Evaluation of Antimicrobial Susceptibility Profile in Salmonella Typhi and Salmonella Paratyphi A: Presenting the Current Scenario in India and Strategy for Future Management.

BACKGROUND: Systematic studies to estimate the disease burden of typhoid and paratyphoid in India are limited. Therefore, a multicenter study on the Surveillance of Enteric Fever in India was carried out to estimate the incidence, clinical presentation, and antimicrobial resistance (AMR) trend. The data presented here represent the national burden of AMR in Salmonella Typhi and Salmonella Paratyphi A. METHODS: Antimicrobial susceptibility testing was performed for S. Typhi and S. Paratyphi A (n = 2373) isolates collected prospectively during a 2-year period from November 2017 to January 2020. RESULTS: Of 2373 Salmonella isolates, 2032 (85.6%) were identified as S. Typhi and 341 (14.4%) were S. Paratyphi A. Approximately 2% of S. Typhi were multidrug-resistant (MDR), whereas all 341 (100%) of S. Paratyphi A isolates were sensitive to the first-line antimicrobials. Among 98% of ciprofloxacin nonsusceptible isolates, resistance (minimum inhibitory concentration [MIC] >0.5 µg/mL) was higher in S. Typhi (37%) compared with S. Paratyphi A (20%). Azithromycin susceptibility was 99.9% and 100% with a mean MIC of 4.98 µg/mL for S. Typhi and 7.39 µg/mL for S. Paratyphi A respectively. Ceftriaxone was the only agent that retained 100% susceptibility. Moreover, beta-lactam/beta-lactamase inhibitors showed potent in vitro activity against the study isolates. CONCLUSIONS: Data obtained from this systematic surveillance study confirms the declining trend of MDR Salmonella isolates from India. The higher prevalence of ciprofloxacin nonsusceptibility enforces to limit its use and adhere to the judicious usage of azithromycin and ceftriaxone for enteric fever management.

Serotypes of Streptococcus pneumoniae in Children Aged <5 Years Hospitalized With or Without Pneumonia in Developing and Emerging Countries: A Descriptive, Multicenter Study.

BACKGROUND: Improving knowledge regarding Streptococcus pneumoniae distribution in pneumonia cases is important to better target preventive and curative measures. The objective was to describe S. pneumoniae serotypes in children with or without pneumonia. METHODS: It was a case-control study carried out in 8 developing and emerging countries between 2010 and 2014. Cases were children aged <5 years admitted to the hospital for pneumonia. Controls were children admitted for surgery or routine outpatient care. RESULTS: In nasopharyngeal samples, S. pneumoniae were detected in 68.2% of the cases and 47.5% of the controls (P < .001). Nasopharyngeal carriage was associated with a higher risk of being a case in 6/8 study sites (adjusted odds ratio ranged from 0.71 [95% confidence interval [CI], .39-1.29; P = .26] in India [Pune/Vadu] to 11.86 [95% CI, 5.77-24.41; P < .001] in Mongolia). The 13-valent pneumococcal conjugate vaccine (PCV13) serotypes were more frequently detected in cases with nasopharyngeal carriage (67.1%) than in controls with nasopharyngeal carriage (54.6%), P < .001. Streptococcus pneumoniae was detected in blood by polymerase chain reaction in 8.3% of the cases. Of 34 cases with an S. pneumoniae serotype detected in blood, 27 (79%) had the same serotype in the nasopharyngeal sample. CONCLUSIONS: The results confirm the assumption that the isolate carrying or causing disease in an individual is of the same serotype. Most serotypes independently associated with nasopharyngeal carriage or pneumonia are covered by PCV13, suggesting that increased PCV coverage would reduce the burden of S. pneumoniae-related pneumonia.

Microorganisms Associated With Pneumonia in Children <5 Years of Age in Developing and Emerging Countries: The GABRIEL Pneumonia Multicenter, Prospective, Case-Control Study.

