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1.  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
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.  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
4.  Development and application of a SYBR green RT-PCR for first line screening and quantification of porcine sapovirus infection 
Sapoviruses are single stranded positive sense RNA viruses belonging to the family Caliciviridae. The virus is detected in different species including the human and the porcine species as an enteric pathogen causing asymptomatic to symptomatic enteritis. In this study, we report the development of a rapid real time qRT-PCR based on SYBR Green chemistry for the diagnosis of porcine sapovirus infection in swine.
The method allows the detection of porcine sapoviruses and the quantification of the genomic copies present in stool samples. During its development, the diagnostic tool showed good correlation compared with the gold standard conventional RT-PCR and was ten-fold more sensitive. When the method was applied to field samples, porcine noroviruses from genogroup 2 genotype 11b were also detected. The method was also applied to swine samples from the Netherlands that were positive for PoSaV infection. Phylogenetic results obtained from the samples showed that PoSaV sequences were genetically related to the currently described genogroup III, to the proposed genogroup VII and also to the MI-QW19 sequence (close to the human SaV sequences).
A rapid, sensitive, and reliable diagnosis method was developed for porcine sapovirus diagnosis. It correlated with the gold standard conventional RT-PCR. Specificity was good apart for genogroup 2 genotype 11b porcine noroviruses. As a first line screening diagnosis method, it allows a quicker and easier decision on doubtful samples.
PMCID: PMC3528410  PMID: 23072668
Sapovirus; Diagnosis; Real time PCR; Porcine; SYBR green; Phylogeny
5.  Comparison of bacterial enriched-broth culture, enzyme linked immunosorbent assay, and broth culture-polymerase chain reaction techniques for identifying asymptomatic infections with Salmonella in swine 
A polymerase chain reaction (PCR) assay was combined with a broth-culture enrichment system to detect Salmonella shed in feces from subclinically infected swine. The effectiveness of the broth culture-polymerase chain reaction (BC-PCR) assay to identify pigs shedding Salmonella in feces was compared with a microbiological culture and a commercial enzyme linked immunosorbent assay (ELISA) kit to detect Salmonella-specific serum antibody. A total of 67 pigs were tested by each of the 3 methodologies. Forty-one pigs tested positive for Salmonella by BC-PCR and ELISA identified 6 positives and 23 suspicious samples. It was shown that the BC-PCR assay is a rapid diagnostic tool for detecting of Salmonella shed by asymptomatic swine compared with current diagnostic technologies.
PMCID: PMC227056  PMID: 12889729
6.  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
7.  Genogroup IIb Norovirus Infections and Association with Enteric Symptoms in a Neonatal Nursery in Southern India▿  
Journal of Clinical Microbiology  2010;48(9):3212-3215.
Noroviruses (NoVs) are increasingly recognized as an important cause of acute gastroenteritis in children worldwide. However, there are limited data on the role of NoVs in neonatal infections and disease. The objectives of the present study were to determine the prevalence of NoVs in neonates with gastrointestinal disease using a case-control study design and to characterize the NoV strains infecting neonates. A total of 309 fecal samples from 161 neonates with gastrointestinal symptoms and 148 asymptomatic controls were screened for genogroup II (GII) NoV using reverse transcription-PCR. A subset of PCR-positive amplicons for the polymerase and capsid regions was sequenced. NoV was detected in 26.2% of samples, with the rate of detection being significantly higher among symptomatic neonates (60/161, 37.2%) than asymptomatic neonates (24/148, 14.1%) (P < 0.001). On the basis of sequencing of 29 strains, a single NoV strain, GIIb, was identified to be the predominant (27/29, 93.1%) cause of neonatal infections. Coinfection with rotavirus was seen in nearly one-third of symptomatic neonates. The study demonstrates a high prevalence of NoV infections in neonates and indicates that coinfection with rotavirus may result in significantly more gastrointestinal disease in this population.
