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1.  Carriage of Mycoplasma pneumoniae in the Upper Respiratory Tract of Symptomatic and Asymptomatic Children: An Observational Study 
PLoS Medicine  2013;10(5):e1001444.
In order to determine the possible asymptomatic carriage of Mycoplasma pneumoniae in the upper respiratory tracts of children, Emiel Spuesens and colleagues investigate the prevalence of M. pneumoniae in symptomatic and asymptomatic children at a hospital in The Netherlands.
Please see later in the article for the Editors' Summary
Mycoplasma pneumoniae is thought to be a common cause of respiratory tract infections (RTIs) in children. The diagnosis of M. pneumoniae RTIs currently relies on serological methods and/or the detection of bacterial DNA in the upper respiratory tract (URT). It is conceivable, however, that these diagnostic methods also yield positive results if M. pneumoniae is carried asymptomatically in the URT. Positive results from these tests may therefore not always be indicative of a symptomatic infection. The existence of asymptomatic carriage of M. pneumoniae has not been established. We hypothesized that asymptomatic carriage in children exists and investigated whether colonization and symptomatic infection could be differentiated by current diagnostic methods.
Methods and Findings
This study was conducted at the Erasmus MC–Sophia Children's Hospital and the after-hours General Practitioners Cooperative in Rotterdam, The Netherlands. Asymptomatic children (n = 405) and children with RTI symptoms (n = 321) aged 3 mo to 16 y were enrolled in a cross-sectional study from July 1, 2008, to November 30, 2011. Clinical data, pharyngeal and nasopharyngeal specimens, and serum samples were collected. The primary objective was to differentiate between colonization and symptomatic infection with M. pneumoniae by current diagnostic methods, especially real-time PCR. M. pneumoniae DNA was detected in 21.2% (95% CI 17.2%–25.2%) of the asymptomatic children and in 16.2% (95% CI 12.2%–20.2%) of the symptomatic children (p = 0.11). Neither serology nor quantitative PCR nor culture differentiated asymptomatic carriage from infection. A total of 202 children were tested for the presence of other bacterial and viral pathogens. Two or more pathogens were found in 56% (63/112) of the asymptomatic children and in 55.5% (50/90) of the symptomatic children. Finally, longitudinal sampling showed persistence of M. pneumoniae in the URT for up to 4 mo. Fifteen of the 21 asymptomatic children with M. pneumoniae and 19 of the 22 symptomatic children with M. pneumoniae in this longitudinal follow-up tested negative after 1 mo.
Although our study has limitations, such as a single study site and limited sample size, our data indicate that the presence of M. pneumoniae in the URT is common in asymptomatic children. The current diagnostic tests for M. pneumoniae are unable to differentiate between asymptomatic carriage and symptomatic infection.
Please see later in the article for the Editors' Summary
Editors' Summary
Pneumonia (a form of acute respiratory infection) is the single largest cause of death in children worldwide, killing an estimated 1.2 million children aged five and under every year, particularly in South Asia and sub-Saharan Africa. In these settings, bacterial infections with Streptococcus pneumoniae and Haemophilus influenzae are the most common causes of bacterial pneumonia. However, in high-income settings, bacterial infection with Mycoplasma pneumoniae is a major cause of upper and lower respiratory tract infections in children: over one-third of childhood cases of community-acquired pneumonia that require admission to a hospital are caused by M. pneumoniae. Currently, diagnosis of M. pneumoniae infections relies on the detection of antibodies against M. pneumoniae in the blood or detection of bacterial DNA in samples from the upper respiratory tract through polymerase chain reaction (PCR) tests.
Why Was This Study Done?
Other bacteria, such as Streptococcus pneumoniae, are commonly present in children without causing infection, a situation known as asymptomatic carriage. However, to date, it is unknown whether M. pneumoniae is also commonly carried in the upper respiratory tract of children without causing symptoms or leading to infection. The possibility of asymptomatic carriage of M. pneumoniae could have major implications for the interpretation of the results of diagnostic tests and also for clinical management. So in this study conducted in The Netherlands, the researchers investigated whether asymptomatic carriage of M. pneumoniae exists and also whether symptomatic infection could be differentiated from asymptomatic carriage by current diagnostic methods.
What Did the Researchers Do and Find?
Between 2008 and 2011, the researchers recruited children aged between three months and 16 years attending a hospital in Rotterdam for an elective surgical procedure (asymptomatic group) or admitted with a respiratory tract infection (symptomatic group). All children had blood tests and respiratory samples (nasopharyngeal swab) taken on admission and were tested for other pathogens. The researchers invited children who tested positive for M. pneumoniae by PCR to attend for further follow-up and tested them monthly for the presence of M. pneumoniae DNA in the upper respiratory tract until the test was negative on two occasions. Using these methods, the researchers recruited 726 children over the study period—405 in the asymptomatic group and 321 in the symptomatic group. The researchers found that the prevalence of M. pneumoniae did not differ between the asymptomatic group and the symptomatic group, with prevalences of 21.2% and 16.2%, respectively (the prevalence of M. pneumoniae also did not differ significantly between those with lower versus upper respiratory infection). There were also no differences in prevalence in the asymptomatic and symptomatic groups when diagnosed using blood tests. The researchers found a high rate of multiple, coexisting bacterial and viral pathogens in both asymptomatic and symptomatic children: two or more pathogens were found in 56% (63/112) of the asymptomatic children and in 55.5% (50/90) of the symptomatic children. Furthermore, season and the year of enrollment affected the prevalence of M. pneumoniae in the asymptomatic group, ranging from 3% during the spring of 2009 to 58% during the summer of 2010. Finally, of the 21 children from the asymptomatic group who participated in the follow-up study, 15 (71%) tested negative within one month, and in the symptomatic group, 19 of 22 children (86%) tested negative after the first visit.
What Do These Findings Mean?
These findings show that M. pneumoniae is carried at high rates in the upper respiratory tracts of healthy children, and that this asymptomatic carriage cannot be differentiated from symptomatic respiratory tract infection by diagnostic tests (serology or PCR). As the prevalence of M. pneumoniae varied between year and season, carriage of M. pneumoniae may follow a cyclic epidemic pattern. This study is from a single study site in one city in The Netherlands, with a relatively small number of children, and so these findings may not be generalizable to other populations. However, as this study suggests that current diagnostic tests do not discriminate between carriage and infection, clinicians may need to reconsider the clinical significance of a positive test result. Future studies are needed to address this diagnostic challenge and also to investigate possible factors that may affect the progression of asymptomatic carriage of M. pneumoniae to symptomatic infection.
Additional Information
Please access these websites via the online version of this summary at
MicrobeWiki has more information on M. pneumoniae
Lab Tests Online explains current tests for M. pneumoniae
PMCID: PMC3653782  PMID: 23690754
2.  Human Cytomegalovirus: detection of congenital and perinatal infection in Argentina 
BMC Pediatrics  2004;4:11.
Human cytomegalovirus (CMV) is one of the most commonly found agents of congenital infections. Primary maternal infection is associated with risk of symptomatic congenital diseases, and high morbidity is frequently associated with very low birth weight. Neonates with asymptomatic infection develop various sequelae during infancy. This is the first Argentine study performed in neonates with congenital and postnatal HCMV infection. The purpose of this study was to evaluate the performance of the polymerase chain reaction (PCR) technique with different pairs of primers, to detect cytomegalovirus isolated in tissue cultures and directly in urine and dried blood spot (DBS) specimens. Results were compared with IgM detection.
The study was performed between 1999 and 2001 on routine samples in the Laboratory. A total of 61 urine and 56 serum samples were selected from 61 newborns/infants, 33 patients whose samples were analyzed during the first two to three weeks of life were considered congenital infections; the remaining 28 patients whose samples were taken later than the third week were grouped as perinatal infections, although only in 4 the perinatal transmission of infection was determined unequivocally
Cytomegalovirus diagnosis was made by isolating the virus from urine samples in human foreskin fibroblast cells.
Three different primer pairs directed to IE, LA and gB genes were used for the HCMV PCR assay in viral isolates. Subsequently, PCR and nested PCR (nPCR) assays with gB primers were performed directly in urine and in 11 samples of dried blood spot (DBS) on Guthrie Card, these results were then compared with serology.