Background: Pneumonia, the leading infectious cause of child mortality globally, mainly afflicts developing countries. This prospective observational study aimed to assess the microorganisms associated with pneumonia in children aged <5 years in developing and emerging countries. Methods: A multicenter, case-control study by the GABRIEL (Global Approach to Biological Research, Infectious diseases and Epidemics in Low-income countries) network was conducted between 2010 and 2014 in Cambodia, China, Haiti, India (2 sites), Madagascar, Mali, Mongolia, and Paraguay. Cases were hospitalized children with radiologically confirmed pneumonia; controls were children from the same setting without any features suggestive of pneumonia. Nasopharyngeal swabs were collected from all subjects; 19 viruses and 5 bacteria were identified by reverse-transcription polymerase chain reaction. Associations between microorganisms and pneumonia were quantified by calculating the adjusted population attributable fraction (aPAF) after multivariate logistic regression analysis adjusted for sex, age, time period, other pathogens, and site. Results: Overall, 888 cases and 870 controls were analyzed; ≥1 microorganism was detected in respiratory samples in 93.0% of cases and 74.4% of controls (P < .001). Streptococcus pneumoniae, Mycoplasma pneumoniae, human metapneumovirus, rhinovirus, respiratory syncytial virus (RSV), parainfluenza virus 1, 3, and 4, and influenza virus A and B were independently associated with pneumonia; aPAF was 42.2% (95% confidence interval [CI], 35.5%-48.2%) for S. pneumoniae, 18.2% (95% CI, 17.4%-19.0%) for RSV, and 11.2% (95% CI, 7.5%-14.7%) for rhinovirus. Conclusions: Streptococcus pneumoniae, RSV, and rhinovirus may be the major microorganisms associated with pneumonia infections in children <5 years of age from developing and emerging countries. Increasing S. pneumoniae vaccination coverage may substantially reduce the burden of pneumonia among children in developing countries.

Compromised Nutritional Status as a Risk Factor for the Incidence of Nosocomial Infections.

Background Poor nutritional status may lead to longer hospital stays, increased mortality and morbidity, increased cost, and higher suffering. Nosocomial infections (NI) are a global health concern, and several risk factors are associated with their higher incidence. This study aimed to reveal that compromised nutritional status is one of the risk factors for developing NIs. Methodology The study was conducted in a tertiary care hospital in Pune, India. This was a prospective cohort study with a sample size of 200 hospitalized participants. Data collection was based on standard tools and structured forms which had two parts. In the first part, the assessment of nutritional status was done for which patients were categorized into two groups, namely, well-nourished and undernourished. Additionally, biochemical parameters (serum albumin) were also assessed. The second part included a follow-up of participants to evaluate the development of NIs including their laboratory investigation. Results were analyzed statistically using R software. Results Among 200 participants, 60 were female, of whom 15% developed NIs. Of the 140 males, 8% had NIs. Among 200 participants, 101 (51%) were well-nourished, of whom two (2%) developed NIs. Of the 99 (49%) undernourished participants, 18 (18%) had NIs. Those who were undernourished (univariate relative risk = 6.10, 95% confidence interval) were more prone to developing NIs compared to the well-nourished group. Conclusions NIs are widespread globally but are less studied and given less emphasis in developing countries. This study reports various types of NIs along with their incidence in well-nourished and undernourished groups. The incidence of NI observed in this study may reflect the higher severity of illness, age, poor nutritional status, and longer hospital stays. Identifying risk factors that can contribute to developing NI may help in their prevention by maximizing patient safety.

In Vitro Susceptibility of Clinical Isolates to Ceftriaxone Alone and Ceftriaxone in Combination With Sulbactam or Tazobactam: A Comparative Study of Broad-Spectrum ß-Lactam Antibiotics in India.