PMCID: PMC2937701  PMID: 20631107
8.  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
9.  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
10.  Detection and Molecular Characterization of Porcine Picobirnavirus in Feces of Domestic Pigs from Kolkata, India 
Picobirnaviruses (PBVs) are small, non-enveloped, 35–41 nm virion with bisegmented double-stranded RNA genome. PBVs are widespread and were detected in feces of humans and a wide variety of animals. Domestic pig, one of the ubiquitous farm animal reported incessant association with a variety of viral zoonoses. The objective of our study is to find out the incidence of PBV infection in healthy domestic pigs. The study was conducted by collecting feces of healthy/asymptomatic pigs from a piggery located in an urban slum at Kolkata, India to detect PBV infections. All the 11 fecal samples were tested by polyacrylamide gel electrophoresis and reverse transcription–polymerase chain reaction assay. In this study, we report the first incidence of detection and molecular characterization of porcine PBV (BG-Por-2/2010 and BG-Por-7/2010) in feces of domestic pigs from India using the human PBV genogroup I specific primer pair: PicoB25(+) and PicoB43(−). Sequence comparison and phylogenetic analysis of partial RNA-dependent RNA polymerase gene of genome segment 2 revealed genetic relatedness to hitherto reported porcine, murine and human genogroup I PBVs from different geographical regions. This warrants a stringent global surveillance to study the potential zoonotic and emerging PBV infections.
Electronic supplementary material
The online version of this article (doi:10.1007/s13337-012-0106-z) contains supplementary material, which is available to authorized users.
PMCID: PMC3550800  PMID: 24293831
Picobirnaviruses (PBVs); Genogroup I PBVs; Domestic pigs; Genomic diversity; Zoonoses
11.  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
12.  Searching for Helicobacter pylori and Chlamydia pneumoniae in primary endodontic infections 
European Journal of Dentistry  2012;6(2):158-162.
The purpose of this study was to search samples from primary endodontic infections for the presence of two common human bacterial pathogens - Helicobacter pylori and Chlamydia pneumoniae.
Genomic DNA isolated from samples taken from 25 root canals of teeth with asymptomatic (chronic) apical periodontitis and 25 aspirates from acute apical abscess was initially amplified by the multiple displacement amplification approach and then used as template in species-specific polymerase chain reaction (PCR) for detection of H. pylori and C. pneumoniae.
All clinical samples were positive for the presence of bacterial DNA. However, no clinical sample was positive for either H. pylori or C. pneumoniae.
Neither H. pylori nor C. pneumoniae were found in samples from primary endodontic infections. These findings suggest that these species are not candidate endodontic pathogens and that the necrotic root canal does not serve as a reservoir for these human pathogens in healthy patients.
PMCID: PMC3327501  PMID: 22509118
Helicobacter pylori; Chlamydia pneumoniae; apical periodontitis; acute apical abscess; polymerase chain reaction
13.  Enteric Viruses in Raw Vegetables and Groundwater Used for Irrigation in South Korea▿  
Applied and Environmental Microbiology  2009;75(24):7745-7751.
Raw vegetables irrigated with groundwater that may contain enteric viruses can be associated with food-borne viral disease outbreaks. In this study, we performed reverse transcription-PCR (RT-PCR) and cell culture-PCR to monitor the occurrence of enteric viruses in groundwater samples and in raw vegetables that were cultivated using that groundwater in South Korea. Samples were collected 10 times from three farms located in Gyeonggi Province, South Korea. RT-PCR and cell culture-PCR were performed to detect adenoviruses (AdVs), enteroviruses (EVs), noroviruses (NoVs), and rotaviruses, followed by sequence analyses of the detected strains. Of the 29 groundwater samples and the 30 vegetable samples, five (17%) and three (10%) were positive for enteric viruses, respectively. AdVs were the most frequently detected viruses in four groundwater and three vegetable samples. EVs and NoVs were detected in only one groundwater sample and one spinach sample, respectively. The occurrence of enteric viruses in groundwater and vegetable samples was not correlated with the water temperature and the levels of indicator bacteria, respectively. Phylogenetic analysis indicated that most of the detected AdVs were temporally distributed, irrespective of sample type. Our results indicate that raw vegetables may be contaminated with a broad range of enteric viruses, which may originate from virus-infected farmers and virus-contaminated irrigation water, and these vegetables may act as a potential vector of food-borne viral transmission.
PMCID: PMC2794108  PMID: 19854919
14.  Asymptomatic carriage of Haemophilus ducreyi confirmed by the polymerase chain reaction. 
Genitourinary Medicine  1995;71(4):224-227.
