Daniela Ram

Mutation D30N Is Not Preferentially Selected by Human Immunodeficiency Virus Type 1 Subtype C in the Development of Resistance to Nelfinavir

ABSTRACT Differences in baseline polymorphisms between subtypes may result in development of diverse mutational pathways during antiretroviral treatment. We compared drug resistance in patients with human immunodeficiency virus subtype C (referred to herein as “subtype-C-infected patients”) versus subtype-B-infected patients following protease inhibitor (PI) therapy. Genotype, phenotype, and replication capacity (Phenosense; Virologic) were determined. We evaluated 159 subtype-C- and 65 subtype-B-infected patients failing first PI treatment. Following nelfinavir treatment, the unique nelfinavir mutation D30N was substantially less frequent in C (7%) than in B (23%; P = 0.03) while L90M was similar (P < 0.5). Significant differences were found in the rates of M36I (98 and 36%), L63P (35 and 59%), A71V (3 and 32%), V77I (0 and 36%), and I93L (91 and 32%) (0.0001 < P < 0.05) in C and B, respectively. Other mutations were L10I/V, K20R, M46I, V82A/I, I84V, N88D, and N88S. Subtype C samples with mutation D30N showed a 50% inhibitory concentration (IC50) change in susceptibility to nelfinavir only. Other mutations increased IC50 correlates to all PIs. Following accumulation of mutations, replication capacity of the C virus was reduced from 43% ± 22% to 22% ± 15% (P = 0.04). We confirmed the selective nature of the D30N mutation in C, and the broader cross-resistance of other common protease inhibitor mutations. The rates at which these mutational pathways develop differ in C and subtype-B-infected patients failing therapy, possibly due to the differential impact of baseline polymorphisms. Because mutation D30N is not preferentially selected in nelfinavir-treated subtype-C-infected patients, as it is in those infected with subtype B, the consideration of using this drug initially to preserve future protease inhibitor options is less relevant for subtype-C-infected patients.

Rapid Detection of blaKPC Carbapenemase Genes by Real-Time PCR

ABSTRACT Carbapenem resistance among Enterobacteriaceae is an emerging problem worldwide. Klebsiella pneumoniae carbapenemase (blaKPC) enzymes are among the most common β-lactamases described. In this study, we report the development and validation of a real-time PCR (q-PCR) assay for the detection of blaKPC genes using TaqMan chemistry. The q-PCR amplification of blaKPC DNA was linear over 7 log dilutions (r2 = 0.999; slope, 3.54), and the amplification efficiency was 91.6%. The q-PCR detection limit was 1 CFU, and there was no cross-reaction with DNA extracted from several multidrug-resistant bacteria. Perianal/rectal swabs (n = 187) collected in duplicate from 128 patients admitted to Sheba Medical Center surgical intensive care units were evaluated for the presence of carbapenem-resistant bacteria by culturing on MacConkey agar-plus-carbapenem disks and for blaKPC genes by q-PCR. Carbapenem-resistant organisms, all K. pneumoniae, were isolated from 47 (25.1%) of the 187 samples collected, while blaKPC genes were detected in 54 (28.9%) of the patient samples extracted by the NucliSENS easyMAG system. Of these, seven samples were positive for blaKPC genes by q-PCR but negative for carbapenem resistance by culture, while all samples in which no carbapenem-resistant bacteria were detected by culture also tested negative by q-PCR. Thus, the sensitivity and specificity of the q-PCR assay after extraction by the NucliSENS easyMAG system were 100% and 95%, respectively. Similar values were obtained after DNA extraction by the Roche MagNA Pure LC instrument: 97.9% sensitivity and 96.4% specificity. Overall, the blaKPC q-PCR assay appears to be highly sensitive and specific. The utilization of q-PCR will shorten the time to blaKPC detection from 24 h to 4 h and will help in rapidly isolating colonized or infected patients and assigning them to cohorts.