Background This study was designed to evaluate the current in vitro susceptibility of clinical isolates to broad-spectrum ß-lactam antibiotics. Methodology Bacterial isolates, cultured from 180 non-repetitive clinical samples between April and November 2022 at three hospitals in India, were used to evaluate the minimum inhibitory concentration (MIC) of broad-spectrum ß-lactam antibiotics using the Epsilometer test (E-test) method. Test antibiotics were ceftriaxone and ceftriaxone in combination with ß-lactamase inhibitors (BLIs) sulbactam and tazobactam. Comparator antibiotics included amoxicillin + BLI clavulanic acid, piperacillin + tazobactam, cefotaxime, and cefepime. The MIC values obtained were used to assess the susceptibility of the isolates and to compute the efficacy ratios (ERs) of the antibiotics. Results Among the 180 clinical isolates, ~89% were gram-negative bacteria, the most prevalent ones being Escherichia coli and Klebsiella pneumoniae. Of the gram-negative isolates, ~37% were susceptible/intermediately susceptible to ceftriaxone, and ~29% were susceptible to ceftriaxone + BLIs. The test antibiotics had ER >10 against 85%-95% E. coli isolates, whereas comparator antibiotics had ER >10 against 31%-68% isolates. The differences between the test antibiotics and piperacillin + tazobactam or cefotaxime were statistically significant. Ceftriaxone, ceftriaxone + sulbactam, and ceftriaxone + tazobactam had ER >10 against 78%, 100%, and 90% of K. pneumoniae isolates, while the corresponding percentages for cefotaxime, piperacillin + tazobactam, and cefepime were 100%, 64%, and 80%, respectively. The difference between ceftriaxone + BLIs and piperacillin + tazobactam was statistically significant. Ceftriaxone + BLIs had ER >10 against all E. coli isolates producing extended-spectrum ß-lactamases (ESBLs); the percentage of isolates was significantly higher than that for piperacillin + tazobactam. Ceftriaxone + tazobactam had ER >10 against all ESBL-producing K. pneumoniae isolates; ceftriaxone and ceftriaxone + sulbactam had ER ranging 6-10. Conclusions Ceftriaxone and ceftriaxone in combination with sulbactam and tazobactam are promising antibiotics to explore against prevalent infectious microorganisms such as E. coli and K. pneumoniae. Ceftriaxone + tazobactam also holds promise against ESBL-producing variants.

Effect of reduced two-dose (1+1) schedule of 10 and 13-valent pneumococcal conjugate vaccines (SynflorixTM and Prevenar13TM)) on nasopharyngeal carriage and serotype-specific immune response in the first two years of life: Results from an open-labelled randomized controlled trial in Indian children.

INTRODUCTION: This study aimed to assess the effect of a reduced dose regime (1 + 1) of PCV10 and PCV13 along with 3-dose regimes on pneumococcal vaccine-type (VT) carriage and immunogenicity in the first two years of life in PCV-naïve Indian children. METHODS: A total of 805 healthy infants aged 6-8 weeks were randomised to 7 groups (n = 115). Six groups received SynflorixTM(PCV10) or Prevenar13TM(PCV13) in the following schedules: 3 + 0 (three primary at 6, 10, and 14 weeks); 2 + 1 (two primary 6 and 14 weeks with booster at 9 months; 1 + 1 (one primary at 14 weeks with booster at 9 months). The 7th group was a PCV-naïve control group. Nasopharyngeal swabs were collected at 6, 18 weeks, 9, 10, 15, and 18 months of age. Venous blood samples were collected at 18 weeks, 9, 10, and 18 months of age for assessment of sero-specific IgG antibodies. Additionally, functional activity using a serotype specific opsonophagocytic assay (OPA) was assessed at 10 and 18 months of age in a subset (20%) of participants. RESULTS: All schedules of PCV13 showed significant 13VT carriage reduction in the second year of life as compared to control. At 15 months of age, PCV13 (1 + 1) showed 45 % reduction in 13VT-carriage compared to the control [OR = 0.55 (95% CI; 0.31-0.97), p= 0.038]. None of the PCV10 schedules showed significant reduction in 10VT carriage in the second year. Although not powered for these outcomes, at 18 months of age, 1 + 1 and 2 + 1 schedules of both vaccines demonstrated higher sero-responders for all serotypes, higher geometric mean concentrations (GMC) for all serotypes except 23F [with both vaccines], higher percent OPA responders and OPA geometric mean titres (GMT) compared to the 3 + 0 schedules for all serotypes. CONCLUSION: The reduced dose schedule (1 + 1) of PCV13 results in significant VT-carriage reduction in the second year of life. Immune protection provided by 1 + 1 schedules of PCV10 and PCV13 in the second year of life is comparable to WHO-recommended 3-dose schedules.