OBJECTIVE--To investigate asymptomatic carriage of Haemophilus ducreyi by using polymerase chain reaction (PCR) on samples from women at high risk of infection. SUBJECTS--213 commercial sex workers (CSWs) recruited in The Gambia, West Africa. METHODS--Genital samples (cervical, vaginal and ulcer) were tested for the presence of H ducreyi by PCR with the technique of "one tube nested primer". RESULTS--12 CSWs were PCR positive for H ducreyi; 8 of these women had genital ulcers on examination. CONCLUSION--Using a simplified PCR technique for detecting H ducreyi we have shown that 2% of CSWs were carrying the organism without clinical symptoms or signs. This has important implications for sexually transmitted disease control programmes in areas with a high prevalence of chancroid.
PMCID: PMC1195517  PMID: 7590712
15.  Polymerase chain reaction assay of parvovirus B19 DNA in clinical specimens. 
Journal of Clinical Microbiology  1989;27(12):2647-2651.
The polymerase chain reaction (PCR) was used to detect parvovirus B19 DNA in a panel of sera from individuals recently infected with B19, as demonstrated by the presence of anti-B19 immunoglobulin M. Of 95 serum samples, 60 (63%) were found positive by PCR, whereas only 1 was also found positive by dot hybridization. In a control panel of 100 serum samples from individuals with other infections, only 1 serum sample was found positive by PCR, and this was also found positive by dot hybridization. This was probably just a fortuitous discovery of viremia. Placental tissues from women (n = 89) who had proven B19 infections in pregnancy but who gave birth to healthy infants at term were also tested. A total of 74 (83%) were found positive for B19 DNA by PCR. The high rate of detection by PCR probably represents "decay" of viral DNA after the peak of viremia and is not a clinically significant phenomenon.
PMCID: PMC267101  PMID: 2556428
16.  Isolation and Typing of Canine Parvovirus in CRFK Cell Line in Puducherry, South India 
Indian Journal of Microbiology  2011;51(4):456-460.
A total of 128 faecal samples/rectal swabs were collected from dogs showing signs of diarrhea/enteritis in and around Puducherry, South India. Eighteen clinical samples, showing high HA titre of 1:512 and above and positivity by polymerase chain reaction (PCR) with CPV-2ab primers, were subjected to virus isolation in CRFK cell line. Of the 18 samples processed, 3 samples (16.6%) were positive for CPV and were confirmed by haemagglutination, dot-ELISA, and IFAT. The three cell culture isolates were characterized as CPV-2b types by multiplex PCR as well as by monoclonal antibody typing.
PMCID: PMC3209934  PMID: 23024407
Cell culture; CPV; CRFK; HA; Dot-ELISA; PCR; IFAT; Monoclonal typing
17.  Post-Arrival Screening for Malaria in Asymptomatic Refugees Using Real-Time PCR 
Malaria is a significant health risk to refugee populations originating from endemic areas, but there is little consensus on screening and/or treatment approaches for malaria in this population. Furthermore, detection of malaria in semi-immune asymptomatic refugees is limited by the sensitivity of diagnostic tests used for screening. We determined the prevalence of malaria by microscopy and real-time polymerase chain reaction (PCR) in a consecutive population of 324 asymptomatic refugees examined in Edmonton, Canada, during 2009–2010. Although all thick and thin blood smear results were negative, 10 subjects (3.1%) tested PCR positive for Plasmodium DNA. Interestingly, 6 of 10 PCR positive subjects are at risk of malaria relapse by P. vivax or P. ovale infections. These results suggest that appropriate guidelines for malaria screening should consider the risk of relapsing infections, and they highlight the potential usefulness of real-time PCR in the diagnosis of asymptomatic malaria.
PMCID: PMC3005492  PMID: 21212221
18.  Nasal swab samples and real-time polymerase chain reaction assays in community-based, longitudinal studies of respiratory viruses: the importance of sample integrity and quality control 
Carefully conducted, community-based, longitudinal studies are required to gain further understanding of the nature and timing of respiratory viruses causing infections in the population. However, such studies pose unique challenges for field specimen collection, including as we have observed the appearance of mould in some nasal swab specimens. We therefore investigated the impact of sample collection quality and the presence of visible mould in samples upon respiratory virus detection by real-time polymerase chain reaction (PCR) assays.