Rapid changes in the expression of a gene encoding a calcium-binding protein in Schistosoma mansoni

Genes expressed in a stage-specific manner may help us understand the molecular events controlling the complex life cycle of schistosomes. cDNA and genomic clones encoding a calcium-binding protein (CaBP) were obtained from cercariae and their sequence determined. The encoded protein (69 amino acids long) shows clear resemblance to the domain structure and organization of CaBP molecules. It contains two typical calcium-binding loops, the distance between which is identical to the length conserved in other CaBP molecules. In addition, the schistosome CaBP shows Ca2+-dependent electrophoretic mobility (increased with Ca2+-ions and decreased with EGTA). Northern blots revealed expression of the CaBP gene in cercariae but not in sporocyst or worm (developmental stages preceding and following cercaria). The preferential expression of this CaBP in the cercaria raises questions as to what cercaria-specific function(s) it performs. The structure of the gene is similar to that in other eukaryotes, and one intron interrupts the coding sequence. The region of the cap site was determined, and there was no evidence of the spliced leader sequence found in the mRNAs of other parasites. The CaBP reveals a rapid change in gene expression, since the mRNA is missing in the parasite residing in infected snails, but is readily detected in cercariae 1 h after shedding. We identified other genes which are turned on (like the CaBP) or shut off within the short period of transition from cercariae in the snail to free-swimming cercariae.

Rapid Detection of Influenza A Pandemic (H1N1) 2009 Virus Neuraminidase Resistance Mutation H275Y by Real-Time Reverse Transcriptase PCR

ABSTRACT The emergence of oseltamivir-resistant influenza A pandemic (H1N1) 2009 virus highlights the need for rapid oseltamivir resistance screening. We report the development and validation of high-throughput real-time reverse transcriptase PCR assays for the detection of the H275Y substitution in the neuraminidase 1 gene that can be accomplished in 3 to 4 h.

High Rate of Human Bocavirus and Adenovirus Coinfection in Hospitalized Israeli Children

ABSTRACT We investigated coinfection of human bocavirus (HBoV) and other respiratory viruses in hospitalized children by real-time PCR. A high rate (69.2%) of adenovirus infection was found among children infected with HBoV. Such high rates of HboV-adenovirus coinfection have not been previously reported, underscoring the need to investigate the contribution of HBoV in patient clinical presentations.

Schistosoma mansoni: Stage-specific expression of muscle-specific genes

It was previously shown that an antigen preparation termed 9B obtained from Schistosoma mansoni cercarial extracts partially (34%) protects mice from challenge infection with cercariae (R. Tarrab-Hazdai et al., J. Immunol. 135, 2772, 1985). To characterize some of the proteins which comprise this preparation, rabbit antibodies to the 9B antigen preparation were used to screen cDNA libraries of cercariae and adult worms. We isolated and sequenced cDNA clones encoding three proteins: calcium-binding protein, paramyosin, and myosin. The calcium-binding protein was previously shown to be expressed in cercariae but not in sporocysts or adult worms (D. Ram et al., Mol. Biochem. Parasitol. 34, 167, 1989). Northern blots showed the presence of paramyosin and myosin mRNAs in sporocysts and adult worms but not in cercariae. Antibodies to paramyosin detected the protein in sporocysts and adult worms as well as in cercariae. These findings explain, in part, the protective activity of the 9B antigen preparation against challenge infection.

Characterization of Large Mumps Outbreak among Vaccinated Palestinian Refugees

During a large mumps virus (MuV) outbreak which occurred in the Palestinian refugee camps of the West Bank, 68.1% (2,636/3,871) of the cases were vaccinated with one dose of trivalent measles, mumps, and rubella (MMR) vaccine. Attack rates by camp ranged from less than 1 case per 1,000 people in the population to 43/1,000 (overall, 11/1,000). The outbreak lasted from December 2003 to June 2005, with two peaks, one from April to May 2004 and the other from March to April 2005. To control the outbreak, a mass MMR vaccination campaign was conducted in May 2005. Evaluation of the immune status of cases (n = 59) and healthy controls (n = 51) revealed high levels of mumps immunoglobulin G (IgG) and a low MuV-specific IgM in clinical cases indicative of a booster immune response. This suggested a secondary rather than a primary infection due to the insufficient protection conferred by the single vaccine dose included in the vaccination program. This prediction was further confirmed by the low seroprevalence (68.6%) found in the healthy control group, which was below the threshold level required for MuV herd immunity. Mumps diagnosis was established mainly by reverse transcription-PCR in clinical samples obtained within 48 h from the onset of disease. Of the parotid fluids and nasopharyngeal aspirates analyzed, 92% were positive for MuV RNA, while only 33% of the urine samples were positive. Phylogenetic analysis of the MuV SH gene identified the outbreak strain as the H genotype, which has been in circulation worldwide at least since 1989.