The main clinical manifestations of the 33 patients with congenital infection were purpura, jaundice, hepatomegaly and anaemia. Three patients presented low birth weight as single symptom, 10, intracranial calcifications, and 2, kidney failure. In the 28 patients grouped as with perinatal infection, anaemia, hepatosplenomegaly and enzymatic alteration were predominant, and 4 patients were HIV positive.
The primers used to amplify the gB region had a PCR positivity rate of 100%, whereas those that amplified IE and LA regions had a PCR positivity rate of 54% and 61% respectively, in CMV isolates.
Amplification by PCR of urine samples (with no previous DNA extraction), using primers for the gB region, detected 34/61 positive samples. Out of the 33 samples from patients with congenital infection, 24 (73%) were positive. When nPCR was used in these samples, all were positive, whereas in the remaining 28 patients, two negative cases were found.
Cytomegalovirus DNA detection in 11 samples was also carried out in DBS: 7 DBS samples were positive and 4 were negative.
Primers directed to the gB fragment region were the best choice for the detection of CMV DNA in positive isolates. In congenital infections, direct PCR in urine was positive in a high percentage (73%) of samples; however, in patients grouped as with perinatal infection only 36% of the cases were positive. With n-PCR, total sample positivity reached 97%. PCR technique performed in DBS allowed identifying congenital infection in four patients and to be confirmed in 3. These results show the value of nPCR for the detection of all cases of CMV infection. The assay offers the advantage that it may be performed within the normal working day and provides reliable results in a much shorter time frame than that required for either traditional tissue culture or the shell-viral assay.
PMCID: PMC449715  PMID: 15214967
3.  Human Caliciviruses in Symptomatic and Asymptomatic Infections in Children in Vellore, South India 
Journal of medical virology  2007;79(5):544-551.
Pediatric gastroenteritis is a major cause of childhood morbidity and mortality worldwide, especially in developing countries. It has been increasingly recognised that human caliciviruses (HuCV), comprising noroviruses (NoV), and sapoviruses (SaV), are important in both outbreak and non-outbreak settings. This study aimed to characterise caliciviruses detected in the faeces of hospitalized children and children in the community in India. This study examined 350 faecal samples from children presenting to the hospital with acute gastroenteritis and 673 samples collected from children in the community, 500 from children with diarrhea, and 173 samples from children without diarrhea. Strain characterisation was performed by reverse transcription-polymerase chain reaction (RT-PCR) and partial sequencing of the gene encoding the RNA-dependent RNA polymerase (RdRp) and/or a region spanning the open reading frames (ORFs) 1 and 2 (ORF1/ORF2) junction. A total of 68 of 350 specimens (19.4%) from hospitalized children were positive, and SaV and NoV accounted for 5.1 and 15.1% of the infections, respectively. Mixed infections of HuCVs with other enteric pathogens were seen in 9.4% of the total children tested. Sixty-eight out of 673 (10.1%) samples collected from children in the community were positive for caliciviruses, and SaV and NoV accounted for 3.4 and 6.6% of the infections. In the community cohort 55/500 (11%) and 13/173 (7.5%) were from symptomatic and asymptomatic children, respectively, and SaVs accounted for 17/500 (3.4%) and NoVs for 38/500 (7.6%) of the symptomatic infections. This is the first report of genotyping of circulating caliciviruses in both hospital and community in India and has increased the evidence for the role of these viruses in pediatric gastroenteritis in India.
PMCID: PMC2473265  PMID: 17385696
gastroenteritis; genotyping; norovirus; sapovirus
4.  Giardia duodenalis Assemblages Associated with Diarrhea in Children in South India Identified by PCR-RFLP 
Giardial diarrhea in a birth cohort of 452 children in an urban slum in South India was characterized. Of the 155 episodes that occurred in 99 children, 73% were acute diarrhea. Children with better educated mothers and a toilet at home had lower odds of acquiring giardial diarrhea, whereas low socioeconomic status and drinking municipal water were associated with greater risk. Children with co-infections tended to have a slightly longer duration of diarrhea (P = 0.061) and showed significantly more wasting after an episode than children with diarrhea resulting from Giardia alone (P = 0.032). Among the 99 cases, 50 diarrheal and 51 asymptomatic Giardia positive samples were genotyped by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) at the triose phosphate isomerase gene. Assemblage B was predominant both in giardial diarrhea (80%) and asymptomatic giardiasis (94%). Children with Assemblage A subgroup-II alone or dual infections with both assemblage A and B had diarrhea more frequently (P = 0.07).
PMCID: PMC3909731  PMID: 19141832
5.  Epidemiological Characteristics of 2009 (H1N1) Pandemic Influenza Based on Paired Sera from a Longitudinal Community Cohort Study 
PLoS Medicine  2011;8(6):e1000442.
Steven Riley and colleagues analyze a community cohort study from the 2009 (H1N1) influenza pandemic in Hong Kong, and found that more children than adults were infected with H1N1, but children were less likely to progress to severe disease than adults.
While patterns of incidence of clinical influenza have been well described, much uncertainty remains over patterns of incidence of infection. The 2009 pandemic provided both the motivation and opportunity to investigate patterns of mild and asymptomatic infection using serological techniques. However, to date, only broad epidemiological patterns have been defined, based on largely cross-sectional study designs with convenience sampling frameworks.
Methods and Findings
We conducted a paired serological survey of a cohort of households in Hong Kong, recruited using random digit dialing, and gathered data on severe confirmed cases from the public hospital system (>90% inpatient days). Paired sera were obtained from 770 individuals, aged 3 to 103, along with detailed individual-level and household-level risk factors for infection. Also, we extrapolated beyond the period of our study using time series of severe cases and we simulated alternate study designs using epidemiological parameters obtained from our data. Rates of infection during the period of our study decreased substantially with age: for 3–19 years, the attack rate was 39% (31%–49%); 20–39 years, 8.9% (5.3%–14.7%); 40–59 years, 5.3% (3.5%–8.0%); and 60 years or older, 0.77% (0.18%–4.2%). We estimated parameters for a parsimonious model of infection in which a linear age term and the presence of a child in the household were used to predict the log odds of infection. Patterns of symptom reporting suggested that children experienced symptoms more often than adults. The overall rate of confirmed pandemic (H1N1) 2009 influenza (H1N1pdm) deaths was 7.6 (6.2–9.5) per 100,000 infections. However, there was substantial and progressive increase in deaths per 100,000 infections with increasing age from 0.66 (0.65–0.86) for 3–19 years up to 220 (50–4,000) for 60 years and older. Extrapolating beyond the period of our study using rates of severe disease, we estimated that 56% (43%–69%) of 3–19 year olds and 16% (13%–18%) of people overall were infected by the pandemic strain up to the end of January 2010. Using simulation, we found that, during 2009, larger cohorts with shorter follow-up times could have rapidly provided similar data to those presented here.
Should H1N1pdm evolve to be more infectious in older adults, average rates of severe disease per infection could be higher in future waves: measuring such changes in severity requires studies similar to that described here. The benefit of effective vaccination against H1N1pdm infection is likely to be substantial for older individuals. Revised pandemic influenza preparedness plans should include prospective serological cohort studies. Many individuals, of all ages, remained susceptible to H1N1pdm after the main 2009 wave in Hong Kong.
Please see later in the article for the Editors' Summary
Editors' Summary
From June 2009 to August 2010, the world was officially (according to specific WHO criteria—WHO phase 6 pandemic alert) in the grip of an Influenza A pandemic with a new strain of the H1N1 virus. During this time, more than 214 countries and overseas territories reported laboratory confirmed cases of pandemic influenza H1N1 2009 with almost 20,000 deaths.
While much is already known about patterns of incidence of clinical influenza, the patterns of infection incidence are much more uncertain, because many influenza infections are either asymptomatic or cause only mild symptoms. This means that it is difficult to obtain accurate estimates of risk factors for infection and the overall burden of disease using only clinical surveillance. However, without accurate estimates of infection incidence across different risk groups, it is not possible to establish the number of infections that give rise to severe disease (the per infection rate of severe disease). Consequently, it is difficult to give evidence-based advice for individuals, groups, and populations about the potential benefits of interventions including drugs and vaccines that might reduce the risk of influenza infection.
Why Was This Study Done?