Anterior nasal swab samples were collected from infants participating in an ongoing community-based, longitudinal, dynamic birth cohort study. The samples were first collected from each infant shortly after birth and weekly thereafter. They were then mailed to the laboratory where they were catalogued, stored at -80°C and later screened by PCR for 17 respiratory viruses. The quality of specimen collection was assessed by screening for human deoxyribonucleic acid (DNA) using endogenous retrovirus 3 (ERV3). The impact of ERV3 load upon respiratory virus detection and the impact of visible mould observed in a subset of swabs reaching the laboratory upon both ERV3 loads and respiratory virus detection was determined.
In total, 4933 nasal swabs were received in the laboratory. ERV3 load in nasal swabs was associated with respiratory virus detection. Reduced respiratory virus detection (odds ratio 0.35; 95% confidence interval 0.27-0.44) was observed in samples where the ERV3 could not be identified. Mould was associated with increased time of samples reaching the laboratory and reduced ERV3 loads and respiratory virus detection.
Suboptimal sample collection and high levels of visible mould can impact negatively upon sample quality. Quality control measures, including monitoring human DNA loads using ERV3 as a marker for epithelial cell components in samples should be undertaken to optimize the validity of real-time PCR results for respiratory virus investigations in community-based studies.
PMCID: PMC3897990  PMID: 24405747
Nasal swab; Respiratory virus; Real-time polymerase chain reaction; Quality control; Mould; Community-based study
19.  Usefulness of Polymerase Chain Reaction to Supplement Field Microscopy in a Pre-Selected Population with a High Probability of Malaria Infections 
This study determines the use of nested PCR as a diagnostic tool to supplement field microscopy in symptomatic individuals suspected of being positive for malaria, and it explores its role in active case detection to identify asymptomatic parasite carriers. In symptomatic individuals, compared with PCR, microscopy had a sensitivity of 86.6% (95% confidence interval [CI] = 77.8–92.4) and specificity of 100% (95% CI = 96.9–100). During active case detection, two asymptomatic persons were diagnosed as having vivax malaria by polymerase chain reaction (PCR) but not microscopy. Currently, PCR is being carried out in Sri Lanka only for population surveys to estimate the hidden reservoir of malaria. Based on the results of this study and because of cost considerations, pooled PCR will be used in the future to screen samples from clinically suspected foci to increase the proportion of malaria cases detected. This strategy will assist the success of the malaria elimination program in Sri Lanka.
PMCID: PMC3122336  PMID: 21734117
20.  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
21.  Genital mycoplasma & Chlamydia trachomatis infections in treatment naïve HIV-1 infected adults 
Background & objectives:
Sexually transmitted infections (STIs) enhance the transmission of human immunodeficiency virus (HIV). Thus, screening for STIs is a routine component of primary HIV care. There are limited data for selective screening guidelines for genital mycoplasmas and Chlamydia trachomatis in HIV-infected adults. The aim of the present study was to determine the frequency of genital infections with Ureaplasma spp., Mycoplasma hominis, M. genitalium and C. trachomatis in treatment naïve asymptomatic HIV-1 - infected adults and study their association with CD4+ T-cell count.
First-void urine samples were collected from 100 treatment-naïve HIV-1-infected adults and 50 healthy volunteers. C. trachomatis and M. genitalium were detected by polymerase chain reaction (PCR). Ureaplasma spp. and M. hominis were detected by both culture and PCR. Circulating CD4+ cell counts of HIV-1-infected patients were determined from peripheral blood by flow-cytometry.
C. trachomatis was detected in 7 per cent of HIV-1-infected adults compared to none in control population. Ureaplasma spp. and M. hominis showed infection rates of 6 and 1 per cent in the HIV group and 2 and 0 per cent in the control group, respectively. None of the individuals from the patient and control groups was tested positive for M. genitalium. A significant association was found between CD4 cell count and detection of C. trachomatis in HIV-infected adults (P = 0.01).
Interpretation & conclusions:
Screening of HIV-infected individuals for C. trachomatis infection could be recommended as a routine component of HIV care. The role of mycoplasmas as co-pathogens of the genitourinary tract in HIV-1 infected patients seems to be unlikely. Further longitudinal studies need to be done to confirm these findings.
PMCID: PMC3284105  PMID: 22310829
CD4 cell counts; Chlamydia trachomatis; genital mycoplasmas; HIV
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.  Human TRIM5α Expression Levels and Reduced Susceptibility to HIV-1 Infection 
The Journal of infectious diseases  2009;199(11):1657-1663.