Clinical evaluation of multiplex real-time PCR panels for rapid detection of respiratory viral infections.

Respiratory viral infections are one of the leading causes of morbidity and mortality, particularly in children, the elderly and immunocompromised persons. Rapid identification of viral etiology is critical in ruling out non-viral infections, initiating antiviral treatment and limiting the spread of the infection. Multiplex assays of more than one viral gene target in a single tube have the advantage of rapid screening of a large number of potential viral pathogens in a short time. A multiplex real-time PCR assay was used in this study for detection of respiratory RNA and DNA viral infections in 728 specimens received from 585 adult and pediatric patients comprised of symptomatic and asymptomatic organ transplant recipients and non-recipients for diagnosis of respiratory illnesses and for routine clinical monitoring. Multiplex PCR was more sensitive than the multiplex immunofluoresence culture assay (R-mix) and also detected additional respiratory viruses that were not covered by the R-mix panel. The number of respiratory viruses detected in symptomatic patients was significantly higher than asymptomatic patients in both adult and pediatric patients. Herpesviral infections were the predominant cause of lower respiratory tract infection in the organ transplant recipients, whereas respiratory syncytial virus was the most common pathogen in non-transplant patients particularly children. Multiplex real-time PCR for detection of respiratory viruses has the potential for rapid identification of viral pathogens. In this era of emerging viral infections, addition of newer viral targets to the multiplex PCR panels will be beneficial in determining both patient management and public health epidemiology.

Emerging Antibiotic Resistance Patterns in a Neonatal Intensive Care Unit in Pune, India: A 2-Year Retrospective Study.

Objective: Treating neonatal bloodstream infections and meningitis in South Asia remains difficult given high rates of antimicrobial resistance (AMR). To evaluate changing epidemiology of neonatal infections, we assessed pathogen-specific and clinical features of culture-proven infections in neonates admitted to a neonatal intensive care unit (NICU) in Pune, India. Materials and Methods: This retrospective cohort study was performed in the King Edward Memorial Hospital and Research Center NICU over 2 years between January 1, 2017 and December 31, 2018. We included all neonates admitted to the NICU with positive blood or cerebrospinal fluid cultures. Demographic, clinical, and microbiologic data were collected from the medical record. We reviewed antimicrobial susceptibility testing (AST) of all isolates. Results: There were 93 culture-positive infections in 83 neonates, including 11 cases of meningitis. Fifteen (18%) neonates died. Gram-negative pathogens predominated (85%) and AST showed 74% resistance to aminoglycosides, 95% resistance to third/fourth generation cephalosporins, and 56% resistance to carbapenems. Resistance to colistin was present in 30% of Klebsiella pneumoniae isolates. Birth weight <1,000 g [odds ratio (OR) 6.0, p < 0.002], invasive respiratory support (OR 7.7, p = 0.001), and antibiotics at the time of culture (OR 4.2, p = 0.019) were associated with increased risk of mortality. Rates of AMR to all major antibiotic classes were similar between early onset and late onset infections. There was no association between carbapenem resistance and mortality. Conclusion: In our NICU in India, there are high rates of AMR among Gram-negative pathogens that are predominantly responsible for infections. Given higher colistin resistance in this cohort than previously reported, hospitals should consider routinely testing for colistin resistance.

