ICR SS protozoan data site-by-site: a picture of Cryptosporidium and Giardia in U.S. surface water.

The U.S. Environmental Protection Agency (USEPA) Information Collection Rule Supplemental Survey (ICR SS) required analysis of Cryptosporidium and Giardia in 10 L surface water samples twice a week for a year by USEPA Method 1623 at 80 representative U.S. public water systems (PWS). The resulting data are examined site-by-site in relation to objectives of the Federal drinking water regulation, The Long-Term (2) Enhanced Surface Water Treatment Rule (LT2), currently under formal 6-year review by the USEPA. The data describe Cryptosporidium and Giardia in watersheds nation-wide over a single annual cycle. Due to limited recovery efficiency measurement results are not fully quantified. In the required sample volumes of 10 L no Cryptosporidium were found in 86% of samples and no Giardia were found in 67% of samples. Yet, organisms were found in enough samples at 34 of 80 sites to detail a specrtum of occurrence and variability for both organisms. The data are shown to describe indivudual site risk essential for guidance of watershed and water treatment management by PWSs. The span of median occurrence for both organisms was about 2 orders of magnitude above the limit of detection (LD), ca. 0.05 raw no's/L for Cryptosporidium and ca. 0.10 raw no's/L for Giardia. Data analysis illustrates key features of Cryptosporidium and Giardia in surface water: presence is continuous not intermittent; zeros indicate presence below the LD; occurrence level and variations depend on watershed sources; risk depends on both magnitude and variability of concentration; accurate estimation of risk requires routine measurement of recovery efficiency and calculation of concentration. The data and analysis illustrate features of Cryptosporidium and Giardia occurrence in surface water relevant to their effective regulation for public health protection.

LT2 Cryptosporidium data: what do they tell us about Cryptosporidium in surface water in the United States?

Beginning in 2006 a United States Federal regulation required public water suppliers using surface water serving more than 10,000 population to analyze for Cryptosporidium in at least 24 consecutive monthly samples from each surface water source. In July 2012, the U.S. EPA released the resulting data consisting of ca. 45,000 records. No Cryptosporidium were found in 93% of samples and no Cryptosporidium were found in any samples analyzed from over half of 1670 locations sampled. Nevertheless, at 250 locations representing every region of the U.S., Cryptosporidium were found in sufficient numbers of samples to provide a picture of their occurrence nationwide. Data from about 100 sites reporting the highest numbers were examined in detail. Although analysis of matrix spikes was required for quality control, the results do not permit estimating organism concentrations. The data reported at each of the individual sample locations were analyzed in the form of cumulative probability distributions to describe key risk-related features of median level and variability. Taken as a whole, the data describe a spectrum of median Cryptosporidium occurrence in surface waters of the U.S. ranging from ca. 0.005 to ca. 0.5 oocysts/L. The variability at individual sites ranged from ca. 1 to 15 r.s.d. Based on the LT2 positive data, comparison to measurements of other water quality parameters, and independent means of estimating organism production from watersheds reported in the literature, the hypothesis is offered that Cryptosporidium may be found in surface water anywhere worldwide continuously and within the spectrum defined above.

Distribution of Cryptosporidium oocysts and Giardia cysts in water above and below the normal limit of detection.

Analysis of water samples for Cryptosporidium oocysts and Giardia cysts is a specialised and demanding pursuit. Understanding and evaluating data resulting from such analyses is equally specialised and complicated by the most common result--not finding any of the target organisms. Coming to an accurate conclusion regarding such monitoring results has been hampered by a lack of pertinent information presented in the context of current monitoring requirements. The work reported here presents laboratory data demonstrating an appropriate skewed distribution model statistical framework. It is shown that the Poisson model provides for understanding how Cryptosporidium oocysts and Giardia cysts are distributed in water at typical ambient concentrations that are near or most commonly below the limit of detection of the most widely used analytical procedure, USEPA Method 1623. From three to six replicate 50-L volumes of particle-free water were seeded with Cryptosporidium oocysts and Giardia cysts each at concentrations of ca. 0.2/L, 1-2/L, and 6-8/L. The seeded 50-L volumes were analysed in five 10-L aliquots to determine the number of oocysts and cysts in each. The data conformed to the Poisson distribution. This supports the interpretation that analysis of 10-L surface water samples resulting in not finding any target organisms is the result of their presence below the limit of detection. This interpretation strongly suggests that analysing fewer larger volume samples would provide more useful information.