During the 2009 pandemic, some countries and territories, such as Hong Kong, were able to investigate patterns of mild and asymptomatic infection using serological techniques, thus providing information that may help to fill this knowledge gap. Given the high levels of polymerase chain reaction (PCR) testing and the robust reporting of hospital episodes, the main H1N1 pandemic wave in Hong Kong (during September 2009) provided an opportunity to implement a prospective cohort study to investigate the incidence of infection.
What Did the Researchers Do and Find? The researchers collected data on the asymptomatic symptoms of influenza by randomly selecting households to participate in the study. Each member of the household willing to participate had a baseline blood sample taken before the main wave of the pandemic (July to September 2009), then, when clinical surveillance suggested that the main peak in transmission had passed, after the main wave (November 2009 to February 2010). During the study period, participants were asked to report any flu-like symptoms in three ways: to phone the study team and report symptoms in real time; to fill out a paper diary with the day and symptoms; and to report symptoms during a follow-up interview. In parallel, the researchers monitored data on every patient with H1N1 admitted to intensive care units or who died while in the hospital. The researchers then estimated the number of H1N1 infections (infection incidence) per severe case by developing a likelihood-based framework. They used a simulation model to investigate alternate study designs and to validate their estimates of the rate of severe disease per infection.
Using these methods, the researchers found that rates of H1N1 infection during the study period decreased substantially with age: for 3–19 years, the attack rate was 39%; 20–39 years, 8.9%; 40–59 years, 5.3%; and 60 years or older, 0.77%. In addition, patterns of symptom reporting indicated that children experienced symptoms more often than adults. The overall rate of confirmed H1N1 deaths was 7.6 per 100,000 infections. However, there was a substantial and progressive increase in deaths per 100,000 infections with increasing age from 0.66 for 3–19 years up to 220 for 60 years and older. Statistical modeling suggested that 56% of 3–19 year olds and 16% of people overall were infected by the pandemic strain up to the end of January 2010.
What Do These Findings Mean?
The results of this study suggest that more children were infected with H1N1 than adults but most of them did not progress to severe disease. Conversely, although fewer older adults were infected with H1N1, this group was much more likely to experience severe disease. Therefore, should H1N1 infection incidence ever increase in older adults, for example by evolving to become more infectious to this group, average rates of severe disease per infection could be much higher than for the 2009 pandemic. Revised pandemic preparedness plans should include prospective serological cohort studies, such as this one, in order to be able to estimate rates of severe disease per infection.
Additional Information
Please access these Web sites via the online version of this summary at
WHO has information about the global response to the 2009 H1N1 pandemic
WHO also provides recommendations for the H1N1 post-pandemic period
The government of Hong Kong's Centre for Health Protection provides information about H1N1 in Hong Kong
PMCID: PMC3119689  PMID: 21713000
6.  Canine Skin and Conjunctival Swab Samples for the Detection and Quantification of Leishmania infantum DNA in an Endemic Urban Area in Brazil 
We evaluated kDNA PCR/hybridization and quantitative real-time PCR (qPCR) targeting the gene of DNA polymerase of Leishmania infantum for CVL diagnosis and assessment of parasite load in clinical samples obtained invasively and non-invasively.
Methodology/Principal Findings
Eighty naturally infected dogs from an endemic urban area in Brazil were used. Animals were divided into two groups based on the presence or absence of CVL clinical sings. Skin biopsies, bone marrow, blood and conjunctival swabs samples were collected and submitted to L. infantum DNA detection. In addition, anti-Leishmania antibody titers were measured by Immunofluorescence antibody test. The symptomatic dogs had increased titers compared to asymptomatic dogs (P = 0.025). The frequencies of positive results obtained by kDNA PCR/hybridization for asymptomatic and symptomatic dogs, respectively, were as follows: right conjunctiva, 77.5% and 95.0%; left conjunctiva, 75.0% and 87.5%; skin, 45.0% and 75.0%; bone marrow, 50.0% and 77.5%; and blood, 27.5% and 22.5%. In both groups, the parasite load in the skin samples was the highest (P<0.0001). The parasite loads in the conjunctival swab and bone marrow samples were statistically equivalent within each group. The parasite burden in conjunctival swabs was higher in the dogs with clinical signs than in asymptomatic dogs (P = 0.028). This same relationship was also observed in the bone marrow samples (P = 0.002). No differences in amastigotes load in the skin were detected between the groups.
The conjunctival swab is a suitable clinical sample for qualitative molecular diagnosis of CVL. The highest parasite burdens were detected in skin regardless of the presence of VL-associated clinical signs. The qPCR results emphasized the role of dogs, particularly asymptomatic dogs, as reservoirs for CVL because of the high cutaneous parasite loads. These results may help to explain the maintenance of high transmission rates and numbers of CVL cases in endemic urban regions.
Author Summary
Visceral leishmaniasis (VL) is considered the most lethal manifestation among the diseases caused by genus Leishmania. The main control measures for VL are: diagnosis and early treatment of human cases, insecticide vector control, and euthanasia of seropositive dogs. Although these strategies have been continuously applied in Brazil, the number of VL cases has increased and this disease still poses as a serious public health problem. Belo Horizonte is an endemic urban area in Brazil and it is considered by the Ministry of Health the third most affected Brazilian metropolitan region by VL. In the context of prophylaxis, the correct diagnosis of infected dogs is critical for the VL control because dogs represent the main domestic reservoir. However, the serological diagnosis techniques used in large-scale for canine screening in Brazil have important limitations including the difficulty of diagnosing asymptomatic dogs. Molecular methods based on Polymerase Chain Reaction (PCR) and real-time PCR (qPCR) have greatly improved VL diagnosis. Based on this, we aimed to evaluate different canine clinical samples for qualitative VL diagnosis by PCR and quantification of parasite load by qPCR in dogs from Belo Horizonte. The possible implications of results are discussed with emphasis on skin and conjunctival swab samples.
PMCID: PMC3323509  PMID: 22506084
7.  Genetic polymorphism of merozoite surface protein 2 and prevalence of K76T pfcrt mutation in Plasmodium falciparum field isolates from Congolese children with asymptomatic infections 
Malaria Journal  2012;11:105.
In order to prepare the field site for future interventions, the prevalence of asymptomatic Plasmodium falciparum infection was evaluated in a cohort of children living in Brazzaville. Plasmodium falciparum merozoite surface protein 2 gene (msp2) was used to characterize the genetic diversity and the multiplicity of infection. The prevalence of mutant P. falciparum chloroquine resistance transporter (pfcrt) allele in isolates was also determined.
Between April and June 2010, 313 children below 10 years of age enrolled in the cohort for malaria surveillance were screened for P. falciparum infection using microscopy and polymerase chain reaction (PCR). The children were selected on the basis of being asymptomatic. Plasmodium falciparum msp2 gene was genotyped by allele-specific nested PCR and the pfcrt K76T mutation was detected using nested PCR followed by restriction endonuclease digestion.
The prevalence of asymptomatic P. falciparum infections was 8.6% and 16% by microscopy and by PCR respectively. Allele typing of the msp2 gene detected 55% and 45% of 3D7 and FC27 allelic families respectively. The overall multiplicity of infections (MOI) was 1.3. A positive correlation between parasite density and multiplicity of infection was found. The prevalence of the mutant pfcrt allele (T76) in the isolates was 92%.
This is the first molecular characterization of P. falciparum field isolates in Congolese children, four years after changing the malaria treatment policy from chloroquine (CQ) to artemisinin-based combination therapy (ACT). The low prevalence of asymptomatic infections and MOI is discussed in the light of similar studies conducted in Central Africa.
PMCID: PMC3349535  PMID: 22463364
Plasmodium falciparum; Asymptomatic infection; Multiplicity of infection; msp2; pfcrt; Brazzaville; Republic of Congo
8.  Determining Risk for Severe Leptospirosis by Molecular Analysis of Environmental Surface Waters for Pathogenic Leptospira 
PLoS Medicine  2006;3(8):e308.
Although previous data indicate that the overall incidence of human leptospirosis in the Peruvian Amazon is similar in urban and rural sites, severe leptospirosis has been observed only in the urban context. As a potential explanation for this epidemiological observation, we tested the hypothesis that concentrations of more virulent Leptospira would be higher in urban than in rural environmental surface waters.