Nasopharyngeal Viral and Bacterial Co-Detection among Children from Low- and Middle-Income Countries with and without Pneumonia.

The role of microbial coinfection in the pathogenesis of pneumonia in children is not well known. The aim of this work was to describe the prevalence of microorganism co-detection in nasopharyngeal samples (NPS) of pneumonia cases and control subjects and to study the potential association between nasopharyngeal microorganism co-detection and pneumonia. A case-control study was carried out from 2010 to 2014 in nine study sites located in low- or middle-income countries. The data from 888 children under 5 years of age with pneumonia (cases) and 870 children under 5 without pneumonia (controls) were analyzed. Nasopharyngeal samples were collected; reverse transcription polymerase chain reaction (RT-PCR) enabled the detection of five bacteria and 19 viruses. Multiple, mixed-effects logistic regression modeling was undertaken to evaluate the association between microorganism co-detection and pneumonia. A single Streptococcus pneumoniae colonization was observed in 15.2% of the controls and 10.1% of the cases (P = 0.001), whereas S. pneumoniae and a single virus co-detection was observed in 33.3% of the cases and in 14.6% of the controls (P < 0.001). Co-detections with rhinovirus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, and influenza virus were more frequent in the cases compared with the controls (P < 0.001) and were significantly associated with pneumonia in multiple regression analysis. The proportion of single virus detection without bacterial co-detection was not different between cases and controls (13.6% versus 11.3%, P = 0.13). This study suggests that coinfection of S. pneumoniae and certain viruses may play a role in the pathophysiology of pneumonia in children.

Nasopharyngeal pneumococcal carriage in South Asian infants: Results of observational cohort studies in vaccinated and unvaccinated populations.

BACKGROUND: Nasopharyngeal pneumococcal carriage (NPC) is a prerequisite for invasive pneumococcal disease and reduced carriage of vaccine serotypes is a marker for the protection offered by the pneumococcal conjugate vaccine (PCV). The present study reports NPC during the first year of life in a vaccinated (with PCV10) cohort in Bangladesh and an unvaccinated cohort in India. METHODS: A total of 450 and 459 infants were recruited from India and Bangladesh respectively within 0-7 days after birth. Nasopharyngeal swabs were collected at baseline, 18 and 36 weeks after birth. The swabs were processed for pneumococcal culture and identification of serotypes by the Quellung test and polymerase chain reaction (PCR). An identical protocol was applied at both sites. RESULTS: Prevalence of NPC was 48% in the Indian and 54.8% in the Bangladeshi cohort at 18 weeks. It increased to 53% and 64.8% respectively at 36 weeks. The average prevalence of vaccine serotypes was higher in the Indian cohort (17.8% vs 9.8% for PCV-10 and 26.1% vs17.6% for PCV-13) with 6A, 6B, 19F, 23F, and 19A as the common serotypes. On the other hand, the prevalence of non-vaccine serotypes was higher (43.6% vs 27.1% for non-PCV13) in the Bangladeshi cohort with 34, 15B, 17F, and 35B as the common serotypes. Overcrowding was associated with increased risk of pneumococcal carriage. The present PCV-13 vaccine would cover 28%-30% and 47%-48% serotypes in the Bangladeshi and Indian cohorts respectively. CONCLUSIONS: South Asian infants get colonised with pneumococci early in infancy; predominantly vaccine serotypes in PCV naïve population (India) and non-vaccine serotypes in the vaccinated population (Bangladesh). These local findings are important to inform the public health policy and the development of higher valent pneumococcal vaccines.