Prevalence of Hepatitis B and Hepatitis C in Migrants from Sub-Saharan Africa Before Onward Dispersal Toward Europe.

Viral hepatitis is a global health care challenge due to its worldwide distribution, chronic persistence, complications, and high prevalence with unchecked conditions in areas like sub-Saharan Africa. A high proportion of asymptomatic infections allows serious complications and poses infection risk to destination populations. This study aimed to determine the prevalence of both HBV and HCV among 3248 migrants from different parts of sub-Saharan Africa newly arrived at Kufra, Libya, a remote agricultural North African city. All these migrants were required by the Libyan authorities to undergo a complete medical check-up for different purposes such as joining new jobs, and obtaining licenses for trade and commerce. UAT sera from 3248 migrants, aged 18-53 years, attending the Al Kufra city hospital from January 01 to December 31, 2019, were screened for HBsAg and anti-HCV antibody by rapid tests and positive samples were further tested by ELISA method. The results showed that 761/3248 (23.4%) of the migrants were positive for HBV and 1014/3248 (31.2%) were positive for HCV. Migrants from sub-Saharan Africa carry high rates of HBV and HCV infection. This suggests the importance of increased attention to actions to deal with findings among positive migrants, and for awareness about risks of transmission to the local population. Study results indicate the value of routine migrant monitoring, the need for awareness in destination country health authorities, and the potential for impact on migrant destination populations.

RT qLAMP-Direct Detection of SARS-CoV-2 in Raw Sewage.

A highly efficient, selective, and sensitive method for analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in raw sewage was developed and tested to illustrate basic characteristics of the procedure. The method uses reverse transcriptase (RT) loop mediated isothermal amplification (LAMP) in a quantitative application, RT qLAMP. The applicability of this procedure to detection of SARS-CoV-2 in clinical samples has been documented in many reports since early 2020. Basic LAMP characteristics depending on the multiple primer design that produce highly selective and sensitive target amplification virtually free of interferences in complex sample media make it ideal for application to target recognition in raw sewage. Three previously described primer sets targeting ORF1a, E- and N-gene regions were selected and tested to define method performance characteristics and performance for SARS-CoV-2 detection in raw sewage samples from a municipal sewage system serving > 600 000, between July and October, 2020. The virus was detected in all samples from each of three independent interceptors near their treatment terminus. Virus quantities varied significantly between samples and between primer targets within samples. Sewage sampling dates corresponded to relatively low COVID-19 incidence rates reported by the local service area health department. The limited number of samples and aggregating downstream sampling locations did not permit resolving concentration differences. The most significant finding was the ability of the RT qLAMP method to detect SARS-CoV-2 in the raw sewage samples directly without preprocessing to isolate or concentrate the virus or to extract and concentrate viral RNA.

Loop-Mediated Isothermal Amplification Detection of SARS-CoV-2 and Myriad Other Applications.

As the second year of the COVID-19 pandemic begins, it remains clear that a massive increase in the ability to test for SARS-CoV-2 infections in a myriad of settings is critical to controlling the pandemic and to preparing for future outbreaks. The current gold standard for molecular diagnostics is the polymerase chain reaction (PCR), but the extraordinary and unmet demand for testing in a variety of environments means that both complementary and supplementary testing solutions are still needed. This review highlights the role that loop-mediated isothermal amplification (LAMP) has had in filling this global testing need, providing a faster and easier means of testing, and what it can do for future applications, pathogens, and the preparation for future outbreaks. This review describes the current state of the art for research of LAMP-based SARS-CoV-2 testing, as well as its implications for other pathogens and testing. The authors represent the global LAMP (gLAMP) Consortium, an international research collective, which has regularly met to share their experiences on LAMP deployment and best practices; sections are devoted to all aspects of LAMP testing, including preanalytic sample processing, target amplification, and amplicon detection, then the hardware and software required for deployment are discussed, and finally, a summary of the current regulatory landscape is provided. Included as well are a series of first-person accounts of LAMP method development and deployment. The final discussion section provides the reader with a distillation of the most validated testing methods and their paths to implementation. This review also aims to provide practical information and insight for a range of audiences: for a research audience, to help accelerate research through sharing of best practices; for an implementation audience, to help get testing up and running quickly; and for a public health, clinical, and policy audience, to help convey the breadth of the effect that LAMP methods have to offer.