Methods and Findings
A quantitative real-time PCR assay was used to compare levels of Leptospira in urban and rural environmental surface waters in sites in the Peruvian Amazon region of Iquitos. Molecular taxonomic analysis of a 1,200-bp segment of the leptospiral 16S ribosomal RNA gene was used to identify Leptospira to the species level. Pathogenic Leptospira species were found only in urban slum water sources (Fisher's exact test; p = 0.013). The concentration of pathogen-related Leptospira was higher in urban than rural water sources (~103 leptospires/ml versus 0.5 × 102 leptospires/ml; F = 8.406, p < 0.05). Identical 16S rRNA gene sequences from Leptospira interrogans serovar Icterohaemorrhagiae were found in urban slum market area gutter water and in human isolates, suggesting a specific mode of transmission from rats to humans. In a prospective, population-based study of patients presenting with acute febrile illness, isolation of L. interrogans-related leptospires from humans was significantly associated with urban acquisition (75% of urban isolates); human isolates of other leptospiral species were associated with rural acquisition (78% of rural isolates) (chi-square analysis; p < 0.01). This distribution of human leptospiral isolates mirrored the distribution of leptospiral 16S ribosomal gene sequences in urban and rural water sources.
Our findings data support the hypothesis that urban severe leptospirosis in the Peruvian Amazon is associated with higher concentrations of more pathogenic leptospires at sites of exposure and transmission. This combined quantitative and molecular taxonomical risk assessment of environmental surface waters is globally applicable for assessing risk for leptospiral infection and severe disease in leptospirosis-endemic regions.
Vinetz and colleagues used a quantitative real time PCR assay combined with molecular taxonomic analysis to quantify Leptospira in environmental surface waters in the Peruvian Amazon region of Iquitos.
Editors' Summary
Humans catch many diseases from animals—so-called zoonotic infections. Often, these occur in limited regions of the world. However, one—leptospirosis—occurs in temperate and tropical climates, and in urban and rural settings, making it the most widespread zoonotic disease. Leptospirosis is caused by Leptospira, a large group of closely related spiral-shaped bacteria that live in both domestic animals (for example, cattle) and wild animals (particularly rats). Millions of humans become infected each year with leptospires through close contact with water, food, or soil contaminated with the urine of infected animals—swimming or wading in contaminated water is particularly hazardous. Some infected people have no symptoms; others develop a flu-like disease that clears up within a few days. However, in 5%–10% of infected people, the disease progresses to a second, sometimes fatal phase. This is usually characterized by jaundice, kidney problems, and an enlarged spleen (it's then called Weil disease) but can also involve the lungs (pulmonary leptospirosis). Leptospirosis can be successfully treated with antibiotics if treatment is started soon after infection.
Why Was This Study Done?
In a recent study in the Peruvian Amazon, half of the people visiting urban hospitals and rural health posts with acute fever had antibodies in their blood to Leptospira, suggesting that they had acute leptospirosis. However, only patients living in urban areas developed pulmonary leptospirosis. In this study, the researchers tested the hypothesis that this pattern arose because more virulent types of Leptospira were present at higher levels in urban environmental surface water than in rural water sources.
What Did the Researchers Do and Find?
Between June 2003 and March 2004, the researchers isolated strains of Leptospira from patients with acute fever who visited a hospital in the town of Iquitos or clinics in nearby villages. Early in 2004, they also collected a large number of different water samples from an urban slum in Iquitos and from a nearby rural community. They measured the concentrations of Leptospira in these samples by using a molecular technique called real-time PCR (polymerase chain reaction) to detect and quantify a type of RNA found only in disease-causing Leptospira. They also identified which specific Leptospira were present in the water samples and the patient samples by sequencing this RNA. The researchers found that leptospires were present in both urban and rural water samples (particularly in samples from gutters and puddles in the urban slum's market area) but that their concentration in the positive water samples from the urban sites was 20 times that in the positive samples from the rural sites. Furthermore, the distribution of different Leptospira types isolated from the patients mirrored that of the bacteria in the local environment. So, one particular type of Leptospira interrogans known as icterohaemorrhagiae—the leptospire most commonly associated with severe leptospirosis in the patients—was found more often in the urban water samples than in the rural ones. Finally, the researchers discovered a new group of Leptospira in the rural environment. This group may contain one or several new species of Leptospira but whether any of them causes human disease is unknown.
What Do These Findings Mean?
These results support the researchers' hypothesis that pulmonary leptospirosis in urban areas of the Peruvian Amazon is associated with high environmental levels of specific disease-causing leptospires. The researchers were able to discover this link only by using molecular techniques—this sort of study is impossible with traditional bacteriological techniques because Leptospira are hard to grow in the laboratory and cannot be isolated efficiently from environmental water sources. Different types can't be identified using a microscope. The researchers' findings need to be validated in other settings, but they suggest that environmental interventions such as reducing sources of standing water and clearing away garbage in urban areas might reduce the number of cases of severe leptospirosis. The distribution of different Leptospira types also suggests that whereas rats may be the main disease reservoir in towns, cattle, pigs, and bats may be more important in rural settings in Peru and presumably elsewhere. Overall, this new information, together with the availability of molecular methods for rapid clinical diagnosis and environmental risk assessment, should aid attempts to control leptospirosis around the world.
Additional Information.
Please access these Web sites via the online version of this summary at
US Centers for Disease Control and Prevention, information for patients and professionals on leptospirosis
The Leptospirosis Information Center, information and advice on human leptospirosis for the public and medical professionals
MedlinePlus encyclopedia entry on leptospirosis
NHS Direct Online, patient information on leptospirosis from the UK National Health Service online encyclopedia
Wikipedia pages on leptospirosis (note: Wikipedia is a free online encyclopedia that anyone can edit)
PMCID: PMC1551915  PMID: 16933963
9.  Low Parasite Load Estimated by qPCR in a Cohort of Children Living in Urban Area Endemic for Visceral Leishmaniasis in Brazil 
An important issue associated with the control of visceral leishmaniasis is the need to identify and understand the relevance of asymptomatic infection caused by Leishmania infantum. The aim of this study was to follow the course of asymptomatic L. infantum infection in children in an area of Brazil where it is endemic. The children were assessed twice during a 12-month period.
In this population study, 1875 children, ranging from 6 months to 7 years of age, were assessed. Blood samples were collected on filter papers via finger prick and tested by ELISA (L. infantum soluble antigen and rk39). Seropositives samples (n = 317) and a number of seronegatives samples (n = 242) were subjected to qPCR. After 12 months, blood samples were collected from a subgroup of 199 children and tested for Leishmania spp. to follow the course of infection.
Principal Findings
At baseline qPCR testing identified 82 positive samples. The prevalence rate, as estimated for 1875 children based on the qPCR results, was 13.9%. The qPCR testing of whole blood samples collected from a cohort of children after 12 months (n = 199) yielded the following results: of the 44 (22.1%) children with positive qPCR results at baseline, only 10 (5.0%) remained positive, and 34 (17.1%) became negative; and of the 155 (77.9%) children with negative qPCR results, 131 (65.8%) remained negative, and 24 (12.1%) became positive at the follow-up measurement. The samples with positive findings at baseline (n = 82) had a mean of 56.5 parasites/mL of blood; and at follow-up the mean positive result was 7.8 parasites/mL.
The peripheral blood of asymptomatic children had a low and fluctuating quantity of Leishmania DNA and a significant decrease in parasitemia at 1-year follow-up. Quantitative PCR enables adequate monitoring of Leishmania infection.
Author Summary
In Brazil, visceral leishmaniasis (VL) is caused by the parasite protozoon Leishmania (Leishmania) infantum (syn chagasi), which is transmitted to humans by sandflies that have widespread wild and domestic animal reservoirs. An important issue associated with the control of VL is the need to identify and understand the relevance of asymptomatic infection in the transmission cycle of the parasite. We evaluated the course of asymptomatic infection in children by using molecular methods to detect parasite DNA and to monitor parasitemia level over time. The prevalence of VL, as detected by means of a molecular method, for 1875 children was 13.9%. The data demonstrated a significant decrease in parasitemia after 1 year. Furthermore, the level of parasitemia was generally lower in asymptomatic children than in sick children. None of the asymptomatic children showed signs or clinical symptoms associated with VL throughout the duration of the study. This molecular method of measuring parasitemia is adequate for monitoring VL transmission in areas where it is endemic.
