Hassan M. Al-Emran

Identification of Serotype in Culture Negative Pneumococcal Meningitis Using Sequential Multiplex PCR: Implication for Surveillance and Vaccine Design

Background PCR-based serotyping of Streptococcus pneumoniae has been proposed as a simpler approach than conventional methods, but has not been applied to strains in Asia where serotypes are diverse and different from other part of the world. Furthermore, PCR has not been used to determine serotype distribution in culture-negative meningitis cases. Methodology Thirty six serotype-specific primers, 7 newly designed and 29 previously published, were arranged in 7 multiplex PCR sets, each in new hierarchies designed for overall serotype distribution in Bangladesh, and specifically for meningitis and non-meningitis isolates. Culture-negative CSF specimens were then tested directly for serotype-specific sequences using the meningitis-specific set of primers. PCR-based serotyping of 367 strains of 56 known serotypes showed 100% concordance with quellung reaction test. The first 7 multiplex reactions revealed the serotype of 40% of all, and 31% and 48% non-meningitis and meningitis isolates, respectively. By redesigning the multiplex scheme specifically for non-meningitis or meningitis, the quellung reaction of 43% and 48% of respective isolates could be identified. Direct examination of 127 culture-negative CSF specimens, using the meningitis-specific set of primers, yielded serotype for 51 additional cases. Conclusions This PCR approach, could improve ascertainment of pneumococcal serotype distributions, especially for meningitis in settings with high prior use of antibiotics.

Surveillance for Invasive Streptococcus pneumoniae Disease among Hospitalized Children in Bangladesh: Antimicrobial Susceptibility and Serotype Distribution

BACKGROUND Vaccines offer the prospect of primary disease prevention of pneumococcal disease in childhood. For introduction of such vaccines in developing countries, information about disease epidemiology is necessary. METHODS We evaluated antimicrobial susceptibility and serotype distribution of invasive Streptococcus pneumoniae disease in children aged <5 years in a network of 7 hospitals in Bangladesh from May 2004 through May 2007. RESULTS Of 17,969 blood cultures and 3765 cerebrospinal fluid cultures, 139 yielded S. pneumoniae isolates; 94 were from meningitis cases, 13 were from pneumonia cases, and 32 were from sepsis cases. Among the children with positive culture results, 73% were aged <12 months and 90% were aged <24 months. Complete resistance against penicillin, chloramphenicol, and cotrimoxazole was found in 0%, 6%, and 32% of isolates, respectively. Of the 37 serotypes observed, the predominant serotypes were 2 (17%), 1 (12%), 14 (7%), 5 (6%), 7F (6%), 45 (7%), and 12A (4%). Serotypes differed between meningitis cases and nonmeningitis cases, especially for serotype 2 (25% of meningitis cases vs. 0% of pneumonia cases; P < .001). The 7-, 10-, and 13-valent vaccines would cover 20% (95% confidence interval [CI], 13%-27%), 43% (95% CI, 35%-51%), and 50% (95% CI, 42%-58%) of these cases of invasive pneumococcal disease overall, with higher coverage of nonmeningitis cases, compared with meningitis cases (7-valent coverage, 23% vs. 18%; 10-valent coverage, 55% vs. 38%; 13-valent coverage, 66% vs. 42%). CONCLUSIONS High levels of nonsusceptibility to cotrimoxazole and susceptibility to penicillin suggest that penicillin may be a drug of choice for treatment of invasive pneumococcal disease. Although serotype distribution is diverse, with changes over time and differences between syndromes observed, implementation of use of the currently available 10- or 13-valent vaccines would have a substantial impact on pneumococcal disease in Bangladesh.