Cryptosporidium and Giardia detection in environmental waters of southwest coastal areas of Thailand.

The aim of this study was to investigate water samples collected in coastal areas of Southern Thailand in the years of 2005 and 2008 for their contamination by the protozoan parasites Cryptosporidium and Giardia. One hundred eighteen water samples of different origin were collected from six Tsunami affected southern provinces of Thailand in early 2005, and they have been analyzed using standardized methodology. Fifteen out of 118 samples (12.7%) were positive for Cryptosporidium spp. and nine (7.6%) positive for Giardia spp. Additional 42 samples from two same areas were examined 3 years later, in the early 2008. Five out of 42 (11.9%) samples were positive for Cryptosporidium spp., and three out of 42 (7.1%) were positive for Giardia spp.. Both protozoans were found in reservoir, river/canal, and pond waters. It appears no significant differences (p < 0.05) between Cryptosporidium and Giardia (oo)cysts levels during the two monitoring periods; however, the number of the investigated areas and samples in the second period was significantly less than in the first period. This is the first description on Cryptosporidium and Giardia (oo)cysts in water sources of Thailand, and it suggests the need for water quality control in the interest of public health safety.

Giardia and Cryptosporidium in children with diarrhea, Kufra, Libya, a North African migration route city.

BACKGROUND: Giardia and Cryptosporidium are common parasitic diarrhea agents of children contributing to childhood morbidity and mortality in developing countries. Major risk factors, based on the international literature, are expected to include domestic animals, fresh vegetables and drinking water and foodstuffs purchased from street vendors. These factors and sub Saharan migrants are common in the study area. Reports elsewhere indicate that person-to-person transmission is also important. OBJECTIVE: To assess the prevalence Cryptosporidium and Giardia in children with diarrhea in the Kufra City hospital and to assess risk factors for cryptosporidiosis and giardiasis. METHODS: A 10-month study, September 2013-June 2014 provided a fecal sample from 505 patients with diarrhea aged from 2 to 17 attending the outpatient clinics of local Kufra hospital, plus100 non symptomatic controls. Specimens were assayed for parasitic infection and for bacterial pathogens. Demographic information was obtained by questionnaire. RESULTS: Giardia was found in nearly 1/3 of the symptomatic population but few had Cryptosporidium. Mixed parasite infections were found in 1/7th of samples including: Shigella, Salmonella, Ascaris ova, E. coli, and E. histolytica. Infection frequency was age related, and risk factors included: domestic animals, foreign workers from Africa, contaminated fresh vegetables and drinking water. CONCLUSION: Cryptosporidium and Giardia were frequently associated with diarrhea in children in a remote desert agricultural community with many opportunities for infection. Contact with animals, foreign workers from Africa, fresh vegetables and drinking water sources contaminated with sewage materials are the likely modes of transmission of both organisms.

Cryptosporidium oocysts and Giardia cysts-A practical and sensitive method for counting and manipulating small numbers.

A rapid and inexpensive method is described for accurate and reproducible counting and manipulating small numbers of Cryptosporidium oocysts and Giardia cysts. From a suspension of oocysts or cysts at concentration from 1000 to 5000/mL (1-5/µL), replicate 5µL droplets are micro pipetted onto the edge of a microscope slide. Unstained oocysts or cysts in each droplet can be counted in a few minutes and replicated for statistical strength. The concentration of the suspension can then be verified by pipetting desired volumes containing approximately desired numbers onto confined 13mm membranes for IFA staining and counting with replication as desired. Requiring only a micropipette, analytical balance, and a microscope, this provides a useful tool accessible to virtually any microbiology laboratory.

Waterborne transmission of protozoan parasites: Review of worldwide outbreaks - An update 2011-2016.