PMCID: PMC3521664  PMID: 23272263
10.  Inaccuracy of Enzyme-Linked Immunosorbent Assay Using Soluble and Recombinant Antigens to Detect Asymptomatic Infection by Leishmania infantum 
One of the most important drawbacks in visceral leishmaniasis (VL) population studies is the difficulty of diagnosing asymptomatic carriers. The aim of this study, conducted in an urban area in the Southeast of Brazil, was to evaluate the performance of serology to identify asymptomatic VL infection in participants selected from a cohort with a two-year follow-up period.
Blood samples were collected in 2001 from 136 cohort participants (97 positive and 39 negatives, PCR/hybridization carried out in 1999). They were clinically evaluated and none had progressed to disease from their asymptomatic state. As controls, blood samples from 22 control individuals and 8 patients with kala-azar were collected. Two molecular biology techniques (reference tests) were performed: PCR with Leishmania-generic primer followed by hybridization using L. infantum probe, and PCR with specific primer to L. donovani complex. Plasma samples were tested by ELISA using three different antigens: L. infantum and L. amazonensis crude antigens, and rK39 recombinant protein. Accuracy of the serological tests was evaluated using sensitivity, specificity, likelihood ratio and ROC curve.
The presence of Leishmania was confirmed, by molecular techniques, in all kala-azar patients and in 117 (86%) of the 136 cohort participants. Kala-azar patients showed high reactivity in ELISAs, whereas asymptomatic individuals presented low reactivity against the antigens tested. When compared to molecular techniques, the L. amazonensis and L. infantum antigens showed higher sensitivity (49.6% and 41.0%, respectively) than rK39 (26.5%); however, the specificity of rK39 was higher (73.7%) than L. amazonensis (52.6%) and L. infantum antigens (36.8%). Moreover, there was low agreement among the different antigens used (kappa<0.10).
Serological tests were inaccurate for diagnosing asymptomatic infections compared to molecular methods; this could lead to misclassification bias in population studies. Therefore, studies which have used serological assays to estimate prevalence, to evaluate intervention programs or to identify risk factors for Leishmania infection, may have had their results compromised.
Author Summary
Visceral leishmaniasis or kala-azar in Brazil, caused by a parasite protozoon, is transmitted to humans by sandflies and has wild and domestic dogs as reservoirs. The disease can become chronic, characterized by fever, weight loss, and enlargement of the liver and spleen. However, the infection in humans can be asymptomatic, and the importance of this phase is unknown. The low levels of immune response and the small number of circulating parasites in the blood are the main problems in identifying the asymptomatic phase. In this study we evaluated the performance of the serology tests compared with molecular tests to identify asymptomatic infection in individuals identified in an urban area of Brazil. Also included were non-infected individuals (healthy controls) and patients (diseased controls). Blood was collected and examined by different serological methods and compared with molecular techniques. The presence of the parasite was confirmed by all molecular techniques and should be the method used to identify true infection. The serology methods showed low sensitivity in detecting the infection. We concluded that the serologic methods are inaccurate in identifying asymptomatic infection when compared to molecular techniques and could underestimate the infection in population studies.
PMCID: PMC2759029  PMID: 19841736
11.  Etiological study of enteric viruses and the genetic diversity of norovirus, sapovirus, adenovirus, and astrovirus in children with diarrhea in Chongqing, China 
BMC Infectious Diseases  2013;13:412.
Enteric viruses are a major cause of diarrhea in children, especially those <5 years old. Identifying the viral agents is critical to the development of effective preventive measures. This study aimed to determine the prevalence of common enteric viruses in children <5 years old presented with diarrhea to the Children’s Hospital of Chongqing Medical University.
Five hundred fecal samples were collected between August and November 2010 from children <5 years of age who presented with acute diarrhea at the Children’s Hospital of Chongqing Medical University. All samples were tested for rotaviruses A, B, and C, noroviruses GI and GII, adenovirus, sapovirus, and astrovirus using enzyme-linked immunosorbent assay, reverse transcription-polymerase chain reaction (RT-PCR), or PCR. Partial sequences of norovirus, sapovirus, adenovirus, and astrovirus were phylogenetically analyzed to determine the genotype.
Enteric viruses were detected in 302 of the 500 children who presented with acute diarrhea (277/477; 58.07%) and persistent diarrhea (5/23; 21.74%). In 277 samples from children with acute diarrhea in whom at least one viral agent was found, rotavirus A was the most frequent virus identified (132 cases; 27.67%), followed by norovirus GII in 130 cases (27.25%), adenovirus in 30 cases (6.29%), sapovirus in 9 cases (1.89%) and astrovirus in one case (0.21%). Twenty-two of the norovirus GII-positive cases were randomly selected for genotyping. GII/4 was the predominant strain, followed by GII/6, GII/2, GII/3, and GII/7. Sapovirus was classified into four genotypes: GI/1 was predominant, followed by GI/2, GII/1, and GIV. The predominant adenovirus was type 41. Mixed infections were found in 25 cases, all of which presented with acute diarrhea (25/477; 5.24%). Viruses were positive in 5/23 (21.74%) cases with persistent diarrhea. Neither rotavirus B, rotavirus C, nor norovirus GI were found in any of the samples.
Enteric viruses are a major cause of diarrhea in children <5 years old in Chongqing. Rotavirus A is the most common etiological agent, follow by norovirus.
PMCID: PMC3766652  PMID: 24004442
12.  Evaluation of the Loop Mediated Isothermal DNA Amplification (LAMP) Kit for Malaria Diagnosis in P. vivax Endemic Settings of Colombia 
Most commonly used malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections which are frequent in low transmission settings. Molecular methods such as polymerase chain reaction (PCR) are highly sensitive but remain too laborious for field deployment. In this study, the applicability of a malaria diagnosis kit based on loop-mediated isothermal amplification (mLAMP) was assessed in malaria endemic areas of Colombia with Plasmodium vivax predominance.
Methodology/Principal Findings
First, a passive case detection (PCD) study on 278 febrile patients recruited in Tierralta (department of Cordoba) was conducted to assess the diagnostic performance of the mLAMP method. Second, an active case detection (ACD) study on 980 volunteers was conducted in 10 sentinel sites with different epidemiological profiles. Whole blood samples were processed for microscopic and mLAMP diagnosis. Additionally RT-PCR and nested RT-PCR were used as reference tests. In the PCD study, P. falciparum accounted for 23.9% and P. vivax for 76.1% of the infections and no cases of mixed-infections were identified. Microscopy sensitivity for P. falciparum and P. vivax were 100% and 86.1%, respectively. mLAMP sensitivity for P. falciparum and P. vivax was 100% and 91.4%, respectively. In the ACD study, mLAMP detected 65 times more cases than microscopy. A high proportion (98.0%) of the infections detected by mLAMP was from volunteers without symptoms.
mLAMP sensitivity and specificity were comparable to RT-PCR. LAMP was significantly superior to microscopy and in P. vivax low-endemicity settings and under minimum infrastructure conditions, it displayed sensitivity and specificity similar to that of single-well RT-PCR for detection of both P. falciparum and P. vivax infections. Here, the dramatically increased detection of asymptomatic malaria infections by mLAMP demonstrates the usefulness of this new tool for diagnosis, surveillance, and screening in elimination strategies.
Author Summary
The ability to detect and treat asymptomatic infections will be fundamental to eliminate malaria. This requires highly sensitive screening tests close enough to cases to enable rapid treatment. Very low parasitemias can be detected by molecular methods such as PCR; however, these techniques require considerable training and are restricted to reference laboratories. A new field-stable diagnostic kit for malaria based on loop-mediated isothermal DNA amplification (LAMP) is now commercially available. This LAMP kit is able to detect down to 1 parasite/µl of blood in less than 1 hour. In order to evaluate the feasibility of this LAMP kit as a tool for the detection of asymptomatic malaria in malaria endemic areas of Colombia with Plasmodium vivax predominance, we conducted field studies using the LAMP kit implemented in remote settings. We found that LAMP sensitivity and specificity were comparable to RT-PCR for detection of both P. falciparum and P. vivax infections and dramatically increased detection of asymptomatic malaria infections. This simple detection method for very low parasitemia raises opportunities and new strategies for malaria elimination.