Invasive Pneumococcal Disease among Children in Rural Bangladesh : Results from a Population-Based Surveillance

BACKGROUND Streptococcus pneumoniae infection is recognized as a global priority public health problem, and conjugate vaccines have been shown to prevent vaccine-type invasive pneumococcal disease (IPD) in children. However, better estimates of the disease burden and reliable population-based data on serotype composition are needed for vaccine development and implementation in developing countries. METHODS We initiated a population-based surveillance in the rural Bangladesh community of Mirzapur, covering a population of approximately 144,000. Village health care workers made weekly visits to approximately 12,000 children 1-59 months of age in the study area. Children with reported fever, cough, or difficulty breathing were assessed by the village health care workers using a clinical algorithm and were referred to the hospital if required. Children from the study area who were seen in the hospital underwent clinical examination and laboratory testing if they met standardized case definitions. IPD was confirmed by blood and/or cerebrospinal fluid culture results. Isolates were identified, tested for susceptibility to antibiotics, and serotyped in accordance with standard laboratory methods. We present here the results from the first 3 years of the surveillance (July 2004-June 2007). RESULTS Village health care workers identified 5020 cases of possible severe pneumonia and/or very severe disease (165 cases per 1000 child-years)and 9411 cases of possible pneumonia (310 cases per 1000 child-years) as well as 2029 cases of suspected meningitis and/or very severe disease (67 cases per 1000 child-years) and 8967 cases of high fever and/or possible bacteremia (295 cases per 1000 child-years). Pneumonia was the single most common form of illness observed among 2596 hospitalizations (found in 977 [38%] of cases). We recovered 26 S. pneumoniae isolates (25 isolates from 6925 blood cultures and 1 isolate from 41 cerebrospinal fluid cultures), which gave an overall IPD incidence of 86 cases per 100,000 child-years. Invasive pneumococcal infection was common during infancy (with infants accounting for 23 of the 26 cases), and 50% of the total isolates were obtained from nonhospitalized patients who received a diagnosis of upper respiratory tract infection and fever. The most prevalent pneumococcal serotypes were serotypes 1, 5, 14, 18C, 19A, and 38. Ten of the 26 isolates were completely resistant to trimethoprim-sulfamethoxazole, and another 10 isolates had intermediate resistance. CONCLUSIONS IPD contributes substantially to childhood morbidity in rural Bangladesh. S. pneumoniae can cause invasive but nonsevere disease in children, and IPD incidence can be seriously under reported if such cases are overlooked. The emerging high resistance to trimethoprim-sulfamethoxazole should be addressed. Data on serotype distribution would help to guide appropriate pneumococcal conjugate vaccine formulation.

Molecular characterization of Cryptosporidium spp. among children in rural Ghana.

Background The relevance of Cryptosporidium infections for the burden of childhood diarrhoea in endemic settings has been shown in recent years. This study describes Cryptosporidium subtypes among symptomatic and asymptomatic children in rural Ghana to analyse subtype-specific demographic, geographical, seasonal and clinical differences in order to inform appropriate control measures in endemic areas. Methodology/Principal Findings Stool samples were collected from 2232 children below 14 years of age presenting with and without gastrointestinal symptoms at the Agogo Presbyterian Hospital in the rural Ashanti region of Ghana between May 2007 and September 2008. Samples were screened for Cryptosporidium spp. by PCR and isolates were classified into subtypes based on sequence differences in the gp60 gene. Subtype specific frequencies for age, sex, location and season have been determined and associations with disease symptoms have been analysed within a case-control study. Cryptosporidium infections were diagnosed in 116 of 2232 (5.2%) stool samples. Subtyping of 88 isolates revealed IIcA5G3 (n = 26, 29.6%), IbA13G3 (n = 17, 19.3%) and IaA21R3 (n = 12, 13.6%) as the three most frequent subtypes of the two species C. hominis and C. parvum, known to be transmitted anthroponotically. Infections peak at early rainy season with 67.9% and 50.0% of infections during the months April, May and June for 2007 and 2008 respectively. C. hominis infection was mainly associated with diarrhoea (odds ratio [OR] = 2.4; 95% confidence interval [CI]: 1.2–4.9) whereas C. parvum infection was associated with both diarrhoea (OR = 2.6; CI: 1.2–5.8) and vomiting (OR = 3.1; 95% CI: 1.5–6.1). Conclusions/Significance Cryptosporidiosis is characterized by seasonal anthroponotic transmission of strains typically found in Sub-Saharan Africa. The infection mainly affects young infants, with vomiting and diarrhoea being one of the leading symptoms in C. parvum infection. Combining molecular typing and clinical data provides valuable information for physicians and is able to track sources of infections.

