2003 were analyzed by QBC malaria test, Giemsa stained thin smear microscopy and nested PCR. Out of 85 samples, 48 (56.47%), 17 (20.00%) and 24 (28.24% ) were positive by malaria QBC test, microscopy and PCR, respectively. Among 24 Plasmodium genus specific PCR positive samples, 12 (50.00%) were P. falciparum, 7 (29.16%) were P. vivax, 4 (25.00%) were mixed infection with P. falciparum, and P. vivax. One sample (4.17%) was positive for malaria by genus specific PCR but was negative for all species specific PCR. Three P.falciparum and one P. vivax microscopic positive samples were found to have mixed infection with both P.falciparum and P. vivax by PCR. Although QBC was able to pick up all microscopy positive cases, a large numbers of QBC positive cases were negative by both microscopy and PCR suggesting that QBC may have a high false positive rate. The results of this study indicated that malaria was seasonal in Nepal, was much more common among males and maximum numbers of malaria positive cases were found in the age group of 15-30 years. As expected, PCR detected much more cases than the microscopy technique and more importantly many more mixed infections were identified by the PCR in this study. However, the nested PCR was a very labor intensive procedure. Since, the existing microscopy has low sensitivity, and the QBC has very high false positive rate, PCR would be the most reliable alternative for clinical and epidemiological diagnosis of malaria in Nepal. However, its routine use in the clinical laboratory in Nepal in the present form does not appear to be feasible because of its high cost, long processing time and requirement to run several PCR cycles for each samples. However, it could be a valuable tool for quality control as it can be used in selective samples to verify the microscopy or QBC test results

Potency of vancomycin against Mycobacterium tuberculosis in the hollow fiber system model

Objectives: To determine whether an inhaled vancomycin formulation resulting in high intrapulmonary 24-h area under the concentration–time curve (AUC0–24) could be optimised for tuberculosis treatment. We also explored vancomycin synergy and antagonism with d-cycloserine and benzylpenicillin. Methods: We determined MICs of two Mycobacterium tuberculosis (Mtb) laboratory strains (H37Ra and H37Rv) and two drug-susceptible and nine multidrug resistant clinical strains. Second, in the hollow fiber system model of TB [HFS-TB] using Mtb H37Ra strain, we recapitulated vancomycin intrapulmonary pharmacokinetics of eight doses administered twice daily over 28 days, mimicking a 6-h half-life. Using the HFS-TB, vancomycin was tested in combination with d-cycloserine and benzylpenicillin to determine synergy or antagonism between drugs targeting the same pathway. Results: Vancomycin MICs were 12 and 48 mg/L in drug-susceptible clinical isolates but >96 mg/L in all MDR isolates.In the HFS-TB, vancomycin killed 3.9 ± 0.6 log10 CFU/mL Mtb. The EC50 was calculated as AUC0–24/MIC of 184.6 ± 106.5. Compared with day 0, 1.0 and 2.0 log10 CFU/mL kill was achieved by AUC0–24/MIC of 168 and 685, respectively. Acquired vancomycin resistance developed to all vancomycin doses tested in the HFS-TB. In the HFS-TB, vancomycin was antagonistic to benzylpenicillin, which works downstream to glycopeptides in peptidoglycan synthesis, but synergistic with d-cycloserine, which inhibits upstream d-Ala-d-Ala ligase and alanine racemase. Conclusion: Our proof-of-concept studies show that vancomycin optimal exposure target for Mtb kill could be achieved via inhalational drug delivery. Addition of drugs synergistic with vancomycin, e.g. d-cycloserine, may lower the vancomycin concentrations required to kill Mtb

Detection of IgM against dengue virus in clinically suspected patients presenting at a tertiary care centre, Narayani Zone, Nepal

Background: The global prevalence of dengue has increased dramatically in recent decades, with currently 50 million clinical cases and up to 5 million hospitalisations annually. Caused by one of five closely related but antigenically distinct virus serotypes (DEN-1 to DEN-5), dengue is an emerging mosquito-borne viral disease and an important public health problem in Nepal. Objectives: This study was designed to determine the occurrence of dengue in clinically suspected patient in Narayani Zone, Central Nepal. Methods: A descriptive cross-sectional study was conducted between January 2010 and December 2011 at Chitwan Medical College Teaching Hospital, Bharatpur, the fifth largest city of Nepal. A total of 590 blood samples were collected and processed for anti-dengue immunoglobulin (Ig)M by antibody isotype-capture enzyme-linked immunosorbent assay. Results: Positive detection of anti-dengue IgM was found in 8.5% of patients (50/590 cases). The highest number of dengue cases was observed in the 21-30 years age group with greater predilection in males than in females. The positive cases showed higher frequency in winter season than at other times of year. There was a significantly greater prevalence of dengue among residents of urban locations compared to those from rural areas. Conclusions: A high percentage of dengue positivity among suspected patients demands early investigation and careful management to prevent significant outbreaks of dengue fever and dengue haemorrhagic fever