Relationship of cytomegalovirus load assessed by real-time PCR to pp65 antigenemia in organ transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) infection in immunocompromised patients can lead to viremia associated with morbidity and mortality. Monitoring of viral loads in blood is critical for initiating and monitoring antiviral treatment. OBJECTIVES: Validate quantitative real-time PCR assay targeting the US17 and UL54 regions of the CMV genome for automated DNA and extraction and amplification. STUDY DESIGN: 3422 blood specimens from organ transplant recipients, including longitudinal specimens from 12 organ transplant recipients, were tested by CMV PCR and pp65 antigenemia. RESULTS: CMV PCR for both US17 and UL54, was more sensitive and detected CMV DNA earlier and for longer than the CMV pp65 antigenemia test. Using antigenemia results as a reference standard, an optimal cutoff of 500 normalized copies was calculated for both US17 and UL54 PCR targets based on high sensitivity, specificity, and positive and negative predictive values. CMV DNA levels tracked well with clinical symptoms, response to treatment, and antigenemia. CONCLUSIONS: Detection of persistent increases in CMV DNA levels above 500 normalized copies by this real-time PCR assay is indicative of symptomatic CMV disease in organ transplant recipients. Quantitative real-time PCR for CMV DNA can be used in lieu of antigenemia for monitoring CMV infection and determining when to initiate preemptive treatment.

Increased expression of interferon-inducible genes in macaque lung tissues during simian immunodeficiency virus infection.

Pulmonary infections and dysfunction are frequent outcomes during the development of immunodeficiency associated with human immunodeficiency virus type 1 (HIV-1) infection, and obtaining a better understanding of the immunologic changes that occur in lungs following HIV-1 infection will provide a foundation for the development of further intervention strategies. We sought here to identify changes in the pulmonary immune environment that arise during simian immunodeficiency virus (SIV) infection of rhesus macaques, which serves as an excellent model system for HIV-1 infection and disease. To examine the gene expression profiles of macaque lung tissues following infection with the pathogenic SIV/DeltaB670 isolate, we performed cDNA microarray hybridizations with lung total RNAs using two commercially available cDNA arrays and a custom-fabricated, immunologically focused macaque cDNA microarray. In situ hybridization and real-time RT-PCR were performed to provide additional analyses of gene expression. Among the genes exhibiting the highest level of induction in lung tissues were the IFN-gamma-inducible chemokines, CXCL10/IP-10 and CXCL9/Mig. In situ hybridization and real-time RT-PCR strongly supported these findings. Correlation analyses revealed that the levels of expression of IFN-gamma, CXCL9/Mig, and CXCL10/IP-10 mRNAs were all strongly positively correlated, and that CXCL10/IP-10 mRNA and Pneumocystis carinii rRNA were positively correlated. Taken together, these findings demonstrate that inflammatory chemokines are among the most differentially expressed mRNAs in macaque lung tissues during systemic SIV infection of rhesus macaques, and provide insight into the complicated events occurring in the lung tissues during HIV-1 infection in humans.

Macaque paneth cells express lymphoid chemokine CXCL13 and other antimicrobial peptides not previously described as expressed in intestinal crypts.

CXCL13 is a constitutively expressed chemokine that controls migration of immune cells to lymphoid follicles. Previously, we found CXCL13 mRNA levels increased in rhesus macaque spleen tissues during AIDS. This led us to examine the levels and locations of CXCL13 by detailed in situ methods in cynomolgus macaque lymphoid and intestinal tissues. Our results revealed that there were distinct localization patterns of CXCL13 mRNA compared to protein in germinal centers. These patterns shifted during the course of simian immunodeficiency virus (SIV) infection, with increased mRNA expression within and around follicles during AIDS compared to uninfected or acutely infected animals. Unexpectedly, CXCL13 expression was also found in abundance in Paneth cells in crypts throughout the small intestine. Therefore, we expanded our analyses to include chemokines and antimicrobial peptides (AMPs) not previously demonstrated to be expressed by Paneth cells in intestinal tissues. We examined the expression patterns of multiple chemokines, including CCL25, as well as α-defensin 6 (DEFA6), ß-defensin 2 (BDEF2), rhesus θ-defensin 1 (RTD-1), and Reg3γ in situ in intestinal tissues. Of the 10 chemokines examined, CXCL13 was unique in its expression by Paneth cells. BDEF2, RTD-1, and Reg3γ were also expressed by Paneth cells. BDEF2 and RTD-1 previously have not been shown to be expressed by Paneth cells. These findings expand our understanding of mucosal immunology, innate antimicrobial defenses, homeostatic chemokine function, and host protective mechanisms against microbial translocation.