PMCID: PMC4287555  PMID: 25569550
13.  Use of a Rapid Test of Pneumococcal Colonization Density to Diagnose Pneumococcal Pneumonia 
In a prospective study, human immunodeficiency virus (HIV)–infected patients with pneumococcal pneumonia had nasopharyngeal colonization densities 5 log10 higher than those in concurrently identified HIV-infected asymptomatic controls, as measured by real-time polymerase chain reaction (rtPCR). A nasopharyngeal lytA density of ≥8000 copies/mL at rtPCR may be a useful diagnostic marker for pneumococcal pneumonia.
Background. There is major need for a more sensitive assay for the diagnosis of pneumococcal community-acquired pneumonia (CAP). We hypothesized that pneumococcal nasopharyngeal (NP) proliferation may lead to microaspiration followed by pneumonia. We therefore tested a quantitative lytA real-time polymerase chain reaction (rtPCR) on NP swab samples from patients with pneumonia and controls.
Methods. In the absence of a sensitive reference standard, a composite diagnostic standard for pneumococcal pneumonia was considered positive in South African human immunodeficiency virus (HIV)–infected adults hospitalized with radiographically confirmed CAP, if blood culture, induced good-quality sputum culture, Gram stain, or urinary Binax demonstrated pneumococci. Results of quantitative lytA rtPCR in NP swab samples were compared with quantitative colony counts in patients with CAP and 300 HIV-infected asymptomatic controls.
Results. Pneumococci were the leading pathogen identified in 76 of 280 patients with CAP (27.1%) using the composite diagnostic standard. NP colonization density measured by lytA rtPCR correlated with quantitative cultures (r = 0.67; P < .001). The mean lytA rtPCR copy number in patients with pneumococcal pneumonia was 6.0 log10 copies/mL, compared with patients with CAP outside the composite standard (2.7 log10 copies/mL; P < .001) and asymptomatic controls (0.8 log10 copies/mL; P < .001). A lytA rtPCR density ≥8000 copies/mL had a sensitivity of 82.2% and a specificity of 92.0% for distinguishing pneumococcal CAP from asymptomatic colonization. The proportion of CAP cases attributable to pneumococcus increased from 27.1% to 52.5% using that cutoff.
Conclusions. A rapid molecular assay of NP pneumococcal density performed on an easily available specimen may significantly increase pneumococcal pneumonia diagnoses in adults.
PMCID: PMC3275757  PMID: 22156852
14.  Prospective Case-Control Study of the Association between Common Enteric Protozoal Parasites and Diarrhea in Bangladesh 
The parasitic causes of diarrhea have historically been identified by use of microscopy; however, the use of this technique does not allow one to distinguish between subspecies or genotypes of parasites. Our objective was to determine, by use of modern diagnostic methods, the proportion of diarrhea cases in Bangladesh attributable to Cryptosporidium hominis, Cryptosporidium parvum, Entamoeba histolytica, and Giardia lamblia assemblages A and B.
A prospective case-control study was performed involving 3646 case patients (both children and adults) who presented with diarrhea to the Dhaka hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh, and 2575 control subjects with asymptomatic infection. Parasitic infection was detected by use of a stool parasite antigen test, and the parasite load and the species and/or genotypes were determined by use of polymerase chain reaction (PCR).
Cryptosporidium species and E. histolytica were more prevalent in patients with acute diarrhea than in healthy control subjects, for all ages (2.1% vs. 1.4%; P =.039) and, specifically, for those 0–12 months of age (2.2% vs. 0.4%; P =.009). G. lamblia assemblage A was also more prevalent in case patients with diarrhea than in healthy control subjects (20% vs. 5%; P <.001). For case patients with diarrhea, the parasite load in feces, as measured by quantitative real-time PCR cycle threshold, was not higher that that for control subjects with asymptomatic infection. Case patients with diarrhea and cryptosporidiosis were less likely to have abdominal pain, compared with control subjects (15% vs. 37%; P <.001); case patients with amebiasis more likely to have visible blood in stool, compared with control subjects (8% vs. 1.6%; P <.001); and case patients with giardiasis more likely to be dehydrated, compared with control subjects (81% vs. 71%; P =.001).
E. histolytica, C. hominis, C. parvum, and G. lamblia assemblage A infections are important causes of diarrheal illness in Bangladesh.
PMCID: PMC2883291  PMID: 19323634
15.  HIV/AIDS-Associated Opportunistic Protozoal Diarrhea 
Human immunodeficiency virus (HIV) infection has altered both the epidemiology and outcome of enteric opportunistic parasitic infections. This study was done to determine the prevalence and species/genotypes of intestinal coccidian and microsporidial infections among HIV/AIDS patients with diarrhea and/or a history of diarrhea alternately with an asymptomatic interval, and their association with CD4 T cell count. This cross-sectional study was done from May 2010 to May 2011 in Shiraz University of Medical Sciences, South of Iran. A blood sample was obtained from HIV-positive patients for a CD4 T cell count upon enrollment. Sociodemographic data and a history of diarrhea were collected by interviewing 356 consecutive participants (273 males and 83 females). Whenever possible more than a fecal sample was collected from all the participants and examined for parasites using direct, physiological saline solution ethyl acetate, an acid-fast trichrome stain, nested polymerase chain reaction, and sequencing techniques for the detection, confirmation, and genotyping of Cryptosporidium spp., Cyclospora cayetanensis, Isospora belli, and intestinal microsporidia (Enterocytozoon bieneusi). The most common opportunistic and nonopportunistic pathogens were Cryptosporidium spp. (C. parvum and C. andersoni), E. bieneusi, Giardia lamblia, Sarcocystis spp., and Blastocystis homonis affecting 34, 8, 23, 1, and 14 patients, respectively. C. cayetanensis, I. belli, Enterobius vermicularis, and Hymenolepis nana were observed in few patients. A CD4 count <200 cells/μl was significantly associated with the presence of opportunistic parasites and diarrhea (p<0.05). Opportunistic intestinal parasites should be suspected in any HIV/AIDS patient with chronic diarrhea. Tropical epidemic nonopportunistic enteric parasitic infections among such patients should not be neglected in Iran.
PMCID: PMC3537293  PMID: 22873400
16.  An outbreak of acute norovirus gastroenteritis in a boarding school in Shanghai: a retrospective cohort study 
BMC Public Health  2014;14(1):1092.
More than 200 students and teachers at a boarding school in Shanghai developed acute gastroenteritis in December, 2012. The transmission mode remained largely unknown. An immediate epidemiological investigation was conducted to identify it.
Using a retrospective cohort design, we investigated demographic characteristics, school environment, and previous contacts with people who had diarrhea and/or vomiting, drinking water conditions, recalls of food consumption in the school cafeteria, hand-washing habits and eating habits. Rectal swabs of the new cases and food handlers as well as water and food samples were collected to test potential bacteria and viruses. Norovirus was detected by real-time reverse transcription-polymerase chain reaction (RT-PCR).
A total of 278 cases developed gastrointestinal symptoms in this outbreak, and the overall attack rate was 13.9%. The main symptoms included vomiting (50.0%), abdominal cramps (40.3%), nausea (27.0%), diarrhea (6.8%) and fever (6.8%). Twenty rectal swab samples were detected as Norovirus–positive, including 11 from student cases and 9 from asymptomatic food handlers (non-cases). Among environmental surface samples from the kitchen, 8 samples were also detected as Norovirus-positive. The genotypes of viral strains were the same (GII) in patients, asymptomatic food handlers and environmental surfaces. Other samples, including rectal swabs, water samples and food samples were negative for any bacteria and other tested viruses. Asymptomatic food handlers may have contaminated the cooked food during the food preparation.
The study detected that the outbreak was caused by Norovirus and should be controlled by thorough disinfection and excluding asymptomatic food handlers from food preparation. Early identification of the predominant mode of transmission in this outbreak was necessary to prevent new cases. Furthermore, good hygiene practices such as regular hand washing and efficient daily disinfection should be promoted to prevent such infection and outbreaks.
PMCID: PMC4221699  PMID: 25335780
Norovirus; Acute gastroenteritis; Outbreak; Asymptomatic food handler
17.  Genital Shedding of Herpes Simplex Virus Among Symptomatic and Asymptomatic Persons with HSV-2 Infection 
Since HSV-2 antibody tests have become commercially available, an increasing number of persons learn that they have genital herpes through serologic testing. The course of natural history of HSV-2 in asymptomatic, seropositive persons is uncertain.
