How many human pathogens are there in Laos? An estimate of national human pathogen diversity and analysis of historical trends

Objective The emergence of infectious diseases pose major global health threats. Estimates of total in-country human pathogen diversity, and insights as to how and when species were described through history, could be used to estimate the probability of new pathogen discoveries. Data from the Lao People’s Democratic Republic (Laos) were used in this proof-of-concept study to estimate national human pathogen diversity and to examine historical discovery rate drivers. Methods A systematic survey of the French and English scientific and grey literature of pathogen description in Laos between 1874 and 2017 was conducted. The first descriptions of each known human pathogen in Laos were coded according to the diagnostic evidence available. Cumulative frequency of discovery across time informed the rate of discovery. Four distinct periods of health systems development in Laos were identified prospectively and juxtaposed to the unmodelled rate of discovery. A model with a time-varying rate of discovery was fitted to these data using a Markov-Chain- Monte-Carlo technique. Results From 6456 pathogen descriptions, 245 discoveries of known human pathogens in Laos, including repeat discoveries using different grades of evidence, were identified. The models estimate that the Laos human pathogen species diversity in 2017 is between 169 and 206. During the last decade, there has been a 33-fold increase in the discovery rate coinciding with the strengthening of medical research and microbiology. Conclusion Discovery curves can be used to model and estimate country-level human pathogen diversity present in a territory. Combining this with historical assessment improves the understanding of the factors affecting local pathogen discovery. PROSPERO registration number A protocol of this work was registered on PROSPERO (ID:CRD42016046728).


Exclusion Criteria
Entries were excluded if: the disease or organisms was not specific to the species level; if the methodology used for the diagnosis was unclear and therefore could not be assigned an appropriate evidence grade; if the outcome of tests or examinations were unclear or returned a negative result; if the discovered organism was known not to be an agent of human infection or disease; if a symptom was described but could not be traced to a single causative organism (specifically referencing clinical diagnoses); if the organism could not be traced to current taxonomic practise or the causative agent could not be derived from the translation (unclear handwriting and or missing text). Reports of infectious diseases among emigrants, travellers and refugees were excluded as there is uncertainty whether the pathogen was contracted in Laos.

Notes on Lao Pathogen Line List Issues
Data line items were individually considered for eligibility on the basis of: likelihood of presence of pathogen, specificity of disease, pathogenic nature of organism (either identified as non-pathogenic or non-pathogenic to humans), taxonomic level available, a likely misspecification or contaminant, unknown sample specimen or a specimen collected outside of Lao PDR. The complied dataset was reviewed multiple times by MCC and PNN. Suspicious line items were followed up by a re-examining of the reference article and if need be further research or specialist consultation.

a. Unlikely Organisms
A line item that was excluded on the basis of likelihood was the Leishmania genus. The report came from the annual national RLG hospital report of 1966 (1). In the report numbers of "consult" referring to patients was listed as 644 and was attended by "consultations" admitted nor received any days of treatment. The laboratory report at the end of the document gave no confirmatory evidence.
Historically there have been investigations into the presence of Leishmania in Laos. A report from 1937 documented a negative report resulting from an investigation of spleen smears of dogs from Vientiane for the presence of Leishmania (2). In even earlier reports, "Kala azar" was mentioned in Rapport Medical Mensuel from 1923 and 1924 which indicate it was being at least looked for in what we presume is now the Microbiology Laboratory at Mahosot Hospital, but without any details of what laboratory tests were performed or clinical details it is impossible to confirm this diagnosis (3,4). A 2012 journal article (5) and searches of Pubmed and Google Scholar revealed no further evidence of Leishmania in Laos. With this uncertainty we decided not to record this disease as present in Laos, However, Leishmania is an emergent threat in this region with a growing number of case reports from its neighbour, Thailand, suggesting that future discoveries are likely (6).
The other line item excluded on this basis, was the Ross River virus. This appeared as a short paragraph in a French publication (7). There was insufficient information in the publication to source the original report. Subsequent searching of Pubmed and Google Scholar suggested that Ross River virus is not known to be in Lao or in any adjacent countries.

b. Unclear diagnosis
An unclear diagnosis was one where either the report was misleading or did not provide detail on the level of the diagnosis or for which no clear inference could be made, as per example, " 1 cas de spirochetose pulmonaire gueri par une injection intramusculaire" appeared in a 1920 report but the specific causative agent is unclear (8).

c. Misidentification
The first reports of schistosomiasis in Lao were as early as 1935 (9). The species that was recorded was stated to be Schistosoma mansoni. Later reports identify the species as Schistosoma japonicum. It was only in 1973, in a paper by Snormani et al., that is was first described as a separate species of a "japonicum-like" appearance (10). The "japonicum-like" species was given the name Schistosoma mekongi in 1978. The identification of the species is recognized in these data as 1973, as "japonicum-like" implies a distinct and separate organism (11). Schistosoma mekongi is clinically similar to Schistosoma mansoni and Schistosoma japonicum, suggesting that the records of these two Schistosoma species were a consequence of misidentification (12).
Isospora hominis and Sarcocystis hominis were recorded as separate organisms in a paper by Giboda et al. (13). Isospora hominis was subsequently identified as a separate stage of the organism Sarcocystis hominis (14). This implies that Giboda et al. double reported the same organism as separate pathogens, resulting from the previous misclassification.

d. Non-pathogenic in humans
For the purpose of this paper the Oxford English Dictionary definition of a pathogen was applied, which states (15): "A microorganism that can cause disease" A combination of PubMED, Google Scholar and textbook searches were used to determine the pathogenic nature of organisms (16). Within the items not meeting the eligibility criterion of pathogenic organisms were: Phthiriasis pubis and Pediculus humanus. These organisms were excluded on the basis of not meeting the definition of being agents of disease. Pubic and head lice are associated with discomfort but we decided not to regarded as causing disease.
However, the anthropods Sarcoptes scabiei, Chrysomya bezziana and Demodex spp. all meet the disease eligibility criterion and are included in the analysis, because they are associated with subsequent pathology post-inoculation.

e. Unspecified organisms associated with a disease, syndrome or pathogen description
A number of the hospital reports and early documentation listed a disease, syndrome or pathogen description. For many diseases, syndromes and pathogen descriptions are sufficient to identify singular causative agent, such as is the case with measles that is caused solely by the measles virus. Two descriptions of herpes simplex virus (HSV) 1 and 2 were categorised by their clinical manifestation (HSV1 is an oral and HSV2 is a sexually associated infection (17)). However, a number of clinical and phenotypic descriptions are common to multiple organisms. The report of "hookworm" in the data, which can refer to multiple organisms, was a frequent example of this. Line items of this nature were excluded, as it was not specific enough to allow for inference as to the causative organism.

d. Probable contaminants
Probable contaminants were identified by the isolate sample and the clinical features by PNN.

e. Samples collected outside Laos
These reports were excluded during the selection of appropriate literature. as, inclusion of samples collected outside of Lao invites uncertainty into the likelihood of the pathogen being present in Lao. There is no way to be sure that the organisms identified were not acquired elsewhere. Temporary resettlement is identified as a contributing factor to communicable disease, making refugees more susceptible to diseases acquired as a result of the displacement (18).

f. Unknown samples
A number of reports cited organisms without the sample from which they were isolated.
Often the isolates were recorded as being sampled from "Divers" which translates from French to English as "various". Without this information it is not possible to assign a grade for the strength of evidence. Since the strength of evidence was the basis of the analysis these organisms were excluded from the analysis.