Virologic and immunologic events in hilar lymph nodes during simian immunodeficiency virus infection: development of polarized inflammation.

Lymphoid tissues are sites of soluble and cell-associated antigen sampling of peripheral tissues, and they are key compartments for the generation of cellular and humoral immune responses. Hilar lymph nodes (HiLNs), which drain the lungs, were examined to understand the effects of simian immunodeficiency virus (SIV) infection on this compartment of the immune system. Histologic and messenger RNA (mRNA) expression profiling approaches were used to determine the numbers, types, and distributions of SIV viral RNA cells and to identify differentially expressed genes in HiLNs during SIV infection. SIV RNA cells were found to be primarily CD68 and localized to paracortical and medullary regions early in infection, whereas they resided mainly in paracortex during AIDS. As SIV infection progressed, CXCL9, CXCL10, interferon-gamma, and Toll-like receptor 3 levels all increased. In contrast, CCL19 increased early in infection but decreased during AIDS, whereas CCL21 decreased progressively throughout infection. Finally, local levels of cellular activation were increased throughout infection. Taken together, these findings indicate that SIV infection leads to an inflammatory environment in lung-draining lymph nodes that is characterized by type 1 cytokines and chemokines and likely has an impact on the nature and strength of immune responses to pulmonary pathogens.

Successful outcome of human metapneumovirus (hMPV) pneumonia in a lung transplant recipient treated with intravenous ribavirin.

Human metapneumovirus (hMPV) has recently been shown to be a prominent cause of respiratory infections in immunocompromised hosts, and is associated with high morbidity and mortality. We report a case of hMPV pneumonia in a lung transplant recipient presenting with respiratory failure and sepsis syndrome. hMPV was diagnosed by polymerase chain reaction, and treated with intravenous ribavirin with a successful outcome.

Diagnosis of human metapneumovirus infection in immunosuppressed lung transplant recipients and children evaluated for pertussis.

Human metapneumovirus (hMPV) is a recently discovered paramyxovirus that is known to cause respiratory tract infections in children and immunocompromised individuals. Given the difficulties of identifying hMPV by conventional culture, molecular techniques could improve the detection of this virus in clinical specimens. In this study, we developed a real-time reverse transcription-PCR (RT-PCR) assay designed to detect the four genetic lineages of hMPV. This assay and a commercial real-time nucleic acid sequence-based amplification (NASBA) assay (bioMérieux, Durham, NC) were used to determine the prevalence of hMPV in 114 immunosuppressed asymptomatic and symptomatic lung transplant recipients and 232 pediatric patients who were being evaluated for pertussis. hMPV was detected in 4.3% of the immunosuppressed lung transplant recipients and in 9.9% of children evaluated for pertussis. Both RT-PCR and NASBA assays were efficient in detection of hMPV infection in respiratory specimens. Even though hMPV was detected in a small number of the lung transplant recipients, it was still the most prevalent etiologic agent detected in patients with respiratory symptoms. In both of these diverse patient populations, hMPV infection was the most frequent viral respiratory tract infection identified. Given our findings, infection with hMPV infection should be determined as part of the differential diagnosis of respiratory illnesses.

Increased expression of TLR3 in lymph nodes during simian immunodeficiency virus infection: implications for inflammation and immunodeficiency.