Extended spectrum beta-lactamase producing Enterobacteriaceae causing bloodstream infections in rural Ghana, 2007-2012.

BACKGROUND High prevalence of Extended Spectrum Beta-Lactamase (ESBL) producing Enterobacteriaceae threatens treatment options for invasive bloodstream infections in sub-Saharan Africa. OBJECTIVES To explore the frequency and genotype distribution of ESBL producing Enterobacteriaceae causing bloodstream infections in a primary health care setting in rural Ghana. METHODS Blood cultures from all patients with fever ≥38°C within 24h after admission (community-acquired) and from all neonates with suspected neonatal sepsis (hospital-acquired) were obtained. ESBL-producing isolates were characterized by combined disc test and by amplifying the blaCTX-M, blaTEM and blaSHV genes. Multilocus sequence typing (MLST) was performed for all ESBL-producing Klebsiella pneumoniae and Escherichia coli isolates, and all K. pneumoniae isolates were differentiated by pulsed-field gel electrophoresis (PFGE). RESULTS Among 426 Enterobacteriaceae isolated from blood cultures, non-typhoid Salmonella (n=215, 50.8%), S. Typhi (n=110, 26.0%), E. coli (n=50, 11.8%) and K. pneumoniae (n=41, 9.7%) were the most frequent. ESBL-producing isolates were restricted to the CTX-M-15 genotype and the species K. pneumoniae (n=34, 82.9%), Enterobacter cloacae complex (n=2, 66.7%) and E. coli (n=5, 10.0%). The rates of ESBL-producers in K. pneumoniae were 55.6% and 90.6% in community-acquired and neonatal bloodstream infections, respectively. MLST and PFGE analysis identified four outbreak clusters among neonates. CONCLUSIONS Considering the rural primary health care study setting, the high proportion of ESBL-producing Klebsiella pneumoniae is worrisome and might be devastating in the absence of second line antibiotics. Therefore, enhanced diagnostic laboratories for surveillance purposes and sustainable hospital hygiene measures must be considered to prevent further spread of multidrug resistant bacteria within rural communities.

Newborn Vitamin A Supplementation Does Not Affect Nasopharyngeal Carriage of Streptococcus pneumoniae in Bangladeshi Infants at Age 3 Months

Nasopharyngeal (NP) carriage of S. pneumoniae (Spn) is a risk factor for pneumococcal disease and its transmission. We assessed the impact of vitamin A (VA) supplementation shortly after birth in reducing Spn colonization in early infancy in rural Bangladesh. We recruited 500 infants participating in a cluster-randomized trial that reported a 15% reduction in mortality following receipt of an oral dose of VA (52.25 μmol) compared to placebo. NP specimens were collected at the age of 3 mo to study the effect of VA on the prevalence of culture-confirmed Spn. Analyses were conducted by intention to treat. Spn carriage prevalence did not differ between VA and placebo recipients [OR = 0.83 (95% CI: 0.55-1.27); P = 0.390]. Spn carriage at the age of 3 mo was not lowered by VA given at birth. Results are similar to those from an Indian study in which impact on Spn carriage was assessed at the age of 4 mo [OR = 0.73 (95% CI: 0.48-1.10); P = 0.128]. The point estimate of the pooled effect size for the 2 studies is OR = 0.78 [(95% CI: 0.58-1.04); P = 0.095], which may imply a modest impact on carriage. If so, then the evidence thus far would suggest that Spn carriage reduction is unlikely to be a primary ancillary benefit of newborn VA supplementation.