This review provides a comprehensive update of worldwide waterborne parasitic protozoan outbreaks that occurred with reports published since previous reviews largely between January 2011 and December 2016. At least 381 outbreaks attributed to waterborne transmission of parasitic protozoa were documented during this time period. The nearly half (49%) of reports occurred in New Zealand, 41% of the outbreaks in North America and 9% in Europe. The most common etiological agent was Cryptosporidium spp., reported in 63% (239) of the outbreaks, while Giardia spp. was mentioned in 37% (142). No outbreaks attributed to other parasitic protozoa were reported. The distribution of reported outbreaks does not correspond to more broadly available epidemiological data or general knowledge of water and environmental conditions in the reporting countries. Noticeably, developing countries that are probably most affected by such waterborne disease outbreaks still lack reliable surveillance systems, and an international standardization of surveillance and reporting systems has yet to be established.

Cryptosporidium and cryptosporidiosis: The Asian perspective.

This review discusses findings of Cryptosporidium and cryptosporidiosis research in Asia and highlights the current situation of Cryptosporidium epidemiology, genetic diversity and distribution, and transmission throughout Asia taking into account all the available papers published for Cryptosporidium research in Asian countries since 2000. This effort will facilitate future research approaches and further developments in the understanding of Cryptosporidium epidemiology in Asia. The intent is to contribute to improvement in protection measures for mitigating the burden associated with this illness in the future.

Modelling of pharmaceutical residues in Australian sewage by quantities of use and fugacity calculations.

A conceptual model is presented for determining which currently prescribed pharmaceutical compounds are most likely to be found in sewage, and for estimating their concentrations, both in raw sewage and after successive stages of secondary sewage treatment. A ranking of the "top-50" pharmaceutical compounds (by total mass dispensed) in Australia over the 1998 calendar year was prepared. Information on the excretion ratios and some metabolites of the pharmaceuticals enabled prediction of the overall rates of excretion into Australian sewage. Mass-balance and fugacity modelling, applied to sewage generation and to a sewage treatment plant, allowed calculation of predicted concentrations of the compounds in raw, primary and secondary treated sewage effluents. Twenty nine of the modelled pharmaceutical residuals were predicted to be present in raw sewage influent at concentrations of 1 microgl(-1) or greater. Twenty of the compounds were predicted to remain in secondary effluent at concentrations of 1 microgl(-1) or greater.

The concentration of Cryptosporidium and Giardia in water--the role and importance of recovery efficiency.

The concentration of Cryptosporidium and of Giardia in surface water is a subject of importance to public health and public water supply. The term concentration is a fundamental property of any water quality parameter having a classical definition as used in chemistry and biology. Analytical methods for measuring the occurrence of Cryptosporidium and Giardia in water find only a fraction of the organisms actually present. This paper collects recently available data to examine the role and importance of recovery efficiency measurement to description of the concentrations of these organisms. Data from Australian sources graphically illustrate the variability of recovery efficiency at individual sites over relatively short time scales. Additional data on replicated recovery measurements establish their reproducibility. The recently released USEPA LT2 data along with those from Australia illustrate the independent variation of Cryptosporidium and Giardia occurrence and recovery efficiency at individual sampling locations. Calculation of concentration from paired raw numbers and recovery efficiency measurements clearly shows the magnitude and importance of taking recovery into account in expressing the concentration of these organisms.

Development and Evaluation of Oligonucleotide DNA Probes for Detection and Genotyping of Shiga-like Toxin Producing Escherichia coli.

Use of antisense oligonucleotide probes (OP) for detection and genotyping of Shiga-like toxin producing Escherichia coli (SLTEC) was evaluated. Based on published deoxyribonucleic acid (DNA) sequences of the A subunits of Shiga-like toxin (SLT) I and II genes, three synthetic antisense OP were constructed (OP-1, -2 and -3). Their use for detection and genotyping of SLTEC was evaluated and the results were compared to those obtained using cloned toxin-gene probe fragments. Both the OP-1 and OP-2 hybridized with all 69 SLT-I and II producing strains. Furthermore, the OP-1 hybridized only with all 18 SLT-I-only producing strains, and the OP-2 hybridized only with all 48 SLT-II-only producing strains. OP-3, a pool of four OP, detected all SLTEC strains regardless of toxin genotype. None of the OP hybridized to any of the 91 SLT-negative strains. These results demonstrate the three OP to be unique reagents for SLTEC epidemiological studies.