As pattern recognition receptors, TLRs signal and induce expression of multiple host defense genes including proinflammatory cytokines and chemokines. To investigate the mechanisms of up-regulation of proinflammatory cytokines and chemokines during SIV infection in rhesus macaques, we measured the relative levels of expression of TLRs 1-10 in lymphoid tissues during different stages of SIV infection. By real-time RT-PCR, TLR3 was determined to be up-regulated in macaque lymph nodes (LN) throughout the course of infection, whereas TLR9 was down-regulated during early stages of infection. CXCL9/Mig, CXCL10/IP-10, IFN-gamma, and IFN-alpha mRNAs were also increased during acute SIV infection and AIDS. Treatment of macaque spleen and LN cells with TLR3 and TLR9 ligands led to the induction of these same genes. TLR3 stimulation had disparate effects on viral transcription and viral replication, because poly(I:C), a model TLR3 ligand, stimulated the viral promoter but potently inhibited SIV replication in primary cultures of macaque spleen and LN cells. These findings identify roles for TLR3 inflammation in lymphoid tissues and in the immunopathogenesis of HIV-1/SIV, and suggest that TLR3 ligands could potentially be used to flush out latently infected cells that persist during antiretroviral therapies.

Genetic analysis of Toll/Interleukin-1 Receptor (TIR) domain sequences from rhesus macaque Toll-like receptors (TLRs) 1-10 reveals high homology to human TLR/TIR sequences.

Toll-like receptors (TLRs) form a major group of pattern recognition receptors of the innate immune system that sense molecular patterns on microbes. The cytoplasmic Toll/Interleukin-1 Receptor (TIR) signaling domain is instrumental in inducing a signaling cascade upon recognition of specific ligands by TLRs. Because nonhuman primates are used as models of infectious and immune processes, we sought to obtain an increased understanding of nonhuman primate TLRs. We obtained the nucleotide sequences of the TIR domains of rhesus macaque TLRs 1-10 and examined their genetic relationships to TLRs from humans and mice. Alignment of the deduced amino acid sequences revealed macaque-specific changes mostly outside the conserved "Box" regions of the TLR/TIR domain. Assessment of mutational biases among TLRs from multiple species revealed a strong overall bias towards synonymous substitutions, with a few short regions showing evidence for positive selection outside the Box regions. This first presentation of the TLR/TIR domain sequences from nonhuman primates indicates that although there are species-specific differences, a high level of sequence homology exists in the critical signaling Box regions of macaque, human, and murine TLR/TIR domains. These findings suggest that animal models, including nonhuman primates, will be useful in modeling human TLR pathophysiology and therapy.

Increased expression of the inflammatory chemokine CXC chemokine ligand 9/monokine induced by interferon-gamma in lymphoid tissues of rhesus macaques during simian immunodeficiency virus infection and acquired immunodeficiency syndrome.

Chemokines are important mediators of cell trafficking during immune inductive and effector activities, and dysregulation of their expression might contribute to the pathogenesis of human immunodeficiency virus type 1 and the related simian immunodeficiency virus (SIV). To understand better the effects of SIV infection on lymphoid tissues in rhesus macaques, we examined chemokine messenger RNA (mRNA) expression patterns by using DNA filter array hybridization. Of the 34 chemokines examined, the interferon gamma (IFN-gamma)-inducible chemokine CXC chemokine ligand 9/monokine induced by interferon-gamma (CXCL9/Mig) was one of the most highly up-regulated chemokines in rhesus macaque spleen tissue early after infection with pathogenic SIV. The relative levels of expression of CXCL9/Mig mRNA in spleen and lymph nodes were significantly increased after infection with SIV in both quantitative image capture and analysis and real-time reverse transcriptase-polymerase chain reaction assays. In addition, in situ hybridization for CXCL9/Mig mRNA revealed that the patterns of expression were altered after SIV infection. Associated with the increased expression of CXCL9/Mig were increased numbers of IFN-gamma mRNA-positive cells in tissues and reduced percentages of CXC chemokine receptor (CXCR) 3(+)/CD3(+) and CXCR3(+)/CD8(+) lymphocytes in peripheral blood. We propose that SIV replication in vivo initiates IFN-gamma-driven positive-feedback loops in lymphoid tissues that disrupt the trafficking of effector T lymphocytes and lead to chronic local inflammation, thereby contributing to immunopathogenesis.