To evaluate the virologic and clinical course of HSV genital shedding among participants with symptomatic and asymptomatic HSV-2 infection.
Design, Setting and Participants
Cohort of 498 immunocompetent HSV-2 seropositive persons enrolled in prospective studies of genital HSV shedding at the University of Washington Virology Research Clinic, Seattle, Washington, and Westover Heights Clinic in Portland, Oregon, between 1992 and 2008. Each participant obtained daily self-collected swabs of genital secretions for ≥ 30 days.
Main Outcome Measurement
The rate of viral shedding measured by quantitative real-time fluorescence polymerase chain reaction (PCR) for HSV DNA from genital swabs.
HSV was detected on 4,753 of 23,683 days (20.1%; 95% CI, 18.3 to 22.0) in persons with symptomatic genital HSV-2 infection compared with 519 of 5,070 days (10.2%; 95% CI, 7.7 to 13.6) in persons with asymptomatic infection, p<0.001. Subclinical shedding rates were higher in persons with symptomatic infection compared with asymptomatic infection (2,708 of 20,735 days (13.1%; 95% CI, 11.5 to14.6) vs. 434 of 4,929 days (8.8%; 95% CI, 6.3 to 11.5), p<0.001. However, the amount of HSV detected during subclinical shedding episodes was similar (median 4.3 [IQR 3.1-5.6] log10 copies in the symptomatic infection group vs. 4.2 [IQR, 2.9-5.5], p=0.27 in the asymptomatic infection group). Days with lesions accounted for 2,045 of 4,753 days (43.0%; 95% CI, 39.8 to 46.5) with genital viral shedding among persons with symptomatic genital HSV-2 infection compared with 85 of 519 days (16.4%; 95% CI, 11.2 to 23.9) among persons with asymptomatic infection, p<0.001.
Persons with asymptomatic HSV-2 infection shed virus in the genital tract less frequently than persons with symptomatic infection, but much of the difference is attributable to less frequent genital lesions, as lesions are accompanied by frequent viral shedding.
PMCID: PMC3144252  PMID: 21486977
18.  Use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms. 
Journal of Clinical Microbiology  1993;31(1):111-117.
Rhinoviruses and enteroviruses are the major members of the picornavirus genus that cause human disease. We compared the polymerase chain reaction and viral culture for the identification of picornaviruses in nasal aspirates from children during episodes of respiratory symptoms and when asymptomatic and from asymptomatic adults. One hundred eight children, aged 9 to 11 years, completed a year-long study. Within 24 to 48 h of a report of respiratory symptoms, a nasal aspirate was taken in the home. Nasal aspirates were also taken from 65 of the children and from 33 normal adults when they had been free of respiratory symptoms for at least 2 weeks. Picornaviruses were isolated by culture for three passages in Ohio HeLa cells in rolling tubes at 33 degrees C and pH 7.0. For the polymerase chain reaction, duplicate 50-microliters samples were amplified with conserved primers from the 5' noncoding region. Picornaviruses generated approximately 380-bp bands in agarose gel electrophoresis; the specificity of these bands was confirmed by filter hybridization with a conserved internal probe. Picornaviruses were isolated by culture in 47 (46 rhinoviruses) of 292 symptomatic episodes (16%), whereas the polymerase chain reaction identified picornavirus genomic material in 146 episodes (50%), including all but one of the culture-positive episodes. As for asymptomatic samples, eight (12%) children and two (4%) adults were positive by the polymerase chain reaction, whereas only one child's specimen was positive by culture. This polymerase chain reaction assay represents a clear advance in the identification of picornavirus infection, with a detection rate threefold greater than the virus culture method.
PMCID: PMC262631  PMID: 8380179
19.  Direct detection of Chlamydia trachomatis in urine specimens from symptomatic and asymptomatic men by using a rapid polymerase chain reaction assay. 
Journal of Clinical Microbiology  1993;31(5):1209-1212.
Screening for Chlamydia trachomatis infection in men has traditionally been limited to men who present with urethral symptoms, thereby limiting the detection of asymptomatic chlamydia infection in men. In order to effectively screen both symptomatic and asymptomatic men, we evaluated a newly developed polymerase chain reaction (PCR) assay, Amplicor C. trachomatis, from Roche Molecular Systems for the detection of C. trachomatis in urine specimens in comparison with urethral culture. A total of 530 male urine specimens were collected from 322 symptomatic and 208 asymptomatic men attending two sexually transmitted disease clinics in Baltimore, Md. The prevalence of C. trachomatis by culture was 9.8% (10.6% in symptomatic men and 8.2% in asymptomatic men). Compared with culture, the sensitivity of the PCR was 92.8%, the specificity was 94.7%, the positive predictive value was 68.4%, and the negative predictive value was 99.1%. Discrepant results between culture and PCR were further analyzed by direct fluorescent-antibody staining of elementary bodies in urine sediment and in culture transport vials and by major outer membrane protein PCR of transport media for specimens with negative culture. The revised sensitivity and specificity of PCR for urine were 95.0 and 99.8%, respectively, and the positive and negative predictive values were 98.7 and 99.1%, respectively. The sensitivity of culture compared with PCR and/or direct fluorescent-antibody staining was 68.4%. These results indicate that the PCR assay is a highly sensitive and specific assay for the detection of C. trachomatis in male urine specimens and provides a noninvasive technique for routine screening of chlamydia infection in both symptomatic and asymptomatic men.
PMCID: PMC262905  PMID: 8501220
20.  Detection of Trichomonas vaginalis Using the Polymerase Chain Reaction in Pregnant and Non-Pregnant Women 
Objective: Trichomonas vaginalis vaginal infections are often both asymptomatic and difficult to detect by current methods. We evaluated the ability of a newly developed polymerase chain reaction (PCR) assay to identify T. vaginalis in vaginal samples from pregnant and non-pregnant women.
Methods: In the 1st study, we compared the prevalence of T. vaginalis detection by PCR and culture using Diamond's medium in 52 women with symptoms of vaginal infection. In the 2nd study, T. vaginalis was detected using PCR and wet mount microscopy in 131 asymptomatic pregnant women.
Results: Among the women with symptoms of vaginitis, 7 (13.5%) were PCR-positive for T. vaginalis. Six of the PCR-positive women, but none of the PCR-negative women, were culture-positive for this organism. All but 1 of the women with candidal vaginitis or bacterial vaginosis were PCR-negative for T. vaginalis. Among the asymptomatic pregnant women, all of whom were negative for T. vaginalis by wet mount, l0 (7.6%) were PCR-positive for T. vaginalis.
Conclusions: PCR offers a rapid and sensitive alternative to culture and microscopy for the detection of T. vaginalis vaginal infections in both symptomatic and asymptomatic women.
PMCID: PMC2364357  PMID: 18475360
21.  Multiple Norovirus Infections in a Birth Cohort in a Peruvian Periurban Community 
Serial norovirus infections with multiple genotypes were found among a Peruvian birth cohort early in infancy. Protection against the subsequent infection was genotype specific, suggesting that norovirus vaccines may need to target multiple genotypes.
Background. Human noroviruses are among the most common enteropathogens globally, and are a leading cause of infant diarrhea in developing countries. However, data measuring the impact of norovirus at the community level are sparse.
Methods. We followed a birth cohort of children to estimate norovirus infection and diarrhea incidence in a Peruvian community. Stool samples from diarrheal episodes and randomly selected nondiarrheal samples were tested by polymerase chain reaction for norovirus genogroup and genotype. Excretion duration and rotavirus coinfection were evaluated in a subset of episodes.
Results. Two hundred twenty and 189 children were followed to 1 and 2 years of age, respectively. By 1 year, 80% (95% confidence interval [CI], 75%–85%) experienced at least 1 norovirus infection and by 2 years, 71% (95% CI, 65%–77%) had at least 1 episode of norovirus-associated diarrhea. Genogroup II (GII) infections were 3 times more frequent than genogroup 1 (GI) infections. Eighteen genotypes were found; GII genotype 4 accounted for 41%. Median excretion duration was 34.5 days for GII vs 8.5 days for GI infection (P = .0006). Repeat infections by the same genogroup were common, but repeat infections by the same genotype were rare. Mean length-for-age z score at 12 months was lower among children with prior norovirus infection compared to uninfected children (coefficient: −0.33 [95% CI, −.65 to −.01]; P = .04); the effect persisted at 24 months.