Stage-specific alternative splicing of the heat-shock transcription factor during the life-cycle of Schistosoma mansoni.

Stage-specific alternative splicing of the heat-shock transcription factor of Schistosoma mansoni (SmHSF) generates isoforms with structural diversity that may modulate the activity of SmHSF at different life-stages, and thus may regulate the expression of different genes at different developmental stages. RT-PCR, cloning and DNA-sequence analyses showed stage-specific alternative splicing inside the DNA-binding domain (DBD) involving introns I1 and I2, and beyond the DBD involving introns I4a and I7. Retention of introns I2 and I4 would inactivate SmHSF since they contain termination codons. Retention of intron I1 would add 11 amino acids inside the DBD and may change the DNA-binding specificity of SmHSF; intron I7 would add 13 amino acids to the effector region of HSF. Retention of introns was more pronounced in cercariae (larval stage living in water) than in adult worms (parasitic form in mammals). The isoforms were expressed in bacteria, but functional evaluation was not feasible, because only the isoform lacking introns was soluble while isoforms with introns were insoluble. However, stage-specific alternative splicing that changed HSF function in vivo was evidenced in intact cercariae. The cercarial SmHSF mRNA was enriched with introns I2 and I4a that contain termination codons. Therefore, translation of the SmHSF mRNA was impaired, and the SmHSF protein was undetectable. Consequently, the HSP70 gene could not be transcribed, and the HSP70 mRNA was missing. Alternative splicing was observed for short DNA segments (33-45 bp) bound by splice signals, located in the coding region. These are not bona fida exons since they are not flanked by introns. Yet, they are not regular introns since they are often found in mature mRNA. Alternative splicing of these DNA segments caused structural diversity that could modulate the function of the gene product.