Human TRIM5α (TRIM5αhu), a member of the tripartite motif protein family, displays some anti–human immunodeficiency virus type 1 (HIV-1) activity in vitro, although it is substantially less potent than its rhesus monkey counterpart (TRIM5αrh). The effects of levels of TRIM5αhu on prevention or control of HIV-1 infection in vivo are unknown.
We used a quantitative real-time polymerase chain reaction (PCR) assay to measure levels of TRIM5αhu expression in peripheral blood mononuclear cells (PBMCs) obtained from a cohort of individuals at high risk for HIV-1 infection in Durban, South Africa. Samples were available from 38 infected subjects (with all these samples obtained within 1 year of infection) and from 57 uninfected persons. Matched preinfection and postinfection samples were available from 13 individuals.
TRIM5αhu messenger RNA levels were lower in the PBMCs of HIV-1–infected subjects than in those of uninfected subjects (P < .001). Seroconverters had lower preinfection levels of TRIM5αhu than did nonseroconverters (P < .001). TRIM5αhu levels did not change significantly after infection. There was no correlation between TRIM5αhu levels and viral loads or CD4+ T cell counts.
High expression of TRIM5αhu is associated with reduced susceptibility to HIV-1 infection. Furthermore, infection is not associated with disregulation of TRIM5αhu. TRIM5αhu expression levels do not contribute to the control of primary HIV-1 viremia.
PMCID: PMC2725358  PMID: 19388851
24.  Clostridium difficile is not associated with outbreaks of viral gastroenteritis in the elderly in the Netherlands 
The coincidental increase in norovirus outbreaks and Clostridium difficile infection (CDI) raised the question of whether these events could be related, e.g. by enhancing spread by diarrhoeal disease outbreaks. Therefore, we studied the prevalence of C. difficile in outbreaks of viral gastroenteritis in nursing homes for the elderly and characterised enzyme immunoassay (EIA)-positive stool samples. Stool samples from nursing home residents (n = 752) in 137 outbreaks of viral aetiology were investigated by EIA for the presence of C. difficile toxins. Positive samples were further tested by a cell neutralisation cytotoxicity test, a second EIA and culture. Cultured isolates were tested for the presence of toxin genes, the production of toxins and characterised by 16S rRNA polymerase chain reaction (PCR) and sequencing. Twenty-four samples (3.2%) tested positive in the EIA. Of these 24 positive samples, only two were positive by cytotoxicity and three by a second EIA. Bacterial culture of 21 available stool samples yielded a toxinogenic C. difficile PCR ribotype 001 in one patient sample only. In conclusion, we found no evidence in this retrospective study for an association between viral gastroenteritis outbreaks and C. difficile. The high rate of false-positive EIA samples emphasises the need for second confirmation tests to diagnose CDI.
PMCID: PMC2871102  PMID: 20339889
25.  Increased number of circulating HTLV-1 infected cells in peripheral blood mononuclear cells of HTLV-1 uveitis patients: a quantitative polymerase chain reaction study. 
AIMS--This study aimed to characterise the status of viral infection in patients with HTLV-1 uveitis (HU) by quantifying the circulating HTLV-1 infected cells in the peripheral blood. METHODS--Genomic DNA samples of peripheral blood mononuclear cells (PBMC) were obtained from 25 patients with HU, 14 patients with tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM), and 21 asymptomatic carriers of HTLV-1. Quantitative polymerase chain reaction (PCR) of the gag region of HTLV-1 provirus DNA was performed on these DNA samples. To confirm the PCR, genomic Southern blot hybridisation was performed to identify integrated HTLV-1 provirus. This procedure detected a few percent of HTLV-1 infected cells in the PBMC. RESULTS--Most of the HU patients had a significantly increased number of circulating HTLV-1 infected cells (mean (SD) 3.84% (4.45%) of the PBMC), whereas the percentage of infected cells in most asymptomatic carriers was less than 1% (0.54% (1.11%)). Most of the TSP/HAM patients also had a relatively high percentage (11.63% (7.67%)). The differences among these three groups were highly significant by the Mann-Whitney U test. CONCLUSION--The results suggested that the increase in the number of HTLV-1 infected cells is one base for the development of inflammatory HU lesions, as it is for TSP/HAM.
PMCID: PMC505078  PMID: 7703209

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