Conclusions. Norovirus infection occurs early in life and children experience serial infections with multiple genotypes, suggesting genotype-specific immunity. An effective vaccine would have a substantial impact on morbidity, but may need to target multiple genotypes.
PMCID: PMC3905757  PMID: 24300042
norovirus; infant diarrhea; gastroenteritis; birth cohort; natural infection
22.  A large proportion of asymptomatic Plasmodium infections with low and sub-microscopic parasite densities in the low transmission setting of Temotu Province, Solomon Islands: challenges for malaria diagnostics in an elimination setting 
Malaria Journal  2010;9:254.
Many countries are scaling up malaria interventions towards elimination. This transition changes demands on malaria diagnostics from diagnosing ill patients to detecting parasites in all carriers including asymptomatic infections and infections with low parasite densities. Detection methods suitable to local malaria epidemiology must be selected prior to transitioning a malaria control programme to elimination. A baseline malaria survey conducted in Temotu Province, Solomon Islands in late 2008, as the first step in a provincial malaria elimination programme, provided malaria epidemiology data and an opportunity to assess how well different diagnostic methods performed in this setting.
During the survey, 9,491 blood samples were collected and examined by microscopy for Plasmodium species and density, with a subset also examined by polymerase chain reaction (PCR) and rapid diagnostic tests (RDTs). The performances of these diagnostic methods were compared.
A total of 256 samples were positive by microscopy, giving a point prevalence of 2.7%. The species distribution was 17.5% Plasmodium falciparum and 82.4% Plasmodium vivax. In this low transmission setting, only 17.8% of the P. falciparum and 2.9% of P. vivax infected subjects were febrile (≥38°C) at the time of the survey. A significant proportion of infections detected by microscopy, 40% and 65.6% for P. falciparum and P. vivax respectively, had parasite density below 100/μL. There was an age correlation for the proportion of parasite density below 100/μL for P. vivax infections, but not for P. falciparum infections. PCR detected substantially more infections than microscopy (point prevalence of 8.71%), indicating a large number of subjects had sub-microscopic parasitemia. The concordance between PCR and microscopy in detecting single species was greater for P. vivax (135/162) compared to P. falciparum (36/118). The malaria RDT detected the 12 microscopy and PCR positive P. falciparum, but failed to detect 12/13 microscopy and PCR positive P. vivax infections.
Asymptomatic malaria infections and infections with low and sub-microscopic parasite densities are highly prevalent in Temotu province where malaria transmission is low. This presents a challenge for elimination since the large proportion of the parasite reservoir will not be detected by standard active and passive case detection. Therefore effective mass screening and treatment campaigns will most likely need more sensitive assays such as a field deployable molecular based assay.
PMCID: PMC2944341  PMID: 20822506
23.  Rotavirus Shedding in Symptomatic and Asymptomatic Children using Reverse Transcription-Quantitative PCR 
Journal of medical virology  2013;85(9):1661-1668.
Reverse transcription-real time polymerase chain reaction (RT-qPCR) for the VP6 gene was used to study group A rotavirus shedding in children with symptomatic and asymptomatic rotavirus infection. Sequential stool samples (n=345) from ten children with rotavirus associated diarrhea and from five children (n=161) with asymptomatic rotavirus infection were collected over a period of two months. A RT-qPCR assay on the samples using a rotavirus VP6 plasmid standard demonstrated high reproducibility, with an inter-assay coefficient of variation (CV) of 1.40–2.97% and an intra-assay CV of 0.03–3.03%. The median duration of shedding was longer in children with diarrhea compared to asymptomatic children (24 days vs. 18 days) (p=0.066). The median quantitation cycle (Cq) at presentation in symptomatic children was 17.21 compared to 30.98 in asymptomatic children (p=0.086). The temporal pattern in symptomatic children consisted of a high initial viral shedding coinciding with the duration of diarrhea, followed by a rapid fall, and then a small increase in secondary shedding 21 days later. Compared to children with rotavirus diarrhea, those with asymptomatic infection shed lower quantities of virus throughout the observation period. No difference was noted between the G and P genotypes of samples collected at onset of infection and during the shedding period. Shedding was intermittent in a subset of children in both groups. RT-qPCR is a useful method to characterize shedding patterns.
PMCID: PMC3758921  PMID: 23775335
rotavirus; RT-qPCR; virus shedding; children
24.  Evaluation of real-time PCR assay to detect Schistosoma mansoni infections in a low endemic setting 
BMC Infectious Diseases  2014;14(1):558.
Schistosomiasis constitutes a major public health problem, and 200 million people are estimated to be infected with schistosomiasis worldwide. In Brazil, schistosomiasis has been reported in 19 states, showing areas of high and medium endemicity and a wide range of areas of low endemicity (ALE). Barra Mansa in Rio de Janeiro state has an estimated prevalence of 1%. ALE represent a new challenge for the helminth control because about 75% of infected individuals are asymptomatic and infections occur with a low parasite load (<100 eggs per gram of feces), causing a decrease in sensitivity of stool parasitological techniques, which are a reference for the laboratory diagnosis of this helminth. The objective of this study was to evaluate the performance of a TaqMan quantitative polymerase chain reaction (qPCR) technique in serum and feces DNA samples using the techniques of Kato-Katz (KK), Hoffman, Pons and Janer (HH) as references, during an epidemiological survey using fecal samples and sera from randomized residents from an ALE.
A cross-sectional study conducted from April to December 2011 using a probabilistic sampling that collected 572 fecal and serum samples. The laboratory diagnostic techniques used were: KK, HH and qPCR (feces and serum).
We obtained the following results using the different diagnostic techniques: KK and HH, 0.9% (n =5); qPCR-feces, 9.6% (n =55); and qPCR-serum, 1.4% (n =8). The qPCR-feces presented the highest positivity, whereas the techniques of HH and KK were the least sensitive to detect infections (0.8%). Compared to HH and KK, qPCR-feces showed a statistically significant difference in positivity (p <0.05), although with poor agreement.
The positivity rate presented by the qPCR approach was far higher than that obtained by parasitological techniques. The lack of adequate surveillance in ALE of schistosomiasis indicates a high possibility of these areas being actually of medium and high endemicity. This study presents a control perspective, pointing to the possibility of using combined laboratory tools in the diagnosis of schistosomiasis in ALE.
Electronic supplementary material
The online version of this article (doi:10.1186/s12879-014-0558-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4210485  PMID: 25338651
Schistosomiasis mansoni; TaqMan® Real-Time PCR system; Laboratory diagnosis; Low endemicity areas; Brazil/epidemiology
25.  Protective Effect of Natural Rotavirus Infection in an Indian Birth Cohort 
The New England journal of medicine  2011;365(4):337-346.
More than 500,000 deaths are attributed to rotavirus gastroenteritis annually worldwide, with the highest mortality in India. Two successive, naturally occurring rotavirus infections have been shown to confer complete protection against moderate or severe gastroenteritis during subsequent infections in a birth cohort in Mexico. We studied the protective effect of rotavirus infection on subsequent infection and disease in a birth cohort in India (where the efficacy of oral vaccines in general has been lower than expected).
We recruited children at birth in urban slums in Vellore; they were followed for 3 years after birth, with home visits twice weekly. Stool samples were collected every 2 weeks, as well as on alternate days during diarrheal episodes, and were tested by means of enzyme-linked immunosorbent assay and polymerase-chain-reaction assay. Serum samples were obtained every 6 months and evaluated for seroconversion, defined as an increase in the IgG antibody level by a factor of 4 or in the IgA antibody level by a factor of 3.
Of 452 recruited children, 373 completed 3 years of follow-up. Rotavirus infection generally occurred early in life, with 56% of children infected by 6 months of age. Levels of reinfection were high, with only approximately 30% of all infections identified being primary. Protection against moderate or severe disease increased with the order of infection but was only 79% after three infections. With G1P[8], the most common viral strain, there was no evidence of homotypic protection.
Early infection and frequent reinfection in a locale with high viral diversity resulted in lower protection than has been reported elsewhere, providing a possible explanation why rotavirus vaccines have had lower-than-expected efficacy in Asia and Africa. (Funded by the Wellcome Trust.)
PMCID: PMC3596855  PMID: 21793745

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