Community-acquired Oseltamivir-Resistant Pandemic (H1N1) 2009 in Child, Israel

To the Editor: During the spring of 2009, a pandemic influenza A (H1N1) virus emerged and spread globally. Initial testing of the virus found it susceptible to neuraminidase inhibitors and resistant to adamantanes (1,2). As of March 5, 2010, only 264 cases of oseltamivir-resistant pandemic (H1N1) 2009 infection had been reported to the World Health Organization, but the number of cases has been steadily increasing (2). These viruses were carrying the H275Y mutation, which conferred resistance to oseltamivir (2). Most of the reported cases were in immunocompromised patients who had prolonged viral shedding or in patients who had received oseltamivir prophylaxis or treatment (1–4). We describe an otherwise healthy 2-year-old boy with oseltamivir-resistant pandemic (H1N1) 2009 infection and a traumatic lung contusion, complicated by acute respiratory distress syndrome (ARDS). He had not received prior chemoprophylaxis or treatment with oseltamivir. In November 2009, a healthy 2-year-old boy was admitted to the pediatric intensive care unit at the Western Galilee Hospital in Nahariya, Israel, after he had been hit by a car. One day before the accident, he had exhibited fever and cough (for which he was treated with acetaminophen). His 4-year-old brother had recovered recently from an influenza-like illness without antiviral treatment. The other household contacts were his parents, who did not have a respiratory illness. On admission, small, bilateral lung contusions, right pneumothorax, and liver lacerations were shown on computed tomographic scan. The patient was treated with a chest tube for drainage, supplemental oxygen, and oseltamivir from hospital day 1 (30 mg 2 ×/day; childs body weight = 13 kg) and was placed in droplet isolation. Respiratory swab specimens, obtained on hospital day 1, were sent to the Israel Central Virology Laboratory (ICVL) and found to be positive for pandemic (H1N1) 2009 by real-time reverse transcription–PCR (RT-PCR). On hospital day 3, the child was intubated because of worsening respiratory distress and hypoxemia, and he required a second chest tube drain. His chest film showed bilateral pulmonary infiltrates. His condition was then treated with nitric oxide, dopamine, and milrinone for ARDS and failure of the right side of the heart. The dosage of oseltamivir was doubled on hospital day 4 because of gastric residuals. Antimicrobial drug therapy with vancomycin and piperacillin-tazobactam was added because sepsis and secondary bacterial lung infection were suspected. Because of the severity of his symptoms and persistence of fever, additional lower and upper airway specimens were sent to ICVL on hospital days 5 and 10; they were positive for pandemic (H1N1) 2009. After these results were received, oseltamivir resistance was suspected, and his respiratory specimens were also checked by ICVL. A mixture of both wild-type and mutant pandemic (H1N1) 2009 was found in the specimens from hospital days 1, 5, and 10 by an in-house q-RT-PCR assay designed to detect the H275Y mutation (4,5). Further testing by sequence analysis of the neuraminidase gene showed a mixed population of wild-type and mutant pandemic (H1N1) 2009; the mutant virus was carrying the histidine-to-tyrosine substitution at position 275, which conferred the quantitative RT-PCR result and the H275Y phenotype of oseltamivir-resistant pandemic (H1N1) 2009. By the time these laboratory results were known, the patient’s respiratory condition was improving without changing the oseltamivir therapy. Cultures of blood and endotracheal specimens were sterile and antimicrobial drug therapy was stopped. On hospital day 15, he was extubated, oseltamivir therapy was ended, and he was weaned off oxygen a few days later. The respiratory specimen on hospital day 20 was negative for pandemic (H1N1) 2009. No secondary influenza cases were detected among healthcare personnel or patients in the unit. In Israel, oseltamivir resistance has been detected by ICVL in 6 cases (5). The fact that our patient had oseltamivir-resistant pandemic (H1N1) 2009 without a previous oseltamivir exposure is surprising because almost all cases of oseltamivir-resistance have been associated with previous oseltamivir prophylaxis or therapy and with prolonged viral shedding (which is often combined with oseltamivir therapy) in immunocompromised patients (1–5). Our patient did not attend daycare and his parents had not been ill recently. Therefore, he likely was infected by his older brother who probably had pandemic (H1N1) 2009 but was neither diagnosed nor treated with antiviral medications. This theory suggests that oseltamivir-resistant viruses circulate in the community with the potential to be transmitted between persons. Lung contusions and pandemic (H1N1) 2009 can cause ARDS (6,7). We do not know the relative role of each in causing the ARDS that our patient had, but the severity of clinical symptoms, although the lung injury was judged to be only of moderate magnitude, suggests that influenza played a major role in the development of his acute lung disease. The infection with oseltamivir-resistant virus, for which he probably did not receive effective therapy, likely had an effect on the duration and severity of his course. Although our patient had a favorable outcome, the possibility of widespread resistance, similar to the phenomenon observed with seasonal influenza in the 2008–2009 season, is alarming and should be monitored. The suspicion of resistance should be based upon compatible clinical scenario, such as continuation of symptoms in spite of antiviral therapy (even in patients who are not immunocompromised), combined with early performance of resistance assays. Early and rapid detection of oseltamivir resistance and a change of antiviral treatment (if feasible) might benefit the patient.

Cloning of the SmSPO-1 gene preferentially expressed in sporocyst during the life cycle of the parasitic helminth Schistosoma mansoni1.

Schistosomes are parasitic helminths with a complex life cycle in human and snail hosts. They express stage-specific genes that conceivably determine distinct properties of the parasite at different developmental stages. Here we report the stage-specific gene SmSPO-1, which is preferentially expressed in sporocysts residing in the snail host. The cDNA and the gene were cloned and sequenced. The cDNA, from cap site to the poly(A) addition site, is 498 bp long. It encodes a protein of 117 amino acids with a hydrophobic signal peptide of 18 residues, indicating that SmSPO-1 is a secreted or a membranal protein. In the gene the cDNA is split into four exons spread over 2.1 kb of chromosomal DNA.