Detection of co-colonization with Streptococcus pneumoniae by algorithmic use of conventional and molecular methods

Detection of pneumococcal carriage by multiple co-colonizing serotypes is important in assessing the benefits of pneumococcal conjugate vaccine (PCV). Various methods differing in sensitivity, cost and technical complexity have been employed to detect multiple serotypes of pneumococcus in respiratory specimens. We have developed an algorithmic method to detect all known serotypes that preserves the relative abundance of specific serotypes by using Quellung-guided molecular techniques. The method involves culturing respiratory swabs followed by serotyping of 100 colonies by either capsular (10 colonies) or PCR (90 colonies) reactions on 96-well plates. The method was evaluated using 102 nasal swabs from children carrying pneumococcus. Multiple serotypes were detected in 22% of carriers, compared to 3% by World Health Organization (WHO)-recommended morphology-based selection of 1 to 3 colonies. Our method, with a processing cost of

Impact of introduction of the Haemophilus influenzae type b conjugate vaccine into childhood immunization on meningitis in Bangladeshi infants.

87, could detect subdominant strains making up as low as 1% of the population. The method is affordable, practical, and capable of detecting all known serotypes without false positive reactions or change in the native distribution of multiple serotypes.

Variations of Invasive Salmonella Infections by Population Size in Asante Akim North Municipal, Ghana

OBJECTIVES Some Asian countries have been reluctant to adopt Haemophilus influenzae type b (Hib) conjugate vaccination because of uncertainty over disease burden. We assessed the impact of introduction of Hib conjugate vaccine into the Expanded Program on Immunization in Bangladesh on purulent and laboratory-confirmed H influenzae meningitis. STUDY DESIGN Within a well-defined catchment area around 2 surveillance hospitals in Dhaka, Bangladesh, we compared the incidence of Hib meningitis confirmed by culture, latex agglutination, and polymerase chain reaction assay among infants 1 year before and 1 year after introduction of Hib conjugate vaccine. We adjusted the incidence rate for the proportion of children who sought care at the surveillance hospitals. RESULTS Among infants, the incidence of confirmed Hib meningitis decreased from 92-16 cases per 100,000 within 1 year of vaccine introduction (vaccine preventable incidence = 76; 95% CI 18, 135 per 100,000). The incidence of purulent meningitis decreased from 1659-1159 per 100,000 (vaccine preventable incidence = 500; 95% CI: 203, 799 per 100,000). During the same time period, there was no significant difference in the incidence of meningitis due to Streptococcus pneumoniae. CONCLUSIONS Introduction of conjugate Hib conjugate vaccine into Bangladesh Expanded Program on Immunization markedly reduced the burden of Hib and purulent meningitis.

A Multicountry Molecular Analysis of Salmonella enterica Serovar Typhi With Reduced Susceptibility to Ciprofloxacin in Sub-Saharan Africa

BACKGROUND The Typhoid Fever Surveillance in Africa Program (TSAP) estimated adjusted incidence rates (IRs) for Salmonella enterica serovar Typhi and invasive nontyphoidal S. enterica serovars (iNTS) of >100 cases per 100 000 person-years of observation (PYO) for children aged <15 years in Asante Akim North Municipal (AAN), Ghana, between March 2010 and May 2012. We analyzed how much these rates differed between rural and urban settings. METHODS Children recruited at the Agogo Presbyterian Hospital and meeting TSAP inclusion criteria were included in the analysis. Towns with >32 000 inhabitants were considered urban; towns with populations <5200 were considered rural. Adjusted IRs for Salmonella bloodstream infections were estimated for both settings. Setting-specific age-standardized incidence rates for children aged <15 years were derived and used to calculate age-standardized rate ratios (SRRs) to evaluate differences between settings. RESULTS Eighty-eight percent (2651/3000) of recruited patients met inclusion criteria and were analyzed. IRs of Salmonella bloodstream infections in children <15 years old were >100 per 100 000 PYO in both settings. Among rural children, the Salmonella Typhi and iNTS rates were 2 times (SRR, 2.2; 95% confidence interval [CI], 1.3-3.5) and almost 3 times (SRR, 2.8; 95% CI, 1.9-4.3) higher, respectively, than rates in urban children. CONCLUSIONS IRs of Salmonella bloodstream infections in children <15 years old in AAN, Ghana, differed by setting, with 2 to nearly 3 times higher rates in the less populated setting. Variations in the distribution of the disease should be considered to implement future studies and intervention strategies.